Effects of Rosiglitazone on Nitroglycerin

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EFFECTS OF ROSIGLITAZONE ON NITROGLYCERIN-

INDUCED ENDOTHELIAL DYSFUNCTION

Kumar Perampaladas B.Sc.

A thesis submitted in conformity with the requirements for the degree of

Master of Science

Graduate Department of Pharmacology and Toxicology

University of Toronto

Supervisor: John D. Parker, MD, FRCP(C)

Effects of Rosiglitazone on Nitroglycerin-Induced Endothelial Dysfunction

Kumar Perampaladas, Master of Science, 2010

Graduate Department of Pharmacology and Toxicology, University of Toronto

ABSTRACT

Sustained nitroglycerin (GTN) therapy impairs endothelial function in healthy volunteers and patients with cardiovascular disease, caused by an increase in vascular oxidative stress. This study aims to estimate the effect of rosiglitazone on vascular endothelial function in healthy volunteers continuously dosed to transdermal GTN (0.6mg/hr) for 7 days. To assess endothelial function, forearm blood flow was measured by venous occlusion strain-gauge plethysmography in response to intra-brachial infusions of acetylcholine. GTN-treated subjects experienced significant attenuation of endothelium-dependent responses to acetylcholine (p<0.05; compared to placebo), but was reversed with vitamin C

(p=ns; compared to placebo). Endothelium-dependent responses to acetylcholine were blunted in groups randomized to rosiglitazone alone (p<0.05; compared to placebo) and rosiglitazone + GTN (p<0.05 compared to placebo). Interestingly, this effect was not modified by vitamin C. In conclusion, rosiglitazone impairs endothelial function and concurrent therapy with rosiglitazone does not attenuate the adverse effects of transdermal GTN on the vasculature.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my supervisor Dr. John Parker for his continuous support during my Master’s Program. Dr. Parker has always given me the space, support, and guidance, needed to explore ideas and take on ambitious projects. The successful completion of the current study, the initiation of a pilot project looking at alternative tools to identify and quantify biomarkers, is in part to Dr. Parker’s unrelenting support. He has instilled in me the values and persistence needed in accomplishing goals that are both research and professional oriented. These past years have truly been an informative and exciting time in my life. I’m truly grateful for the opportunity to study under the guidance of Dr. John Parker.

I would also like to thank Sue Kelly, Andrew Liuni, Mary Clare DeLuca, Wilson

Kwong, Becky Pipes, Diane Locke, Diana Vasiliu, Tom Benson, Wilson Chan,

Drs. George Thomas, Gary Newton, Susanna Mak, Alan Barolet, Amar Uxa,

Rajesh Dhopeshwarkar, Justin Mariani, and Tommaso Gori, for their support and assistance in completing this study.

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TABLE OF CONTENTS

TITLE i ABSTRACT ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS iv LIST OF TABLES vi LIST OF FIGURES vii ABBREVIATIONS viii

CHAPTER 1.0 INTRODUCTION 1 1.1 Problem and Purpose of the Study 2 1.2 Thiazolidinediones 4 1.3 Rationale and Objectives of the Present Study 6 1.3.1 Nitrate-Induced Model of endothelial dysfunction 6 1.3.2 Hypothesis 9 1.4 Literature Review: Fundamentals of Nitrate Tolerance 10 1.4.1 Nitric Oxide Synthesis 10 1.4.2 Nitroglycerin and Organic Nitrates 12 1.4.3 Nitrate Tolerance and Proposed Mechanisms 15 1.4.4 Neurohormonal Activation 16 1.4.5 Plasma Volume Expansion 17 1.4.6 Vascular Free Radical Hypothesis 18 1.4.7 GTN-Induced Abnormalities in the Endothelium 20 1.4.8 Abnormalities in Nitrate Biotransformation 22 1.4.9 Intermittent Nitrate Dosing 25

iv 2.0 METHODOLOGY 27 2.1 Assessment of Endothelial Function 27 2.1.1 Plethysmography 27 2.1.2 Flow-mediated Dilation 29

2.2 Methods 30 2.2.1 Study Population 31 2.2.2 Study Design 32 2.2.3 Vascular Function Procedures 33 2.2.4 Experimental Sessions 36 2.2.5 Side Effects and Risks of the Study 38 2.2.6 Statistical Analysis 39

3.0 RESULTS 40 3.1 Responses to Blood Pressure and Heart Rate 40 3.2 Effect of Acetylcholine on Endothelium-dependent vasodilation 40 3.3 Effect of Vitamin C on Endothelium-dependent vasodilation 41 3.4 Tables and Figures 43

4.0 DISCUSSION 54 4.1 Nitrate-Induced Endothelial dysfunction 55 4.2 Rosiglitazone-Induced Endothelial dysfunction 55 4.3 Limitations of the Current Study 59

5.0 CONCLUSIONS 63 6.0 REFERNCES 65

LIST OF TABLES

3.0 RESULTS

Table 1: The effect of GTN on blood pressure and heart rate on healthy volunteers at baseline, 3 hours, and 7 days after treatment.

Table 2: The effect of intra-arterial infusions of acetylcholine and vitamin C after 7 days of treatment with placebo, rosiglitazone, GTN, or both rosiglitazone and GTN.

LIST OF FIGURES

3.0 RESULTS

Figure 1 : Study protocol outlining the sequence of events for this study.

Figure 2: Schematic of experimental sessions conducting vasomotor responses using venous occlusion plethysmography.

Figure 3: Graphic presentation of plethysmographic curve in response to acetylcholine.

Figure 4: Setup of forearm blood flow assessments, including placement of strain-gauge.

Figure 5: Baseline (saline infusion) forearm blood flow ratios for subjects randomized to placebo, rosiglitazone, GTN, and both GTN + rosiglitazone

Figure 6: The effect of acetylcholine on forearm blood flow in subjects randomized to placebo, rosiglitazone, GTN, and both GTN + rosiglitazone.

Figure 7: Saline re-control to ensure forearm blood flows returned to baseline after acetylcholine infusions.

Figure 8: The effect of vitamin C (co-infused with acetylcholine) on forearm blood flows in subjects randomized to placebo, rosiglitazone, GTN, and both GTN + rosiglitazone.

vii ABBREVIATIONS

ADMA Asymmetric Dimethylarginine ANOVA Analysis of Variance GTN Glyceryl trinitrate, Nitroglycerin NOS Nitric Oxide Synthase ISDN Isosorbide dinitrate ISMN Isosorbide Mononitrate NAD/NADPH Nicotinamide Adenine Dinucleotide Phosphate PPAR-γ Peroxisome Proliferator Activated Receptor gamma

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1.0 INTRODUCTION

1 1.0 INTRODUCTION

1.1 Problem and Purpose of the study

The endothelium is a complex organ influencing the delivery of and flow of nutrients that include proteins (growth factors, coagulation and anti-coagulation factors), lipid-transporting particles, metabolites, and hormones.(1-3) While contributions to the regulation of vasomotor tone involve production of mediators such as prostacyclin and endothelium-derived hyperpolarizing factor, the vasodilatory actions of nitric oxide (NO) is believed to be the most potent mediator in regulatory blood flow.(1-3) A healthy endothelium produces NO continuously and is responsible for vascular homeostasis by regulating important physiological functions such as platelet and leukocyte adherence, thrombolysis, inflammation, blood flow, and vascular smooth muscle cell proliferation and migration. (1;3) In contrast, a reduction in the bioavailability of NO results in endothelial dysfunction, which has been implicated in the underlying pathophysiology of several cardiovascular diseases including atherosclerosis, hypertension, heart failure, and stroke.(4-6) Reduced NO bioavailability, brought on by a reduced capability of the endothelium to produce NO or by an increased inactivation of NO, leads to disruption of NO-mediated signaling pathways that regulate homeostasis.(7;8)

Organic nitrates such as nitroglycerin (glyceryl trinitrate; GTN) have long been used as a means to improve NO bioavailability by acting as NO donors, and are routinely used in the clinical management of angina pectoris, myocardial

2 infarction, and heart failure.(9;10) When administered acutely, GTN provides rapid hemodynamic and anti-ischemic benefits, but their clinical utility rapidly diminishes with continuous dosing. This is observed as a loss of their hemodynamic and anti-ischemic effects, a phenomenon termed ‘nitrate tolerance.’(10;11) Sustained nitrate therapy has been documented to cause endothelial dysfunction in animals and patients with angina pectoris and heart failure.(12-15) A plethora of evidence has demonstrated that the etiology of nitrate tolerance is multifactorial,(16;17) with an increase in vascular superoxide anions having a prominent role.(17;18) Sources of reactive oxygen species production may include xanthine and nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidases, nitric oxide synthase (NOS), cyclooxygenase, and the mitochondrial respiratory chain.(18) Increased activity of these complexes by GTN may react with nitrate-derived NO, leading to increased NO clearance, dysfunction of the

NOS complex, and inhibition of mitochondrial aldehyde dehydrogenase – the enzyme responsible for GTN bioactivation. (16;17)

Rosiglitazone is an insulin sensitizing agent that has been demonstrated to improve vascular function in patients with diabetes mellitus and metabolic syndrome.(19-21) Rosiglitazone improves glycemic control by modulating transcription factors involved in the regulation of glucose and lipid metabolism,(22) and has been widely used in the management of type 2 diabetes mellitus.(22;23)

Beyond effect on insulin sensitivity, rosiglitazone has been documented to have anti-oxidative properties in cardiovascular animal models, a result that indicates

3 rosiglitazone may act independent of its effect on glycemic control.(24-27) Since oxidative stress have an important role in the development of nitrate tolerance and nitrate-induced endothelial dysfunction,(16;17) we explored whether the potential beneficial effects of rosiglitazone on endothelial function would extend to healthy volunteers randomized to continuous GTN treatment.

