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ROLES AND MECHANISMS OF OXIDATIVE PATHWAYS IN
CARDIOVASCULAR DISEASE
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of
Philosophy in the Graduate School of the Ohio State University
By
Suvara Kimnite Wattanapitayakul, M.S.
* * * *
The Ohio State University 2000
Dissertation Committee: Approved by Associate Professor John A. Bauer, Advisor
Professor Lane J. Wallace Adviser Professor Anthony P. Young College o f Pharmacy
Assistant Professor Dale G. Hoyt UMI Number 9962459
Copyright 2000 by Wattanapitayakul, Suvara Kimnite
All rights reserved.
UMI
UMI Microform9962459 Copyright 2000 by Bell & Howell Information and Leaming Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Aitor, Ml 48106-1346 Copyright by Suvara Kimnite Wattanapitayakul 2000 ABSTRACT
The primary goals of this thesis work were to further define the roles and mechanisms of oxidant related events during cardiac and vascular disease, particularly with respect to nitric oxide related actions, and to define potential anti-oxidant therapeutic strategies. In initial studies angiotensin II (ANGII) was administered to rats with a short term (3-day) sub-pressor dose and vascular tissues were then studied. Selective reductions in NO-dependent endothelial dysfunction was observed in ANGII treated animals, and this dysfunction was associated with endothelium specific prevalence of protein 3-nitrotyrosine (3-NT, a stable biomarker of tissue peroxynitrite formation). A novel image analysis technique was developed to quantify the extent of endothelial immunostaining, and a significant inverse correlation between endothelium mediated relaxant response and 3-NT immunoreactivity was observed. Endothelial peroxynitrite formation may therefore participate in (or initiate) early vascular effects of ANGII.
Isolated endothelial cells showed that ANG II induced concentration and time dependent increases in protein nitration, demonstrating the direct in vitro action of ANGII in eliciting this response. Time dependent protein nitration was also observed in isolated endothelial cells in the absence of ANG II stimulation, and endothelial cell lysates were shown to possess enzymatic activity in modifying nitrated protein requiring a serum cofiactor(s). These data suggest that nitration of protein tyrosine residues may be a regulated process through nitration/denitration mechanisms rather than a contemporary view of it being a cytotoxic phenomenon.
Cardiac remodeling is a key event in progressive heart disease involving selective myocyte dropout and fetal gene expression, but the initiating events are not well defined. Short-term (3-day) administration ANG II or TNF alone at physiologically relevant concentrations induced significant TUNEL positive nuclei
(apoptosis marker) and 4-fold increases in LV atrial natriuretic peptide mRNA
(fetal gene marker). In contrast, the combination of ANG II and TNF caused only changes in fetal gene expression. These changes in vivo were independent of hemodynamic or cardiac size changes, and not related to cardiac NOS II induction. Our findings suggest that important interactions between ANGII and
TNF exist in vivo, and that induction of NOS II is not associated with these early hormonal influences.
Ill In memory o f my mother and my father... who have fathomed me to the true meanings of love, loss, and .... life
IV ACKNOWLEDGMENTS
Many people have participated in assisting me building up my dissertation the way it is now. They are also being part of my appreciation of certain elements and aspects of sciences and the world during my long joumey in the graduate school. First of all, I wish to express my special gratitude to my adviser,
Dr. John Anthony Bauer, for his valuable guidance, intellectual support, zealous inspiration, and encouragement. These not only made this dissertation possible but also provided me a life-long foundation of becoming a scientist.
My parents have given me imperiled strength and supports. Their encouragement would remain in my mind eternally. Many thanks go to my sisters and brothers for their consistent supports throughout all these years.
I am in debt to Dr. Anthony Young for transfection experiment and his co advice on the cardiac gene therapy project. I also thank Dr. Terri Riffe and
Shawn Pierson for their assistance and companionship at the Neurobiotech.
Center on west campus. I wish to thank Or. Dale Hoyt for his kind advice and assistance in endothelial cell culture. Assistance from Dr. Cynthia Carnes in preparing Langendorff hearts is greatly appreciated. I am also in debt to Dr.
Bethany Holycross who provided me an initial guidance and assisted me to become proficient in rat surgery and Northem blot analysis. V Thank all the pharmacology colleagues and friends (Alysia Chaves. Mike
Mihm, Dave Weinstein, Brandon Schanbacher, and others) who provided a very favorable environment and enchantment during my graduate school years.
Special appreciation for ourfrierxtship is sent to Rosario Rojas who always resurrects my passion on sciences.
I am grateful to the Royal Thai Government for providing me the opportunity to study abroad, and for the major financial support in my graduate school training.
VI VITA
September 11,1968 ...... Bom - Bangkok. Thailand
1991 ...... B.S. Pharmacy (Hon.), Mahidol University
1991 -1 9 9 4 ...... Medical Scientist,
National Institute of Health,
Nonthaburi, Thailand
1994 - 1998 ...... The Royal Thai Government Scholar
1998 ...... M.S. Pharmacy Administration,
The Ohio State University
1998 - present ...... Graduate Teaching and Research
Associate, The Ohio State University
PUBLICATIONS
1. SK. Wattanapitayakul, DM. Weinstein, BJ. Holycross, and JA. Bauer,
“Endothelial dysfunction and peroxynitrite formation are early events in
angiotensin-induced cardiovascular disorders”. FASEBJ 14(2):271-278,
(2000).
VII 2. S. Wattanapitayakul, DM Weinstein, BJ. Holycross, and JA. Bauer, “Cardiac
apoptosis and iNOS expression following angiotensin II, TNF-a, or their
combination” FASEB J 13(5), Pt II, A753, (1999).
3. S. Wattanapitayakul and JO. Schommer, “The human genome project:
benefits and risks to society”. Drug Information J.33(3),729-35, (1999).
4. S. Wattanapitayakul, DM. Weinstein, BJ. Holycross, and JA. Bauer,
“Angiotensin II induced peroxynitrite formation in rat vasculature”. Circulation
98(17), SuppI 1395-1396, (1998).
FIELDS OF STUDY
Major Field: Pharmacy
VIII TABLE OF CONTENTS
Page
Abstract...... ii Dedication ...... Iv Acknowledgments ...... v V ita ...... vii List of Tables...... xi List of Figures ...... xii
Chapters; 1. Introduction: Oxidative pathways in cardiovascular disease ...... 1 2. Endothelial dysfunction and peroxynitrite formation are early events in angiotensin ll-induced cardiovascular disease ...... 49 3. Digital image analysis for immunohistochemical evaluation ...... 80 4. Angiotensin II induces endothelial cell protein nitration in vitro ...... 93 5. Interaction of angiotensin II and TNF-a in vivo ...... 118 Part I: Cardiac TUNEL staining and NOS II induction ...... 119 Part II: Neurohormones and gene expression ...... 152 6. Cardiac gene therapy: recent development and a new approach ...... 167 7. Conclusions ...... 214
Bibliography ...... 220
IX Appendix I. The human genome project: benefits and risks to society ...... 247 Appendix II. The role of prescription drug Insurance type and drug cost Information on physician antibiotic selection for the elderly ...... 265 Appendix III. Genetic variations In nitric oxide synthase Isoforms: evidence, significance, and therapeutic Implications ...... 287 LIST OF TABLES
Table Page
1.1 Evidence of increased ROS production in cardiovascular disease ...... 4 1.2 Antioxidant supplements in cardiovascular disease ...... 30 3.1 Sufficiency of sampling 25% of the endothelial perimeter area ...... 88 5.1 Organ weights of rats treated with saline vehicle (VEH) angiotensin II (ANG II), tumor necrosis factor-a (TNF). or combination of ANG II and TNF (AT) ...... 164 6.1 Review of cardiac gene transfer attempts ...... 175-177 6.2 Infusion of transfected fibroblasts into Langendorff hearts ...... 208
XI LIST OF FIGURES
Figure Page
2.1 Plasma renin activity and hemodynamic parameters measurement in saline control (vehicle, VEH), angiotensin II (ANG II), tumor necrosis factor-a (TNF), and the combination of ANG II and TNF (AT) ...... 69 2.2 Vascular contractile responses in isolated aortic rat rings ...... 70 2.3 Vascular relaxation responses in isolated aortic rat rings ...... 71 2.4 Semi-quantitative digital image analysis method for rat aortic endothelial 3-nitrotyrosine immunoreactivity ...... 72 2.5 Extent of 3-NT immunoreactivity in rat aortic endothelium and correlation to vascular function ...... 73 3.1 Sampling regions in the aorta ...... 89 3.2 Semi-quantitative digital image analysis method for rat aortic endothelial 3-nitrotyrosine immunoreactivity ...... 90 3.3 Inter-observer correlation analysis of 3NT immunoprevalence ...... 92 4.1 Endothelial cell growth curve ...... 113 4.2 Nitrated BSA concentration and 3NT immunoreactivity curve ...... 114 4.3 Angiotensin ll-induced protein nitration in endothelial cells in vitro ...... 115 4.4 Quantitative 3NT immunoprevalence in angiotensin ll-induced endothelial cell nitration ...... 116 4.5 Endothelial cell denitration ...... 117
XII 5.1 Plasma renin activity (FRA) and hemodynamic parameters measurement in saline control (vehicle, VEH), angiotensin II (ANG II), tumor necrosis tector-a (TNF), and the combination of ANG II and TNF (AT) ...... 144 5.2 Measurement of body weight and cardiac mass (left ventricle, LV; total heart weight) following infusion of VEH, ANG II, TNF and AT ...... 145 5.3 Representative photomicrographs of TUNEL positive nuclei (CardioTACS system) in the left ventricles of rats infused VEH, ANGII, TNF, and AT on day 3 ...... 146 5.4 Representative photomicrographs of NOS II immunohistochemisty in left ventricles of rats infused with ANGII, TNF, or their combination on day 3 ...... 147 5.5 Quantitative digital image analysis for in situ nick end-labeling (TUNEL) nuclei and NOS II immunoprevalence in rat myocardium after 3-day Infusions of VEH. ANGII, TNF, or AT ...... 148 5.6 Statistical associations between NOS II immunoprevalence and numt>er of apoptotic nuclei in cardiac tissues ...... 150
5.7 Effects of ANG II and/or TNF infusion on plasma levels of renin activity, aldosterone, and atrial natriuretic peptides ...... 165 5.8 Effects of ANG II and/or TNF infusion on gene expression. Northem blot analysis showed alteration in mRNA expression of AOGEN in the liver, renin in the kidney, and ANP in the left ventricle of rats infused with ANG and/or TNF ...... 166 6.1 Schematic representation of transfection procedure ...... 209 6.2 Photomicrographic representation of enzymatic and immunohistochemical detection of rat skin fibroblast expressing p-galactosidase ...... 210 6.3 Diagram of growth curves of normal rat skin fibroblasts and fibroblasts stably transfected p-galactosidase gene ...... 211 xiii 6.4 Percentage of infused fibroblasts remaining in the Langendorff hearts ...... 212 6.5 Immunohistochemical detection of rat skin fibroblasts expressing p-galactosidase in the Langendorff heart ...... 213
XIV CHAPTER 1
INTRODUCTION:
OXIDATIVE PATHWAYS IN CARDIOVASCULAR DISEASE
This chapter will provide an introduction to the overall research problem of my research project and will address the following questions:
Do oxidative mechanisms play a mie in cardiovascular disease?
Where do the oxidants come from?
What are the relevant oxidative events?
What are the therapeutic implications? INTRODUCTION
Despite some recent declines, cardiovascular disease remains the
principle cause of death in both developed and developing countries, accounting
for roughly 20% of all deaths (14 million worldwide) per year. In the United States
cardiovascular disease represents the leading killer of men over 45 years old and
women over 65 years old, is responsible for one in three deaths, and accounts
for approximately 750,000 mortalities each year (Hennekens, 1998). These
conditions include essential hypertension, coronary artery disease, myocardial
infarction, and cardiac failure. Cardiac disease is progressive and often
associated with inter related disease states that typically require many years of
therapy and are associated with extensive health care costs (more than $10
billion annually).
Evolving therapeutic strategies of cardiovascular disease
Since cardiovascular disease has been viewed as a hemodynamic
disorder, traditional approaches for the treatment of cardiovascular disease
states rely heavily on the use of small molecule drug therapies. These typically
include agents that reduce fluid retention and/or systemic blood pressure,
enhance cardiac contractility, or lower blood lipid levels. Thus, many of these
strategies were historically intended to alleviate of symptoms of disease rather than addressing mechanistic processes. Interestingly, several agents used to
control symptoms in the short term (such as diuretics and direct acting vasodilators) have not t)een shown to affect long term survival or disease 2 progression in large clinical trials (Levinson et al, 1999). Thus, hemodynamic status may not be a sole determinative factor in disease progression. As the mechanisms of cardiovascular disease are increasingly defined, therapeutic strategies have evolved. For example, more recent approaches in cardiovascular disease management involve inhibition of pathways that contribute to disease progression. For example, angiotensin-converting enzyme (ACE) inhibitor captopril and its analogs have shown symptomatic improvement (e.g. improved cardiac performance and reduction of cardiac hypertrophy) and significant improvement in survival (Sharpe, 1999). ACE inhibitors are now established as standard initial and maintenance treatment for congestive heart failure in combination with diuretics.
Oxidative events In cardiovascular disease
Several recent studies have demonstrated that altered oxygen utilization and/or increased formation of reactive oxygen species (ROS) contribute to cardiovascular disease progression. Shown in Table 1.1 is a brief listing of evidence for oxidative mechanisms in these conditions. Condition Evidence Reference (as of January 2000) Hypertension -VSMC proliferation induced by ROS in - Griendling and vitro and in vivo Ushio-Fukai, 1998 -Ang II promotes oxidant production via - Fukai, 1999 NADPH oxidase -Superoxide production-mediated - Kanani et al, endothelial dysfunction 1999 Coronary artery disease -Superoxide production-mediated -Britten et al, 1999 (atherosclerosis) endothelial dysfunction -oxidized LDL -Jimi et ai. 1998 Myocardial infarction -ischemia/reperfusion injury driven by ROS -Ferrari et al, 1998 formation -oxidant-derived myocyte necrosis and/or - Anversa 1998 apoptosis Heart failure -cardiac dysfunction induced by increased -Sawyer et al, NO production 1998 -cardiac apoptosis induced by cytokines- -Pulkki, 1997 derived ROS
Table 1.1 Evidence of increased ROS production in cardiovascular disease
(representative examples, not intended to be exhaustive)
The initial suggestions of oxidative mechanisms during cardiovascular disease were described in the acute settings of ischemia/reperfusion injury and myocardial infarction. These conditions are associated with a sudden reduction of coronary perfusion and oxygen availability, leading to altered myocardial metabolism, ROS production and cell death. Interestingly, ROS production and associated cellular damage is higher in cardiac tissue during tissue reperfusion relative to ischemic conditions.
The acute (and relatively severe) paradigm of cardiac ischemia and reperfusion has provided insight into the mechanisms of ROS-induced alteration of cardiac function and disease progression. In fact, it is now recognized that
ROS may contribute to the progression of other more chronic cardiovascular conditions that are not related to acute oxygen deprivation. Supportive evidence of these phenomena are briefly listed in Table 1.1. In addition to cell and/or tissue evidence of oxidative processes, increased plasma levels of oxidative stress markers have t>een commonly observed in patients with several chronic pathologic conditions, including congestive heart failure (CHF). diat)etes, and
AIDS. Elevated plasma levels of oxidative stress markers are detected in several pathologic conditions of cardiovascular disorders, including ventricular hypertrophy, atherosclerosis and coronary heart disease, and congestive heart failure (Hatjai, 1999;Boaz et al, 1999; Gokce et al, 1999; 1997;Keith et al, 1998).
It has been shown that increased oxygen radical production in endothelium is involved in pathology of hypertension and atherosclerosis (Carlos et al,
1998;Suzuki H. etal, 1998;Milleretal, 1998)
In summary, despite the diverse etiology of cardiovascular conditions, the enhanced production of ROS and altered oxygen utilization is apparently a common phenomenon and a participant in disease progression. Further understanding of the events that contribute to these changes, and the cellular adaptations involved may provide new opportunities fo r rational therapeutic strategies. SOURCES OF REACTIVE OXYGEN SPECIES (ROS) DURING
CARDIOVASCULAR DISEASE
The general phenomenon of increased ROS production in cardiovascular disease is becoming increasingly evident, but the actual sources of these species and mechanisms involved may not be identical in all conditions. Important ROS in mammalian cells include superoxide anion (O 2"'), hydroxyl radical (OH*), and hydrogen peroxide (H 2 O 2). ^ described below, there are several potential contributors to cellular ROS increases during cardiovascular disease:
UncouDlina of mitochondria electron transport
This is a classical mechanism of intracellular oxidant production. Under normal physiological conditions these potentially toxic species are formed intracellularly, primarily by mitochondria for oxidative phosphorylation reactions and production of ATP. During normal cellular respiration these reactive species are controlled by several antioxidant pathways that include enzymes (superoxide dismutase, catalase, glutathione peroxidase) and small molecule antioxidants
(tocopherols, ascorbate, thiols, others). In contrast to these highly ordered conditions in healthy cells, during conditions of hypoxia mitochondrial electron transport is uncoupled, leading to accumulation of toxic metabolites, increased membrane permeability and accumulation of mitochondrial Ca^*(Ferrari, 1996).
This disruption of mitochondrial electron transport leads to uncontrolled and disordered production of ROS that escape antioxidant control mechanisms.
6 Induction of oxIdmUve enzvme*
While "escape" of ROS from mitochondria has been a classical mechanism of intracellular oxidation, many recent studies suggest that non- mitochondrial sources may be equally or perhaps more important in some cardiovascular conditions. Activation and/or induction of cytosolic oxidases (such as NADH/NADPH oxidase, xanthine oxidase, and nitric oxide synthase isoforms) have been demonstrated during several physiological stress conditions. The potential significance of these processes is described below.
Xanthine oxidase (XO): Under normal physiological conditions XO is a key enzyme in the purine degradation pathway. The enzyme generates the final product uric acid (excreted by urine), and the byproduct superoxide anion. Under normal physiological conditions this enzyme is localized almost exclusively in the liver and the small intestinal mucosa. However, oxidative stress conditions such as prolonged hypoxia or increased inflammatory cytokines can enhance XO activity and its release into plasma. It has been demonstrated that significantly increased XO activity (and superoxide production) in the mesenteric tissue may responsible for escalating vascular tone in the animal model of essential hypertension (Suzuki et al, 1998). The circulating XO may bind to target tissues and can be concentrated more than thousand-fold at the cell surface or interstitial matrix of the vasculature (Houston et al, 1999). Clearly, ongoing investigations of this accumulation of endogenous oxidative enzyme would expand the knowledge of ROS generating systems in cardiovascular disease. 7 NADH/NADPH oxitiasm: The enzyme NADPH oxidase is generally found in phagocytic cells. It plays an important role in non-specific host defense during
Infection by generating large quantity of superoxide (millimolar). Recently, it has been demonstrated that vascular NADH/NADPH oxidase significantly contribute to superoxide production in all components of the vasculature, i.e. endothelium, medial smooth muscle, and adventitia (Bayraktutan et al, 1998; Wang et al,
1999). Participants in cardiac disease such as angiotensin II may activate superoxide production in vascular tissue via this enzyme, and it has been implicated in the pathogenesis of angiotensin ll-induced vascular hypertrophy and endothelial dysfunction (Griendling et al, 1994; Rajagopalan et al,
1996;Ushio-Fukai et al, 1996).
N itric oxide synthases (NOS): It has been recently recognized that several cytokines such as IL-1p, IL-6, IFN-y, and TNF-a are pro-oxidants .and they are commonly found elevated in plasma of patients with heart disease.
Increased nitric oxide (NO) production via induction of NOS has been suggested as a major mechanism by which cytokines mediate cardiac contractile dysfunction and development of heart disease (Shulz, 1995; Wildhirt,
1995;Sawyer, 1998). Additionally, other potent oxidants can be generated from reactions of NO with other ROS. For example, NO avidly reacts with superoxide anion at diffusion-limited rates to form the potent oxidant peroxynitrite (ONOO ).
The presence of peroxynitrite and its biological marker 3-nitrotyrosine has been associated with several oxidant-related pathologic conditions, including 8 atherosclerotic lesion, endothelial dysfunction, reperfusing-ischemic damage, cardiac sepsis, and myocarditis (Kooy. 1997; Wattanapitayakul. 2000). Reactions of peroxynitrite with pathologically relevant molecules have been implicated in oxidative-related cardiovascular disorders (see below).
The cytokine-induced increases in oxidants are regulated by various stimuli in several cell types engaged in tissue repair and restoration of homeostasis as well as immune response. For example, increased cytokine release was found in acute hypoxia followed by reperfusion and in myocardium stunning. These cytokines enhance expression of a variety of cell adhesion molecules (e.g. ICAM-1, VCAM, and MCP-1) in myocardium leading to transient leukocyte sequestration and its transmigration to the areas of cardiac injury
(Matsumori et al. 1997;Kacimi et al. 1998). In addition to acute activation of cytokines in myocardial infarction, elevated plasma levels of proinflamatory cytokines along with high oxidase activities are commonly observed in a more chronic pathologic condition of the cardiovascular disorders such in heart failure patients (Leyva etal..1998;Torre-Amione. 1996;Katz. 1994; Milani, 1996).
Recent studies suggest that the heart per se is capable of synthesizing biologically active TNF-a which may responsible for the progression of heart diseases. (Kapadia et al. 1995; Bergman and Holycross, 1996).
9 CONSEQUENCES OF CARDIAC OXIDATIVE STRESS
Cardiac and Vascular Dvfunction
Not only are oxidants associated with cardiovascular disease, they may actually mediate some aspects of cardiac and vascular dysfunction. For example,
In both experimental and pathologic conditions impaired vascular function and decreased cardiac performance are mediated by oxygen-derived free radicals such as H 2 O2 , peroxynitrite, superoxide anion, and hydroxyl radical (Rubanyi and
Vanhoutte, 1986; Matsuura and Shattock, 1991 ; Terada, 1996; Bouloumié et al,
1997;Miller et al, 1998;Ferdinandy et al, 1999). ROS have direct impact on myocardial function through inhibition of the sarcoplamic reticulum Ca^* pump in cardiac contraction-relaxation cyde (Matsubara and Dhalla, 1996;Temsah et al,
1999). Cardiac depression (cardiac stunning) during ischemic/reperfusion and tissue damage in acute myocardial infarction was prevented by using superoxide dismutase and antioxidant vitamin E (Nagel et al, 1997; Carrasquedo et ai,
1999).
Proinflamatory cytokines and immune cell activation are modulators of cardiovascular functions by a variety of mechanisms, induding the generation of oxygen-derived free radicals and production of nitric oxide (Cheng et al, 1999).
Elevated plasma and tissue levels of the cytokine TNF-a are commonly observed
In severe cardiac depression along with increased NO production (Torre-Amione etal, 1996, Wildhirt et al, 1995; Fukuchietai, 1998). Inhibition of superoxide production prevented cytokine-induced cardiac dysfunction (Cheng et al, 1998). 10 Endothelial-derived NO plays a key role in the local regulation of vasomotor tone and prevention of thrombus formation. Studies from our laboratory and others have suggested that reduction in bioavaliability of NO and endothelial dysfunction may be the initial event in atherosclerosis and cardiovascular disorders (Ferrari etal, 1998; Lyons, 1997; Wattanapitayakul,
2000).
C e ll D eath
Two types of cell death-necrosis and apoptosis-are both implicated in the oxidative-related cell loss in cardiovascular tissue. Generally, necrotic cell death is associated with inflammatory cell infiltration and subsequent collagen deposition and scar formation, while apoptotic cell death is differentiated by ultrastructure and biochemical features such as cyptoplasmic and nuclear condensation, formation of membrane-bound apoptotic body, and DMA fragmentation ('-ISO bp). Although recognized since 1972, the apoptotic phenomenon has just become center of attention in cardiovascular disease in the past decade. It appears that cardiac and vascular cell loss observed during remodeling process occurs in the absence of necrosis. The role of ROS involved in both types of cell death are discussed below.
N e cro sis: Prolonged ischemic condition may cause irreversible myocardial cell injury and cell death via necrosis. Transmigration of immune cells from vasculature into the myocardium results in a release of toxic mediators 11 that induce myocardial cell dysfunction and necrotic cell death (Grisham et al.
1998). Unlike apoptosis, necrosis does not generally appear in more chronic oxidative conditions.
A p o p to s is Recently, : the recognition of a different cell death phenomenon ‘apoptosis’ has become a major clinical interest since it accounts for a great proportion of cell death assodated with myocardial infarction and/or myocardial ischemia/reperfusion. Cell loss through apoptosis contributes to the impairment of cardiac performance and also plays an important role in myocardium and vascular remodeling processes. Induction of apoptosis is implicated in atherogenesis and cardiac dysfunction. Not only ROS per se but also their oxidative products and other secondary messenger molecules generated by ROS can trigger the programmed cell death. For example, oxidized low density lipoprotein (oxLDL) has shown to induce DMA fragmentation and apoptosis in macrophages (Kinsherf et al, 1998). The antioxidant N- acetylcysteine (NAG) prevented apoptosis induced by oxLDL, ceramide, TNFa, and H2O 2 (Kinsherfetal, 1998).
ROS-induced cardiac apoptosis is mediated through several signaling systems including intracellular calcium, cytokines, lipid oxidation, and proto oncogene activation. The perturbation of intracellular Ca^* homeostasis by cellular redox state can aggravate free radical reactions and activate endonucleases such as caspases (key enzymes in apoptotic pathway) that contribute to the programmed cell death. 12 It has been shown that the saturated fatty adds palmitate and stearate induce apoptosis in neonatal rat myocytes (DeVries JE, et al, 1997). Both palmitate and stearate are precursors of de novo synthesis of ceramide-a second messenger of the sphingomyelin signaling pathway initiated by hydrolysis of plasma membrane sphingomyelin to ceramide. Ceramide and sphingolipid metabolites are involved in the antiproliferative responses and apoptosis in several cell types induding cardiac myocytes. Several stimuli known to trigger sphingolipid-induced apoptosis indude TNFa, y-IFN, ionizing irradiation, and ischemia/reperfusion (Sparagna and Hickson-Bick, 1999). The cytokine TNF-a is apparently pro-apoptotic via several signaling pathways, induding activation of
Fas ligand, and binding of TNF to the death domain of TNF receptors. Binding of
TNFa and the Fas ligand to their receptors results in degradation of sphingomyelin to ceramide which mediates apoptosis through induction of
JNK/SAPK (see section “Altered gene expression”).
Induction of the proto-oncogene p53 and/or inhibition of bd-2 is indicated in the several stimuli that induce apoptosis. Intrinsic degree of oxidative stress regulates susceptibility to apoptosis induction through p53-dependent and p-53- independent pathways (Lotem et al, 1996).
The stimulus NO can send both proapoptotic and antiapoptotic signals depending on level of antioxidants and predominant regulatory pathways endowed in different cell types (Kim et al, 1999). For example, it appears that
NO-induced apoptosis in vascular smooth muscle cells is mediated by cGMP accumulation while intracellular elevation of cGMP in response to NO activation 13 protects apoptosis in PC12 cells and hepatocytes. The formation of peroxynitrite from Interaction of NO and superoxide may account for the proapoptotic effects
of NO. Peroxynitrite is a potent oxidant that induce DNA fragmentation and p53-
dependent apoptosis. Accumulation of the tumor suppressor protein p53 is a
crucial and early event in NO-mediated apoptosis (Kim et al, 1999).
