Derived Resuscitation Fluids on Euvolemic and Hypovolemic Rats

PHYSIOLOGIC CHANGES INDUCED BY INTRAVENOUS INFUSION OF KERATIN-

DERIVED RESUSCITATION FLUIDS ON EUVOLEMIC AND HYPOVOLEMIC RATS

FIESKY A. NUÑEZ JR, MD

A Dissertation Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

In Partial Fulfillment of the Requirements

For the Degree of

DOCTOR OF PHILOSOPHY

Physiology and Pharmacology

May 2012

Winston-Salem, North Carolina

Approved by

Thomas L. Smith, PhD, Advisor

Mark Van Dyke, PhD, Chair

George Christ, PhD

Patricia Gallagher, PhD

Ann Tallant, PhD DEDICATION

To my wife: Alejandra, your infinite patience and understanding allowed me to mature as a person and as a scientist. Without you I would not have accomplished this feat. I love you

To my parents, for your unconditional support that allowed this road to be filled with joy and success.

To my sister: Mary, your wisdom and emotional support gave me the strength and hope I needed in order to achieve many things. You always make me think of a better tomorrow and a better me.

ACKNOWLEDGEMENTS

Special Thanks to:

Tom: For your impeccable mentorship and disposition to always put my best interests first.

Mark: For your dedication and patience to teach such an impatient subject.

Mike: For your knowledge and hard work in day-to-day lab efforts.

Maria: Thank you for your patience in the lab and for sharing your immense knowledge of keratin with me.

Keratin Krew and Biomaterials core: Roche, Bailey, Chris, Lauren, Mary, Julie, Jill,

Carmen, Deepika. Your collaborations allow individual contributions to flourish into an extraordinary lab.

Orthoapedic Lab: Beth, Eileen, Martha, Jan, Casey. All of you make this workplace pleasant and successful, I hope for years to come it remains this way because it’s my second home.

Keranetics Personnel: Thank you for your assistance and support during our collaboration.

iii

TABLE OF CONTENTS

LIST OF ILLUSTRATIONS AND TABLES……………………………………………………………… V

LIST OF ABBREVIATIONS……………………………………………………………….………………… VII

ABSTRACT……………………………………………………………………………..………………………….. IX

CHAPTER I – Introduction…………………………………………………………………………………… 1

CHAPTER II – Vasoactive Properties of Keratin-Derived Compounds……………..…… 28

CHAPTER III – Hemodynamic Recovery After Hypovolemic Shock

and Resuscitation with Lactated Ringer’s and

Keratoe Resuscitation Fluid;

A Novel Colloid………………………………………………………………………….… 58

CHAPTER IV – Conclusions……………………………………………………………………………… 93

APPENDICES…...... 104

SCHOLASTIC VITA………………………………………………………………………………………… 112

iv LIST OF ILLUSTRATIONS AND TABLES

CHAPTER I - Introduction

Figure 1 – Renin-Angiotensin-Aldosterone system………….…………………………………... 6

Figure 2 – Shear stress-mediated release of NO from the endothelium………….…..…8

Table 1 – Safety tests conducted on a Keratose biomaterial…………………..…………… 16

CHAPTER II - Vasoactive Properties of Keratin-Derived Compounds

Figure 1 - Size exclusion chromatography of keratose samples………………………… 52

Figure 2 - Video stills of the same vessel before and after KRF administration……53

Figure 3 - Change in arteriolar diameter with i.v. administration of Fluid…………… 54

Figure 4 - Effect on arteriolar diameter with and without L-nitro-arginine…………. 55

Figure 5 - Average arteriolar diameter increase with KRF

of different viscosities……………………………….……………………………………… 56

Figure 6 - Average arteriolar diameter increase with

infusion of Alpha and KAP containing fluids………………………………….…… 57

CHAPTER III - Hemodynamic Recovery After Hypovolemic Shock and Resuscitation with Lactated Ringer’s and Keratoe Resuscitation Fluid; A Novel Colloid

Figure 1 – Hemodynamic changes after hypovolemic shock and

treatment with low-volume resuscitation fluids ………………… 83

Figure 2 - Circulating blood volume changes after hypovolemic shock and

v treatment with low-volume resuscitation fluids...... 84

Figure 3 – Hemodynamic changes after hypovolemic shock and

treatment with full-volume resuscitation fluids……………………...…… 85

Figure 4 - Circulating blood volume changes after hypovolemic shock and

treatment with full-volume resuscitation fluids...... 86

Figure 5 – Tissue Histology………………………………………………………………………...... … 87

Supplemental Figure 1 - Ultrasound Transit Flow Probes...... 88

Supplemental Figure 2 - Mean Arterial Pressure and Heart Rate Monitoring…….89

Supplemental Table 1 - Hemodynamics and Arterial Blood Gases:

Low Volume Resuscitation, Short Term Assessment……………………….… 90

Supplemental Table 2 - Hemodynamics and Arterial Blood Gases:

Full Volume Resuscitation, Short Term Assessment………………………..… 91

Supplemental Table 3 - Hemodynamics, Arterial Blood Gases and Electrolytes:

Long Term Assessment………………………………………………………………………. 92

CHAPTER IV - CONCLUSIONS

Figure 1 - Blood Pressure Recovery After Hypovolemic Shock

and Treatment with KIF-KRF and AK-KRF…………………………………..…… 98

APPENDICES

Table 1 - Different Fractions of Keratose:

vi Classification by components,

their respective biologic activity and potential clinical uses……………….. 107

Figure 1 - Concentration of Keratose needed to prepare a compound

with a viscosity of 3.88cP at 10dynes/cm2...... 109

vii LIST OF ABBREVIATIONS

Abs Absorbance

ACV Actively circulating volume

ANOVA Analysis of variance

CBV Central blood volume

CI Cardiac Index

CO Cardiac output cP Centipoise

DI Deionized

Hb Hemoglobin

HCT Hematocrit

HEPES Hydroxyethyl- piperazine ethanesulfonic acid

HR Heart rate i.v. or IV Intravenous

IEx Ion exchange

KAP Keratin-associated protein

KDa Kilo Dalton

KIF Keratose Intermediate Filament

KRF Keratin resuscitation fluid l-NA l-nitroarginine

MAP Mean Arterial Pressure

viii mmHg Millimeters of mercury

NLMWCO Nominal low molecular weight cutoff

NO Nitric oxide

PBS Phosphate buffered saline

Phe Phenylephrine

SEC Size exclusion chromatography

SNP Sodium Nitroprusside

ULF Unit length filament.

ix ABSTRACT Fiesky A. Nuñez Jr. MD PHYSIOLOGIC CHANGES INDUCED BY INTRAVENOUS INFUSION OF KERATIN- DERIVED RESUSCITATION FLUIDS ON EUVOLEMIC AND HYPOVOLEMIC RATS Research Problem: The field of cardiovascular research has evolved significantly over the last decade, and yet cardiovascular related deaths remain a primary cause of trauma related death. Shock and resuscitation are topics of high importance in the field of emergency medicine due to the high mortality that they carry, which has not been properly addressed in the past decades. The development of better treatment options for this entity is essential. Methods: 1) The vasoactive properties of intravenously infused keratin- derived compounds were assessed, with the use of cremaster muscle microvascular observations in the Sprague-Dawley rat. 2) After assessing feasibility for intravenous infusion in the microvascular model, hemodynamic and metabolic recovery after hemorrhage and resuscitation with commercially available crystalloids and keratin compounds was studied. Ultrasound dilution method was used to determine changes in cardiac index, stroke volume and circulating volumes. Femoral cannulation was used to determine changes in arterial blood pressure and heart rate. Arterial blood gases were measured to compare acid-base profile changes.

Conclusions: The initial keratin compounds tested in this research elicited an unexpected vasodilatory effect on the microvasculature of the cremaster muscle, which can be further developed for other uses. Further purification allowed the formulation of keratin compounds without vasodilatory effects, improved osmotic effects, and enhanced viscosity. These compounds are biocompatible colloids for resuscitation purposes. These enhancements will allow the use of this compound with lower volumes than the current gold standard and significantly reduce complications.

x CHAPTER I

INTRODUCTION

Fiesky A. Nunez Jr. MD

1 Clinical Significance

Traumatic injuries account for 9% of annual global mortality and it is estimated that half of these deaths occur in individuals between 15 and 44 years of age (1).

Hemorrhagic shock is the leading cause of death among trauma patients worldwide.

50% of patients that suffer bleeding severe enough to cause shock will die despite aggressive and prompt treatment (2). Although not reliable as an isolated measure of disease severity, a systolic blood pressure below 100mmHg at emergency department admission increases hospital mortality threefold and sudden death risk tenfold (3, 4). However, continuous research has proven that the only factor that decreases mortality is early diagnosis and prompt restoration of volume and cardiac function (5-7).

Shock is a state of severe impairment in tissue perfusion that results in diminished tissue oxygen delivery and utilization as well as impaired removal of metabolic waste byproducts. Untreated, shock triggers a chain of pathologic events that result in localized cellular death followed by multiple organ dysfunction and death of the individual. The primary treatment for all types of shock remains proper fluid resuscitation and specific efforts to correct the factors that precipitate shock.

Etiology of Shock

Shock is defined as the incapacity of the cardiovascular system to maintain proper tissue perfusion (8, 9). Causes of shock are variable and are grouped into four specific categories according to the pathologic mechanisms involved:

2 1. Cardiogenic Shock: Incapacity of the heart to maintain proper cardiac output

due to intrinsic abnormalities of the myocardium. It is seen during

myocardial infarction, cardiac dysrhythmias or due to chronic development

of congestive heart failure.

2. Obstructive Shock: As the name implies it is caused by obstruction of blood

flow. Its causes are commonly found outside the myocardium: Pulmonary

embolism, tension pneumothorax and cardiac tamponade.

3. Distributive Shock: Caused by uncontrolled and generalized vasodilation

which greatly increases the size of the vascular system and, the myocardium

cannot maintain proper blood pressure of such a large volume. It is often

triggered by the release of potent vasodilators that may be intrinsic

(histamine during anaphylactic reactions) or extrinsic (endotoxemia during

septic shock) (10).

4. Hypovolemic Shock: Defined by insufficient circulating volume to fill the

heart chambers and maintain proper cardiac output. It is most commonly

caused by uncontrolled hemorrhage, which also carries the highest mortality.

It is also seen during severe vomiting and diarrhea, burns and high urinary

output states. Some clinicians use the etiologic name in order to easily clarify

the clinical scenario: hemorrhagic shock, traumatic shock, surgical shock,

burn shock, but these are all subsets of hypovolemic shock.

3 Pathopysiology of Hypovolemic Shock

When a pathologic insult causes circulating volume to decrease in the cardiovascular system, blood pressure and flow to organs inevitably decrease causing impaired venous return. These changes cause inadequate preload and heart contractility is diminished, which further drops blood pressure and exacerbates the pathologic cycle.

Inadequate tissue flow causes increased anaerobic glycolysis with large production of lactic acid. The resulting lactic acidosis has a negative inotropic effect, decreases vascular reactivity to catecholamines, and, in severe cases, can cause coma. The progressive metabolic acidosis and localized necrosis due to ischemia can result in multi-organ failure and death (11).

Compensatory Mechanisms: Neural Control of Homeostasis

Decreased mean arterial pressure is detected by aortic arch and carotid sinus baroreceptors decreasing the number of impulses sent to the brain stem. This signaling pattern produces modulating changes in the autonomic nervous system, which consist of increased sympathetic tone and decreased parasympathetic tone.

Decreased parasympathetic tone allows the sympathetic stimulus to predominate in the cardiovascular system:

1. Beta-1 adrenergic stimulation on the heart causes increased inotropism

and chronotropism. The combination of a more powerful myocardial

contraction and increased heart rate causes a significant increase in

cardiac output and blood pressure (8).

4 2. Alpha-1 adrenergic stimulation on the vascular system causes

widespread vasoconstriction to raise blood pressure. Few organs are not

affected by this vasoconstrictive reflex; increase metabolism in the

myocardium causes coronary vasodilation, cerebral blood flow is

maintained due to CNS autoregulation, kidneys are spared initially but as

hypovolemia worsens they become underperfused causing decreased

glomerular filtration rate and prerenal azotemia (nitrogenous product

accumulation in the blood). Kidney hypoperfusion marks the triggering

point of the humoral control of homeostasis, which is explained in detail

below (8).

Compensatory Mechanisms: Humoral Control of Homeostasis

Decreased renal perfusion is detected in the juxtaglomerular apparatus triggering the release of renin, which hydrolyzes angiotensinogen secreted from the liver into angiotensin I. Angiotensin I is further cleaved by the angiotensin converting enzyme

(ACE) into angiotensin II (Figure 1). Both these hormones aid in restoring blood pressure through stimulation of vascular smooth muscle constriction and increased peripheral vascular resistance. Angiotensin II has other actions that aid in normal restoration of blood pressure and circulating blood volume:

1. Adrenal gland stimulation to release aldosterone, a mineralocorticoid

hormone that acts by increasing sodium and water reabsorption from the

distal convoluted tubule and collecting ducts (9, 12).

5 2. Stimulation of the posterior pituitary gland to release Antidiuretic

Hormone, which acts by increasing water retention from the renal

collecting duct as well as increasing peripheral vascular resistance (13).

3. As mentioned above angiotensin II has an intrinsic vasoconstrictor effect,

however, it also causes increased sympathetic activity, as well as water

and sodium reabsorption form the distal convoluted tubules (9).