1.2 Thiazolidinediones

Thiazolidinediones are a class of drugs that are commonly used for improving insulin sensitivity in patients with type 2 diabetes mellitus.(28) Thiazolidinediones are agonists of the peroxisome proliferator-activated receptor-gamma (PPAR-γ), a class of nuclear hormone receptors that are capable of regulating gene expression related to glucose homeostasis, inflammatory responses, and adipogenesis. (28-31) PPAR-γ is predominately expressed in adipose tissue and skeletal muscle, the main site of insulin sensitization. (31) Currently available treatments for diabetes mellitus include sulfonylureas and biguanides, which work by promoting pancreatic insulin secretion and reduce hepatic glucose production respectively. (31;32) But thiazolidinediones act by improving insulin sensitivity and improve glucose uptake in peripheral tissues and reducing hepatic glucose output.(23;33-36)

The emerging interest in rosiglitazone – a member of the family of thiazolidinediones - on endothelial function is motivated by recent clinical observations that therapy with rosiglitazone has been shown to improve vascular

4 function in patients with metabolic syndrome, diabetes mellitus, and non- diabetics with risk factors for cardiovascular disease.(19;21;37;38) In non-diabetic patients with metabolic syndrome, Wang et al. reported a significant improvement in both flow-endothelium-dependent and independent vasodilatory responses after 8 weeks of treatment with rosiglitazone.(21) Patients also experienced a 40% reduction in fasting insulin levels and a 30% reduction in the inflammatory marker C-reactive protein. (21) Similar findings were observed with

Campia et al who observed an improvement in forearm blood flow responses to bradykinin (measured by venous occlusion plethysmography) with pioglitazone in non-diabetic patients with cardiovascular risk factors such as hypertension and hypercholesterolemia.(37) Patients in this study also experienced a 28% decrease in C-reactive protein. (37)

Further, it is now recognized that rosiglitazone’s receptor - PPAR-γ, is expressed in smooth muscle cells and endothelial cells of the vasculature and is thought to mediate effects on the vasculature independent of its effect on glycemic control.(39-41) In support of this, an uncontrolled trial in patients with diabetes mellitus treated with pioglitazone for 3 months demonstrated an increase in the levels of extra-cellular superoxide dismutase, an important enzyme used to regulate free radical species.(42) Animals studies have demonstrated rosiglitazone can reduce oxidative stress in a setting of both diabetes and hypercholesterolemia by modulating the activity of NAD(P)H oxidases. (25;27;43)

One report in hypercholesterolemia-induced rabbits found that treatment with

5 rosiglitazone inhibited expression of gp 91 phox, a critical NAD(P)H oxidase subunit, resulting in the suppression of superoxide anions from NAD(P)H oxidase.(27)

It is also important to note that a number of studies have shown that rosiglitazone therapy improves endothelial function in patients with diabetes mellitus and metabolic syndrome, but the PPAR-γ stimulation effects of thiazolidinediones have not been evaluated in a nitrate-induced model of endothelial dysfunction or extensively tested in a cardiovascular population for that matter. Further, it remains unclear if the improvements in endothelial function are secondary to changes in the components of metabolic syndrome, or attributed to direct changes in net NO bioavailability. These questions have important clinical implications, particularly in a population at high risk for cardiovascular events.

Despite these limitations, rosiglitazone’s purported beneficial effect on vascular function could be useful in combating the adverse events of organic nitrates therapy.

1.3 Rationale and Objectives of the Present Study

1.3.1 Nitroglycerin-Induced Model of Endothelial Dysfunction

With this knowledge as our starting point and under the leadership of Dr. John

Parker, our laboratory has developed a human model of nitrate-induced endothelial dysfunction that allows for the study of mechanisms and potential interventions that modify the development of nitrate tolerance, including the use

6 of antioxidants. The laboratory has demonstrated that healthy volunteers treated continuously with transdermal GTN (0.6mg/hr/day) for 7 days develop impaired endothelium-dependent responses to acetylcholine.(14;44;45) Our laboratory has also demonstrated that the development of nitrate tolerance in healthy volunteers can be reversed with intra-brachial infusions of vitamin C, (44) showing the increases in vascular free radicals is the fundamental casual factor in the development of nitrate tolerance. This research suggests that the mechanisms believed to contribute to endothelial dysfunction are centered on abnormalities in endothelial NOS function and vascular free radical generation.(14;44;45)

The dose of vitamin C (24mg/min) used in this protocol stem from findings reported by Joseph Vita et al., who demonstrated that under physiological conditions, normal extracellular concentrations of vitamin C (30 to 150 umol/L) are unlikely to prevent the interaction of NO with superoxide and that the concentration of vitamin C should exceed that of NO by a factor of 10.5(46;47) To interfere with the reaction kinetics of NO and superoxide, Vita et al demonstrated that a very high local concentration of 10 mmol/L of vitamin C (~24mg/min) would modify the bioactivity of NO in vivo in humans.(46) In fact, clinical studies using acute intra-arterial infusions of vitamin C at this dose in patients with risk factors for cardiovascular disease improved vasomotor function. (48-50) Taken together, these observations help aid in explaining why studies evaluating oral vitamin C for longer durations have for the large part failed to change cardiovascular outcomes, (51-53) as the concentration of oral vitamin C being absorbed into the

7 plasma is affected by absorption kinetics and mediated by the availability of its intestinal transport receptors. (54-56) Whereas low doses are taken up effectively, larger doses or frequently repeated doses down-regulate the intestinal receptors, leaving excess vitamin C in the intestinal lumen.(54;56)

Endothelial dysfunction, in broad terms, can manifest as a loss of vasodilator response to a muscarinic agonist like acetylcholine and result in reduced endothelium-dependent vasodilation. (1;57;58) The mechanisms of endothelial dysfunction in our model of nitrate-induced endothelial dysfunction is similar to that observed in patients with cardiovascular disease, where hyperactivity of membrane oxidases, NOS uncoupling, or pro-inflammatory activation all contribute to superoxide anion generation. While we understand that the results obtained in healthy volunteers may not translate to a clinical population who have overt cardiovascular disease, using this model of nitrate-induced endothelial dysfunction allows us to induce endothelial dysfunction with a similar pathophysiology to patients with vascular disease, whose endothelial dysfunction is brought about by risk factors for cardiovascular disease that include age, smoking, hypertension, hyperlipidemia, and diabetes. (6;59) This model also allows us to test specific interventions in healthy volunteers without being confounded by variables like the above described risk factors for cardiovascular disease.

8 1.3.2 Hypothesis

We set out to test our hypothesis that co-treatment of rosiglitazone (4mg bid) with continuously applied GTN (0.6mg/hr/day) for 7 days will reverse the impaired endothelium-dependent vasodilation in response to acetylcholine. This will provide evidence that rosiglitazone does have anti-oxidative properties independent of its effect on glycemic control.

Our objectives are as follows:

1. Conduct vasomotor responses of the forearm vasculature in response

to intra-brachial acetylcholine infusions in healthy volunteers, assessed

by measuring forearm blood flow with venous occlusion strain gauge

plethysmography (Section 2 – Methodology)

a. Baseline forearm blood flows will be determined with intra-

brachial saline infusions.

b. Vasomotor responses will be assessed in response to intra-

brachial infusions of acetylcholine.

c. Nitrate tolerance will be induced in healthy volunteers through

continuous administration of transdermal GTN patch

(0.6mg/hr/day) for 7 days. Subjects will be asked to apply a

replacement patch every 24 hours at approximately 8AM

including the morning of visit 2.

9 d. Acute intra-brachial infusion of vitamin C will be co-infused with

acetylcholine to assess vascular oxidative stress causes of

endothelial dysfunction.

2. To study the independent effects of GTN on endothelial function, with

and without the presence of vitamin C.

3. To study the independent effects of rosiglitazone on endothelial

function, with and without the presence of vitamin C.

1.4 Literature Review: Fundamentals of Nitrate Tolerance

1.4.1 Nitric Oxide Synthesis

NO is produced from the conversion of the amino acid L-arginine into L-citrulline by endothelial NOS.(60-62) Three distinct NOS enzyme isoforms have been identified and contribute to NO synthesis. (63-66) Neuronal NOS produces NO in the nervous tissue; in contrast, the inducible NOS is involved in inflammatory responses and its activity is implicated in host immunity. (67) Endothelial NOS is expressed constitutively in vascular endothelial cells and production of NO is tightly regulated by various endothelial NOS cofactors. (67)

The optimal activity of endothelial NOS is dependent on substrate and cofactor availability.(68) Substrates required for NO synthesis include the amino acid L- arginine, NAD(P)H oxidase, and molecular oxygen.(69;70) L-arginine is an essential substrate for NO synthesis, as proven by the series of important

10 biological and spectroscopy experiments conducted at the laboratory of Salvador

Moncada. (61;71;72) By using primary endothelial cells from pig aortas and chromatographic columns with aortic strips with the endothelium removed,

Moncada et al. showed that NO originated from the endothelium as the relaxation was observed in aortic strips devoid of the endothelium, which were placed underneath the endothelial cells with an intact endothelium. (2) Mass spectroscopy was then used to show that NO originated from the terminal guanidine nitrogen atoms of L-arginine. (61;71;72) In addition to its substrates, endothelial NOS also requires a number of necessary cofactors and include tetrahydrobiopterin,(73;74) calmodulin, (75) prosthetic heme groups, flavin mononucleotide, (68) and flavin adenine dinucleotide. (65;73;76)

Activation of the endothelial NOS complex through shear stress or pharmacological stimuli (acetylcholine, bradykinin) leads to NO biosynthesis. The production of NO from L-arginine involves two mono-oxygenation steps requiring molecular oxygen and the electrons of NAD(P)H.(3;68;77;78) The first step is the initial hydroxylation of L-arginine to generate N-hydroxyarginine and the second step, is an oxidation leading to the formation of NO and citrulline, with molecular oxygen being reduced.(3;68;77;78) NO released from vascular endothelial cells rapidly diffuses into neighboring vascular smooth muscle cells and triggers vasodilation through a NO-mediated signaling mechanism.(79;80) The NO/cyclic guanosine monophosphate pathway is the primary route which NO transmits it biological effects.(78;81) Once entering the smooth muscle cells, NO initiates a

11 cascade of events beginning with NO binding to the prosthetic heme group of soluble guanylyl cyclase. (81-83) This increases intracellular levels of cyclic guanosine monophosphate, a second messenger that activates a family of protein kinases called cyclic guanosine-monophosphate-dependent protein kinases, which mediates a reduction in cytosolic Ca 2+ concentration leading to vascular smooth muscle cell relaxation.(3;78-80)

The role of endothelial NOS in regulating multiple biological systems (71;78) is supported by findings that eNOS-deficient knockout mice develop hypertension, insulin resistance, and hyperlipidemia.(84) Further, the constant release of NO in vivo from endothelial cells contributes to maintaining basal vasodilatory tone, (85) and inhibition of endothelial NOS (utilizing a NOS inhibitor such as N G- monomethyl-L-arginine) results in vasoconstriction, as NO is unable to enter smooth muscle cells to activate soluble guanylyl cyclase, in addition to increasing blood pressure. (86) Unfortunately, in disease states like heart failure the endothelium is incapable of producing sufficient quantities of endogenous NO, and exogenous NO supplementation is required to improve NO bioavailability.

