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Endothelial Dysfunction and Arterial Stiffness in Heart Transplant Recipients

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PAGE 1

ENDOTHELIAL DYSFUNCTION AND ARTERIAL STIFFNESS IN HEART TRANSPLANT RECIPIENTS By GARY L. PIERCE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2005

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Copyright 2005 by Gary L. Pierce

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This dissertation is dedicated to my wife Cathy. Her unwavering support, patience, and unconditional love for the past 4 years has made this accomplishment the most rewarding of my life. I am happy I coul d share it with the woman I l ove. I want to thank her for being a remarkable friend, spouse, career wo man, and caring and dedi cated mother to our daughter Carolyn.

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ACKNOWLEDGMENTS No project this size can be completed without help from many people. I first thank my supervisory committee chair and mentor (Randy Braith, Ph.D.) who gave me invaluable support and guidance through this 3-year project. I am also indebted to Dr. Braith for giving me the freedom to explore my research ideas, encouraging my teaching style, and being an advocate for my career development. I look forward to continued future collaboration and friendship with him. I would also like to thank my committee members (Scott Powers, Ph.D., Christiaan Leeuwenburgh, Ph.D., and Wilmer Nichols, Ph.D.). They offered valuable encouragement and advice during this project, and were instrumental in instilling in me the enthusiasm for scientific research. I would like to thank several individuals in the Heart Transplant Program in the Division of Cardiology at the University of Florida College of Medicine. In particular, Rich Schofield, M.D. helped me recruit patients for the study, supervised the graded exercise tests, and offered valuable advice on clinical issues involving heart-transplant recipients. I also thank James Hill, M.D. (the director of the Heart Transplantation program) for his full support of this project from its conception. Randy Harris, CVT, unselfishly offered his time and knowledge and taught me about high-resolution vascular ultrasound. I thank the heart transplant nurse practitioners at Shands Hospital (particularly Suzanne Conrad, Suzy Holder, Tim Cleeton, Tracy Walker, and Alex Price) for graciously responding to my frequent calls and emails. iv

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I would like to thank several individuals in our laboratory. My colleagues, David Edwards, Ph.D. and Peter Magyari, Ph.D. offered me valuable advice and friendship during my first year at the University of Florida. I also thank Darren Casey, M.S. and Scott Hamlin, M.S. for their camaraderie in the laboratory during the last 2 years and for their help in supervising the graded exercise tests, processing blood samples, and with biochemistry assays. I also thank Louise Perras and Kim Hatch (Center for Exercise Science) for their endless administrative help for the last 4 years. I would like to thank my parents Al and Rita Pierce; my brother Mark and his family; and my sister Margie and her family for their encouragement and support to pursue my professional goals. I thank my in-laws, Dr. and Mrs. John and Judy King for supporting my academic pursuits and for their love and support of me and Cathy during these past 4 years. I need to thank several colleagues and mentors in Boston who encouraged me to pursue doctoral training and a career in academic research: in particular, L. Howard Hartley, M.D.; Kyle McInnis, Sc.D; Avery Faigenbaum, Ed.D.; Gary Balady, M.D.; William Gillespie, Ed.D.; and Joe Libonati, Ph.D. Finally, I thank the heart-transplant recipients who participated in this project. Many of them inspired me with their will for life, positive attitude, and unselfish willingness to contribute to scientific research. I am grateful for the trust they placed in me. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iv LIST OF TABLES .............................................................................................................ix LIST OF FIGURES ...........................................................................................................xi ABSTRACT .....................................................................................................................xiv CHAPTER 1 INTRODUCTION........................................................................................................1 Rationale for the Study.................................................................................................6 Specific Aims and Hypotheses.....................................................................................8 2 REVIEW OF LITERATURE.....................................................................................14 Normal Endothelial Function.....................................................................................15 Nitric Oxide.........................................................................................................15 Nitric Oxide Synthase..........................................................................................15 Mechanism of NO Release..................................................................................16 Basal release of NO......................................................................................16 Agonist-mediated release of NO..................................................................16 Shear stress-mediated release of NO............................................................17 Pleiotropic Actions of NO...................................................................................17 Other Endothelial Vasodilators...........................................................................18 Endothelial Vasoconstrictors...............................................................................19 Endothelin-1.................................................................................................19 Angiotensin II...............................................................................................19 Vasoconstrictor prostaglandins....................................................................20 Vascular Endothelial Dysfunction..............................................................................20 Decreased NO Synthesis by eNOS......................................................................21 NO Degradation by Reactive Oxygen Species....................................................23 Vascular Endothelial Dysfunction before Heart Transplantation.......................24 Vascular Endothelial Dysfunction after Heart Transplantation..........................26 Cyclosporine and vascular endothelial dysfunction in heart transplant recipients...................................................................................................31 vi

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Inflammation and vascular endothelial dysfunction in heart transplant recipients...................................................................................................32 Asymmetric dimethylarginine and vascular endothelial dysfunction in heart transplant recipients.........................................................................33 Arterial Stiffness.........................................................................................................33 Arterial Stiffness and Cardiovascular Risk.........................................................37 Arterial Stiffness before Heart Transplantation..................................................37 Arterial Stiffness and Hypertension after Heart Transplantation........................39 Role of Exercise Training in HTR..............................................................................41 Exercise Training and Functional Capacity........................................................41 Exercise Training and Endothelial Dysfunction..................................................42 Exercise Training and Arterial Stiffness.............................................................44 Exercise Training and Nitric Oxide Synthesis............................................................46 Exercise Training and Oxidative Stress..............................................................48 Exercise Training and Vasoconstrictors..............................................................49 Exercise Training and Inflammation...................................................................50 3 METHODS.................................................................................................................52 Subjects.......................................................................................................................52 Inclusion Criteria.................................................................................................52 Exclusion Criteria................................................................................................53 Group Assignments.............................................................................................53 Exercise Training Protocol.........................................................................................53 Specific Measurements...............................................................................................54 Arterial Stiffness Testing.....................................................................................55 Endothelial Function Testing..............................................................................57 Brachial artery flow-mediated dilation........................................................57 Forearm and calf flow-mediated vasodilation..............................................59 Graded Exercise Test...........................................................................................61 Blood Collection..................................................................................................62 Plasma Biochemical Analysis.............................................................................62 Vasoactive balance.......................................................................................62 Lipid peroxidation........................................................................................62 Extracellular antioxidant enzyme activity....................................................63 Inflammatory markers..................................................................................63 Endogenous NO inhibition...........................................................................63 Blood hemoglobin, hematocrit, serum lipids, glucose, creatinine, white blood cell count, cyclosporine, and cytomegliovirus status.....................64 Endocardial biopsy rejection history............................................................64 Statistical Considerations............................................................................................64 4 RESULTS...................................................................................................................66 Subject Characteristics before and after Heart Transplantation.................................66 Serum Metabolic Parameters before and after Heart Transplantation........................68 Brachial Artery Endothelial Function before and after Heart Transplantation..........69 vii

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Blood Pressure and Pulse Wave Analysis before and after Heart Transplantation....70 Forearm and Calf Resistance Artery Endothelial Function before and after Heart Transplantation........................................................................................................72 Vasoactive Balance before and after Heart Transplantation......................................73 Plasma Lipid Peroxidation, Antioxidant Defense, and Endogenous Nitric Oxide Inhibition before and after Heart Transplantation...................................................73 Inflammatory Markers before and after Heart Transplantation..................................74 Baseline Subject Characteristics before Exercise Training or Control......................75 Body Weight, Serum Metabolic Parameters, and Endocardial Rejection History after Exercise Training............................................................................................76 Brachial Artery Endothelial Function after Exercise Training...................................77 Blood Pressure and Pulse Wave Analysis after Exercise Training............................78 Forearm and Calf Resistance Artery Blood Flow after Exercise Training.................79 Vasoactive Balance after Exercise Training...............................................................79 Lipid Peroxidation, Antioxidant Enzyme Activity, and Endogenous Nitric Oxide Inhibition after Exercise Training...........................................................................80 Inflammatory Markers after Exercise Training..........................................................80 Peak Cardiopulmonary Exercise Testing Variables after Exercise Training.............81 5 DISCUSSION.............................................................................................................95 Peripheral Conduit Artery Endothelial Function and Heart Transplantation.............96 Peripheral Resistance Artery Endothelial Function and Heart Transplantation.........98 Pulse Wave Analysis and Heart Transplantation......................................................100 Endothelial-Derived Vasoactive Balance and Heart Transplantation......................101 Lipid Peroxidation, Antioxidant Enzyme Activity, Endogenous Nitric Oxide Inhibition and Heart Transplantation....................................................................103 Inflammatory Markers and Heart Transplantation...................................................105 Peripheral Conduit Artery Endothelial Function and Exercise Training..................108 Peripheral Resistance Artery Endothelial Function and Exercise Training.............109 Pulse Wave Analysis, Blood Pressure, and Exercise Training.................................110 Endothelial-Derived Vasoactive Balance and Exercise Training.............................112 Lipid Peroxidation, Antioxidant Enzyme Activity, Endogenous Nitric Oxide Inhibition and Exercise Training...........................................................................114 Inflammatory Markers and Exercise Training..........................................................117 Exercise Capacity and Exercise Training.................................................................120 Conclusions...............................................................................................................120 Limitations and Future Research..............................................................................122 LIST OF REFERENCES.................................................................................................123 BIOGRAPHICAL SKETCH...........................................................................................141 viii

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LIST OF TABLES Table page 4-1 Patient characteristics before and after heart transplantation...................................67 4-2 Serum metabolic parameters before and after heart transplantation........................68 4-3 Brachial artery flow-mediated dilation before and after heart transplantation........69 4-4 Blood pressure components and pulse wave analysis before and after heart transplantation..........................................................................................................71 4-5 Forearm and calf flow-mediated vasodilation before and after heart transplantation..........................................................................................................72 4-6 Vasoactive balance before and after heart transplantation.......................................73 4-7 Lipid peroxidation, antioxidant enzyme activity, and endogenous nitric oxide inhibition before and after heart transplantation......................................................74 4-8 Inflammatory markers before and after heart transplantation..................................75 4-9 Baseline patient characteristics before exercise training or control.........................76 4-10 Body weight, serum metabolic parameters, and endocardial rejection episodes at baseline and after exercise training or control.........................................................77 4-11 Brachial artery flow-mediated dilation at baseline and after exercise training or control.......................................................................................................................78 4-12 Blood pressure components and pulse wave analysis at baseline and after exercise training or control.......................................................................................78 4-13 Forearm and calf flow-mediated vasodilation at baseline and after exercise training or control.....................................................................................................79 4-14 Vasoactive balance at baseline and after exercise training or control......................80 4-15 Lipid peroxidation, antioxidant enzyme activity, and endogenous nitric oxide inhibition at baseline and after exercise training or control.....................................80 ix

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4-16 Inflammatory markers at baseline and after exercise training or control.................81 4-17 Peak cardiopulmonary graded exercise testing variables at baseline and after exercise training or control.......................................................................................82 x

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LIST OF FIGURES Figure page 3-1 Study design.............................................................................................................55 3-2 Ascending aortic pressure waveform.......................................................................57 4-1 Brachial artery flow-mediated dilation before and after heart transplantation........83 4-2 Brachial artery flow-mediated diameter dilation before and after heart transplantation..........................................................................................................83 4-3 Aortic augmentation index (AI a ) corrected for heart rate=75 b/min before and after heart transplantation.........................................................................................83 4-4 Roundtrip travel duration of reflected wave (t p ) before and after heart transplantation..........................................................................................................84 4-5 Aortic systolic tension-time index (A s TTI) before and after heart transplantation..84 4-6 Forearm blood flow (FBF) before and after heart transplantation...........................84 4-7 Calf blood flow (CBF) before and after heart transplantation.................................85 4-8 Nitrate/nitrite (NOx) before and after heart transplantation.....................................85 4-9 Endothelin-1 (ET-1) before and after heart transplantation.....................................85 4-10 Eight (8)-iso-prostanglandin-F 2 (PGF 2 ) before and after heart transplantation....86 4-11 Superoxide dismutase (SOD) activity before and after heart transplantation..........86 4-12 Asymmetric dimethylarginine (ADMA) before and after heart transplantation......86 4-13 C-reactive protein (CRP) before and after heart transplantation..............................87 4-14 Log-transformed C-reactive protein (logCRP) before and after heart transplantation..........................................................................................................87 4-15 Interluekin-6 (IL-6) before and after heart transplantation......................................87 4-16 Tumor-necrosis factor-alpha (TNF-) before and after heart transplantation.........88 xi