Figure 1: Renin-Angiotensin-Aldosterone System

Aria Rad, 2006 from http://en.wikipedia.org/wiki/File:Renin-angiotensin-aldosterone_system.png

Microcirculation:

The microcirculation is defined as the system of vessels embedded within organs, which are responsible for the maintenance and distribution of blood flow within that organ. Several hormones and neurotransmitters are actively involved in the regulation of the cardiovascular system but local metabolic changes and specific paracrine hormones more actively control the microvasculature. Local metabolic

6 product buildup causes arteriolar vasodilation to match metabolic activity with proper blood flow. During high metabolic demand, flow is increased and, during hypoperfusion, toxic material buildup, as well as adenosine production from oxygen deprived cells causes vasodilation in order to restore proper flow (14). During physiologic conditions blood viscosity and normal blood flow maintain shear stress on the endothelium, stretch-sensitive Ca+ channels are activated, which signals the production of nitric oxide (NO) from arginine, a reaction that is metabolized by nitric oxide synthase (NOS) (14, 15). The NO that is produced in the endothelium diffuses to the neighboring smooth muscle cells and mediates relaxation through activation of soluble guanylyl cyclase and increased cyclic guanosine monophosphate (cGMP) (15). Other hormones such as Acetylcholine, Bradykinin,

Vasoactive Intestinal Peptide, and Substance P can also stimulate NO synthesis.

However, the shear stress mechanism is believed to be involved in reducing the normal net resting tone of arterioles (Figure 2). During hypovolemic shock increased water reabsorption from the renal tubules as explained previously causes hemodilution and decreased blood viscosity. These changes disrupt the normal homeostasis of nitric oxide and microvascular perfusion control is lost (16, 17).

7 Figure 2: Shear stress-mediated release of nitric oxide from the endothelium

Figure 2A: Normal viscous blood maintains shear stress on the microvascular endothelium. IC = Intracellular space, which represents vascular smooth muscle in this specific diagram

Figure 2B: Shear stress triggers Ca+ sensitive stretch channels and stimulates NO production.

Figure 2C: NO diffuses into vascular smooth muscle cells and causes relaxation through increased cGMP. Vasodilation allows improved tissue flow

8 Therapy

The pathologic changes exposed above present challenging circumstances for treatment; in order to replace lost volume and re-instate tissue perfusion, resuscitation fluids must be infused. However, exaggerated use of these fluids during the pre-hospital course may exacerbate uncontrolled active bleeding, dislodge partially formed clots and dilute existing clotting factors. Surgery to stop active bleeding and replacement of lost components with packed red blood cells and fresh frozen plasma remain the mainstay of treatment. Consequently, during transit between the accident scene and surgical care, proper fluid resuscitation and blood pressure monitoring remains a necessary procedure.

In hypovolemic shock due to hemorrhage, no treatment has proven to be more beneficial than another or as effective as whole blood in re-instating normal tissue oxygenation. When blood products are not readily available, crystalloid or colloid fluid infusion is used.

Isotonic Crystalloids:

Crystalloids are solutions that contain water and electrolytes. These are the most commonly used resuscitation fluids today, among which two specific ones are used for the treatment of hypovolemic shock:

• Isotonic 0.9% NaCl (AKA normal saline or physiologic solution) is composed

of 154meq/L of Na+ and 154meq/L of Cl- with an osmolarity of 290 Osm/L.

9 • Lactated Ringer’s solution contains 130meq/L of Na+, 109meq/L of Cl-,

28meq/L of Lactate, 4meq/L of K+ and 3meq/L of Ca+ with an osmolarity of

273 Osm/L.

These two solutions have been used interchangeably with similar results over the past decades. They have the advantage of low cost, availability, and relatively low incidence of side effects. However, large volumes of such solutions often are necessary because the water they contain readily diffuses out of the vascular space and into the interstitium. The estimated replacement requirement is threefold the volume of hemorrhaged blood, it is commonly thought that 20% of the infused volume remains in the intravascular space (18). Normal saline contains higher Cl- concentration than the extracellular space and may induce hyperchloremia, but it is preferred to Lactated Ringer’s Solution when brain injury is suspected or hyponatremia is present due to its higher concentration of Na+ (154meq/L vs

130meq/L).

Hypertonic Crystalloids:

Hypertonic crystalloids may lower the necessary volume of resuscitation fluid, although the increased tonicity may lead to electrolyte disturbances and hemolysis

(19, 20). These fluids have a sodium concentration that ranges from 250 to

1200meq/L, lower volumes are needed with higher concentrations of sodium due to the osmotic effect it produces. 250ml of 7.5% saline solution produces volume restoration similar to 3L of normal saline (21). Hypertonic crystalloids improve blood pressure, decrease systemic vascular resistance and reduce subsequent need

10 for further resuscitation fluids (22-24). Although some clinical trials and animal studies have shown improved mortality, these results are not consistent and large randomized controlled studies are needed to fully determine their advantages (21,

25).

Colloids

Colloids such as polysaccharides (dextran, hydroxyethyl starch) and proteins

(albumin) are viable alternatives with hyperosmotic properties. There is no doubt that these expand intravascular volume more than crystalloids (26). However, similar outcomes including mortality have been reported with crystalloids and colloids, these results make the justification of their use a difficult one, given their side effect profile (27). Synthetic starch induces coagulation problems due to direct factor VII inhibition and unexplained erythrocyte lysis (28), while albumin has been shown to induce bronchospasm, pulmonary edema, has a negative inotropic effect, and may precipitate congestive heart failure (29, 30).

Keratose

Although no treatment has proven to be as beneficial as human blood products, its lack of availability during the transit time between trauma and surgical care warrants the need for viable resuscitation fluid options. Hemorrhagic shock however poses significant challenges for treatment. A resuscitation fluid must be infused without dislodging partially formed clots, diluting the reserve of clotting factors and red blood cell mass, or further amplifying uncontrolled active bleeding.

11 As such, in the field of trauma surgery, it remains a fundamental task to develop a biocompatible resuscitation fluid, which adequately restores tissue perfusion and cardiac dynamics (11, 19).

In order to improve outcomes of treatment for hypovolemic shock, there is a need to develop a biocompatible resuscitation fluid that re-instates tissue perfusion and improves cardiac dynamics with minimal side effects. Keratin-derived compounds, more specifically keratose, have recently been identified as potential biocompatible resuscitation fluids (31). Keratoses are sulfonic acid-functional keratin proteins obtained using disulfide bond scission with peracetic acid. They can be formulated at various concentrations resulting in fluids with variable viscosities and oncotic characteristics.

Intravenous hyperviscous fluids are believed to restore normal shear stress on vessel walls, which may help restore normal homeostasis of vasodilators such as NO and prostaglandins (PGs) (17, 32). In addition, the hydrophilicity of the sulfonic acid residue resulting from keratin oxidation renders keratose highly soluble in water.

This high tonicity may be beneficial in helping withdraw water from the interstitial space into the vasculature. Using this tonicity, the need for high-volume resuscitation fluids and their resulting electrolyte disturbances would be decreased.

Lower volumes would also decrease logistics requirements for those treating hypovolemic shock.

12 There are two key elements of innovation offered by the use of a keratose-based biomaterial (further defined below) for fluid resuscitation: 1) keratose is viscous, oncotic, and has been shown to produce vasodilation and increase blood flow (33)

(see preliminary data section) and 2) keratose biomaterials are safe, biocompatible, and inexpensive to produce. The goal of this proposal is to expand our extensive preliminary work to investigate the underlying mechanism of keratose’s vasodilation properties in order to optimize the use of this unique biomaterial as a resuscitation fluid.

The disulfide crosslinks in human hair cortical proteins can be broken down by highly controlled methods using either oxidative or reductive chemistries, and soluble keratins can be extracted and purified in high yield (i.e. >80%)(34). If oxidative chemistry is used, the resulting keratins are referred to as “keratoses”. If reductive chemistry is used, the resulting keratins are referred to as “kerateines”.

Kerateines are hemostatic, keratoses are not, and hence the sole focus of this project is on keratoses. Throughout this proposal, the term “keratin(s)” is used to refer generally to human hair keratin proteins. The term “keratose” is intended to refer to the oxidized, non-hemostatic form of extracted keratins. Keratins are highly biocompatible and non-toxic. Research at WFUSM has utilized human hair keratins as implants in numerous animal models including mouse, rat, rabbit, pig, and non- human primates, with no adverse outcomes. Moreover, the keratose used in this proposal as been extensively tested in several safety assays conducted under ISO

10993 standards (Table 1). These tests demonstrate a complete lack of cell or

13 tissue toxicity, genotoxicity, cardiovascular incompatibility, immunogenicity, or pyrogenicity.

Keratin biomaterials typically have been described in the literature in terms of the procedures and processes used to extract them. Extracts of hair, wool and feathers often were “characterized” as heterogeneous mixtures, with estimates of more than

100 protein homologs being proposed as potentially present in wool extracts (35).

More recently, human hair fibers have been described as consisting of 17 type I and type II alpha keratins that exist as obligate heterodimers (36), as well as 85 matrix proteins termed keratin-associated proteins (KAPs)(37). During the past three years we have worked to evaluate the potential of these compounds for resuscitation fluids. In so doing, we have been able to reproducibly manufacture highly purified keratin biomaterials and thoroughly characterize them. As a result, several highly unique characteristics of these purified sub-types have been identified that are not evident in the more heterogeneous fractions. For example, alpha keratin proteins produced in their laboratory have been separated using techniques such as dialysis and chromatography to the level of K81/K31 dimers.

These dimers have been shown to have the capability to form stable tetramers in solution (38), a property that likely contributes to the viscosity characteristics of these materials.

Human hair can be purchased from commercial vendors and is clean, free of contaminants, and ethnically homogeneous. There are no cellular or nuclear

14 materials present in end-cut human hair extracts (as opposed to hair follicles that are used for forensic analysis) so the risk of disease or DNA transmission is eliminated. Moreover, the batch-to-batch variability that is inherent in most naturally-derived biomaterials is not a problem for keratin biomaterials production.

Variability in the appearance of human hair (e.g. color, texture, thickness, curliness, etc.) is the result of the specific keratins present and their relative ratios within the hair cortex. Specific keratins that comprise an individual’s hair fiber are dictated largely by diet and ethnicity. By obtaining human hair from a consistent commercial source, these variables can be eliminated. In the case of our technology, hair is purchased from Asia where diet and ethnicity are more homogeneous than other parts of the world. Most importantly, the relative ratio of different keratose sub- types can be normalized during the biomaterials production process. Our methods rely on separation of keratose sub-types and their de novo recombination into resuscitation fluid formulations. Following this strategy of “synthesizing” the keratose in each lot of fluid, the relative ratio of the sub-types can be highly controlled. Control over the composition of keratose biomaterials leads to predictable chemical, physical, and biological properties. This is another facet of innovation in our approach that lends itself to high translational potential for this research.

15 Table 1. Safety tests conducted on a keratose biomaterial

ISO 10993 Test Description Outcome

ISO 10993-5 Cytotoxicity The biological reactivity of mouse L929 Passed

fibroblasts toward aqueous solutions of

keratose was tested in culture. The viability

of cells exposed to several concentrations of

extract for 24 hours was tested using an

MTT assay.

ISO 10993-10 Guinea pigs are inoculated with saline Passed

Sensitization solutions of keratose plus adjuvant by

intradermal injection. After 7 days, a

challenge was administered by topical

application to the injection sites and signs

of a reaction monitored

ISO 10993-11 Acute Saline solutions of keratose were Passed

Systemic Toxicity administered intravenously or

intraperitoneally, respectively, at 50ml/kg

in mice. Clinical observations, animal

weight, and mortality were noted at 1, 2,

and 3 days.

ISO 10993-3 Genotoxicity Four strains of S. typhimurium and one Passed

16 strain of E. coli were treated with saline

solutions of keratose and the ability to

induce reverse mutations in histidine and

tryptophan genes was assessed.

ISO 10993-10 Irritation Saline solutions of keratose were Passed

administered by intracutaneous injection of

0.2ml in New Zealand White rabbits and the

sites were observed for signs of erythema

and edema at 1, 2, and 3 days.

ISO 10993-11 Determined the presence of chemical Passed

Pyrogenicity pyrogens by measuring an increase in

temperature in New Zealand White rabbits

following intravenous injection of 10ml/kg

of an aqueous solution of keratose.

17 Keratose fraction processing and nomenclature:

Throughout this research on keratose and its application in experimental subjects, the process by which keratose is processed has been refined. With significant advances in these processes, the reproducibility of specific fractions of keratose has been achieved. Further explanation of these processes can be found in chapter 2 and further interpretation of the purification processes is explained in Appendix 1 but the basic nomenclature of keratose will be clarified here in order to ease the readability and understanding of the research.

• “Crude” keratose is the primary result obtained after oxidative chemistry is

applied to “free up” proteins and extract them from the human hair fiber

cortex. This fraction contains alpha helical proteins, gamma proteins, and

KAPs. Alpha proteins are typically extracted in the form of molecular

complexes that contain monomers, dimers, and tetramers, and these are

often further complexed to the KAP molecules.

• Alpha/KAPs keratose as its name implies contains alpha monomers, dimers,

tetramers, and KAPs. This is the compound that was initially slated for study

in this doctoral dissertation but as more specific techniques of isolation were

developed, this compound was studied only in chapter 2. Due to editorial

requests and our lack of definite characterization of these molecular

complexes at the time of publication of Chapter 2, it was referred to as “crude

keratose” in said chapter.

• Keratose Intermediate Filaments (KIFs): This fraction of keratose exclusively

contains alpha dimers and tetramers. Its name derives from the fact that it

18 lacks smaller alpha monomers. Ionic exchange separation was used to obtain

this fraction; this procedure is thoroughly explained in chapters 2 and 3.

During chapter 2 it was referred to as “alpha keratose” and the name was

changed as more characterization information became available during the

course of this research.

• KAPs are also obtained by ion exchange separation and are studied in

chapter 2 alongside alpha/KAPs keratose.

19 SPECIFIC AIMS AND HYPOTHESIS:

Overarching Hypothesis

Human hair derived keratin compounds can be used as a biocompatible hyperviscous and hyperoncotic resuscitation fluid for hemorrhagic shock.

Specific Aim #1:

To determine the vasoactive effects of keratin-derived hyperviscous compounds

(Keratose Resuscitation Fluid [KRF]) on the microvasculature and to determine whether these effects are mediated through the nitric oxide synthase pathway.