1.4.2 Nitroglycerin and Organic Nitrates

For more than 100 years nitroglycerin GTN and amyl nitrate have been used to treat cardiovascular disease. (18;87;88) The pharmacological effects of GTN dates back to the 19 th century when William Murrell prescribed a patient GTN to relieve symptoms of angina pectoris. (87) When administered in an acute manner,

12 sublingual GTN preparations are rapidly absorbed and exert their anti-ischemic effect within minutes.(89;90) Patients with heart failure or stable angina however, require long-acting GTN therapy in an effort to prevent symptoms of myocardial ischemia. (87) As a result, long-acting preparations have been developed and include isosorbide-2,5-dinitrate (ISDN), isosorbide-5-mononitrate (ISMN), and transdermal GTN preparations.(87) These formulations were designed to have a slower onset of action and improve systemic bioavailability, leading to constant plasma concentrations over 24 hours.(87;91-93) Depending on the preparation, nitrates can act relatively rapidly as they are absorbed from the skin and mucous membranes, and achieve peak plasma concentrations in a short period of time before being subject to extensive hepatic and vascular clearance.(87;88)

Organic nitrates are pro-drugs that must undergo metabolic biotransformation to exert their therapeutic benefits. (87;88) This transformation involves denitrification - a detachment of a nitrate group from the molecule that results in the liberation of

NO. (94) NO is thought to be the primary mediator of organic nitrates and transmits its effects through the NO signaling pathway, where it triggers the formation of cyclic guanosine monophosphate causing vascular smooth muscle relaxation. (81;82;95) The importance of the NO/cyclic guanosine monophosphate pathway in mediating the effects of GTN is supported by the observation that isolated vessels exposed to GTN cause an increase in the production of cyclic guanosine monophosphate. (81) Of note, endothelial vasodilation can be induced

13 by the pharmacological actions of NO donors or endogenously produced NO, and thus can be independent or dependent vasodilation, respectively.(3;18)

The underlying mechanism by which GTN transforms and release NO has remained a mystery for several decades with several hypotheses being suggested.(94;96) Early reports by Needleman and colleagues suggest that reduced sulfhydryl groups were essential for GTN biotransformation.(97;98) This hypothesis suggested that nitrates react with reduced sulfhydryl groups in the vascular smooth muscle cells, leading to the formation of disulphide bridges before liberating NO from GTN. (99) More recently, in vitro studies have shown that mitochondrial aldehyde dehydrogenase is present in smooth muscle cells and is responsible for the biotransformation of GTN. (100) However, less is known about the mechanisms of other nitrates. (100)

The beneficial vasodilatory effects of nitrates are attributed to dilation of various vascular beds and its ability to optimize myocardial oxygen consumption. (87;88)

Dilation of large coronary arteries leads to improved blood supply to the heart, whereas dilation of the capacitance veins leads to a reduction in cardiac preload and a subsequent decrease in myocardial oxygen demand. (87;88) Although there is no question that the acute administration of GTN yields clear beneficial therapeutics effects (such as the acute relief of symptoms of cardiac ischemia and reduction in blood pressure), continuous use of organic nitrates over a period of approximately 24 hours is more controversial. (18;88;99)

Given the importance of NO in cardiovascular physiology, a long-standing hypothesis is that continuous use of long-acting nitrates would be beneficial as it is thought that patients with vascular disease have reduced endogenous NO bioavailability.(87;88) However, within 24 to 48 hours of continuous application their hemodynamic and anti-ischemic effects of these drugs is diminished.(87;94) What was thought to be beneficial (exogenous NO replacement) is now questionable.

1.4.3 Nitrate Tolerance and Proposed Mechanisms

Despite the theoretical benefit of organic nitrates as exogenous NO donors, their sustained use is associated with loss of efficacy – a phenomenon termed nitrate tolerance.(88;101) Nitrate tolerance was first observed more than a 100 years ago by Stewart, who reported the necessity to increase the dose of GTN in an attempt to maintain therapeutic efficacy in a patient with Bright’s disease. (88;102)

The mechanisms that contribute to the development of nitrate tolerance are still unclear, but over four decades of research have yielded a number of hypotheses explaining the phenomenon of nitrate tolerance. This includes activation of neurohormonal factors, expansion of plasma volume, vascular free radical hypothesis, abnormalities in GTN biotransformation, and GTN-induced abnormalities to the endothelium.(18;103)

15 1.4.4 Neurohormonal Activation

The neurohormonal activation hypothesis centers around the observations that various vasoconstrictor molecules are released during GTN therapy, thereby overcoming the vasodilatory effects of NO.(104;105) These include norepinephrine, angiotensin-II, and endothelin-1.(106;107) Support for this comes from observations by Münzel et al., who demonstrated that GTN therapy is associated with counterregulatory responses fashioned by an increase in the production of endothelin-1 and angiotensin-II. (12;108) The vasoconstriction brought on by these mediators may negate or prevail over the vasodilator effects of nitrates, thereby possibly contributing to tolerance. Further, nitrate-induced neurohormonal responses also play a role in the generation of free radicals during continuous nitrate therapy,(109) as neurohormonal stimulation of angiontensin-II during nitrate therapy can activate NAD(P)H oxidases and increase superoxide generation and bioavailability. (110;111)

Endothelin-1 has potent vasoconstricting capabilities and can be stimulated by angiotensin-II and free radicals in endothelial cells.(108;112) In normal vessels exposed to low concentrations of endothelin-1, significant vasoconstriction occurs, comparable to that of nitrate tolerant vessels.(108;113) The increases in endothelin-1 have also been documented in nitrate tolerant animals, and inhibition of the endothelin-1 receptor reduced superoxide anion production and the development of nitrate tolerance in rabbits. (12;108) Finally, endothelins can also

16 activate protein kinase C, which has been shown to stimulate NAD(P)H oxidase- mediated superoxide anion generation. (108)

Given the impact nitrates can have on sympathetic activity, efforts to increase the clinical utility of nitrates by inhibiting the production of vasoconstrictors through therapy with angiotensin-converting enzyme inhibitors, beta-blockers, and angiotensin-II type 1 antagonists have shown to reduce the development of nitrate tolerance in animal models. (112;114-116) However, findings in patients with cardiovascular disease have yielded negative results, (105;117;118) as a study by

Parker et al. demonstrated that concomitant treatment with benazepril, a non- thiol angiotensin-converting enzyme inhibitor did not modify the hemodynamic responses to continuous GTN therapy.(105)

1.4.5 Plasma Volume Expansion

A number of clinical studies have reported plasma-volume expansion during sustained GTN therapy, observed as a decrease in hematocrit levels in healthy volunteers and patients with heart disease. (106;109;119-121) In heart failure patients, intravascular volume increased by as much as a liter within 24 hours of intravenous GTN therapy. (119) The plasma volume expansion hypothesis states that the increases in intravascular volume would counteract nitrate-induced decreases in ventricular preload contributing to the loss of hemodynamic effects, and thus nitrate tolerance.(87) While the role of plasma volume in the development of tolerance is still not completely understood, it seems that the mostly likely

17 explanation is a shift in fluid from the extravascular to the intravascular space likely due to changes in Starling’s force. (18;122;123) The use of diuretics – in an attempt to counteract the increase in intravascular volume – has had no effect on the development of nitrate tolerance in patients with stable angina.(12;124;125)

These findings limit the importance of this hypothesis in the development of tolerance.

1.4.6 Vascular Free Radical Hypothesis

The free radical hypothesis is the most recent and current explanation for the development of nitrate tolerance.(10;18) This hypothesis was first reported by

Thomas Münzel, who demonstrated that aortic segments of rabbits treated with sustained GTN therapy was associated with a large increase in vascular superoxide production, primarily in the form of superoxide anions.(12) Thomas

Münzel went on to show that the superoxide anions was normalized (attenuation of nitrate tolerance) by co-treatment with superoxide dismutase. (12) Of note, the bioavailability of superoxide anions is balanced by anti-oxidant defense mechanisms such as intra and extracellular mechanisms like superoxide dismutase.(126) Abnormally high concentrations of superoxide can overwhelm these mechanisms, allowing GTN-derived NO to react with circulating superoxide anions to form peroxynitrite.(47;109) Peroxynitrite may thwart the vasodilatory effect of NO donors. (109;127) Additional support for a superoxide anion-mediated role in the development of tolerance stem from an in vitro study, where co-incubation

18 with superoxide dismutase restored GTN’s vasodilatory effect in vascular tissues. (12)

Sources of free radical generation induced by GTN that could offset the vasodilatory effects of NO include xanthine and NAD(P)H oxidase, mitochondria, cyclooxygenase, cytochrome, and endothelial NOS.(128) NAD(P)H oxidase is a multi-component enzyme that generates superoxide upon stimulation.(129)

Importantly, Münzel et al. demonstrated in animals that hydralazine – an inhibitor of membrane bound NADH and NAD(P)H oxidases - could attenuate the development of nitrate tolerance.(130;131) In this report, rabbits either received

GTN patches or both hydralazine and GTN patches for 3 days. Aortic segments were isolated to study vascular superoxide anion production with chemiluminescence and relaxation was assessed with infusions of acetylcholine.

Rabbits co-treated with hydralazine prevented the development of nitrate tolerance compared to rabbits treated with GTN patches alone.(130) Further, isolated vessels showed that superoxide anion generation was NADH- dependent, as the activity of this oxidase increased three-fold in rabbits treated with GTN compared to controls (no treatment).(130) Similar increases in superoxide anion generation were seen with xanthine oxidase during continuous

GTN treatment. (130-132)

Despite hydralazine’s ability to inhibit the activity NAD(P)H oxidase during continuous GTN treatment and modify the development of nitrate tolerance in

19 animal models,(131;132) Parker et al. found that hydralazine did not prevent the loss of systemic effects or counter-regulatory responses to continuous GTN treatment in healthy volunteers.(133;134) The role of membrane oxidases in the development of nitrate tolerance however, is primarily based on objective evidence from animal studies with some antioxidant interventions in humans. (133;134)

1.4.7 GTN-Induced Abnormalities in the Endothelium

While hyperactivity of membrane oxidases have been implicated in nitrate tolerance, sustained nitrate therapy can also have a detrimental effect on endothelial NOS, the complex responsible for endogenous production of NO and controlling vascular tone. (12) Despite the increased expression of endothelial NOS during prolonged nitrate exposure, its activity is decreased during continuous

GTN treatment resulting in an increase in superoxide anion generation. (12;135)

This paradoxical observation in the function of endothelial NOS can be explained by the observations made by Dr. Thomas Rabelink. (17;77;136) The pioneering investigations by his group clarified the importance of molecular oxygen transfer through the endothelial NOS domain.(68;77) In a setting of nitrate tolerance or dysfunctional endothelial NOS, molecular oxygen is reduced to form superoxide anions and is not followed by oxidation of L-arginine and NO synthesis. (68;77) The term used to describe this event is ‘NOS uncoupling’ and can be prompted by a reduction in the availability of endothelial NOS cofactors tetrahydrobiopterin and