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4-17 Soluble intercellular adhesion molecule-1 (sICAM-1) before and after heart transplantation..........................................................................................................88 4-18 Brachial artery flow-mediated dilation (FMD) at baseline and after 12 weeks of exercise training or control.......................................................................................88 4-19 Brachial artery absolute diameter dilation at baseline and after 12 weeks of exercise training or control.......................................................................................89 4-20 Aortic augmentation index (AI a ) normalized for heart rate at 75 b/min at baseline and after 12 weeks of exercise training or control.....................................89 4-21 Roundtrip travel time of reflected wave (tp) at baseline and after 12 weeks of exercise training or control. .....................................................................................89 4-22 Peak and total area under curve (AUC) forearm blood flow (FBF) at baseline and after 12 weeks of exercise training or control...................................................90 4-23 Peak and total area under curve (AUC) calf blood flow (CBF) at baseline and after 12 weeks of exercise training or control..........................................................90 4-24 Nitrate/nitrite (NOx) at baseline and after 12 weeks of exercise training or control.......................................................................................................................91 4-25 Endothelin-1 (ET-1) at baseline and after 12 weeks of exercise training or control.......................................................................................................................91 4-26 Eight (8)-iso-prostaglandin-F 2 (PGF 2 ) at baseline and after 12 weeks of exercise training or control.......................................................................................91 4-27 Superoxide dismutase (SOD) activity at baseline and after 12 weeks of exercise training or control.....................................................................................................92 4-28 Asymmetric dimethylarginine (ADMA) at baseline and after 12 weeks of exercise training or control.......................................................................................92 4-29 C-reactive protein (CRP) at baseline and after 12 weeks of exercise training or control.......................................................................................................................92 4-30 Interleukin-6 (IL-6) at baseline and after 12 weeks of exercise training or control.......................................................................................................................93 4-31 Tumor necrosis factor-alpha (TNF-) at baseline and after 12 weeks of exercise training or control.....................................................................................................93 4-32 Soluble intercellular adhesion molecule-1 (sICAM-1) at baseline and after 12 weeks of exercise training or control.......................................................................93 xii

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4-33 Peak exercise oxygen uptake (VO 2 ) on graded exercise test at baseline and after 12 weeks of exercise training or control..................................................................94 4-34 Peak exercise duration on graded exercise test at baseline and after 12 weeks of exercise training or control.......................................................................................94 xiii

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xiv Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ENDOTHELIAL DYSFUNCTION AND ARTERIAL STIFFNESS IN HEART TRANSPLANT RECIPIENTS By Gary L. Pierce August 2005 Chair: Randy W. Braith Major Department: Applied Physiology and Kinesiology Heart transplantation (HT) has become a life-extendi ng intervention for patients with end-stage heart failure (HF). However, frequent complications after HT, such as hypertension and coronary artery vasculopa thy (CAV), have been linked to vascular endothelial dysfunction (VED) a nd arterial stiffness (AS), and jeopardize the long-term survival of heart transplant recipients (H TR). Recent studies suggest that endurance exercise training modifies VED and AS in chronic HF and hypertensive individuals. Therefore, the purpose of this study was to i nvestigate the effects of HT on VED and AS in end-stage HF patients, and the effects of endurance exercise training early after HT. Twelve (n=12) end-stage HF patients awai ting orthotopic HT at Shands Hospital at the University of Florida were recru ited in the observational part of the study. Peripheral endothelial function, AS, and plas ma vasoactive balance, lipid peroxidation, antioxidant enzyme activity, inflammation and nitric oxide ( NO) inhibition were measured before and 8 weeks after HT. HT R were randomly assigned to an exercise

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training group (TRAINED; n=9) who performed 12 weeks of supervised endurance exercise training beginning at 8 weeks post-HT, or to a non-exercise control group (CONTROL; n=7). All vascular and plasma measurements were performed before and after 12 weeks. Brachial artery flow-mediated dilation (+49.5%) and calf (+34.8%) hyperemic blood flow (BF) improved after HT. Superoxide dismutase activity (SOD), C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-), and soluble intercellular adhesion molecule (sICAM-1) decreased significantly (p<0.05) 17, 23, 63, and 35% after HT, respectively. In the exercise training study, brachial artery FMD did not change in TRAINED, however there was a significant decrease in FMD (-29%) in CONTROL after 12 weeks. Peak forearm (+34%) and calf (+17%) hyperemic blood flow increased significantly in TRAINED, but did not change in CONTROL. Furthermore, there was a significant increase in TNF(+53%) in CONTROL, but no change in TRAINED after 12 weeks. We found that HT improved peripheral endothelial function and partially reduced the hyperinflammatory state in end-stage HF subjects. Furthermore, endurance exercise training attenuated a progressive decline in brachial artery endothelial function, improved endothelial function of limb resistance vasculature, and attenuated a progressive increase in TNFin HTR. xv

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CHAPTER 1 INTRODUCTION Heart transplantation (HT) has become a life-extending intervention for patients with end-stage heart failure (HF). Although HT reverses many of the primary symptomatic and physiological derangements of the chronic HF syndrome, lifelong immunosuppressive therapy and cardiac denervation result in de novo physiological and clinical sequelae that jeopardize long-term survival of the heart-transplant recipient (HTR). Post-transplant hypertension and coronary artery vasculopathy (CAV) may be the most frequent complications in HTR surviving greater than one year (Ventura et al. 1997; Davis et al. 1996; Hollenberg et al. 2001), and have been linked to vascular endothelial dysfunction (VED) and arterial stiffness (Davis et al 1996; Hollenberg et al. 2001; Schofield et al. 2003). One of the earliest events in the pathophysiology of cardiovascular disease is VED. VED is associated with traditional risk factors for cardiovascular disease before manifestations of clinical atherosclerosis, and is a key contributor in the progression of advanced symptomatic disease. VED refers to a pathological phenotype of the vascular endothelium including pro-inflammatory and thrombotic properties, enhanced platelet aggregation, impaired inhibition of vascular smooth-muscle growth, and impaired endothelial-dependent vasodilation (EDV) (Cai and Harrison 2000). Clinically, impaired EDV of the coronary (Halcox et al. 2002; Suwaidi et al. 2000) and peripheral (Gokce et al. 2003; Heitzer et al. 2001) circulation are independent predictors of adverse cardiovascular events in individuals with (or at risk for) cardiovascular disease. 1

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2 Physiologically, impaired EDV of coronary arteries may contribute to impaired myocardial perfusion and myocardial ischemia (Halcox et al. 2002), whereas impaired EDV of peripheral conduit and resistance arteries may contribute to increased vascular resistance (Hambrecht et al. 2000), large-conduit artery stiffness (Wilkinson et al. 2004), and reduced exercise muscle blood flow during exercise (Kao 1994). Thus, pharmacological or non-pharmacological interventions that preserve endothelial function should be a major therapeutic goal for individuals at risk for cardiovascular disease. EDV occurs from the release of endothelial-derived vasodilators including nitric oxide (NO), prostacyclin (PGI 2 ), and endothelial-derived hyperpolarizing factor (EDHF) (Mombouli and Vanhoutte 1999). Of the three vasodilators, endothelial-derived NO has been the most widely studied because impaired synthesis or enhanced degradation of NO plays a key role in VED. NO is synthesized by a 5-electron oxidation of the amino acid L-arginine by the endothelial isoform of nitric oxide synthase (eNOS) enzyme. NO induces EDV via agonist stimulation of a muscarinic receptor on the endothelial membrane, or by a mechanical shear stress-mediated mechanism from increased laminar blood flow along the endothelial wall. One mechanism responsible for impaired EDV is decreased bioavailability of endothelial-derived NO (Vallance and Chan 2001). Cai and Harrison (2000) hypothesized that reduced bioavailability of endothelial-derived NO occurs due to several potential mechanisms. First, decreased synthesis of NO may be due to decreased eNOS gene transcription, increased post-transcriptional degradation of eNOS mRNA, or post-translational modification eNOS enzyme activity. Moreover, increased asymmetric dimethylarginine (ADMA), an endogenous intracellular

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3 competitive inhibitor of eNOS, may also contribute to decreased NO synthesis (Tran et al. 2003). A second potential mechanism of impaired EDV is increased inactivation of NO by reactive oxygen species (ROS), such as superoxide anion (Cai and Harrison 2000). In states of vascular homeostasis, superoxide is rapidly dismutated to hydrogen peroxide (H 2 0 2 ) and water by intracellular Cu/Zn superoxide dismutase (SOD) in the endothelial cytosol (Cai and Harrison 2000). In the vascular wall, superoxide produced is quickly dismutated by the primary extracellular isoform of SOD (ecSOD), which is strategically located on the endothelial membrane between the endothelial and smooth muscle layer (Fukai et al. 2002). However, when excess superoxide overwhelms the primary antioxidant defenses, superoxide reacts quickly with NO to generate the potent oxidant, peroxynitrate (ONOO-), and the metabolite nitrate, both of which have minimal vasodilating properties (Cai and Harrison 2000). In addition, ROS (such as superoxide and ONOO-), inactivate dimethylarginine dimethylaminohydrolase (DDAH), the enzyme that degrades intracellular ADMA, thus increasing ADMA and further inhibiting NO synthesis (Sydow and Munzel 2003). A third mechanism that contributes to VED and impaired EDV is increased vasoconstrictor peptides endothelin (ET-1) and angiotensin II (ANG-II), which oppose the vasodilator action of NO and promote vasoconstriction (Nickenbig and Harrison 2002). ET-1 is produced by the endothelial cells and other tissues when exposed to cytokines such as TNFand the immunosuppressive agent cyclosporine (Bunchman and Brookshire 1991). ANG II is increased in plasma and vascular tissues in diseased

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4 conditions such as acute and chronic HF due to overactivation of the renin-angiotensin system and sympathetic activity to the kidney (Nickenbig and Harrison 2002). In HTR receiving immunosuppressive therapy, EDV is impaired in the coronary arteries early after HT (Fish et al. 1988; Mills et al. 1992; Davis et al. 1996; Hollenberg et al. 2001), and is an independent predictor of the development of CAV (Davis et al. 1996; Hollenberg et al. 2001) and cardiac death (Hollenberg et al. 2001). In the peripheral vasculature, impaired EDV is present in conduit brachial artery of HTR (Patel et al. 2002; Lim et al. 2003; Saxonhouse et al. 2000), particularly in HTR with antecedant ischemic HF etiology (Patel et al. 2001). EDV of forearm resistance arteries is improved after HT in end-stage HF patients (Sinoway et al. 1988; Kubo et al. 1993; Cavero et al. 1994), however it is currently unknown whether it returns to that of age-matched controls. Physiologically, impaired EDV contributes to increased vascular resistance (Kao et al. 1994), arterial stiffness (Schofield et al. 2002), decreased exercise capacity (Kao et al. 1994), and decreased exercise muscle blood flow in HTR (Kao et al. 1994). Clinically, impaired peripheral EDV may partly contribute to the development of de-novo hypertension after HT, which jeopardizes the long-term success of the cardiac allograft (Lim et al. 2002; Caveo et al. 1994). Impaired peripheral EDV may also contribute to increased stiffness (reduced compliance) of large elastic and muscular conduit arteries (Wilkinson et al. 2004). Increased arterial stiffness of large elastic and muscular conduit arteries increases the amplitude of the forward (incident) pressure wave during LV ejection, and increases pulse wave velocity of reflected pressure waves returning to the ascending aorta. This change in amplitude and timing of forward and reflected pressure waves causes the

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5 reflected waves to return early to the ascending aorta during systole instead of diastole, augmenting aortic systolic pressure. Thus, augmented aortic systolic and pulse pressure due to arterial stiffness of large conduit arteries contributes to increased LV afterload, myocardial oxygen demand, and wasted energy by the heart (Nichols and Singh 2002). Large-artery stiffness has been reported in primary aging (Tanaka et al. 2000), hypertension (Nichols and Singh 2002), renal failure (Laurent et al. 2003), and chronic HF (Nichols and Pepine 1992; Lage et al. 1994; Mitchell et al. 2001) population. Arterial stiffness is also an independent predictor of adverse cardiovascular events in patients with renal failure (Laurent et al. 2003) and coronary artery disease (Weber et al. 2003). To date, only one cross-sectional study has evaluated arterial stiffness in HTR. Schofield et al. (2002) reported that that 82% of 53 HTR had an elevated aortic augmentation index, elevated aortic pulse pressure, and a decreased time of reflected wave (inverse of pulse wave velocity), compared to age-matched controls, despite normal brachial systolic and mean blood pressure controlled by antihypertensive medications. Although there are currently no data on the prognostic implications of increased arterial stiffness in HTR, Schofield and colleagues show that HTR may be at increased cardiovascular risk despite having optimally managed brachial blood pressure. Chronic cyclosporine therapy in HTR results in increased ROS in the vascular wall (Dietrich et al. 1994) and elevated circulating ET-1 and ANG II (Lerman et al. 1992; Haas et al. 1993;Grief et al.1993; Perez-Villa et al.2004). ET-1 has been implicated in the development of post-transplant hypertension and vascular remodeling of smooth muscle. ANG II is elevated secondary to chronic cardiac denervation (Braith et al. 1996; Perez-Villa et al 2004) and cyclosporine-induced overactivation of the renin-angiotensin