Cellular Taroete of Reactive Oxvoen Specie»
Depletion of GSH and other enttogenous oxidative Oxidative stress is a reflection of intracellular concentration of oxidants such as H2O2 and 02~* as well as antioxidants defense, such as glutathione (GSH). Glutathione, a tripeptide consisting of glutamate, cysteine and glycine, is important for cellular defense against ROS toxicity. It is the key cellular reductant to maintain redox state of cysteine-thiol linkage in proteins. The intracellular levels of oxidized GSH (GSSG) increase by the metabolism of H 2 O 2 and by glutathione peroxidase, but decrease by extracellular export and glutathione reductase. Depletion of protective the form of GSH by excess amount of ROS leads to increased protein oxidation at cysteinyl-thiol linkage and subsequent changes in protein conformation and protein function. These alterations can occur to important proteins that function as receptors, enzymes or signaling proteins thus impairing normal cellular processes. Additionally, GSH participates in maintaining antioxidant ascorbic acid (vitamin 0 ) in its reduced form (the active form). Other important functions of GSH include coenzyme activities and detoxification process (forming GSH conjugates and excreted through urine). 14 Low levels of GSH are assodated with a number of disease conditions known to generate high amount of ROS. as observed in atherosderosis, heart failure, diabetes, neurodegenerative disorders, and AIDS. Increased oxidative stress in CNF patients is assodated with increased glutathione peroxidase and decreased plasma antioxidant vitamins, e.g. vitamin E and C (Keith et al. 1998). It has been shown that the proinflammatory cytokine T N F -a decreased cellular GSH level and many antioxidants abolished the T N F -a effects (Suzuki et al. 1997). In addition to GSH. the most important endogenous antioxidant defense against superoxide is superoxide dismutase (SOD). Three isoforms of SOD have been cloned and identified as: mitochondrial manganese-containing SOD (MnSOD); cytosolic copper-zinc SOD (Cu-Zn SOD); and extracellular SOD (EC- SOD). Cu-Zn SOD plays an important role in protecting NO from destruction by superoxide in the endothelium (Harrison. 1997). NO is important in reducing ischemic/reperfusion-induced adhesion of monocytes to post-ischemic endothelium and inhibiting induction of proinflammatory cytokine and adhesion molecule synthesis (Grisham et al. 1998). Endothelial dysfunction is one of the earliest events in the pathogenesis of myocardial reperfusion injury. The early decline in coronary NO release occurs simultaneously with the oxygen-derived free radical burst observed in ischemic/reperfused heart (Zweier et al. 1987). Additionally. NO plays an important role in cardioprotection during reperfusion by directly enhancing coronary blood flow and preventing adhesion of immune cells. 15 Upfd metabolism and lipid porwddation: Lipids, both in free or bound forms, are vulnerable and represent one of the immediate targets of ROS. The overall effects of lipid peroxidation include diminishing membrane fluidity, increasing membrane permeability, destabilizing membrane receptors, and inducing immune response to altered phospholipids. Described below are three major lipid categories that contribute to pathogenesis of cardiovascular disease: Long chain free fattv acids (FFAh Elevated plasm a levels o f FFA are implicated in many pathologic conditions, as observed in myocardial ischemia, diabetes, hyperlipidemia, and cardiac hypertrophy (Sparagna and Hickson-Bick, 1999). Under normal physiological condition of the heart, FFA are the preferred source of energy generated via p-oxidation within the mitochondrial matrix. FFA are estimated to account for 60-70% of oxygen consumption for energy production (Grynberg and Demaison, 1996). However, p-oxidation cannot proceed under oxygen-deprived conditions such in hypoxia or ischemia where FFA and their metabolites become harmful. When FFA metabolism is inhibited metabolic intermediates are accumulated and insert themselves into lipid bilayer membrane, i.e. sarcolemma, sarcoplasmic reticulum and mitochondrial membrane. The lipid metabolic intermediates interfere with membrane integrity and function of membrane-bound enzymes by altering their conformation and ion pumps. High levels of free fatty acid and metabolites impair calcium homeostasis and ion gradients, which may lead to cardiac arrhythmias during ischemia/reperfusion. (Hendrickson et al, 1997). 1 6 Membrane lioids: In addition to oxidation of FFA in the cytosolic compartment, ROS also react with membrane-bound lipids leading to ‘lipid peroxidation’. Generally, reperfusion injury appears when oxygen is exposed to reactive intermediates compounds formed during the ischemic condition. The interaction of those compounds leads to the generation of oxygen free radicals and highly active molecules such as superoxide anion, hydroxyl radical, and hydrogen peroxide that can react with polyunsaturated lipids in membranes. Lipid peroxidation products can inhibit protein synthesis and alter enzyme activities. Their effects can be accentuated by high levels of FFA intermediates. Oxidation of polyunsaturated fatty acid moieties of membrane phospholipids can cause membrane disintegration, mitochondrial dysfunction and calcium overload. It has been shown that high redox state and increased lipid peroxidation are associated with the transition of cardiac hypertrophy to heart failure (Dhalla et al, 1996). Elevated levels of plasma lipid peroxides were also observed in CHF patients (Keith etal, 1998;Diaz-Vélezetal,1996). Upoproteins: It is widely accepted that hypertriglyceridemia and abnormal lipoprotein profile are associated with an increase in cardiovascular risk. Polyunsaturated fatty acid residues in lipoproteins are chemically vulnerable for free radical oxidation. Unlike superoxide anion, other oxidizing species such as hydroxyl, peroxyl and alkoxyl radicals are capable of entering the hydrophobic membrane interior and initiating free radical chain reactions (Maxwell and Lip, 1997). Therefore, endogenous antioxidants in the hydrophilic cellular compartment cannot prevent the propagating of carbon-centered radical and 17 oxygen reaction leading to long chain lipid hydroperoxide (peroxide). These peroxides are unstable and degrade rapidly to form secondary products which are toxic to cells. These adducts may interact with the apolipoprotein moiety of lipoprotiens such as apoproteinB (apoB) moiety in low density lipoprotein (LOL) resulted in modified apoB. The oxidized LDL represents not only abnormal cellular recognition but also becomes chemotactic for circulating monocytes. After monocytes enter the arterial wall, they differentiate to macrophages and take up oxidized LDL due to its immunogenidty. The unregulated uptake of LDL leads to the formation of foam cells and ultimately results in fatty streak-the first phase of an atherosclerotic lesion. Additionally, oxidized LDL promotes production of several cytokines and growth factors, and increases platelet aggregation that can further aggravate the lesion and cause arterial wall thickening. Protein modifications: In addition to their direct impact on cardiac function, ROS generated during pathophysiology of the heart can oxidize amino acid side chains and protein backbone leading to protein-protein cross-linkage and protein fragmentation (Berlett and Stadtman, 1997). As a consequence of these modifications, signaling proteins may not function properly which may lead to organ malfunction and even cell death. Two major protein derivatives have been used as markers of oxidative stress-mediated protein modifications-protein carbonyl derivatives and protein nitrotyrosine derivatives. The presence of carbonyl groups in proteins has been referred as ROS-mediated protein 18 oxidation while nitrated tyrosine residues (3NT) have been identified as a product of peroxynitrite-dependent nitration (ONOO", mainly derived from the interaction o f O 2" and NO). Elevated levels of oxidized protein and nitrated proteins are observed in both animal models and patients under various conditions of oxidative stress, including ischemia-reperfusion, CHF, sepsis, and pulmonary fibrosis (Liu et al, 1997; Kooy et al, 1997; Szabo et al. 1997; Saleh et al, 1997.) Altered signal transduction C a ^ S ig n a lin g : is a second messenger that regulates a broad array of biological processes in cardiac tissue, including contraction, neurotransmission, gene expression, and cell growth. Two sources of Ca^^ — influx across the sarcolemmal membrane and Ca^* from sarcoplasmic reticulum (SR) of cardiac cells—are critical for excitation-contraction coupling in cardiac tissue. Oxidative stress mediated by ischemia-reperfusion induces excessive intracellular calcium accumulation (Tani, 1990;Kaneko, 1994). Suggested mechanisms of oxidants-induced Ca^* influx from extracellular to intracellular space include increased membrane ATP-dependent Ca^* binding, activation of Ca^* and IC channels, changes in Na*, NaV Ca^* exchangers and Na*/lC ATPase activities, and stimulation of the adrenergic system (Chakraborti et al, 1998). Additionally, an increase in cytosolic Ca^* is transiently enhanced by the inhibition of Ca^*-pump of the SR, resulting in a passive moving of SR Ca^* to cytosolic space (Suzuki et al, 1997). Other cellular components such as 19 mitochondria and Ca^^-binding proteins have also been suggested as sources of oxidant-induced Ca^* release. ProiBin phosphorylation: Protein phosphorylation regulates a wide array of cellular signaling pathways that carry signals through different functional proteins, including enzymes, receptors, tanscription factors, and contractile elements (Suzuki et al, 1997). Generally, oxidant-related stimulation of protein phosphorylation acts through the enzymes that regulate the balance of phosphorylation (kinases) and dephosphorylation (phosphatases) of specific amino acid residues of signaling proteins. The overall increases in protein phosphorylation generally reflect stimulation of the signaling pathway. Two well- defined subtypes of protein phosphorylation are discussed below: Tyrosine Dhosohon/lation: Most of growth factor receptors are transmembrane tyrosine kinases (RTKs) with exception of TGF-p and IFG-II. These kinases are sensitive to ROS stimulation, leading to activation of downstream signaling pathways such as the protein kinases of the MARK cascade (see below), phospholipase C, and phosphatidylinositol-3-kinase (Kamata and Hirata, 1999). Serine/threonine phosphorylation: The mitogen-activated protein kinase (MARK) superfamily of protein serine/threonine-kinases is a group of enzymes that are involved in the regulation of cell growth, proliferation, and differentiation as well as oxidative responses. There are three major subfamilies of MARKs, including the extracellular responsive kinases (ERKs), the c-Jun N-terminal kinases (JNKs, also known as stress-activated protein kinases-SARKs), and the 20 p38-MAPKs. The ERKs appear to be associated with cell growth and differentiation while the JNKs and the p38-MAPKs are involved in responses to cytotoxic insults. In the heart, the ERKs are primarily activated by G protein- coupling receptor agonists such as a-adrenergic receptor agonists, angiotensin II, and endothelin-1, leading to activation of phospholipase C cascade and ultimately activation of protein kinase C (PKC) (Yamazaki et al, 1998; Choukroun et al, 1998). The release of ROS, proinflammation cytokines, and humoral factors during ischemia-reperfusion activate ERKs which are known to be responsible for the cardiac hypertrophy in the non-ischemic zone of the heart to compensate for myocyte loss (ventricular remodeling). Additionally, JNKs and p38-MAPKs are activated during global cardiac ischemia and after incubation of H 2 O2 (an example of ROS), TNF-a, or IL-lp with cultured myocytes (Sugden and Clerk, 1997,1998). Inhibition of p38-MAPK attenuated ischemia-induced cardiac apoptosis and decreased TNF-a production in both experimental animal model and human cardiac tissue (Cain etal, 1999; Mackay and Mochly-Rosen, 1999; Ma et al, 1999) Altered aene expression It has become increasingly evident that ROS are more than simply cellular toxicants and may be important modulators of cellular gene expression patterns. For example, redox cycling of cysteinyl residues is one of important mechanisms that ROS regulate activity of transcription factors and signaling molecules. Disruption (reduction) or formation (oxidation) of a disulfide bone plays a central 21 part in protein conformation and fonction established for protein-protein and protein-DNA interactions that determine several aspects of signal transduction. ROS activate a wide variety of cellular signaling molecules and pathways including Ca^*, protein tyrosine kinases (PTKs), serine/threonine kinases, and phospholipases. ROS-induced alteration in gene expression is mediated by the activation of transcription activators such as nuclear factor -kB (NF-kB) and activation protein-1 (AP-1), and the responses are rapid. Changes in early response genes such as egr-1, hsp70, c-fos, c-jun, c-myc are detected within 30 min of to a hypertrophic stimulus exposure and stimulate re-expression of fetal genes such as p-MHC, a- skeletal actin, and ANP within 6-12 hr (Hefti et al, 1997). ROS- induced increases in intracellular calcium homeostasis may be a significant initial step in the activation of NF-k B (Sen and Packer, 1996). Additionally, lipid peroxidation products can activate NF-k B. At least two important transcription factors, nuclear factor -k B (NF-k B) and activation protein-1 (AP-1) are controlled by the intracellular redox state. These redox-regulated transcription factors bind to several promotor regions of genes that are directly responsible for the pathology of diseases such as atherosclerosis, diabetic complications, cancer, and AIDS (Sen and Packer, 1996). Transcription activator NF-k B is important in inflammatory responses since it regulates a number of cytokine genes and their receptors, including TNF- 22 a. IL-1, IL-2, and MHC class I. NF-k B binding sites were also found in a variety of cell adhesion molecules such as CSF-1. MCP-1, and VCAM-1. AP-1 proteins are a family of leucine zipper transcription foctors that specifically regulate gene expression upon binding to ds-acting transcriptional control DNA element AP-1 genes are indudble by a broad range of stimuli including ROS, and they function as mediators of transcriptional regulators in signal transduction processes leading to proliferation and transformation. Examples of AP-1 transactivators are heterodimers of c-fos and c-jun immediate- early response gene families or homodimers of c-jun proteins. The activity of AP-1 is regulated at different levels induding transcriptional, posttranscriptional, and postranslational mechanisms. Activation of c-fos/c-jun transcription is promoted by oxidants, cytokines and growth factors. Redox regulation of AP-1 binding appears to occur at posttranslational level. Oxidants can modify the redox state of cysteine residues located in the DNA-binding domain of each fos/jun protein that is crudal of their DNA binding activity and subject to redox regulation. Under oxidative stress, the loss of redox regulation of cysteine residues in AP-1 proteins inhibits DNA binding, whereas treatment with redudng agents such as DTT, NADPH, GSH, or p-mercaptoethanol increased AP-1 DNA binding (Sun and Oberiey,1996). The field of oxidant related stress genes and signaling is rapidly expanding and a great deal of new insight into cellular responses to physiological stress has emerged. The regulation of transcriptional activators by oxidants and 23 reductants is complex and the ultimate biological effects are dependent upon the net results of cross-talk between these redox-mediated transcriptional activators. THERAPEUTIC IMPLICATIONS In general, currently "conventional" therapies for cardiovascular disease primarily include drugs that primarily affect vascular tone, cardiac contractility, and/or fluid status. However, given emerging evidence of oxidative processes in cardiac and vascular disease, it seems reasonable to suggest that anti-oxidant therapeutic strategies may have value. Below is a brief review and current perspective regarding antioxidants and cardiovascular disease management. Currently employed Pharmaceuticals Interestingly, several of the most prominent pharmaceuticals used in cardiovascular medicine are known to have some anti-oxidant properties that may contribute to their long-term efficacy. For example, the first commercially available angiotensin converting enzyme (ACE) inhibitor, captopril, contains a sulfhydryi moeity in its chemical structure. While this has been linked to a unique side effect of patient coughing, the sulfhydryi structure has also been suggested as a chemical scavenger of free radicals that may contribute to captopril's actions (Anning et al, 1997; Mittra and Singh, 1998). Several recent reports also suggest that the long-term value of ANG II inhibition for cardiovascular disease is mediated by non-hemodynamic actions, rather than acute vasorelaxant effects per se. It has been recognized that angiotensin II itself is a stimulator of cardiac 24 and vascular oxidative pathways via induction of NADH/NADPH oxidase (see above). Therefore, inhibitors of the renin-angiotensin system (ACE inhibition or ANG II receptor antagonism) may actually sen/e as indirect antioxidants by blocking this pathway, and some aspects of their long term efficacy may be related to this effect. ACE inhibition has recently been shown improve endothelial function in patients with coronary artery disease or its risk factors (TREND; Trial on reversing Endothelial Dysfunction). While the mechanism of this clinical benefit is unclear, inhibition of angiotensin ll-mediated superoxide production may be a protective mechanism associated with preservation of endothelial health (Rajagopalan et al, 1996; Laursen et al, 1997, chapter 2). The recently introduced and novel vasodilating p-blocker, carvedilol has also been shown to possess antioxidant effects in vitro and in vivo. This agent has emerged as a valuable therapeutic for long term survival enhancement in heart failure, for prevention of diabetes related renal failure, and in long term hypertension control. The antioxidant actions of carvedilol (and its primary metabolite) significantly contribute to cardioprotective effects in hypertension and congestive heart failure (Moser etal, 1998; Feuerstein etal, 1998). Carvedilol administration significantly increased the resistance to LDL oxidation in patients with essential hypertension (Maggi et al, 1996). Inhibition of ROS in the myocardium may prevent consequences of oxidative damages such as cardiac remodeling, activation of transcription factors, and apoptosis (Feuerstein and Ruffolo, 1998; Feuerstein et al, 1998). 25 L-Arainine After the recognition of the important preventive role of NO in cardiovascular disorders (i.e. increases blood flow, inhibits key processes involved In atherogenesis including platelet aggregation, monocyte adherence and infiltration, vascular smooth muscle proliferation, oxidative enzyme activity), its natural metabolic donor L-arginine has drawn a massive intention form medical scientists. As we've described above, the bioavailability of NO is known to be inversely related to the presence of other ROS. One approach to augment NO bioavailability is through enhanced provision of NOS substrate arginine. Clinical studies showed that of L-arginine supplement improved coronary and peripheral blood flow in healthy and heart failure patients (Lerman et al 1998; Rector et al, 1996). Although L-arginine supplement has shown therapeutic value but its low efficacy has become a drawback in patient compliance. The lowest effective dosage ranged from 6-8 g/day (up to large 20 capsules/day), whereas the maximum effective dose is 18-20 g/day (Tenenbaum et al, 1998). In addition to the large dose required, L-arginine has a bitter unpleasant taste. Therefore, the on-going development of L-arginine-enriched nutrient bar (the HeartBar) may promote the use of this promising natural nutrient (Cooke, 1998). While there has been some suggestion that this supplementation may have value, large scale, double blind, placebo controlled trials have not been conducted, and the small scale data available suggest only minor efficacy. 26 Antioxidant Vitamin Supplm ent» Having recognized the significant role of ROS in the pathogenesis of cardiovascular disease, several antioxidants from dietary and supplements have been proposed to prevent or reduce risk of heart disease. The term cardiovascular ‘nutriceuticals’ has been introduced to describe agents that have been shown to enhance cardiovascular health based on their naturally presence, strong scientific foundation, and support from clinical trials of their uses (Cooke, 1998). Discussion below is limited to three important exogenous antioxidants- vitamin 0 (ascorbic acid), vitamin E (a-tocopherol), and p-carotene (provitamin A )- because little information is available on benefits of other potential nutritional antioxidants in human subjects. The potential value of antioxidant vitamin supplements has become an area of interest for cardiovascular and other disease management Table 1.2 shows completed large-scale trials and some small clinical outcome studies of antioxidant effects on cardiovascular disease. In the last decade several studies have been conducted using highly variant experimental designs, populations, vitamin supplements and/or combinations. In general targeted therapy in high risk populations (secondary prevention trials) have shown more consistent value for antioxidants than large scale primary prevention trials. Vitam in C: Vitamin 0 is the primary antioxidant water-soluble vitamin that can directly scavenge singlet oxygen, superoxide and hydroxyl radicals. The Food and Nutritional Academy of Sciences now proposes the new recommended 27 dietary allowance (RDA) for vitamin C as 120 mg/d (previously 60 mg/d) based on new biochemical, molecular, epidemiologic and clinical data (Levine et al, 1999). Hydrophilic antioxidants (e.g. ascorbic acid and glutathione) appear to be the first line of antioxidant defenses against reperfusion damages during the return of blood flow while lipophilic antioxidants (e.g. ubiquinol 9 and vitamin E) play an important role in protecting the integrity of cellular membranes from oxidative damage at a later time point (Haramaki et al, 1998). A significant decrease in plasma vitamin C is associated with the presence of unstable coronary syndrome in patient with coronary artery disease (Vita et al, 1998) V itam in E : Vitamin E appears to be the most effective lipid soluble antioxidant in biological systems (Nagel et al, 1997). It inhibits lipid peroxidation and regenerates reduced vitamin C and glutathione (GSH). Myocardial phospholipid membrane protection may be the mechanism by which vitamin E inhibits membrane peroxidative damage. The cardioprotective effect of oral vitamin E against oxidative damage has been well recognized from both animal studies and clinical trials (Nagel et al, 1997). For instance, plasma levels of a- tocopherol correlate well with endothelial health (Kinlay et al, 1999). The decreases in plasma levels of lipophilic antioxidants such as a- and p-carotene, and vitamin E have been suggested as indicative of atherosclerosis in patients with coronary heart disease (Kontush et al, 1999). It has been shown that administration of vitamin C or vitamin E restored endothelial function in patients 28 with coronary heart disease (Ito et al. 1998; Motoyama et al. 1998; Gokce et al, 1999).Therefore, vitamin E may preserve endothelial function in patients with coronary atherosclerosis. fi-carot9ne and Vitamin A : Antioxidant potential of p-carotene and vitamin A has been demonstrated by their ability to quench singlet oxygen and interrupt generation of ROS at a very early stage (Nagel et al. 1997; Palace et al. 1999). Most of the provitamin A carotenioids absort)ed across the brush border are packaged into chylomicrons and transported in the plasma in lipoprotein particles. Their uptake and cellular distribution and metabolism varies widely among species. Thus, studies from animal models cannot be postulated to humans. Despite the antioxidant mechanisms involved is not well understood, some epidemiological studies in humans provide supportive evidence of their antioxidant value (Table 1.2). 29 Study/Trial study AndwddantOoeageamd Outcome RefMence Population OufaUono# Study Large Scale ObeervaUonal Studies The First National Health and 11,384 US Vitam in C -Significant lower risk o f death Enstrom et Nutrition Examination Survey adults Duration:>10 year from CHD al, 1992 Nurses’ Health Study 87,245 Vitam in E ^ 30 lU/d) -Significant lower risk o f CHD Stampferet female nurses Duration: 8 years al, 1993 Health Professionals Follow-up 39,910 male 1) Vitamin C (1,162 mg/d)* -Vitamin C: no benefit on CHD Rimm e t al, Study (HPFS) health 2) Vitamin E(>60IU/d) -Vitamin E: significant lower risk 1993 proféssionals 3) p-carotene (13.5 mg/d)* o f CHD Duration: 4 years -p-carotene: significant lower risk o f CHD in smokers Dietary antioxidant vitamins 34,486 1) Vitamin A (> 20,000 lU/d) -No tmnefit from vitamin A or C K u sh ie ta l, and death from coronary fieart postmenopau 2) Vitamin C (>390 mg/d) -Vitamin E significant lower 1996 disease in postmenstrual sal women 3) Vitamin E (> 35 lU/d) mortality risk of CHD women Duration: 7 years Primary Prevention Trials Chinese Cancer Prevention 29,584 men Comtxnation: p-carotene (15 -Reduction in risk of stroke Blot etal, Trial and women mg/d), vitamin E (30 mg/d) and 1993 selenium (50 uo/d) Atpha-Tocopherol. Beta- 29,133 male 1) Vitam in E (50 lU/d) -Vitamin E no trenefit on risk of the ATBC Carotene Cancer Prevention smokers 2) p-carotene (20 mg/d) CHD; increased risk o f death study (ATBC) 3) combination from hemorrhagic stroke group, Duration: 5-8 years -p-carotene: increased mortality 1994 from ischemic heart disease Beta-Carotene and Retinol 18,314 men ComlM'nation: p-carotene (30 -Increase risk of CVD Omenn et Efficacy Triai (CARET) smokers and mg/d) and reb'nyl palmitate al, 1996 astjestos (25,000 lU/d) workers Duration: 4 years U.S. Physicians' Health Study 22,071 male 1) p-carotene (50 mg/d) -No benefit on Risk of CVD Hennekens (PHS) physicians 2) aspirin 325 mg/d e ta l, 1996 3) combination Duration: 12 years Skin Cancer Prevention Study 1,805 skin p-carotene (50 mg/d) -No benefit on Risk of CVD Greentierg cancer Duration: median of 4.3 years e ta l, 1996 patients Secondary Prevention Trials Cambridge Heart Antioxidant 2002 men Vitamin E (400 or 800 lU/d) -Significant decrease in risk of Ml Stephens Study (CHAOS) and women Duration : median of 510 days -Apparent increase in e ta l, 1996 with coronary (3-981 days) cardiovascular death artery disease Clinical Outcome Studies Indian Experiment of in^rct 125 patients Combination: vitamin A (50,000 -Significant reduction in infarct Singh et al, Survival Study with lU/d), vitamin C (1,000 mg/d), size and post-MI complications 1996 suspected vitamin E (400 lU/d), and p- acute Ml carotene (25 mg/d) Duration: 28 days post-MI Multivitamins and Probucol 317 patients 1) probucol (1000 mg/d) -No benefit on prevention of Tardif etal, Study (MVP) 2) combination of p ro b r^ and restenosis after angioplasty 1997 multivitamins Duration :1 mo. Pre- and 6 mo. post-angioplasty Postangioplasty Restenosis 119 patients Vitam in C (500 mg/d) -Significant reduction of Tom odaet Prevention Duration: 4 mo. Post-angioplasty restenostt incidenoe al, 1996 Cholesterol Lowering 146sul)iects 1) colestipol/niacin (drug) or -Significant less IMT progression Azen etal, Atherosclerosis Study (CLAS) with previous vitamin E (>100 lU/d) in vitamin E group 1996 coronary 2) drug+vitamin E -No benefit o f vitamin E in artery tjypass 3) drug*vitamin C (>250 mg/d) combination with the drug graft surgery Duration: 4 years -No benefit of vitamin C on IMT * median Intake of the quintile that showed significant efltoct; CHD, coronary heart disease; CVD, cardiovascular disease; Ml, myocardial infarction; IMT, intima-medial thickness; multivitamins. (P-carotene.100 mg/d; vitamin C.1,000 mg/d; vitamin E.1,400 lU/d) Table 1.2 Antioxidant supplements in cardiovascular disease 30 Although inconsistent, the observational epidemiologic studies provide data suggesting that consumption of foods rich in antioxidant vitamin reduces the risks of developing heart disease. The available data from large-scale randomized, controlled trials are also inconclusive. Among the lipid-phase antioxidants studied, vitamin E shows the most preventive benefits on coronary heart disease while p-carotene shows very little benefit or even harmful effects. Although both vitamin E and p-carotene (and vitamin A) are carried within LDL particles, an apparent superior preventive effects of vitamin E may be explained by the fact that vitamin E increases LDL oxidative resistance while p- carotene shows little protective effect in vitro (Trible, 1999) and in human subjects (van het Hof et al, 1999). Conflicting outcomes from the studies of antioxidant intake may result from their fluctuating bioavailability from each preparation. Since antioxidant supplements fall into the dietary supplements category, the quality control of the products has not been regulated by the US Food and Drug Administration. Vitamin E provides an important aspect of how vitamin isomers appeared in commercially available preparations affect bioavailability. Gamma-tocopherol is the predominant tocopherol that accounts for 70% of the total vitamin E intake in the United States while its antioxidant activity and steady state concentration in plasma and tissue is only 10-20% of the a-tocopherol (Cohn, 1997). Other factors also influence plasma levels and bioavailability of each antioxidant. For example, plasma level of p-carotene may not reflect the 31 absorbability différences between natural sources and supplementation due to the modification or processing prior to its enter to the blood steam and the matrix in which it locates (van het Hof, 1999). In the study of antioxidant vitamin and death form coronary heart disease in postmenopausal women, Kushi et al (1996) found no association between vitamin E supplements (up to >250 lU/day) and mortality from coronary heart disease whereas a significant inverse correlation was presented in vitamin E derived from food (>10 lU/d). Additionally, metabolism of vitamin E and free radical defenses are altered in different pathologic states which can influence the bioavailability of tissue vitamin E, as observed in the tissues of patients with different manifestations of atherosclerosis such as aortic occlusion, aneurysma, and peripheral occlusive disease (Kilion et al, 1996). Dosage differences may account for the incongruent outcomes of the vitamin studied. Based on kinetic studies of vitamin E transport in humans, it is recommended that the amount of vitamin E required for maintaining a steady state level is 135-150 lU/d, and the intake of 40 lU/d is the minimum amount effective for protecting LDL oxidation (Weber et al, 1997). Moreover, the myocardium is well recognized as one of the most difficult biological organ compartments to be reached by certain drugs and molecules. It has been suggested that 5-10 days are required to achieve two-fold rat myocardium vitamin E content at the dose of 200 lU/kg/d (Machlin and Gabriel, 1982). Tissue level of vitamin E in the myocardium of infarcted rat receiving 2-week vitamin E 32 supplement was decreased despite no change in plasma levels of vitamin E (Palace et al, 1999). Using antioxidant vitamins in combination may be of importance in maximizing antioxidant defenses through synergistic effects and replenishment of antioxidant reservoirs in both aqueous and lipid compartments. For instance, in addition to its aqueous phase antioxidant property, vitamin C serves as a reducing agent in regeneration of antioxidant vitamin E (and vice versa) from a- tocopheroxyl radical formed from reaction of vitamin E and radicals. Niki et al (1995) demonstrated that in the presence of both vitamin E and p-carotene in oxidation of fatty acid in liposome preparation, vitamin E was predominantly consumed at first while p-carotene was spared and rapidly used after depletion of vitam in E. In summary, despite some evidence from cohort studies suggest benefits of antioxidants in prevention and therapies of cardiovascular disorders, additional reliable randomized controlled trials are in need in order to fully evaluate the role of antioxidant supplements. More forthcoming data from ongoing large-scale trials will further define the role of antioxidants (p-carotene, vitamin C, and vitamin E) in prevention and treatment of cardiovascular disease. 33 CONCLUSIONS Cardiovascular disease is prevalent and represents huge costs to the U.S. health care system. Despite diverse etiologies and symptoms, increased prevalence of reactive oxygen species is an apparent unifying mechanism of most forms of cardiac and vascular dysfunction and disease. Several pro-oxidant pathways have been identified, and these oxidant mechanisms may be important in structural and functional changes that occur in cardiovascular disease initiation and/or progression. Targeting the inhibition of these pathways (and/or associated cell responses) may have therapeutic value for cardiovascular disease management, although currently available data using vitamin supplements have been less than convincing. It is obvious that the roles of oxidative mechanisms in disease is a rapidly emerging research field. It is likely that these pursuits will lead to a better understanding of these biological phenomena, and will hopefully provide new opportunities for therapeutic interventions. 34 MY THESIS OBJECTIVES The central goals of my thesis project are to further the understanding of oxidant related mechanisms in cardiac and vascular disease, particularly with respect to nitric oxide control mechanisms. In chapters 2,3, and 4 we Investigated mechanistic aspects of oxidative events in vascular tissue, whereas chapters 5 and 6 were focused on oxidant-related cardiac consequences and potential interventions. Collectively, these investigations were conducted at the whole animal, isolated tissue, cellular and subcellular levels. As described above, recent studies by others have suggested that angiotensin II promotes superoxide formation in isolated endothelial cells. In chapter 2 we studied the actions of ANGII in vivo, particularly with respect to changes in endothelial performance and peroxynitrite formation (a byproduct of superoxide and nitric oxide interactions). Our findings suggest that impaired endothelium function and cell-specific peroxynitrite formation (and related protein nitration) are early events during elevated angiotensin levels in vivo. This chapter has recently been published in FASEB J 2000;14:271-278. Immunohistochemical techniques have become valuable as a research tool and in situ evaluation of tissues in disease states, but quantitative analysis using this approach is not well established, particularly in vascular tissues. We developed a novel digital image analysis approach for the evaluation of vascular immunohistochemical results. We observed endothelial cell specific staining for 3-nitrotyrosine (the biomarker of peroxynitrite formation, see chapter 2), and developed a novel digital image analysis approach for statistical comparison 35 among treatment groups. Description and validation of this novel method is described in chapter 3. In chapter 4 we further studied the effects of ANG II on erxlothelial cells. In these studies we investigated this interaction using isolated cells in culture. We found that angiotensin II induced protein nitration in vitro in a concentration and time-dependent manner. In addition, we found evidence of nitrated protein modification capabilities in cultured endothelial cells. This novel finding suggests that modification of nitrated protein may be important as a mechanism of cell defense against the adduct formation generated by the potent oxidant peroxynitrite. Furthermore, it suggests that nitration may be a regulated event that modulates tyrosine chemistry (analagous to phosphorylation reactions). While increased levels of ANG II and IN F have been commonly observed in CHF, their combined influences and interactions in vivo have not been extensively investigated with respect to their relationships of cardiac NOS II expression and apoptosis. In addition, altered gene expression in the renin- angiotensin axis in response to the cytokine and the combination of the cytokine and angiotensin II has not been investigated. In chapter 5 we studied the interactions of these important agents in vivo, at the levels of cellular signaling and gene expression. Gene therapy is emerging as a potentially valuable therapeutic approach for specific settings of cardiac disease. Chapter 6 is a detailed review of the current state of this field. In addition, we have proposed a novel approach for cell-based gene delivery system using autologous fibroblasts. Provided in this 36 chapter is both the rationale for our approach and some preliminary data supporting the plan and providing an initial evaluation for “proof of concept" in using fibroblasts as a therapeutic tool for gene product delivery for cardiac tissue. Finally, a general overview and summary of the important findings from my thesis project is provided in chapter 7. 37 REFERENCES Anning PB, Grocott-Mason RM, Lewis MJ. Effects of sulphydryi- and non- suiphydryl-containing ACE Inhibitors on left ventricular relaxation in the isolated guinea pig heart. Endothelium 1997; 5:265-75. Anversa P, Cheng W, Liu Y, Leri A, Redaelli G, Kajstura J. Apoptosis and myocardial infarction. Basic Res CardioM998; 93 (SuppI 3): 8-12. Azen SP, Qian K, Mack WJ, et al. Effect of supplementary antioxidant vitamin intake on carotid arterial wall intima-media thickness in a controlled clinical trial of cholesterol lowering. Circulation 996:94:2369-2372. Bayraktutan U, Draper N, Lang D, Shah AM. 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Flaherty JT, W eisfeldt ML. Observation o f free radical generation in the post-ischemic heart. Proc Natl Acad Sci USA 1987;84:1404-1407. 48 CHAPTER 2 ENDOTHELIAL DYSFUNCTION AND PEROXYNITRITE FORMATION ARE EARLY EVENTS IN ANGIOTENSIN-INDUCED CARDIOVASCULAR DISORDERS This manuscript has been published in FASEB J 2000;14:271-278 , and is presented here in the format identical to the published manuscript, with minor changes. 49 ABSTRACT Angiotensin II (ANG 11} is a well-established participant in many cardiovascular disorders, but the mechanisms involved are not dear. Vascular cell experiments suggest that ANG II is a potent stimulator of free radicals such as superoxide anion, an agent known to inactivate nitric oxide and promote the formation of peroxynitrite. Here we hypothesized that ANG II reduces the efficacy of NO- mediated vascular relaxation and promotes vascular peroxynitrite formation in vivo. ANG II was infused in rats at sub-pressor doses for 3 days. Systolic blood pressure and heart rate were unchanged on day 3, despite significant reductions in plasma renin activity at this time. Thoracic aorta was isolated for functional and immunohistochemical evaluations. No difference in isolated vascular contractile responses to KCI (125 mM), phenylephrine or ANG II was observed between groups. In contrast, relaxant response to acetylcholine (ACh) was decreased 6- fold without change in relaxant response to sodium nitroprusside. Extensive prevalence of 3-nitrotyrosine (3-NT. a stable biomarker of tissue peroxynitrite formation) immunoreactivity was observed in ANG II treated vascular tissues and was specifically confined to the endothelium. Digital image analysis demonstrated a significant inverse correlation between ACh relaxant response and 3-NT immunoreactivity. These data demonstrate that ANG II selectively modifies vascular NO control at sub-pressor exposures in vivo. Thus, endothelial dysfunction apparently precedes other established ANG II induced vascular pathologies and this may be mediated by peroxynitrite formation in vivo. 50 Keywords: nitric oxide, vascular, peroxynitrite, endothelium, angiotensin II, oxid a tio n 51 INTRODUCTION Angiotensin II (ANG II) is a well-recognized participant in many cardiovascular diseases, and its production or activity are currently important targets in cardiovascular pharmacology and medians (ACE Inhibitors and ANG II receptor antagonists) (1,2). Early studies of this hormone demonstrated its potent vasoconstrictive actions, while more recent studies have defined important effects on vascular cell growth, differentiation and gene expression (3,4,5). Very recent studies using isolated vascular cells demonstrate that ANG II promotes the production of oxygen radicals (particularly superoxide anion) (6.7), and this may be an important component of ANG II mediated cardiovascular disease (8.9). While a role for ANG II in cardiovascular disease is well established, the mechanisms by which it participates have not been elucidated. Particularly, the early and initiating events during elevated ANG II levels in vivo are not well de fine d. In the last decade, recognition of the importance of vascular endothelial cells for maintenance and regulation of vascular health has emerged (10,11). Nitric oxide (NO) derived from vascular endothelial cells has been shown to be a critical modulator of local vascular tone and thrombus formation, and deficient endothelial NO production has been demonstrated in a wide array of cardiovascular diseases, including hypertension, atherosclerosis, unstable angina and congestive heart failure (10,11.12). The chemistry of NO in biological matrices is very complex, and several biochemical pathways other than NO 52 production can influence NO actions (13,14). For example, superoxide anion interacts with NO, reduces its efficacy as a signal transduction agent, and promotes the formation of peroxynitrite, a highly reactive intermediate known to nitrate protein tyrosine residues and cause cellular oxidative damage (15,16). The reaction of NO with superoxide anion occurs at a diffusion-limited reaction rate, and NO is the only molecule known to compete with superoxide dismutase for its substrate in a biological setting (17,18,19). Thus, excess or uncontrolled superoxide anion formation can shift the actions of available NO from a useful cellular signal to peroxynitrite and redox-related toxic products. While dysfunction of vascular endothelium and elevated blood concentrations of ANG II are each commonly observed in many forms of cardiovascular disease, few studies have evaluated the relationships between these phenomena. Here we investigated the actions of ANG II in vivo, testing the hypotheses that sub-pressor ANG II causes selective vascular dysfunction, and that vascular peroxynitrite formation (and attendant dysregulation of NO control) participates in this phenomenon. 