This compound can be formulated in a viscosity similar to blood (4cP at 10 dynes/cm2) and infused intravenously to exert shear stress on vascular walls NO and improve tissue perfusion. Nitric oxide production can be blocked locally by reversibly inhibiting eNOS with L-Nitro-Arginine (L-NA). It is important to determine the physiologic effects and the pharmacologic pathways by which

Keratose elicits its effects. Using a cremaster muscle microvascular preparation and videomicroscopy, Specific Aim #1 will evaluate the vasoactive effects of keratose as well as the physiologic pathway it uses to elicit changes (Chapter 2). The cremaster microvascular preparation has been thoroughly established in the literature.

Specific Aim #2

To determine KRF’s suitability as a resuscitation fluid for the treatment of hemorrhagic shock by demonstrating KRF’s capacity to restore normal hemodynamics and decrease long-term morbidity and mortality after hypovolemic shock.

20 Using arterial pressure-guided controlled hemorrhage, rats will be maintained at levels of hypovolemic shock for a determined time and then provided with resuscitation treatment. Hemodynamic parameters of blood pressure and cardiac output will be measured by using femoral arterial cannulation with fluid-filled catheters and ultrasound dilution methods, respectively. Changes in these hemodynamic parameters will be measured at normovolemia, during shock, and following resuscitation. Additional blood withdrawals will be used to measure arterial blood gas profiles. Rats will be randomly assigned to one of two treatment groups: KRF or the current clinical gold standard (Lactated Ringer’s solution), and hemodynamic parameters compared between groups.

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DK, Billiar TR, Dunn DL, Hunter JG, Matthews JB, et al., editors. Schwartz's Principles of Surgery. 9th ed. New York: McGraw-Hill; 2010.

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23 13. Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, Knepper MA.

Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad

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17. Cabrales P, Tsai AG, Intaglietta M. Microvascular pressure and functional capillary density in extreme hemodilution with low- and high-viscosity dextran and a low-viscosity Hb-based O2 carrier. Am J Physiol Heart Circ Physiol. 2004

Jul;287(1):H363-73.

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Oct;95(4):858-65.

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31. Widra A, inventor Alpha-keratose as a blood plasma expander and use thereof. USA Patent: 2004 04-23-2001.

32. Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science. 1985 Mar

22;227(4693):1477-9.

26 33. Nunez FT, S; Callahan, M; Van Dyke, M; Smith, T;. Intravenous Infusion of

Keratose-based Fluid Induces Arteriolar Vasodilation in Cremaster Muscle of Rats

[abstract]. . FASEB J. 2010 April 2010;24 (meeting abstract supplement):593.3.

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38. Bernot KM, Lee CH, Coulombe PA. A small surface hydrophobic stripe in the coiled-coil domain of type I keratins mediates tetramer stability. J Cell Biol. 2005

Mar 14;168(6):965-74.

27 CHAPTER II

VASOACTIVE PROPERTIES OF KERATIN DERIVED COMPOUNDS

Fiesky A. Nunez Jr. MD, Simon Trach, Luke Burnett PhD, Rahul Handa, Mark

Van Dyke PhD, Michael Callahan PhD, Thomas L. Smith PhD

Wake Forest University School of Medicine, Winston Salem, NC 27157

This manuscript was published in Microcirculation 2011 Nov;18(8):663-9, which is

a peer-reviewed Medline indexed journal. Stylistic variations are due to

requirements of the journal.

28 ABSTRACT

Objective: Keratin proteins have been utilized as biomaterials for decades, and are currently under investigation for a variety of tissue regeneration and trauma applications. It has been suggested that certain keratins may have the capacity to act as a colloid in fluid resuscitation applications, providing viscosity and oncotic properties that may be beneficial during acute ischemic events. Oxidized keratin derivatives, also known as keratoses, show good blood and cardiovascular compatibility and thus are the subject of this study.

Methods: The effects of keratose compounds will be assessed using a topload i.v. infusion model and observation of changes in the microvasculature of the cremaster muscle of rats.

Results: Keratose resuscitation fluid (KRF) administration resulted in significant vasodilation in the cremaster muscle. This effect was blocked with pretreatment of l-NA to inhibit NO. Another keratin fraction, alpha-keratose, which is the primary viscosic compound, was not found to induce vasodilation.

Conclusions: The apparent mechanism of vasodilation was found to be NO- mediated and isolated to a particular purified fraction, the KAP.

Key words: keratin, biomaterial, keratin-associated protein, vasodilation, resuscitation, ischemia

Abbreviations used: Abs, Absorbance; ANOVA, analysis of variance; cP, centipoise;

DI, deionized; HEPES, hydroxyethyl- piperazine ethanesulfonic acid; i.v. or IV,

intravenous; IEx, ion exchange; KAP, keratin-associated protein; KDa, kilo Dalton;

29 KRF, keratin resuscitation fluid; l-NA, l-nitroarginine; NLMWCO, nominal low

molecular weight cutoff; NO, nitric oxide; PBS, phosphate buffered saline; SEC, size

exclusion chromatography; ULF, unit length filament.

30 Introduction

Human hair keratins can be classified into three broad groups, alpha- and beta-keratins, as well as the keratin associated proteins or KAP fraction (often referred to as gamma-keratin). Alpha-keratins are the main structural component in hair fibers, have a relatively high molecular weight and low sulfur content, and are the most abundant [18]. KAP are globular, lower in molecular weight, higher in sulfur content, and act as a matrix that holds the cortical super structure together

[16]. The beta-keratins are primarily protective and constitute most of the cuticle

[15]. They are significantly more difficult to extract and do not form useful reconstituted structures.

The keratins found in hair fibers are characterized by generally high cysteine content and disulfide crosslinking. In order to extract useful keratins from end-cut human hair fibers, these disulfide crosslinks must be broken without appreciable disruption of amide bonds, then the strong hydrogen bonding also present in keratinous tissues must be overcome. If hydrogen peroxide or peracetic acid oxidant is used for this disulfide bond scission, cysteine side groups are converted to sulfonic acid and do not readily react with other materials. These keratin derivatives are referred to as “keratose”, which has been investigated as a biomaterial for a number of medical applications and shown to have interesting properties including excellent cell and tissue compatibility [5]. Keratoses can be formulated as a biocompatible fluid with various viscosities and have previously been identified as potential constituents in a resuscitation fluid [21]. In a preliminary experiment

31 described within the patent, Widra showed that intravenous injection of keratose- containing fluid did not induce untoward effects in dogs [21]. The patent states that after 25% blood volume hemorrhage and keratin-containing fluid replacement, the animal showed no signs of anaphylaxis, toxicity, red cell damage, or elevated counts of eosinophils, basophils, lymphocytes, or monocytes.

High viscosity fluids may help release potent vasodilators such as nitric oxide

(NO) and prostaglandins when infused intravenously (i.v.). The release of these mediators is induced through vascular wall shear stress [3, 6]. The mechanism for

NO release has been linked to mechanoreceptors within endothelial cells that are stimulated by shear stress and appears to be linked to a G protein-coupled receptor mechanism [10]. Furthermore, endothelial cells exposed to laminar shear stress have demonstrated enhanced eNOS transcription factors such as eNOS

3’polyadenylation resulting in eNOS expression [13]. Keratose also possesses high tonicity, which may induce net transport of water from the extravascular space into the vascular system. Small molecules can be eliminated from the final product through dialysis in order to avoid leakage of these compounds out of the vasculature. Keratoses are also compatible with the cells and tissues of the cardiovascular system. While the full therapeutic potential of keratins remains to be determined, several clinical pathologies may benefit from treatment with a fluid having these characteristics. We hypothesized that hyperviscous keratose fluids would provide the most resuscitative benefit, simply due to their mechanical effects and hygroscopic nature, and endeavored to study their behavior in the microcirculation. Keratose-based fluids formulated at different viscosities were

32 compared to another high viscosity compound, Hetastarch 6% in saline solution.

Surprisingly, the keratins were observed to have vasoactive properties, so a potential physiologic mechanism and the source of keratose-induced vasodilation was also investigated in this study.

33 Methods

Keratose was prepared as previously described [20]. Briefly, human hair was obtained from a commercial vendor and treated with peracetic acid to break protein disulfide crosslinks. Residual oxidant was removed by rinsing the retted hair and soluble keratins extracted into excess tris base and deionized (DI) water. The extracted keratose solution was dialyzed with a 30 kiloDalton NLMWCO cartridge in order to eliminate small molecules from the final product, thus avoiding leakage of these compounds out of the vasculature. The dialyzate was concentrated, neutralized, lyophilized, and sterilized by gamma irradiation (1M Rad).

Resuscitation fluids were prepared by aseptic reconstitution of the keratose powder with phosphate buffered saline at the desired concentration. Viscosity was determined with a DV-II+ Pro cone and plate viscometer at 37 °C and shear rate of

275 sec-1(Brookfield Engineering Laboratories, Inc., Middleboro MA).

In order to assess the native molecular weight of the keratin compounds isolated by the process described above, size exclusion chromatography [19] was performed. Samples were dissolved at 1-2 weight/volume percent (depending on viscosity) in 10 mM HEPES + 130 mM sodium chloride, pH 7.4 buffer. A Biologic LP chromatography system (Bio-Rad Laboratories, Hercules CA) using a 1x30cm

Sephacryl S-300 column (GE Healthcare, Piscataway NJ) running at 0.17 mL/min was injected with approximately 800 µL of sample. Detection of proteins was at 250 nm. Standards at 340, 230, 128, and 64KDa molecular weight were analyzed under

34 identical conditions and molecular weights of the keratose samples calculated from the Log(10) molecular weight-peak elution time relationship.

Under a protocol approved by the Wake Forest Institutional Animal Care and

Use Committee, male Sprague-Dawley rats (Harlan Laboratories, Dublin VA) were anesthetized with urethane diluted in 0.9% sodium chloride solution and injected intraperitoneally at a dose of 1-1.5 gram per kilogram of body weight. Urethane was chosen because it is easy to use, long lasting, and does not contribute appreciable physiologic effects in the vasculature. The drug was administered in incremental doses until adequate anesthesia was achieved, this schedule of administration avoids overdosing and normal cardiovascular physiology is maintained. Animals were positioned on the surgery table and the proximal trachea was isolated. A small incision was made between tracheal cartilage rings and a polyethylene tube (PE205) was inserted into the trachea to ensure sufficient and adequate airflow throughout the experiment. The incision was then extended laterally towards the right clavicle until the jugular vein was isolated. A catheter was inserted and secured for later infusion of treatments into the venous bloodstream.

The animals were repositioned and an incision was made through the lateral aspect of the scrotum for cremaster muscle isolation. The fascia overlying the cremaster was resected and the muscle incised through the midline until its inner contents were exposed. The spermatic cord and its components were ligated and resected. The muscle was completely isolated from other tissues and spread over a

35 glass pedestal, secured with 6-0 silk sutures and a glass cover slip was placed over it for efficient transillumination.

The animals were transferred to a compound microscope equipped for videomicroscopy. A nomenclature system was used in which arterioles are numbered according to their branch order, A1 being the first branches off the main feeding artery to the tissue, second order branches are named A2 and so on. Chosen for study were A2 (45-65 µm) and A3 (20-45µm) arterioles with clearly defined walls.

Animals were allowed to stabilize for one hour to allow the vasculature to recover from any transient changes the surgical trauma and environment temperature exposure might have induced. The muscle was maintained inside the confines of the glass cover slip on top and gauze strips secured on the sides with silk sutures. This allowed for physiologic tissue temperature control to take over. Our preliminary studies showed that tissue temperature was maintained within physiologic levels and therefore temperature-controlled chamber was not necessary. Infusion of a defined volume of resuscitation fluid (2.25 ml/100 g of body weight) was given without hemorrhage (this method is referred to as a “topload”).

The fluid was warmed to body temperature prior to injection and infused through the jugular access over a period of two minutes. This slow infusion rate was used to avoid a localize increase in blood viscosity. The videomicroscopy equipment was used to measure arteriolar diameter prior to infusion and at five-minute intervals up to thirty minutes after infusion. One to three vessels were chosen per rat

36 depending on how clearly the vessel walls were visualized. When several vessels were measured, the changes were averaged for that rat thus, every “n” in this study represents the changes in one study subject.

The animals were randomly assigned daily to a different treatment group to received intravenous infusion. A pilot study was originally done to compare KRF 1X,

Hetastarch and PBS. These data were included in this study, hence the difference in sample sizes between treatment groups.:

1. Crude keratose extract solution dialyzed with a 30 KDa NLMWCO

cartridge to exclude all compounds below that molecular weight and

formulated half as viscous as normal human blood (Keratose

Resuscitation Fluid or “KRF” 0.5X).

2. Same as solution #1 but formulated at a viscosity similar to that of

normal human blood (KRF 1X).

3. Same as solution #1 but formulated at a viscosity one hundred percent

higher that that of normal human blood (KRF 2X).

4. KRF 1X after bathing the muscle with L-Nitro-Arginine (L-NA) in order to

inhibit NO Synthase activity.

5. 6% Hetastarch in 0.9% Sodium Chloride solution.

6. Control group treated with the vehicle only, phosphate-buffered saline

(PBS).

37 In order to determine the origin of the compound(s) responsible for vasodilation, a follow up experiment was conducted in which all low molecular weight keratose molecules (i.e. hydrolysis by-products) were eliminated by dialysis and a high molecular weight fraction of alpha+KAP complexes were fractionated.

This was accomplished by removing the crude keratose extract immediately after

100KDa NLMWCO dialysis, titrating the solution to pH 6.0, and loading the sample onto a glass column containing anion exchange resin (Q Sepharose, General Electric

Healthcare, Fairfield, CT). The resin was used according to the manufacturer’s instructions and was conditioned with three volumes of 10mM tris at pH 6.0. After loading the sample, the column was rinsed with an additional three volumes of

10mM tris buffer. The flow through and rinse solutions representing the alpha fraction were collected, dialyzed at 100KDa NLMWCO, and processed to a dry powder as previously described. The sample bound to the resin representing the

KAP fraction was eluted with three volumes of 100mM tris at pH 8.0 + 2M sodium chloride, dialyzed at 30KDa NLMWCO and further processed to a dry powder as previously described.