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6 system (Julien et al. 1993). ANG II is a potent vasoconstrictor and stimulates production of ROS in the vascular wall via activation of membrane-bound NADPH oxidase. As such, ANG II contributes to impaired EDV and hypertension in HTR (Nickenbig and Harrison 2002), and has been implicated in the development of coronary artery vasculopathy in HTR (Yousufuddinn and Yamani 2004). Thus, interventions that reduce ANG II and ET-1 levels may have important physiological and clinical benefit in HTR. Cardiovascular disease has recently gained widespread acceptance as an inflammatory disease. As such, inflammatory mediators C-reactive protein (CRP) and soluble intercellular adhesion molecule (sICAM-1) have been reported to be elevated in HTR (Laberrere et al. 2000) and are strong predictors of coronary artery vasculopathy (Pethig et al. 2000), allograft failure (Eisenburg et al. 2000), and mortality in HTR (Laberrere et al. 2002). Besides being a marker of future cardiovascular outcomes in HTR, in vitro studies have recently confirmed that elevated CRP actively contributes to VED by activating expression of the endothelial adhesion molecule sICAM-1 (Pasceri et al. 2000), and decreasing eNOS mRNA, protein, and bioactivity (Venupogal et al. 2002). Proinflammatory cytokines IL-6 and TNF(known hepatic stimulants of CRP) are also elevated in HTR and may be involved in VED in HTR (Katz et al. 1994; Holm et al. 2000; Weis et al. 2001). Thus, interventions that attenuate basal levels of inflammatory mediators in HTR may improve endothelial function. Rationale for the Study Alterations in endothelial function in humans were first described by Ludmer et al. (1986) who observed paradoxical vasoconstriction of the left coronary artery in response to acetylcholine in patients with CAD. Hambrecht et al. (2000b) reported that lower

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7 body-dynamic endurance-exercise training improved this paradoxical vasoconstriction of the coronary arteries in response to acetylcholine in patients with CAD, suggesting that lower body exercise training can alter endothelial function systemically. In addition, other studies report that lower body exercise training improves peripheral VED in the upper limbs in individuals with essential hypertension (Higashi et al. 1999), CAD (Edwards 2004a et al.; Walsh et al. 2003), and chronic HF (Linke et al. 2001). This supports the theory of a systemic improvement in endothelial function, since lower body exercise improved upper-body limb vasculature. This theory was further supported by Anderson et al. (1995) who reported that brachial artery flow-mediated EDV is correlated with acetlycholine-induced coronary EDV in patients with CAD (r=0.36). Furthermore, Takase et al. (1998) reported a strong positive correlation between brachial artery flow-mediated EDV and coronary artery flow-mediated EDV (r=0.79) in CAD patients. Such studies suggest that endothelial function testing in the peripheral vasculature of the upper limbs may be a good adjunct method to detect changes in systemic endothelial function in response to a therapeutic intervention. As mentioned, impaired coronary artery EDV is a strong predictor of coronary artery vasculopathy (Davis et al. 1996; Hollenberg et al. 2001) and cardiac death in HTR (Hollenberg et al. 2001). Therefore, restorating endothelial function (such as with exercise training) should be a major therapeutic goal to potentially decrease long-term cardiovascular risk in HTR. However, there have been no prospective reports in the literature on the modulating effects of an endurance-exercise training intervention on endothelial function in HTR.

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8 Specific Aims and Hypotheses Our study is the first prospective, randomized, controlled study to investigate the effects of endurance exercise training on endothelial function, arterial stiffness, oxidative stress, and inflammation in HTR. Our study is also the first longitudinal, prospective study on the effects of HT on endothelial function and arterial stiffness in end-stage HF patients, and comparing them to an age-matched healthy control group. Thus, our experiments are novel and will further the understanding of the mechanisms contributing to VED in HTR. Specific Aim 1: To measure EDV of limb conduit and resistance arteries, systemic arterial stiffness, plasma vasoactive balance, oxidative stress, antioxidant enzyme activity, and endogenous NO inhibition before and 2 months after HT in patients with end-stage HF, and in an age-matched healthy control group. Hypothesis 1: Brachial artery flow-mediated dilation (FMD), forearm/calf flow mediated vasodilation (peak FBF/CBF), aortic augmentation index (AI a ), plasma nitrate/nitrite (NOx), ET-1, 8-iso-prostaglandin-F 2 (PGF 2 ), ecSOD activity, inflammatory cytokines, and ADMA will improve 2 months after HT, but will remain abnormal compared to age-matched healthy controls. Rationale: Although it is accepted that coronary artery VED exists in HTR (Fish et al. 1988; Mills et al. 1992), data are conflicting on whether endothelial function of peripheral limb vasculature improves in end-stage HF patients after HT. Several studies suggest that endothelial function of brachial artery does not improve after HT in HTR patients of ischemic HF etiology, and that it does improve in those HTR of non-ischemic HF etiology (Patel et al. 2001). Other studies report reduced brachial artery FMD in

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9 HTR compared to controls irrespective of etiology of HF (Saxonhouse et al. 2000; Lim et al. 2002; Schmidt et al. 2002; Cuppoletti et al. 2003). However, there have been no longitudinal studies of brachial artery FMD before and after HT in the same cohort compared to age-matched, healthy controls. Three studies have evaluated endothelial function of forearm resistance arteries before and after HT (Sinoway et al. 1988; Kubo et al. 1993; Cavero et al. 1994). In a prospective study before and after HT, Sinoway et al. (1988) reported that forearm EDV in response to reactive hyperemia did not improve within several weeks of HT, but improved by 4 months. Kubo et al. (1993) reported that EDV in response to reactive hyperemia in forearm resistance arteries improved 4 months after HT. Cavero et al. (1994) reported an increase in forearm EDV 24-36 hours after HT, and then no change 1 week and 6 weeks after starting cyclosporine therapy. However, these studies did not have an age-matched, healthy control group for comparison, so it is unclear whether endothelial function in peripheral resistance arteries HTR was restored to normal. Furthermore, no studies have investigated the effects of HT on systemic arterial stiffness in end-stage HF patients, or on the potential mechanisms involved in VED such as oxidative stress, enzymatic antioxidant capacity, NO inhibition, and inflammation. Specific Aim 2: To measure EDV of limb conduit and resistance arteries, systemic arterial stiffness, plasma vasoactive balance, oxidative stress, antioxidant enzyme activity, and endogenous NO inhibition in HTR before, and after 12 weeks of supervised endurance-exercise training or a 12-week control period.

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10 Hypothesis 2: In HTR, 12 weeks of supervised endurance exercise training will increase brachial-artery FMD, peak forearm and calf BF, plasma NOx, and ecSOD activity; and decrease aortic AI, plasma ET-1, 8-iso-PGF 2 and ADMA. Rationale: Several studies have reported that exercise training improves EDV in both conduit (Hambrect et al. 1998; Linke et al. 2001) and resistance arteries (Katz et al. 1997) in chronic HF patients, and that the improvement is NO-mediated. In a design similar to ours, subjects with essential hypertension, Higashi et al. (1999) reported a 22% increase in peak FBF during reactive hyperemia using plethysmography, after a 12 week exercise training intervention. The increase in FBF was abolished by the NO inhibitor, L-NMMA, suggesting that the increase in EDV of forearm resistance arteries was NO mediated. In HTR, one cross-sectional study has investigated the effects of exercise on endothelial function. Schmidt et al. (2002) reported higher brachial artery FMD in exercise-trained HTR than sedentary HTR. No prospective studies have yet reported the effects of endurance-exercise training on either peripheral-conduit or resistance-artery endothelial function in HTR. Cross-sectional studies suggest that exercise capacity is positively associated with increased arterial compliance of large arteries in healthy older individuals (Vaitkevicius et al. 1993) and persons with dilated cardiomyopathy (Bonapace et al. 2003). Several prospective studies report that exercise training decreased arterial stiffness in sedentary young (Cameron et al. 1994), sedentary aged (Tanaka et al. 2000), individuals with CAD (Edwards et al. 2004), and chronic HF (Parnell et al. 2002). However, no prospective studies have reported the effects of endurance-exercise training on arterial stiffness in HTR.

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11 Endurance-exercise training increased aortic expression of eNOS mRNA and eNOS protein in both animal (Sessa et al. 1994, Fukai et al. 2000; Woodman et al. 1999) and human models (Hambrecht et al. 2003). Concominant increases in agonist-mediated EDV suggest an increased NO synthesis after chronic exercise training. Plasma NOx, the stable end product of NO metabolism in plasma, also is increases after 8 weeks of exercise training in healthy humans (Jungersten et al. 1997; Maeda et al. 2001) and in CAD patients (Edwards et al. 2004a). Together, these data support the hypothesis of increased systemic NO synthesis after chronic exercise training. ET-1 is elevated in HTR due to chronic exposure of the endothelium to cyclosporine and may contribute to hypertension and impaired EDV in HTR (Haas et al. 1993; Greiff et al. 1993). Endurance exercise training reduces reduce plasma ET-1 levels in young, healthy subjects (Maeda et al. 2001), and in older women (Maeda et al. 2003), but not in chronic HF patients (Callaerts-Vegh et al. 1998). Plasma ET-1 levels are also inversely correlated to increased plasma NOx after exercise training in young healthy subjects, suggesting that NOx has a modulating effect on ET-1 levels (Maeda et al. 2001). However, the effects of endurance exercise training on circulating ET-1 levels in HTR has not been investigated. The F 2 isoprostane isomer, 8-iso-prostaglandin-F 2 (PGF 2 ), a stable and specific marker for in vivo oxidative stress-induced lipid peroxidation, can be measured in plasma or as its metabolite in urine (Roberts and Morrow 2000). Plasma or urinary levels of 8-iso-PGF 2 ), are elevated in patients with cardiovascular risk factors such as smoking, hypercholesterolemia, diabetes (Pratico 1999; Patrono and Fitzgerald 1997), coronary artery disease (Vassalle et al. 2003), and chronic HF (Polidori et al. 2004). Also, 8-iso

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12 PGF 2 has biological vasoconstrictor action (Roberts and Morrow 2000) and is an independent predictor for development of cardiovascular disease (Schwedhelm et al. 2004). Furthermore, Edwards et al. (2004a) found that 12 weeks of endurance exercise training reduced plasma levels of 8-iso-PGF 2 and increased plasma NOx and ecSOD activity in patients with CAD. However, no prospective study has yet reported the effects of endurance exercise training on plasma levels of 8-iso-PGF 2 NOx, and ecSOD activity in HTR. Moreover, no human studies have tested the effects of endurance exercise training on ADMA. We hypothesize that by attenuating production of ROS and preventing inactivation of DDAH, exercise training may lower ADMA levels and thus preserve NO synthesis. Specific Aim 3: To measure plasma levels of inflammatory cytokines in HTR before and after 12 weeks of supervised endurance-exercise training or 12 week control period. Hypothesis 3: In HTR, 12 weeks of supervised endurance-exercise training will decrease plasma levels of CRP, TNF-, IL-6, and sICAM-1. Rationale: In addition to being markers of future cardiovascular risk, experimental evidence shows that CRP, TNF-, and sICAM-1 actively contribute to VED (Blake and Ridker 2003). Several prospective exercise-training studies have tested the effects of endurance-exercise training on inflammatory mediators in patients with CAD (Milani et al. 2004; Edwards 2002) and chronic HF (Larsen et al. 2001; Adamopoulos et al. 2003). Milani et al. (2004) reported a 41% decrease in CRP in a cohort of 277 CAD patients who completed 12 weeks of exercise training as part of cardiac rehabilitation. Edwards et al. (2002) reported that 12 weeks of exercise training as part of cardiac rehabilitation

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13 (in patients with CAD) lowered CRP by 45% and IL-6 by 32%. In 28 patients with chronic HF, Larsen et al. (2001) reported a 12.5% decrease in TNFbut no change in IL-6. Furthermore, in 24 chronic HF patients, Adamopoulos et al. (2003) reported that 12 weeks of endurance exercise training significantly lowered IL-6, TNF-, sVCAM-1 and sICAM-1. However, no prospective studies have tested the effects of endurance-exercise training on inflammatory mediators in HTR.