53 METHODS A nim at m o d e/and anglotansin adminlatntlon Osmotic mini-pumps (Alza Corp., Palo Alto, CA) were used to provide continuous ANG II administration for 3 consecutive days in rats (male Sprague Dawley, 325-350 g). Intraperitoneal implantation was conducted during pentobarbital anesthesia using sterile surgical technique. ANG II (200 ng/kg/min) or saline vehicle was administered at a flow rate of 1.0 pi/hr. The dose employed here has been shown by others to be a "slow-pressor dose" in rats, producing no significant elevation of systemic blood pressure until 7 days of continuous administration (4,5). ANG II was kindly supplied by Bachem Biosciences Inc, (King of Prussia, PA). Prior to pump implantation (Day 0), and on Day 3 of infusion, systemic blood pressure and heart rate were evaluated in conscious rats using a tail cuff apparatus (Stoelting Instruments, Wood Dale, IL). Following pentobarbital overdose (75 mg/kg ip), blood was rapidly collected by cardiac puncture and vascular tissues were rapidly isolated. Plasma renin activity Blood was collected by cardiac puncture at sacrifice, and plasma was rapidly collected. Plasma renin activity (PRA) was measured by a commercially available radioimmunoassay kit adapted for low volume samples (Incstar, Stillwater, Minnesota). Activity was determined as nanograms of angiotensin I produced per ml/hr. Intra-day and inter-day assay variability was less than 10%. 54 Isolated vascular studies After animal sacrifice, thoracic aorta was rapidly isolated for functional evaluations using methods sim ilar to those previously described (20,21). Vascular segments (2-3 mm) were mounted on isometric force transducers (Grass Instruments, Quincy MA) and incubated in 10-mL organ baths containing Krebs' buffer bubbled with 95% 02 at 37° C. Following 90 min equilibration (resting tone 1.0 g). maximal contractile force was determined for each segment using a high potassium concentration (modified Kreb s buffer containing 14.4 mM NaCI and 125 mM KCI). This complete depolarization was used to define maximal contractile response and was considered 100% contraction. Segments were then consecutively washed with Krebs’ buffer four times with two 5-min intervals and two 10-min intervals, and allowed to equilibrate for 30 min. Cumulative responses to cumulative concentrations of phenylephrine were then determined. In parallel studies in vitro responses to ANG II were evaluated by single exposure of concentrations ranging InM to 1 ^iM (thus preventing development of tachyphylaxis). After precontraction with phenylephrine at 80% of maximum, relaxant responses to cumulative acetylcholine were assessed. In preliminary experiments acetylcholine relaxation was completely blocked by 200 pM I- Nitroarginine (a non-specific NOS inhibitor), thus the vasodilatory response is apparently endothelium dependent and NO mediated in this vascular tissue. Relaxant responses to cumulative sodium nitroprusside were also determined (an endothelium independent but NO-mediated response) (22). Contractile and 55 relaxant response data were fit to the 4-parameter logistic equation using GraphPad Prism Software (GraphPad Software, inc., San Diego, CA) (20,21). Immunohistoehemistry Immunohistochemlstry was performed to determine the relative extent of protein nitration (a stable biomarker of endogenous peroxynitrite formation), using an antitxxfy raised against 3-nitrotyrosine (3-NT). A section of aorta from each rat, separated from the isolated vascular tissue studies, was fixed in formalin and emt)edded in paraffin. Aorta cross sections were cut into 5-pm sections, deparaffinized and rehydrated. Tissues were then exposed to a 3% hydrogen peroxide/methanol solution for 10 min to block endogenous peroxidase activity. To restore antigenicity, slides were immersed for 15 min in a 10 mM citrate buffer (pH6) preheated to boiling. Sections were then incubated for 30 min in a 10% normal goat serum/PBS solution to block non-specific antibody binding. Tissue sections were then incubated with rabbit anti-mouse polyclonal antibody raised against 3-NT (1:200 dilution. Upstate Biotechnology, Lake Placid, NY) for 1 hr. Biotinylated secondary anti-rabbit serum was then applied (1:200), followed by horseradish peroxidase complex reagent (ABC Elite, Vector Laboratories, Burlingame, CA). Positive immunoreactivity was visualized through the development of diaminobenzidine (DAB) chromogen. Harris' Modified hematoxylin was used for nuclear counterstaining. Preliminary experiments were conducted to verify the specificity of immunostaining in our laboratory. Preincubation of primary antibody with free 3NT (Im M ) completely quenched 56 positive tissue staining, whereas tyrosine (1mM) had no effect Non-immune serum (isotypic) controls also showed no detectable immunoreactivity in any treatment group. Digital image analysis Images of immunostained tissue were captured using an Olympus microscope (BX-40) and a high-resolution digital camera (Fixera. Inc., 1260x960 pixel resolution). Unmodified images were then analyzed using research-based image analysis software (ImagePro Plus, Media Cyt)emetics, Silver Spring, MD). Six endothelial regions of each aortic ring were systematically captured at 200x, representing approximately 45% of the total vascular endothelium circumference. Relative extent of 3-NT immunoreactivity was assessed using digital color image analysis. Similar to other previous reports (23), we observed the most striking difference in "positive" vs. background staining regions in the blue channel of RGB color profile (i.e., red, green, blue). There was a well defined segmentation of the intensity profile such that brown DAB staining registers between 0-100 intensity units, while shades of blue or white background were consistently greater than 100 intensity units as shown in Fig. 4. For each vascular image the percentage of positive pixels in the captured images was used as an objective and semi-quantitative measure of immunoreactivity (24), thus allowing for statistical comparisons among groups (25). Intra-observer variability was consistently less than 12% (coefficients of variation from 3 daily measurements), whereas inter-observer variability was less than 10%, (coefficients of variation of 57 average measurements between 3 observers collecting 6 images on three different days). S ta tis tic a l A n alyses All data are presented as mean+SEM. Statistical evaluations were performed using Sigmastat software (Jandel Scientific Inc., San Rafael. CA). Comparisons among treatment groups were performed using Students t-tests or analysis of variance where appropriate (26). Spearman non-parametric correlation analysis was used to test for significant association of functional and immunohistochemical data (26). In all cases, p<0.05 was deemed statistically significant. 58 RESULTS Shown in Figure 2.1 are changes in plasma renin activity, hemodynamics and cardiac mass following ANG II or vehicle infusion for 3 days. Statistically significant reductions in plasma renin activity were observed by day 3 following ANG II administration relative to vehicle infusion, illustrating hormonal feedback of the renin-angiotensin axis and demonstrating successful infusion of low-dose ANG II. There was no significant change in plasma aldosterone concentrations (data not shown). In contrast, ANG II did not cause significant changes in systolic blood pressure or heart rate by day 3 (Figure 2.1). Similarly, no significant change in bodyweight, total heart weight, or left ventricular weight (all parameters known to be affected at higher Ang-ll exposures) was observed by day 3. Aortic cross sectional areas were also unaffected by ANG II infusion (data not shown). Isolated vascular contractile responses are shown in Figure 2.2. Maximal vascular contractile response to total depolarization (125 mM KCI) was not different between ANG II and vehicle infused groups. Contractile responses to the a-receptor agonist phenylephrine were also unaltered (EC50 49 ±12 vs 73 ± 14nM; Emax 1.09 ± 0.06 vs 0.99 ± 0.06g; ANG II vs control respectively). Contractile response to ANG II in vitro was also not statistically affected by 3 day infusion (EC50 280 ± 110 vs 183 ± 41 nM; Emax 0.41 ± 0.12 vs 0.34 ± 0.8g; ANG II infused vs control respectively). Despite unaltered vascular contractile properties, significant alteration of acetylcholine induced relaxation was observed (Figure 2.3). A statistically significant 6-fold increase in EC50 (390 ± 12 vs 64 ± 13nM, ANG II infused vs 59 vehicle infused) was obsenæd with no change in maximal relaxant response (Emax 93±4 vs 109±4). No significant differences in extent of phenylephrine- induced precontraction were observed between treatment groups. In contrast to diminished acetylcholine responses, no significant change in vasorelaxant response to the endothelium independent and “spontaneous” NO donor sodium nitroprusside was observed between treatments (Figure 2.3, EC50 1.11 ± 0.3nM vs 0.84 ± 0.2nM; Emax 117±9 vs 107±1; ANG II vs vehicle, respectively). Representative photomicrographs of vascular immunostaining for 3-NT are shown in Figure 2.4. Extensive protein nitration was obsenred in the vasculature from ANG II treated animals and was found nearly exclusively in the intimai layer. In contrast, little evidence of 3-NT was found in control (vehicle infusion). Figure 2.4 also shows the distinct color distribution patterns for diaminot>enzidine (positive signal for 3-NT immunohistochemistry) and hematoxylin counterstain. This distinction was used to determine the percentage of the cross-sectional area that was considered a positive signal using digital image analysis (see methods description). Digital image analysis demonstrated statistically significant increases in vascular 3-NT immunoprevalence from ANG II treated animals relative to vehicle infusion controls (Figure 2.4). This difference was confined to the endothelial layer (no significant differences were observed when regions of exclusively vascular smooth muscle were compared, see Figure 2.5). In addition to comparing the two treatment groups, controls were used to evaluate sensitivity 60 and selectivity of the primary 3-NT antibody. The positive immunostaining signal could be completely abolished by pre-incubation of primary antibody with free nitrotyrosine in solution (5mM), or by replacing it with pre-immune serum. Significant 3-NT immunoprevalence was observed in the vehicle treatment group when compared to these staining controls, demonstrating a slight but detectable protein nitration (and corresponding peroxynitrite production) under control conditions (Figure 2.5, top panel). The relationship between the prevalence of endothelial protein nitration and endothelium dependent functional response to acetylcholine is shown in Figure 2.5 (bottom panel). Measurements of 3-NT immunoprevalence (using image analysis) and acetylcholine EC50 were related for each animal in the study. A highly statistically significant inverse correlation was observed between the extent of intimai protein nitration and endothelium dependent acetylcholine functional response (p<0.02, Spearman non-parametric correlation analysis). 61 DISCUSSION First identified as a potent vasoconstrictor in the 1950’s, angiotensin II now represents a pivotal therapeutic target for the management of many forms of cardiovascular disease. For example, Inhibition of ANG II formation (through the use of angiotensin converting enzyme inhibitors), or selective blockade of ANG II receptors, has been shown to be valuable for the treatment of essential hypertension, congestive heart failure, and coronary artery disease (1,2). W hile ANG II was first recognized as a vasoconstrictor, its roles are now known to be diverse, including stimulation of cardiac and vascular cell growth and division, and activation of vascular NADH/NADPH oxidase activity (1-6). Given these diverse hormonal influences, the long-term value of ANG II inhibition for cardiovascular disease may be mediated by non-hemodynamic actions rather than acute vasorelaxant effects per se (2). The vascular endothelium plays a key role in the local regulation of vasomotor tone and prevention of thrombus formation. Using agents like acetylcholine or changes in flow to stimulate the release of NO (e.g., endothelium derived relaxing factor, EDRF), clinical studies have demonstrated the importance of EDRF/NO in both basal and stimulated control of vascular tone (10-12). Dysfunction of vascular endothelium (particularly decreased activity of NO dependent pathways) has been associated with a wide array of cardiovascular risk factors, including chronic smoking, hypercholesterolemia, hypertension, and chronic heart failure (10-12, 27). Loss of endothelial integrity is known to promote vascular remodelling and thrombus formation, impair tissue 62 perfusion (particularly during stress), and to result in vasoconstriction (11). Thus, endothelial dysfunction is associated with a diverse array of cardiovascular disease states, and may be an important initiator of these progressive conditions. Inhibition of angiotensin-converting enzyme (ACE) has recently been shown improve endothelial function in patients with coronary artery disease or its risk factors (TREND; Trial on reversing Endothelial Dysfunction). In this large scale clinical trial, treatment with quinapril (an ACE inhibitor with affinity for vascular tissue) for 6 months was associated with significantiy improved vasodilator response to acetylcholine in coronary artery segments (28). Similar improvements were also observed in microvasculature (29). These clinical findings suggest that angiotensin has important influence on endothelium in vivo, and that some benefit of ACE inhibitor therapy is mediated by improving endothelial function in vivo, but the mechanisms involved have not been established. Recent studies have demonstrated that ANG II is an activator of superoxide production in several cell types, including fibroblasts (30), mesangial cells (31), and endothelial cells (32). This stimulation is apparently mediated by the AT 1 receptor subtype activation, leading to increased gene expression of NADH/NADPH oxidase subunits p67phox (33), p22phox (34), and perhaps others. The role of this enhanced oxidase expression in ANG II mediated actions is not clear, but it may contribute to the other known cellular responses (e.g. proliferation, hypertrophy, etc). While isolated endothelial cell studies have demonstrated the selective presence of ATI receptors (and an apparent absence 63 of the AT 2 subtype) (32). whether the endothelium plays a direct role in ANG II related cardiovascular disease in vivo has not been established. Given previous reports of ANG II promotion of vascular superoxide formation and the newly recognized importance of endothelial NO for vascular health, here we hypothesized that ANG II in vivo would selectively modify endothelium dependent function. Recent investigations have demonstrated that the biological activities of NO are highly dependent on both production and destruction related pathways (13). A chemical pathway of biological importance appears to be the formation of peroxynitrite through the interaction of NO with superoxide anion. This reaction is exceedingly rapid and efficient (1.9 x10^° M '^s'\ see reference 19), leading to reduced levels of available NO and formation of a highly reactive oxidant in vivo (17-19). Peroxynitrite and related species aggressively nitrate protein tyrosine residues, leading to a chemically stable biomarker, 3-nitrotyrosine (3-NT) (16). We and others have demonstrated the value of this marker as evidence of NO dysregulation in disease, including advanced atherosclerotic lesions (35), sepsis related organ failure (36), and renal transplant rejection (37), and others. The ANG II infusion employed in our studies (200ng/kg/min) caused significant inhibition of plasma renin activity but no change in systemic blood pressure or cardiac mass on Day 3. This dosing strategy appears to be physiologically relevant since it has been shown to produce steady state ANG II blood levels similar to those observed in hypertensive animals and humans (approximately 100 pg/ml) (38). This strategy of a short-term, “slow pressor dose” administration allowed us to evaluate early vascular changes during ANG II 6 4 elevations in the absence of detectable cell growth or hemodynamic influences. No significant change in vascular response was observed for total depolarized contraction, a-adrenergic stimulation, or ANG II contractions. Using an identical animal model but more prolonged ANG II dosing (120ng/min/kg of 21 days), Dowell et al. observed significant potentiation of adrenergic contractile activities, likely due to increased vascular smooth muscle cell growth over 3 weeks (39). In contrast, we observed significant reduction in acetylcholine induced vasorelaxation prior to any changes in contractile responses, and after only 3 days of elevated blood ANG II. This endothelium dependent relaxant response is mediated by NO formation from the constitutively expressed nitric oxide synthase within endothelial cells (NOS III) (10). Once formed, endothelium derived NO apparently diffuses to vascular smooth muscle cells and elicits relaxation via cyclic GMP dependent pathways (22). In contrast to diminished acetylcholine response, no change in nitroprusside action was found. Nitroprusside is an exogenous agent that generates NO through non-enzymatic and enzymatic pathways in smooth muscle cells and does not require functional endothelium or NOS enzymes for activity. Since vascular smooth muscle response to an exogenous NO source (nitroprusside) was not altered, the diminished response to acetylcholine was apparently related to either decreased endothelial production of NO or reduced bioavailability to effector smooth muscle cells. Consistent with selective endothelium dysfunction, we also observed intima-specific staining for protein 3-NT residues. Given the extremely rapid reaction rates for peroxynitrite formation from NO and superoxide (known to be 65 diffusion rate limited) and protein nitration, the staining pattern observed suggests that peroxynitrite was concentrated in or near the endothelial layer. In addition to serving as a biomarker of peroxynitrite, nitration of protein tyrosine residues is known to be an inhibitor of several biochemical pathways, including mitochondrial respiration (40), high energy phosphate utilization (41), and prostagladin systhesis (42), and superoxide dismutase activity (43). Peroxynitrite also can induce DMA strand breakage in human umbilical vein endothelial cells (44). Thus, endothelial formation of peroxynitrite (through interaction of NO with superoxide) is likely to reduce availability of NO to vascular smooth muscle as well as have functional consequences to endothelial cell biochemistry. Interestingly, we have recently reported a distinctly different vascular 3-NT staining pattern during development of organic nitrate pharmacodynamic tolerance (widespread distribution throughout smooth muscle) (45). Thus peroxynitrite formation and protein nitration may exist in a variety of vascular disorders, but the mechanisms involved and cellular distributions may be distinct and dependent on the stimuli involved. While recent studies have suggested the existence of other potential biological pathways of tyrosine nitration, these studies have demonstrated that the chemistries responsible are dependent upon neutrophil infiltration and activation (46,47). Since we did not observe immune cell involvement in our vascular studies, the protein nitration observed here is most likely derived from ONOO formation. Additionally, previous studies demonstrate that cardiovascular tissue homogenates require exogenous addition of neutrophil myeloperoxidase (5pM), ImM concentrations of nitrite and 66 hydrogen peroxide, and long Incubation times (>1hr) to produce detectable protein nitration (46). In contrast. NO and superoxide anion are known to form OONO at a diffusion limited rate, even at very low concentrations. Therefore, under our experimental conditions the observed protein nitration may be most simply explained by increased peroxynitrite formation, rather than more complicated or less efficient chemical processes. In summary, we found that low dose and short-term administration of ANG II caused a selective reduction in endothelium dependent (nitric oxide mediated) relaxant effects. These changes occurred prior to any other established consequences of elevated ANG II in vivo, including enhanced vascular contractile responses, development of hypertension, or increase in cardiac or vascular mass. Further evaluations revealed that this endothelial dysfunction was associated with increased production of peroxynitrite in vivo. Protein nitration in the endothelial layer correlated with the extent of functional impairment observed. Thus, selective perturbation of normal endothelial function is an apparent early or initiating event during ANG II exposure in vivo. Furthermore, vascular peroxynitrite formation (and attendant NO dysregulation) may mediate these early events during ANG II induced vascular dysfunction in vivo. Our findings may help to explain the results of the recently published TREND trial (see above) and suggest that evaluation of selective endothelial therapies for the prevention of disease appears warranted. 67 Acknowledgements This work was supported in part by grants from the American Heart Association, Ohio-West Virginia Affiliates, and the National Institutes of Health (HL59791 & H L63067). 68 200 500 • VEH • VEH o ANG II O ANG II 5 .4 0 0 i 150 >- 30 k 200 X 100 VEH ANG il D A YO DAY 3 DA YO DAY 3 1.5 ^ 300 ^ 1 . 0 O K 0.5 0.5 O 100 0.0 VEH ANG II VEH ANG II V EH ANG II Figure 2.1 Plasma renin activity (PRA) and hemodynamic parameters measurement after vehicle saline control (vehicle,VEH) and angiotensin II (ANG II) infusion. Top, le ft panel: PRA was measured by radioimmunoassay at day 3 of infusion. Data are mean+SEM, n=6-8. * p<0.05. Top,middle and right panel: Systolic blood pressure and heart rate were monitored using the tail-cuff method at day 0 and day 3 of infusion. Bottom panel: No significant change in body weight, heart weight and left ventricle (LV) weight at day 3 of infusion (see text). 69 1.5 e VEH 0.6 • VEH O ANG II ^ O ANG II w 1.0 1.0 0.4 ■ ^ 0.5 0.2 • / 0.0 . y 0.0 VEH ANG -9 ^ 7 ^ -6 -9 -8 -7 -6 -5 KCI (125 mM) PHENYLEPHRINE ANGIOTENSIN II (log M) (log M) Figure 2.2 Vascular contractile responses in isolated aortic rat rings. Left panel: Maximal contractile responses were obtained by exposure of rings to a total depolarizing concentration of 125 mM KCI (n=6-8). Middle and right panels: Concentration-response relationships for phenylephrine and angiotensin II (1 nM to 1 pM) in vessels obtained from ANG II (open circles) and vehicle-treated rats (closed circles). No differences in EOso or Emax values were observed (see text). 70 ANG II “9 -8 "7 “6 ACH (log M) O ANG II -10 -9 -8 -7 SNP (log M) Figure 2.3 Vascular relaxation responses in isolated aortic rat rings. Top panel: Change in vascular endothelium-dependent relaxation response to acetylcholine (ACH, 1 nM to 1 pM) obtained form ANG II (open circles) and vehicle-treated rats (closed circles). Relaxation responses were presented as percent relaxation of phenylephredine precontraction (80% maximum contraction), n-6-8. Bottom panel: Endothelium-independent relaxation response to nitric oxide donor sodium nitroprusside (SNP) was generated in the same manner as ACH (n=6-8). 71 m Ê&Fj4S-/ £2150 100 150 200 250 Blue Channel Pixel Intensity Figure 2.4 Semi-quantitative digital image analysis method for rat aortic endothelial 3-nitrotyrosine immunoreactivity. Top and middle panel: Representative photomicrograph of aortic endothelium from a vehicle-treated (top panel) and an ANG ll-treated rat (middle panel) were captured as described in Methods (400x mag.). Tissue section immunostained with rabbit polyclonal 3- nitrotyrosine, demonstrating specific immunoreactivity of intimai layer (A) but not medial smooth muscle cells (B). Bottom panel: Example of pixel intensity distributions in the blue color channel representing regions delineated by boxes A (endothelium). 8 (smooth muscle) and C (endothelium). Color patterns for A and B are overiayed. Note the clear differentiation of frequency distributions between perceived “brown” immunoreactive (A) and “blue" counterstain (B and 0). Extent of positive immunoreactivity was determined as frequency of pixels registering in the 0-100 range, as described in Methods. 72 3-NT IMMUNOPREVALENCE 3-NT IMMUNOPREVALENCE (% positive pixels) (% positive pixels) o -& N w ^ oi m 6 b 0» m £ = I I.S «I * o il Figure 2.5 Extent of 3-NT immunoreactivity in rat aortic endothelium and correlation to vascular function. Top panel: Quantitative measure of aortic 3-NT immunoreactivity and isotypic staining controls from ANG II- (open bars) and vehicle-treated rats (closed bars). A rabbit, anti-mouse, polyclonal, 3- nitrotyrosine primary antibody was used to detect endothelial protein nitration. Pre-immune rabbit IgG serum was utilized in place of the primary antibody for isotypic controls, f, p<0.05 for both treatment groups vs. isotype staining controls; t , p<0.05 for ANG II treatment vs. vehicle, via one way ANOVA with SNK post-hoc. Bottom panel: Significant positive correlation between aortic endothelial 3-NT immunoprevalence and endothelial-dependent relaxant responses. Data plotted as mean 3-NT immunoreactivity per rat (n=6 independent determinations) vs. mean EC50 values per rat (n=4-6 rings) from ANG ll-treated (closed circles) and vehicle-treated (open circles) rats. P<0.02 via Spearman's non-parametric correlation analysis. 74 REFERENCES 1. Stroth, U. and Unger, T. 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Sampson, J.B., Ye, Y., Rosen, H., Beckman, J.S. (1998) Myeloperoxidase arxl horseradish peroxidase catalyze tyrosine nitration in proteins from nitrite and hydrogen peroxide. Arch Biochem Biophys 356,207-13 47. Eiserich, J.P., Hristova, M., Cross, G.E., Jones, A.D., Freeman, BA., Halliwell, B., van der Vliet, A. (1998) Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391, 393-7 79 C H A PTER 3 DIGITAL IMAGE ANALYSIS FOR IMMUNOHISTOCHEMICAL EVALUATIONS INTRODUCTION Recent developments of in situ immunodetection methods have advanced histochemical procedures from a diagnostic tool primarily used in pathological evaluations to a valuable research approach. An important advantage of immunohistochemistry (INC), relative to Westem and Northern blotting approaches, is the preservation of tissue structure, allowing detection of regional distributions of proteins and providing cellular localization and identification. However, these in situ methods have often been limited by providing only qualitative visual information rather than statistical comparisons to control tissues, etc (Jones, 1998; Raleigh, 1998). Efforts to overcome this have typically included investigator-defined scoring systems to characterize the intensity or pattern of tissue staining (Zhou, 1997; Abberton, 1999). While these scoring approaches have been useful, issues of subjective evaluations and reproducibility in other laboratories are limitations. Furthermore, investigator- defined scoring generally provides discontinuous parameters (making statistical comparisons among groups difficult), and often evaluates only small regions of 80 tissue (thus increasing potential sampling bias). Recent advances in digital image capture and analysis methods have provided opportunity for new and improved image evaluations, allowing for objective, reproducible, and continuous parameters for statistical comparisons. Here we describe our recent development of a convenient and objective approach to employ digital image analysis for immunohistochemical studies of vasculature, particularly for evaluation of selective endothelial cell immunostaining. This method has obvious utility for evaluation of other cell-specific or localized tissue staining as well. 81 MATERIALS AND METHODS In preliminary studies we evaluated the formation of vascular protein nitration (a marker of nitric oxide-related oxidative damage) in a well-described rat model of hypertension, using immunohistochemical methods. Angiotensin II was infused to rats for 3 days (ANG II, 200ng/kg/min), and thoracic aortic tissue was harvested on day 3 (see chapter 2). Vascular segments were fixed in formalin and processed for immunohistochemistry as previously described (Mihm and Bauer, 1999). Following fixation in 10% formalin for 48 hours, vascular tissue was then transferred to tissue cassettes for standard dehydration and paraffin infiltration in an automated tissue processor (Fisher Histomatic Model 166, Fisher Scientific, Pittsburgh, PA). Five micron sections were mounted onto Fisher Scientific ProbeOn Plus slides for immunohistochemical treatment. Tissue sections were heated to 60 C for 30 min followed by immersion in clearing fluid (Hemo-D, Fisher Scientific) to remove paraffin wax. The tissue was partially rehydrated by alcohol gradient (100% to 70%), then immersed in 3% hydrogen peroxide/methanol solution for 10 min to block endogenous peroxidase activity. Slides were rinsed and reheated in citrate buffer (pH 6.0) to recover antigenicity. Tissues were blocked in 10% goat serum (Vector Laboratories, Burlingame CA)/PBS blocking solution (BS) for 30 min, then incubated with rabbit anti-mouse polyclonal antibodies directed against 3-nitro-L-tyrosine (anti-3NT, Upstate Biotechnology, Lake Placid. NY 1:400 dilution). Staining (isotypic) control tissues were exposed for the same duration to non-immune rabbit IgG (Vector Labs, 1 ;200) in place of primary antibody. Following primary antibody incubation, 82 tissues were washed, then exposed to biotinylated goat anti-rabbit secondary antibody (Vector Labs) for 20 min. Peroxidase enzymes were linked to the antibody complex by incubation in a 1:200 dilution of ABC Elite reagent (Vector Labs) in PBS. Diaminobenzidine (DAB. 0.06% w/v) was used to provide visualization of immunoreactivity, followed by nuclear counterstaining by Harris' modified hematoxylin. Preliminary experiments verified the specificity of immunostaining in our laboratory (Mihm and Bauer, 1998). Preincubation of primary antibody with free 3NT (ImM) completely quenched positive tissue staining, whereas tyrosine (ImM) had no effect. Non-immune serum (isotypic) controls also showed no detectable immunoreactivity in any treatment group. Immunostained tissues were visualized using light microscopy at 400x magnification using an Olympus BX40 microscope. Photomicrographs were captured using a digital camera with 1440x960 pixel resolution (Pixera Corporation, Los Gatos, CA). Color mapping and digital image analysis was conducted using Image Pro Plus software (Media Cybernetics, Sliver Springs, MD). 83 RESULTS AND DISCUSSION In our preliminary examinations, we visually observed endothelium- specific positive staining for protein 3-nitrotyrosine residues in ANG II treated animal tissues relative to vehicle control (see representative photomicrographs in Figure 3.2, upper panels). In an attempt to develop an objective approach to compare relative staining Intensities in different tissues, we studied the digital characteristics of the positive "brown" DAB color versus background color of the hematoxylin counterstain, using Image Pro Plus software. This Image analysis software provides data as the intensity of any given image pixel in any color channel (i.e., red, green, or blue), and a profile as a function of intensity (from 0- 255) in any color channel. Similar to other previous reports (Fritz, 1995), we observed the most striking difference in "positive" vs. background staining regions was observed in the blue channel. There was a well defined segmentation of the intensity profile such that brown staining registers between 0-100 intensity units, while shades of blue (or white background) were consistently greater than 100 intensity units (see Figure 3.2, middle panels, note that peaks of line A and B are superimposed). Thus, in any area of the captured image, the percent of the area that has greater or less than 100 intensity units could be considered a sensitive discriminator between DAB-negative and DAB- positive signals respectively, regardless of perceived colors. To employ this approach, 6 non-overlapping digital images were captured from each vascular cross section (representing 40-50% of the total circumference, Figure 3.1)) For each image, a 100 x 100 pixel box was positioned along the endothelial surface 84 in a sequential manner such that essentially all of the endothelial area of the image was evaluated. The percentage of the tissue area within each 100x100 pixel box was recorded and combined to provide average endothelial staining for each captured image (see box representations in Figure 3.2, lower panels). These objective image staining intensities were then combined for average staining immunoprevalence for each control and treated vascular segment. Several observers were employed in a blinded fashion to evaluate the reproducibility and accuracy of this approach. Each observer individually recaptured 6 images/ring and applied the blue channel box method on 3 separate days. The intra-observer variability was consistently less than 12% (coefficients of variation from 3 daily measurements), whereas inter-observer variability was less than 10%, (coefficients of variation of average measurements between the 3 observers). Shown in Figure 3.3 are tissue immunoprevalence determinations compared from 2 blinded observers. The solid line shown is fitted to the presented data using linear regression, whereas the dotted line is the unity line. These lines are overlaid, demonstrating the high degree of reliability of this image approach and a lack of subjective influence in evaluating tissue segments in this manner. The robustness of this approach is further supported by the observation that sampling only one-half of the observations per ring (i.e., 3 image captures/ring, -25% of endothelial linear area) consistently resulted in quantitative values that were within 5% of those obtained from sampling 6 images (-50% of area) per ring (Table 3.1). 85 These results further demonstrate the utility o f image analysis as a valuable approach for quantitative evaluation of immunohistochemical data. Some previous image analysis approaches have employed complicated color segmentation and/or contrast thresholding techniques to identify positive immunoreactivity. but these methods are often subject to investigator bias since some form of image manipulation is frequently necessary to generate optimal color contrasts for successful color segmentation (Kuyatt, 1993; Ruifrok. 1997). Importantly, the method described herein offers a non-biased alternative to other segmentation or thresholding approaches and is amenable to automation for high throughput data analyses. 8 6 REFERENCES Abberton KM, Taylor NH, Healy DL, Rogers PA (1999). Vascular smooth muscle cell proliferation in arterioles of the human endometrium. Human Reproduction 14:1072. Fritz P, Wu X, Tuczek H, Multhaupt H, Schwarzmann P (1995). Quantitation in immunohistochemistry. A research method or a diagnostic tool in surgical pathology? Pathologica 87:300. Jones GT, van RA, Packer SG, Walker RJ, Stehbens WE (1998). Venous endothelial changes in therapeutic arteriovenous fistulae. Atherosclerosis 137:149. Kuyatt BL, Reidy CA, Hui KY, Jordan WH (1993). Quantitation of smooth muscle proliferation in cultured aorta. A color image analysis method for the Macintosh. Anal Quant Cytol Hist 15:83. Mihm MJ, Coyle CM, Jing L, Bauer JA (1999). Vascular peroxynitrite formation during organic nitrate tolerance. J Pharmacol Exp Ther 291(1 ):209. Raleigh JA, Calkins-Adams DP, Rinker LH, Ballenger CA, Weissler MC, Fowler WJ, Novotny DB, Varia MA (1998). Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker. Cancer Res 58:3765. Ruifrok AC (1997) Quantification of immunohistochemical staining by color translation and automated thresholding. Anal Quant Cytol Hist 19:107. Zhou M, Wang P, Chaudry IH (1997). Endothelial nitric oxide synthase is downregulated during hyperdynamic sepsis. Biochimica Et Biophysica Acta 1335:182. 87 Aortic Regions 3NT 3NT Statistical (endothelial perimeter immunoprevalence immunoprevalence Significance area, %) in Control group in ANG group Regions 1-6 (-’50%) 2.11+0.26 4.42+0.37 p<0.05 Regions 1-3 (-’25%) 2.01+0.40 4.24+0.51 p<0.05 Regions 4-6 (~25%) 2.22+0.35 4.60+0.56 p<0.05 Table 3.1 Sufficiency of sampling 25% of the endothelial perimeter area. 8 8 Figure 3.1 Sampling regions in the aorta. The number 1-6 represents the systematical sampling regions from each aortic ring. 89 CONTROL TRKATED m- A* t. > ISO I— A 100 I— B l£ » 8 0 1 00 180 2 0 0 280 0 8 0 100 180 2 00 280 BLUE CHANNEL PIXEL INTENSITY BLUE CHANNEL PIXEL INTENSITY 90 Figure 3.2 Semi-quantitative digital image analysis method for rat aortic endothelial 3-nitrotyrosine immunoreactivity. Right and Left Panels: Representative photomicrographs of aortic endothelium from a vehicle-treated (left panels) and an ANG ll-treated rat (right panels) were captured as described in Methods (400X). Tissue sections immunostained with rabbit polyclonal 3- nitrotyrosine demonstrate specific immunoreactivity of intimai layer in treated animals (C) vs control animals (A), but not medial smooth muscle cells (B). Middle panels: Example of pixel intensity distributions in the blue color channel representing regions delineated by boxes A (endothelium). B (smooth muscle) and C (endothelium). Color patterns for A and B are overlayed. Note the clear differentiation of frequency distributions between perceived “brown” immunoreactive (A) and “blue” counterstain (A and B). Extent of positive immunoreactivity was determined as frequency of pixels registering in the 0-100 range, as described in Methods. Bottom Panels: Five to six 100x100 pixel boxes were positioned along the endothelial for pixel intensity analysis. 91 r=0.99 p<0.001 0 2 4 6 8 10 12 IMMUNOPREVALENCE Observer #1 (% pixel area) Figure 3.3 Inter-observer correlation analysis of 3NT immunoprevalence. Tissue immunoprevalence determinations were compared from 2 blinded observers (opened circle ^observer #1 ; closed circle=observer #2). The solid line shown is fitted to the presented data using linear regression, whereas the dotted line is the unity line. Note that these lines are overlaid. 92 C H A PTER 4 ANGIOTENSIN II INDUCES ENDOTHELIAL CELL PROTEIN NITRATION IN VITRO ABSTRACT Angiotensin II (ANG II) is a recognized participant in the progression of many cardiovascular disease states, but its roles in disease initiation are not fully defined. Peroxynitrite is a reactive by product of nitric oxide and superoxide anion and is known to be toxic to endothelial cells. We have recently described selective endothelial cell dysfunction following short term ANG II treatment in rats. This dysfunction strongly correlated with increased endothelium specific protein nitration (evidence of peroxynitrite formation). Here we tested the hypothesis that ANG II directly induces endothelial cell peroxynitrite formation in vitro. Confluent cells were incubated with ANG II (0,100,250 |jM), and cell specific nitrated protein was assessed by dot blot analysis for 3-nitrotyrosine (3NT). ANG II caused time and concentration dependent increases in 3NT above control levels. Endothelial cell lysates were incubated with nitrated protein standard (3NT-albumin) to assess cellular capacity for enzymatic denitration. Significant denitrating ability was observed but only from lysates exposed with 20% serum 93 (50% conversion at 12 hr). These data demonstrate that protein nitration occurs In normal healthy endothelial cells in culture and ANG II can directly stimulate protein nitration in vitro. Furthermore, endothelial cells possess denitrating abilities requiring some cofactor(s), and nitration may be a regulated intracellular signaling process. 