The following additional fluids were prepared and tested in the rat cremaster model through intravenous infusion as well:

7. Purified alpha-keratose fraction described above, formulated at a

viscosity similar to that of normal human blood (Alpha)

8. Purified KAP fraction described above, formulated at a viscosity 2.5 times

that of normal human blood (KAP)

38 KAP was applied both i.v. as described above, and topically. Topical application was done using a 27G hypodermic needle (BD PresicionGlidetm), which was inserted between the muscle and the glass pedestal. This allowed the fluid to be applied without lifting the glass cover and the puncture was small enough that bleeding was avoided.

Statistical Analysis: A two-way analysis of variance (ANOVA) was conducted on vessel diameter measurements and Bonferroni post-hoc comparisons were performed when appropriate to determine differences between the treatments at each time point. All graphs indicating arteriolar diameter change show increase over baseline (e.g. 1.2 indicates a 20% increase in diameter) and represent the mean with error bars indicating standard error of the mean (SEM) unless otherwise stated.

Significant differences are noted in the figures.

39 Results

SEC analysis of the keratose samples used in these experiments showed a heterogeneous mixture of proteins. Crude keratose extract prepared by 30KDa

NLMWCO dialysis had a bimodal distribution with peak apexes at approximately 60 and 200KDa, indicating the presence of monomeric and tetrameric forms of keratin

(Figure 1A). Purification by IEx resulted in a notable shift in this distribution. While the 100K dialyzed sample before IEx fractionation (Figure 1B) showed multiple peaks, the alpha fraction (Figure 1C) showed a bimodal distribution but with peak apexes at 67 and 270KDa, indicating a shift to higher molecular weight compared to

Figure 1A. The KAP fraction (Figure 1D) was comprised primarily of a large peak at

47KDa with a slight shoulder at 28KDa, indicating the presence of monomeric alpha keratin and KAP, respectively.

KRF 1X i.v. administration induced vasodilation that was apparent under optical microscopy (Figure 2). The effect plateaued 15 minutes following infusion and was statistically significant at all time points after 5 minutes, with a maximum average increase in diameter of 37.4% observed at 15 minutes (Figure 3).

Hetastarch administration induced vasodilation that plateaued after 20 minutes with a maximum averaged effect of 12.4% increase in vessel diameter observed at

20 minutes. Equivalent volumes of PBS did not elicit significant changes in vessel diameter (Figure 3). Moreover, there were no statistically significant differences between responses elicited by Hetastarch and PBS (Figure 3). Cremaster muscles pre-treated with topical L-NA were observed for 30 minutes after application and

40 exhibited a small decrease in diameter within the first 5 minutes; this decrease plateaued after 5 minutes and was not statistically different from levels before application. After 30 minutes KRF 1X was administered i.v. and no significant change in vessel diameter was observed (Figure 4). Altering the viscosity of KRF produced differing effects on vasodilation after i.v. infusion. KRF 0.5X did not induce statistically significant changes but KRF 2X did elicit statistically significant changes in microvascular diameter similar to KRF 1X (i.e. KRF 1X and 2X were not statistically different; p = 0.87)(Figure 5).

The preceding experiments using KRF demonstrated the effect of a fluid containing solute molecules composed of monomers, dimers and higher oligomers, which exist in solution as associated molecules. The experiments using the purified fluid were intended to find individual effects of these compounds. Interestingly, the purified fluid (Alpha) only induced a 2.3% increase in vessel diameter even though it was formulated at the same viscosity as the unfractionated sample and infused in the same manner (Figure 6). This observation spurred further investigation of the

KAP fraction as a potential mediator of vasodilation. An additional experimental group was tested wherein a fluid at 2.5X viscosity (200 mg/ml) containing only the purified KAP fraction (KAP) in PBS was injected IV. All other experimental parameters were as described previously. Intravenous administration of the KAP fluid resulted in a rapid, transient increase in vessel diameter of 7.6% (Figure 6).

The effect was substantially diminished by 20 minutes and it was noted by a change in urine coloration that the keratose was quickly cleared through the renal system.

These experiments showed that the KAP fraction induced statistically different

41 changes from the Alpha fraction (p<0.0001), but the statistically significant difference is lost at the 30 minutes time point with the observation of rapid clearance. To further confirm the KAP fraction’s potential effect, the KAP solution was administered topically, directly to the cremaster muscle. This was done using a

27G hypodermic needle (BD PresicionGlideTM), which was inserted between the muscle and the glass pedestal; this allowed the fluid to be applied without lifting the glass cover and the puncture was small enough that bleeding was avoided. In these experiments, enhanced vasodilation was achieved (17.75% increase in vessel size,

Figure 6), this effect was statistically different compared to the same compound when infused intravenously (F=18.03, p = 0.0001) as well as to the Alpha (F=58.59, p = 0.0001).

In all cases, rats did not demonstrate any adverse reactions to the infusion of the treatments such as sudden death, seizures, blood flow alterations (i.e. emboli) or increased neutrophil adherence as noted on microvascular observations of venous flow. Animals were euthanized according to the protocol after the experiments were concluded.

42 Discussion

Keratins are a family of fibrous, intermediate filament forming structural proteins that self-assemble by coordination of type I (acidic) and type II (basic) monomers into obligate heterodimers [14]. These dimers self-assemble to form stable tetramers, two tetramers form an octomer [2] and four octamers complex to form a cylindrical unit length filament (ULF). End-to-end coordination of ULFs can result in the formation of 10nm diameter intermediate filaments [7]. In the samples tested in this study, monomers, dimers, and higher ordered oligomers were evident from SEC analysis. Interestingly, the KAP fraction appeared to remain complexed to these oligomers during extraction and dialysis. Only after IEx fractionation was a lower molecular weight fraction of these associated proteins able to be isolated.

The rat experiments demonstrate that KRF formulated at a viscosity similar to blood viscosity induces arteriolar vasodilation on the skeletal muscle vascular bed in a normovolemic rat This vasodilation is mediated, at least in part, through the

NO pathway as demonstrated by the fact that after inhibiting NO synthase with L-

NA, KRF 1X did not induce any change in arteriolar size (Figure 4). Low viscosity

(i.e. 0.5X) KRF induced less vasodilation compared to fluid with increased viscosity

(achieved by increasing concentration), which resulted in significantly greater vasodilation (Figure 5). This relationship suggests that one or both of the following mechanisms may mediate vasodilation:

1. vascular shear stress at the vessel wall, or

2. pharmacologic stimulation of endothelial NO production

43 In order to investigate the source of the keratin responsible for this activity, chromatographic separation was performed so that the alpha and KAP fractions could be studied separately. The alpha fraction is made up of large molecules that contribute viscosity to the solution. The KAP materials bound to the anionic exchange column are small molecules so they would be expected to contribute little to no viscosity to the fluid. Data in Figure 3 demonstrate that persistent vasodilation is achieved with the fluid containing alpha plus KAP, but eliminating

KAP from this molecular complex significantly reduces the vasoactive effect, as shown in Figure 6. Moreover, when a KAP only fluid is infused, a transient vasodilation effect is noted with rapid clearance of these smaller molecules. This suggests a pharmacologic-mediated action from the KAP fraction. To further confirm the KAP fraction’s potential vasoactivity, the KAP solution was administered topically, directly to the cremaster muscle. In these experiments, a mean increase of 17.75% in vessel diameter was observed (Figure 6). However, this effect was muted when compared to the KRF 1X fluid but was potent enough to demonstrate a statistically significant difference compared with the same compound infused intravenously (F=18.03, p = 0.0001). This suggests that the KAPs’ efficacy is dependent on it being able to form a molecular complex with the larger alpha keratose molecules.

Whether the vasoactive effect is in fact achieved by one of the mechanisms mentioned above or a summative effect of both will require further investigation.

However, the fact that the Alpha compound failed to elicit vasodilation when formulated at the same viscosity and infused i.v. in the same manner, points toward

44 a receptor-mediated mechanism rather than a viscosity effect. Moreover, the fact that the effect was more pronounced when KAP was complexed with the larger

Alpha molecules suggests that the Alpha is either serving to increase the circulating half-life of the KAP compound, or the formation of the molecular complex itself renders the KAP molecules more active, perhaps through some specific folding interaction.

Changes in the concentration of alpha-keratose allow for adjustment of solution viscosity and oncotic characteristics in the KRF. Data from fluids ranging in viscosity from 0.5X (1.7 cP) to 2X (9.7 cP) demonstrated more of a threshold effect than a dose response (Figure 5). This suggests that viscosity plays a smaller role relative to the pharmacologic effect of the KAP fraction. However, the KAP fraction appeared to have been cleared quickly, thereby diminishing the persistence of vasoactivity. Interestingly, topical application appeared to induce stronger vasodilation than i.v. injection, although it was weaker than infusion of the combined alpha+KAP, KRF 1X. These observations again suggest that the complex of alpha and KAP is important for vasoactivity. Binding to a keratin oligomer would appreciably increase the effective molecular weight of the alpha+KAP complex, thereby increasing its circulation half-life. Preserving structural integrity of these complexes may also be important for presenting active sites that are needed for pharmacologic efficacy. Interestingly, other keratin preparations have been shown to have hemostatic characteristics [1], whereas keratoses seem to be completely blood and cardiovascular compatible. This further suggests that tertiary structure of the alpha+KAP keratose complex is an important aspect of vasoactivity.

45 While these observations for keratins are novel, vasoactive peptides are not a new discovery. Angiotensin is a strong vasoconstrictor, kinins are vasodilators, and vasoactive intestinal peptide has several roles, including vasodilation. All of these compounds have been reviewed elsewhere and have complex mechanisms [4, 8, 11].

Interestingly, antihypertensive effects have been associated with several materials including peptides derived from dietary sources [22], as well as another biomaterial, chitin [9]. In a similar investigation to the present work, Miguel et al. showed vasodilator activity associated with several peptides from egg whites [12]. The peptide sequences IVF, RADHPFL, YAEERYPIL, YPI, and RADHP all induced vasodilation in intact aortic rings, up to a maximum of >50%. However, none of these sequences are present in the human trichocytic keratin proteome with the exception of YPI, which is found in one KAP molecule, KRTAP7-1 [17], a KAP not found in our human hair keratin extracts [5].

46 Conclusions

This work represents the first observations of vasodilation activity associated with human trichocytic keratins. KRF administered intravenously caused rapid, sustained vasodilation when the form of the keratin was a supramolecular complex comprised of both alpha and KAP keratose derivatives. The vasoactivity appeared to be more pharmacologic than shear mediated, and could be isolated to a purified KAP fraction consisting of a relatively low molecular weight fraction. Both i.v. and topical application of this material induced vasodilation. Experiments using

L-NA implicated the NO pathway, although a definitive mechanism deserves further study.

Acknowledgements

The authors thank Dr. Olga Greengauz-Roberts, Dr. Roy Hantgan and the

Macromolecular Interactions Core Laboratory at Wake Forest School of Medicine for assistance with size exclusion chromatography.

Funding was provided by KeraNetics LLC and the National Institutes of

Health (NIH/NHLBI, 1R43HL099010-01A1, PI: Burnett L).

Author Mark Van Dyke holds stock and is an officer in the company,

KeraNetics LLC, who has provided partial funding for this research. Wake Forest

School of Medicine has a potential financial interest in KeraNetics through licensing agreements.

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Jan;292(1):C82-97.

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3;140(3):131-5.

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FIGURES

A B

C D

Figure 1. Size exclusion chromatography of keratose samples. The sample used in test groups 1-4 (A) indicates the presence of monomeric and tetrameric forms of alpha keratin. Before purification, many peaks are present in the 100K dialyzed crude extract (B), indicating multiple protein species. After fractionation by ion exchange chromatography the higher molecular weight alpha (C) and lower molecular weight KAP (D) fractions are evident (K = KDa, Abs = Absorbance, nm = nanometers).

Figure 2. Video stills of the same vessel before and after KRF administration.

The image on the left shows a blood vessel before KRF administration while the one on the right shows the same vessel after i.v. injection of KRF 1X. Magnification is the same in both images.

Figure 3. Change in arteriolar diameter with i.v. administration of Fluid. KRF

1X induced a maximum vasodilation of 37%. Hetastarch induced vasodilation of

12%; the difference in relative response between these compounds was statistically significant. There was no statistically significant difference between the effects of hetastarch and PBS. Data is presented as a percentage of baseline (statistical difference noted is between treatment groups).

Figure 4. Effect on arteriolar diameter with and without L-nitro-arginine. The vasodilator effect of KRF 1Xwas blocked with L-Nitro-Arginine. This demonstrates that NO production plays a role in the vasodilator effects of KRF administered intravenously (statistical difference noted is between treatment groups).

Figure 5. Average arteriolar diameter increase with KRF of different viscosities. Low viscosity KRF induced less vasodilator effect while increasing viscosity resulted in an increase in vasodilation (0.5X [n=5], 1X [n=11], 2X [n=5] when administered intravenously (statistical difference noted is between groups, no difference was noted between KRF 1X and KRF 2X).

Figure 6. Average arteriolar diameter increase with infusion of Alpha and KAP containing fluids. The KAP fraction showed a modest, transient increase when applied intravenously which was statistically higher than the Alpha fraction. Alpha compound did not induce a significant increase in vessel diameter. The KAP fraction’s relative effects were increased when applied topically, which is demonstrated by a statistical difference when compared to the KAPs IV.

57 CHAPTER III

HEMODYNAMIC RECOVERY AFTER HYPOVOLEMIC SHOCK AND

RESUSCITATION WITH LACTATED RINGER’S AND KERATOSE RESUSCITATION

FLUID; A NOVEL COLLOID

Fiesky A. Nunez Jr. MD, Simon Trach, Luke Burnett PhD, Michael Callahan PhD,

Victor Kislukhin PhD, Thomas L. Smith PhD, Mark Van Dyke PhD

Wake Forest University School of Medicine, Winston Salem, NC 27157

This manuscript was submitted to Resuscitation, which is a peer-reviewed Medline

indexed journal. Stylistic variations are due to requirements of the journal.