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CHAPTER 2 REVIEW OF LITERATURE Before the 1980s, the endothelial layer of the vasculature was believed to be a physiological inert layer of epithelial cells acting as a barrier between the blood and medial layer of the vascular wall. This changed with Furchgott and Zawadzkis (1980) discovery that vasorelaxation of vascular smooth muscle cells in response to acetylcholine is dependent on an endothelial-derived relaxing factor released from endothelial cells. They reported that if the endothelial layer was removed from rabbit aorta, the vessel vasoconstricted in response to acetylcholine, but its vasodilatory response to nitrates was preserved. In the late 1980s, this endothelial-relaxing compound was discovered to be the free radical gas, NO (Palmer et al. 1987; Ignarro et al. 1987). Furthermore, over the last decade it has been discovered that the endothelial layer is not inactive, but is intimately involved in regulating vascular tone and homeostasis. NO has proven to be a critical component of vascular health such that the decreased bioavailability of NO results in the phenomenon of ED (Cai and Harrison 2000). ED is present in individuals with primary cardiovascular risk factors such as hypercholesterolemia, hypertension, diabetes, and obesity; and with primary aging and in those with documented cardiovascular disease (Drexler 1997). As mentioned, VED develops years before clinical evidence of atherosclerosis develops and remains evident in individuals with occult cardiovascular disease (Drexler 1997). In particular, VED is evident in individuals with both ischemic and non-ischemic HF (Kubo et al. 1991; Patel et al. 2001), and is believed to partly contribute to increased peripheral vascular 14

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15 resistance (Hambrecht et al. 2000), arterial stiffness (Arnold et al. 1991), and impaired muscle-blood flow and exercise capacity in HF patients (Hambrecht et al. 1998; Linke et al. 2001). Furthermore, both coronary and peripheral ED persists in end-stage HF patients who undergo orthotopic HT (Fish et al. 1988; Mills et al. 1992; Patel et al. 2001; Schmidt et al. 2003), however, the mechanisms have not been elucidated. Normal Endothelial Function Nitric Oxide Endothelial-derived NO is synthesized from the amino acid L-arginine which undergoes a five-electron oxidation to NO and L-citrulline by the endothelial isoform of the nitric oxide synthase (eNOS) enzyme (Moncada and Higgs 1993). Since NO has a short biological half-life (3-10 seconds) at physiological pH, and is rapidly oxidized to nitrate (NO 2 ) and then nitrite (NO 3 ) by oxygenated hemoglobin (Moncada and Higgs 1993), the primary biological signaling activity of endothelial NO occurs a short diffusion distance across the endothelial wall into the smooth muscle layer. NO binds to the heme moiety of the enzyme guanylate cyclase activating it to catalyze the conversion of GTP to the second messenger cyclic guanosine 3, 5-monophosphate (cGMP). cGMP mediates vascular smooth muscle relaxation via increase Ca +2 extrusion from the smooth muscle cells (Moncada and Higgs 1993). Nitric Oxide Synthase There are three isoforms of nitric oxide synthase (NOS): constitutively expressed neuronal NOS (nNOS) and endothelial NOS (eNOS), and the inducible NOS isoform (iNOS). nNOS and eNOS activation are Ca +2 -dependent and are located in neurons and vascular endothelial cells, respectively (Mayer and Hemmens 1997). Endothelial cells constitutively express eNOS which is an NADPH-dependent oxygenase that requires the

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16 cofactors tetrahydrobiopterin (BH 4 ), FAD, and FMN (Mayer and Hemmens 1997). In endothelial cells, eNOS, is located in special invaginations in the cell membrane called caveolae, and is associated with a specialized protein, caveolin, which interacts with signaling proteins, such as eNOS, and inhibits its activity (Feron et al. 1998). Stimulation of endothelial cells by agonists such as acetylcholine or bradykinin, dissociates the caveolin/NOS complex and allows Ca +2 /calmdulin complex to bind to NOS and activate NO synthesis (Feron et al. 1998). This compartmentalization of eNOS allows rapid conversion of L-arginine to NO since the y+ transporter for L-arginine is also located in the cell membrane near the caveolae (Harrison 1997). Mechanism of NO Release Basal release of NO There is a continuous basal release of NO from vascular endothelium to maintain resting vascular tone. The first evidence of this was in 1989 by Vallance et al. (1989) who demonstrated that by infusing an inhibitor of eNOS, N G -monomethyl-L-arginine (L-NMMA), into the brachial artery of the human forearm, there was a dose-dependent reduction in resting blood flow. L-NMMA is a methylated analogue of L-arginine which prevents the synthesis of NO and when systemically infused into experimental animals and humans (Vallance et al. 1989), results in an increase in mean arterial pressure. Thus, these data demonstrate the importance of NO in maintaining tonic peripheral arterial vasodilation and blood pressure in vivo. Agonist-mediated release of NO Several substances can stimulate muscarinic receptors on the endothelial membrane and activate eNOS to increase NO synthesis via a Ca +2 -dependent mechanism. In particular, acetylcholine, bradykinin, and substance P, can stimulate EDV of resistance

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17 and conduit arteries in humans, which can be partially inhibited by L-NMMA. In addition, other endothelial vasodilators, such as prostacyclin and EDHF may also be involved in EDV but to a lesser extent. Shear stress-mediated release of NO Laminar pulsatile flow of blood along the endothelial wall causes a mechanical shear stress which provides the stimulus for both short-term and long-term regulation of eNOS and NO synthesis. In vitro studies suggest that specific potassium ion channels respond immediately to increase shear stress and induce increase intracellular calcium within one minute and increase short-term eNOS acitivity and NO synthesis (Cooke et al. 1991). After one hour exposure of endothelial cells to increased shear stress, serine-threonine protein kinase B (Akt) phosphorylates eNOS activating the enzyme and increasing NO synthesis six-fold independent of increase in intracellular calcium (Dimmeler et al.1999). Prolonged exposure of increased shear stress for 24 hours, induces increased eNOS mRNA expression in a dose dependent manner in bovine and human endothelial cells (Uematso et al. 1995), and in isolated soleus feed arteries from rats exposed to increased luminal shear stress (Woodman et al. 2004). Furthermore, human studies suggest that flow-mediated dilation of forearm brachial (Lieberman et al. 1996) and resistance arteries (Meredith et al. 1996) is partially attenuated by the eNOS inhibitor, L-NMMA, suggesting that shear-stress mediated vasodilation is NO dependent. Pleiotropic Actions of NO Endothelial-derived NO not only modulates vascular tone, but also has antiatherosclerotic, antithrombotic, and anti-inflammatory functions on the endothelial wall (Vallance and Chan 2001). Specifically, NO suppresses platelet aggregation, leukocyte migration and adhesion to the endothelial wall, and prevents vascular smooth

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18 muscle migration and proliferation into the subendothelial space (Mombouli and Vanhoutte 1999). Thus, the decrease in bioavailability of NO not only promotes an endothelial phenotype of vasoconstriction, but also of platelet aggregation, leukocyte migration and adhesion to the endothelial layer, and smooth muscle migration and proliferation into the subendothelial layer (Mombouli and Vanhoutte 1999). Other Endothelial Vasodilators The endothelium also was discovered to release several other vasodilating compounds including prostacyclin and an unknown endothelial derived hyperpolarizing factor (EDHF). The production of prostaglandins is regulated by the availability of membrane-bound arachadonic acid (AA) and the activity of the enzyme cyclooxygenase (COX). AA is derived from the phospholipid membrane which is enzymatically released via action of the enzyme phospholipase A 2 and is converted to prostaglandin H 2 by COX and peroxidase (Savidge 2001). Prostacyclin is the major endothelial metabolite derived from arachadonic acid (AA) and diffuses into vascular smooth muscle and activates the enzyme adneylate cyclase. Adenylate cyclase converts ATP to 3, 5 cyclic adenosine monophosphate (cAMP) and induces vasorelaxation of vascular smooth muscle (Savidge 2001). Endothelial hyperpolarizing factor (EDHF) is less well characterized but is believed to act through activation of calcium-activated K+ channels on the smooth muscle membrane resulting in hyperpolarization and vasorelaxation (Mombouli and Vanhoutte 1999; Triggle et al. 2004). Although identification of EDHF is still unclear, possible EDHFs include hydrogen peroxide, isoprostanes, potassium, or the AA metabolite, epoxyeicosatrienoic acid (Triggle et al. 2004)

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19 Endothelial Vasoconstrictors The endothelial wall also secretes vasoconstrictors ET-1, ANG II, and vasoconstrictor prostaglandins which compete with NO for the vasoactive balance. Endothelin-1 ET-1, the major endothelin isoform, is produced by the endothelium and is derived from the precursor big ET-1 by the enzyme endothelin converting enzyme (ECE). ET-1 can act in a paracrine or autocrine manner via ET type A or ET type B receptors on adjacent endothelial and smooth muscle cells. ET A and ET B receptors are on smooth muscle cells and both mediate vasoconstriction, cell proliferation, and hypertrophy. ET B exist on endothelial cells as well, and mediate vasodilation via release of NO and prostacyclin (Taddei et al. 2001). Although ET-1 is generally considered to be a potent vasoconstrictor, this activity can be attenuated by the increased expression or activity of ET B receptor mediated release of NO by the endothelium (Taddei et al. 2001). However, systemic infusion of ET-1 into animals results in a decrease in glomerlular filtration rate, renal blood flow, and an increase in mean arterial blood pressure (Goetz et al. 1988). In humans, infusion of an ET-1 receptor antagonist significantly decreased peripheral vascular resistance and blood pressure (Haynes et al. 1996). Taken together, these data support the idea that ET-1s vasoconstrictor activity on the smooth muscle predominate and strongly contributes to basal vascular tone and blood pressure. Angiotensin II ANG II is a strong vasoconstrictor peptide that also has direct and indirect salt and water regulatory actions on the kidney (Nickenig and Harrison 2002). ANG II is formed in the circulation when angiotensinogen production is increased via increased adrenergic stimulation release of renin from juxtaglomerular cells in the afferent arteriole of the

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20 kidney. Angiotensinogen is converted to angiotensin I, which is quickly converted to ANG II by angiotensin converting enzyme (ACE). Besides being a potent vasoconstrictor, ANG II stimulates kidney tubule absorption of salt and water directly, as well as indirectly via stimulation of aldosterone from the adrenal cortex. In addition, the endothelial and smooth muscle wall contain a vascular form of ACE which is responsible for production of ANG II in the vascular wall (Nickenig and Harrison 2002). Furthermore, ANG II is major stimulus of NADPH oxidase production of superoxide anion in the vascular wall (Nickenig and Harrison 2002; Cai and Harrison 2000), thus, in conditions of elevated levels of ANG II such as chronic HF, ANG II may be partially responsible for increased vascular oxidative stress. Vasoconstrictor prostaglandins As mentioned earlier, the production of prostaglandins is regulated by the availability of AA and the activity of the enzyme COX (Savidge 2001). Under certain pathophysiological conditions, increased thromboxane A 2 is formed from its precursor prostaglandin H 2, both of which bind to endoperoxide/thromboxane receptors on vascular smooth muscle and induce vasoconstriction (Mombouli and Vanhoutte 1999). However, in states of vascular homeostasis, NO, EDHF, and prostacyclin, override any influence of endothelial vasoconstrictors, promote vasodilation and an antithrombotic phenotype of the endothelium. (Mombouliand Vanohoutte 1999). However, when the endothelial-NO pathway is disrupted, the vasoconstrictor, pro-thromobotic, and proinflammatory phenotype of the endothelium prevails. Vascular Endothelial Dysfunction Several mechanisms have been implicated in VED including: 1) decreased NO synthesis due to decreased expression or activity of nitric oxide synthase (eNOS); 2)