L-arginine. (137;138) Dr. Rabelink demonstrated in vascular tissue that

20 tetrahydrobiopterin can be readily oxidized by peroxynitrite to dihydrobiopterin, a form that is not utilized in the biosynthesis of NO.(68)

The importance of tetrahydrobiopterin lies in its responsibility to couple with the eNOS dimer, and along with molecular oxygen, facilitate the transfer of electrons from the endothelial NOS heme group to L-arginine, which is then converted to

NO and L-citrulline.(68;137;139;140) In support of this concept, infusions of tetrahydrobiopterin into the brachial artery of the forearm resulted in improved endothelial-dependent vasodilation and restored endothelial NOS activity in those with risk factors for endothelial dysfunction. (141-145)

Diminished bioactivity of endothelial NOS can also be attributed to increased levels of asymmetric dimethylarginine (ADMA), an endogenously produced competitive inhibitor of L-arginine, and can inhibit the biosynthesis of NO.(146-148)

ADMA degradation occurs via dimethylarginine dimethylaminohydrolase,(149) an enzyme that hydrolyzes ADMA to L-citrulline and dimethylamine. (150;151)

Dimethylarginine dimethylaminohydrolase has an important role in preventing the accumulation of ADMA,(152) but its oxidation results in impaired enzymatic activity and elevated levels of ADMA.(153-156) Plasma levels of ADMA are increased 2 to

10 fold in individuals with risk factors for cardiovascular disease, which have been shown to reduce net NO bioavailability.(70;157;158) Another explanation in support of the nitrate-induced NOS uncoupling leading to increases in superoxide anion generation is intracellular L-arginine depletion. A report by Abou-Mohamed

21 et al. demonstrated in cultured bovine aortic endothelial cells that continuous transdermal GTN therapy causes oxidation of L-arginine transporters and induce abnormalities in L-arginine transport, leading to L-arginine depletion. (159-162)

1.4.8 Abnormalities in Nitrate Biotransformation

Impaired nitrate biotransformation have been proposed to contribute to nitrate tolerance.(163) The concept finds support in biochemical studies demonstrating that tolerance is associated with reduced bioactivity of NO from GTN or some NO adjunct(s).(163;164) In a report by Sage et al., patients undergoing coronary artery bypass received 24 hours of intravenously infused GTN.(163) Segments of the mammary artery were isolated and evaluated for bioconversion of GTN and vascular responsiveness. Patients treated with nitrates had reduced vascular responses and reduced formation of 1,2-glyceryl dinitrate in the nitrate group when compared to controls. (163-166) In line with this impaired nitrate biotransformation, this group also found no difference in NO clearance via a reaction with circulating superoxide.

Cytochrome P540, glutathione transferases, NAD(P)H and xanthine oxidases, and NOS have been proposed to be sites of nitrate biotransformation.(167-170)

Using an in vivo model of nitrate tolerance in isolated rat aorta however, the activity of NAD(P)H oxidase could not account for the biotransformation of GTN, as the reduced activity of this enzyme was comparable between control and nitrate therapy, showing nitrate tolerance is not associated with reduced activity

22 of this enzyme.(171) Indeed, the enzymatic mechanism of nitrate (GTN and other organic nitrates) denitrification has remained a mystery for decades. However, recent advances made by the laboratory of Jonathan Stamler demonstrated that mitrochondrial aldehyde dehydrogenase is responsible for the biotransformation of GTN.(100) This group went on to demonstrate that inhibition of the mitochondrial aldehyde dehydrogenase enzyme led to a blunting of the acute hemodynamic effects of GTN in animals.(100;172;173) The activity of mitochondrial aldehyde dehydrogenase has also been evaluated in healthy volunteers prescribed clinically relevant doses of GTN. (174) Results of this study demonstrated that healthy volunteers with an inhibitor mutation (glu504lys) in the mitochondrial aldehyde dehydrogenase gene had a significantly lower forearm blood response to intra-arterial infusions of GTN compared to humans without the mutation.

Forearm blood flow responses were also blunted after administration of disulfiram, an inhibitor of mitochondrial aldehyde dehydrogenase.(174) While mitochondrial aldehyde dehydrogenase plays an important role in bioactivation of

GTN, the role of this enzyme in other organic nitrates has not been fully explored.

It is also worthy to note that one of the original hypothesis for the development of nitrate tolerance is the sulfhydryl-depletion hypothesis.(87;175) Based on this hypothesis, it is was thought that depletion of reduced thiols was responsible for the loss of clinical efficacy observed with sustained nitrate therapy, as it was thought that decreased bioavailability of reduce thiols was necessary for GTN biotransformation. (97;175) The role of thiol groups in the biotransformation of GTN

23 is controversial. Boesgaard et al.(176) and others demonstrated that venous and arterial thiol levels were not significantly different in arterial vessels exposed to both sustained nitrate therapy and acute nitrate therapy, suggesting that thiols are not essential in the liberation of NO from GTN and that they do not contribute to the development of nitrate tolerance. (97;98;177) In contrast, Fung and colleagues found that infusions of N-acetylcysteine, an exogenous sulfhydryl group donor, partially reversed tolerance to nitrates in nitrate-tolerant rats.(178-180) Despite these observations, it is thought to be that nitrate-based responses in the presence of sulfhydryl group donors occur through an extracellular pathway independent of nitrate tolerance as previous reports have demonstrated that there is no correlation between intracellular thiol levels and tolerance. (180-182)

While thiol depletion is not an explicit cause of nitrate tolerance, oxidation of protein-bound thiols from circulating superoxide anions can impair the cellular- antioxidant defenses. (18;183) Glutathione and glutathione-S-transferases protects membranes from oxidant-induced damage, and in a setting of nitrate tolerance these enzymes are found to be inhibited. (184;185) The NO-derived byproduct peroxynitrite can also inhibit the activity of superoxide dismutase, an enzyme that converts superoxide back to hydrogen peroxide and molecular oxygen.(186;187)

Increased bioavailability of peroxynitrite or free radicals can impair the function of heme proteins, which are routinely used in physiological processes of the cytochrome P450 and endothelial NOS systems. (18;188;189)

24 1.4.9 Intermittent Nitrate Dosing

The clinical effectiveness of nitrate therapy is significantly diminished with continuous dosing. (190) Further, the development of nitrate tolerance is not dependent on pharmacokinetics, as reports have shown that continuous nitrate therapy actually leads to an increase in plasma drug concentrations compared to those observed during initial acute therapy.(191;192) A meta-analysis of 1042 patients enrolled in the Multi-centre Study of Myocardial Ischemia demonstrated a negative effect of long-acting nitrates on cardiovascular outcomes.(193) The analysis revealed that nitrates were associated with a significantly increased mortality risk in patients who had survived an acute coronary event and increased risk for cardiac death in those receiving continuous nitrate therapy.(193)

To date the only clinically proven method of preventing nitrate tolerance has been the use of an intermittent dosing schedule or a nitrate formulation that incorporates a nitrate-free interval.(190;194;195)

A number of complications are associated with intermittent dosing regimens.

First, withdrawal from GTN exposure has been associated with an increase in risk for myocardial infarctions, angina pectoris, and death.(190;196;197) As well, the use of an intermittent regimen in patients with coronary artery disease is also associated with worsening of endothelial dysfunction during the nitrate-free period, observed as a paradoxical increase in vasoconstrictor responses to intra- coronary infusions of acetylcholine.(197-199) Evidence from numerous studies suggest that during the nitrate-free interval, the ischemic threshold of these

25 patients is paradoxically reduced, a scenario termed ‘rebound angina.’ (190;198) The prevailing increase in vasoconstriction activity during the nitrate-free period appears to be explained by an exacerbation of endothelial dysfunction caused by the rapid loss of exogenous NO, although this is entirely working hypothesis.(190;197) Despite the adoption of intermittent therapy, concerns remain over its long-term effects.(190;197)

26 2.0 METHODOLOGY

2.1 Assessment of Endothelial Function

The assessment of endothelial function in humans initially focused on the quantification of endothelium-dependent vasomotor responses to pharmacological stimuli. (200;201) These responses can be made in conduit vessels

(from the epicardial coronary vessels or the resistance vessels of the forearm).(202;203) These measures provide an index of endothelial function that is reflective of the bioavailability of NO.(200) This is an important concept since reduced NO bioavailability is implicated in cardiovascular risk factors and overt disease.(17;204) Commonly used methods of measuring vascular function include venous occlusion strain gauge plethysmography, intra-coronary infusions of vasoactive agents, and an ultrasound-based method evaluating changes in vessel diameter during flow-mediated vasodilation.(200;205;206) Collectively, these methods have discriminatory abilities to separate those with normal vascular health and those that have impaired endothelium-dependent responses.

2.1.1 Plethysmography

For the purpose of the current study, we adopted the use of venous occlusion plethysmography to measure forearm blood flow in response to intra-arterial infusions of acetylcholine.(14) Acute intra-arterial infusions of vitamin C were infused to assess the impact of oxidative stress on the development of nitrate tolerance.(14) The use of venous occlusion strain gauge plethysmography to measure blood flow in organs dates back more than 80 years when Hewlett and

27 colleagues first introduced the technique.(200;207) The method is based on the principle that if venous return from the arm is obstructed - achieved through the inflation of pressure cuffs – and arterial inflow is unaltered, the forearm volume swells at a rate proportional to the rate of arterial inflow. (200;207-209) It is also important to note that a critical element of the venous occlusion plethysmography technique is that at any given time 90% of the blood within the forearm blood is venous blood in the capacitance venous system, and forearm blood flow is measures the total flow in the forearm from the upper arm cuffs to the wrist cuffs.(200;203) Venous occlusion strain gauge plethysmography is a well-described technique that we and other laboratories have employed.(14;48;141;203)

Venous occlusion strain gauge plethysmography can be an invasive method when incorporating with intra-brachial infusions of a pharmacological stimuli into the brachial artery, usually a muscarinic agonist like acetylcholine that can stimulate the release of NO from forearm resistance vessels.(210;211) The use of venous occlusion plethysmography in assessing endothelial function can provide mechanistic insight into the various pathways that influence endothelial function.(212) The use of vasoactive agents such as acetylcholine allow for the construction of dose-response curves to assess endothelial function and allow for comparisons between different treatment groups.(200) An example can be found in the report of Panza et al. who demonstrated blunted endothelium-dependent vasodilation in hypertensive patients when compared with healthy controls.(213)

These observations have been extended to patients with hypertension,

28 hypercholesterolemia, and diabetes when compared to healthy controls who demonstrate normal endothelial function.(214-217) We elected not to use flow- mediated dilation (FMD) measures responses to conduit arteries (207;208;218;219) since continuous transdermal GTN therapy leads to persistent conduit artery dilation,(218) making the use of FMD an unsuitable technique to study nitrate- induced models of endothelial dysfunction. (Details of the FMD technique will be described below)