58 Abstract

Review: Death after severe hemorrhage remains an important cause of mortality in people under 50 years of age, but remarkably the most common fluids used to treat hypovolemia were invented over 100 years ago. Keratin resuscitation fluid (KRF) is a novel resuscitation solution made from keratin protein that may restore cardiovascular stability while reducing the need for large volume infusions.

Methods: This postulate was tested in Sprague-Dawley rats that were instrumented and exsanguinated to 40% of their estimated blood volume. Test groups received either low or high volume resuscitation with either KRF or lactated Ringer’s solution (LR) and were studied under acute recovery conditions. One group receiving high volume resuscitation was also assessed after one week of recovery.

Results: KRF low volume resuscitation was more effective than LR in recovering cardiac function, blood pressure and blood chemistry. Furthermore, in contrast to LR-treated rats,

KRF-treated rats exhibited vital signs that resembled normal controls at 1-week post recovery.

Conclusions: These results, combined with the extensive cell and tissue compatibility data for keratin biomaterials, show that KRF may be a suitable fluid for the treatment of hypovolemia.

59 Introduction

Though it was invented over one hundred years ago, lactated Ringer’s (LR) solution remains the most widely used fluid for the treatment of hypovolemic shock [1].

However, there is a significant and persistent debate on whether colloid solutions provide better outcomes than crystalloids. Despite the lack of clarity, emergency medicine and intensive care textbooks continue to advocate crystalloids, even if large volumes must be infused [1, 2]. When such fluid overloading is performed, perfusion is restored at the expense of resulting hemodilution, risking dilutional coagulopathy, diminished oxygen carrying capacity of the blood, persistent hypo-oxygenation of tissues, and organ failure.

Regardless of the fluid used, overly aggressive resuscitation has the potential to dilute clotting factors, dislodge clots, and thus interfere with the clotting cascade and exacerbate bleeding [1]. Clinical studies have not found any difference in mortality between critically ill patients randomized to receive infusion of saline or 4% albumin [3].

However, human albumin solution may impair cardiac function due to a reduction in ionized calcium and in the ionized calcium/total calcium ratio [4]. Another colloidal option, hetastarch, is considered relatively safe at low dosages. Gan et al. defined a dosing limit of up to 5 L as determined in their clinical trial [5]. However, it’s labeling establishes a safe limit of 20 ml/kg, which can be quite limiting in the emergency setting.

Investigators have also reported prolonged partial thromboplastin time and decreased circulating factor VII after infusion of hetastarch, even to euvolemia [6, 7]. Patients with active hemorrhage have an increased probability of bleeding following starch based colloid treatment, thus limiting effective dosing and resuscitation.

60 The United States military employs a colloid primed resuscitation protocol on the battlefield, with an initial infusion of 500ml of 6% hetastarch for unstable patients, followed by a bolus of 500ml of 6% hetastarch if hemodynamic stability is not achieved

[8]. However, if cardiovascular stability is still not attained, further infusion of hetastarch is avoided to prevent complications. LR infusion is then started in large volumes until blood transfusions can be given. Although no wide consensus has been reached regarding the proper use of colloids such as hetastarch, several studies have demonstrated significant advantages at the cellular level when compared to conventional crystalloids

[9]. Recent debate in the field has proposed damage control resuscitation (DCR) as the solution to mortality after severe hemorrhage. This approach is based on a timely diagnosis of incoming patients at high risk for developing coagulopathy and allows prompt treatment of the so-called “lethal triad” of hemorrhage: hypothermia, acidosis and coagulopathy [10, 11]. However, DCR is a protocol for in-hospital use, and therefore large volume isotonic crystalloids continue to be the “gold standard” in pre-hospital resuscitation [1].

More broadly, cardiovascular research has produced insights that have significantly impacted clinical practice over the last decade, yet cardiovascular related mortality remains a primary cause of trauma-related death. Shock and resuscitation are important topics in this field and the development of better treatment options is essential.

Previous experiments in our laboratory demonstrated the viability of human keratin protein solutions for intravenous use [12]. Keratins represent a novel colloid that can be formulated at a desired viscosity with the potential to diminish the need for large volumes of resuscitation fluids by improving blood’s osmotic profile, thereby reducing the risk for

61 side effects described with the use of other colloid compounds [13-16]. Keratin proteins have been investigated more broadly as biomaterials for several medical applications and have shown excellent cell and tissue compatibility [17]. Our recent study investigating keratin colloids for intravenous use showed encouraging results[12]. Nunez et al. showed that specific fractions of keratin were found to have a vasodilation effect while further separation of the molecules yielded a purified compound of high molecular weight dimers and tetramers that could serve as a hypotensive resuscitation fluid. These purified keratins offer potential advantages as a colloid including:

• High molecular weight prevents fast clearance from the circulation during

periods of vascular pathology

• Hyperviscosity aids in the restoration of normal microcirculatory

perfusion

• Purified keratin fractions are not vasoactive, which avoids further

hypotension

• Keratin is compatible within the circulatory system and does not cause

immunologic reactions or untoward acute reactions with blood

The aim of the present study was to examine the potential of KRF to recover hemodynamic parameters after hypovolemic shock. Our goal was to recreate conditions similar to pre-hospital course of treatment in which emergency medical technicians rely on crystalloids and for which there is no disagreement that new treatment options are needed. The hypothesis tested was that intravenous infusion of KRF would restore cardiovascular stability after maintained hypovolemic hypotension with less volume than

LR solution.

62 Materials and Methods:

Fluid Preparation: Alpha-keratin (the keratin used in KRF) was prepared as previously described [12] and the solution prepared by aseptic reconstitution of the lyophilized keratin powder with phosphate buffered saline at a concentration to match human blood viscosity (3.88cP at 10 dynes/cm2). Viscosity was determined with a DV-

II+ Pro cone and plate viscometer (Brookfield Engineering Laboratories, Inc.,

Middleboro, MA). To ensure cardiovascular compatibility, KRF was clarified prior to infusion. This could not be accomplished via filtration because of the protein’s high molecular weight, so solutions were repeatedly (i.e. 10X) centrifuged at 2000 rpm for 5 minutes and only the supernatant used for infusion.

Surgical Preparation: The animal research protocol was approved by the Wake

Forest School of Medicine Institutional Animal Care and Use Committee. Thirty-seven male Sprague-Dawley rats (S-D; Charles River Laboratories International Inc,

Wilmington, MA) weighing 327-528 g were studied. Anesthesia was induced with isoflurane at 3 volume % concentration and maintained at 1.5% isoflurane using a nose cone (95% O2/5%CO2). A femoral artery catheter (PE50) was inserted for blood pressure measurements, and a carotid artery to jugular vein extracorporeal loop was established and driven through a mechanical pump (Instech Laboratories, Plymouth Meeting, PA) at

10ml/min. Transit time flow probes continuously monitored the carotid outflow and the jugular inflow for changes in blood density (Transonic Systems, Inc, Ithaca, NY).

Automatic cardiac output measurements using the ultrasound dilution method were triggered with a 0.3ml bolus injection of saline into the jugular line (Supplemental

Figure 1) [18].

63 Parameters:

• Blood pressure and heart rate were continuously monitored and stored

with IOX2 Software (EMKA Technologies, Falls Church, VA).

• Cardiac output was calculated using Transonic ICU software (Transonic

Systems, Inc, Ithaca, NY) software at baseline, 1 minute before treatment

was given, and at 5, 15, 30, 60, 90 and 120 minutes after treatment was

initiated. Stroke volume was calculated by dividing cardiac output by the

heart rate at the time of each measurement.

• Cardiac index was obtained by dividing cardiac output by the total body

surface area, which was obtained using the Meeh constant formula [19].

• Blood-filled microhematocrit tubes (Fisher Scientific, Pittsburgh, PA)

were spun at 3000 rpm for 5 minutes and hematocrits measured using an

International Microcapillary Reader (International Equipment Company,

Boston, MA) at baseline, 1 minute before treatment, 30 minutes after

treatment, and 120 minutes after treatment. An estimate of oxygen

delivery was obtained by multiplication of cardiac output and hematocrit.

• Blood gases were analyzed 120 minutes after initiation of resuscitation

treatment using an IRMA Trupoint blood analysis system (ITC, Edison,

NJ).

• Actively circulating volume (ACV) and central blood volume (CBV) were

calculated posthoc by Transonic Systems Inc (Ithaca, NY) [20]. These

parameters correlate with severity of cardiac instability during

hypovolemia [21].

64 • Volume restoration measurements were obtained by dividing the restored

ACV by the volume of resuscitation fluid infused intravenously (IV).

Restored ACV was obtained by averaging ACV after 15 minutes of

treatment and subtracting the ACV level before treatment.

1. Low-volume resuscitation, short-term assessment (experiment #1):

Eleven Male Sprague-Dawley rats were studied. Blood was withdrawn from the carotid arterial catheter until a mean arterial blood pressure of 40mmHg was reached.

This pressure was maintained for 30 minutes (Supplemental Figure 2). After 30 minutes of hypovolemic shock, rats were given the equivalent of 20% of their total blood volume of a randomly assigned fluid, LR or KRF. Total blood volume was calculated as 6.5% of body weight. Two hours after treatment arterial blood gases were measured and the rats were euthanized.

2. High-volume resuscitation, short-term assessment (experiment #2):

Ten male Sprague-Dawley rats were treated as described previously and were randomized to receive either total volume resuscitation of shed blood (total blood removed during hemorrhage was replaced by KRF) or 3X the volume of shed blood replaced by LR. Outcomes measured were the same as in the low volume group described above and animals were euthanized after 2 hours.

3. Long-term assessment of recovery (experiment #3)

Sixteen male Sprague Dawley rats were anesthetized and treated as described in experiment #1. Treatment conditions consisted of 3X the shed blood volume replaced by

LR (i.e. same as high volume treatment) or 20% of total blood volume replaced by KRF

65 (i.e. same as low volume treatment). After two hours, instrumentation was removed and the rats were returned to the vivarium. Seven days later the rats were re-instrumented and hemodynamic parameters, arterial blood gases, hemoglobin, hematocrit and electrolytes were assessed. Arterial oxygen content was obtained using values obtained from arterial blood gas analysis [22]. Comparisons with healthy controls were made for reference purposes but no statistical comparison was made to the treatment groups.

Histology: At the end of experiment #3, the rats were infused with saline solution followed by 10% neutral buffered formalin. Brain, heart, liver and kidney were harvested for histologic evaluation. The tissues were embedded in paraffin blocks, cut and stained with hematoxylin and eosin (H&E) for optical microscopy examination.

Statistical Analyses: A two-way repeated measures analysis of variance

(ANOVA; treatment vs time) was performed to compare the treatment groups for all parameters measured repeatedly during the experiments. Specific time points were analyzed using Bonferroni post-hoc tests when appropriate. A one-tailed Student’s test was performed to determine mean differences between parameters measured at the end of the experiment.

Results

1. Low-volume resuscitation, short-term assessment (experiment #1):

Hypovolemic Shock: Rats were hemorrhaged 10.9ml equivalent to 40% of estimated total blood volume (± 6.22%) to a mean arterial pressure of 38 mmHg (±

5.6mmHg) for 35 minutes (± 2 minutes). There was no significant difference in volumes of hemorrhage or time spent in a hypotensive state between experimental groups. The

66 mean volumes given IV were 5.94ml and 5.63ml in the LR and KRF groups, respectively.

Cardiac Index: No significant main effect due to treatment was found (F=1.43, p

= 0.26) (Figure 1A). A statistically significant interaction between time and treatment group showed that the KRF group had an increasing slope in the recovery of cardiac index from 11.9 L/min/m2 at 15 minutes to 13.9L/min/m2 at 120 minutes, while the LR group had a decreasing slope from 13.3L/min/m2 to 10.8L/min/m2 (F=7.75, p<0.0001) during the same period. This finding indicates that there was an improving trend in cardiac index in the KRF group compared to the LR group, which was in decline (Figure

1A).

Stroke Volume: There was no difference between treatment groups in recovery of stroke volume following resuscitation (Figure 1B).

Heart Rate: There was no difference between treatment groups in recovery of heart rate following resuscitation.

Mean Arterial Pressure (MAP): Blood pressure was significantly elevated after

KRF treatment compared to treatment with LR (Figure 1C; F=16.33, p=0.003). A statistically significant interaction was noted between time and treatment group (F=8.37, p<0.0001),

Hematocrit and Oxygen Delivery: There was no difference in hematocrit between treatment groups. However, calculated oxygen delivery showed a trend toward greater improvement in the KRF group, evidenced by a significant interaction between time and treatment group (Figure 1D; F=12.93, p =0.0003).

67 Changes in ACV and CBV were similar between the two groups (p=0.73 and p=0.99 respectively). However, a statistically significant interaction was found between time and treatment group in recovery of CBV (F=2.97, p=0.01) (Figure 2).

Blood Gases: A one-tailed Student’s test of blood gases after 120 minutes of treatment showed that the KRF group had arterial blood gas values more closely resembling normal than did the LR group: pH 7.16 vs. 7.09 (Normal = 7.35 –

7.45)(t=2.43, p=0.02) and pCO2 89.0 vs. 108.2 (NL = 35 – 45mmHg) (t=2.46, p =0.02).

No significant differences were found among other blood gas parameters (Supplemental

Table 1).

Volume Restoration: KRF restored higher ACV per ml of resuscitation fluid infused but the difference was not significant (p=0.16) (Supplemental Table 1).

2. High-volume resuscitation, short-term assessment (experiment #2):

Hypovolemic Shock: Rats were hemorrhaged 11.1ml equivalent to 39% of estimated total blood volume (± 7.6%) to a mean arterial pressure of 39 mmHg (±

2.5mmHg) for 39 minutes (± 2 minutes). There was no significant difference in volumes of hemorrhage or time spent in a hypotensive state between the groups. The mean volumes given IV were 34.5ml and 10.6ml in the LR and KRF groups, respectively. The high volume of resuscitation treatment took longer to infuse, hence the difference in resuscitation time between the first and second set of experiments.