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21 decreased NO synthesis due to increased competitive inhibition by ADMA; 3) post-translational inactivation of NO by ROS such as superoxide radical; and 4) enhanced production of vasoconstrictor substances such as ET-1, ANG II, and vasoconstricting prostaglandins, which oppose vasodilatory effects of NO. Decreased NO Synthesis by eNOS Decreased synthesis of NO can occur via several distinct mechanisms. Pathophysiological factors such as TNF-, hypoxia, oxidized LDL (Harrison 1999), and reduced blood flow and shear-stress in heart failure (Smith et al. 1996), have been shown to decrease eNOS levels through both transcriptional regulation and post-transcriptional modifications in half-life of eNOS mRNA (Harrison 1999). In contrast, shear stress increases gene transcription of eNOS mostly by transcriptional regulation (Uematsu et al. 1995; Harrison 1999). Thus, although eNOS is constitutively expressed, eNOS undergoes various degrees of expression under different physiological and pathophysiological conditions (Harrison 1999). NO synthesis can be also can be decreased when eNOS becomes uncoupled due to reduction of the essential eNOS cofactor, BH 4 (Cai and Harrison 2000). Decreased BH 4 results in eNOS transferring electrons to molecular oxygen instead of L-arginine, resulting in increased production of superoxide radical. As such, pathophysiological conditions such as insulin resistance, cigarette smoking, and hypercholesterolemia can cause impaired EDV due to BH 4 depletion, and supplementation with BH 4 restores EDV in these clinical conditions (Vallance and Chan 2001). Lastly, ADMA, an endogenous inhibitor of eNOS, can also decrease NO synthesis. ADMA is derived from methylation of the side chain nitrogen of arginine residues in

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22 nuclear proteins by enzymes called protein-arginine methyltransferases (PRMTs) (Tran et al. 2003). Specific PRMTs methylate L-arginine residues on intracellular RNA binding proteins in endothelial cells, which are released into the cytoplasm upon normal cellular protein turnover (Tran et al. 2003). DDAH, the enzyme responsible for degradation of ADMA into L-citrulline and dimethylamine (Tran et al. 2003), is responsible for 85-90% of ADMA degradation with only a small amount of ADMA excreted in the urine (Tran et al. 2003). As such, impairment of this DDAH may be a key mechanism for ADMA accumulation under certain pathophysiological conditions (Tran et al. 2003). Vallance et al. (1992) first described the in vivo effects of ADMA on EDV in healthy humans. They infused ADMA into brachial artery of healthy subjects and caused a dose-dependent vasoconstriction that was completely reversed with infusion of L-arginine. In the same study, ADMA was reported to be elevated in chronic renal failure patients which was attributed to reduced renal excretion. However, ADMA has been reported to be elevated in clinical populations with normal renal function including, CAD, peripheral artery disease (PAD), hyperhomocysteinemia, hypercholesterolemia, primary aging (Tran et al. 2003), chronic HF (Usui et al. 1998), and HT (Fearon et al. 2004), suggesting that elevated ADMA must be due to some other mechanism than poor renal function. As such, in vitro studies report that DDAH contains a reactive cysteine residue in the active site which can be reversibly inhibited by s-nitrosylation by NO -derived oxidants or via oxidation by superoxide radical (Sydow and Munzel 2003). Additionally, there is some evidence that suggests that ADMA may be a direct mediator of oxidative stress by causing uncoupling of eNOS from oxidation of the eNOS cofactor, BH 4 (Sydow and Munzel 2003). Hence, ROS may contribute to decreased NO

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23 bioavailibility indirectly by inhibiting DDAH activity, thus promoting the accumulation of ADMA, or directly, by enhanced degradation of synthesized NO (Sydow and Munzel 2003). NO Degradation by Reactive Oxygen Species Vascular redox homeostasis involving superoxide levels are normally controlled by intracellular superoxide dismutase (SOD) isoforms copper/zinc (Cu/Zn) SOD and manganese (Mn) SOD; and the extracellular isoform of SOD (ecSOD), which is located on the endothelial membrane and in extracellular space between endothelial and smooth muscle cells (Fukai et al. 2002). However, during states of increased production of superoxide, excess superoxide reacts rapidly with NO because this reaction has a rate constant three times faster than with SOD. This results in loss of bioactivity and forming of peroxynitrite anion (ONOO-) which is a potent oxidant and has minimal vasodilating properties (Cai and Harrison 2000). There are several potential sources of superoxide production in the vascular wall. Membrane-bound NADH/NADPH oxidase is postulated to be the major source of ROS in endothelial and smooth muscle vasculature (Fukai et al. 2002; Cai and Harrison 2000). NADH/NADPH oxidase uses NADH and NADPH as substrates for electron transfer to molecular oxygen. Experimental evidence shows that NADH/NAPDH oxidase activation can occur via stimulation by ANG II and TNF-, both of which are elevated in HF and HTRs. In vitro studies show that ANG II is a primary stimulus of NADH/NADPH oxidase activity, and in vivo studies report that chronic infusion of ANG II in rats results in increased superoxide production and impaired EDV (Harrison 1999).

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24 Another potential enzymatic source of superoxide production in the vascular wall is xanthine oxidase, which catalyzes conversion of hypoxanthine to xanthine in endothelial and smooth muscle cells (Cai and Harrison 2000). Xanthine oxidase is synthesized as xanthine dehydrogenase and under normal conditions uses NAD + as an electron acceptor. However, under pathophysiological conditions such as exposure to TNFor ONOOin endothelial cells, xanthine dehydrogenase is converted to xanthine oxidase (Landmesser et al. 2002). Thus, increased xanthine oxidase activity transfers electrons to molecular oxygen instead of NAD + resulting in excess superoxide production and impaired EDV (Harrison 1999). Moreover, supplementation with the xanthine oxidase inhibitor, allopurinol, results in improved EDV in smokers (Guthikonda et al. 2003), diabetics (Butler et al. 2000), and chronic HF patients (Farquharson et al. 2002), suggesting that xanthine oxidase contributes at least, in part, to impaired EDV in these conditions. Vascular Endothelial Dysfunction before Heart Transplantation Impaired EDV of peripheral conduit and resistance arteries exists before HT in chronic HF patients (Kubo et al. 1991; Katz et al. 1992; Hornig et al. 1996; Hambrecht et al. 1998; Linke et al. 2001). Kubo et al. (1991) was the first to demonstrate that EDV of forearm resistance vasculature was attenuated in patients with chronic HF in response to a muscarinic agonist, methacholine, compared to healthy controls. The mean increase in forearm blood flow to three dosages of methylcholine using strain gauge plethysmography was significantly attenuated in HF patients. Katz et al (1992) then demonstrated that endothelium-dependent blood flow velocity of conduit femoral artery was attenuated in HF patients compared to healthy controls in response to infusion of

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25 acetylcholine. Taken together, these early studies confirmed that EDV was impaired in both resistance and conduit arteries of HF patients. The mechanisms for impaired EDV in HF likely include several pathophysiological mechanisms. First, Smith et al. (1996) reported a significant decreased expression of eNOS and COX-1 mRNA and a 70% reduction in eNOS protein in aortas of dogs after 1 month left ventricular pacing-induced HF suggesting regulation at the transcriptional or post-transcriptional level. Second, HF is associated with increased levels of circulating TNF(Levine et al. 1990) which, in vitro, post-transcriptionally degrades eNOS mRNA (Yoshizumi et al. 1993). In vivo evidence to support this, Katz et al. (1994) found that elevated TNFlevels to be highly correlated with impaired forearm EDV in response to acetylcholine. Thirdly, in humans, administration of vitamin C, a known scavenger of superoxide anion radical, reverses impaired radial artery EDV in HF, suggesting that superoxide plays a significant role in endothelial dysfunction in HF patients (Hornig et al. 1999). Excess superoxide production may be due to hyperactivity of the RAAS in HF, which results in increased levels of ANG II via the enzyme angiotensin-converting enzyme (ACE) (Nickenbig and Harrison 2002). However, not only does ANG II and TNFstimulate NADPH oxidase production of superoxide anion, but vascular ACE also degrades bradykinin, which stimulates release of NO and endothelial-derived hyperpolarizing factor (EDHF) from the endothelium (Drexler 1997). TNFalso upregulates ET-1 production, which is also elevated in HF, and therefore competes with endogenous vasodilators and promote systemic vasoconstriction and impairesd EDV. Indeed, ET A receptor blockade improves EDV of the brachial artery in chronic HF patients (Berger et al. 2001). Taken together, decreased eNOS and COX-1 gene

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26 expression, increased ANG II-stimulated superoxide degradation of NO, and increased vasoconstrictors ET-1 and ANG II likely all contribute to impaired EDV in chronic HF. ADMA levels are also elevated in chronic HF patients (Usui et al. 1998). Usui et al. (1998) found that chronic HF patients had elevated plasma levels of ADMA and NOx compared to healthy controls. In addition, ADMA and NOx were significantly associated with NYHA functional HF class and NOx inversely associated with ejection fraction (r=-0.33, p=0.004) (Usui et al. 1998). Moreover, there was a significant positive relationship between plasma ADMA and NOx in only the moderate and severe HF patients (r=0.41, p=0.01). Thus, the authors suggested that increased NOx may be due to inflammatory cytokine-induced excessive NO production in severe HF, which may have negative inotropic effects on the myocardium. Therefore, increased ADMA may be a compensatory mechanism against hyperactive systemic or myocardial NO synthase activity and NO production. Mechanisms for increased ADMA in HF are unclear, but may include decreased renal excretion of ADMA due to renal failure, since renal plasma flow and excretion decline as HF progresses. However, Usui et al. (1998) excluded all HF patients with renal dysfunction making this hypothesis unlikely. Finally, as mentioned earlier, it is postulated that increased ROS in endothelial cells in severe HF may contribute to increased ADMA by oxidatively inactivating DDAH, the enzyme that degrades intracellular ADMA. However, the effects of ROS on ADMA in HF patients has not been investigated. Vascular Endothelial Dysfunction after Heart Transplantation Fish et al. (1988) were the first to demonstrate that HTR had impaired coronary EDV early after HT. They demonstrated paradoxical vasoconstriction to acetylcholine in

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27 12 of 13 HTR who were 12 months post-HT. Using intravascular ultrasound, Mills et al. (1992) also described vasoconstriction of conduit coronary arteries in response to acetylcholine in HTR who had no evidence of coronary vasculopathy one year after HT. Davis et al. (1996) reported that impaired coronary artery EDV predicts development of coronary allograft arteriosclerosis by one year. Furthermore, Hollenburg et al. (2002) not only reported that impaired coronary EDV is an independent predictor of development of coronary allograft arteriosclerosis but also of cardiac death in HTR. Thus, coronary ED develops early after HT, and provides valuable prognostic information on long-term risk of the cardiac allograft in HTR. In contrast, similar prognostic data using peripheral endothelial function testing in HTR is not available at this time. However, Anderson et al. (1995) reported that peripheral endothelial function and coronary endothelial function correlate, albeit modestly, and therefore may provide some valuable clinical information in HTR if coronary endothelial function testing is not available. As such, peripheral endothelial dysfunction is present after HT and persists indefinitely (Saxonhouse et al. 2000; Patel et al. 2001; Lim et al. 2002; Schmidt et al. 2002; Cuppoletti et al. 2003). Several cross-sectional studies report that brachial artery FMD is impaired in HTR compared to healthy controls (Saxonhouse et al. 2000; Patel et al. 2001; Lim et al. 2002; Schmidt et al. 2002; Cuppoletti et al. 2003). Saxonhouse et al. (2000) reported that brachial artery FMD in HTR one to seven years post-transplant, was similar to stable class IV HF patients, and was decreased compared to age-matched healthy controls. Patel et al. (2001) compared brachial FMD in a group of ischemic vs. non-ischemic HF patients, to two groups of HTR with antecedent ischemic and non-ischemic HF etiology. Ischemic and non

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28 ischemic HF patients did not differ in ejection fraction, duration of heart failure illness, total cholesterol, or ACE inhibitor use. Brachial artery FMD was the same in both HF groups (3.6% vs. 5.1%, p=NS), but significantly less than controls (13.9%, p<0.001). Ischemic vs. non-ischemic HTR did not differ in time since transplant, duration of pre-transplant heart failure, ejection fraction, cyclosporine levels, lipid lowering therapy, and cardiac risk factors. HTR with ischemic HF etiology had significantly decreased brachial FMD compared to non-ischemic HTR (5.5% vs. 13.0%, p=0.002). FMD in non-ischemic HTR did not differ from healthy, age-matched controls (13.0% vs. 13.9%, p=NS). Thus, this data suggests that EDV of conduit brachial artery is restored after HT in non-ischemic HTR, but not in HTR with ischemic etiology. Although this was not a prospective follow-up of the same patients before and after transplant, this study illustrates that etiology of heart failure can influence endothelial function after HT. Lim et al. (2002) reported reduced brachial artery FMD in14 young HTR (mean age 18 years) with non-ischemic HF, etiology compared to ageand gender-matched healthy controls (3.0% vs. 15.5%, p<0.05). Interestingly, there was no relationship in HTR between impairment in brachial artery FMD and gender, time since transplantation, number of rejection episodes, cyclosporine levels, or presence of hypertension. Thus, this suggests that other factors may contribute to impairment of brachial FMD, however, the small size of the study limits the generalizability of the results. Schmidt et al. (2002) reported a reduced brachial FMD in sedentary HTR (age 60 6 years) six years post-transplant compared to age-matched sedentary, healthy control group (1.4% vs. 8.4%, p<0.05). Lastly, Cuppoletti et al. (2003) measured brachial FMD at one and six months after heart transplant in the same 12 HTR. They reported that 10