Measuring forearm blood flow requires precision as absolute responses to vasoactive agents can vary in response to sympathetic tone.(200) In order to minimize the variability in measurements (due to systemic changes that include release of vasoactive hormones and sympathetic output), the results are expressed as a ratio of flow between the infused and the non-infused arm.(200;203)

Furthermore, measurements are taken in a quiet temperature controlled room to avoid fluctuations in temperature and ambient noise. (203;220) Despite its limitations, venous occlusion plethysmography also shows good within-subject reproducibility (221;222) and as a whole, this technique discriminates between impaired endothelial function and those with normal intact endothelial function.(200;223)

29 2.1.2 Flow-mediated Dilation (FMD)

A more recently adopted technique that has gained considerable popularity is the use of an ultrasound-based method that is commonly referred to as flow- mediated dilation (FMD).(205) This technique provides an in vivo assessment of endothelial function and evaluates changes in blood vessel diameter in the conduit artery of the forearm during hyperemia using ultrasound guided measures of brachial or radial artery diameters.(206;224)

The principle is based on inducing ischemia in the forearm via the use of a pressurized cuff inflated above the elbow for 4-7 minutes, followed be a release of the pressure cuff and subsequent quantitative assessment of vasodilation post-ischemia.(205;206;225) When the cuff is released, there is an increase of flow in the forearm causing an increase in shear stress via a number of mechanisms that stimulates NO release and subsequent vasodilation.(205;206;223) The dilation is expressed as the percentage increase in vessel diameter as compared to baseline (226) and the relative increase in diameter of the brachial artery is used as an indication of endothelial function.(227)

FMD based methods have achieved similar results when compared with venous occlusion plethysmography, showing that this endothelium-dependent response is reduced with age and in patients with risk factors for cardiovascular disease such as hypertension, hypercholesterolemia, smoking, and diabetes as compared to healthy controls.(205;228-232) However, some limitations do exist with

30 FMD-based assessments. First, no specific signaling pathways have been reported and precise mechanisms that modulate vasomotor tone with this method is not fully understood (205) and second, several other endothelial mediators other than NO are capable of acting as signals between the endothelium and vascular smooth muscle.(232) Its widespread adoption of this technique is in part, due to its simplicity, non-invasive nature, and its high correlation to invasive measurements of endothelial function. As a result, FMD- based assessments are increasingly used in measuring endothelial function, but will not be used in this study for the above described reasons.

2.2 Methods

2.2.1 Study Population

This study was designed as a randomized, investigator-blinded, placebo- controlled study. Study subjects recruited through university advertisements, and were non-smoking males aged 18 to 30 years, having an average age of 24 years old. Prior to enrollment, subjects underwent a physical examination.

Subjects were required to abstain from alcohol and any drugs, including supplemental vitamins, for the duration of the study. The study protocol was approved by Mount Sinai Hospital Research Ethics Board. Study objectives and procedures were explained to individual subjects, and written informed consent was obtained from all subjects. The exclusion criteria for this study are listed below.

Inclusion Criteria Exclusion Criteria

• Healthy males between the ages of 18 and • Insulin resistance 30 years • Hyperlipidemics • Abstain from alcohol and supplemental • Vascular disease vitamins for duration of study • Diabetes mellitus • Smoking

2.2.2 Study Design

Forty-four healthy volunteers aged between 18 to 30 years participated in this

study. Subjects were asked to fast overnight (no food or drink after 8pm) and

upon admission into the study, standing blood pressure and heart rate

measurements was obtained using an automatic, calibrated,

sphygmomanometer (Critikon Company LLC, Tampa, Florida). The mean value

of 3 measurements was determined. Subjects were then randomized in an

investigator-blinded fashion to receive placebo (2 pills orally a day, n=12),

rosiglitazone alone (4 mg, bid.; n=11, a dose shown to improve endothelial

function in healthy volunteers (233) ), transdermal GTN patch alone (0.6

mg/24hrs/day with the patch placed on the upper shoulder, n=11) or the

combination of both drugs (rosiglitazone+GTN) at the same dosages (n=10) for 7

days. A nurse not involved in the study procedures randomized all volunteer and

instructed them to wear the transdermal GTN patch continuously for 24 hours a

day and replace them each morning with a new patch until their return visit in 7

days. Three hours after the first dose of the study medication, repeat standing

32 blood pressure and heart rate measurements were taken. This was to ensure that those randomized to GTN did experience a decrease in blood pressure, but did not become hypotensive. Rosiglitazone was administered 4mg twice a day as this was the dosage previously used to show an improvement in endothelium function in healthy normal volunteers.(233)

During all the procedures of the study, investigators were blinded to the treatment group of the subjects and only the research nurse not involved in the analysis of the data remained unblended. The protocol is outlined below .

Visit 1 Visit 2 (7 days later)

- History/Physical, baseline blood pressure and - baseline blood pressure and heart rate heart rate measurements measurements

- Randomization to Placebo, rosiglitazone (4 mg, - Forearm blood flow measurements in bid), transdermal GTN (0.6mg/24 hrs/day) response to intra-brachial infusions of continuously, GTN+ rosiglitazone for 7 days acetylcholine - Co-infusion with Vitamin C to assess role of - Subjects returned 3 hours after repeat blood oxidative stress pressure and heart rate measurements

2.2.3 Vascular Function Procedures

Vascular function measurements were performed at 9:00AM after an overnight

12 hour fast with the subjects lying supine in a quite, room temperature controlled room. After 15 minutes in a supine position and under local anesthesia (1% xylocaine), the non-dominant arm was cannulated with a 21-gauge intravascular catheter (Cook Inc., Bloomington, Indiana) passed into the brachial artery using standard over the wire aseptic techniques. The catheter serves as a port for drug

33 infusion. The catheter is connected to a pressure transducer (Gould, Inc.,

Oxnard, CA, USA) which allows continuous monitoring of arterial blood pressure before and after transfusions. (200;201)

Forearm blood flow was obtained simultaneously in both forearms (experimental and countra-lateral) using venous occlusion strain-gauge plethysmography (D. E.

Hokanson Inc., Bellevue, Washington) (220) and recorded on a multi-channel recorder (Gould Instrument Systems, Inc., Valley View, Oh, USA). Forearm blood flows were performed approximately 30 minutes after cannulation. During the experiments the subjects remained in supine position with both arms extended and stabilized slightly above the level of the heart with the assistance of a

Styrofoam block.(200;201)

Prior to each forearm blood flow measurement session, blood pressure cuffs (on both upper arms and wrist) were positioned around the midpoint of the subject’s upper arm and a second blood pressure cuff was positioned on the tip of the wrist. The strain-gauges, which were 2-3 cm less than the forearm circumference was placed around the forearm approximately 10 cm distal cubital fossa.(200;207;234;235) Additionally, in order to obtain forearm blood flow, a manual cuff inflator was used to apply a cuff pressure of 240 mm Hg to the wrist to exclude circulation in the hand for 3 minutes. The purpose of this is to exclude the hand circulation which has conduit vessels (i.e. non-resistance vessels directly connecting the brachial and ulnar conduit artery distributions).(200;236;237)

34 In parallel, six flow measurements each lasting 10 seconds were made via serial upper-arm cuff inflations (inflated to 40 mm Hg) by using an automatic cuff inflator.(14;44;207;238) This pressure is used to prevent venous outflow and is the standard venous occlusion pressure used with this technique.(200) Each inflation lasts 10 seconds (when forearm blood flow is measured) and repeated after 10-

15 second interval when forearm volume returns to baseline.(207) After 3 minutes the wrist cuff was deflated and the serial inflation-deflation of the upper-arm cuff ceased. Each forearm blood flow session lasted 6 minutes. Forearm blood flow was recorded as the average of five consecutive measurements. Vasomotor responses in response to intra-arterial infusions took approximately 4 hours to complete.

The response to intra-arterial infusions of vasoactive agents is a well described technique in assessing endothelial function.(239-242) Forearm blood flow is calculated by taking the initial slope of the arterial inflow, expressed in units of ml

(of blood) per 100 ml (of volume) per minute [ml/100ml forearm volume/min] (203;207) and is normalized by presenting it as the ratio of infused to non-infused arm.(209) The slope is measured over the highest peak because we are measuring the linear increase in forearm volume over time, which is proportional to arterial blood inflow, until venous pressure rises towards the occluding pressure.(207)

35 The control arm serves as a control for any systemic changes in blood flow such as changes in arterial blood pressure, release of vasoactive hormones, or changes in sympathetic activity.(203;207;209) To minimize error, flows are expressed as a ratio of infused to non-infused arm. (203;207;209) Interpreting responses to intra- arterial infusions is done by analyzing forearm blood flows as percent changes from baseline.(207) This approach has been widely adopted by others in the field and is considered the most accurate way of evaluating forearm blood flow.(243-245)

The ratio of blood flow between arms approaches unity at baseline,(209) and as such, any change in ratio of blood flow in the infused arm is the result of the infused agonist.(200) It’s also important when comparing between groups that baseline ratio’s are equivalent across all groups, as differing baseline forearm blood flow can lead to erroneous conclusions.(200) When assessing interventions, the results are described as percent or absolute changes from baseline (saline infusion) when comparing multiple groups, as some interventions can result in depressed responses when compared to placebo.(50;200;246)

2.2.4 Experimental Sessions

All subjects returned to the laboratory after 7 days of continuous therapy of GTN, rosiglitazone, placebo, or both GTN + rosiglitazone. Standing blood pressure and heart rate measurements were repeated and recorded.

Baseline forearm blood flow measurements were recorded with infusions of normal saline at a flow rate of 0.4ml/min. To evaluate whether continuous GTN

36 therapy can cause endothelial dysfunction, endothelium-dependent vasodilation was estimated by performing a dose-response curve to intra-arterial acetylcholine (cumulative increase in infusion rates: 7.5 ug/min, 15 ug/min, and

30 ug/min) . Acetylcholine was diluted in the control vehicle with normal saline and was infused for 6 minutes at a flow rate of 0.4 ml/min using a precision pump

(Harvard apparatus, South Natick, Massachusetts) (44) and forearm blood flow measurements were initiated during the last 3 minutes when the wrist cuff was inflated to 240 mm Hg. Once cuffs were deflated the acetylcholine infusion is stopped but normal saline was continued. The infusions of acetylcholine were done in sequential order and periods of 3 minutes were allowed to pass before beginning the next incremental acetylcholine infusion.