Cardiac Index: Main effect analysis showed that administration of LR resulted in improved overall recovery of cardiac index compared to KRF (F=9.93, p = .01).

However, a significant interaction between time and treatment group showed that the

68 KRF group maintained a steadily increasing cardiac index from 12.2L/min/m2 5 minutes after treatment to 13.2L/min/m2 2 hours after treatment, while the LR group showed a decreasing slope from 15.9L/min/m2 to 14.4L/min/m2 (F=3.56, p=0.005) during the same period (Figure 3A).

Stroke Volume: There was no significant main effect of treatment on stroke volume. However, a significant interaction between time and treatment group was found

(F=3.17, p=0.01) (Figure 3B). This interaction statistically describes a steadily increasing slope in stroke volume in the KRF group versus a decreasing slope in the LR group.

Heart Rate: There was no difference between treatment groups in recovery of heart rate following resuscitation.

Mean Arterial Pressure: Blood pressure was significantly increased after KRF treatment compared with LR (F=11.38, p < 0.01) (Figure 3C).

Hematocrit and Oxygen Delivery: There was no difference between treatment groups in hematocrit or oxygen delivery (p=0.4 and p=0.9 respectively) (Figure 3D).

Actively Circulating Volume and Central Blood Volume: No main effect was found between treatment groups in ACV or CBV. LR produced a rapid increase in ACV and CBV determined at 5 minutes, which was followed by a steep drop. ACV and CBV equilibrated at levels similar to those in the KRF group (p>0.05). A significant interaction between time and treatment group was found in both ACV and CBV recovery analyses

(F=4.02, p=0.002 and F=4.19, p=0.002 respectively) (Figure 4). This interaction indicates that KRF was able to maintain steady circulating volumes while circulating

69 volumes in the LR group decrease significantly over two hours. This effect is expected given that LR tends to escape into the interstitium [1].

Volume Restoration: KRF showed a significantly higher ratio of ACV compared to LR (p=0.01) (Supplemental Table 2).

Blood Gases: There were no differences between treatment groups in any blood gas parameters (Supplemental Table 2).

3. Long-term assessment of recovery (experiment #3)

Hypovolemic Shock: Rats were hemorrhaged 10.9ml equivalent to 41.8% of their total blood volume (± 5.9%) to a mean arterial pressure of 39mmHg (± 4mmHg) for 35 minutes (± 2 minutes). There was no significant difference in volumes of hemorrhage or time spent in hypotension between groups. The mean volumes given IV were 32.4ml and

5.6ml in the LR and KRF groups, respectively.

Cardiac Index: No difference was found between the two groups and both exhibited CI similar to healthy controls (Supplemental Table 3).

Stroke Volume: KRF-treated rats recovered to a higher stroke volume compared to the LR rats (t=2.86, p=0.006). The KRF group was similar and the LR group slightly lower than healthy controls (Supplemental Table 3).

Heart Rate: The KRF group trended toward a statistically lower mean heart rate than the LR group (t=1.59, p=0.06). The KRF group had similar mean heart rates whereas the LR group was significantly higher compared to healthy controls

(Supplemental Table 3).

70 Mean Arterial Pressure: No difference in mean arterial pressure was found between the two groups. Both groups exhibited mean arterial pressures similar to healthy controls (Supplemental Table 3).

Actively Circulating Volume and Central Blood Volume: The KRF group showed slightly higher ACV and CBV than the LR group, but the difference was not statistically significant (p=0.15 for ACV and p=0.09 for CBV). The LR group had similar ACV and

CBV and the KRF group higher compared to healthy controls (Supplemental Table 3).

Blood Gases: There were no significant differences in arterial blood gases between groups (Supplemental Table 3).

Electrolytes: No abnormalities were noted in any treatment group (Supplemental

Table 3).

Arterial Oxygen Content and Myocardial Oxygen Delivery: No difference was noted between groups in arterial oxygen content. Myocardial oxygen delivery was higher in the KRF group (t=2.91, p=0.006).

Histology: Histologic features of the brain, heart, liver and kidney were similar between the two treatment groups and resembled normal healthy tissue (Figure 5).

Discussion

Data demonstrating the efficacy of in-hospital DCR supports lower volume usage pre-hospital, and recent trends suggest that mean resuscitation fluid volume infused before packed red blood cells are given started to decline after 2007 [23]. However, there

71 is consensus opinion that the lack of an ideal colloid has clouded the debate around pre- hospital resuscitation protocols and that a search for new colloids is certainly warranted.

With that objective in mind, the present study shows that low volume resuscitation with KRF proved to be more effective in recovering cardiac function, blood pressure, and blood chemistry than equivalent volumes of LR solution. This result was not surprising given LR’s isotonicity and the tendency for the fluid to leak out of the vasculature. KRF, on the other hand, contains dimers and tetramers with molecular weights well above that of serum albumin (i.e. >120K Da). At 3X volume resuscitation,

LR showed improved capacity to restore some cardiovascular parameters in the short term, but a significant interaction between time and treatment suggests that the LR volume likely moves into the interstitium, thus diminishing the ability of LR to maintain proper preload over time. This finding may explain why LR needs to be infused at three times the shed volume to maintain cardiovascular stability. KRF infusion at a level equivalent to shed blood volume maintained cardiac index similar to that obtained with high-volume LR after 2 hours.

KRF quickly and steadily increased MAP from immediate post-hemorrhage values and although cardiac index was also improved, the increase in cardiac index was not equivalent in amplitude to the increase in pressure. An increase in circulating volume usually restores cardiac function to a greater extent than it does blood pressure. This finding might suggest that KRF has some other mechanism to raise pressure while not significantly improving cardiac contractility, aside from restoration of intravascular volume. This observation is counter-intuitive because in previous research, fractions of keratin different from that used in KRF induced significant vasodilation and did not

72 increase vascular resistance[12]. Cardiac index was slightly improved when KRF was given in lower volumes (13.9L/min/m2 vs 13.2L/min/m2 at 2 hours), but the difference was not statistically significant. During low volume resuscitation with KRF, blood pressure increased more quickly and was maintained at a higher level than with 1X volume resuscitation using KRF. No significant difference was found but a trend was evident (p=0.06). Although no explanation for these phenomena can be found in these data, it is possible that higher doses (volumes) of intravenous keratin proteins could blunt cardiac dynamics and diminish its advantages in low volume replacement. Further research is necessary to delineate the mediators/mechanisms of hemodynamic effects of

KRF over a range of volumes.

During 1X volume resuscitation, KRF did not improve cardiac index as well as

LR. However, LR caused a profound dilution of hematocrit such that estimated oxygen delivery was equivalent in both treatments. This phenomenon was also reflected by the fact that blood gases, direct markers of tissue perfusion and multi-organ function, did not differ with either treatment.

The rationale behind low volume resuscitation is to restore hemodynamics just enough to provide tissue perfusion to vital organs while diminishing complications from resulting dilution, including dilution of clotting factors and exacerbation of bleeding.

Low volume resuscitation with KRF might not be as beneficial when compared to 3X volume LR in the short term, but this comparison is irrelevant if the most important long- term outcomes are achieved. Long-term assessment of resuscitation provides information on the course of hospitalization and some of this information can be extrapolated to assess how recovery, discharge and return to normal activities might happen. This

73 experiment provided important information on survival as well as effectiveness of the treatments.

The results of long-term assessment demonstrated that low volume KRF provides sufficient cardiovascular support to allow the system to compensate for hemorrhage and recover from a loss of 40% of estimated blood volume. The fact that the KRF group in this set of experiments had higher stroke volume and lower heart rate than the LR group suggests that the animals in the KRF group were recovering more completely. When compared to healthy controls, the LR rats show equal cardiac index but at the expense of a higher heart rate (Supplemental Table 3). During periods of hypovolemia, energy conservation becomes essential to survival. Therefore, the hemodynamic changes observed in the LR group could exacerbate myocardial oxygen deficits and trigger heart failure.

For the short-term assessments, low-volume KRF did not restore ACV to the same degree that 1X volume KRF did (p=0.04). However, for the long-term assessments

1X KRF actually restored ACV and CBV to slightly higher levels than high-volume LR.

This effect on ACV suggests two things: 1) overly aggressive volume expansion might not be necessary for proper recovery after hypovolemia, and 2) KRF has a longer lasting effect than previously anticipated. This finding is supported by the observation that seven days after treatment, the KRF group showed higher ACV than healthy controls. ACV provides proper preload and cardiac output, thus preventing the required tachycardia to maintain adequate heart function that is seen during hypovolemia. This improved cardiac function provides efficient oxygen delivery and allows the restoration of normal myocardial oxygen balance based on our calculations.

74 A 3X volume of LR would be expected to contribute to an exacerbation of active bleeding due to dilution of clotting factors [1]. However, under the conditions of a controlled hemorrhage the deleterious effect of dilutional coagulopathy might not be observed. Several studies state that colloids restore cardiac function better than some crystalloids but survival is not improved [24]. These observations, along with the higher costs of colloids are the main reasons why crystalloids continue to be employed as the primary resuscitation fluid in civilian emergency departments.

Conclusions

Our studies evaluated a novel keratin colloid that allows easy manipulation for improved viscosity and osmotic parameters. Keratins are inexpensive to isolate and purify from human hair fibers and KRF does not cause electrolyte disturbances

(Supplemental Table 3). During hematocrit measurements, visual inspection of supernatant plasma did not exhibit pink/red hue or unexpected changes in hematocrit, both of which indicate hemolysis. In addition, KRF is not toxic and does not stimulate chronic inflammatory reactions such as those frequently elicited by some synthetic polymers [25].

Although our results suggest that there is a measurable benefit to low-volume

KRF resuscitation, life-threatening hemorrhage might benefit even further from additional fluid infusion. Further studies should be designed to develop a more severe form of hemorrhage to determine: 1) the most efficient volumes of KRF for resuscitation following life threatening blood loss, 2) the difference in mortality between low-volume and full-volume KRF resuscitation, and 3) whether a combined KRF/crystalloid protocol

75 for resuscitation is beneficial. Moreover, an active bleeding protocol should be designed to contrast the behavior of both KRF and LR in a realistic trauma scenario.

76 Conflicts of Interest

Author Mark Van Dyke holds stock and is an officer in the company, KeraNetics LLC.

Wake Forest School of Medicine has a potential financial interest in KeraNetics through licensing agreements. Author Luke Burnett is currently an employee of KeraNetics LLC but participated substantially in the planning, execution and manuscript sections of the research study.

Acknowledgements

Partial funding for this project was provided by KeraNetics LLC and NIH/NHLBI (grant no. 1R43HL099010-01A1).

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[12] Nunez F, Trach S, Burnett L, Handa R, Van Dyke M, Callahan M, et al. Vasoactive properties of keratin-derived compounds. Microcirculation. 2011;18:663-9.

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37-132.

FIGURES

Figure 1. Changes after hypovolemic shock and treatment with low-volume resuscitation fluids (Solid Line=KRF, Dashed Line=LR). A) Cardiac index, B) Stroke volume, C) Mean arterial pressure, D) Oxygen delivery

Figure 2. Changes after hypovolemic shock and treatment with low-volume resuscitation fluids (Solid line=KRF, Dashed Line=LR). A) Actively Circulating Volume, B) Central Blood Volume

Figure 3. Changes after hypovolemic shock and treatment with full-volume resuscitation fluids (Solid Line=KRF, Dashed Line=LR). A) Cardiac index, B) Stroke volume, C) Mean arterial pressure, D) Oxygen delivery

Figure 4. Changes after hypovolemic shock and treatment with full-volume resuscitation fluids (Solid Line=KRF, Dashed Line=LR). A) Actively Circulating Volume, B) Central Blood Volume

Figure 5: Tissue Histology. 5.1-5.2 Cardiac Muscle Histology. Endocardial simple squamous epithelium marked by black arrow, intercalated disk marked by white arrow, normal muscle striations are evidence throughout the tissue. 5.3-5.4 Renal Histology. Simple squamous epithelium (black arrow), Bowman space (B), glomerular basement membrane (white arrow tip), loop of Henle tubule (A) and proximal convoluted tubule (C), arterial pole (D). 5.5-5.6 Liver Histology. Portal triad composed of Portal vein (V), hepatic artery (black arrow) and bile duct (white arrow). 5.7-5.8Brain Histology. Coronal cut through the medial thalamus (A) and surrounding third ventricle (B).

Supplemental Figure 1. Ultrasound Transit Flow Probes. A constant flow of 10ml/min extracts blood from the carotid artery and returns it into the jugular vein. Saline injections into the venous line lowers blood density, which is detected by the venous flow probe and triggers automatic cardiac output measurement.

Supplemental Figure 2. Mean arterial pressure and heart rate monitoring. Hypotension was maintained for 30 minutes around 40 mmHg by periodically withdrawing blood.

89 Supplemental Table 1. Hemodynamics and Arterial Blood Gases: Low Volume Resuscitation, Short Term Assessment

LR (n=6) KRF (n=5) Group Time Int.

CI (l/min/m2) 10.81 ± 7.4 13.85 ± 11.6 0.26 0.0001 0.0001

SV (µl) 234.4 ± 16.5 261.5 ± 15 0.52 0.0001 0.15

Heart Rate (bpm) 262 ± 19 292 ± 20 0.76 0.0001 0.33

MAP (mmHg) 58 ± 5 93 ± 6 0.003 0.0001 0.0001

ACV (ml) 12.1 ± 1.03 11.8 ± 1.3 0.73 0.0001 0.88

CBV (ml) 2.17 ± 0.1 2.35 ± 0.1 0.99 0.0001 0.01 pH 7.092 ± 0.02 7.161 ± 0.02 0.02 NA NA pCO2 (mmHg) 108.2 ± 7.3 89 ± 4 0.02 NA NA p02 557.4 ± 12.6 619.9 ± 44 0.13 NA NA

Base Excess (mEq/L) -0.98 ± 1.4 -0.38 ± 0.93 0.36 NA NA

O2 Delivery 18.6 ± 0.83 24.8 ± 2.3 0.15 0.0001 0.0003

Hematocrit 30.92 ± 0.6 32.4 ± 0.9 0.66 0.1 0.23

Vol. Restoration* 0.32 ±0.18 0.59 ± 0.17 0.16 NA NA Values presented were taken 120 minutes after treatment, refer to figures for changes over the 8 timepoints measured. All values represent mean and SEM. Group = main effect for group mean difference, Time = main effect for time mean difference, Int. = Interaction between group and time means.*Vol. Restoration: ml of ACV restored by every ml of resuscitation fluid infused IV

90 Supplemental Table 2. Hemodynamics and Arterial Blood Gases: Full Volume Resuscitation, Short Term Assessment

LR (n=5) KRF (n=5) Group Time Int.