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29 of HTR (83%) had a brachial FMD < 4% at one month (0.4% vs. 9.9%, p=0.01), and at six months, brachial FMD remained < 4% in all 10 HTR and as well as the remaining two HTR suggesting that VED persists early after HT. In a longitudinal study, Sinoway et al. (1988) measured EDV of resistance arteries of forearm using strain-gauge plethysmography during reactive hyperemia following 5 minutes of upper arm occlusion with a blood pressure cuff. They measured EDV before, 18 days after HT, and four months after HT in 10 HTR who had severe HF. Basal forearm blood flow did not significantly increase immediately after transplantation, but increased four months after-transplant. Similarly, peak hyperemic forearm blood flow following arterial occlusion did not increase immediately after transplant, but increased significantly at four months. This suggests that impaired forearm blood flow is not directly related to normalizing cardiac output, but that it is increased after several months probably as a result of resumption of daily physical activities. However, because there was no ageand weight-matched healthy group, it was unknown whether the four month peak blood flow was completely normalized. In a cross-sectional study and longitudinal design, Kubo and associates (1993) investigated EDV of forearm resistance arteries using measurement of forearm blood flow by strain gauge plethysmography. In the cross-sectional design, forearm blood flow was measured during infusion of the muscarinic agonist metacholine in three doses in a group of HF and HTR. Forearm blood flow at three doses of methacholine was higher in HTR than CHF patients. In addition, they measured EDV in the forearm during reactive hyperemia in both groups as well. Reactive hyperemia forearm blood flow following upper arm occlusion was not statistically different in HTR than HF patients. This data

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30 suggests that the EDV following brief ischemia of forearm resistance vasculature is similar, but agonist-mediated vasodilation is impaired in HF compared to HTR. Furthermore, endothelium-independent vasodilation (EIV) using an NO donor, nitroprusside, showed that smooth muscle vasculature was not different between groups. In the longitudinal design, forearm blood flow was measured after methacholine and reactive hyperemia in the same six patients before and 4 months after HT. Patient characteristics before and after HT had similar resting forearm blood flow (3.3 1.1 vs. 3.7 1.5 ml/min/100 ml) and forearm vascular resistance (30.5 11 vs. 37 16 U). After HT, HTR had higher blood pressure, cholesterol, and cyclosporine levels, but a lower pulmonary wedge pressure and norepinphrine. Forearm blood flow increased significantly at four months after HTX at each doses of methacholine. Also, reactive hyperemia forearm blood flow increased significantly after HTX from 19.0 3.7 to 44.8 6.4 ml/min/100 ml. This data suggests that both agonist-mediated (methacholine) and flow-mediated (reactive hyperemia) forearm vasodilation is increased after HT. However, subjects did not have nitroprusside EIV mediated forearm blood flow measured, so it is not known if this increase in blood flow after transplant was partially mediated by improvement in forearm vascular smooth muscle. Furthermore, it is not known whether this increase in blood flow is normalized since there was no age-matched healthy control group. The discordant results in forearm EDV to reactive hyperemia may be due to the inherent limitations in cross-sectional study and small number of subjects studied. In a longitudinal study, Cavero et al. (1994) investigated the effects cyclosporine on peripheral vascular EDV in end-stage HF patients before and after HT. Peak FBF

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31 during reactive hyperemia was measured in HF patients 1) before HT; 2) 24-36 hours after HT but before initiation of cyclosporine therapy; 3) 6-8 days after HT in presence of therapeutic cyclosporine levels; and 4) 4-6 weeks post-transplantation on cyclosporine. Forearm blood flow to reactive hyperemia after 10 minutes of upper arm cuff occlusion, increased significantly after HT (11.2 vs. 21.2 ml/min/100 ml, p<0.05), but did not change significantly after 6-8 days (22.3 ml/min/100ml), or after 6 weeks (22.7 ml/min/100ml) on cyclosporine therapy. Taken together, this suggests that HT results in an immediate increase in peak forearm blood flow during reactive hyperemia which does not change in the ensuing several weeks. However, there was no age-matched healthy control group to compare to so it is unknown if peak FBF is completely normalized. Cyclosporine and vascular endothelial dysfunction in heart transplant recipients Cultured endothelial cells exposed to cyclosporine increase expression of eNOS mRNA (Navarro-Antolin et al. 2000), but also increase production of ET-1 (Bunchman et al. 1991) and superoxide anion levels (Navarro-Antolin et al. 2001). Diederich et al. (1994) reported that pretreatment of mesenteric arteries with SOD, normalized acetylcholine-induced impaired vasodilation in cyclosporine treated rats, suggesting that superoxide was a contributing mechanism for impaired NO mediated vasodilation. Sudhir et al. (1994) showed attenuated acetylcholine-induced vasodilation of epicardial and conductance coronary arteries of dogs treated with cyclosporine. Vasoconstriction induced with L-NAME (NO inhibitor) was exacerbated by cyclosporine suggesting that cyclosporine attenuates release of, or increases degradation of, NO in coronary arteries. Furthermore, human studies show that cyclosporine is associated with increased production of ET-1 (Grief et al. 1993; Lerman et al. 1992), and sympathetic nervous hyperactivity and hypertension in HTR (Scherrer et al. 1990). Thus, taken together these

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32 data suggest that despite increased eNOS expression in endothelial cells, cyclosporine therapy may partly contribute to impaired EDV via increased superoxide anion release, ET-1 production, and increased sympathetic hyperactivity in HTR. Inflammation and vascular endothelial dysfunction in heart transplant recipients CRP is an acute phase protein released by liver cells in response to inflammatory cytokines IL-6 and TNF-. CRP is a strong predictor of cardiovascular events in previously healthy men and women, and in individuals with existing cardiovascular disease (Blake and Ridker 2003). However, recently inflammatory proteins have been suggested as a contributing mechanism to VED by in vitro studies which suggest that CRP is directly involved in the development of VED (Pasceri et al. 2000; Venupogal et al. 2002). Venupogal et al. (2002) demonstrated that endothelial cells incubated with CRP decrease expression of eNOS mRNA, eNOS protein, and eNOS bioactivity, and increase expression of vascular adhesion molecules VCAM-1, ICAM-1, and P-selectin (Pasceri et al. 2000). Decreased NO and increased ROS also activate expression of endothelial VCAM-1 and sICAM-1 which initiates an inflammatory response and activate T-lymphocytes, monocytes, and macrophages into the endothelial wall to release proinflammatory cytokines such as IL-6 and TNF(Blake and Ridker 2003). Furthermore, Fichtlscherer et al. (2000) reported that elevated CRP correlated inversely with impaired EDV in a cohort of CAD patients, and a reduction in CRP after 3 months was associated with improved EDV. In HTR, CRP and has been reported to be a strong independent predictor of coronary artery vasculopathy, cardiac allograft failure, and mortality (Pethig et al. 2000; Eisenberg et al. 2000; Labarrare et al. 2002). Holm et al. (2001) found that elevated

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33 levels of IL-6 and TNFcorrelated negatively with acetylcholine-induced peripheral EDV. Moreover, Weis et al. (2001) reported that treatment with simvastatin lowered IL-6 and TNFand was associated with improved acetylcholine-induced coronary EDV. Taken together, these in vitro and clinical data suggest that CRP may not solely a marker of future cardiovascular risk in HTR, but may be intimately involved in the development of VED. Asymmetric dimethylarginine and vascular endothelial dysfunction in heart transplant recipients Lastly, recent accumulating evidence suggests that a contributing mechanism for VED in HTR may due to elevated intracellular and plasma ADMA. A recent study reported a 200% increase in plasma ADMA levels in HTR compared to healthy controls (Weis et al. 2004) and was slightly more elevated in CMV-positive HTRs (Weis et al. 2004). In addition, in vitro, human endothelial cells infected with CMV, had decreased DDAH activity and produced more ADMA (Weis et al. 2004). Furthermore, a recent study demonstrated that the degree of impaired EDV of coronary arteries of HTR was more profound in those with elevated ADMA levels (Fearon et al. 2004). Therefore, ADMA may also be a key contributor of decreased NO bioavailability and impaired EDV in HTR, particularly in those who are CMV-positive. Arterial Stiffness The arterial system can be divided into three anatomic regions with distinct physiological functions. Large elastic arteries of the central circulation, such as the aorta and carotids, act as a buffering or cushioning function to absorb pressure and flow pulsations from LV ejection. This Windkessel effect, allows blood (and potential energy) to be stored in the large arteries during systole, and then expelled to the

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34 peripheral circulation during diastole (Nichols and Singh 2002). This allows for continuous blood flow in the capillaries throughout the cardiac cycle, and dampening of pressure oscillations from intermittent ejection from the LV (Safar et al. 2003). Second, large muscular conduit arteries, such as femoral, brachial, or radial, have a thicker layer of smooth muscle (Wilkinson et al. 2004) and are about twice as long as the elastic arteries (Nichols and Singh 2002). The muscular conduit arteries can alter smooth muscle tone and therefore, modify the speed of forward and reflected pressure waves, also known as pulse wave velocity. Third, the small arterioles, or resistance vessels, control blood flow into tissues and can affect mean arterial pressure by altering their diameter. Additionally, the state of arteriolar tone can affect the distance of reflecting sites, whereby increased tone can result in reflecting sites closer to the ascending aorta and an early return of reflected waves from the periphery to the heart (Nichols and Singh 2002). Arterial compliance (inverse of stiffness) is a function of structural elements of the vessel wall (elastin/collagen/smooth muscle) and distending pressure or mean arterial pressure (Wilkinson et al. 2004). During aging, decreased arterial compliance (increased arterial stiffness) in large elastic central arteries can occur due to passive alterations in elastin/collagen matrix resulting in the inability to absorb pulsations from LV ejection and increased pulse wave velocity of forward and reflected traveling pressure waves. Increased arterial stiffness in the central elastic arteries results in increased central systolic pressure and pulse pressure because they are dependent on LV stroke volume and compliance of the proximal ascending aorta. In contrast, active increases in peripheral muscular conduit artery and arteriolar tone, contribute to arterial stiffness by increasing

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35 pulse wave velocity of forward and reflected traveling pressure waves, and by decreasing the distance to peripheral reflecting sites, respectively (Nichols and Singh 2002). Increased pulse wave velocity results in early return of reflected pressure waves to the ascending aorta during systole instead of diastole. Reflected waves merge with forward (incident) pressure waves resulting in augmentation of systolic pressure. This alteration in arterial reflected wave properties creates an undesirable mismatch between the LV and the arterial system (ventricular/vascular coupling), thus increasing LV afterload and myocardial oxygen demand. Moreover, increased LV afterload or wasted energy, increases the risk of LV hypertrophy and myocardial ischemia (Nichols and Singh 2002). In 1980, Murgo et al. published a detailed description of invasively recorded ascending aortic pressure and flow waves in humans. Murgo et al. (1980) characterized the aortic pressure and flow wave reflection in young and older individuals. In young individuals, they reported that the aortic pressure augmentation occurs in diastole after LV ejection and closing of the aortic valve. This aortic pressure waveform was designated a type c wave, in which augmentation occurs in late systole or diastole after peak pressure and flow ejection. Therefore, augmentation is negative and the augmented pressure wave corresponded to the peak flow wave. In middle-aged individuals, augmented pressure occurred earlier in the cardiac cycle during mid-late systole resulting in a type b wave. Type b wave had an augmentation between 0 and 12% of pulse pressure, and also did not affect aortic flow. Lastly, older individuals had a type a wave which showed augmented pressure in early systole, resulting in augmentation >12% of pulse pressure (ORourke and Pauca 2004). Calculation of augmentation, also known as AI, is the ratio of the reflected wave amplitude or augmented pressure divided by pulse

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36 pressure. Thus, AI is a measure of amplitude and timing of reflected pressure waves arriving at the large central arteries, therefore, is an indirect measure of pulse wave velocity and arterial stiffness of the entire arterial tree (Nichols and Singh 2002). Furthermore, AI of the carotid or aorta can be measured noninvasively using a high fidelity transducer (e.g., applanation tonometry) to record the pressure waveform of the carotid directly, or the aorta indirectly by recording the radial pressure wave and using a generalized transfer function to obtain an aortic pressure waveform (ORourke et al. 2001). A primary mechanism for a change in arterial stiffness in both large elastic and muscular arteries is acute changes in distending or mean arterial pressure (Wilkinson et al. 2004). However, smooth muscle tone of muscular conduit arteries, are also influenced by circulating and local vasoactive substances, and sympathetic nervous tone. In particular, endothelial-derived NO can influence arterial stiffness through its vasodilatory properties. Systemic studies to stimulate or inhibit release of NO can be confounded by changes in mean arterial pressure (Wilkinson et al. 2004). However, recent in vivo studies in sheep and in humans, show that NO released by local intraarterial infusion of acetylcholine, indicate that NO release decreases arterial stiffness (Wilkinson et al. 2004). Furthermore, exogenous NO donors (e.g., nitroglycerin) and phosphodiesterase inhibitors (e.g., sildenifil), reduce arterial reflected pressure waves and arterial stiffness, independent of any change in mean arterial pressure (Wilkinson et al. 2004). Thus, arterial stiffness may be present in clinical conditions in which endothelial dysfunction and reduced NO bioavailability are present.