After the completion of the acetylcholine infusions, which lasted approximately 90 minutes, a 20 minute wash-out period was allowed to ensure that the effects of acetylcholine infusions had completely dissipated. Subsequently, repeat baseline forearm blood flow measurements (saline infusion) were performed to ensure that forearm blood flow had returned to levels comparable to that of baseline

(saline infusion).

To evaluate whether loss of vasodilatory effect was attributed to oxidative stress, intra-arterial vitamin C (24 mg/min) was infused. This concentration was chosen as previous reports Intra-arterial infusions of vitamin C at this concentration is expected to achieve a physiological concentration in the range of ~10mmol/L (i.e.

37 approximately 50 times the normal concentration) which was previously shown in vitro to protect human plasma from free radical oxidation.(46;49;247) Vitamin C was infused with saline as the control for 15 minutes before being co-infused with the various doses of acetylcholine. At the end of the study, the arterial line was removed, all study medications discontinued, and subjects were discharged from the laboratory . Analysis was performed offline by a blinded investigator (graduate student).

Figures 1, 2, 3, and 4 provide schematics for study protocol, setup, and methods.

2.2.5 Side Effects and Risk of Study

Rosiglitazone is an insulin sensitizing agent and does not alter blood glucose levels in normal healthy volunteers.(31) This can be attributed to its mechanism of action, in that rosiglitazone does not increase insulin levels directly but rather acts as an insulin receptor sensitizer in diabetic patients and increases insulin sensitivity.(35) Rosiglitazone (Avandia, GSK) side effects include back pain, fatigue, headaches and upper respiratory tract infections. GTN can also cause headaches and hypotension. Adverse events with nitrate therapy include headaches and skin irritation at the site of transdermal GTN patch. Subjects suffering side effects were withdrawn from the study (this occurred in about 8% of subjects).

38 2.2.6 Statistical Analysis

The primary endpoint of this current study was the measure of forearm blood flow in response to acetylcholine in healthy volunteers, which was therefore used to determine the study sample size. To calculate the number of subjects required to detect a significant change in forearm blood flow due to treatment, we used previously reported values from our lab, for healthy volunteers randomized to either no treatment or prolonged exposure to transdermal GTN.(14;45) The mean relative % change compared to treatment was approximately 180% at the highest acetylcholine dose, resulting in 11 subjects per group to receive statistical significance with 80% power (2-sided α of 0.05).

All data are expressed as mean±SE. A value of p<0.05 was set as the threshold for significance. Differences in blood flow responses and blood pressure responses were tested using a 2-way repeated measures ANOVA on the % change and absolute changes as compared with the corresponding baseline

(saline). If the difference was significant, intra-treatment comparisons were made by a Student Newman-Keuls post-hoc test. Statistical analysis was performed using Statview 4.0 (SAS Institute, Cary, USA).

39 3.0 RESULTS

3.1 Responses to Blood Pressure and Heart Rate

Baseline assessment of standing heart rate and systolic blood pressure did not differ between groups (Table 1). When compared with baseline values, a significant decrease in standing systolic blood pressure was observed in groups randomized to GTN alone and GTN + rosiglitazone 3 hours after the administration of the first patch (p<0.05, multi-group ANOVA, Table 1). A corresponding significant increase in heart rate was also observed in the same groups (p<0.05, multi-way ANOVA, Table 1). After seven days of continuous dosing, the hemodynamic effects of GTN were no longer evident in either group

(p=ns as compared to placebo and to initial baseline assessment) and returned to baseline. Blood pressure and heart rate in the placebo and rosiglitazone groups were similar at all time points.

3.2 Effect of Acetylcholine on Endothelium-Dependent Vasodilation

Baseline (saline infusion) forearm blood flow levels, expressed as a ratio of infused to non-infused arms, were comparable between groups (Table 2). The mean basal forearm blood flow in the experimental arm for subjects randomized to placebo, rosiglitazone, GTN, and GTN + rosiglitazone were 3.2±0.3, 2.6±0.2,

3.2±0.3, and 2.8±0.3 mL/min per 100 mL, respectively (p=ns compared to placebo). When normalized with the control arm, the ratios were 1.1±0.9,

1.1±0.1, 1.3±0.1, and 1.2±0.1 respectively (p=ns compared to placebo; Figure 5).

40 Responses to intra-brachial infusions of acetylcholine caused a significant dose- dependent increase in forearm blood flow in all groups (p<0.05, Table 2, Figure

6). The responses to acetylcholine were significantly attenuated in the groups randomized to GTN, rosiglitazone, and GTN+ rosiglitazone when compared with placebo (p<0.05, multi-way ANOVA, Table 2, Figure 6), which exhibited the most pronounced effects. Subjects randomized to placebo experienced significant increases in forearm blood flow ratios from 137% to 300% at the maximum acetylcholine dose and when compared with rosiglitazone, GTN, and GTN

+rosiglitazone, respectively their increases in forearm blood flow were significantly blunted (41% to 116%, 63% to 175%, and 33% to 145%, respectively; p<0.05; Figure 6).

The ANOVA demonstrated a significant difference in the responses to acetylcholine between the four treatment groups (effect of group: p<0.05, Table

2, Figure 5). There was also a significant dose response relationship in the placebo group (effect of infused Ach concentration: p<0.05, Figure 6, Table 2).

Acetylcholine-induced vasodilation was significantly impaired in the GTN, rosiglitazone, and GTN+ rosiglitazone treatment groups.

3.3 Effects of Vitamin C on Endothelium-Dependent Vasodilation

A 20 minute re-control period occurred prior to infusing vitamin C. During this re- control period, saline was infused to ensure that forearm blood flows returned to levels comparable to that of baseline (p=ns compared to baseline, Figure 7,

Table 2). Forearm blood flow ratios at baseline and at re-control did not differ

41 showing re-control was successfully achieved. The mean basal forearm blood flow ratio during this re-control period for subjects randomized to placebo, rosiglitazone, GTN, and GTN+ rosiglitazone were 1.3±0.1, 1.1±0.1, 1.2±0.1,

1.2±0.1, respectively ( p=ns compared to baseline saline; Table 2, Figure 7).

Vitamin C administration did not change basal forearm blood flow in any of the treatment groups (p=ns compared to baseline saline and placebo; Table 2).

When normalizing their flows, forearm blood flow ratios in response to vitamin C alone were 1.1±0.1, 1.1±0.1, 1.2±0.1, and 1.2±0.1 for subjects randomized to placebo, rosiglitazone, GTN, and GTN rosiglitazone respectively (Table 2).

Vitamin C did not change the response to acetylcholine in subjects randomized to placebo, where forearm blood flow in the experimental arm rose from 3.5 ±0.6 to 11.4 ±61.7 at the maximum acetylcholine dose (Table 2, p=ns between the response to acetylcholine in the absence and presence of vitamin C). In subjects treated with GTN, vitamin C co-infusion was associated with the normalization of acetylcholine-induced vasodilation rising from 3.4±1.0 to 12.4±5.5 ml/100ml forearm volume/min (p=ns compared corresponding placebo, p<0.05 compared to rosiglitazone + GTN in the presence of vitamin C, Table 2). The response to acetylcholine during vitamin C co-infusion remained significantly blunted in the group randomized to GTN+ rosiglitazone when compared to placebo and GTN alone (p<0.05, Table 2, Figure 8).

42 In subjects randomized to rosiglitazone alone, vitamin C co-infusion improved forearm blood flow but this was not significant (p=ns compared to rosiglitazone in the absence of vitamin C; compared to corresponding placebo; Table 2).

Forearm flows increased from 2.7 ± 0.2 to 6.5 ± 1.0 ml/min per 100ml in response to vitamin C and did not differ in the absence of vitamin C (2.6 ± 0.2 to

6.1 ± 1.0, Table 2).

When data was normalized and expressed as percent change from baseline, responses to intra-arterial infusions of vitamin C in subjects randomized to placebo experienced forearm blood flow ratios increases from 107% to 230% at the maximum acetylcholine dose. These responses were similar to subjects randomized to GTN as their increases in forearm blood flow ratios rose from 68% to 245%. By contrast, the impact of vitamin C were significantly blunted in the group randomized to GTN+ rosiglitazone when compared to placebo and GTN respectively (p<0.05; multi-way ANOVA; Table 2; Figure 8). Subjects randomized to GTN+ rosiglitazone experienced forearm blood flow ratios increases from 30% to 78%, showing vitamin C had no effect on subjects randomized to endothelium- dependent vasodilation. Subjects randomized to rosiglitazone alone exhibited a non-significant increase in forearm blood flow that rose from 68% to 155% (p=ns; compared to corresponding placebo; compared to rosiglitazone in the absence of vitamin C).

3.4 TABLES AND FIGURES

Table 1: Blood Pressure and Heart Rate Characteristics Characteristics Placebo, n=12 ROSI, GTN, n=11 GTN+ROSI, n=10 n=11 Blood Pressure (mmHg) Visit 1 Systolic Pressure 116 ± 4 119 ± 3 124 ± 1 124 ± 3 Heart Rate, beats/min 82 ± 4 82 ± 7 72 ± 2 80 ± 1

After 3 hrs Systolic Pressure 115 ± 4 117 ± 4 103 ± 4 * 106 ± 4* Heart Rate, beats/min 82 ± 4 88 ± 3 83 ± 5* 91 ± 4*

Visit 2 Systolic Pressure 117 ± 4 118 ± 2 124 ± 2 122 ± 4 Heart Rate, beats/min 81 ± 4 86 ± 6 77 ± 4 78 ± 3

Values are mean ± SE. *p< 0.05 as compared with baseline (multi-way ANOVA). ROSI - Rosiglitazone

Table 2: Forearm Blood Flow Responses Infusions Placebo, n=12 ROSI, n=11 GTN, n=11 GTN+ROSI , n=10

Forearm Blood Flow (ml/100ml forearm volume/min) in the Infused Arm

Saline 3.2 ± 0.3 2.6 ±0.2 3.2 ± 0.3 2.8 ±0.3 Ach 7.5 µg/min 6.9 ± 1.1 3.8 ± 0.5 4.9 ± 0.7 4.3 ± 0.7 Ach 15 µg/min 9.1 ± 1.8 4.6 ± 0.7 * 6.0 ± 1.0 5.1 ± 1.0 Ach 30 µg/min 11.5± 2.0 6.1 ± 1.0 9.5 ± 1.3 7.4 ± 1.1

Vit C + Saline 3.5 ± 0.6 ‡ 2.7 ± 0.2 ‡ 3.4 ± 0.3 ‡ 3.5 ± 0.6 ‡ Vit C + Ach 7.5 µg/min 6.9 ± 1.2 4.1 ± 0.6 5.7 ± 0.9 4.6 ± 0.8 Vit C + Ach 15 µg/min 9.7 ± 1.3 4.8 ± 0.7 * 8.7 ± 1.2 5.3 ± 1.0 * Vit C + Ach 30 µg/min 11.4± 1.7 6.5 ± 1.0 12.4 ± 1.7 5.9 ± 0.9