94 INTRODUCTION Angiotensin II (ANG II) is a weil-recognized participant in many cardiovascular diseases, and alteration of its production or activity are currently important targets in cardiovascular pharmacology and medicine (ACE inhibitors and ANG II receptor antagonists) (Stroth and Unger, 1999; Lonn etal, 1994). Initially identified as a potent vasoconstrictor in the mid 1950s, ANG II is now recognized as a regulator of vascular cell growth, differentiation and gene expression (Brink et al, 1996;Griffin et al, 1991; Ratajska, 1994). Very recent vascular cell experiments by others suggest that ANG II is a potent stimulator of free radical such as superoxide anion via stimulation of NADH/NADPH oxidase activity. This pro-oxidant effect may contribute to several aspects of cell growth and altered gene expression (Griendling et al, 1994; Rajagopalan et al, 1996; Laursen etal, 1997). In the last decade, recognition of the importance of vascular endothelial cells for maintenance and regulation of vascular health has emerged (Drexler and Homig, 1999; Britten et al, 1999). Nitric oxide (NO) derived from vascular endothelial cells has been shown to be a critical modulator of local vascular tone and thrombus formation, and deficient endothelial NO production has been shown in a wide array of cardiovascular complications from various origins, including hypertension, atherosclerosis, heart failure, diabetes, and AIDS (Boulanger, 1999; Shimokawa, 1999; Cavallaro et al, 1997;Laightet al, 1999). The chemistry of NO in biological matrices is very complex, and several 95 biochemical pathways other than NO production can influence NO actions. For example, superoxide anion destroys NO, reduces its efficacy as a signal transduction agent, and promotes the formation of peroxynitrite, a highly reactive intermediate known to nitrate protein tyrosine residues and cause cellular oxidative damage (Pryor and Squadrito, 1995; Beckman, 1996). The reaction of NO with superoxide anion occurs at a diffusion-limited reaction rate, and NO is the only molecule known to compete with superoxide dismutase for its substrate in a biological setting. Thus, excess or uncontrolled superoxide anion formation can shift the actions of available NO from a useful cellular signal to a toxic free radical. ANG II is a well-known effector of vascular smooth muscle cells, but only a few reports have investigated its actions on endothelial cells. In previous studies we have shown that low-dose and short-term administration of ANG II reduced the efficacy of NO-mediated vascular relaxation in the absence of changes in contractile function in vivo. Endothelial dysfunction was associated with the extent of protein nitration confined to the endothelial layer (chapter 2). Our studies suggested to us that selective actions of ANG II on vascular endothelium occur and these changes apparently precede other established ANG II actions on vascular smooth muscle in vivo. These findings have demonstrated newly identified actions of ANG II in vivo and perhaps explain some aspects of ANG II inhibitor effects in humans, but the mechanisms involved are not established. In an attempt to more directly evaluate ANG II effects on endothelium, here we conducted in vitro experiments using cultured mouse endothelial cells. We 96 specifically tested the hypothesis that ANG II can promote protein nitration in endothelial cells directly and in the absence of other physiologic stimuli in vivo. Furthermore we conducted preliminary investigations o f enzymatic capacity for denitration in endothelial cells. 97 MATERIALS AND METHODS Cell isolator! and culture of endothelial cells. Pulmonary endothelial cells were isolated from male C57BL mice and graciously provided for our investigations by Dr. Dale Hoyt (Assistant Professor, Division of Pharmacology). The methods employed were as previously described (Gerritsen et al, 1995). Briefly, sixteen hours prior to sacrifice mice were injected ip with 50 ^g of gram- negative bacterial endotoxin (E. coli, serotype 0111:84, Sigma, UPS). The right and left lungs from 6-10 mice were dissected and briefly dipped in 70% ethanol, washed with PBS, and finely minced with scissors in Dulbecco's Modified Eagles Medium: Nutrient Mixture F I2 (DME/F12, Gibco-BRL Grand Island, NY) containing 0.1% collagenase. After 40 min incubation at 37 °C, the tissue mince was homogenized using a Dounce homogenizer (Kontes, Vineland, NJ) and filtered through 80-pm Nitex nylon screen (Small Parts Inc., Homestead, FL). The endothelial cells were selected by panning in flasks coated with phycoerythrin-conjugated anti-VCAM antibody, and subsequent fluorescence activated cell sorting (FACS) analysis. Cell were maintained in modified murine lung endothelial cells (MLEC) medium containing (per 500 mL) DMEM, 200 ml; Hams F I2 ,200 ml ; Penn/Strep , 8 ml of 200U+200ng/ml; ECGS, 15 mg; Heparin 5,000 U (Gibco-BRL, Grand Island, NY). Angiotensin II incubations: Cultured endothelial cells were seeded onto 6-well plates at the concentration of 3x10^ cells/mL. After establishing 90% confluence 98 fresh media containing 0,100 or 250 angiotensin II (Calbiochem- Novabiochem Corp., San Diego, CA) was added to each well. Degradation of angiotensin II during incubation was prevented by replacing the media every 12 hr thereafter. After 0, 48, and 72 hr of incubation, media was removed, cells were washed with PBS and then collected using lysis buffer (1% SDS, 1.0 mM sodium ortho-vanadate, 10 mM Tris pH 7.4). In a parallel set of experiments we incubated cells with tetranitromethane (TNM), a selective protein nitrating agent (Sokolovsky, 1966) as a positive control treatment group. Cells were washed with PBS and then incubated with 1 mM TNM for 15 min. After removing excess TNM by several PBS washes, cells were harvested in lysis buffer for 3NT immunoblot analysis. 3NT immunohistochemistry: Immunohistochemistry was performed to determine the relative extent of protein nitration (a stable biomarker of endogenous peroxynitrite formation), using an antibody raised against 3- nitrotyrosine (3-NT). Following various cell treatments (PBS, Angiotensin , or TNM) endothelial cells were trypsinized and wash twice with PBS. Cells were fixed in 10% buffered formalin for 15 minutes, then washed once with PBS and resuspended in 70% EtOH for seeding on slides. To restore antigenicity, slides were immersed for 15 min in a 10 mM citrate buffer (pH6) preheated to boiling. Cells were then incubated with rabbit anti-mouse polyclonal antibody raised against 3-NT (1:200 dilution. Upstate Biotechnology, Lake Placid, NY) for 1 hr. Biotinylated secondary anti-rabbit serum was then applied (1:200), followed by 99 horseradish peroxidase complex reagent (ABC Elite. Vector Laboratories, Burlingame, CA). Positive immunoreactivity was visualized through the development of diaminobenzidine (DAB) chromogen, and methyl green was used for nuclear counterstaining. Preliminary experiments were conducted to verify the specificity of immunostaining In our laboratory. Preincubation of primary antibody with free 3NT (1mM) completely quenched positive staining, whereas tyrosine (1mM) had no effect. Non-immune serum (isotypic) controls also showed no detectable immunoreactivity in any treatment group. Dot blot analyses for enrlothelial cell protein nitrabon: To aid in quantification we developed a dot blot assay for protein 3NT in cellular lysates. Twenty micrograms of protein from each cell lysate sample were adjusted to 500 with PBS and then blotted onto Protran®BA nitrocellulose membrane (Schlelcher&Schuell, Keene, NH) with the aid of 96-well Bio-Dot® Microfiltration Apparatus (Bio-Rad, Hercules, CA). Total protein amount on the nitrocellulose membrane was visualized using BLOT-FastStain^(Geno Technology, Inc., St. Louis, MO). Following destaining the same blot was processed for Immunodetection of 3NT (preliminary experiments demonstrated this did not affect 3NT measurement). Membrane was incubated for 1 hr in blocking solution (5% dry milk in TBST, 0.01 M Tris, 0.14 M NaCI, pH 8.0), then incubated with rabbit anti-mouse polyclonal antibody raised against 3-NT (1:200 dilution. Upstate Biotechnology, Lake Placid, NY) for 1 hr in blocking solution. After incubation, membrane was washed four times with TBST at 5-minute intervals 100 with agitation. Biotinylated secondary anti-rabbit serum was then applied (1:5000). followed by alkaline phosphatase-streptavidin conjugate (Vector Laboratories, Burlingame, CA). Positive immunoreactivity was visualized through the development of the purple product of BCIP/NBT (5-bromo-4-chloro-3-indolyl phosphate/nitroblue tétrazolium) substrate. Digital images of the protein stained and immunostained membrane were obtained using a flatbed scanner (2400 dpi resolution, Hewlett Packard ScanJet 63000) and then analyzed using research-based image analysis software (ImagePro Plus, Media Cybernetics, Silver Spring, MD). Gray scale intensity values were determined for the protein stain and the 3NT immunostaining for each dot on the membrane. Preliminary experiments were conducted using nitrated bovine serum albumin (BSA) to demonstrate linearity of our analytical methods (see results). SIN 1 was used as a BSA protein nitrating agent, under conditions reported to cause selective tyrosyl nitration (Kamisaki et al, 1998). BSA (lOmg/mL) was dissolved in PBS, and incubated for 4 hr at 37°C. The nitrated protein product was then dialyzed in PBS (3/4” dialysis tubing, 12-14 kD molecular weight exclusion limit. Life Technologies, Inc, Gaithersburg, MD) overnight to remove excess TNM. Previous studies by others have demonstrated that this nitration method provides 10% conversion of available BSA tyrosine residues, this efficiency was used for subsequent standard curve formation. 1 0 1 D e n itra tio n s U id ie s :We conducted preliminary studies to test the hypothesis that endothelial cells can denitrate proteins. After washing with PBS. confluent cells were harvested by scraping from 75-cm^ culture flasks in PBS buffer containing cocktail of protease inhibitors (10 pg/mL each of soybean trypsin inhibitor, aprotinin, leupeptin, prostatin A; 200 pM phenylmethanesulfonyl fluoride, 100 pM EDTA). Cells were then disrupted using an ultrasonic probe for ten 5-second intervals on ice (Fisher Scientific, Pittsburgh, PA). Three mL of cell lysate (total protein concentration 2 mg/mL) were incubated with 0.5 mL of nitrated BSA (1 mg/mL) at 37"C with or without 20% fetal bovine serum. Samples from this incubation mixture (SOOpI each) of cell lysate and nitrated BSA were collected at different time points (1 hr, 6 hr, 12 hr, and 24 hr) and heated to boiling for 10 min in the presence of 2% SDS. Protein concentration in each sample was then determined by a Bicinchoninic Acid (BOA) protein assay (Walker, 1994). These samples were then evaluated for total protein nitration using the dot blot assay described above. 102 RESULTS Shown in Figure 4.1 is a normal growth curve of endothelial cells in culture media. Initial seeding of 1x10* cells/mL medium resulted in cell population doubling time (2X) on day 3, and cells reached 90% confluence on day 5. In these studies we focused on studying endothelial cells at (or near) confluence and therefore only employed cells after 5 days of culture. Initial studies using immunohistochemistry demonstrated evidence of ANG II induced cell nitration. Representative photomicrographs are shown in Figure 4.2. Using these in situ methods, positive signal for 3NT was observed in the ANG II treated cells relative to PBS incubation and isotypic staining control. High magnification showed that the observed 3NT immunoreactivity was predominantly intracellular and cytosolic. As expected, cell incubation with TNM (ImM for 15 min) also caused significant evidence of 3NT formation (serving as a positive control). In an attempt to allow quantification of protein 3NT in cell lysates, we developed a dot blot analysis method. Shown in Figure 4.3 (top) are representative dot blots for protein standards with respect to dot protein quantities and 3NT immunoprevalence. Linearity of this method was established using nitrated BSA protein standards. Digital image analysis of staining intensity for each dot was assessed, and a standard curve for protein nitration was created. 3NT signal for each dot was normalized to protein signal, and a linear relationship was established between pM nitrated BSA and 3NT 103 immunoreactivity (3NT immuncprevalence/protein prevalence, Figure 4.3 lower panel). Figure 4.4 shows the effects of ANG II incubation on endothelial cell protein nitration. Cell lysates were evaluated using dot blot analyses and pmoles 3NT/mg protein were measured using the standard curve methods described above. Incubation of endothelial cells in the absence of ANG II produced detectable 3NT after 48 hr, and this was further increased at 72 hr. Furthermore, time and concentration dependent increases in endothelial protein nitration was observed during ANG II incubation (100 and 250pM). In preliminary experiments we assessed the ability of endothelial cells to modify nitrated protein in vitro, using nitrated BSA as a substrate. Our preliminary results are shown in Figure 4.5. Cell lysates were incubated with nitrated BSA (143|jg/ml) in the presence and absence of fetal bovine serum (20% by volume). Samples were collected for dot blot analysis at various times during incubation at 37'C. All cell lysates contained protease inhibitors in PBS solution (see methods). Significant denitrating ability was observed in cell lysates containing fetal bovine serum. An apparent zero-order rate was observed with approximately 50% converted in 12hr. In contrast, no significant cellular denitration was observed in the absence of serum, and incubation of nitrated BSA in serum alone also had no significant affect on 3NT quantities within the 24 hr study. 104 DISCUSSION Enhanced superoxide production and peroxynitrite-mediated tyrosyl protein nitration are implicated in oxidative-related pathologies of ischemia/reperfusion injury and cardiovascular disorders (Sakurai et al. 1998;Kooy et al, 1997;Oyama et al, 1998). Studies from our laboratory and others suggest that the presence of nitrotyrosine residues in the vasculature and myocardium is associated with significant organ malfunction (Depre etal, 1999;Wang and Zweier, 1996, chapter 2). Although the direct causative consequences of protein nitration on cellular function has not been extensively Investigated, this post-translational protein modification may participate in the dysregulation of several cellular signaling pathways mediated by tyrosine residues, including receptor protein tyrosine kinases, i.e. cytokine, growth factors and insulin receptors. Gow et al (1996) demonstrated that peroxynitrite-mediated protein nitration Inhibited tyrosine phosphorylation of the important tysosine kinases (c-src and v-abi) responsible for growth regulation. Thus, a cellular capacity to reverse protein nitration may be beneficial to tissues and organs that are vulnerable to oxidative damage such as endothelial cells. Our previous study (Chapter 2) has shown that low dose angiotensin II promoted tyrosyl protein nitration selectively in the endothelium in vivo. We now demonstrate that this phenomenon also occurs in an isolated cell setting. Thus the complex multi-cell environment and presence of complex physiological regulation are apparently not required for ANG II induced endothelial protein nitration. Recent studies have demonstrated that ANG II is an activator of 105 superoxide production in several cell types, including fibroblasts (Pagono et al, 1997), mesangial cells (Jaimes et al, 1998),and endofiielial cells (Pueyo et al, 1998). This stimulation is apparently mediated by the AT 1 receptor subtype activation, leading to increased gene expression of NADH/NADPH oxidase subunits p67phox (Pagono et al, 1998), p22phox (Marumo et al, 1998), and perhaps others. The role of this enhanced oxidase expression in ANG II mediated actions is not clear, but it may contribute to the other known cellular responses (e.g. proliferation, hypertrophy, etc). While isolated endothelial cell studies have demonstrated the selective presence of AT1 receptors (and an apparent absence of the AT 2 subtype) (Pueyo et al, 1998), whether the endothelium plays a direct role in ANG II related cardiovascular disease in vivo has not been established. Recent investigations have demonstrated that the biological activities of NO are highly dependent on both production and destruction related pathways (Freeman, 1994). A chemical pathway of biological importance appears to be the formation of peroxynitrite through the interaction of NO with superoxide anion. This reaction is exceedingly rapid and efficient (1.9 x10^° M*^s'\ Kissner et al, 1997), leading to reduced levels of available NO and formation of a highly reactive oxidant in vivo (Becman and Koppeno, 1996; Huie and Padmaja, 1993; Kissner etal, 1997). Peroxynitrite and related species aggressively nitrate protein tyrosine residues, leading to a chemically stable biomarker, 3-nitrotyrosine (3-NT) (Beckman, 1996). We and others have demonstrated the value of this marker as evidence of NO dysregulation in disease, including advanced atherosclerotic lesions (Beckman, 1994), sepsis 106 related organ failure (Grouser et al. 1998), and renal transplant rejection (MacMlllan-Crow et al, 1996), and others. Here we found the extent of ANG II induced endothelial cell protein nitration was both time and concentration dependent. This effect is not likely to be mediated by changes in cell populations since we focused on the use of confluent cells during a plateau growth phase. While detailed mechanistic studies have not yet been conducted, we postulate that the observed increases in protein nitration occur via ANG II activation of NADH/NADPH oxidase, promotion of superoxide, leading to enhanced peroxynitrite formation. Further studies to address these hypotheses are planned. While increased protein nitration has been observed by us and others in a variety of disease states, the consequences of this phenomenon are not clear and the roles of nitration mechanisms in cellular responses are not well studied. Recently, Kamisaki etal (1998) have shown that rat tissue homogenates have the ability to modify nitrated protein within 30 minutes. Additionally, Greenacre et al (1999) has described biphasical degradation of peroxynitrite-induced protein nitration in rat skin. The first clearance phase was rapid {VA - 2 hr) while the later phase appeared to be a slow process (t!4 = 5 hr). We also found significant evidence of specific endothelial cell denitrating ability and that some cofactor(s) was required for this reaction to occur. This denitration pathway that may be a protective mechanism for refreshing redox proteins. Further studies identifying the enzyme(s) responsible are warranted. 107 Interestingly, we also observed readily detectable and time dependent protein nitration in the absence of ANG II stimulation. Thus, nitration may be a phenomenon that occurs during normal cellular aging (Stadtman and Berlett, 1998). Altematively, our findings suggest that perhaps nitration is a normal cellular event, rather than one that occurs only during cell toxicity and/or death. Previous chemical studies have shown that an adjacent nitro- functional group decreases a phenolic pKa by by roughly one log unit Thus tyrosine nitration would likely influence phosphorylation reactions at the adjacent phenolic moeity. Our findings of a basal presence of tyrosine nitration and evidence of denitration suggest that perhaps nitration mechanisms participate as an intracellular signaling mechanism. Altematively perhaps it serves as an indirect modulator of tyrosine kInase/phosphatase signaling pathways. 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J Mol Cell Cardiol 1999;31:23-37. 8tadtman ER and Berlett B8. Reactive oxygen-mediated protein oxidation in aging and disease. Drug Metab Rev 1998;30: 225-243. 8troth U and Unger T (1999) The renin-angiotensin system and its receptors. J Cardiovascular Pharmacol 33 8uppl 1, 821-8 Walker JM. The bicinchoninic acid (BCA) assay for protein quantitation. In Methods in Molecular Biology, Vol. 32:Basic Protein and Peptide Protocols. Ed. JM Walker, Humana Press Inc., Totowa, NJ, 1994, pp. 5-8. Ill Wang P and Zweier JL Measurement of nitric oxide and peroxynitrite generation In the postischemic heart. J Biol Chem 1996;271:29223-29230. 1 1 2 E 3 " a £ 1e+5 ® 1e+4 0 ) O 8 W—— LAG LOG PLATEAU Figure 4.1 Endothelial cell growth curve. Cultured endothelial cells reached 90% confluence on day 5 (plateau phase). Population doubling time (2X) was achieved on day S.There was no more than 10% change in cell number during 0- 72 hr incubation beginning on day 5 (plateau phase). 113 o 0.100 s , s c g 0.025 O) % 0.000 150 300 450 600 3NT (pmoles) Figure 4.2 Nitrated BSA concentration and 3NT immunoreactivity curve. A linear correlation of nitrated BSA and 3NT immunoreactivity ratio was assessed by gray scale intensity values, i.e. gray scale intensity of BSA determine by FastStain divided by gray scale intensity of nitrated BSA determined by western blot analysis. 114 Q A D J# * * % * ' f * ' ««» C D • * * * 0 #■ ^ # V ^ # % # ^ # % * * 4# t # # # t Figure 4.3 Angiotensin ll-induced protein nitration in endothelial cells in vitro. Photographic representation of 3NT immunoreactivity in endothelial cells incubated with PBS (A) or 100 pM angiotensin II (B) for 72 hr. Positive control for 3NT immunoreactivity was generated by Incubation of endothelial cells with 1 mM TNM for 15 min (D). Panel C showed isotypic control (without primary antibody) using cells treated with 1 mM TNM as shown in panel D. 115 c 140 - BHI 48hr £ . 1--- 1 72hr o Q. 120 - t G) T CO 3 g 100 o E Q. 80 L# 0 100 250 Angiotensin il (^M) Figure4.4 Quantitative 3NT immunoprevalence in angiotensin ll-induced endothelial cell nitration. Cells were incubated with angiotensin II (100 or 250 |jM) for 48 and 72 hr. Total protein nitration was detected by immunoblot as described in methods. *, p<0.05 compared with 0 pM angiotensin II; f. p<0.05 compared to 0 pM angiotensin II at 48 hr. 116 125 100 CO § 75 CO I- o Z o O 50 O 25 • no serum O with serum cell free 10 20 30 HOURS Figure 4.5 Endothelial cell dénitration. Decreases in 3NT immunoprevalence in endothelial cell lysates incubated with nitrated BSA (no serum and with 20% serum) were detected by slot blot immunoreactivity analysis at 1, 6, 18, 24, and 48 hr. Serum control was performed by incubation of 20% serum and nitrated BSA without cell lysate. 117 CHAPTER 5 INTERACTION OF ANGIOTENSIN II AND TNF-a IN VIVO: PART I. CARDIAC TUNEL STAINING AND NOS II INDUCTION PART II. NEUROHORMONES AND GENE EXPRESSION 118 PART I. CARDIAC TUNEL STAINING AND NOS II INDUCTION ABSTRACT O bjective: Angiotensin II (ANGII) and TNF-a have each been shown to promote superoxide and nitric oxide production and induce apoptosis in vitro. While both agents have been implicated in cardiac failure and remodeling, few studies have evaluated their interactions in vivo. We tested the hypothesis that ANGII or TNF promote early cardiac apoptosis when administered alone or in combination. We also tested if NOS II is a participant in these early cardiac changes. M ethods: Osmotic mini-pumps were implanted in Sprague Dawley rats to deliver ANGII (288 |ag/kg/day), TNF-a (22 pg/kg/day), or their combination, for 3 days. Systolic blood pressure and heart rate were recorded on days 0 and 3. At sacrifice, cardiac tissues were fixed in formalin for TUNEL staining and immunohistochemistry for NOS II and digital image analyses. R esults: No alterations in blood pressure or heart rate were observed in any treatment group on day 3. Cardiac and left ventricular mass was also unaltered relative to vehicle controls. ANGII or TNF-a alone induced increases in cardiac TUNEL staining (%posltive nuclei/mm^=8.4+1.6 and 8.5+1.6; ANGII and TNF, respectively) relative to vehicle control (3.8+0.7,p<0.05), but combined infusion resulted in no change from control (2.1+0.6). No significant change in total nuclei/mm^ was observed in any treatment group. Immunoprevalence of NOS II was not significantly different between groups. Correlation analysis of regional NOS II immunoprevalence and frequency of TUNEL positive cells showed no statistically 119 significant relationships in any treatment groups. Conclusion: These results demonstrate that ANGII and TNF-a alone promote cardiac apoptosis in vivo independent of detectable hemodynamic or cardiac size changes, but their combined administration does not. These changes are apparently not related to NOS II induction, but suggest important interactions between ANGII and TNF exist in vivo. 1 2 0 INTRODUCTION Chronic congestive heart failure currently affects more that 5 million individuals and represents a significant health problem in the United States (Sharpe, 1999). This condition is characterized by progressive reduction in cardiac performance, ventricular remodeling, and activation of various neurohormonal pathways. It is now recognized that the renin-angiotensin system plays an important role in CHF progression, and inhibition of this pathway has provided significant therapeutic value. The primary effector of this system, angiotensin II (ANG II), is an important stimulant of cardiac remodeling as well as vascular contractility and smooth muscle proliferation (Brilla, 1990;Griffin 1991;Ratajska 1994;Brilla 1996;Liu 1998). Thus, experimental and therapeutic outcomes from the use of ACE inhibitors and angiotensin type I (ATI) receptor blockade have illustrated the importance of ANG II in the progression of heart failure (Childs, 1990; Brilla 1996;Yonezawa 1996,Yang 1997;Griffin 1991;Ratajska 1994). Despite these significant therapeutic gains, five year mortality in CHF patient populations remains approximately 40-50% even with optimized currently available therapies (Sharpe, 1999). In addition to elevated levels of angiotensin II (ANGII), increased plasma levels of the tumor necrosis factor-a (TNF) have been commonly observed in heart failure patients. This cytokine has been implicated in the progression of cardiac failure (Levine 1990;Dutka 1993; Katz 1994;Torre-aminone1995;Milani 1996), and TNF serum levels have been correlated to severity of cardiac dysfunction (Ferrari 1995;Torre-Aminone 1996). While the roles of TNF activation 121 in heart failure progression are not defined, this cytokine is known to promote cardiac gene expression of the inducible isoform of nitric oxide synthase (NOS II) and stimulate cellular apoptosis (Wildhirt et al, 1995; Luss et al, 1997). Interestingly, ANG II also has been shown to elicit similar induction of NOS II and promote apoptosis in vitro. While increased levels of ANG II and TNF have been commonly observed in CHF, their combined influences and interactions in vivo have not been extensively investigated. Additionally, the relationships of cardiac NOS II expression and apoptosis in the presence of these modulators have not been defined. Here we evaluated the in vivo effects of ANG II or TNF on cardiac tissue, when infused alone or in combination, with respect to DNA strand breaks suggestive of apoptosis (TUNEL staining, in situ terminal deoxynucleotydyltransferase nick-end labeling), and NOS II induction. 1 2 2 MATERIALS AND METHODS Animal model Alzet osmotic pumps (Aiza Corp., Palo Alto, CA) were implanted intraperitoneally to rats (Sprague Dawley, 325-350g). Vehicle (saline, n=8), or ANGII (288 pg/kg/day, n=6; Bachem Biosciences Inc, King of Prussia, PA), or TNF-a (22 ^g/kg/day, n=6; NCI, Bethesda, MD), or their combination were continuously administered for 3 days. On Day 0 and Day 3 systolic blood pressure (SBP) and heart rate (HR) were recorded using tail cuff methods. At sacrifice infusion pumps were isolated and weighed to assure reliable delivery of infusion solution. The ANG II dose we employed is considered a "sub-pressor" dose and appears to be physiologically relevant since it has been shown to produce steady state ANGII blood levels similar to those observed in hypertensive animals and humans (approximately 100 pg/ml. Tan et al, 1991). To confirm reliable delivery of a biologically active ANGII dose we measured plasma renin activity in the various treatment groups (see below). The TNF dose employed in this study was approximately equivalent to the doses used in human studies (Gaskill, 1988). Previous studies in rats showed this dose to have no influence on heart weight and hemodynamic parameters after one-week infusion (Steven 1993). This is a short-term nontoxic TNF administration when compared to a reported 20% mortality in animals infused with 100 jjg/kg/day TNF for 1 week (Gaskill 1988). Plasma levels of TNF on day 3 (at sacrifice) were less than 100 pg/ml. Since previous studies have 123 demonstrated TNF concentrations to be 20-100 pg/ml in heart failure patients the dose we employed has apparent physiological relevance. Plasma renin activity Rats were sacrificed on day 4 by pentobarbital (Nembutal, 75 mg/kg) injection. Blood was withdrawn by cardiac puncture and plasma was separated for plasma renin activity (PRA> radio-immunoassay (Incstar, Stillwater, Minnesota). Intra-day and inter-day assay variability was less than 10%. Plasma concentrations of TNF Plasma TNF concentration was determined using cytotoxicity assay as previously described (Bergman and Holycross, 1996). Briefly, L929 cells were seeded in 96-well plate and cultured in RPM11640 medium containing 5% fetal bovine serum and antibiotics (GIBCO BRL). The next day, actinomycin D (1^g/mL) was added to each well in the presence of TNFa standards or plasma samples. Following overnight incubation, cells were fixed with 5 % buffered formaldehyde for 5 min and then incubated with 0.5% crystal violet for 5 min. After several washes cells were solubilized in 150 pL of 33% glacial acetic acid, and the absorbance at 580 nm was obtained using SLC Spectra plate reader. Standard curve was generated and the extent of cytotoxicity was determined. 124 Cardiac tissue collection At sacrifice, cardiac tissues were rapidly weighed, cut transversely at the midline, and fixed in 10% buffered formalin for 24 hours. Tissues were then dehydrated and embedded in paraffin blocks. Five-micron tissue sections were used for general morphological evaluations and immunohistochemical studies using standard methods (described below). Immunohistochemistry for NOS II Tissues were then exposed to a 3% hydrogen peroxide/methanol solution for 10 min to block endogenous peroxidase activity. To restore antigenicity, tissues were incubated for 15 min in a 10 mM citrate buffer (pH6) preheated to boiling. Sections were then incubated for 30 min in a 10% normal goat serum/PBS solution to block non-specific binding. Next, tissues were incubated with a 1 ;200 dilution of rabbit anti-mouse polyclonal antibody raised against NOS II (Transduction, Lexington, KY) for 1 hr. Biotinylated secondary anti-rabbit serum was then applied to the tissues at a dilution of 1 ;200. Then, a horseradish peroxidase complex reagent (ABC Elite, Vector Laboratories, Burlingame, CA) was added, and positive immunoreactivity was visualized through the development of diaminobenzidine chromogen. Methyl green was used for nuclear counterstaining. 125 In situ detection of DNA damage Nuclear 3 -OH DNA strand breaks were detected in cardiac tissue by the in situ CardioTACS™ kit (Travigen, Gaithersburg, MD) according to manufacturer instructions. Nuclease-treated control was generated in order to confirm and optimize the labeling procedure. The cells undergoing apoptosis exhibited blue nuclear staining, while negative nuclei could not be observed under this staining condition. Using this commercially available kit, only TUNEL positive nuclei are identifiable (blue) with a high-contrast red cellular counterstain. Therefore a serial 5pm section of each tissue was obtained and stained with hematoxylin and eosin (H&E), allowing determination of total number nuclei per square millimeter using digital image analysis. Digital Image analysis Images were captured using a high-resolution digital camera (Fixera, Inc., 1260x960pixels) and Olympus BX-40 microscope Unmodified images were analyzed using research based image analysis software (Image Pro Plus, Media Cybernetics, MD), using methods previously described (Wattanapitayakul et al, 2000). Six regions of each heart tissue were systematically captured at 400x magnification, representing approximately 5% of the total left ventricle area. Cardiac apoptosis was reported as percent TUNEL positive nuclei per square millimeter. 126 NOS II immunoreactivity was assessed at the same tissue regions as the detection of TUNEL positive nuclei (in serial 5pm sections). Extent of NOS II immunoreactivity in LV was determined in the tissues by applying intensity thresholding analysis, similar to that described by others (Kuyatt et al.. 1993). Briefly, the captured cross sectional images were background corrected (to reduce stray illumination artifact) and converted to gray scale. We observed that immuno-positive staining of NOS II due to DAB deposition provided a distinct gray scale intensity distribution when compared to methyl green counter stain. Gray scale intensity values representing the brown color consistently exhibited values less than 170 units, whereas pixel intensity units representing methyl green and background staining where consistently above this value. Thus, the quantity of pixels falling in the range of 0-170 provided a quantitative estimate of positive immunoreactivity. Statistical Analvsis All data are presented as mean+SEM. Statistical comparisons were performed with Kruskal-Wallis One Way ANOVA on Ranks (Sigmastat software), and Spearman non-parametric correlation was generated by SigmaPlot software (Jandel Scientific Software, San Rafael, CA). Criterion for statistical significance was set at p<0.05. 127 RESULTS Shown in Figure 5.1 are hormonal and hemodynamic measurements after 3-day infusions of saline (vehicle control, VEH), ANGII. TNF, or their combination (AT). ANGII administration, either alone or in combination with TNF, caused significant reductions in PRA by day 3, demonstrating hormonal feed back of renin-angiotensin axis and illustrating a successful infusion of ANGII (Figure 5.1, top panel). Despite this early evidence of physiological actions, no change in systolic blood pressure or heart rate were observed on day 3 in any of the treatment groups (lower panel). No significant change in plasma aldosterone was observed in any treatment group, and plasma TNF levels were below 100 pg/mL (assay detection limit) in all animals (data not shown). Figure 5.2 shows measurements of cardiac mass following ANGII, TNF, or their combination on day 3. No significant changes in bodyweight were observed among these groups. Similarly, total heart/body weight and left ventricular/body weight ratios were not altered by any treatment at day 3, suggesting no detectable cardiac hypertrophy at this time. Figure 5.3 shows representative photomicrographs of TUNEL positive neclei. The CardioTACS system employed provided excellent contrast of positive (blue) nuclei against red cytosolic counterstain. Positively stained cells included myocytes and nonmyocytes. Serial 5 micron tissue sections were used to count total nuclei per tissue cross sectional areas. Identical tissue regions were studied as TUNEL measurements. Hematoxilin & Eosin staining revealed no obvious immune cell infiltration or significant changes in total number of nuclei per square 128 millimeter in any treatment groups (VEH= 2021+57; ANGIf=2256+44; TNF=2160+71; ANG/TNF=2285+71 nuclei/mm^). Figure 5.4 demonstrates the photomicrographs of cytoplasmic NOS II immunoreactivity (slightly brown staining representative of DAB deposition) against methyl green nuclei counterstain. Shown in Figure 5.5 are averaged data for in situ nick end-labeling (TUNEL) nuclei and NOS II immunoprevalence in cardiac tissues after 3-day infusions of saline (vehicle control, VEH), ANGII, TNF, or their combination (ANG/TNF). ANGII and TNF both induced statistically significant increases in TUNEL positive nuclei but their combination did not (top panel). Immunohistochemistry showed that NOS II expression was slight but detectable in all treatment groups and statistically increased from isotypic staining controls (bottom panel). Digital image analysis showed no significant changes in NOS II immunoprevalence following ANG II and/or TNF infusion. In Figure 5.6 we additionally tested for statistical associations between NOS II immunoprevalence and number of apoptotic nuclei in cardiac tissue using 5pm serial tissue sections and digital imaging of identical tissue areas. No statistically significant association of NOS II immunoprevalence with frequency of TUNEL positive cells was observed for any treatment group. (Spearman non- parametric correlation analyses, NS for all panels). 