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37 Arterial Stiffness and Cardiovascular Risk Several recent studies investigated the effects of arterial stiffness on cardiovascular risk. Weber et al. (2003) recently reported that aortic AI was an independent predictor for developing premature coronary artery disease in men undergoing coronary angiography. London et al (2001) reported that increased carotid AI to be an independent predictor of all cause and cardiovascular mortality in renal failure patients. Furthermore, Laurent et al. (2001) reported that aortic pulse wave velocity was an independent predictor of all-cause and cardiovascular mortality in patients with essential hypertension. Thus, measures of arterial stiffness may provide important prognostic in populations at risk for cardiovascular disease. However, the long-term prognostic implication of arterial stiffness in HTR has not been determined. Arterial Stiffness before Heart Transplantation Systemic measures of arterial stiffness such as aortic input impedance are increased are increased in chronic HF patients (Nichols and Pepine 1992; Mitchell et al. 2001). Regional measures of arterial stiffness, such as in the carotid (Lage et al. 1994), iliac (Ramsey et al. 1995), and brachial (Arnold et al. 1991; Ramsey et al. 1995; Nakamura et al. 2004) conduit arteries are also elevated in HF patients (Nichols and Pepine 1992; Ramsey et al. 1995; Nakamura et al. 2004). Using invasive simultaneous measures of ascending aortic pressure and flow, Nichols and Pepine (1992) reported an increased aortic impedance and resistance, the pulsatile and nonpulsatile component of LV afterload, respectively, in HF patients compared to ageand pressurematched controls. Elevation of aortic input impedance, consisting of aortic elastance (stiffness) and wave reflection at various oscillatory frequencies, suggests that arterial stiffness of the arterial tree is increased in HF patients (Nichols and Pepine 1992).

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38 Ramsey et al. (1995) found decreased pulse wave velocity in the common iliac artery in response to intraarterial infusion of acetylcholine in healthy subjects but not patients with HF. This suggests that distensibility of the iliac artery in healthy subjects is influenced by stimulated release of NO, and that decreased release of NO may be contributing to increased stiffness of large muscular conduit artery in HF patients. This was further supported by noninvasive measures of brachial artery flow-mediated dilation and distensibility during reactive hyperemia, which both were significantly decreased in HF patients compared to healthy controls. Using high resolution ultrasound, Nakamura et al. (2004) and Arnold et al. (1991) also found decreased compliance of the brachial artery in HF patients, and Lage et al. (1994) reported reduced carotid artery compliance and increased wall thickness in non-ischemic HF patients. Taken together, these data support the hypothesis that regional muscular conduit artery stiffness is present in chronic HF patients. However, systemic noninvasive measures of arterial stiffness such as aortic-femoral pulse wave velocity, total arterial compliance, and AI have yielded conflicting results (Mitchell et al. 2001). Using noninvasive applanation tonometry and high-resolution ultrasound, Mitchell et al. (2001) reported that pulse wave velocity and total arterial compliance was not different than age-matched controls who had coronary artery disease or risk factors. However, they reported a lower carotid AI in the HF patients (8 vs. 21%, p=0.001) than in controls, but a higher central pulse pressure, characteristic impedance, and lower proximal aortic compliance. This discordant results do suggest that proximal aortic stiffness is elevated, but that systemic measures of AI and pulse wave velocity may not be able to detect these changes. As such, during chronic HF, reduced cardiac output

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39 and reduced left ventricular ejection duration lead to reduced mean arterial pressure, pulse pressure of incident pressure wave, and decreased perfusion pressure of organs (kidney, skeletal muscle, etc). Thus, return of reflected waves to the ascending aorta from the periphery reduces the aortic blood flow wave during deceleration phase, rather than add to second systolic peak of the aortic pressure wave (Nichols and ORourke 1998). Moreover, augmented pressure and AI may appear normal in HF patients, but ascending aortic flow is reduced due to the increased arterial stiffness. Arterial Stiffness and Hypertension after Heart Transplantation Post-transplant hypertension is a common complication in HTR occurring in 72% of HTR by one year, and 95% HTR by five years (Hertz et al. 2002). Post-transplant hypertension jeopardizes the long-term survival of the allograft by increasing left LV afterload and LV mass, and increases the risk of the of coronary artery vasculopathy, the leading cause of death in HTR surviving one year (Hertz et al. 2002). Mechanisms proposed for this de novo hypertension in HTR include 1) cyclosporine-induced sympathetic system hyperactivity and nephrotoxicity (Scherrer et al. 1990); 2) failure of the renin-angiotensin-aldosterone axis to reflexly suppress volume expansion-induced hypertension due to cardiac denervation (Braith et al. 1996); and 3) arterial stiffness due to structural changes in large elastic arteries and increased peripheral conduit arterial tone due to endothelial dysfunction. However, it is currently unknown the relative contribution of each of the above mechanisms on post-transplant hypertension. Schofield et al. (2002) recently reported that 82% of 53 HTR had elevated aortic AI and decreased time of the reflected wave (T p ), despite being on optimal hypertensive therapy as indicated by brachial blood pressure measured by standard cuff

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40 sphygmomanometry. In addition, when HTR were stratified by Murgo aortic wave types, this revealed significantly different aortic pulse pressure, reflected wave amplitude, and aortic AI between the three groups, despite having similar mean arterial blood pressure. This data suggests that a subgroup of HTR have increased aortic augmented systolic and pulse pressure and arterial stiffness which cannot be identified by standard brachial sphygmomanomtry assessment. Although it is currently unknown whether elevated aortic augmented pressure and AI is a predictor of future cardiovascular risk in HTR, it is conceivable that any intervention that can attenuate these physiological parameters of arterial stiffness may have beneficial prognostic implications. There may be several mechanisms for increased arterial stiffness in HTR. Structural changes in the large proximal elastic arteries (e.g. aorta, carotids) during chronic HF prior to HT may occur due to chronic neurohormonal and sympathetic hyperactivity. Increased chronic salt and water retention in the vascular wall, and the hypertrophic effects of elevated ANG II and ET-1 on the vascular smooth muscle layer may contribute to increased proximal arterial stiffness. As such, increased stroke volume, cardiac output, and mean arterial pressure from HT in this setting, may lead to increased pulse pressure and amplitude of the forward traveling pressure wave and increased pulse wave velocity (Pierce et al. 2004). ED and cyclosporine-induced sympathetic hyperactivity (Scherrer et al. 1990) may also contribute to increase stiffness in HTR due to elevated vascular resistance via increased tone of peripheral muscular conduit and resistance arteries. Increased vascular tone leads to increased pulse wave velocity of forward and reflected pressure waves, while increased tone of small resistance vessels decreases distance to reflecting sites.

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41 The cumulative effect contributes to increased timing and amplitude of forward and reflected waves to the peripheral reflecting sites and back to the ascending aorta. Thus, increased LV afterload due to increased vascular resistance and AI due to alterations in wave reflections, may lead to increased myocardial oxygen demand and long-term deleterious effects on the cardiac allograft. Role of Exercise Training in HTR Exercise Training and Functional Capacity Exercise capacity and peak heart rate increase in the first year after transplantation, but remain approximately 60-70% of age-matched normals for the subsequent five years (Givertz et al. 1997). The reasons for this persistent subnormal exercise capacity in HTR is related to several mechanisms. First, cardiac output may be limited during peak exercise due to impaired chronotropic reserve and mild allograft diastolic dysfunction (Kao et al. 1994). Chronic cardiac denervation limits heart rate during submaximal and peak exercise and mild diastolic dysfunction of the cardiac allograft reduces end-diastolic volume and stroke volume during peak exercise (Kao et al. 1988). Thus, at low workloads submaximal cardiac output is maintained by augmenting stroke volume in plasma volume expanded HTR via the Frank Starling mechanism, however peak heart rate and stroke volume contribute to a reduced cardiac output at peak exercise compared to age-matched controls (Braith et al. 1998a). Second, myopathy of peripheral skeletal muscle due to chronic deconditioning and glucocorticoid therapy may also contribute to reduced exercise capacity (Braith et al. 1998b). Reduced muscle girth, muscle strength, and metabolic enzyme activity of muscle (Braith et al. 2005), contribute to decreased oxygen utilization and aerobic ATP production during exercise (Kao et al. 1994). Lastly, impaired EDV of peripheral vasculature persists after transplantation in HTR, and may

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42 contribute to reduced muscle blood flow and a-VO 2 difference during peak exercise (Kao et al. 1994). There has been only one randomized, controlled study of the effect of endurance exercise training in HTR. Kobashigawa et al. (1999) randomized twenty-seven HTR two weeks after HT to an exercise or control group. They reported that 24 weeks of supervised exercise training as part of a cardiac rehabilitation program increased peak VO 2 by 49% and exercise duration 59%, compared to 18% and 18% in the non-supervised control HTR, respectively. To date there is no evidence that exercise training improves central hemodynamic parameters such as LV ejection fraction or stroke volume at rest or during exercise in HTR, so it reasons that improvement in exercise capacity in HTR following chronic exercise training likely involves peripheral mechanisms. As such, peripheral improvements in metabolic capacity of muscle or increased muscle blood flow due to improvement in shear-stress mediated EDV of peripheral conduit and resistance arteries, likely play a significant role improved exercise capacity in HTR. Exercise Training and Endothelial Dysfunction Several recent randomized, controlled studies in chronic HF patients report that chronic lower-body endurance training improves EDV of peripheral conduit arteries (Hambrecht et al.1998; Linke et al. 2001). Linke et al. (2001) reported that 4 weeks of lower body cycle training improved radial artery EDV in response to acetylcholine. In addition, they reported that change in EDV of the radial artery after the exercise intervention correlated positively with the change in peak VO 2 (r=0.63, p<0.05). Hambrecht et al. (1998) also reported a high positive correlation (r=0.64) between the increase in EDV of conduit femoral artery blood flow velocity and the increase in peak oxygen uptake (VO 2 ) following an exercise training program in HF patients. The results

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43 of these studies suggest that lower body exercise training results in a systemic improvement in endothelial function in chronic HF, since EDV was improved in both an upper limb conduit artery (Linke et al. 2001), and lower body conduit artery (Hambrecht et al. 2000). Moreover, the studies suggest that peripheral endothelial dysfunction contributes significantly to impaired exercise capacity in HF because improvement in EDV correlated highly with improvement in peak VO 2 Brachial artery FMD has become an accepted non-invasive test of EDV of upper limb conduit artery function (Corretti et al. 2002). Edwards et al. (2004a) showed that 12 weeks of treadmill walking as part of a cardiac rehabilitation program resulted in an improvement in brachial artery FMD (7.9 vs. 11.2%, p<0.05). In contrast, Goyce et al. (2002) showed a trend but no statistical difference in brachial FMD after 12 weeks of cardiac rehabilitation in patients with coronary artery disease (6.4% vs. 8.3%, p>0.05), but they did report a significant increase in posterior tibial artery FMD after the training period (9.7% vs. 11.7%, P<0.05). Furthermore, Walsh et al. (2003) recently reported that 8 weeks of cross-training (aerobic/resistance training) in CAD patients resulted in improved brachial artery FMD (3.0% to 5.7%, p<0.05). In a similar design to our study, Higashi et al. (1999) reported a 24% increase in peak FBF during reactive hyperemia using plethysmography in 20 patients with essential hypertension after a 12-week exercise training intervention. In eight of the 20 exercise patients who showed an improvement in peak FBF, the increased FBF was abolished by the NO inhibitor, L-NMMA, suggesting that the increase in EDV during reactive hyperemia of forearm resistance arteries was NO mediated.