Forearm Blood Flow Ratio (Infused to Non-Infused)

Saline 1.1 ± 0.1 1.1 ± 0.1 1.2 ± 0.1 1.2 ± 0.1 Ach 7.5 µg/min 2.4 ± 0.3 1.5 ± 0.2 1.9 ± 0.3 1.6 ± 0.2 Ach 15 µg/min 3.1 ± 0.5 1.7 ± 0.2 * 2.2 ± 0.3 * 1.7 ± 0.2 * Ach 30 µg/min 4.0 ± 0.6 2.5 ± 0.5 3.7 ± 0.7 2.8 ± 0.5

Re-control with saline 1.3 ± 0.1‡ 1.1 ± 0.1‡ 1.2 ± 0.1‡ 1.2 ± 0.1‡

Vit C + Saline 1.2 ± 0.1 ‡ 1.0 ±0.1 ‡ 1.2 ± 0.1 ‡ 1.2 ± 0.1 ‡ Vit C + Ach 7.5 µg/min 2.4 ± 0.3 1.7 ± 0.2 2.1 ± 0.5 1.6 ± 0.2 Vit C + Ach 15 µg/min 3.2 ± 0.4 1.8 ± 0.2 † Ψ 2.9 ± 0.5 † 1.8 ± 0.3 * Vit C + Ach 30 µg/min 3.8 ± 0.8 2.8 ± 0.4 4.2 ± 0.6 2.0 ± 0.2

Values are mean ± SE. Forearm Blood Flows are expressed as ml/100ml of forearm volume/min

Significance refers to absolute, forearm blood flow in infused arm, % change in response to Ach *p<0.05 compared to placebo ‡ p=ns; compared to baseline saline † p=ns; compared to placebo + vitamin C Ψ p=ns; compared to rosiglitazone (without vitamin C infusion) Ach = Acetylcholine; Vit C =Vitamin C; ROSI - rosiglitazone

44 Healthy Volunteers

Visit 1 - day 1 Blood Pressure measurements Randomization 4 Treatment Groups

Placebo (n=12) GTN (0.6mg/hr/day; (n=11) Rosiglitazone (n=11; 4 mg (bid)) GTN + rosiglitazone (n=10)

3 hour follow - up Blood Pressure measurements

Visit 2 - day 8 Blood Pressure measurements Vascular function measurements with intra-brachial infusions of acetylcholine Re-control with intra-brachial infusions of saline Intra-brachial infusions of vitamin C (assess role of oxidative stress)

Figure 1: Study Protocol. Subjects were randomized to either placebo, rosiglitazone, transdermal GTN for 7 days, or the combination of both transdermal GTN and rosiglitazone. Blood pressure and heart rate measurements were recorded at visit 1, 3 hour follow-up, and at visit 2. Vascular function measurementes were conducted at visit 2.

Ach co-infused with saline Vitamin C co-infused with saline (15 mins)

Recontrol Baseline (saline) C C FBF (saline; 10 mins) B B

A A

FBF recorded during last 3 Vitamin C co-infused with minutes of each infusion rate Ach; FBF recordings repeated Intra-brachial needle insertion

Ach A = 7.5 ug/min; Ach B = 15 ug/min; Ach C = 30 ug/min; Vitamin C = 24mg/min FBF = Forearm Blood Flow

Figure 2: Schematic of Infusion Procedures. Subjects enter the laboratory and are asked to rest on a bed after taking standing blood pressure measurements. The non-dominant arm is cannulated. After a period of 20 minutes, baseline values are recorded with saline infusions. Vasomotor response to acetylcholine is conducted in a stepwise fashion in incremental doses. Before initiating vitamin C infusions, saline is re-infused to ensure return to baseline values. Forearm blood flow recordings are taken during the last 3 minutes of each dose. A – Acetylcholine (7.5 ug/min); B – Acetylcholine 15 ug/min); C – Acetylcholine (30 ug/min); Vitamin C – 24 mg/min.

Venous occlusion released

Venous return Upper arm cuff

occluded inflated (10 second) Upper arm cuff deflated (10 seconds) Experimental Arm (Infused – Acetylcholine) 10 ml/100ml forearm volume/min Wrist cuff inflated (3 minutes)

Control Arm (Non-Infused) 2 ml/100ml forearm volume/min

Time (seconds)

(Infused Arm) Forearm Blood flow Ratio: ------= 10/2 = 5 (Non-infused Arm)

Vasoactive ratio – baseline ratio Calculating Percent Change in forearm blood flow ratio: ------* 100 Baseline ratio

Figure 3: Plethysmographic curve. Forearm Blood Flow measurements are calculated by taking slope of the arterial inflow response (increase in volume during venous occlusion). Forearm blood flow is expressed as ratio of infused over non-infused arm.

Figure 4: Visual display of forearm blood flow assessment. The picture to the left (full body) demonstrates the intricate setup of venous occlusion plethysmography with both upper arm and wrist cuffs in place. The picture to the right shows the placement of the strain gauge, used to measure forearm blood flow.

Figure 5: Baseline forearm blood flow ratios . Data points are individual subjects and are organized into their respective intervention groups. Horizontal bars represent mean ratio values. Vertical bars represent standard deviations. Forearm blood flow ratio is expressed as ratio of infused to non-infused arm. Saline infusion constitutes baseline forearm blood flow (p=ns compared to control group; multi-way ANOVA)

* 400 * * Placebo ROSI 350 * *p<0.05 GTN 300 * * GTN + ROSI 250 * * 200 *

150

100 % Change in Forearm Blood Flow Forearm Flow in Change Ratio Blood % 50

0 Ach 7.5 ug/min Ach 15 ug/min Ach 30 ug/min

FIGURE 6: Forearm blood flow responses to intra-brachial acetylcholine. Forearm blood flow ratio (expressed as absolute percent change from baseline) during intra-brachial infusions of acetylcholine. The response to acetylcholine was significantly attenuated in subjects randomized to GTN, ROSI, GTN+ROSI as compared to placebo (repeated measures ANOVA, effect of group: * p<0.05 compared to placebo). ROSI – rosiglitazone

FIGURE 7: Re-control forearm blood flow ratios. Normalized forearm blood flows at baseline and re-control were similar after acetylcholine-induced vasodilation. (p=ns compared to control group; multi-way ANOVA).

52 400 350 Placebo ns ROS I 300 Effect of group (compared to placebo): * p<0.05 ns GTN GTN + ROSI 250 *

200

150

100

% Change in Forearm Forearm Blood Ratio in Flow Change % 0 Vit C + Vit C + Vit C + Ach 7.5 ug/min Ach 15 ug/min Ach 30 ug/min

FIGURE 8: Forearm blood flow Responses to vitamin C and acetylcholine. In the presence of vitamin C, endothelial responses remained significantly attenuated in subjects randomized to rosiglitazone + GTN; in contrast, forearm blood flow responses were normalized in the subjects randomized to GTN.

4.0 Discussion

The study was designed to determine if the effect of PPAR-γ activation with rosiglitazone could prevent endothelial dysfunction induced by continuous GTN therapy.(14;233) In the current study, we tested the hypothesis that rosiglitazone would prevent the loss of endothelium-dependent vasodilation to acetylcholine in healthy volunteers randomized to sustained therapy with transdermal GTN

(0.6mg/hr/day) for 7days.

The results of this study demonstrate that our hypothesis was rejected, as concurrent therapy with rosiglitazone did not reverse the impaired intra-brachial responses to acetylcholine to sustained therapy with transdermal GTN. The study documents that healthy volunteers treated with transdermal GTN alone,

GTN combined with rosiglitazone and rosiglitazone alone develop significant endothelial dysfunction, assessed by a loss of endothelium-dependent vasodilation to acetylcholine. The abnormalities in vascular function were normalized in the GTN alone group with intra-brachial infusions of vitamin C, suggesting reductions in NO bioavailability were caused, at least in part, by an increase in vascular superoxide anion production and subsequent inactivation of

NO. Surprisingly, rosiglitazone treatment alone also resulted in a significant loss of endothelium-dependent vasodilation to acetylcholine, which was no longer significant with intra-brachial infusions of vitamin C (p=ns compared to corresponding placebo). This suggests that rosiglitazone’s negative impact on endothelial function appears to be mediated by oxidative stress.

54 4.1 Nitrate-Induced Endothelial Dysfunction

After 7 days of treatment with GTN, subjects randomized to GTN alone showed evidence of endothelial dysfunction. This evidence is consistent with previous reports from our laboratory, which demonstrated that continuously applied transdermal GTN therapy is associated with an increase in vascular oxidative stress.(14;44;135) As described in literature review, several mechanisms have been described to explain GTN-induced vascular superoxide anion production in the development of nitrate tolerance and include activation of membrane oxidases, and NOS uncoupling. (12)

4.2 Rosiglitazone-Induced Endothelial Dysfunction

Thiazolidinediones are a novel class of insulin sensitizing agents used to improve glycemic control. (31) Human studies have demonstrated that rosiglitazone improves endothelial function, observed as improvements in endothelium- dependent vasodilation after 8 to 52 weeks of treatment in patients with diabetes and metabolic syndrome.(19;21;37;248) It has been suggested that rosiglitazone may have a direct anti-oxidative effect on the vasculature and this may be independent of its effect on glucose control by modulating the activity of membrane oxidases and inflammatory pathways.(27) In light of this, we hypothesized that rosiglitazone’s beneficial effect on endothelial function could prevent endothelial dysfunction induced by sustained GTN therapy.

55 The results of this study indicate that co-treatment with rosiglitazone did not attenuate the development of nitrate tolerance. Interestingly, treatment with rosiglitazone alone caused endothelial dysfunction, assessed as a loss of endothelium-dependent vasodilation to acetylcholine. The observed impaired vasomotor responses with rosiglitazone therapy could partly be explained by the parallel relationship between endothelial function and insulin sensitivity. Previous studies demonstrating an improvement in endothelial function in patients with diabetes and metabolic syndrome were also paralleled by an improvement in insulin sensitivity, characterized as an improvement in fasting insulin or indexes of insulin sensitivity. Further, in non-diabetic patients with coronary artery disease, rosiglitazone failed to improve endothelial function or modify intercellular adhesion molecule-1 or vascular cell adhesion molecule-1 levels, despite reducing markers of inflammation that include E-selectin, Von Willebrand factor,

C-reactive protein, fibrinogen.(249) The relationship between insulin sensitivity and endothelial function is further supported by the work of Pistrosch et al., who demonstrated a 60% increase in insulin sensitivity and a corresponding improvement in endothelial function with a 12 week treatment of rosiglitazone in patients recently diagnosed with type 2 diabetes mellitus. (19) Taken together, this suggests that rosiglitazone’s purported benefit on vascular function is likely mediated by an improvement in insulin sensitivity rather then changes in vascular

NO bioavailability. This could possibly explain why we did not see improvements in vasodilator responses to acetylcholine in subjects randomized to GTN and

56 rosiglitazone or rosiglitazone alone, as we adopted the use of healthy volunteers with what is thought to be normal insulin sensitivity indexes.