129 DISCUSSION While increased levels of ANGII and TNF have been commonly observed in CHF, their combined influences have not been extensively investigated. Recent investigations have suggested that cardiac cell apoptosis and/or induction of NOS II may play an important role in ventricular remodeling and progressive contractile depression in heart failure. In vitro experiments have shown that ANGII or TNF are each capable of promoting apoptosis and/or stimulating NOS II gene expression in a variety of cell types, including neuronal PC12 cells, Hela cells, hepatocytes, and cardiac myocytes (Heneka et al, 1998; Dickinson et al, 1995; Leist et al, 1995; Cigola et al, 1997;Kajstura et al, 1997). Here we investigated the effects of ANGII and TNF in vivo in an attempt to define interactions between these agents. To our knowledge this is the first study designed to evaluate such interactions in vivo. The doses employed herein were designed to provide blood concentrations that are physiologically relevant and comparable to those found in heart failure patients and animal models. Recent investigations have suggested cardiac cellular apoptosis may play a role in ventricular remodeling and progressive myocardial dysfunction. For example, positive TUNEL nuclei and/or DNA fragmentation has been demonstrated in human dilated cardiomyopathy, pacing induced heart failure in dogs, and a genetic rat heart failure mode (Hamlet et al, 1995; Narula et al, 1996; Mallat et al, 1996; Li et al, 1997; Capasso et al, 1990, Tomanek and Aydelotte,1992). Thus, selective loss of cardiac myocytes, and perhaps other cell types, are apparently a common phenomenon in a variety of human and animal 130 models of cardiac remodeling and failure. While the in vivo significance and mechanisms involved in cardiac cell dropout have not been fully elucidated, apoptosis may be an initial event of remodeling process resulting from neurohormonal and/or cytokine actions. These include ANG II and TNF, which are known mediators of cardiovascular disease progression and likely participants in the process of remodeling. In vitro studies have shown that each of these agents alone promote programmed cell death in a variety of cells, including cardiac myocytes, although the signaling pathways involved are agent specific. ANGII-induced apoptotic signaling appears to be diversified among cell types, and requires specific AT 1 and/or AT2 angiotensin receptor subtypes (Yamada 1996,1998). In cultured cardiac myocytes, the ATI subtype is solely responsible for triggering apoptosis (Kajstura 1997, Cigola 1997). Suggested mechanisms for ANG ll-induced apoptosis include activation of protein kinase, MAP kinase, and STAT pathways (Hiriuchi 1997a,c). Studies with angiotensin converting enzyme inhibitors or AT 1 receptor antagonists have demonstrated favorable effects in heart failure therapy and have also been shown to reduce cardiac myocyte apoptosis. Thus, some of the benefit derived from these ANGII- based therapies may be the prevention of apoptosis related myocyte dropout and decreased cardiac remodeling (Goussev et al, 1998). TNF is apparently pro-apoptotic via several signaling pathways, including activation of Fas ligand, and binding of TNF to the death domain of TNF receptors (Wong et al, 1994; Leist et al, 1995). Several studies in the last decade have illustrated that TNF is commonly elevated during cardiac dysfunction and 131 failure. As a Th1 (T cell helper type 1) cytokine it has potent effects on both immune and parenchymal cells. In vitro studies have shown that TNF depresses cardiac myocyte contractility, alters electrophysiological functions, and promotes apoptosis (Sarter et al, 1996; Cain et al, 1999; Alloatti et al, 1999; Edmunds et al, 1999). Thus, elevated TNF may participate in heart failure progression and ventricular remodeling. Interestingly, suppression of TNF production by phosphodiesterase III inhibitors has been shown to provide some hemodynamic benefit and improved survival in the treatment of heart failure (Feldman et al, 1993). Given the recognition that ANGII and TNF are commonly elevated in cardiac disease and that they employ differing apoptosis signaling pathways in cardiac myocytes, the studies described herein were originally designed to test the hypothesis that ANGII and TNF would synergize with respect to pro-apoptotic effects. We chose to test this hypothesis in vivo to provide a higher degree of physiological relevance to the studies and to attempt to relate the changes to functional (hemodynamic) data. We focused on the effects of these agents at only 3 days during administration to investigate the early events in the cardiac remodeling process. Here we found that both ANGII and TNF, when administered alone, caused significant increases in the frequency of TUNEL positive nuclei. These changes occurred after only 3 days of administration in vivo and at doses that yield pathophysiologically relevant blood concentrations (see description in methods section above). While other in vivo reports have demonstrated cardiac 132 apoptosis during pressure overload in vivo (Teiger etal, 1996 and Condorelli et al, 1999), the changes we observed were independent of hemodynamics (no change In blood pressure or heart rate were observed). While the pro-apoptotic actions of these agents have been previously observed in vitro, to our knowledge this is their first demonstration cardiac tissue in vivo. The cell types involved included both myocytes and non-myocytes fudged by size and shape of TUNEL positive nuclei). The general morphological characteristics in these treatment groups were unremarkable, and H & E and Masson’s trichrome staining revealed no appearance of immune cell infiltrates or fibrosis development by day 3. These rapid effects are generally consistent with isolated cell experiments that have shown ANGII orTNF induced apoptosis within 24hrs of exposure. Interestingly, we observed no significant change in TUNEL positive nuclei following administration of combined ANGII and TNF. Thus, the apoptotic effects of each agent alone were apparently cancelled out by their combined presence in vivo. This combined inhibition is not likely due to rapid and extensive apoptosis that occurred prior to day 3 since total nuclei and general morphological characteristics (H & E and Masson’s trichrome staining) were not different from the vehicle treatment group. Recent studies have suggested that positive detection of nuclear 3 -OH groups using TUNEL methodologies may not exclusively detect cells undergoing apoptosis. For example, Kanoh et al. (1999) recently described a detailed study to evaluate DNA fragmentation in a small population of late-stage dilated cardiomyopathy (DCM) autopsy samples. While TUNEL-positive nuclei were 133 detected in these tissues, these were not confirmed by a Taq polymerase-based DNA in situ ligation assay, which detects double strand breaks. Additionally, most TUNEL positive nuclei did not have typical ultrastructural abnormalities consistent with apoptosis. These authors concluded that while apoptosis likely contributes to the considerable myocyte loss during DCM. the TUNEL positive nuclei were actually indicative of DNA repair rather than apoptotic processes. This report therefore raises concerns about the specificity o f the TUNEL method, at least in late stage DCM tissues. While we cannot rule out DNA repair as a mechanism involved in our conditions, a direct relation of our experimental conditions (otherwise healthy rats receiving infusions of ANGII and/or TNF) to the DCM tissues described above is not likely. Therefore, given previous demonstration that these agents are pro-apoptotic in various cell types, we have cautiously interpreted TUNEL positive nuclei as suggesting apoptotic events. We did not perform DNA gel electrophoresis in the present study because of limited tissue samples available and because DNA laddering is generally only detectable when a high percentage of cells are simultaneously undergoing apoptosis. Additionally, it is impossible to differentiate the cell types involved using ladder methods, and the actual sensitivity of ethidium bromide staining can be quite low. For these reasons the general value of DNA laddering techniques for cardiac tissue studies is still unclear. The apparent inhibition of apoptosis from ANGII and TNF combination is interesting and unexpected. Given the fact that these agents stimulate apoptosis via differing signaling cascades, it is possible that a dual message may cause net 134 inhibition. Several previous studies have suggested complex interactions between the renin-angiotensin system and TNF both in vivo and in vitro, but no previous studies have investigated apoptosis in these interactions. For example, TNF administration has been shown to modulate renal gene expression of aldosterone and secretion of renin (Antonipillai et al, 1990 and Natarajan et al, 1989). TNF also stimulated angiotensionogen gene expression in hepatocytes via the transactivating protein Rel A (Brasier et al, 1996). In addition, ACE inhibitors dose-dependently suppressed IL1-induced TNF synthesis in human peripheral blood mononuclear cells (Schindler et al, 1995). Further studies investigating this potentially important observation and defining the mechanisms responsible are warranted, particularly using isolated cell conditions. In addition to promoting apoptosis in cardiac myocytes, both ANGII and TNF are known to cause induction of NOS II gene expression (Saito, 1996; Hennington, 1998; Schena, 1999). This enzyme, the “inducible” nitric oxide synthase, is recognized as a high capacity and calcium independent isoform involved in conversion of arginine to nitric oxide and citrulline. First identified as an important contributor to immune system function and host-defense reactions, NOS II has recently been investigated in various cardiac pathologies, including cardiomyopathy, cardiac graft rejection, and heart failure. Although at this time the significance of cardiac NOS II induction during heart failure is somewhat controversial in human heart failure (Drexler et al, 1998 and Stein et al, 1998), high concentrations of nitric oxide produced from exogenous chemicals or NOS II are known to cause DNA damage and apoptosis in cardiac myocytes (Shimojo et 135 al, 1999 and ing et al, 1999). TNF-medîated induction of NOS II also appears to be a potential signaling pathway of cardiomyocyte apoptosis in vitro (Schulz, 1995). For these reasons we in we investigated the potential contribution o f NOS II to the observed TUNEL changes after ANGII and/or TNF. Using immunohistochemical methods we observed slight but detectable NOS II presence in all treatment groups (statistically increased from Isotypic staining controls). In previous studies others have also observed slight but detectable levels of this isoform in cardiac tissue during control conditions (Rouet-Benzineb et al, 1999, and Brahmajothi and Campbell, 1999). Using identical immunohistochemical methods described herein, we have also recently demonstrated significant increases cardiac myocyte NOS II expression in a rat model of congestive heart failure induced by myocardial infarction. Here we found no statistically significant elevations in any treatment group relative to control, despite significant changes in cardiac TUNEL detection. Thus, the early remodeling induced by ANGII and/or TNF apparently do not involve NOS II isoform expression (at least at the physiologically relevant concentrations we employed). As described above, some in vitro studies have suggested that NOS II induction is required for TNF induced apoptosis, but our in vivo findings do not support this conclusion. Additionally, the observed interaction of ANGII and TNF with respect to frequency of TUNEL positive cells we observed (apparently combined inhibition) was not related to NOS II changes in vivo. In summary, ANGII and TNF are two established agents involved in cardiac remodeling and heart failure. Here we found that short-term (3-day) 136 administration of these agents at physiologically relevant concentrations induced significant TUNEL positive nuclei alone (suggesting apoptosis), but no change when combined in vivo. This apparent apoptosis in vivo was independent of detectable hemodynamic or cardiac size changes and an apparently early event in the remodeling process. TUNEL frequency was apparently not related to cardiac NOS II induction. 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Yonezawa t, Umemoto S, Fujii a, Katayama K, Matsuzaki M. comparative effects of type I angiotensin ll-receptor blockade with angiotensin converting-enzume Inhibitor in left ventricular distensibility and collagen metabolism in spontaneously hypertensive rats. J Cardiovasc Pharmacol 1996;27:119-124. 143 45 30 sfG) < 15 O) VEH ANG II TNF AT 200 E 500 Q. a 400 O) 150 X liJ E 300 E 100 1 Q. K- 200 CO • VEH CK • VEH CO 50 O ANG < O ANG ▼ TNF IIJ 100 ▼ TNF V Afr X V AH- 0 0 Day 0 Day 3 Day 0 Day 3 Figure 5.1 Plasma renin activity (PRA) and hemodynamic parameters measurement in saline control (vehicle, VEH), angiotensin II (ANGII), tumor necrosis factor-a (TNF), and the combination of ANGII and TNF (AT) infusion. Top panel: PRA was measured by radioimmunoassay at day 3 of infusion. Data are mean+SEM, n=6-8 animals/group (*, p<0.05). Bottom panel: Systolic blood pressure (SBP) and heart rate were determined using the tail-cuff method at day 0 and day 3 of infusion. 144 400 S 3 200 VEH ANG II TNF AT O lU VEH ANG II TNF AT 5 — 0.4 VEH ANG II TNF AT Figure 5.2 Measurements of body weight and cardiac mass (total heart weight and left ventricle, LV), following infusion of saline control (vehicle, VEH), angiotensin II (ANGII), tumor necrosis factor-a (TNF), and the combination of ANGII and TNF (AT) on day 3. 145 J': ' Figure 5.3 Representative photomicrographs of TUNEL positive nuclei (CardioTacs system) in the left ventricles of rats infused with saline control (vehicle, VEH), angiotensin II (ANGII), tumor necrosis factor-a (TNF), and the combination of ANGII and TNF (AT) on day 3. (400X) 146 ' A ^ -- _ / VEH ANG If TNF a t Figure 5.4 Representative photomicrographs of NOS II immunohistochemisty in left ventricles of rats infused with ANGII, TNF, or their combination on day 3. (400X) 147 NOS II IMMUNOPREVALENCE POSITIVE TUNEL STAINING (%) (#NUCLEI/0.2 mm^) Figure 5.5 Quantitative digital image analysis for in situ nick end-labeling (TUNEL) nuclei and NOS II immunoprevalence in rat myocardium after 3-day infusions of saline (vehicle control, VEH), ANGII, TNF, or their combination (AT). Top panel: Quantitative measurement of TUNEL positive nuclei in saline control saline (vehicle, VEH), angiotensin II (ANGII), tumor necrosis factor-a (TNF), and the combination of ANGII and TNF (AT) infusion; *, p<0.05 for ANG II or TNF treatment vs. VEH using one way ANOVA with Kruskal-Wallis post-hoc. Bottom panel: Extent NOS II immunoreactivity in rat left ventricle. Quantitative digital image analysis was employed in the measurement of cardiac NOS II immunoreactivity in saline control saline (vehicle, VEH), angiotensin II (ANGII), tumor necrosis factor-a (TNF), and the combination of ANGII and TNF (AT) infusion. A rabbit, anti-mouse, polyclonal, NOSH primary antibody was used to detect NOS II expression. NOS II immunoreactivity was measured using gray scale intensity units (see methods). 149 VEH ANG II 3 lU 2 Oz LU -J 1 ê 0 Q. 0 10 20 30 40 50 0 10 20 30 40 50 O TNF AT 3 2 (O O 1 0 0 20 40 60 80 0 10 20 30 40 50 # TUNEL POSITIVE NUCLEI 150 Figure 5.6 Statistical associations between NOS II immunoprevalence and number of apoptotic nuclei in cardiac tissues. Five pm serial tissue sections were stained for each marker and identical tissue areas were investigated and digital imaging of identical tissue areas. Data plotted as NOS II immunoreactivity in individual region from rats treated with saline control saline (vehicle, VEH), angiotensin II (ANGII), tumor necrosis factor-a (TNF), and the combination of ANGII and TNF (AT). No statistically significant association of NOS II immunoprevalence with frequency of TUNEL positive cells was observed for any treatment group. (Spearman non-parametric correlation analyses, NS for all panels). 151 PART il. NEUROHORMONES AND GENE EXPRESSION INTRODUCTION It is commonly observed that the components of renin-angiotensis- aldosterone (RAA) axis, a crucial hormone system that regulates blood pressure and fluid homeostasis, are activated during pathological states of cardiovascular disease. Elevated plasma levels of renin, ANG II. aldosterone (ALOO), and atrial natriuretic peptide (ANP) have been observed in hypertension and heart failure patients (Levine et al, 1990; Mugoyama et al, 1990; MacFadyen et al, 1999). The regulation of RAA system is complex, involving not only its own components of RAA axis that consist of renin, angiotensinogen (AOGEN), angiotensin- converting enzyme (ACE), the angiotensin peptides, and aldosterone (ALDO), but it is regulated by mechanical receptors and other hormonal systems as well. Shown below is a schematic presentation of the RAA axis and other hormones involved in its regulation. Renin is originally produced in kidney then secreted into the renal arterial circulation. Its substrate is the circulating AOGEN that is primarily produced in the liver. This enzyme is a significant rate-determinant of ANG II production (Jackson and Garrison, 1996). Angiotensin II (ANG II) is also known to activate atrial production and release of the fluid retention hormone atrial natriuretic peptides (ANP). 162 ANGIOTENSINOGEN (LIVER) RENIN (KIDNEY) ACTIVATION ANGIOTENSIN I INHIBITION ACE (ENDOTHELIUM) ANGIOTENSIN II ACTIVATION ANP ALDOSTERONE (ATRIA) (ADRENAL CORTEX) DIURESIS AND RETENSION OF SALT NATRIURESIS AND W ATER Schematic presentation of the renin-angiotensin-aldosterone system and other related hormones. 153 In addition to the activation of RAA system, recent studies have shown that in progressive and late pathologic conditions of the cardiovascular disorders increases in plasma levels of proinflammatory cytokines such as TNFa were observed. Moreover, changes in cardiac structure, cell type adaptation, and gene expression are associated with disease progression. The hallmark of genetic modulation during cardiac hypertrophy or end-stage of heart failure is the expression of “fetal” cardiac genes. It has t)een shown that selected mRNAs such as p-actin and ANP were substantially re-expressed in human heart failure (their expression are normally ceased after birth) (Feldman et al.. 1991). However, the mechanisms involved in the pathology and progression of cardiovascular disease at the early state of ANG II and TNF elevation have not been defined. Additionally, although the coexistence of these two systems is commonly observed, whether there is a synergistic interaction between these two important systems has not been well studied. Here we tested the hypothesis that early elevation of ANG II modulates the regulation of RAA axis and its related hormonal pathways at the level of protein and gene expression, and interaction between cytokine TNF and RAA system exists. 154 MATERIALS AND METHODS Plasma and rat tissues were collected from the same groups of animals as described in PART I. Blood collection and plasma hormone analysis At sacrifice blood samples were collected by cardiac puncture and rapidly transferred to tubes containing 10 pL heparin (100 Units/mL) and 10 pL aprotinin (10 mg/mL), with an exception of samples for renin activity analysis which were collected in tubes containing 20 |iL of 20% EDTA. Plasma renin activity (PRA) was measured as described in PART I. Commercially available radioimmunoassay kits were employed to quantified plasma levels of aldosterone (ALDO) (Coat-A-Count Aldosterone, Diagnostic Product Corporation, Los Angeles, CA), and atrial natriuretic peptide (ANP) (Peninsular Laboratories, Inc., Belmont, CA). RNA isolation Using standard protocol for RNA isolation (Sambrook et al, 1989) total RNA was extracted from fresh frozen tissue from left ventricle, kidney and liver for analysis of ANP, renin, and angiotensinogen (AOGEN) gene expression, respectively. Briefly, one hundred milligrams of frozen tissues were homogenized in guanidinium thiocyanate buffer (4 M guanidinium thocyanate, 0.1 M Tris (pH 7.5), 1% (3-mercaptoethanol) and the homogenate was then layered on a cushion of dense solution of CsCI (5.7 M). Isolation of RNA from 155 other cellular components was performed by 24-hr centrifugation in a high speed ultracentrifuge using SW41 rotor at 32,000 rpm. RNA pellets were washed and dissolved in Tris-EDTA (TE) buffer (pH 7.6), reprecipitated, and stored at -70 “C for northern blot analysis. Northern blot analysis Northern ael efectmohomsis and transfer of denatured RNA to nitrocellulose filters: Twenty micrograms of RNA from each sample were denatured in a formamide and formaldehyde buffer at the ratio of 7:23. The buffer contains 10X northem buffer (0.2 M morpholinopropanesulfonic acid, MOPS; 0.05 M sodium acetate; 0.01 M EDTA), 37% formaldehyde, and 99% formamide (purified by mixed bed ion exchange resin) at the ratio o f 3:5:15. The samples were then loaded on a 1.2% agarose with 6% formaldehyde gel and subjected electrophoresis. The RNA bands on the gel were transferred onto a MSI nitrocellulose membrane (0.45 pm; MagnaGraph, Westborough, MA) with an aid of capillary action (Sambrook et al, 1987). The quality and uniformity of RNA on the blots were visualized under ultraviolet (UV) light. The major ribosomal RNA bands 28S (4.7 kb) and 18S (1.9 kb) were then located and coarsely used as RNA molecular weight markers. Following fixing RNA on the membrane under UV transillumination (1.5 min, 254 nm, 500 pW/cm^) the blots were then baked at 80°C for 2 hr to remove formaldehyde adducts. Labeling the Probes: Full-length cDNA probes for rat renin (1.4 kb), and rat AOGEN (1.7 kb) were generously provided by Dr. Kevin Lynch, University of 156 Virginia, Charlottesville. VA (Burnham et al, 1987). Dr. Christopher Glembotski of San Diego State University kindly provided the full-length ANP probe (Glembotski et al, 1987). Expression of RNA was normalized to a constitutively expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a 905-bp GAPDH cDNA length purchasing from Ambion (Austin, TX). Labeled DNA probes were prepared using Promega enzyme reagents. (Promega, Madison, Ml). Each cDNA template was randomly primed with radioactive [a-^^P]dCTP. The specific activity of labeled probes were 10® to 10® counts/min/pg DNA, and the percent incorporation was higher than 50 for all probes. To further purify the labeled probes, the reaction mixes were filtered through a Sephadex G-50 column. Hybridization and mRNA signal analysis: For renin gene expression analysis, RNA blots were prehybridized using standard protocol previously described by Church and Gilbert (1984). Briefly, RNA blots were incubated at 42°C for 24 hr in prehybridizing buffer containing 10 mL 99% formamide, 5 mL 20X SSC, 1 mL 20% SDS, and 4 mL 50X Denhardfs solution. Prehybridizing RNA blots for AOGEN and ANP analysis were performed by method described by Chen et al (1992), excepted that yeast transfer RNA was not included. Hybridization was conducted by adding the labeled probes in hybridization solution and incubated at 42°C overnight. To decrease background signals, blots were washed twice for 10 min at room temperature with IX SSC containing 0.1% SDS, then twice for 10 min at room temperature with 0.2X SSC containing 0.2% SDS, and finally once for 10 min at 50°C with 0.2X SSC containing 0.2% SDS. 157 After PBS rinses and air-drying .blots were wrapped in plastic sheets and placed in a Molecular Dynamics Phosphorlmager cassette ovemight Then phosphoimages were obtained through Molecular Dynamics Scanner. The extent of gene expression was then evaluated by ImageQuant Software. Following the initial analysis for renin, AOGEN, and ANP, the label probes were stripped for 15 min in a boiling 0.1X SSPE buffer. Absolute removal of the labeled probes was tested by phosphoimage analysis described above. The stripped blots were then ready for normalization of mRNA loads using labeled GAPDH probe . The conditions for prehybridization and hybridization were employed according to the manufacturer’s instructions (Ambion, Austin, TX). The ratios of specific probing signals (renin, AOGEN, and ANP), and GAPDH signals were representatives of relative mRNA expression expressed in arbitrary unit. 158 RESULTS Table 1 shows body weight, heart weights and other organ weights that have been demonstrated as target organs modulated during progressive heart disease. No significant differences were found in any of the organ weights among the treatment groups. Shown in Figure 5.1 are plasma levels of neurohormones associated with renin-angiotensin-aldosterone (RAA) axis. Infusion of either ANG II alone or in combination with TNF caused a significant reduction in PRA whereas no change in PRA was observed in TNF infusion alone (top panel). A physiological phenomenon of ANG ll-activated the down stream modulator of the RAA axis ALDO was also observed in the ANG II administration groups (see scheme of the RAA system in the introduction section). Slightly increases in plasma ALDO were observed in either ANG II infusion alone or in combination with TNF while infusion of TNF alone had no effect (middle panel). ANP is a family of hormones involved in the regulation of blood pressure and body fluid, and ANG is its known activator. We observed increases in plasma ANP in ANG II infused groups but there was no change in TNF infused group (bottom panel). The combined infusion significantly increased ANP plasma levels. Interestingly, we found that short-term administration of ANG II alone showed a substantial variance of ANP plasma levels (two outliers' value were beyond detection limit at 2,560 pg/mL while others’ levels were comparable to those of the controls). Therefore, the increases of plasma ANP in ANG II treated group did not reach statistical significance. 159 Figure 5.8 demonstrated changes in gene expression of ANG II precursor AOGEN in the liver, expression of renin in the kidney, and expression of ANP gene in the left ventricle. No significant changes in AOGEN expression were observed in any treatment group (top panel). In contrast, only the combined infusion of ANG II and TNF showed significant reduction in renin mRNA expression (middle panel). Interestingly, while expression of ANP in the left ventricle is normally ceased after birth we observed approximately 4-foid increases in its RNA signals following infusion of ANG II, TNF or their combination (Figure 5.8, bottom panel). 160 DISCUSSION Left ventricular remodeling is a crucial process occurring during the progression of heart disease. Several neurohormone systems and cytokines have been implicated in the remodeling process, including RAA system and TNF. Although co-elevation of these agents during pathologic states of the heart are commonly observed, the effects of the cytokine TNF on the homeostatic regulator RAA system and/or their interactions have not been defined in a complex setting in vivo. Therefore, the intent of our studies was to investigate whether interaction of TNF and RAA system exists in a short-term (3 days) administration of ANG II and/or TNF in vivo. Our results suggest that plasma renin activity was highly sensitive to the physiological relevant increases in ANG II levels (see PART I), while plasma ALDO levels were only slightly changed. TNF has very little effect on the plasma levels of PRA and ALDO. Although we cannot rule out the effect of ANG II on ANP plasma level (due to the outliers), its co-administration with TNF significantly increased plasma ANP. An interaction of ANG II and TNF on the RAA component renin was also observed at the level of gene expression. The initial suppression of renin gene expression may lead to compensatory feed back and eventually dysregulation of RAA system and other hormones if the blood pressure and fluid homeostasis is not under control. Despite the fact that TNF caused no change in plasma ANP, this cytokine induced more than 3-fold increases in ANP gene expression. These results 161 suggest that perhaps TNF has very minimal effect on the secretion process of the peptide hormone, but it highly regulates the hormone at trascriptional level. While re-expression of left ventricular ANP in adult is an indicative of cardiac remodeling in late state heart disease, our findings suggest that ANG II and TNF each alone or in combination participate in an early event of cardiac remodeling (only 3 days infusion). in summary, interaction of ANG II and TNF exists at the transcription and post-transcription levels of certain RAA components and the peptide hormone ANP. Short-term administration of ANG II and/or TNF promoted the fetal ANP gene expression in the absence of hemodynamic changes (pressure overload), suggesting that ANG II and TNF may participate in the initiation of cell type adaptation and cardiac remodeling process. 162 REFERENCES Burnham G, Hawelu-Johnson G, Frank B, Lynch K. Molecular cloning of rat renin cDNA and its gene. Proc Natl Acad Sci USA 1987;84:5605-5609. Ghen Y, Naftilan A, Oparil 8. Androgen-dependent angiotensinogen and renin messenger RNA expression in hypertensive rat. Hypertension 1992;19:456-463. Ghurch G, Gilber W. Genomic sequencing. Proc Natl Acad Sci USA 1984;81:1991-1995. Glembotski GG Oronzi ME, Li XB, Shields PP. JohnstonJF, Kallen RG, Gibson TR. The characterization of atrial natriuretic peptide (ANP) expression oby primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe. Endocrinolgy 1987'121:843-852. 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Gold Spring Harbor, New York, Gold Spring Harbor Laboratory Press,1989 . 163 W eight VEH ANGTNFAT (g) BODY 315.5+4.7 316.3+7.2 323.0+5.6 316.8+4.1 TOTAL 1.0421+0.0199 1.0817+0.0287 1.0531+0.0297 1.1077+0.0297 HEART RIGHT 0.0266+0.0009 0.0275+0.0017 0.0248+0.0032 0.0258+0.0022 ATRIUM LEFT 0.0246+0.0017 0.0257+0.0025 0.0261+0.0014 0.0286+0.0012 ATRIUM RIGHT 0.2168+0.0038 0.1985+0.0089 0.2191+0.0089 0.2234+0.0117 VENTRICLE LEFT 0.7742+0.0172 0.8300+0.0224 0.7872+0.0207 0.8299+0.0105 VENTRICLE LIVER 11.94+0.41 11.68+0.39 12.95+0.44 12.13+0.32 KIDNEY 2.32+0.06 2.22+0.04 2.33+0.04 2.1+0.04 LUNG 1.39+0.03 1.42+0.07 1.40+0.06 1.42+0.04 Table 5.1 Organ weights of rats treated with saline vehicle (VEH), angiotensin II (ANG II), tumor necrosis factor-a (TNF), or the combination of ANG II and TNF (AT). The values represent mean+SEM. 164 y . VEH ANG H TNF AT 0 | 300 < g 200 kiâiVEH ANG II TNF AT 1500 1000 400 < ? kmVEH ANG II TNF AT Figure 5.7. Effects of ANG II and/or TNF infusion on plasma levels of renin activity (PRA), ALDO, and ANP. *, p<0.05 vs. vehicle control (VEH). 165 il. VEH ANG TNF AT 0.15 0.10 z | ggo.0. 0.00 VEH ANG TNF AT Z 0 .1 0 a 0.05 0.00 VEH ANG TNF AT Figure 5.8. Effects of ANG il and/or TNF infusion on gene expression. Northern blot analysis showed alteration in mRNA expression of AOGEN in the liver, renin in the kidney, and ANP in the left ventricle of rats infused with ANG and/or TNF. *,p<0.05; #, p<0.1 vs. vehicle control (VEH). 1 6 6 CHAPTER 6 CARDIAC GENE THERAPY: RECENT DEVELOPMENTS AND A NEW APPROACH Introduction Despite decades of extensive research, cardiovascular disease still remains the leading cause of death in the U.S. and consumes a tremendous amount of health care resources and expenditures. Recent studies suggest that some forms of cardiovascular disease are on the decline (e.g., stroke, ischemic heart disease and hypertension), but the incidence of chronic congestive heart failure (CHF) continues to rise (U.S. Department of Health and Human Services, 1994; Ghali et al, 1990). Approximately 400,000 new CHF cases are diagnosed each year, and this condition of progressive pump failure often requires intensive and continuous therapies for many years. Over $10 billion is spent annually on therapeutic management of CHF treatments alone but the overall outcome is not highly successful. Even with currently optimized therapy, typical mortality in large clinical trials remains approximately 50% within 5 years (Schocken et al, 1992;Yusuf and Abbott; 1989). Thus, the rising incidence of CHF, combined with 167 the limited success of current therapies, suggests that new approaches for treatment may have value. Advances in molecular biology and the emergence of the human genome project have revealed opportunities for novel strategies for CHF therapy. These include the identification of new targets for traditional small molecule therapies and gene-based therapeutic strategies. In many ways, gene therapy in general represents the ultimate therapeutic strategy- fixing the disease problem rather than managing symptoms. Thus, the development of molecular technologies have great potential for CHF, and several lines of investigation are already underway. The focus of this thesis chapter is to review current state of the genetics-based therapy for cardiac disease. In addition, description of a novel concept for cell based CHF therapy and initial preliminary findings are provided. General aspects of cardiac gene transfer The global intent of cardiac gene transfer is to produce favorable effects on cardiac performance, and/or patient survival. While thus far no studies have conclusively demonstrated value of any of these approaches, several recent studies have suggested potential value in model systems and/or small trials. Shown in Table 6.1 is a comprehensive review of cardiac gene transfer attempts described in the published literature (as of January 2000). Discussion of specific aspects required for successful implementation and review of published experiences is provided below 1 6 8 Transfection methodologies for cardiac gene transfer A critical aspect of successful cardiac gene transfer is the reliable and efficient incorporation of new genetic material into target cells. Early studies have focused on finding target genes and evaluations of various vectors/promoters and methods for transferring those target genes into myocardium. Naked DMA, liposome-aided, and viral vectors (adenovirus, retrovirus, adeno-associated virus) have been employed to study transfection efficiency and expression kinetics of transgene using reporter genes both in vitro and in vivo. The most commonly used reporter genes in gene transfer include p-galactosidase, luciferase, and chloramphenicol acetyl transferase (CAT). Beta-galactosidase provides histochemical advantage over luciferase and CAT in that cell types expressing p-gal activity in cardiac tissue can be morphologically identified (e.g. cardiac myocytes vs non-myocytes). Discussed below are vectors used to carry target DMA and strategies for gene delivery to cardiac tissue. Viral vectors: Cellular uptake of viruses involves the first step of binding to a specific membrane receptor followed by endocytosis. These viruses then require endosome escape of their genetic material from degradation in cytosol and nucleus of host cell. Transferred DNA can either integrate into the host genome or independently replicate depending on type of viruses. Retroviral vectors: Retroviral (single-stranded RNA virus) gene transfer causes potentially stable integration of DNA into chromosome of target cells. 169 These vectors are appropriate for ex vivo gene transfer where removal of target cells or organs is involved in the process of gene therapy. Retroviruses are able to transduce only actively replicating cells, thus limiting its efficacy for gene transfer into fully differentiated cells such as cardiac myocytes. Their uses are also encumbered by some limitations such as the size of inserts is constraint to 9 kb (Feldman and Steg. 1997). Additionally, the safety of retroviral vectors is inherently of concern. Introducing replication-competent retrovirus in to a cell with a random insertion pattern can generate a potential induction of insertional mutagenesis resulting in a disruption of normal pattern of cellular gene expression (Onions, 1996). Adenoviral vectors: Recombinant adenoviruses (double-stranded DNA viruses) are currently the most often used for gene transfer in cardiovascular tissue. Unlike retroviruses, adenoviruses infect both replicating and non replicating cells in vitro and in vivo. Viral DNA exists in unintegrated form in the nuclei of host cells, thus reducing potential for insertional mutagenesis. Despite adenoviruses are one of the most promising vectors for gene transfer into rat heart, the short half-life (one week) of gene expression is the major drawback (Schneider, 1993). However, this short-term delivery of gene products could be sufficient and appropriate for treatments under which long term exposure would lead to toxicity, such in the case of angiogenesis factors. This sharp decline in the levels of transgene expression may be due to host inflammation response against viral encoded proteins, hence repeated administrations are unfeasible (Yang, 1995). Second and third generation 170 adenoviral vectors are under an ongoing developmental process to overcome these problems. Adeno-associated virus (AAV) vectors: AAV is a single-stranded linear DNA virus. Viral DNA replication requires a helper virus resulting in viral genome amplification and progeny virion production. However, AAV persists in episome or integrates into the host cell genome in an absence of a helper virus (latency). Therefore, AAV gene transfer can occur during the latent phase of AAV life cycle. Although the advantage of its capability to infect both dividing and non-dividing cells, the limited size of DNA insert (4.4 kb) has become primary consideration using AAV in gene transfer. Nonvirai vectors: Generally, nonvirai vectors are relatively safe but inefficient for gene transfer to myocardium and other tissues. Examples of nonvirai vectors include naked plasmid DNA, gene gun (using bioballistic bombardment of DNA-coated gold particles to penetrate into target organs or single cell layer), plasmid- liposome complexes (lipoplex), polymer-DNA complexes (polyplex), and liposome-polymer-DNA complexes (lipopolyplex) (Huang and Viroonchatapan, 1999). Unlike viral vector DNA, internalization of naked DNA or DNA-conjugates can occur either in a random manner or upon binding to a specific receptor depending on design of conjugates. Recently, a controlled-release DNA-polymer coating delivery system has been investigated for transfection of skeletal and cardiac tissues (Labhasetwar et al, 1998). This system provides a prolonged 171 DNA availability and protects DNA from nuclease cleavages, subsequently leading to a prolonged duration of gene expression. Another recent strategy in nonvirai vector gene transfer includes transcription factor decoy using antisense oligodeoxynucleotides (ODNs). These ODNs have been used in gene therapy and studies of gene expression in cardiovascular disease, including restenosis after angioplasty, hypertension, transplant vasculopathy, myocardial reperfusion injury, and graft failure (Morishita et al, 1997). Transcription factor decoy ODNs act at the transcriptional control level. Since the binding of transcription factors to its endogenous cis element is competed by double-stranded ODNs, the initiation of transcription and the subsequent protein synthesis is inhibited. This strategy has been used as a tool to understand mechanisms of myocardial infarction and allograft rejection. Previous investigations have suggested that NF-KB-mediated upregulation of various cytokines and adhesion molecules participate in myocardial infarction and graft rejection. Using ODNs Morishita et al (1997) were able to demonstrate that transfection of NF-kB decoy ODN into rat coronary arteries prior to occlusion attenuated myocardial infarct size following reperfusion. Practically, this strategy may initiate tolerance development after a certain period of time because it relies upon competitive reaction between the endogenous DNA sequence and the decoy ODNs. Compensation and upregulation of certain signaling pathways may occur across the networking of cellular transcriptional regulators due to the complexity and redundancy of cellular signals. 172 Hemagglutinating virus of Japan (HJV) conjugates: H JV-liposome conjugate is a product of a recent development of a novel vector to overcome the limitations of individual vectors by combining liposomes with fusion proteins derived from the hemagglutinating virus of Japan (HVJ, also known as Sendai virus). DNA and DNA-binding protein are incorporated into liposome. The fusigenic viral liposomes then fuse to plasma membrane of target cells and deliver DNA-protein complex into cytoplasm. Fusion DNA-binding proteins enhance the efficiency of gene transfer to the host nuclei. This system induces lesser immune response than viral vector system. Thus, repeated injections are possible. Gene Delivery Techniques Once a specific gene is identified and a vector has been optimized, the actual delivery of this gene cassette to cardiac tissue is an additional challenge. Methods in delivery of a transgene into the myocardium have included direct injection, intracoronary infusion, pericardium perfusion, perfusion of the donor heart, and systemic injection. Thus far, efficient cardiac gene delivery requires an invasive procedure such as opened chest surgery as seen in myocardial direct injection employed in clinical trials for cardiac gene therapy (Table 6.1). However, this technique can cause cardiac injury and severe fibrosis to the areas of injection. 173 Ex vivo gene transfer through perfusion strategy is practicable only for transplanted heart, despite this method provides a more efficient gene delivery and a widespread distribution of transgene expression compared to direct injection (Wang et al. 1996). 174 1. Evaluations of vectors and gene delivery strategies for cardiac gene transfer Gene Vector Model Studied Outcomes Comments References CAT and naked -cultured neonatal rat cardiac -successful gene transfer in vitro -in vivo peak of expression and Lin et ai, p-gal plasmid DNA myocytes (CAT transfection) -in vivo transgene expression was transfection efficiency were not 1990 -in vivo adult rat heart (direct detectable up to 4 weeks determined injection of P-gal plasmid DNA) CAT Adenovirus -cultured fetal rat cardiac - in vitro CAT expression was dose- -transfected both myocytes Kass-Eisier myocytes dependent and detectable at 4 hr, and nonmyocytes e ta l, 1993 -in vivo adult rat heart (direct peaked at 48 hr -induced inflammation response intramyocadial injection) -in vivo transgene expression peaked at 5 days and still detectable at 55 days DNA- -adult mice (iv injection) -transgene expression was detectable -systemic transfection Zhu et al, liposome up to 9 wks. 1993 complex -transplanted rabl)jt hearts -detectable CAT activity at 24 hr after -only one time point (24 hr) was Dalesandro cardiac transplantation studied et ai. 1996 p-gal and naked -rabbit hearts (intramyocardial -direct injection showed higher gene -necrosis was observed at the Gal et al, Luciferase plasmid DNA injection) transfer efficiency but perfusion areas of injection 1993 provided a better transfection distribution adenovirus -porcine hearts ((intramyocardial -transgene expression was detectable -severe immune cells Infiltration French et al, Injection) at day 3, peaked at day 7, and decline -extremely high efficient 1994 -4 en after day 14 adenovirus gene transfer compared to plasmid DNA -rat hearts (intramyocardial -expression stable for 1 month -adenoviral vector provided > 10- Wang et al injection or aorta perfusion) fold higher luciferase activity than ,1996 plasmid vector p-gal, HVJ. -in vivo rat heart (intramyocardial -direct injection showed detectable -although direct injection showed Aoki et al, Luciferase, and liposome injection, pericardium Incubation gene expression at day 3 with 80% the most efficient method but 1997 ACE and coronary infusion) efficiency, and 100% successful myocardial injuries and severe -in vivo infarct rat heart transfection whereas coronary fibrosis were found at the sites of (pericardium incubation) infusion showed 56% success direct injection -obtained gradient transfection using pericardium incubation p-gal Adenovirus -neonatal and adult rat (iv and im -obtained only 0.2% transfected -low efficiency gene transfer but Stratford- injection) cardiac cells with systematical prolong gene expression Perricaudet, infection 1992 Table 6.1. Review of cardiac gene transfer attempts. (Continued) Gene Vector Model Studied Outcome Comments Reference p-gal Adenovirus -neonatal and adult rat (iv and im -obtained only 0.2% transfected -low efficiency gene transfer but Stratford- injection) cardiac cells witti systematical prolong gene expression Perricaudet, infection 1992 -detectable expression up to 12 mo. Adenovirus -cultured rabbit cardiac myocytes -red blood cell interfered gene transfer -provided optimal conditions for in Donahue, -Langendorff infection (coronary -ex vivo and in vitro gene transfer vitro and ex vivo adenoviral gene 1997 perfusion) were most effective at 37 °C witti t)igh transfer virus concentrations in crystalloid form adeno- -cultured neonatal rat -expression of AAV-mediated gene -AAV can integrated into host Maeda et al, associated cardiomyocytes transfer peaked at day 3, tialf-life 14 genome, perhaps long-term 1998 virus -ex vivo rat ventricular papillary days, and 60% efficiency evaluation for mutagenesis is muscles -effective ex vivo gene transfer (80%) essential and detectable at 24 fir II. Target genes for cardiac gene transfer mlc-2v Adenovims -cultured neonatal rat cardiac -relative tieart-specific gene transfer - detectable level of viral DNA in Rothmann myocytes and in vivo intracardiac other organs (diaphragm, lung et al, 1996 cavity injection thymus and intercostal muscle) mlc-2v and Adenovirus -neonatal rat tiearts (intracardiac -mlc-2v provided more specific gene -transfection of a-mhc also Franz et al, a-mhc cavity injection) transfer to ventricle wfiereas a -mtic appeared in lung and liver 1997 transfection occurred lx>tti in ventricle and atria o>«a HRE naked -cultured rabbit cardiac myocytes -gene transfer specifically to fiypoxic -expression was very short Prentice et plasmid DNA -in vivo intramyocardium injection cardiac myocytes (undetectable after 8 hr) al, 1997 MyoD Adenovirus - rat Itearts (freeze-ttiaw cardiac -cardiac injury area sliowed -required high viral dose to induce Murry et al, injury model ) expression of MyoD mRNA and gene In vivo cell transformation 1996 -cultured cardiac fibroblasts product -induced cultured cardiac fibrobtasts to differentiate to skeletal muscle ShK Adenovirus -cultured canine failing myocytes -reverse abnomial prolong action -in vivo cardiac performance has Nuss et al, potential not been tested. 