CI (l/min/m2) 14.36 ± 2.3 13.15 ± 9 0.01 0.0001 0.005

SV (µl) 289.4 ± 3.1 261.9 ± 3 0.18 0.0001 0.01

Heart Rate (bpm) 265 ± 29 283 ± 13 0.39 0.0001 0.07

MAP (mmHg) 77 ± 9 91 ± 4 0.01 0.0001 0.61

ACV (ml) 15.2 ± 1.6 17.2 ± 1.4 0.96 0.0001 0.002

CBV (ml) 2.54 ± 0.2 2.65 ± 0.3 0.42 0.0001 0.002 pH 7.100 ± 0.03 7.127 ± 0.06 0.35 NA NA pCO2 (mmHg) 107.3 ± 8.9 98.1 ± 15 0.32 NA NA p02 634.6 ± 38 628.4 ± 57 0.47 NA NA

Base Excess (mEq/L) -0.75 ± 0.7 -1.9 ± 0.3 0.16 NA NA

O2 Delivery 22.9 ± 3.8 23.7 ± 3.9 0.94 0.0001 0.37

Hematocrit 29.6 ± 1.6 31.7 ± 2.2 0.62 0.004 0.47

Vol. Restoration* 0.13 ± 0.04 0.41 ± 0.09 0.01 NA NA Values presented were taken 120 minutes after treatment; refer to figures for changes over the 8 timepoints measured. All values represent mean and SEM. Group = main effect for group mean difference, Time = main effect for time mean difference, Int. = Interaction between group and time means. *Vol. Restoration: ml of ACV restored by every ml of resuscitation fluid infused IV

91 Supplemental Table 3. Hemodynamics, Arterial Blood Gases and Electrolytes: Long Term Assessment

Controls (n=32) LR (n=7) KRF (n=9) Sig.

CI (l/min/m2) 19.53 ± 4.9 20.53 ± 12.4 20.36 ± 6.5 0.45

SV (µl) 314.4 ± 7.9 286 ± 9.8 331.9 ± 11.9 0.006

Heart Rate (bpm) 332 ± 8 365 ± 16 335 ± 10 0.06

MAP (mmHg) 89 ± 1.5 96 ± 3.8 97 ± 3 0.91

ACV (ml) 18.1 ± 0.4 19.1 ± 1.2 20.8 ± 1.1 0.15

CBV (ml) 3.6 ± 0.1 3.61 ± 0.2 4 ± 0.2 0.09

Ca02 (ml) NA 19.4 ± 0.3 19 ± 0.4 0.44

Myocardial 02 delivery NA 10 ± 0.3 11.9 ± 0.5 0.006 pH NA 7.34 ± 0.02 7.33 ± 0.01 0.65 pCO2 (mmHg) NA 57.9 ± 2.1 58.1 ± 1.9 0.92 p02 NA 584.7 ± 18.7 599.1 ± 26.6 0.67

Base Excess (mEq/L) NA 4.9 ± 0.7 4.3 ± 0.4 0.47

HCO3 (mEq/L) NA 30.7 ± 0.6 30.3 ± 0.3 0.53

Na+ (mEq/L) NA 138.2 ± 0.8 137.8 ± 0.7 0.72

K+ (mEq/L) NA 4.19 ± 0.14 4.16 ± 0.09 0.88 iCa+ (mEq/L) NA 1.43 ± 0.01 1.46 ± 0.008 0.06 tHb (g/dL) NA 12.6 ± 0.43 12.8 ± 0.25 0.67

Hematocrit 44.9 ± 0.3 37.2 ± 1.2 37.8 ± 0.7 0.68

All values represent mean and SEM. Sig. column represent t-test p value between LR and KRF. Healthy Control values are presented for reference purposes.

92 CHAPTER IV

CONCLUSIONS

Fiesky A. Nunez Jr. MD

93 Biologic Activity of Keratose Fractions

The data in chapter 2 demonstrate that the alpha fraction of keratose possesses a vasodilatory activity, which was translated into arteriolar vasodilation in the cremaster muscle bed. Further experimentation demonstrated this activity to be exclusive to the alpha/KAP’s fraction and the KAP’s fraction alone while the KIF fraction was completely devoid of any vasoactive property. Furthermore, the activity was blocked when pretreating the muscle with L-Nitro-Arginine (L-NA), which blocks nitric oxide synthase activity. These data suggested that the alpha/KAP’s compound was not well suited for resuscitation after hypovolemic shock because its vasoactive properties might exacerbate preexisting hypotension.

However, the KIF compound elicited no such vasodilation and, a lower concentration was required to formulate a given viscosity. Due to their high- molecular weight, the KIFs were thought to possess higher oncotic pressure than their counterparts. The KIFs were then tested for resuscitation in a hypovolemic hypotensive state (Chapter 3).

94 KIF-KRF: A Novel Colloid for Hypovolemic Resuscitation

The results presented in chapter 3 substantiate our hypothesis that KIF would produce efficient resuscitation after hypovolemic hypotension. However, a disparity emerged between low-volume resuscitation and full-volume resuscitation with KIF.

Low-volume resuscitation restored cardiac dynamics similarly to full-volume resuscitation. In addition, low-volume resuscitation was also effective in restoring cardiac function after 7 days, measured during a long-term assessment of recovery.

Full-volume resuscitation did however restore actively circulating volume better than low-volume resuscitation (main effect p = 0.04, interaction p = 0.9).

Because circulating volume is maintained, cardiac index and systemic blood pressure are both expected to be restored following volume depletion and restitution. This assumption is maintained as long as the hemodynamic recovery after resuscitation is dependent on restitution of circulating volume and no direct pharmacologic effect is exerted on the heart or the blood vessels. Pure volume resuscitation without pharmacologic interactions is seen with LR, Hetastarch and low-dose albumin solutions (high-dose albumin causes negative cardiac inotropism).

It is expected that a larger volume of protein compound would have a greater effect on volume restoration due to a greater osmotic effect but it should also produce a similar recovery in cardiac function. Recovery of cardiac index was expected to correlate with volume recovery. Our data did not show a correlation between these two parameters, which might be unmasking a yet unknown negative inotropic effect

95 caused by the higher concentration of keratinous proteins in the circulating bloodstream.

During low-volume resuscitation with KIF-KRF, recovery of mean arterial pressure correlated with the recovery of cardiac index (r=0.88, p=0.03). However, during full- volume KIF-KRF resuscitation, recovery of blood pressure did not correlate with recovery of cardiac index (r=-0.31, p=0.31). During low-volume resuscitation with

Lactated Ringer’s, this correlation was not present (r=0.52, p=0.29), and this response was anticipated due to the low physiologic impact such a small volume of

LR has on the system. However, during full-volume resuscitation there was a very strong significant correlation between CI and MAP (r=0.81, p=0.05). Furthermore, there was also a very strong significant correlation between CI and ACV (r=0.95, p=0.006), suggesting that during full-volume LR resuscitation, the hemodynamic recovery was caused by a recovery of circulating volumes. ACV recovery did not correlate with CI during low or full-volume KIF-KRF resuscitation.

These findings suggest that KRF has some other mechanism to raise blood pressure while not significantly improving cardiac contractility, aside from restoration of intravascular volume. This observation is counter-intuitive because, while alpha/KAP’s keratose induced significant vasodilation in chapter 2, KIF-KRF did not increase vascular resistance and was not vasoactive. If our conclusion from Chapter

2 is feasible, in which we state that there was a pharmacologically mediated mechanism for NO release, then we must put forward the possibility that KRF may also interact with other molecules such as alpha-1 and beta-1 adrenergic receptors;

96 this would explain the rise in blood pressure while blunting the beta-1 effect due to increased peripheral vascular resistance and afterload. A more interesting finding would be that KRF is mediating release of myocardial NO. NO released from the myocardium has been known to regulate cardiac function; it has a slightly positive inotropic effect on isolated myocytes but has only shown negative inotropic effect in vivo (1, 2). The combination of these effects would explain the significant rise in blood pressure seen after resuscitation with KRF with a moderate increase in heart function.

During the chapter 3 experiments, the vasodilatory AK-KRF keratose was also infused IV after hypovolemic shock. This was done to confirm our conclusion from chapter 2 that AK-KRF would not be beneficial for hypovolemia but KIF-KRF would.

The result of this experiment was not submitted for publication as part of chapter 3 because it was not the scope of the manuscript. The result was expected; AK-KRF showed an early recovery in mean arterial pressure, which can be attributed to volume restoration, followed by a decreasing slope in pressure that correlates to

AK-KRF’s vasodilatory effect demonstrated in chapter 2. In fact, the onset of pressure decline started 15 minutes after AK-KRF infusion, which correlates with the onset of vasodilation seen in Chapter 2 (Figure 1). When compared to KIF-KRF, there was a statistically significant difference in mean arterial pressure (F=8.1, p=0.02) as well as a significant interaction (F=8.02, p<0.0001) that proves the steady rise in pressure after KIF-KRF infusion and the decreasing slope seen after

AK-KRF infusion (Figure 1).

Figure 1. Blood Pressure Recovery After Hypovolemic Shock and Treatment with KIF-KRF and AK-KRF

98 Low-volume vs full-volume resuscitation

Full-volume resuscitation was not the main goal of these studies. Overly aggressive resuscitation is used less frequently clinically, and more permissive hypotensive resuscitation is used (3-6). High-volume resuscitation tends to overload the interstitium with fluids, causing interstitial distention that results in exacerbation of tissue ischemia. Traditional polytrauma resuscitation procedures promote aggressive fluid resuscitation aimed at restoring lost intravascular blood volume.

This approach may contribute to continued bleeding by increasing the hydrostatic pressure on blood clots, causing dilution of coagulation factors, and aggravating hypothermia (7, 8). Furthermore, after volume balance is achieved, fluid that initially escaped into the interstitium tends to return to the bloodstream causing further dilutional coagulopathy and hemodilution, these complications usually present after the first 2 hours of resuscitation may appear up to 24 hours later. All these features of high-volume resuscitation can cause further clinical problems and may lead to lethal complications.

Low-volume Keratose resuscitation used in the present study represents a novel finding in the science of trauma and resuscitation. We expect the outcomes obtained with this compound to be extensively studied in the near future in more translational scenarios where further applications will become apparent.

The microvascular observations found in Chapter 2 are important for several areas, including the study of hypertension, ischemic diseases and thrombotic events. We

99 expect increased study of the vasodilatory keratoses to occur in the near future and for the aforementioned fields to benefit from it.

We cannot yet hypothesize what effect, if any, these compounds may have on circulating inflammatory cytokines. Several studies have noted changes in the inflammatory response following resuscitation for hemorrhagic shock as well as ischemic events (9-15). An inhibitory effect on these inflammatory cytokines could translate into improved outcomes and therefore should be studied under the effect of all fractions of keratose employed in this study.

100 References

1. Hare JM. Nitric oxide and excitation-contraction coupling. J Mol Cell Cardiol.

[Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Review]. 2003

Jul;35(7):719-29.

2. Massion PB, Feron O, Dessy C, Balligand JL. Nitric oxide and cardiac function: ten years after, and continuing. Circ Res. [Research Support, Non-U.S. Gov't Review].

2003 Sep 5;93(5):388-98.

3. van der Vliet JA, van Aalst DL, Schultze Kool LJ, Wever JJ, Blankensteijn JD.

Hypotensive hemostatis (permissive hypotension) for ruptured abdominal aortic aneurysm: are we really in control? Vascular. [Evaluation Studies]. 2007 Jul-

Aug;15(4):197-200.

4. Bickell WH, Wall MJ, Jr., Pepe PE, Martin RR, Ginger VF, Allen MK, et al.

Immediate versus delayed fluid resuscitation for hypotensive patients with penetrating torso injuries. N Engl J Med. [Clinical Trial Comparative Study

Controlled Clinical Trial]. 1994 Oct 27;331(17):1105-9.

5. Dutton RP. Low-pressure resuscitation from hemorrhagic shock. Int

Anesthesiol Clin. [Review]. 2002 Summer;40(3):19-30.

6. Heidary B, Bell N, Ngai JT, Simons RK, Chipperfield K, Hameed SM. Temporal trends in the treatment of severe traumatic hemorrhage. Am J Surg. 2012 Mar 12.

7. Stahel PF, Smith WR, Moore EE. Current trends in resuscitation strategy for the multiply injured patient. Injury. [Review]. 2009 Nov;40 Suppl 4:S27-35.

101 8. Beekley AC. Damage control resuscitation: a sensible approach to the exsanguinating surgical patient. Crit Care Med. [Review]. 2008 Jul;36(7 Suppl):S267-

74.

9. Hierholzer C, Kalff JC, Billiar TR, Tweardy DJ. Activation of STAT proteins in the lung of rats following resuscitation from hemorrhagic shock. Arch Orthop

Trauma Surg. [Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.].

1998;117(6-7):372-5.

10. Velmahos GC, Spaniolas K, Tabbara M, Duggan M, Li Y, De Moya M, et al.

Abdominal insufflation decreases blood loss without worsening the inflammatory response: implications for prehospital control of internal bleeding. Am Surg.

[Research Support, U.S. Gov't, Non-P.H.S.]. 2008 Apr;74(4):297-301.