Further, our results are in contrast to Hetzel et al. who reported an improvement in flow-mediated dilation in healthy volunteers randomized to short-term rosiglitazone therapy (21 days), despite no change in glycemic status or significant changes in tumor necrosis factor-α, interlukin-6, intercellular adhesion molecule-1, or vascular adhesion molecule-1.(233) The explanation for differences between our findings and those of Hetzel et al (233) are likely explained by differences in techniques used to evaluate endothelial function (venous occlusion plethysmography versus flow-mediated dilation), age-related differences (18 to

30 years versus 25 to 40 years), duration of treatment (7 versus 21 days), and a lack of a placebo-control group in the Hetzel et al. protocol. The contradictory results between ours and those of Hetzel et al. and others are likely due to technique and scope of the experiment. Flow-mediated dilation used by Hetzel et al. is unsuitable in our model of nitroglycerin-induced endothelial dysfunction, as we have previously shown that sustained therapy with GTN persistently dilates the conduit arteries, making it difficult to assess changes in NO bioavailability with various interventions.(218) Our results also indicate that our healthy volunteers had normal glycemic status, and another possible explanation for the differences in rosiglitazone’s effect on endothelial function seen in our study here and those seen in patients having risk factors for cardiovascular disease

(excluding diabetes), could possibly be due to rosiglitazone’s direct effect on NO bioactivity. Campia et al. (37) evaluated non-diabetics with cardiovascular risk

57 factors (hypertension and hypercholesterolemia) and found that improved endothelial function was only correlated to changes in total cholesterol and

Hetzel et al. found improvements in endothelial function were likely due to beneficial changes inflammatory markers. While we know rosiglitazone can beneficially act on these parameters to improve endothelial function via modulation of genes associated with lipid and glucose metabolism, our results show that in a setting of nitrate tolerance, rosiglitazone has a direct negative effect on the availability of NO, which was improved with vitamin C co-infusion

(p=ns compared to corresponding placebo).

A possible explanation of rosiglitazone’s effect on NO bioavailability stem from reports that suggest rosiglitazone can modulate the activity of NOS. In support of

Hetzel et al., findings Ikejima et al. demonstrated that treatment with pioglitazone can improve intracellular levels of tetrahydrobiopterin, an essential NOS cofactor in NOS function.(250) This is in contrast to Linscheid et al., who demonstrated that rosiglitazone can reduce intracellular levels of tetrahydrobiopterin by inhibiting guanosine triphosphate cyclohydrolase, an enzyme involved in tetrahydrobiopterin synthesis.(251) This effect may reproduce and/or further propagate the negative effects of GTN therapy on this important cofactor. IN agreement with this, rosiglitazone inhibited cytokine-induced NO synthesis in

3T3-L1 adipocyte cells.(251) To the best of our knowledge there is no experimental evidence to date that has evaluated rosiglitazone on endothelial NOS cofactors in endothelial vessels, and this observation by Linscheid et al could explain the

58 loss of vasodilatory effect in response to acetylcholine in subjects randomized to rosiglitazone alone. This finding may aid in explaining the harmful effects of rosiglitazone on vascular NO bioavailability observed in our study in healthy volunteers devoid of risk factors for cardiovascular disease.

Interestingly, the impaired endothelial responses to acetylcholine induced by therapy with rosiglitazone were partially reversed with vitamin C, suggesting its negative impact on endothelial function is dependent on oxidative stress.

Similarly, subjects randomized to both continuously applied transdermal GTN and rosiglitazone also experienced endothelial dysfunction, which was not normalized with vitamin C. This effect was a complete surprise, and we are not certain as to why vitamin C had no effect on vasomotor responses in this group.

As described above, this could be due to a combination of reduced intracellular levels of tetrahydrobiopterin and increased vascular oxidative stress.

4.3 Limitations of the Current Study

Within this thesis, the studies regarding nitrate tolerance, nitrate-induced endothelial dysfunction, and the effects of rosiglitazone on endothelial function have been reviewed in detail. The results of our study examining the effects of

GTN on forearm blood flow have been presented. This ambitious project took more then two years to complete and given its invasive nature (brachial artery procedures) required the assistance of licensed cardiologists and registered nurses.

As this was an observational study, we did not attempt to elucidate molecular and physiological mechanisms of the observed effects of GTN and rosiglitazone on the forearm vasculature. However, we proposed several potential mechanisms that could possibly account for these effects based on findings in our lab and those of the literature.(17;18) An important limitation in this study is the use of healthy volunteers. The rationale for the use of healthy volunteers is based on their consistent vasomotor responses to vasoactive medications. By contrast, patients with cardiovascular disease have much more heterogeneous responses based on both their disease state and their drug regiments. As a result, in patients, interventions that cause endothelial dysfunction would make studying endothelium-dependent consequences difficult. Although our results may not be transferable to a clinical setting, our results are consistent with our previous findings that have shown that nitrates cause severe ROS-mediated endothelial dysfunction and NOS uncoupling in healthy volunteers with fully functional antioxidant systems. (14;44;252)

Due to rosiglitazone’s surprising effect on vasomotor function, we were motivated to measure fasting insulin and glucose levels from stored plasma of the healthy volunteers recruited for this study. Blood samples were obtained overnight fast one each visit and were drawn into EDTA anti-coagulated vacutainer tubes.

Samples were assayed for fasting glucose by glucose oxidase technique on an automated analyzer. Specific insulin was measured by an ultra-sensitive

60 electrochemiluminescence immunoassay kit. This assay shows 0.01% cross- reactivity to intact human pro-insulin. An estimate of insulin sensitivity, the homeostasis model assessment of insulin resistance was derived with the fasting insulin and glucose values obtained. The results of our analysis revealed neither rosiglitazone nor GTN treatment modified fasting insulin, glucose, or the homeostasis model assessment for insulin resistance. Baseline fasting glucose levels were 5.0±0.3, 4.8±0.7, 5.0±0.2, and 5.1±0.2 mmol/L for subjects randomized to placebo, rosiglitazone, GTN, and GTN + rosiglitazone. After treatment, fasting glucose levels were 5.3±0.1, 5.6±0.2, 5.1±0.1, and 5.1±0.1 mmol/L for subjects randomized to placebo, rosiglitazone, GTN, and GTN + rosiglitazone, respectively. In all cases the homeostasis model of assessing insulin resistance did not significantly differ between treatment visits.

Another important limitation to our study was that it was specifically powered to see significant differences in response to intra-brachial infusions of acetylcholine between placebo and sustained GTN at the highest infusion rate (30 ug/min).

Based on our previous work, a treatment effect size of 50% revealed that a sample size of 11 per group to achieve 80% power was required. In agreement with this finding, we found a statistically significant difference between placebo and sustained GTN in response intra-brachial infusions of acetylcholine, in addition to a blunted response in the groups randomized to rosiglitazone and the combination therapy. The intra-brachial infusions of vitamin C was incorporated into this protocol to help assess the physiologic processes involved in the loss of

61 vasodilatory responses to acetylcholine in a setting of nitrate tolerance, however.

Further, we also did not adequately control for compliance to treatment regimens.

This is due to difficulties associated with measuring levels of NO metabolites, primarily attributed to their stability and rapid interactions with superoxide anions.

As a result, we relied on the responses to intra-brachial responses to acetylcholine as a proxy for compliance in the subjects randomized to sustained

GTN alone.

Although clinical endothelial dysfunction observed in vascular diseases differs from endothelial dysfunction induced by chronic GTN treatment, we believe the results from this study have clinical significance. As the prevalence of diabetes in cardiovascular patients is on the rise,(253) evaluating interventions that can attenuate the development of nitrate tolerance is of clinical importance. The use of long-term nitrate therapy in patients with chronic coronary artery disease is associated with an increase in adverse events, and current intermittent therapy is linked to higher incidences of myocardial infarctions and worsening of endothelial dysfunction. (193;197)

Furthermore, recent meta-analyses from the FDA and Nissen et al. observed that rosiglitazone use is associated with a higher risk of myocardial ischemic events when compared with placebo, or other anti-diabetic drugs in patients with type 2 diabetes.(254-256) The risk is even greater in those with underlying cardiovascular disease, and those taking concomitant nitrate therapies.(256) These conclusions

62 were based on a retrospective analysis of short-term trials that were not designed to evaluate rosiglitazone’s impact on cardiovascular outcomes. Our results presented here are hypothesis generating and more studies are necessary, especially in patients with coronary artery disease to assess the benefit of rosiglitazone on endothelial function and in those prescribed nitrate therapy.

5.0 Conclusion

The results of this clinical study examining the effects or rosiglitazone and GTN on endothelial function have been presented. Taken together, our study results suggests that continuous transdermal GTN (0.6mg/hrs/day) therapy for 7 days causes significant endothelial dysfunction, assessed as a loss of endothelial- dependent vasodilation to acetylcholine. Abnormalities in vascular function induced by chronic GTN treatment were brought on by an increase in vascular superoxide anion production, as they were normalized with vitamin C. Short-term treatment with rosiglitazone had a detrimental impact on endothelial function, observed as impaired vasodilatory responses to acetylcholine. These responses were partly mediated by an increase in vascular ROS, as the blunting associated with intra-brachial responses to acetylcholine were no longer significant with vitamin C co-infusion (compared to corresponding placebo). Similarly, continuous transdermal GTN therapy co-treated with rosiglitazone had a similar negative impact on endothelial function, which was again not reversed by intra-arterial infusions of vitamin C.

These results add to the growing body of evidence that GTN therapy is associated with oxidative stress. Moreover, this data also augments the evidence in the literature supporting rosiglitazone’s effect on endothelial function. While the majority of studies support rosiglitazone having a positive effect on endothelial function in patients with diabetes mellitus, its effect on endothelial function in non-diabetics with cardiovascular disease is not fully explored. Further studies must attempt to quantify rosiglitazone’s role in a cardiovascular population and evaluate its effect on cofactors endothelial NOS. An intriguing hypothesis that warrants attention is that if glycemic control is established in patients with cardiovascular disease, then the beneficial impact of drugs like rosiglitazone is lost. Additional research will need to be done given the observation that this class of drugs appears to be have adverse effects in patients with cardiovascular disease, which seem to be further augmented in patients receiving organic nitrates. (257)

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