1996 Pz-AR and Adenovirus -failing rabbit tiearts -increased basal cAMP accumulation -replenished p-AR Akhter et al, pARKct -increased cAMP production downregulation 1997 responded to isoproterenol -evidence of benefits from pARKI inhibition (pARKct overexpression),i.e. reversed p- AR coupling defects (Continued) Gene Vector Model Studied Outcome Comments Reference Pz-AR naked -in vitro mouse myocyte -increase myocyte contractility -target for atria-specific Pz-AR Edelberg et plasmid DNA -in vivo intraatrial DNA injection -increased heart rate gene transfer to regulate cardiac ai, 1998 -heart transplantation pacemaking activity Adenovirus -normal rabbit hearts -Increase hemodynamic function and -cross-damped aorta during Maurice et (Intracoronary bolus injection) contractility response to p-agonist gene transfer may induce al, 1999 -isolated failing rabbit myocytes isoproterenol ischemic-reperfusion damages -increased cAMP production -transgene expression reached responded to isoproterenol peak after 6 days and dedined within 21 days A ll receptor Retrovirus -spontaneous hypertensive rats -prevented increase in BP at 120 •no in vivo evaiuation of cardiac Martens et antisense ODNs days postinjection function al, 1998 -prevented increased vascular -gene transfer appears only in contractile response to KCI and dividing ceils, i.e. applicable to phenylephrine cultured neonatal hearts (5 days -prevented cardiac hvpertrophv old) VEGF165 naked -angina patient (intramyocardial -improved angina class -first VEGF gene transfer Losordo et plasmid DNA Injection) evaluation in humans (only 5 al, 1998 patients) VEGF121.10 Adenovirus -pig heart (intramyocardial -enhance collateral vessel formation, -expression of VEGF was Mack et al, injection) improve regional perfusion and evaluated only one time point at 1998 .«a - a reduce ischemic area day 3. VEGF121.10 Adenovirus -angina patients—Phase 1 clinical -Improved angina class and -3 of 21 patients died within 40 Rosengarl trial (intramyocardial injection) increased treadmill exercise tolerancedays post-operation et al, 1999 -increased detedable serum anti- Ad neutralizing antibodies - cannot exdude confounding effeds of conventional adjunct therapy due to small sample size IL-6 and ICAM-1 naked -rat hearts (ex vivo gene transfer) -reduced IL-6 and ICAM-1 expression -altemative approach in gene Mann et al, antisense ODNs plasmid DNA transfer using pressure mediated 1999 transfection -aim for minimizing allotransplantation rejection CAT, catecholamine acetyl transferase; p-gal, p-galactosidase; ACE, angiotensin converting enzyme; HJV, hemagglutinating virus of Japan; mic-2, myosin light chain-2; mlc-2v, ventricle-spedfic myosin light chain-2; a-mhc, a-myosin heavy chain; HRE, hypoxia response element; MyoD, myogenic determination gene; ShK,Drosophila Shaker 8 potassium channel; Pz-AR, Pz-adrenergic receptor; pARKct, p-AR kinase carboxyl-terminal 194 amino acids; AT«, angiotensin type I receptor; VEGF, vascular endothelial growth factor; IL-6, Interleukin-6; ICAM-1, intercellular adhesion molecule-1 Review of cardiac aene transfer attempts As of June 1998, 244 gene therapy protocols have been reviewed by the Recombinant DNA Advisory Committee (RAC). While this novel facet of therapeutic strategy is in its infancy and has not been proven to be a triumphant remedy, useful information has been obtained from early attempts in cardiac gene transfer using several animal models including mouse, rat, rabbit, dog, and pig. Previous studies in both animal models and in humans have provided valuable information for each component of gene therapy strategies as discussed below. V ectors: Despite all the setbacks, most of previous studies described in Table 6.1 employed viral vectors in cardiac gene transfer because of their high efficiency of transfection. For instance, adenoviral vectors are far more superior over naked DNA or plasmid DNA in incorporating target DNA into cardiac myocytes, i.e. 50 to i 00-fold higher transduction efficiency in rat hearts (Kass et al, 1993). In contrast to adenoviral vectors, the use retroviral vectors is intimidating due to potential mutagenesis and their limited capability of transfecting only dividing cells. Therefore, retroviral vectors may be useful for ex vivo gene transfer rather than in vivo trials. Although recombinant adenoviral vectors have been the most frequent used in cardiac gene transfer and showed prominent outcomes, these vectors are short-lived and usually induce host immune response. The recent development of HVJ-hemagglutinin-Ebstein Barr virus (EBV)-liposome complexes has integrated advantages of using viral vectors (high efficiency) and 178 liposome-based nonvirai vectors (low immunogenicity) in gene transfer to augment transgene expression in vivo (Kaneda et ai, 1999). First generation HVJ-liposomes is not integrated into host genome and is degraded within two weeks. The introduction of EBV has stabilized the retention of extrachromosomal transgene expression from two weeks to five weeks in mouse liver, and from one month to four months in mouse skeletal muscle (Kaneda et al, 1999). Nevertheless, this strategy has not been tested in cardiac tissue. Using antisense ODNs has become an attractive altemative when large quantity synthesis is easily obtainable. However, the use of ODNs for therapeutic implications in humans have been overriden by their lack of specificity and reproducibility of biological effects. Additionally, ODNs have relatively short half-life in vivo, which plays an important role in their mechanism of action because they reversibly abrogate gene expression only when they reach a certain therapeutic ratio of specific to nonspecific biological effects. Gene delivery techniques: In addition to the significance of vector(s) selected for gene transfer, gene delivery strategies are important in achieving highly efficient gene transfer and extending duration of transgene expression. Most studies have shown successful gene transfer via intravenous injection, intracoronary injection/perfusion, pericardium incubation, ex vivo transplanted heart transfection, intracardiac cavity injection, and direct injection into the myocardium. Unless tissue-specific gene targeting techniques have been developed, intravenous injection is unlikely to be the choice of gene delivery due to systemic infection and low efficiency of gene transfer (0.2%). Direct myocardial 179 injection is the most frequently method used for in vivo gene transfer. Although injecting target gene into myocardium provide high efficiency and local gene transfer, tissue injuries and host immune cells filtration are commonly observed. Other model of gene transfer in allograft cardiac transplantation have apparently become auspicious due to supportive evidence of preventing allograft rejection using transfection both vitro and ex vivo. However, this strategy thus far has not been applied to human trials pertaining to the use of gene transfer to donor hearts. Despite intramyocardial injection has demonstrated successful gene transfer, cardiac injuries are usually observed. Therefore, cardiac-specific promoters have been developed to obtain a more precise gene transfer to target cells and to allow for lesser invasive gene delivery. Using cardiac specific promoters such as mlc-2v and a-mhc in association with intracardiac cavity injection technique increases global distribution of transgene expression and minimizes mechanical tissue injuries form direct injection. Another approach to achieve localized expression of the disease phenotype cells is to use hypoxia response elements (HREs) incorporated into minimal a-mhc promotor to regulate expression of hypoxic and ischemic myocytes. However, expression efficiency and half-life of transgene largely impinge on the advance and improvement of adenovirus vectors. The concepts of cardiac gene therapy are not limited to supplying missing proteins in pathologic state but also embrace a striking idea of converting cellular phenotype of noncontractile units such as fibroblasts into muscle-like cells via 180 genetic manipulation. Cardiac myocytes represent only 30-40% of total myocardium tissue, and they can not significantly replicate to replace cell loss after tissue injuries or in pathologic conditions. As described in Table 6.1, a resistance to phenotype conversion of MyoD-expressing cells has daunted the use of myogenic determination gene (MyoD) transfer in cardiac fibroblasts. This problem is only partially solved by using near-cytotoxic doses of adenovirus to increase number of MyoD copies per cell. Therefore, increasing transfection efficiency and using enhancer(s) for phenotype conversion are challenging for further development. Additionally, there is a considerable concern emerging from electrophysiological differences between skeletal muscle and cardiac muscle. Skeletal muscle lacks automaticity and has a very short action potential duration and refractory period, but it has 10-fold higher conduction velocity. These disparities in electrical properties are important in the synchronism of overall contractile units, and may cause cardiac arrhythmias. Target genes: The rationales of selecting target genes for CHF gene therapy rely upon an endeavor to restore cardiac function and contractile performance. The p-AR signaling system plays a crucial role in inotropic and chronotropic effects mediated through the sympathetic system. It is well established that losses in receptor subpopulation along with coupling dysfunction of p-ARs are commonly observed in failing heart. Conventional therapy for CHF engages administration of p-AR agonists in acute conditions to stimulate inotropy. but using p-AR blockers for chronic therapy to reserve contractile energy and oxygen consumption. The treatment outcomes are not highly 181 successful due to diminished efficacy by the losses in receptor populations, i.e. 50% downregulation of PiAR in CHF (Bristow et al, 1986). Although increasing p-AR density and inhibition of G protein-coupled p-AR kinases (PARK, the enzyme that degrades the coupling of G-protein and p-AR ) by virtue of gene transfer in experimental animal models deems merit and amendable for replenishing lost receptors, studies of transgenic mice overexpressing Pi-AR in the heart have not shown favorable potential outcomes when phenotype switching was observed at all ages. These transgenic mice developed myocardial remodeling at young age and subsequently demonstrated the signs of dilated cardiomyopathy at the age of 8 months (Bisognano et al, 1997; Port et al, 1998). Transgenic modulation of other receptor-G-protein-adenyl cyclase complex (RGC) components such as Gsa resulted in activation of adenyl cyclase activity but alterations in cardiac morphology and increases in mortality were also observed. Therefore, there are only two components of RGC—Pz-AR and PARK Inhibitor (pARKct)—have been thus far proven to be beneficial for cardiac gene transfer. In addition to abnormality of p-adrenergic responsiveness in heart failure, a deficiency of voltage-dependent potassium channels has also contributed to the impairment of cardiac contractility observed in failing human myocytes (Nuss et al, 1996). Despite the success of reversing the electrophysiologic abnormality of myocytes using adenoviral-mediated gene transfer, the viability of this 182 candidate gene is again confided to the efficiency and half-life of adenoviral vectors. Recent evidence of therapeutic benefits from angiotensin II inhibition (ATI antagonists and ACE inhibitors) now represents a pivotal therapeutic target for the management of many forms of cardiovascular disease, including essential hypertension, congestive heart failure, and coronary artery disease (Stroth and Unger, 1999; Lonn et at, 1994). Administration of angiotensin It type I receptor antisense at the neonatal state is a novel approach to prevent development of hypertension and to attenuate hemodynamic-independent pathophysiologies mediated by angiotensin II in genetically predisposed spontaneous hypertensive rat (Martens et al, 1998). While recent studies have defined important effects of angiotensin II on pathophysiologies of cardiovascular disease, other investigations have shown that this agent is essential for cell growth, differentiation and normal organ development (Alcorn et al, 1996; Price et al, 1997). Therefore, removing angiotensin II at the early state of life may have disintegrative outcomes far beyond beneficial effects. Additionally, hypertensive determinant genes in humans are multifactorial and phenotype prediction is complex due to ethiological heterogeneity. Environmental factors are as important as genetic predisposition in underlying physiological derangement in individuals harboring particular genetic variants (Lifton, 1996). Coronary artery disease is a common cause of CHF while the conventional therapies for cardiac ischemia (percutaneous angioplasty and bypass surgery) provide little benefit. A new therapeutic angiogenesis strategy 183 using vascular endothelial growth factor (VEGF) has shown to promote revascularization of peripheral ischemia and ischemic tissues resulted in improvement of myocardial perfusion and function (Mack et al, 1998). The human VEGF gene expresses in four isoforms posttranscriptional splicing, including 121, 165, 189, and 206 amino acid residues. The peptides VEGF121 and VEGF165 show the most important effects on angiogenesis. VEGF gene transfer appears to be the first cardiac gene therapy trial in humans for the treatment of coronary artery disease (Mack et al, 1998;Losordo et al, 1998). While systemic administration of VEGF is prohibited due to a broad abnormal angiogenesis, direct gene transfer to the myocardium resulted in death-related gene therapy (3 out of 15). The therapeutic outcomes were striking as such the adverse effects are daunting. S um m ary In summary, gene therapy approaches for the treatment of cardiovascular diseases have been moving forward with improvement in strategies to deliver target genes specifically to cardiac tissue, and to prolong transgene expression. Specific promotor elements such as mlc-2v and a-mhc for gene delivery specifically to myocytes and specifically to ventricle chambers of the heart have been introduced and accretive to cardiac gene therapy evolution. Furthermore, not only is specificity of gene transfer exclusively to cardiomyocytes the ultimate goal of gene therapy, but the attempts of using hypoxia-responsive enhancer 184 (HRE) have also strengthen the tendency of exclusive repair of injured myocytes, i.e. a phenotype-specific gene transfer in hypoxic and ischemic myocardium. While it is an on-going process following initial attempts to develop strategies for cardiac-specific gene delivery, target genes chosen for restoration of cardiac contractile function in the failing heart are concurrently in advance and have been recognized through gene transfer of p-adrenergic receptors and potassium channels. The p-adrenergic signaling dysfunction plays an important role in adaptive mechanisms in compromised cardiac function. The potential usefulness of these gene transfers has become partially limited by the shortcoming of adenoviral vectors employed. Cardiac gene therapy would step out from its infancy if improvement in gene delivery strategies and more efficient, immune-tolerance, and persistent vectors are developed. 185 A novel strategy for cardiac aene therapy: autologous fjbroblast-mediatad gene therapy Problems of current cardiac gene therapy approaches: General problems with cardiac gene transfer strategies discussed above are the unstability of transgene expression and the utilization of invasive techniques for gene delivery. Transgene expression in cardiac tissue is typically short (up to 4 weeks) as demonstrated in rat hearts expressing recombinant P-gal reporter gene via direct intracardiac injection (Lin et al, 1990, Wang et al, 1996). Further issues include immune responses to foreign agents and a lack of controlled expression in specific cardiac cell types since cardiac myocytes actually comprise only about 30-40% of the total cells in the heart. Proposal of a new strategy (conceptual developments): In recognition of the weaknesses of current cardiac gene therapy approaches, we propose a novel strategy for cell based cardiac treatments. In this approach human fibroblasts would be isolated from the patients (from a biopsy sample) and transfected with target genes for potential therapy in cardiac tissue. These stably transformed cells would then be implanted to myocardium via minimally invasive technologies (angioplasty catheterization). Shown below are potential advantages and disadvantages of our proposed approach: 186 Potential advantages Potential disadvantages -reduced immune response (autologous cells) -cells may not survive -stable transfection (not transient expression) -cells may proliferate and cause -no viral vectors involved problems (fibroblasts' properties e.g. -cell types involved can be selected producing collagen, growth factors, etc) -cells can be used as production sites (variety -not applicable to vascular of genes could be inserted) administration (no movement of cells -target genes are not limited to antioxidants out of vasculature) -this technique could be done fairly routinely (in clinical setting) The main strength of our approach hinges upon the advance in molecular immunology that has provided an insight into the concepts of tissue compatibility and graft rejection through the discovery of major histocompatibility (MHC) complexes. Immune cells recognize foreign bodies through MHC mismatch and subsequently initiate cellular and/or humoral immune responses. The advantages of using autologous tissue include immune tolerance to self proteins/tissues and the subsequent disappearance of graft rejection. The first autologous gene therapy applied in humans was initiated in the late 1990 when adenosine deaminase gene-modified autologous lymphocytes were injected back to patients. Thereafter, clinical trials in cancer gene therapy with autologous tumor cells have become the major category in gene therapy protocols reviewed by the RAC and/or FDA through 1998 (Pilaro and Serabian 1999). Not only are simplicity and accessibility to autologous cells important in clinical practice, the duration and efficiency in delivering the gene products has also supported the use of autologous skin fibroblast-mediated gene therapy. While introducing this new approach and its applications to cardiac tissue is at 187 our spear, genetically modified skin fibroblast cell transplantation has been evaluated in certain disease models both in vitro and in vivo. Sangalli et al (1998) applied transfected fibroblasts in metachromatic leukodystrophy syndrome. Genetically modified fibroblasts were implanted back into the kidneys of cats with mucopolysaccharidosis type VI syndrome (Yogalingam et al 1999). In a human study, fibroblasts from skin biopsies were obtained from hemophilia B patients and were reimplanted back to the patients after obtaining fibroblasts with transgenes (Xinfang et al 1996). Fibroblasts are easy to isolate and propagate well in culture media after a small skin biopsy. The advantage of their rapid growth allows for efficient retroviral vector gene transfer as well. While randomized controlled trials have shown inconclusive benefits of using antioxidants against cardiovascular disease, evidence form our laboratory and others have strongly suggested the significance of oxidative free radicals in the pathologies of cardiovascular disorders (Harjai, 1999; Boaz et al, 1999; Gokce et al, 1999; Keith et al, 1998). Most clinical trials in preventive uses have been too short, and the antioxidant doses and forms of delivery have been varied greatly. Furthermore, cardiac tissue is kinetically difficult to penetrate; thereby, tissue bioavailability depends on the local concentration of drug surrounding. We foresee the potential advantage of using stable transfected cell lines to generate such a local tissue-specific drug delivery at therapeutic levels. The crux of ex vivo transfection clings upon the combination of obtaining a) manipulatable/controllable cell lines, i.e. using high efficient viral vectors to select only desired transgene expression levels and drop out insertional mutagenesis 188 clones; b) a prolong transgene expression due to stable transfection. Therefore, we propose here an alternative delivery of gene products applied to cardiovascular disease using cell transplantation of autologous skin fibroblasts ex vivo transfected with therapeutic genes such as SOD, NOS, NF-kB, etc. In this chapter, we introduce a preclinical evaluation process in gene therapy as a proof of concept’ to evaluate the autology and kinetics of the cells applied into the cardiovascular system. Only reporter genes including luciferase and p-galactosidase were utilized in this study in order to provide kinetic insights into this autologous cell transplantation system. We initially evaluated cell growth pattern, cell distribution and localization, and transplantation efficiency. The results from our initial attempts will define the strength, weakness, and future directions of this approach. 189 PRELIMINARY INVESTIGATIONS We have conducted preliminary investigations regarding the approach descrit)ed above. Below are descriptions of our findings thus far. Preparation of Stable Transfected Fibroblast Cell Lines: Our initial experiments included the establishment of a fibroblast cell line in our laboratory and evaluating preliminary strategies for stable transfection. A schematic presentation of the approach we used is shown in Figure 6.1. C e lls : We chose to employ rat skin fibroblasts in these initial experiments since rat heart experiments were planned for initial in vivo tests. These cells were acquired from the American Type Culture Collection (ATCC 1213) and maintained in Dulbecco’s Modified Eagles Medium (DMEM) containing sodium pyruvate, 0.1 % glucose, 10% fetal bovine serum, and 50 units/mL penicillin and 50 mg/mL streptomycin (Gibco-BRL). Cells were grown at 37 °C with 5% CO 2 and the media were replaced every 3-4 days. Cell passages were performed at 70% confluence using 0.25% trypsin and 1 mM EDTA in PBS. P la s m id : Large-scale plasmid DMA was prepared as described by Sambrook et al. (1989). Electroporation was employed to transform plasmid DNA into competent E. colL Electric charge (2.5 kV) was applied to a mixture of plasmid DNA (1 ng) and competent cells (40 pL) in a chilled cuvette (1 mm cell gap) using BTX electroporation apparatus (Electro Cell Manipulator 600). The assumed transformation efficiency is 10® transformants/pg DNA. Immediately after electric charge was applied, 500-1000 pL of LB media was added to the 190 mix. The cell suspension was then transferred to a 500-mL cultured flask with LB medium. Amplification of plasmids was obtained after 12-16 hours of incubation at 37 °C with vigorous shaking. Purified plasmid DNA was obtained by equilibrium centrifugation in CsCI-ethidium bromide gradients. Transfection: Cells were seeded at a density of 1X10^ cells/100-mm plate using hemocytometer cell count. Calcium phosphate mediated gene transfer method (Sambrook et al, 1989) was employed for the transfection of rat skin fibroblasts. Cells were incubated overnight to allow for reattachment to the plate. Fresh medium was replaced to the plate four hours prior to performing transfection. Calcium-DNA precipitate was prepared by mixing the 1.35 mL plasmid DNA (containing 2.5 pg of plasmid cDNA neo (Invitrogene), 25 pg of Rous sarcoma virus (RSV)-driven luciferase promotor and/or 25 pg of RSV-(3- galactosidase (kindly provided by Dr. Anthony Young, Professor, Biotechnology Center) with 150 pL of 2.5 M CaClz and 1.5 mL of 2X MBS, pH 7.05. 2X MBS consists of 280 mM NaCI, 10 mM KCI, 1.5 mM Na 2 HP0 4 .2 H2 0 , 12 mM Dextrose, and 50 mM HEPES. The mixture was incubated for 20 minutes at room temperature. Then, the precipitate (1 mL per plate) was added drop by drop on to medium and swirl to obtain a nice and even spread. After more than eight- hour incubation at 37 °C with 5% CO 2 , plates were washed twice with PBS and replaced with a new media. At this point, transient transfection was obtained. Cell selection: Stable transformed cell lines were selected using 400 pg/mL Genecitin™ (G418) containing media. During this time the media were changed every four days. After 14 to 21 days, the plates were washed with PBS, 191 and individual stable cell lines were isolated using cloning cylinders coated with silicone grease to form a tightly sealed well around each colony. Cells from each colony were then trypsinized and transferred with a glass Pasteur pipette to a twenty-four well polystyrene plate. Once the cells began to proliferate, they were moved to twelve- or six-well polystyrene plates and maintained in media with 200 jag/mL G418 (see Figure 6.1). Assessment of transfection success: After cell selection, isolated colonies were evaluated for marker gene expression. Transfected fibroblasts were first washed twice with 5 mL of 1X PBS. Next, cells were harvested using cell scraper and transferred into 1 mL of IX PBS. The cells were spun down for five minutes at 12,000 x g in a microfuge. Cell pellets were then washed with IX PBS and suspended in 300 pL of 0.25 M Tris, pH 7.8. The cells were stored on ice and sonicated with seven pulses using Branson Sonifier 450 set at 50% duty, 6.5 output and equipped with a microtip. Following sonication, the cell debris was pelleted by centrifugation at maximum velocity for 10 minutes in a microfuge. The supemate was used for protein assay, luciferase and/or (3-galactosidase assays. Protein Assay: Protein concentration was measured by Bradford method (1976). Bradford reagent was supplied by Sigma, St. Louise, Ml. Bovine serum albumin (BSA) was used as protein standards (0-1 mg/mL). Ten microlitters of samples were mixed with 300 pL Bradford reagent (Sigma, Saint Louis, MO), and incubated at room temperature for 30 minutes. Standard curves were generated 192 by measuring the optical density at 595 nm using Spectra MAX Microplate Spectrophotometer System (Molecular Devices Corp., Sunnyvale, CA). Luciferase Assay: Luciferase enzyme activity was determined by method described by DeWet et al (1987) with minimum modification. Immediately before the assay, 40 pL of cell extract was mixed with 350 pL of substrate 2 (25 mM gly-gly buffer (pH 7.8), 5 mM Naz -ATP (pH 6.8), 15 mM MgS 0 4 ) in a 12x75 plastic test tube. Luminescence was measured (Berthold Luman Luminometer Model 9801) following 100 pL injection of 1 mM Luciferin (substrate 1, Molecular Probes, Inc. Eugene, OR). The emitted light was monitored for 10 seconds and recorded as relative light units (RLU). After background correcting from RLU, the reading value was normalized for the total amount of protein used in the measurement. Final values are expressed as RLU/pg protein. fi-galactosldase (fi-ga!) Assay: p-gal assay was performed using a colorimetric assay as described by Sambrook et al (1989). A 100-pL cell extract protein was mixed with 3 pL of 100X Mg solution (0.1 M MgClz, 4.5 M p- mercaptoethanol), 66 pL of IX ONPG (4 mg/mL o-nitrophenyi p-D- galactopyranoside in 0.1 M sodium phosphate, pH 7.5) and 0.1 M sodium phosphate. pH 7.5 (total volume = 300 pL). The reaction mixture was incubated at 37 °C until a faint yellow color appeared, then the reactions were stopped with 500 pL of 1 M NaaCOa. Samples were then measured at 420 nm on Spectra Max microplate reader. A unit o f p-gal activity equals the amount of enzyme needed to hydrolyze 1 nM of ONPG in one minute at 37 °C, and is calculated as (380 * A 42o)/time in minutes). 193 Alternatively, monitoring the transfection of p-gal was performed by a histochemical stain using X-gal (MacGregor et al, 1987). This method allows for detection of p-gal activity in situ within cells in culture or organ tissues. The X-gal substrate (5-bromo-4chloro-3indolyl-p-0-galactoside) is hydrolyzed by p- galactosidases to generate galactose and soluble indoxyl molecules, which in turn are oxidized to insoluble indigo. The deep blue color generated facilitates subcellular localization of the p-gal activity. Histochemical staining was performed by washing the plates twice with PBS and then plates were incubated with 2.5% glutaraldehyde in PBS for 10 minutes at room temperature. Next, the plates were again washed twice with PBS and incubated at 37 °C for 6-16 hours with 8 mL of staining solution (200 of 40 mg/mM X-gal in N,N-dimethylformamide, 200 of 200 mM potassium ferrocyanide/fem'cyanide (8.45 g K4Fe(CN)6.3H20, 7.84 g K 3 pe(CN ) 6 and 100 mL distilled water), 16 pLof 1 M MgClg, and 7.584 mL PBS. Fibroblast Cell Transplantation Preparation o f fibroblasts fo r perfusion: The chosen stably transfected fibroblasts were grown in the culture media to reaching 70% confluence. Cells were trypsinized and resuspended in the culture medium at the concentration of 5.2 million cells/mL. Langendorff heart preparation: We employed a classical in vitro cardiac preparation to evaluate the ability of transfected fibroblasts to be retained in 194 cardiac tissue after intracoronary infusion. The experimental setup Dr. Cynthia Games (Assistant Professor, Pharmacy Practice) provided valuable assistance in conducting these methods. Male Sprague-Dawley rats (350-370 grams) were injected with 0.2 mL of Heparin (1000 units/mL i.p.) 30 min prior to anesthesia (Nembutol 100 mg/kg, n=4). The hearts and lungs were rapidly explanted en bloc and placed in ice cold buffer containing (mM) NaCI, 118; KCI, 4.8; MgS 0 4 , 1.2; KH2PO 4,1.2; CaClz, 1.2; glutamine, 0.68; pH 7.35. Following the elimination of lungs and connective tissues, the ascending aorta was cannulated and tied firmly with suture thread. The hearts were then flushed with 1 mL of cold buffer. Perfusion of the hearts was performed in a retrograde manner using a Langendorff apparatus with 50 mL Krebs-Henseleit buffer composed of (mM) NaCI,118; KCI, 4.8; MgS 0 4 , 1.2; KH 2 PO4 , 1.2; glutamine, 0.68; dextrose, 11; NaHCOs, 25; pyruvate, 5. The perfusate was kept warm at 37 “C and oxygenated with 95% oxygen and 5% carbon dioxide. The pH was maintained at 7.30+0.05, and the rate of perfusion was set at 10 mL/min. Shown in Table 6.2 is the study design we employed in our initial trials. Two parameters, cell number and infusion rate, were studied for these preliminary studies, to allow for maximizing transplantation efficiency. After 70 mL of buffer was collected, the bulk rate of perfusion was then set at 2 mL/min. Fibroblast concentrate (5.2 million cells/ml) was coinfused at various flow rates via an injection side port into the bulk flow, using a highly accurate infusion pump (50 or lOOpl/min, Harvard Pump 22). This coinfusion approach allowed for convenient control of actual fibroblast concentrations in infusate and had no 195 affect on buffer pH, etc. Actual cell concentrations in infusate solution were 1.3x10^, and 2.6x10^ cells/ml (see Table 6.2). ImmunohistochemieallyîÊetBetion of cell expressing p^eh Immunohistochemistry was performed to localized fibroblast cells expressing p- gal using an antibody raised against p-gal. Cultured fibroblasts were washed twice with PBS and fixed in 10 % buffered formalin for 15 min. Cells were then washed again with PBS and resuspended in 70 % EtOH. These cell preparations represented negative control (wild type) and positive control (transfected fibroblasts) for p-gal immunoreactivity. Following infusion of transfected fibroblasts, the perfused hearts were immediately formalin fixed and cut into 4 serial sections. The embedded sections were sliced into 5-pm sections, deparaffinized and rehydrated. Slides from cell preparation were processed at the same time as the heart sections. Subsequently, all slides undenA/ent standard protocol of immunohistochemistry as previously described (Materials and Methods,Chapter 2), except the primary antibody was substituted for anti-p-gal antibody (Molecular Probes, Inc, Eugene, OR). Digital image analysis: Images of immunostained tissue were captured using an Olympus microscope (BX-40) and a high-resolution digital camera (Pixera, Inc., 1260x960 pixel resolution). Unmodified images were then analyzed using research-based image analysis software (ImagePro Plus, Media Cybernetics, Silver Spring, MD). RESULTS 196 stable Transfection of rat skin fibroblasts Shown in Figure 6.3 are growth curves of normal rat skin fibroblasts (ATCC 1213) and fibroblasts transfected with p-galactosidase (B62199-30). Transfected cell line showed growth pattern similar to the wild type. The growth cycle consists of lag phase, exponential or log phase, and stationary or plateau phase. These patterns provide important information about characteristics of each cell line. The lag phase, day 0 to day 1, is the time following subculture and reseeding. This is a period of adaptation when only little increases in cell number is observed. Although the transfected line showed a lower cell number at the lag phase, the log and plateau phases are comparable to the wild type (day 1-5 and day 5-8, respectively). No statistical differences in cell number were observed either at the log or at the plateau phases. Within these phases represent crucial information about characteristic of the cell line—the population doubling time (2X), and the maximum cell density achieved at plateau. Approximately 10-15 stable transformants were obtained per 100-mm plate. Of 18 cell lines transfected with RSV-luciferase promoter gene, one cell line showed a very strong signal at 44,448 RLU/mg protein. Fibroblasts expressing p-gal activity after stable transformation were obtained and detected using X-gal histochemical staining (Figure 6.3, A-B) and immunohistochemistry (Figure 6.3, C-0). After X-gal incubation, cells expressing P-gal showed blue cytosol due to the deposition of insoluble indigo blue product from hydrolyzation of X-gal substrate. Although more than 60% of cell lines 197 selected for stable p-gal transformants expressed in situ histochemical blue stain by X-gal, most of these lines showed p-gal activity less than 0.5 unit/mg protein with an exception of one stable line that expressed p-gal activity at 6.5 units/mg protein. This cell line was selected for further ex vivo cell transplantation experiment (cardiac infusion experiments). Intracoronary infusion of fransfected fibroblasts Stably transfected fibroblasts were infused in isolated working rat hearts at different concentrations and various total cell numbers (Table 6.2). In all preparations more than 90% of the fibroblasts were retained in the hearts (Table 6.2 and Figure 6.5). Immunohistochemical staining for fibroblast p-gal revealed that the fibroblasts were distributed throughout in the myocardium both in left (Figure 6.5, A-B) and right ventricles (Figure 6.5, C). Interestingly, these cells were not confined to intravascular space, but rather appeared to be interspersed in among cardiac myocytes (Figure 6.5, D-F). 198 DISCUSSION AND FUTURE DIRECTIONS Gene therapy for cardiac disease has some potential, but currently available approaches have some limitations. We have proposed an approach that may have some advantages over existing technologies; particularly reliable and stable cell transformation, specific selection of altered cells, and (potentially) less invasive administration techniques. Our preliminary investigations show successful stably grown transfected fibroblasts whose growth patterns are comparable to those of the wild type. The initial attempts for fibroblast cell kinetics study in the myocardium revealed that the hearts are capable of retaining fibroblast cells from 1.3 to 7.8 million cells. The six-fold increase in cell number infused had no impact on the percent retention, i.e. more than 90% of cells infused through the rat hearts were retained. Larger number of cells were used for implantation in larger animals and in humans. Yogalingam et al (1999) have implanted 24 to 32.4 million autologous fibroblasts into cat kidneys. Others used 100 to 500 million cells to inject into the back or abdomen of patients who underwent fibroblast-mediated gene therapy for hemophilia B (Xinfang et al, 1996). In addition to different total number of cells infused to the hearts, we have also evaluated the impact of rate of infusion on the cell kinetics. At infusion rates used in this study (50 and 100 pL/min) there was no significant difference in the transplantation efficiency both in % retention and cell distribution pattern. Immunohistochemical detection of fibroblasts expressing p-gal strikingly demonstrated that cells were randomly distributed throughout the perfused 199 hearts. No cell clumps were observed In all 4 serial sections of the hearts. Moreover, it appeared that the cell wall of some infused fibroblasts were merged with or attached to the myocardium. This suggests that a portion of cells retained in the hearts had a tendency toward surviving in the myocardium. The losses of gene expression through passages or down regulation are a general concern in maintaining transduced cell lines. Thus, in vitro cell line evaluations and in vivo kinetics studies are important check points in the “proof of concept” preclinical fibroblast gene transfer. Essential parameters for evaluations include; a) dose (number of cells used), b) half-life of cells and half- life of gene products, c) potential adverse effects e.g. collagen formation, impacts on cardiac performance, distribution and deposition of fibroblasts to peripheral organs. Benefits form using fibroblasts are not limited to their assigned gene product delivery but might also be used in cardiac gene manipulation. Leor et al (1997) demonstrated that transfer of myogenic determination (MyoO) gene can convert cardiac fibroblasts into cells that express skeletal fast myosin heavy chain and cells behave like contractile units. Additionally, fibroblasts may enhance the efficiency of cell transplantation by providing benefit growth factors such as bFGF (Mar et al, 1996). Recent studies have shown the benefits of using fibroblast growth factors (FGF) to mediate increases of free intracellular calcium, to induce cardiac myocyte proliferation, and to stimulate angiogenesis in ischemic heart in humans (Sheikh et al, 1999; Merle et al, 1997; Schumacher et 200 al, 1998). Thus, fibroblast cell transplantation may provide a new synergistic therapeutic outcome for the genes transferred to the myocardium. Our initial studies thus fa r are encouraging, but much more work is left to be done! Future experiments necessary for validation include: 1) a thorough evaluation of fibroblast survival in cardiac tissue and cardiac responses in vivo, 2) Identification and expression relevant genes of interest in fibroblasts, and ultimately 3) evaluation of human cells and tissues. 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Science 1993;261: 209-211. 207 Langendorff Rate o f Time o f Volume of Total C ell count % Retention Preparation infusion Infusion infusate number o f from th e (uL/min) (m in) cells (mil) effluence Heart 1 100 5 0.5 2.6 3000 99.8846 Heart 2 100 15 1.5 7.8 1000 99.9872 Hearts 50 15 0.75 3.9 6500 99.8333 Heart 4 50 5 0.25 1.3 125000 90.3846 Table 6.2 Infusion of transfected fibroblasts into the Langendorff hearts. 208 OR RSV-luciferase RSV-p-gal Neo* Calcium-DNA precipitate G418 selection V Transfer to 24-well, 12- well, and 6-well plate & p-gal and/or Luciferase assay Figure 6.1 Schematic representation of transfection procedure. Calcium phosphate was used to form calcium-DNA precipitate as described in text. 209 l ü k .4 3 . > ■•»:• ;~a.,.. - -.Hv s Figure 6.2 Photomicrographic representation of enzymatic and immunohistochemicai detection of rat skin fibroblasts expressing p- galactosidase. Dark blue product of X-gal degradation indicated expression of p- gal gene products (B) compared to normal rat skin fibroblasts (ATCC1213, A) (250X). Likewise, immunohistochemistry method showed deposition of DAB chromogen in the cells expressing p-gal (D) compared to the wild type (C) (400X). 210 "O o S 1e+5 o Q. • ATCC1213 (0 O B62199-30 Ô) 1e+4 O 2 4 6 8 10 2 x D a y LAG LOG PLATEAU Figure 6.3 Diagram of growth curves of normal rat skin fibroblasts (ATCC 1213) and fibroblasts stably transfected with p-galactosidase gene (B62199-30). LAG, the lag phase; LOG, the log phase; 2X, population doubling time; PLATEAU, the plateau phase (see text). 211 Total cells in 99.9% 8e+6 I I Effluent Cells Û: LU CD 6e+6 99.9% 4e+6 99.9% Ü 2e+6 90.3% Heart 4 Heart 1 Heart 3 Heart 2 PREPARATION Figure 6.4 Percentage of infused fibroblasts remaining in the Langendorff hearts. Total number of cells delivered to the hearts was calculated from the rate and time of infusion (see Table 6.2). After deduction from number of cells in the effluent perfusate, the percentages of retained fibroblasts were obtained. 212 ~ ® Ia1 W B j / J r . * % Î/-' I ' i .1 *• < -:W i ISil # 4 % # # m # 3 ■S'"-' . V , .'•■ , f--,* ' # & x ' ■ * r ** S.L :#.S " 'f ' . *"1 k % f^. l i i l # C'..W !W ^Z mÊËËgl Figure 6.5 Immunohistochemicai detection of rat skin fibrobiasts expressing p-galactosidase in the Langendorff heart. Top Panel: Distribution of fibroblasts retained in the heart. Pointed by arrows are cells positive to p-gal immunoreactivity. Cells were found in both ventricles (left ventricle, A-B; right ventricle, 0) (200X). Bottom Panel: Morphologically evidence of fibroblasts attached to cell surface of the myocardium that were infused for 15 min (400X). 