11. Honma K, Koles NL, Alam HB, Rhee P, Rollwagen FM, Olsen C, et al.

Administration of recombinant interleukin-11 improves the hemodynamic functions and decreases third space fluid loss in a porcine model of hemorrhagic shock and resuscitation. Shock. [Clinical Trial Randomized Controlled Trial Research Support,

U.S. Gov't, Non-P.H.S.]. 2005 Jun;23(6):539-42.

12. Hsieh YC, Frink M, Thobe BM, Hsu JT, Choudhry MA, Schwacha MG, et al.

17Beta-estradiol downregulates Kupffer cell TLR4-dependent p38 MAPK pathway and normalizes inflammatory cytokine production following trauma-hemorrhage.

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102 13. Klingenberg R, Luscher TF. Inflammation in Coronary Artery Disease and

Acute Myocardial Infarction - is the Stage Set for Novel Therapies? Curr Pharm Des.

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103 APPENDICES

Fiesky A. Nunez Jr. MD

104 APPENDIX 1

EXTRACTION AND ISOLATION OF HUMAN-HAIR KERATIN PROTEINS:

CHEMICAL AND VISCOELASTIC PROPERTIES

Fiesky A. Nunez Jr. MD, Mark Van Dyke PhD

Wake Forest University School of Medicine, Winston Salem, NC 27157

105 Keratose Processing

Human-hair keratin proteins can be extracted and isolated into distinguishable fractions that represent compounds for potential clinical applications. Through the use of oxidative chemistry, disulfide bond scission allows the collection of keratose proteins for further processing.

By microdialysing the collected sample at 30Kda with nominal low molecular weight cutoff (NLMWCO) cartridge further separation of fragments can be accomplished. After this step, the dialysate is devoid of gamma proteins, which are globular in shape and provide little viscosity.

The sample obtained is composed mainly of alpha monomers, dimers and tetramers, and KAPs (table 1). Size-exclusion chromatography (1) studies demonstrate two peaks; one at 60Kda and another one at 200Kda (alpha monomers and tetramers respectively). The presence of alpha dimers is difficult to assess given that a peak in the graph around 128Kda may be hidden between the aforementioned peaks (see chapter 2). In this fraction of keratose the KAPs would be strongly associated to alpha helical structures and thus would not appear as a separate fraction. In Chapter

2, this fraction was termed crude due to the mixture of proteins it contains. Crude keratose is actually composed of alpha helical proteins, gamma globular proteins and KAPs. After further separation, which will be explained later in this section, we found this fraction to contain mostly alpha peptides with scant amounts of KAPs.

This finding prompted the labeling of this fraction “alpha/KAPs” compound or AK-

KRF (table 1).

106 In order to further purify and characterize our compound, for the following keratose extraction process, the 30Kda dialysate was concentrated, titrated to a pH of 6.0 and loaded onto a glass column containing Q Sepharose anion exchange resin (Q

Sepharose, General Electric Healthcare, Fairfield, CT). The resin was used according

Table 1. Different Fractions of Keratose: Classification by components, their

respective biologic activity and potential clinical uses

Fraction Composition Activity Potential Uses

Crude Alpha monomers, dimers and Stimulate tissue tetramers, Gamma, KAPs healing Burn healing Size: 3-300Kda Mostly Alpha monomers, Arteriolar Ischemic dimers and tetramers, scant vasodilation, some disorders: MI, Alpha/KAPs KAPs venodilation Sickle Crises, acute ischemic Size: 60-200Kda bowel Arteriolar Ischemic vasodilation, some disorders: MI, KAPs Alpha monomers, KAPs venodilation Sickle Crises, Size: 25-50Kda acute ischemic

bowel No vasoactive Hypovolemic properties. resuscitation, KIF Alpha Dimers and Tetramers dehydration, third Highly viscous Size: 120-280Kda space losses to the manufacturer’s instructions and was conditioned with three volumes of

10mM tris at a pH of 6.0. After loading the sample the column was rinsed with an additional three volumes of 10mM tris buffer. The effluent was collected, dialyzed at

30KDa NLMWCO, and processed to a dry powder as previously described (2).

107 This sample that did not bind to the exchange column was initially referred to as

“unbound fraction of keratose”. With the use of size exclusion chromatography, it was later proven to contain alpha dimers and tetramers exclusively (see chapter 2).

After several nomenclature discussions, this fraction was termed Keratose

Intermediate Fraction (KIFs) due to its lack of smaller alpha monomers (table 1).

The sample bound to the resin, representing the negatively charged KAP fraction, was eluted with three volumes of 100mM tris at pH 8.0 + 2M sodium chloride. This sample was dialyzed at 3KDa NLMWCO. Size exclusion chromatography of this fraction demonstrated two peaks; one at 28Kda and another one at 47Kda. These peaks represent alpha monomers and KAPs. This fraction of keratose was initially labeled “bound fraction of keratose” and, after SEC studies, was termed KAP fraction

(table 1).

All solutions were neutralized to a pH of 7.4, lyophilized, and sterilized by gamma irradiation (1M Rad).

Resuscitation fluids were prepared by aseptic reconstitution of the keratose powder with phosphate buffered saline at a concentration to match human blood viscosity

(3.88cP at 10 dynes/cm2). Viscosity was determined with a DV-II+ Pro cone and plate viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA).

108 The viscoelastic properties of these fractions are quite different from each other. As expected, the KIF component is the most viscous and the KAPs are the least viscous.

The initial compound composed of alpha/KAPs that was studied in chapter 2 reconstitutes similarly to human blood viscosity at a concentration of 40.3mg/ml ±

2.6, while the KIF component reconstitutes at the same viscosity with a concentration of 26.4mg/ml ± 1.3 (Figure 1). The KAPs fraction needed a much

Figure 1. Concentration of Keratose needed to prepare a compound with a viscosity of 3.88cP at 10dynes/cm2. * = 0.01, ** = 0.001 higher concentration to approximate human blood viscosity but a specific point was more difficult to assess due to small changes in concentration increasing viscosity in large units (i.e. a non-linear relationship between concentration and viscosity in that area of the curve). We found the range to be between 120-150mg/ml. One-way

109 ANOVA determined a statistically significant difference between the three compounds (F=498.2, p<0.0001). Bonferroni post-test showed a statistical difference between KIF and alpha (t=4.19, p<0.01), between KIF and KAPs (t=30.9, p<0.001), and between alpha and KAPs (t=23.12, p=0.001) (Figure 1).

Throughout this study, the extraction and purification methods for keratose were improved. These improvements allowed us to separate specific fractions of keratose until we obtained a sample with optimized viscoelastic properties. The KIF-KRF sample represents the most appropriate solution for resuscitation after hypovolemia, but we believe that fine-tuning the separation process can allow further optimization. The continuity of this research will keep yielding very important information to the research of biocompatible biomaterials for resuscitation.

110 REFERENCES

1. Secher NH, Van Lieshout JJ. Normovolaemia defined by central blood volume and venous oxygen saturation. Clin Exp Pharmacol Physiol. 2005 Nov;32(11):901-

10.

2. Sierpinski P, Garrett J, Ma J, Apel P, Klorig D, Smith T, et al. The use of keratin biomaterials derived from human hair for the promotion of rapid regeneration of peripheral nerves. Biomaterials. 2008 Jan;29(1):118-28.

111 SCHOLASTIC VITA

Fiesky A. Nunez Jr. MD

Wake Forest School of Medicine

Medical Center Blvd.

Winston-Salem, NC, 27157 [email protected] [email protected]

Education

2012 Ph.D. in Physiology and Pharmacology

Wake Forest School of Medicine, Winston Salem, NC

2004 Doctor of Medicine

Universidad de Carabobo, Valencia, Venezuela

Research and Teaching Experience

2011-Present Group Moderator: Scientific Integrity Course

Wake Forest School of Medicine

2011-2011 Group Moderator: Phys/Pharm Journal Club

Postbaccalaureate Premedical Program

Wake Forest School of Medicine

2009-Present Research Fellow

Wake Forest School of Medicine

112 Department of Orthopaedic Surgery

2007-2009 Clinical Research Fellow

Massachusetts General Hospital

Hand and Upper Extremity Service

2005-2007 Clinical Research Fellow and Surgery Intern

Centro Medico Guerra Mendez

Upper Extremity Service

Honors and Awards

2004 First place national award.

Venezuelan Society for Surgery of the Hand.

Lateral approach for the treatment of unstable distal fractures

of the radius: corrective osteotomies of radial malunions.

2004 First Place national scientific awards. Medical Doctors College

of Aragua, Venezuela. Scaphoid Pseudoarthrosis: Comparison

of surgical techniques.

Peer-Reviewed Published Articles

• Nunez F, Barnwell J, Li Z, Nunez Sr. F, Metaphyseal Ulnar Shortening Osteotomy

for the Treatment of Ulnocarpal Abutment Syndrome Using Distal Ulna Hook

Plate: A Case Series. J Hand Surg. [In Print].

• Nunez F, Trach S, Burnett L, Handa R, Van Dyke M, Callahan M, Smith T.,

Vasoactive Properties of Keratin Derived Compounds. Microcirculation.

2011;18:663-9

113 • Nunez F, Trach, S., Van Dyke, M., Callahan, M., Smith, T. Intravenous Infusion Of

Keratose-Based Fluid Induces Arteriolar Vasodilation In Cremaster Muscle Of

Rats (Abstract). Shock. 2010;33(7):14-87.

• Nunez F, Vranceanu AM, Ring D. Determinants of pain in patients with carpal

tunnel syndrome. Clin Orthop Relat Res. Dec;468(12):3328-32.

• Nunez F, Cariello, C., Blanco, A., Chuecos, J., Sosa, N., Nunez, F. Treatment of

forearm pseudoarthroses with wave plate and autologous bone graft. .

Venezuelan Society for Hand Surgery Journal. 1999;1(2):63-9.

• Nunez F, Guilliod, M., Figueroa, O., Chuecos, J. Writer’s Cramp: A New Etiologic

Approach. Venezuelan Society for Hand Surgery Journal. 2000;2(1):1-7.

• Nunez F, Perdomo, L., Sosa, N., Chuecos, J., Nunez, F., Guilliod, M., Figueroa, O.

Vicious Consolidation of Radius Fractures: Corrective Osteotomies. . Venezuelan

Society for Hand Surgery Journal. 2000;2(1):27-31.

• Nunez F, Trach, S., Kislukin, V., Van Dyke, M., Callahan, M., Smith, T. Comparison

Of Hemodynamic Changes After Hemorrhage And Treatment With Hyperviscous

Fluids (Abstract). Shock. 2010;33(7):14-87.

• Nunez F, Martinez, J., Guilliod, M., Nunez, F. Fifth Metacarpal Fractures:

Anterograde intramedullary mailing. Venezuelan Society for Hand Surgery

Journal. 2002;3(2).

Podium and Poster Presentations

• Nunez F, Callahan M, Burnett L, Van Dyke M, Smith T. Hemodynamic Circulatory

Changes After Hypovolemic Shock and Resuscitation with Low-Volume 6%

114 Hetastarch and High-Volume Lactated Ringer’s Solution. WFIRM Retreat, 2012.

Poster Presentation.

• Nunez F, Callahan M, Burnett L, Van Dyke M, Smith T. Hemodynamic Circulatory

Changes After Hypovolemic Shock and Resuscitation with Low-Volume 6%

Hetastarch and High-Volume Lactated Ringer’s Solution. Wake Forest School of

Medicine, Surgical Sciences Research Day, 2011. Poster Presentation.

• Nunez F, Trach S, Burnett L, Handa R, Van Dyke M, Callahan M, Smith T

Intravenous Infusion of Keratose-based Fluid Induces Arteriolar Vasodilation in

Cremaster Muscle of Rats. Wfirm Retreat 2011. Poster Presentation.

• Nunez F, Trach S, Van Dyke M, Kislukhin V, Callahan M, Smith T, Koman LA.

Comparison of Hemodynamic Changes after Life Threatening Hemorrhage and

Treatment with Hyperviscous Fluids: Keratose Resuscitation Fluid (KRF) vs

Hetastarch. Wfirm Retreat 2011. Poster Presentation.

• Nunez F, Trach S, Burnett L, Handa R, Van Dyke M, Callahan M, Smith T

Intravenous Infusion of Keratose-based Fluid Induces Arteriolar Vasodilation in

Cremaster Muscle of Rats. North Carolina Tissue Engineering and Regenerative

Medicine Annual Meeting, 2010. Poster Presentation

• Nunez F, Trach S, Burnett L, Handa R, Van Dyke M, Callahan M, Smith T

Intravenous Infusion of Keratose-based Fluid Induces Arteriolar Vasodilation in

Cremaster Muscle of Rats. Experimental Biology, 2010. Poster Presentation

• Villa J, Nunez F, Callahan M, Smith T. Intravenous Injection of Keratin

Biomaterials Restores Arteriolar Diameter in Striated Muscle of Hemorrhaged

Rats. Wake Forest School of Medicine, Surgical Sciences Research Day, 2010.

115 • Trach S, Nunez F, Callahan M, Van Dyke M, Smith T. Assesment of Cardiac Output

by Extracorporeal Ultrasound Dilution Measurement. Wake Forest School of

Medicine, Surgical Sciences Research Day, 2010.

• Nunez F, Vranceanu AM, Ring D. Determinants of Pain in Patients with

Idiopathic Median Nerve Dysfunction at the Carpal Tunnel. American Association

of Orthopaedic Surgeons Annual Meeting, 2010. Podium Presentation.

• Nunez F, Vranceanu AM, Ring D. Determinants of Pain in Patients with

Idiopathic Median Nerve Dysfunction at the Carpal Tunnel. American Society for

Surgery of the Hand, Annual Meeting, 2009. Podium Presentation.

• Nunez F. Use of Autologous Bone Graft in Complex Fractures: Osteoconductive

vs Osteoinductive properties in the healing process of fractures and

pseudoarthroses. Medical School Graduation Thesis.

Graduate Coursework

• Physiology and Pharmacology 701: Principles of Pharmacology

• Physiology and Pharmacology 702: Systems Physiology and Pharmacology

• Hypertension Journal Club (4 semesters)

• Statistics

• Introduction to Professional Development (2 semesters)

116