213 CHAPTER 7 CONCLUSIONS Studies presented herein were conducted to further the understanding of oxidant related mechanisms in pathologic events in cardiovascular disease, particularly with respect to nitric oxide (NO) control mechanisms. In addition, a novel therapeutic strategy for oxidant-related cardiovascular disorders is proposed that may overcome some existing problems associated with current gene therapy regimens and protocols. Information from these studies is in a hope to provide a better understanding of etiology and to suggest therapeutic or preventive strategies for the cure of cardiovascular disease. Provide below are brief summaries of the primary conclusions in this dissertation; 1) Cardiovascular disease is prevalent and represents huge costs to the U.S. health care system. It has become clear that increased prevalence of reactive oxygen species is an apparent unifying mechanism of most forms of cardiac and vascular dysfunction and disease. Several pro-oxidant pathways have been identified, and these oxidant mechanisms may be important in structural and functional changes that occur in cardiovascular disease initiation and/or 214 progression. Targeting the Inhibition of these pathways (and/or associated cell responses) using antioxidants may have therapeutic value for cardiovascular disease management, although currently available data using vitamin supplements have been less than convincing. It is likely that these pursuits will lead to a better understanding of these biological phenomena and will hopefully provide new opportunities for therapeutic interventions. 2) Since angiotensin II was first recognized as a vasoconstrictor, its roles are now known to be diverse and involve regulation of cell growth and development, and activation of superoxide production. While increased blood levels are associated with many cardiovascular disease states its actions in the initiation of disease are have not been well defined. We found that at low dose and short term administration of ANG II caused a selective reduction in endothelium dependent (nitric oxide mediated) relaxant effects. These changes occurred prior to any other established consequences of elevated ANG II in vivo, including enhanced vascular contractile responses, development hypertension, or increase in cardiac or vascular mass. This endothelial dysfunction was associated with increased production of peroxynitrite in vivo. Protein nitration in the endothelial layer correlated with the extent of functional impairment observed. Thus, selective perturbation of normal endothelial function is an apparent early or initiating event during ANG II exposure in vivo. Our findings may help to explain the results of the recently published TREND trial (see above) and suggest that 215 evaluation of selective endothelial therapies for the prevention of disease appears warranted. 3) Having recognized that angiotensin II promoted tyrosyl protein nitration selectively in the endothelium in vivo, we then investigated whether this phenomenon also occurs in an isolated cell setting. Incubation of ANG II in endothelial cell culture demonstrated that ANG II induced endothelial protein nitration in a concentration and time-dependent manner. Thus, the complex multi-cell environment and presence of complex physiological regulation are apparently not required for ANG II induced endothelial protein nitration. In addition, we also observed readily detectable and time dependent protein nitration in the absence of ANG II stimulation. Thus, nitration may be a phenomenon that occurs under normal cellular conditions and/or during cellular maturation or aging. In a separate experiment we found that endothelial cells possess denitration capability in the presence of cofactor(s) from serum. The basal presence of tyrosine nitration and evidence of denitration suggest that perhaps nitration mechanisms participate as an intracellular signaling mechanism. Alternatively it may serve as an indirect modulator of tyrosine kinase/phosphatase signaling pathways. The roles of protein nitration in intracellular cellular signaling pathways are clearly worthy of further investigations. 216 4) ANGII and TNF are two established agents Involved in cardiac remodeling and heart failure. Our studies were conducted to investigate the role of these two agents and their interactions in early events of cardiac remodeling. We found that short-term (3-day) administration of these agents at physiologically relevant concentrations induced significant TUNEL positive nuclei alone (suggesting apoptosis), but no change when combined in vivo. This apparent apoptosis in vivo was independent of detectable hemodynamic or cardiac size changes and an apparently early event in the remodeling process. TUNEL frequency was are apparently not related to cardiac NOS II induction. Our findings suggest that important interactions between ANGII and TNF exist in vivo. Further evaluations of alteration in plasma neurohormones and gene expression of renin-angiotensin system components and atrial natriuretic peptide (ANP) revealed that these interactions of ANG II and TNF occur at the levels of transcription and post- transcription. Short-term administration of ANG II and/or TNF promoted 4-fold increases in fetal ANP gene expression in the absence of hemodynamic changes (pressure overload), suggesting that ANG II and TNF each alone or in combination may participate in the initiation of cell type adaptation and cardiac remodeling process. 5) Genetic approaches for the treatment of cardiac disease is a rapidly developing field with great potential. The potential usefulness of these gene strategies has become partially limited by the shortcoming of adenoviral vectors employed. In recognition of some limitations of current cardiac gene therapy 217 approaches, we proposed a novel strategy for cell based cardiac treatments. In this approach human fibroblasts would be isolated from the patients and stably transfected in vitro with target genes. These stably transformed cells would then be implanted to myocardium via minimally invasive technologies (angioplasty catheterization). In our preliminary studies we performed transfection experiments using p-gal as a marker gene. We observed successful and stable transfection of rat skin fibroblasts, and cellular growth pattems were comparable to wild type cells. The initial attempts for fibroblast cell kinetics study in the rat myocardium revealed that the hearts are capable of retaining fibroblast cells from 1.3 to 7.8 million cells. The six-fold increase in cell number infused had no impact on the percent retention, i.e. more than 90% of cells infused through the rat hearts were retained. In addition to different total number of cells infused to the hearts we have also evaluated the impact of rate of infusion on the cell kinetics. At infusion rates used in this study (50 and 100 pL/min) there was no significant difference in the transplantation efficiency both in % retention and cell distribution pattern. Immunohistochemicai detection of fibroblasts expressing p-gal demonstrated that cells were widely distributed throughout cardiac cross- sections. Our studies thus far are encouraging, but much more work is left to be done. Future experiments necessary for validation include: 1) a thorough evaluation of fibroblast survival in cardiac tissue and cardiac responses in vivo, 2) identification and expression relevant genes of interest in fibroblasts, and ultimately 3) evaluation of human cells and tissues. Despite these reservations, 2 1 8 further evaluations of this potentially valuable approach for cardiac gene therapy are clearly warranted. The work form this dissertation has provided a better understanding of the roles of nitric oxide related oxidative events in the early states of cardiovascular disease. 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Systemic gene expression after intravenous DNA delivery into adult mice. Science 1993,261: 209-211. Zou M, Martin C and Ullrich V. (1997) Tyrosine nitration as a mechanism of selective inactivation of prostacyclin synthase by peroxynitrite. Biol Chem 378,707- 13. 245 Zweier JL, Flaherty JT, Weisfeldt ML. Observation of free radical generation in the post-ischemic heart. Proc Natl Acad Sci USA 1987;84:1404-1407. 246 A PPE N D IX I THE HUMAN GENOME PROJECT: BENEFITS AND RISKS TO SOCIETY This appendix is the result of a collaborative work with Dr. Jon 0. Schommer, Associate Professor, Department of Pharmaceutical Care and Health System, University of Minnesota. This manuscript has been published in Drug Information Joumal 33(3), 729-735 (1999), and is presented here in the format of the Joumal. The authors of the manuscript are Suvara K. Wattanapitayakul and Jon 0. Schommer. 247 The Human Genome Project: Benefits and Risks to Society A b strac t The human genome project was initiated in 1990 to study the structure and characteristics of human DNA that are important for understanding gene functions and their relation to diseases. The large scale genome research has driven the technology advancement in genetic testing, drug design, gene therapy, and other genetic related areas such as pharmacogenetics. Although the project reveals potential benefit, it raises ethical, legal, and social issues. The outcomes of individuals' genetic information disclosure may lead to confidentiality and genetic discrimination issues. In addition, clinical relevance to genetic testing and psychological effect from the results are debatable. This article discusses the potential benefits and risks from the human genome project. Keywords : Human Genome Project, Public Policy 248 Introduction The human genome project was launched in 1990 by a joint commission between the Department of Energy (DOE) and the National Institutes of Health (NIH) to identify all the estimated 80,000 genes in human DNA and to determine the sequences of the 3 billion chemical bases that make up human DNA, store this information in databases, and develop tools for data analysis. It is a 15-year initiative to provide detailed information about the structure and characteristics of human DNA that are critical to understanding the functions of genes and their relation to etiology of diseases (1). More than 60 disease-linked genes have been identified and are believed to be advantageous to gene-based therapy development when the study of human genetics is completed. Potential implications of genetic information and issues raised from the use of that knowledge is discussed in this article. Therapeutic Implications Why the Human Genome Project (HGP)? There are several rationales endorsing the intensive investment and endeavor in HGP. First, researchers will obtain benefits from economy of scale. In general, the estimated cost of searching for a inherited disease gene (e.g. cystic fibrosis) is more than $100 million (2). The total cost of finding all of the human genes, one at a time, according to the estimated cost would be astronomical and prohibitive. However, the systematic finding approach (all at 249 once) helps reduce the cost of finding all genes to $3 billion, or $30,000 per gene. The second reason is to improve the technology for DNA mapping and sequencing, and data manipulation. Major goals of informatics development are to improve data management, analysis, and distribution. Focus has been placed on accessibility and user-friendly tools to organize, and interpret a large amount of data. The large-scale genome research approach has driven the technology advancement toward enhancing capacity and reducing size. The first five-year plan has shown a development in sequencing DNA at reducing cost and increasing rate. The original 1996 goal of $0.50/base pair has been met and an additional annual budget of $100 million will be devoted to develop sequencing technology in order to achieve the entire human genome sequences by 2005 (3). Only a decreased cost of one cent per base pair could save $30 million in sequencing the entire human genome (2). In addition, the “chromosome painting” technology developed by this project in 1995 was licensed to a private company for the research or disease detection in many chromosomal abnormalities such as Down’s syndrome and cancers (4). Moreover, the Advanced Technology Program, National Standards and Technology, has funded several companies to engage in developing diagnostic DNA arrays and adapting diagnostic tools for analyzing human tissues. Other projects that are developed as a high potential for diagnosis and treatment tools include applications of DNA “superchip” technology that have a capacity of rapid sequencing, and industrial and environmental monitoring. 250 Genetic testing Who would benefit firom genetic testing? If genetic testing becomes effective and available in the near future, there would be potential benefits to patients, patients' families, physicians, insurance companies, business, society, and government (5). For instance, patients can decide an appropriate treatment and life planning while physicians make the best use of time and medical resources to treat patients due to more accurate diagnosis and testing. Some genetic screenings of newborns are greatly useful for treatment of disease that can be successfully treated. Insurance companies will have good profiles of medical procedures in each disease state, which will help provide for expected long-term complication actuary and coverage. Business and society would benefit from having a healthy and productive population and the government could effectively allocate funds for detecting and treating diseases as demanded by citizens. The HGP has reformed traditional epidemiological research by giving a better understanding and more precise etiology of genetic disease, especially chronic diseases. It augments the understanding by mapping the genes responsible for the diseases and develops models to predict onset of chronic disease with late onset (6). In addition, the offspring's impending disease from the carriers of recessive genes for untreatable diseases could be avoided by providing genetic counseling about reproductive options. Examples of such tests are Tay-Sachs, HbS, and 0-thalassemia (7). 251 The HGP contributes to a better understanding of etiology and risk factors of breast cancer. It reveals that genetic predisposition and environmental factors play more significant roles than the previous hypothesis of environmental carcinogens and hormonal influences (8). As age is one of the risk factors for breast cancer, the identification of breast cancer susceptibility genes and development of testing for breast cancer will reduce the mortality of women who undergo annual mammography. Breast cancer screening and therapy have been improved dramatically in the past decade. Recently, tamoxifen, has been approved by the Food and Drug Administration as a preventive medicine for certain types of breast cancer. Early identification of high-risk women will help design strategies for prevention and intensive surveillance. Pharmaceutical industry Since the Human Genome Project was launched in 1990, a group of outstanding scientists related to pharmaceutical industry have discussed the trend and potential breakthrough for drug design and therapeutic-based pharmaceutical development at the September 30 satellite meeting to Genome Sequencing Conference II (9). It is estimated that more than 50,000 genes to be identified are the potential targets for pharmacological intervention. The HGP will help scientists understand the etiologies of multigenic diseases including heart disease, diabetes, hypertension, and cancer. Thus, applying the genomic data will facilitate pharmaceutical interventions in alleviating or even eradicating these 252 diseases. As a part of such a drug design revolution, information from the project databases will expedite the design of oligonucleotide drugs that modulate target DNA transcription. In addition, utilizing the DNA sequencing database to predict 3-dimensional protein structure and function is substantially advantageous to protein-based drug design. Moreover, the HGP will assist in identification and evaluation of potential chemopreventive agents in cohorts at genetic risk, and will help develop strategies for specific drug targeting (10). Pharmacogenetics Although a wide variety of drug categories that work at different target organs are available, they fall behind their maximum effectiveness partially due to the variability in individual genetic differences that have a great Impact on drug metabolism. In the past, we had observed and experienced the consequences of responses to drugs from different ethnic groups and people who have impaired metabolism for those drugs. In the late 1930s and early 1940s, monitoring sulfonamide blood levels was recognized as a tool to assure the effectiveness of antibacterial therapy. The observation of isoniazid-induced neuritis that is selective among individuals had demonstrated the significance of inter-individual differences in the metabolic fate of the drug (11 ). In addition to therapeutic failure and drug toxicity due to variation in drug metabolism, genetic polymorphism also plays a role in disease susceptibility as seen in G6PD deficiency. Previously, the pivotal role of pharmacogenetics relied on the limited 253 information on genetic markers for traits and the use of family history, racial and ethnic background. The new technology of physical mapping and functional cloning' will enhance the understanding of an aberrant metabolic pathway, i.e. an insight into the relevant gene(s) encoding the aberrant protein products (12). The genetic polymorphisms that have the most clinical relevance are enzymes related to C-oxidation by cytochrome P450 (CYP2D6) and 2Cmeph, acétylation by N-acetyltransferase, S-methytation by thiopurine methytransferase, and ester hydrolysis by pseudocholinesterase (13). Metoprolol poor metabolism is a prototype of the debrisoquine/separteine polymorphism that embraces a broad list of drugs acting on either the central nervous system or cardiovascular system. The livers of poor metabolizers lack active CYP2D6 enzyme due to mutations, resulting in potentiation of adverse drug reactions. Other prototypes involved in genetic polymorphism include mephenytoin, isoniazid, sulphadiazine, 6-Mercaptopurine, and suxamethonium. The mephenytoin metabolic deficiency is initially observed and studied in a family who had unusual sedation after regular doses of mephenytoin (14). It appears that the genetic polymorphism presents in 2-6% of Caucasians, and 14- 22% of populations in Far Eastern countries such as Japan, China or India. However, the therapeutic/toxic consequences of polymorphic enzymes impinge on the pharmacokinetic and pharmacodynamic outcomes. The determinative factors for the significance of polymorphic enzymes include the effectiveness of the parent drug or its primary metabolite or both, the overall 254 contribution of the impacted pathway, the availability and efficiency of alternative clearance pathway. Nonetheless, the knowledge obtained from genetic studies would lead to allele-specific DNA amplification tests and genotyping. and provide a benchmark for drug dosing and toxicity prevention, especially for low therapeutic index drugs. Gene th e ra p y Gene therapy is a challenge for 21st century healthcare professionals. It involves the transfer of genes, mostly in a form o f an expression cassette, into patients who have “defective genes”. The transfer of an expression cassette into target cells could be accomplished via ex vivo (isolate the target cells and transfer the expression cassette in laboratory), or in vivo (directly introduce the expression cassette into target cells within the individual (15). Human gene therapy is an ongoing research area, and is being developed for a wide variety of diseases such as genetic disorders, cancer, and AIDS. Genetic-based diseases using bone marrow hematopoietic stem cells that are currently in clinical trials include hemophilia B, severe combined immunodeficiency, familial hypercholesterolemia, cystic fibrosis, Gaucher disease, and cancer vaccine. Examples for current gene transfer tools are retroviral vectors, adenovirus and adeno-associated viral vectors, mammalian artificial chromosomes, and cationic lipids and liposomes. In many cases, if temporary expression or transduction is more favorable, only the introduction of DNA or RNA in to the cells is sufficient 255 without incorporation of genes into the host genome. This method is considered safer because it avoids insertional mutagenesis, and the DNA or RNA is ultimately eliminated from the host cells. Preparation and administration of gene delivery systems could be an attractive opportunity for healthcare practitioners, especially pharmacists when special care is required for product delivery in forms of a liposomal system, ffeeze-dried plasmid, and/or a preparation of unstable virus (16). R isks Ethical, Legal, and Social Issues Deciphering the entire blue print of the human genome may raise ethical, legal and social issues (ELSI) when the genetic information becomes available. The HGP has pondered these potential emerging issues and devoted 3-5% of the genome project budget for ELSI study. The study concems five major areas; 1) genetic testing. 2) fairness in the use of genetic information. 3) privacy and confidentiality, 4) clinical application, and 5) professional and public education. Although genetic testing has potential benefits to society, it leads to other issues and concems regarding who should have access to the genetic information and how it is interpreted, clinical relevance, and the psychological outcomes from the tests. 256 Privacy and Confidandality It is important that the privacy of the genetic testing is assured. In other words, individuals who undergo genetic testing have the ability to control, restrict, or refuse unauthorized access to their information (17). However, if genetic information becomes part of a patient’s health history and medical record. It will be accessible by insurance companies (18). It is possible that once the genetic information is revealed, patients may receive discrimination in employment and insurance coverage. Employers would prefer the most efficient employees who have low sick days, and no expected serious illness that may lead to increased insurance premiums. The provisions of the Americans with Disabilities Act of 1990 (ADA) for the relevance of the HGP to the emerging legal, empirical, and policy implications are discussed by Blanck and Marti (19). It is very likely that persons with known onset of disease and potential complications of disease would receive higher premium or coverage denial because of "pre-existing condition”. Clinical relevance Manifestation of genetic diseases is varied at different ages from prenatal life to old age (18). For example, Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, breast and ovarian cancers become more prevalent in advanced age. However, children are the most vulnerable population affected 257 by the social risks of genetic testing. The impact of testing results is primarily imposed on the children's families, especially the parents, due to anxiety, guilt, psychological distancing, over-protectiveness, and financial investment for a child with a possibly limited life span (20). In most cases, parents are the decision-makers for genetic testing. Therefore, it is important that the competency for medical decision making and understanding of genetic information are addressed. Despite the fact that informed consent intends to emphasize the voluntary nature of testing, occasionally it lacks sufficient information for the participant to make an appropriate decision by weighing the risk and benefits of participation, hence pre-test genetic counseling is essential (21). Undoubtedly, there is no necessity to perform genetic tests in children on the late onset genetic susceptible diseases if there is no therapeutic implication or surveillance available. For example, the 1990 committee of the International Huntington Association and the World Federation of Neurology did not recommend a test for HD for children under the age of 18 due to the lack of benefit from testing (20). Likewise, breast cancer genetic screening in children should not be performed with the absence of appropriate surveillance recommendations from routine procedures such as mammograms. Pap smear, or PSA test. The impact of genetic information influencing women's decisions on reproduction, i.e. decision to continue or terminate a pregnancy, is the most challenging issue emerging from the HGP while the treatment of unborn 258 abnormal fetuses is yet available. At present, the only treatment for many fetal genetic abnormalities pursuant to prenatal diagnosis is abortion (22). It seems worthless for prenatal genetic testing for individuals who would continue the pregnancy regardless of the results (23) unless the information would allow preparation for the caregivers of the child. Moreover, low income and minority women are at risk of a denial of access to genetic technology hence increasing genetic- baring disease children (24). Ideally, genetic counseling should be provided before any testing is performed. Ironically, Medicaid programs in some states provide prenatal genetic tests without providing counseling and coverage for abortion (22). Psychological effect Although most single-gene disorders probably have a strong genotype- phenotype relationship, the genes are not insulated from others nor can they be influenced by other genetic influences such as their immediate sequence environment or other gene loci. For example, precise genetic abnormalities information in sickle-cell anemia (homozygous parents with 0-globin gene lesion) fails to predict the exact clinical phenotype (25). The variability in severity of the disease is due to the effects of genetic variation either within the regulatory regions that are linked to the gene or the far upstream regions. In addition, other factors also account for the difficulty in predicting the phenotype, including the proportion of cells bearing the lesion (mosaicism), the activity state of the 259 chromosome carrying it (X-inactivation and imprinting), the effects of other loci and their protein products (epistasis), the status of the other allele and the variable expression of the wild type or mutant allele. Clearly, it is more difficult to achieve perfect manifestation patterns of multifactorial genetic diseases such as heart diseases, familial hypercholesterolemia, cancers, and Alzheimer disease. Therefore, the result of genetic testing can lead to devastating psychological effects rather than benefits to patients in terms of therapy. Genetic testing outcomes could result In worries about one's health, marital problems, anxiety, sorrow, depression, and even suicidal thoughts (26). Thus, it is pointless to have genetic testing for a disease with late onset and no available therapy. Salkovskis (26) requests an urgent study on the likely type and intensity of psychological reactions to different types of testing. In addition, a study of sensitivity and specificity of genetic testing is warranted prior to any public policy of the testing is implemented. The ultimate goal of genetic disease treatment involves the restoration of functional homeostasis. However, meta-analysis of selected 65 Inborn metabolic errors demonstrated that only 8 of the errors were cured while the remainder were far from treatment or experienced only partial treatment (27). The stigma of carrying a genetic disease could be a life-long calamity and disgrace for those who lack distinction in knowledge of genotype and phenotype. Concluding Remarks 260 There are a wide variety of benefits that are potentially gained from the HGP on the premise of a genetic revolution. Improvement in mapping and sequencing technologies, and data manipulation will lead us to the era of informatics and its use in health care. The study of the entire human genome provides insight into how differences in genetic make up influence manifestations of genetic-linked diseases. In addition, the pharmaceutical industry will benefit from applying the genome project information and introducing new pharmaceutical interventions. However, before the genetic information is available, we need to prepare in essence for emerging issues in the advent of the HGP. In light of preventing, ameliorating and eradicating diseases, there are prices related to those interventions and the problem is who will pay? Currently, the growth of health care expenditures is exceeding the inflation rate partially due to an introduction of high-cost new therapeutic interventions. Moreover, the use and misuse of the informatics' will have a huge impact on individuals and society as a whole. The ELSI study has been initiated in the hope of addressing potential emerging issues. Prior to applying any informatics to health insurance and public access, the ELSI should exhaustively explore the issues as well as provide a well-established set of guidelines and regulations that are ready to approach of all potential issues. Public education is the primary and crucial foreground to be considered, especially genetic counseling. In general, most multifactorial genetic-linked diseases are not solely caused by inherited susceptibility. A positive test does not indicate that person will develop the 261 disease and vice versa, a negative result does not guarantee a disease-free future because the risk remains the same as that of the average population such as in the case of cancer. It is still an ongoing study whether the environment or genetics play more important role in triggering onset of certain diseases even for those caused by a single-gene mutation. Paradoxically, there is a concern for potential malpractice suits for failure to inform about genetic risk regarding a wrongful birth. Therefore, there is a real need for how to design a meaningful genetic testing in medical practice. In addition, focus should be meticulously placed on the employment provisions of the American with Disabilities Act (1990) with regard to genetic discrimination. Similar Acts will be necessary in order to prevent averse selection by insurance companies and discrimination by social institutes. In summary, public health policy development needs to address all issues in the arena of informatics and its impact prior to implementing population-based programs designed to minimize morbidity and mortality associated with genetic susceptibility diseases. 262 References 1. Fink L, Collins FS. The Human Genome Project: View from the National Institutes of Health. JAMW 1997; 52(1):4-7.15. 2. Cantor CR. A brief sketch of the Human Genome Project and its implications for pharmaceutical technology. Pharmaceutical Technology 1993;17:22,24,26. 3. The Human Genome Project. Revised 5-year research goals of the U.S. Human Genome Project. Human Genome News 1993;5(4):1-5. 4. The Human Genome Project. Technology transfer—Commercializing genome resource. Human Genome News 1995; 7(3-4):15. 5. Kadlec JV, McPherson RA. Ethical issues in screening and testing for genetic diseases. Clin in Lab Med 1995;15(4):989-999. 6. Ellsworth DL, Hallman DM, Boerwinkle E. Impact of the Human Genome Project of epidermiologic research. Epidermiologic Rev 1997; 19(1):3-13. 7. Scriver CR. Genetic screening, testing and treatment: How far can we go? J Inher Metab Dis 1996;19:401-411. 8. Olopade 01. The Human Genome Project and breast cancer. Women's Health Issues 1997;7(4):209-214. 9. Mansfield BK. The Genome Project and the Pharmaceutical Industry. Human Genome News 1990:2(4):1. 10. Kelloff GJ, Hawk ET, Karp JE, Crowell JA et al. Progress in clinical chemoprevention. Seminars in oncology 1997;24(2):241-252. 11. Kalow W. Pharmacogenetics: Its place in medicine and biology. J Pharmacy Practice 1992;6:312-316. 12. Hildebrand CE, Stallings RL, Tomey DC, Fickett JW et al. Human genome mapping and sequencing: Applications in Pharmaceutical science. In :Biotechnoiogy and Pharmacy. Pezzuto JM, Johnson ME, Manzsse HR, Editors, New York, Chapman&Hall, 1993. 13. Tucker GT. Clinical implications of genetic polymorphism in drug metabolism. J Pharm Pharmacol 1994;46(Suppl 1):417-424. 14. Meyer UA. The molecular basis of genetic polymorphisms of drug metabolism. J Pharm Pharmacol 1994;46(Suppl 1):409-415. 263 15. Sandhu JS, Keating A, Hozumi N. Human gene therapy. Crit Rev Biotech 1997;17(4):307-326. 16. Smith TJ. Gene therapy: Opportunities for pharmacy in the 21st century. Am J Pharmaceutical Education 1996;60:213-215. 17. Beauchamp TL, Childress JF. Principles of Biomedical Ethics. 4th ed. New York, Oxford University Press, 1994. 18. Kopala B. The Human Genome Project: Issues and ethics. MON 1997;22:9- 12. 19.Blanck PD, Marti MW. Genetic discrimination and the employment provisions of the Americans with Disabilities Act: Emerging legal, empirical, and policy Implications. Behavioral Sci and the Law 1996;14:411-432. 20. Patenaude AF. The genetic testing of children for cancer susceptibility: Ethical, legal, and social issues. Behavioral Sci and the Law 1996;14:393-410. 21. Wilfond BS, Noland K. National policy development for the clinical application of genetic diagnostic technologies. JAMA 1993;270(24):2948-2954. 22. Cassel CK. Policy implications of the Human genome Project for women. Women’s Health Issues 1997;7(4):225-229. 23. Mahowald MB, Levinson D, Cassel C, Lemke A et al. The new genetics and women. Milbank Quaterly 1996;74(2):239-283. 24. Nsiah-Jefferson L. Reproductive genetic services for low-income women and women of color: Access and sociocultural issues. In: Women and prenatal testing: facing the challenges of genetic technology. Rothenberg KH, Thomsom EJ, editors. Ohio State University Press, Columbus(OH), 1994. 25. Ravine D, Cooper DN. Adult-onset genetic disease: mechanisms, analysis and prediction. Q J Med 1997;90:83-103. 26. Salkovskis PM, Rimes KA. Predictive genetic testing : psychological factors. J Psychosomatic Res 1997;43(5):477-487. 27. Scriver CR. We mean well: Treatment of Mendelian disease. Acta Paediatr Japonica 1988;30:385-389. 264 APPENDIX II THE ROLE OF PRESCRIPTION DRUG INSURANCE TYPE AND DRUG COST INFORMATION ON PHYSICIAN ANTIBIOTIC SELECTION FOR THE ELDERLY This appendix is the result of a collaborative work with Dr. David A. Mott, Assistant Professor, Department of Pharmacy Administration, University of Wisconsin (Madison). Presented herein is a draft of the manuscript of this work. The authors of the manuscript are Suvara K. Wattanapitayakul and David A. Mott. 265 Introduction Research has documented discrepancies in the use of health care goods and services between the insured and uninsured. The uninsured are less likely to use new, high cost, and discretionary health care items. Research related to prescription drugs suggest the uninsured are less likely to use brand name drug products, high cost drug products and newer drug products . Three factors may cause the discrepancies in use of health care goods and services between the insured and the uninsured (Mort et al.). First, there may be differences in the health status and clinical need of insured and uninsured patients. Second, insured and uninsured patients may have differences in preferences for care. Third, physicians may recommend different goods and services for insured and uninsured patients. Past research examining differences in drug therapy between insured and uninsured patients mainly have focused on drug use . However, differences in drug use may reflect actions taken as a result of different preferences for care such as generic substitution or not dispensing a prescribed medication. The focus o f this study is factors influencing physicians' recommendations for drug therapy thereby addressing differences before preferences influence drug use behavior. Past research has shown that the presence of insurance influences physicians’ selection of drug products. Theoretically, insurance coverage lowers the cost of health care goods and services resulting in increased demand for more or higher cost items by patients or physicians acting as agents of patients 266 (Newhouse). However, different types of insurance coverage may influence drug selection due to the amount of out-of-pocket expenditures patients must pay. For example, service benefit insurance plans require copayments that typically range from $0 to $15 or more. Conversely, indemnity coverage requires patients to pay the full amount out-of-pocket and then be reimbursed. Pauly showed. It is unknown whether different types of prescription drug insurance result in physicians selecting therapeutically similar drugs with different costs. Prescription drug expenditures have increased from x to y since 19xx. Several mechanisms have been employed to influence drug selection. Past research also has shown that physicians' knowledge of prices of services (list) Influence physicians' selection of health care goods and services. Although physicians' knowledge of absolute drug prices is poor, relative price knowledge has not been assessed and may be more important when selecting among alternative treatments. Research has shown that when physicians are provided relative price Information their selection of drug products Is altered. Knowledge of the effects of insurance coverage and price knowledge on physician selection of drug products is Important since new drug products are being introduced which are increasingly more expensive than existing therapies and changes In FDA policy toward DTCA have Increased pharmaceutical manufacturers' promotion of new, brand name products to patients. The role of Insurance coverage and price knowledge In drug selection for the elderly is Important since the population is aging, the elderly use more drug therapy and 267 elderly are being switched into managed care arrangements which offer drug coverage. Knowledge of the impacts o f insurance type and drug price knowledge on drug selection may help control costs in drug insurance programs for the elderly thus preventing the termination o f drug coverage in managed care health plans. The purpose of this study was to examine the impact of patient prescription drug insurance type and drug price knowledge on physician selection of therapeutically equivalent drug products. We wanted to determine whether insurance types that require different levels of patient payment for prescription drugs influenced physician selection of high cost and low drugs. Additionally, we examined whether providing prices of the drug products physicians were to select influences the selection of high and low cost products. Methods A 2x3 factorial experimental design was used to collect and analyze data. We developed 6 questionnaires describing the same clinical scenario and offering the same treatment options. The subject of the clinical scenario was a 65-year-old male patient with moderately severe chronic obstructive pulmonary disease (COPO). Following a description of the patients clinical presentation, a study participant was asked to select one of seven listed antibiotics to prescribe to treat the patient’s COPO. The questionnaires differed in prescription drug price 268 information (two levels) and patient prescription drug insurance type (three levels). The dependent variable was the selection of a high cost or low cost antibiotic from a list of seven antibiotics (three high cost, four low cost). The three high cost antibiotics were Cipro® (ciprofloxacin), Cedax® (ceftibuten) and Biaxin® (clarithromycin). The four low cost antibiotics were sulamethoxazole/trimethoprim, amoxicillin, erythromycin and tetracycline. The antibiotics were listed in the same order on each questionnaire. The brand name was listed first followed by its generic name in parentheses. The first independent variable was prescription drug price information that had two levels, drug price information provided and drug price information not provided. When drug price information was provided we listed the cost per ten- day course of treatment after the antibiotic name. We used the average wholesale price (AWP) listed in the Red Book for brand name antibiotics (Cipro, Cedax, and Biaxin). For antibiotics with generic equivalents (sulfamethoxazole/trimethoprim, amoxicillin, erythromycin, tetracycline) we used the Health Care Financing Administration (HCFA) Federal Financial Participation Upper Limit (FFP-UL) amounts. Price information did not include dispensing fees. The second independent variable was patient prescription drug insurance type. We categorized prescription drug insurance type into three types, Private Third Party (PTP), Indemnity and Uninsured, based on patient payment amount 269 when obtaining prescriptions. PTP represented service benefit prescription drug coverage requiring patients to pay a copayment for prescription drugs. The copayment amount for PTP insurance coverage in the scenario was $5.00 for all prescriptions. Nationally in 1998, the average copayment for brand name drugs was $9.96 and $5.53 for generic drugs (Navarro. 1998). Indemnity insurance required the patient to pay the full retail price and submit a claim to the insurer for reimbursement. Under indemnity insurance the ultimate cost to the patient is the time value of money delayed in the payment-reimbursement cycle and the percentage of the retail price not reimbursed. A patient who was uninsured was required to pay the full retail price of the prescription drug and receive no reimbursement. Other independent variables included respondent gender, years of experience, and solo/group practice. We also controlled for the health insurance mix of a respondent’s practice with five categorical variables. The health insurance types were Uninsured, Managed Care Private, Non-Managed Care Private, Medicare, and Medicaid. Each insurance variable had three levels, less than 25%, greater then 25% and not available (N/A). The sampling frame for this study was a list of internal medicine physicians practicing in a midwestem state obtained from a national mailing list company. We chose internal medicine physicians since a majority of elderly patients receive primary care from internal medicine physicians. Following sample size calculations, a total of nine hundred physicians were selected 270 randomly from the sampling frame and assigned randomly to one of the six experimental groups. There were a total of 150 physicians in each group. Each physician was sent a survey packet consisting of a cover letter introducing the study, a one-page survey form, a postage paid return envelope, and a lottery card for a $75.00 gift certificate for dinner for two at the restaurant of the winner’s choice. Survey packets were mailed in April. 1998 and a follow-up postcard was mailed one week after the initial mailing. Fisher’s exact tests were used to test for differences in respondent characteristics across the six groups. The influence of each independent variable on the selection of a high cost antibiotic was tested with univariate logistic regression. Contrasts between the coefficients for the insurance type variable were examined to determine significant differences between the three levels of insurance type. The significance of each variable was assessed using -2 log likelihood at a significance level of 0.10. We used multivariate logistic regression analysis to examine the influence of the independent variables on the selection of high cost or low cost antibiotics. The statistical significance of parameters was assessed using -2 log likelihood at a significance level of 0.10. Contrasts between estimated logistic regression coefficients for the insurance type variables were performed to test for differences in the probability of selecting a high cost or low cost drug product. The statistical significance of insurance type coefficients was tested at the 0.07 significance level due to multiple contrasts (Keppel. 19xx). 271 Results O f the 900 questionnaires mailed, 26 were returned as undeliverable. A total of 239 surveys were returned and one response was removed due to no information. The response rate was 27.2%. The number of respondents in each group ranged from 29 to 45. Table 1 contains a summary of the characteristics of respondents across the six groups. A majority of respondents were male (74.5%>, roughly 29% had been practicing for 14 or fewer years and a majority were practicing in a group practice. In terms of health insurance mix of patients, 92.5% of respondents saw less than 25% uninsured patients, and approximately 60% of respondents saw greater than 25% of both managed care and non-managed care patients. Approximately 80% of respondents saw less than 25% Medicaid patients and 84% of respondents saw more than 25% Medicare patients. The only difference in respondent characteristics across the six groups was in the proportion of managed care patients seen. Table 2 contains a summary of the number of respondents choosing expensive antibiotics categorized by price information shown and prescription drug insurance type. Overall, expensive antibiotics were chosen by 48.3% (n=115) of the respondents. When price information was shown, 33% of respondents chose an expensive antibiotic compared to 62.1% of respondents when price was not shown. An expensive antibiotic was chosen by 50.6% and 54.8% of respondents when the patient had private third party and indemnity 272 insurance, respectively. When the patient was uninsured for prescription drugs 37.3% of respondents chose expensive antibiotics. The number of respondents choosing expensive antibiotics ranged from 7 (24.1%) when patients were uninsured and prices were provided to 31 (72.1%) when patients had indemnity coverage and prices were not provided. Results of univariate logistic regression showed three variables significantly influenced the selection of high cost antibiotics. The percent of patients covered by Medicaid significantly influenced the selection of high cost antibiotics (-2 log likelihood = 4.97, 2 df, p < 0.10). Physicians with more than 25% of their patients covered by Medicaid were 0.5 times as likely to select a high cost antibiotic relative to physicians with less than 25% of their patients covered by Medicaid. Prescription drug price knowledge (-2 log likelihood = 19.97,1 df, p < 0.10) and prescription drug insurance type (-2 log likelihood = 4.87, 2 df, p < 0.10) significantly influenced the selection of high cost antibiotics. When price information was provided physicians were .31 times as likely to select a high cost antibiotic. In terms of prescription drug insurance type, physicians were 2.03 times more likely to select a high cost antibiotic when the patient had private third party insurance relative to patients without prescription drug insurance. Table 3 contains the results of multiple logistic regression analysis for the probability of choosing a high cost antibiotic. The table contains odds ratios for insurance type and price knowledge since these variables were the only factors 273 which significantly influenced the probability o f a physician selecting a high cost antibiotic. Contrasts for odds ratios for insurance type variable also are contained in the table. The insurance type variables were dummy coded and uninsured was the excluded insurance type in the logistic regression model. Therefore the odds ratios for the insurance type variables are interpreted relative to uninsured patients. When price information was provided physicians were 0.28 times as likely to select high cost antibiotics controlling for patient prescription drug insurance type. Physicians were 1.99 and 2.34 times more likely to select a high cost antibiotic when the patient had Indemnity and private third party insurance, respectively relative to the patient without prescription drug insurance. There was nor difference in the odds of a physicians selecting a high cost antibiotic when the patient had indemnity and private third party prescription drug insurance coverage. Since their was no difference between the insurance types in the odds of physicians choosing a high cost antibiotic we combined private third party insurance and indemnity insurance to create a new prescription drug insurance variable. This variable represented the presence of prescription drug insurance. Multivariate logistic regression results showed that both price knowledge (- 2 log likelihood = 19.97,1 df, p < 0.10) and presence of prescription drug insurance (- 2 log likelihood = 6.29, 1 df, p <0.10) significantly influenced the selection of high cost antibiotics. When price information was provided physicians were 0.28 times 274 as likely to select a high cost antibiotic, controlling for presence of prescription drug insurance. When the patient had prescription drug insurance a physician was 2.16 times more likely to select a high cost antibiotic controlling for price knowledge. 275 Discussion One objective of this study was to examine the influence physician’s knowledge of drug price had on the selection of high cost antibiotics. Our results suggest that providing price information to physicians significantly reduced the probability that a high cost antibiotic would be selected. Our results are consistent with past results showing the impact of price knowledge on physician decision making in general and drug therapy decisions in particular. Hux and Naylor found that physicians who were provided price information were .368 times as likely to select high cost antibiotics controlling for presence of drug insurance. We found that physicians were 0.28 times as likely to select high cost antibiotics when prices were provided controlling for presence of insurance. Our results provide validity to earlier results and suggest the importance of Informing physicians about the cost of drug products when physicians select antibiotics. The responsiveness of physicians to price information highlights the potential usefulness of this method in controlling prescription drug costs. Past research suggests physicians want and would use price information (Kotzan) but a critical question is how and by whom drug price information should be provided. Try to avoid basing prescribing decisions on cost alone. Should provide a balance between clinical information and cost information. Is there a role for public policy to include comparative prices in drug ads? It may be more effective to inform physicians of drug prices at a national level. Such a policy would have 276 implications for managed care firms attempting to control prescription drug costs. OUR work showing how price information is provided. The second objective of this study was to examine the influence of prescription drug insurance type on the selection of high cost antibiotics. Our results showed a strong influence of insurance on the selection of high cost drug products. When both insurance types were combined, a physician was 2.16 times more likely to select a high cost antibiotic for the patient with prescription drug insurance controlling for price information. Past research showed that physicians were 2.6 times more likely to choose a high cost antibiotic for a patient with prescription drug insurance controlling for price information (Hux and Naylor, 1994). The results are consistent with theory suggesting that insurance results in the demand for higher cost health care items by physicians acting as agents of patients (Newhouse, Pauly). We did not find a difference in the probability of a physician selecting a high cost antibiotic between indemnity and private third party prescription drug insurance types controlling for drug price information. We hypothesized that the probability of physicians selecting low cost antibiotics would be higher for the indemnity insured patient due to the large out of pocket costs associated with this type of insurance relative the private third party insurance type. One explanation for this result is that physicians may not understand how different prescription drug insurance types influence patient spending amounts for prescription drugs. Physicians may only be concemed with whether patients 277 have prescription drug insurance coverage, not the type of coverage. Although research has shown that physicians know the insurance status of their patients, little is known concerning physicians knowledge of out-of-pocket expenditures for patients. Insurers increasingly are relying on increased patient cost sharing to control costs in prescription drug programs . Increased cost sharing is hypothesized to control cost by shifting a certain dollar amount of the cost of a prescription to patients and by decreasing demand for higher cost drugs due to the burden of out-of-pocket expenditures. The lack of effect on physicians drug selection of increased cost chairing associated with indemnity insurance in this study highlights the role of the patient when using cost sharing to control costs. Past research has shown that the effect of increased cost sharing on controlling the demand for equally effective, higher cost health care items is questionable. Future research could examine elderly patients’ behaviors resulting from increased expenditures for drug therapy. For example research could examine the elderly ability and willingness to discuss therapy changes with physicians, nurses or pharmacists or the elderly reactions to the burden of the cost of therapy such as not obtaining medication and foregoing activities due to the cost of therapy. The effect of insurance on the selection of high cost antibiotics could be could be due to physicians' uncertainty about the benefits of the high cost drug products. Past research has shown that insurance impacts physician decision 278 making more when a procedure or drug therapy is more discretionary (benefits are less certain). Mort showed that patients with insurance coverage were more likely to receive more discretionary procedures relative to uninsured patients. Lessler and Avins (1992) found that physicians were more likely to choose a more expensive drug despite evidence of its superiority for patients with insurance relative to uninsured patients. Future research should examine the role of insurance on drug selection when the benefits of a drug therapy are known with various levels of certainty. Discrepancies in access to beneficial therapies across insurance types would be cause for public policy solutions. The lack of effect between indemnity and private third party insurance could be due to the nature of demand for antibiotics. Antibiotics typically are indicated for acute condition where demand is more inelastic and physicians and patients are more concemed with producing an outcome regardless of cost. Contrast this with a chronic condition in which patients may be more involved in the drug selection process, weighing benefits of therapies with the economic burden of out-of-pocket costs, resulting in the selection of lower cost drug products. Future research should examine whether discrepancies between insurance types such as indemnity and private third party exist for conditions where demand may be more elastic. The results for groups of physicians which were not shown drug prices are likely most reflective of current prescribing for the elderly. A common problem for patients and insurers is ensuring that physicians will choose drugs that are the 279 most appropriate therapy in terms of clinical effectiveness and cost. The high cost antibiotic products in this study are not clinically better at treating COPD than the low cost antibiotics . It appears that physicians may act as economic agents of their uninsured patients, selecting drug products which result in low out of pocket costs for uninsured patients. Surprisingly, over 47% of the physicians choose high cost antibiotics for uninsured patients. However, the selection of equally effective, high cost antibiotics for insured patients imply that physicians do not act as economic agents for a patient's insurers. Our results hint at the impact of drug advertising and the role of insurance coverage in promoting use of high cost drug products. Future research should examine therapeutic categories of drugs that frequently are used by the elderly to examine the scope of these two effects. 280 T ab le 1 Characteristics o f Respondents Variable B D E Total G en der Male 30 35 34 26 29 24 178 Female 13 10 9 12 8 5 57 N/A 0 0 0 3 1 0 4 Total 43 45 43 41 38 29 Fisher’s Exact Test p = 0.38 Years of Experience 1-14 12 15 14 9 11 7 68 (28.5) (33.3) 0 0 0 0 15-23 12 14 11 11 8 6 62 >23 10 8 10 11 10 12 61 N/A 8 8 8 10 9 4 47 Total 42 45 43 41 38 29 238 Fisher’s Exact Test p - 0.93 281 Practice Environment Group 20 17 19 21 9 16 Solo 14 22 16 13 20 9 N/A 9 5 8 7 9 4 Fisher’s Exact Test p = 0.25 Health Insurance Mix Uninsured Patients <25% 39 39 41 40 36 26 >25% 3 3 2 0 1 1 N/A 1 2 0 1 1 2 Fisher's Exact Test p = 0.68 Managed Care Patients <25% 12 14 15 19 20 3 >25% 30 28 28 21 17 24 N/A 1 2 0 1 1 2 Total 43 44 43 41 38 29 238 Fisher’s Exact T est p = 0.01 282 Non-Managed Care Insured Patients <25% 16 16 18 17 8 12 >25% 26 26 25 23 29 15 N/A 1 2 0 1 1 2 Fisher’s Exact Test p = 0.48 Medicaid Patients <25% 34 33 38 33 31 22 (79.1) >25% a 9 5 7 6 5 (18.6) N/A 1 2 0 1 1 2 (2-3) Fisher’s Exact Test p = 0.90 Medicare Patients <25% 7 5 8 4 4 2 >25% 35 37 35 36 33 25 N/A 1 2 0 1 1 2 Fisher’s Exact Test p = 0.81 283 A= Drug prices not provided, Indemnity prescription drug insurance B= Drug prices provided , Indemnity prescription drug insurance C= Drug prices not provided . Private third Party prescription drug insurance D= Drug prices provided, Private Third Party prescription drug insurance E= Drug prices not provided , no prescription drug insurance F= Drug prices provided, no prescription drug insurance 284 T a b le 2 Number (percent) of Physicians Selecting Expensive Antibiotics by Price Knowledge and Prescription Drug Insurance Type Prescription Drug Insurance Type Private Third Partv Indem nitv Uninsured Total Prices Shown 16 15 7 38 (36.4) (36.6) (24.1) (33.3) Prices Not Shown 28 31 18 77 (65.1) (72.1) (47.4) (62.1) Total 44 46 25 115 (50.6) (54.8) (37.3) (48.3) 285 Table 3 Results of Multivariate Logistic Regression Model Odds Ratio of Choosing High Variable Cost Antibiotics Constant 1-63 (se = -1881)a Drug Prices Provided 1 .28 (se = .2786)a Insurance type2 PTP 2.34 (se = .3532)b Indemnity 1.99(se = .3497)b Uninsured excluded Insurance type contrasts Odds ratio difference Indemnity - PTP .85 (se = .3219)b Note: All variables are dummy coded; = 1 if they possess the characteristics, and = 0 otherwise. Confidence intervals are in parentheses. 1 -2 log likelihood = 21.65, 1 df, p < 0.10 2 -2 log likelihood = 6.54, 2 df, p < 0.10 a 90% confidence interval b 93.3% confidence interval due to multiple contrasts 286 APPENDIX ill Genetic variations in nitric oxide synthase isoforms: Evidence, significance and therapeutic implications This manuscript has been accepted for publication in Pharmaceutical News, 2000 (in press). The authors of the manuscript are Suvara Kimnite Wattanapitayakul\ Anthony P. Young^’^, and John Anthony Bauer^ Division of Pharmacology, College of Pharmacy(1), and OSU Neurobiotechnology Center(2), The Ohio State University, Columbus OH 43210 287 Introduction Nitric oxide (NO) is now recognized as an important participant in a diverse array of physiological and cellular processes, including local regulation of vascular tone, neuronal signaling, immune defenses, and many others. NO is a simple molecule with limited stability in biological settings (t1/2 of several seconds) and is known to participate in a variety of chemical reactions with metals, thiols and other reactive oxygen species. Given the numerous physiological roles for NO and its rapid reaction and inactivation in cellular systems, strict local control of NO production is critical for selective actions. Important components of cell-specific production of NO are the expression and regulation of three discrete gene products, the nitric oxide synthases (see Table 1) In general, these three NOS isoforms are similar in primary structure (50-90% sequence identity, depending on the species) and they conduct the same biochemical reaction, namely the oxido-reduction of arginine to liberate NO and citrulline. However, genetic variations within these three isoforms have been detected and these variations may affect the normal physiological roles, cellular distributions, and levels of expression of the NOS isoforms. Furthermore, several very recent studies suggest that genetic polymorphisms of each of the NOS isoforms occur in humans, and that these molecular variations may play a role in disease risk. The goals of this brief manuscript are to review the current evidence regarding genetic polymorphisms of NOS isoforms and their potential significance in disease incidence and therapeutics. 288 Involvement of NOS isoforms In disease: too little or too much NO? Since the identification of the NOS isoforms in the late 1980’s many investigations have helped to define their roles in normal physiology and participation in disease. Studies using ‘knock-out mice have proven most illuminating. In these transgenic models genetic techniques are exploited to disrupt (and inactivate) individual NOS genes. Shown in Table 2 is a brief summary of physiological characteristics observed in mice that fail to express each of the NOS isoforms due to their targeted disruption. While knockout models have certain liabilities, these animals have been useful to illustrate physiological roles of individual NOS isoforms and suggest potential disease consequences of reduced expression in humans. General characteristics of neuronal NOS (NOS1) knockout mice include deficits in learning and memory, impaired motor coordination, and other behavioral alterations [1]. Important urinary bladder structural changes have also been observed illustrating an important role of this isoform in the genitourinary tract as well as the central nervous system [2]. Primary changes in NOS2 knock-outs involve the immune system and pathogen resistance. These animals are highly susceptible to even minor bacterial or viral infections, demonstrating an important role of NOS2 in host defense reactions. The NOS3 deficit is associated with important changes in cardiovascular physiology. The absence of this isoform, primarily present in vascular endothelium, leads to significant elevation of systemic blood pressure. It is interesting that targeted disruption of a NOS isoform tends to produce both negative and positive physiological outcomes. For example, NOS1 knockout mice have several characteristics consistent with neuronal impairment, but also show markedly attenuated responses to cerebral ischemia [3] and resistance to NMDA [4], and MPTP 289 [5] elicited neurontoxicity. Similarly. NOS2 knockouts are more vulnerable to pathogen infection but have been shown in some studies to be less likely to develop hypotensive shock during sepsis. This phenomenon complicates some aspects of transgenic animal investigations and illustrates the complexities of NO in physiology. Thus, the actions of NO in vivo may be govemed not only by production capabilities but also by the setting and chemical environment in which it is formed. Human NOS genetic polymorphisms Since the mid 1990’s more than 50 published evaluations of NOS isofdrm polymorphisms have been described. These studies have typically employed a candidate gene approach, in which patients are genotyped for a specific variation in an NOS gene sequence using PCR reaction from blood leukocyte genomic DNA. Patient genotypes are then related to the incidence of a disease and/or outcome, to test for statistically significant associations (parametric and/or nonparametric linkage analyses). A summary of the various investigations published to date is provided in Table 3, and further discussion is provided below. NOS1 : First identified in neurons, this constitutive isoform is now known to exist in many non-neuronal cells. Recent studies suggest that excessive NO production from NOS1 may play a role in a variety of neurological disorders, leading to neuronal injury in the central and peripheral nervous systems. NOS1 is co-localized with the Huntington- associated protein (HAP1), and may also play a role in Duchenne muscular dystrophy [6,7]. Dinucleotide repeat elements of variable length have been identified within the 290 untranslated regions of human N0S1 mRNA [8]. NOS1 splice variants have also been identified [9,10]. Finally. NOS1 mRNA is transcribed from a large number of alternative promoters [11] giving rise to N0S1 mRNAs with at least nine different 5’ terminal exons [12]. None of these elements have been linked to human disease. Moreover, the biologic rationale for encoding the NOS1 gene with this enormous structural and regulatory complexity remains to be elucidated. Using linkage analysis Chung et al. [13] recently identified two intragenic polymorphisms (NOSIa and NOS 1b) as a susceptibility locus for infantile pyloric stenosis, a condition leading to hypertrophy of pyloric smooth muscle and gastric outflow obstruction. Furthermore, the diagnosis of asthma has been linked to the chromosomal region that contains the NOS1 gene (12q24.2), and an association between an NOS1 gene polymorphism and asthma has been reported [14]. Importantly, highly significant differences in N0S1 allelic frequencies have been observed between American-Caucasian and African-American populations. This suggests that differences in study populations could be misinterpreted if ethnic diversity is not carefully considered and accounted for. NOS2: This isoform was first identified in macrophage cells and is commonly known as the “inducible” isofdrm (although the other isoforms are now known to upregulated with various stimuli). The primary inducers of N0S2 expression are cytokines, including IL1, TNF, and others. The NOS2 protein is tightly bound to calmodulin and is not calcium dependent, and thus is a high-capacity source of NO production. Interestingly, polymorphic variations of the NOS2 gene has thus far been 291 identified only in its promoter region and this may have consequences in induction capabilities in vivo. For example, Kun et al. [15] recently studied the recurrence rate and severity of malarial infection in Gabon, a geographical area hyperendemic for this infection where survival still is largely dependent on a child innate immune response. A matched-pair case control study observed that heterozygotes with a single point mutation of the NOS2 promoter (-969, G C) was statistically associated with a more mild form of infection and a decreased risk of reinfection. These authors speculate that this mutation mediates an enhanced malarial defense (perhaps enhanced induction), but this has not been experimentally tested. A statistically significant association was also observed between malarial severity and the frequency of sickle-cell trait, but not for reinfection rates. Relative to the constitutively expressed isoforms, genetic variation in NOS2 may be easier to associate with disease risk. This inducible and high-capacity isoform is primarily regulated by its expression rather than receptor mediated signal transduction and intracellular calcium concentrations. Thus, a change in protein sequence and/or structure may lead to more obvious changes in function. In an elegant series of investigations Warpeha et al. [16] have recently evaluated the role of a NOS2 polymorphism in diabetic retinopathy. This condition is associated with initial local increases in retinal blood flow, endothelial cell dysfunction, and subsequent structural loss of capillaries. Type 1 and 2 diabetics were evaluated for associations with individual repeat alleles of a polymorphic 5-nucleotide repeat located in the 5' promoter region of the N0S2 gene. A statistically significant association was observed, in that the risk of retinopathy in a diabetic patient (either type 1 or 2) carrying the 14 repeat allele 292 was estimated to have 0.21 times the risk of retinopathy compared to the wild type gene. They also had fewer renal and cardiovascular complications. These investigators further supported this concept by conducting in vitro cell experiments in which the variant gene was transfected into cells (human colon carcinoma line DLD-1) and induction capabilities were assessed using cytokines in the absence/presence of high glucose. Significant enhancement of NOS induction was observed with the variant form vs. wild-type form of the gene, under all experimental conditions. Similar to the conclusions of the anti-malarial report above, these authors concluded that polymorphic alteration of the NOS2 promoter (in this case a 5-nucleotide repeat) caused an augmented induction capability, and that this variation afforded enhanced protection against disease. Interestingly, these same investigators have shown no association of N0S3 variants and diabetic retinopathy, suggesting an isoform-specific genetic advantage in this condition. NOS3: This isoform has been the most frequently investigated with respect to genetic variations and risk of disease, and the available results are conflicting and often confusing. Commonly described as the constitutive isofdrm in vascular endothelium, it also is present in cardiac tissue (both endothelial cells and cardiac myocytes) and other cell types. All of the polymorphism investigations thus far with this isoform have been confined to cardiovascular disease risk. As summarized in Table 3, the data available thus far are divergent and often contradictory. So far 5 specific polymorphisms have been identified and tested for association with various cardiovascular conditions, these include single nucleotide alterations as well as variable tandem nucleotide repeats 293 (VTNRs, see Table 3). Miyamoto et al. [17] conducted a large scale study In nearly 900 subjects, simultaneously evaluating these 5 different NOS3 genetic variations with respect to Incidence of essential hypertension In Japanese subjects. The frequency of a missense variant Glu298Asp (In exon 7) was found to have statistically significant disequilibrium in hypertensive vs. normotenslve subjects, suggesting an association between genotype and disease Incidence. No other genetic variant was found to be different in the two populations. These Investigators have also demonstrated an association of this missense variant with unstable vasospastic angina and myocardial infarction. In contrast, several similar studies have demonstrated no association to various disease risk for this polymorphism, and/or suggest other genetic variants are relevant. One important study by Tsukada [18] attempted to relate NOS3 gene polymorphism to plasma NO byproducts In 413 healthy subjects. They investigated the VNTR variant (four or five 27-bp repeats, a and b respectively) with respect to concentration of inorganic nitrate and nitrite (NOx) In plasma. Slight but statistically significant reductions (10-20%) in mean plasma NOx concentrations were observed in a-subtype homozygotes, and a statistically significant association between the alleles of the VNTR polymorphisms and plasma NOx levels. Although many aspects complicate these findings, and the magnitude of NO metabolite differences are not great, this has been one of only a few attempts to relate NOS Isofdrm genetic variations to actual protein functional characteristics In vivo (see further discussion of this point below). Another challenge in associating NOS3 genetic variations with cardiovascular conditions is the recognition of several other candidate genes already Identified as 294 contributors. These include angiotensinogen. angiotensin-converting enzyme, angiotensin II type 1 receptor, and the atrial naturetic peptide genes [17]. It has been suggested that genetic variation within an individual gene has little effect on blood pressure unless adjoined with other abnormal genes or with environmental factors such as sodium in diet [19]. Since most forms of cardiovascular disease are clearly multi factorial, further studies to test for relationships among these diverse genetic polymorphisms may be more valuable and insightful than studying any one of them alone. Important considerations and therapeutic implications Given the importance of NOS isoforms in various normal physiological processes, it is plausible to postulate that genetic polymorphisms would play a role in the risk or outcome of certain diseases. Despite this rationale, only about half of the published reports have demonstrated an association between a NOS genotype and disease risk (see Table 3). In some cases the primary findings of some studies are inconsistent or directly contradictory with others. Some reports suggest important relationships, but why are the published results of NOS genotypes and disease so inconsistent? One explanation is related to the typical study designs employed in most studies. The candidate gene approach for mapping complex human diseases requires some important assumptions and conclusions rely upon adequate sample sizes and power to minimize type 1 and 2 statistical errors. As recently described by Speer [20], the assumptions involved merit consideration. For linkage disequilibrium to be detectable, the disease phenotype in 295 unrelated individuals must have arisen from a single mutation in a common ancestor long ago. Any other mutation to the same gene will not show the same marker allele and would tend to dilute the effects of a single major mutation (if one exists). Since several well-defined disease genes have already been shown to have more than one mutation (e.g., cystic fibrosis, breast cancer), it is obvious that the linkage disequilibrium approach is not universally appropriate. Furthermore, the study of populations with diverse ethnicity etc. (a common situation in US trials) greatly reduces the value of linkage analysis. Several studies have described NOS genotype distributions that are different in healthy individuals with various ethnic backgrounds. Including a mixture of diverse ethnicity in a gene-disease linkage analysis reduces statistical power and may lead investigators to erroneous conclusions, purely due to unaccounted for population differences. The total sample size involved in statistical testing is also obviously important for detecting genotype-disease associations. A sample size of at least 194 is needed to approach statistical significance for the linkage of disease and marker loci, assuming a 2.9-fold increase in disease risk and 80% power [20]. Published studies of NOS genes have used populations ranging from 74 to 1460, but few studies defined the actual statistical power of their investigations. Separate from statistical issues, biochemical and physiological issues may explain the divergent conclusions of NOS genotyping and disease. At best linkage analysis can suggest statistical associations, but in most cases functional consequences of NOS polymorphisms remain to be identified. Of note is the fact that very few studies have directly documented the consequences of a mutated NOS gene 296 with respect to actual performance of the protein product, in one case where a VNTR variant of NOS3 was functionally assessed, it was associated with only a 10-20% difference in plasma NOx (an indirect marker of NOS activity). More direct comparisons of NOS isoform enzyme characteristics from the identified polymorphisms would help. Particularly studies of enzyme structure, function, and intracellular distributions would provide mechanistic insight and enhance the credibility o f statistical associations with disease risk. This approach has been successful in identifying the consequences of a NOS2 polymorphism associated with diabetic retinopathy (see above). It is now recognized that in physiologic settings the activities of NOS isoforms are influenced by many variables, including availability of arginine substrate, biopterin cofactors, the presence of calcium/calmodulin, co-expression of other NOS isoforms, etc. Furthermore the consequences of NO production, when formed, is dependent upon the environmental conditions involved. Interaction with superoxide anion reduces the bioavailability of NO and generates peroxynitrite, a powerful oxidizing species that may participate in a wide array of conditions ranging from heart disease to neuronal degeneration. These complex and multi-level controllers suggest that variations of NOS isoform genes is not the only variable worthy of investigation. Rather than singular study of NOS polymorphisms, perhaps relating NOS genotypes with other NO modulating pathways will provide better understanding of genes and disease risk. For example, Adachi and Wang [21] have recently described an interaction of a NOS3 polymorphism (the VNTR 4a allele) with plasma levels of extracellular superoxide dismutase (EC- SOD), a principal scavenger of superoxide in the extracellular space. Low EC-SOD levels were related to the NOS3 variant allele. Molecular genetic studies have shown 297 that a single base substitution in the EC-SOD gene (R213G) causes greatly increased plasma levels of this enzyme due to its dissociation from heparin. Adachi and Wang observed a highly significant association between plasma EC-SOD and the NOS3 polymorphism in an Australian Caucasian population. These studies suggest that Important interactions, either genetic or environmental, may co-regulate these interacting enzymes in humans, further studies relating these phenomena to disease risks are awaited. Are there therapeutic implications from these NOS genotype investigations? Perhaps a few more years are required to determine the answer to this question, but exciting potential certainly exists. Many of the candidate gene studies described thus far have focused on complex and multifactorial diseases, many of which already have other defined genetic influences. Therefore, further investigations of NOS genetic variations in combination with other relevant genes will likely provide more mechanistic insight about disease processes. Additional identification of how the genetic variation is translated to altered enzyme function will also be informative. If enzyme performance is reduced, perhaps something as simple as dietary arginine supplementation may have value. This approach has already shown some value in treatment of coronary artery disease, perhaps it would work best in patients with specific genotypes. Alternatively, selective gene therapies may have value in certain well-defined conditions. Summary and conclusions The NOS enzymes are now recognized as an important family of isoforms with important and varied physiological roles. Molecular genetics have identified 298 polymorphisms for each of the NOS genes. Although some reports have recently associated specific NOS isoform genotypes with increased risk o f specific disease states, in many cases findings are equivocal with respect to the significance of NOS genotypes and disease risk. Despite the less than unanimous findings thus far, this field of functional genomics is rapidly emerging and will provide new and exciting information. Further study of the relationships of NOS genetic variations and disease, and functional consequences of such variations, will provide new insight into disease mechanisms and provide new opportunities for therapy. 299 TABLE 1. Characteristics off human NOS isofforms Isofonn MW Chromosoma Gana Expraaaionai Calcium Call (kOa) Localization Siza Ragulation dapandanca prototype [Gene Structura] (kb) NOS1 (nNOS) 160 12q24.2 >200 constitutive but yes neurons [29 exons.28 introns] highly regulated NOS2 (iNOS) 130 17q11.2-q12 37 inducible by no macrophages, [26 exons.25 introns] cytokines hepatocytes NOS3 (ecNOS) 135 7q35-q36 21 constitutive but yes endothelial [26 exons,25 introns] highly regulated cells 300 TABLE 2. Evidence of physiologic changes and disease susceptibility in NOS knock-out animals Animal Model Physiological Changes and Responses to Diseases References NOS1 knock-out mice Resistance to cerebral ischemia 3 Reduction in dendrite numbers 2 2 Resistance to NMDA-induced neurotoxicity in vitro and in vivo 4 2 3 Resistance to MPTP-induced neurotoxicity in the brain 5 Enhanced aggression in male mice 1 Reduced aggression in female mice 2 4 Deficiency in nocturnal motor coordination 2 5 Altered urinary bladder structure 2 Pyloric sphincter hypertrophy, enlarged stomach 2 6 NOS2 knock-out mice Increased susceptibili^to bacterial infection 2 7 increased susceptibilify to tuberculosis 28 Susceptible to experimental autoimmune encephalitis 2 9 Susceptible to endotoxin-induced uveitis 3 0 Increased mortality in sepsis (conflicting data with others) 31 (32) Neuroprotecthre after endogenous traumatic brain injury 33 NOS3 knock-out mice Hypertension and decreased heart rate 3 4 .3 5 Increased plasma renin activity Lack of response to Ach and calcium ionophore Increased sensitivity to phenylephrine, serotonin, and nitroglycerin Impaired ovulation and oocyte meiotic maturation 3 6 301 TABLE 3. NOS polymorphisms and associations with human disease G enetic V a ria n t 1 Location 1 Condition Evaluated 1 PoiNilation IStadslieal Association 1 RafiwencM NOS1 (CA)nand (AAT)„ exon 29 Healthy suttjects African-American and Yes 37 Caucasian (Différent homozygote distribution between ethnic groups) (TG)„ exon 29 Hypertension Japanese No 38 NOS1a/b 5" flanking Multiple Sclerosis Swedish No 39 region o f exon 1 NOS1 12q14-24.2 Asthma susceptibility loci in African -American no 14 chromosome ethnic diverse populations Caucasians yes linkage region Hispanic yes N O SIa/b 5' flanking In^ntile Pyloric Stenosis Caucasian Yes 13 region o f exon 1 NOS2 (CCTTT)„ promotor region Reduced prevalence of diabetic Caucasian Yes 16 retinopathy G-“ -> C promotor region Less susceptible to Malaria African Yes 15 G C promotor region Chagas’ disease Peruvian No 40 NOS2 17p11.lKt11^ Asthma susceptibility loci in African -American yes 14 chromosome ethnic diverse population Caucasians no linkage region Hispanic no NOS3 VNTR. (CA)„ intron 13.23 Diabetic Retinopathy Caucasian No 16 intron 13 Migraine Caucasian No 41 Hypertension Caucasian No 42 Japanese Yes 43 Coronary artery disease Caucasian Yes 44 A ^-> C intron 18 Hypertension Caucasian No 42 Japanese No 17 Caucasian No 45 G '°-> T intron 23 Hypertension Japanese No 17 VNTR, (27 bp)« intron 4 Plasma Nitric Oxide and Nitric Caucasian Y es* 46 Oxide Products Japanese Yes 18 Plasma EC-SOD Caucasian Yes 21 Hypertension Japanese No 7 Yes 47 Coronary artery disease Caucasian No 48 Caucasian Y e s f 46 Japanese No 49.50 Myocardial Infarction African-American Yes 51 Japanese Yes 52.53 Primary glomeruonephritis Caucasian No 54 IgA nehpropathy Japanese No 55 End-staoe renal disease Japanese Yes 56 Ischemic cerebrovascular Japanese No 57 disease/ischemic stroke Turkish Yes 58 Venous Thromboembolism African-American Yes 51 Deep Vein Thromtx)sis Turkish No 58 Glu -» Asp298 exon 7 Coronary artery disease Caucasian Yes 59 Japanese Yes 60 Hypertension Caucasian No 45 Japanese No 61 Japanese Yes 17 Myocardial Inferction Caucasian No 62 Caucasian Yes 59 Japanese No 52 Japanese Yes 63 Heart Failure Caucasian Yes* 64 Ischemic cerebrovascular Caucasian No 65,66 diseaseTisctwmic stroke iiivcioc idouuiioiiip, I diiiuivc;i*vicpeiiudii a^uvficiuuii, vi^ i r\, vtfiiauio iciiiuciii icpeoia, v i^irx , (2 7 bp >4 and VNTR. 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