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44 In HTR, there is a paucity of data on the effects of exercise training on endothelial function. Only, one cross-sectional study has evaluated brachial artery FMD in trained and untrained HTR six years after HT (Schmidt et al. 2002). Trained HTR participated in 6 months of cycling for 40 minutes 2 to 3 times per week. Brachial artery FMD was significantly higher in trained HTR compared to sedentary HTR (7.1% vs. 1.4%, p<0.05). Patel et al. (2003) reported that brachial artery FMD was significantly correlated to maximal exercise treadmill time and to duration of exercise after ventilatory threshold, but not with time to threshold in HTR four years after transplantation. Thus, these observational data suggest that peripheral artery conduit function may be a valuable therapeutic target for improving exercise capacity in HTR. However, there have been no prospective, controlled studies on the effects of lower body dynamic exercise training (e.g., walking, cycling) on brachial artery FMD or resistance vessel EDV in HTR. Exercise Training and Arterial Stiffness Cross-sectional studies indicate that elevated large artery stiffness is associated with reduced exercise capacity in healthy, sedentary individuals (Vaitkevicius et al. 1993), middle-aged athletes (Kingwell et al. 1995), and individuals with chronic HF (Bonapace et al. 2003). In a large cohort of sedentary, healthy individuals (mean age 55 years) in the Baltimore Longitudinal Study of Aging, Vaitkevisius et al. (1993) reported that aortic AI (men: r=-0.34; women: r=-0.49) and aortic pulse wave velocity (men: r=-0.54; women: r=-0.74) were inversely correlated to peak VO 2 even after controlling for age. In another study, Kingwell et al. (1995) found an inverse correlation between aortic -stiffness index and peak VO 2 (r=-0.44) in aerobically trained middle-aged athletes (age 30-59). Lastly, in a study of 78 patients with stable, chronic HF and dilated

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45 cardiomyopathy, Bonepace et al. (2003) reported an inverse correlation between peak VO 2 and aortic pulse wave velocity (r=-0.39). Thus, these data suggest that large artery stiffness, at least partially, affects exercise capacity in various populations. Several prospective, controlled exercise training studies have reported an improved arterial stiffness in healthy sedentary, young men (Cameron and Dart 1994), healthy, sedentary middleaged and older men (Tanaka et al. 2000), men with coronary artery disease (Edwards et al. 2004), and chronic HF patients (Parnell et al. 2002). Cameron and Dart (1994) reported increased systemic arterial compliance and aortic -stiffness index in previously, healthy young men after 30 minutes of cycling 3 days per week for 4 weeks. Tanaka et al. (2000) studied twenty middle-aged and older men who exercise trained for three months of walking 3 to 4 days per week at 60% of maximal heart rate, and progressed to 4 to 6 days per week at 70-75% of maximal heart rate. They reported a 5% increase peak VO 2 and a 25% increase in dynamic carotid arterial compliance and 20% decrease in carotid -stiffness index after the exercise intervention. Lastly, Edwards et al. (2004) investigated the effects of 12 weeks of endurance exercise training (walking) in men with CAD. Twenty patients with previous myocardial infarction or documented CAD via coronary angiography were assigned to supervised exercise as part of cardiac rehabilitation program or a non-exercise control group. The 12-week training intervention resulted in a decrease in aortic AI and an increase in duration of the reflected wave (inverse of pulse wave velocity) in the exercise group, but with no change in brachial systolic or mean arterial blood pressure. Furthermore, there was no change in the time-control group, thus suggesting that regular exercise training decreased wave

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46 reflection and systemic arterial stiffness of large arteries in patients with CAD and reduced the dynamic components of LV afterload. In a prospective, controlled study using simultaneous noninvasive measures of aortic blood flow velocity and right carotid arterial pressure, Parnell et al. (2002) reported that 8 weeks of endurance exercise training in chronic HF patients improved systemic arterial compliance. However, other indices of arterial stiffness, such as aortic pulse pressure, aortic AI, and aortic pulse wave velocity, did not change following the exercise intervention. The authors explained the discordant results by suggesting that the total arterial compliance measurement was specific for changes in the ascending aorta independent of any changes in pulse wave reflection, and thus explaining the lack of change in pulse wave velocity or AI. Taken together, these studies indicate that regular endurance exercise training may be a valuable adjunct to attenuate arterial stiffness and thus may be one mechanism by which exercise reduces long-term cardiovascular risk. Furthermore, the mechanism for improved large artery stiffness with regular exercise training are currently unknown, but may include reduced vascular smooth muscle hypertrophy, reduced connective tissue cross linking, reduced sympathetic nervous tone, or improvement in endothelial function (Joyner 2000). Exercise Training and Nitric Oxide Synthesis Regular endurance exercise training results in increased aortic expression of eNOS and EDV of aorta (Sessa et al. 1994; Fukai et al. 2000), coronary (Woodman et al. 1997), and peripheral resistance vessels in animal models (Spier et al. 2004), and in human models using the left internal mammary coronary artery (LIMA) (Hambrecht et al. 2003). Sessa et al. (1994) reported increased eNOS mRNA and eNOS protein content in aortas of dogs after 10 days of treadmill training. Woodman et al. (1997) reported that 6 weeks

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47 of exercise training in miniature swine resulted in increased eNOS mRNA in coronary resistance arteries. Fukai et al. (2000) reported a 3-fold increase in aortic eNOS protein expression in mice after 3 weeks of treadmill exercise. Furthermore, Spier et al. (2004) recently reported increased eNOS mRNA and eNOS protein were increased rat skeletal muscle soleus arterioles after 10-12 weeks of treadmill exercise in aged rats. Thus, in animals, there is clear evidence in the aorta, coronary arteries, and the peripheral resistance arteries, that chronic exercise training increases expression of eNOS, the enzyme responsible for synthesis of vascular NO. In humans, a recent elegant study by Hambrecht et al. (2003), stable coronary artery disease patients undergoing elective coronary artery bypass surgery were randomly assigned to a 4 week exercise training program or control period. EDV and average peak velocity in response to acetylcholine (agonist-mediated EDV) and adenosine (flow-mediated EDV) were measured invasively before and after the 4-week period in both groups. Additionally, during bypass surgery, part of the LIMA tissue was harvested and measured eNOS mRNA, eNOS protein, and phosphorlayed eNOS at serine 1177. After the exercise intervention, LIMA average peak velocity and EDV in response to acetylcholine and adenosine was significantly increased. Additionally, the trained group had 96% higher eNOS mRNA, 200% higher eNOS protein expression, and 300% higher phosphorlated eNOS in the explanted LIMA compared to controls. Furthermore, phosporylated eNOS levels was significantly correlated to change in LIMA average peak velocity. Thus, this was the first study in humans to demonstrate increased eNOS mRNA, eNOS protein content, and phosphorylated eNOS and its relationship to impaired agonist-mediated EDV after exercise training in humans.

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48 Finally, several studies in humans have reported an increase in plasma nitrate, the major metabolite of NO in vivo (Jungersten et al. 1996), in response to acute exercise and after chronic exercise training (Jungersten et al. 1997; Edwards et al. 2004). Jungersten (1997) reported higher resting plasma nitrate in athletes compared to sedentary controls (45 vs. 34 uM, p<0.01), and an 18% and 16% increase in plasma nitrate following two hours of cycling in athletes and controls, respectively. Edwards et al. (2004) found a 22% increase in plasma nitrate (28.2 vs. 34.4 uM, p<0.05) after 12 weeks of endurance exercise training (walking) in patients with CAD. Together, these animal and human studies suggest that acute and chronic exercise result in increased NO availability in part due to increased NO synthesis. Exercise Training and Oxidative Stress CuZn SOD is the major intracellular isoform of SOD in the cytosol that responds to increased shear stress and likely plays an important role in preventing intracellular superoxide accumulation and NO degredation. As such, laminar shear stress in cultured endothelial cells increased Cu/Zn SOD mRNA in a time and dose-dependent manner, and increased Cu/Zn SOD protein and enzyme activity as well (Inoue et al. 1996). In isolated soleus feed arteries, exposure to intraluminal shear stress resulted in an increase Cu/Zn SOD protein, Cu/Zn SOD mRNA, and Cu/Zn SOD activity in coronary arterioles of pigs. (Woodman et al. 1999). However, the major isoform of SOD in the vessel wall is the extracellular form (ecSOD). ecSOD is believed to be the principal regulator of endothelial-derived NO bioactivity in the vascular wall (Fukai et al. 2002). ecSOD is produced by smooth muscle and is bound to heparin sulfate proteoglycans on the endothelial wall between endothelial and smooth muscle, and is in equilibrium with plasma ecSOD (Faraci et al.

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49 2004). Fukai et al. (2000) reported that three weeks of exercise training in mice increased ecNOS protein in aortas, but aortic Cu/Zn SOD was not changed. Additionally, they studied the effects of exercise training in eNOS knockout (eNOS -/-) mice, and its potential role in modulating ecSOD expression. At baseline, ecSOD expression was decreased in eNOS -/mice, suggesting that basal NO modulates ecSOD. Interestingly, in control eNOS +/+ mice, aortic eNOS protein was increased 3 fold after exercise training, which was paralleled by a 3-fold increase in aortic ecSOD. However, this increased ecSOD protein expression was not observed in aortas from eNOS -/mice. Thus, this study strongly suggest that endogenous NO production modulates ecSOD expression in the vascular wall, both under basal conditions and in response to exercise training. The authors speculated that enhanced NO formation serves as a feed-forward mechanism by increasing ecSOD expression, therefore increasing its own biological effects. To date there has been only one human study that has investigated the effects of exercise training on ecSOD activity in plasma. Edwards et al. (2004) observed an 8.3% increased in ecSOD activity in CAD patients who completed 12 weeks of endurance training. In addition, they found an increase in plasma nitrate, a reduction in lipid peroxidation, and an improvement in brachial artery FMD, but no change in non-exercise controls. This data, along with the studies by Fukai et al. (2000), suggests that ecSOD may be an important modulator of oxidative stress in vivo, however more studies in humans are needed to confirm this hypothesis. Exercise Training and Vasoconstrictors Several studies have evaluated the effects of exercise training on ET-1 and ANG II. Endurance exercise training has been reported to reduced plasma ET-1 levels in young,

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50 healthy subjects (Maeda et al. 2001), and older women (Maeda et al. 2003). However, Callaerts-Vegh et al. (1998) reported that 12 weeks of exercise training did not alter ET-1 in chronic HF patients. Vanhees et al. (1984) reported that 3 months of endurance exercise training reduced plasma renin activity, but did not change ANG II levels in patients with ischemic heart disease. In chronic HF patients, Braith et al. (1998) reported that 16 weeks of exercise training lowered basal levels of ANG II and aldosterone levels, suggesting that exercise training can modify renin-angiotensin-aldosterone activation in HF patients. However, the effects of endurance exercise training on circulating ET-1 and ANG II levels in HTR has not been investigated. Exercise Training and Inflammation Several cross-sectional studies report that higher exercise capacity is associated with lower CRP levels in men and women at risk for cardiovascular disease (LaMonte et al. 2002; Church et al. 2003). In addition, there have only been two prospective studies on effects of exercise training on CRP in patients with CAD (Milani et al. 2004;Edwards et al. 2004). Milani et al. (2004) reported a 41% decrease in CRP in a cohort of 277 CAD patients who completed 12 weeks of exercise training as part of cardiac rehabilitation. Additionally, Edwards et al. (2003) reported that 12 weeks of cardiac rehab lowered CRP by 45% and IL-6 by 32% in CAD patients. Thus, these studies suggest that endurance exercise training has a modulating effect on CRP levels in patients with documented CAD. There have been two prospective studies on the effects of exercise training on inflammatory cytokines in chronic HF patients. Adamopoulos and colleagues conducted two 12-week, randomized, controlled, cross-over design studies in 24 chronic HF patients and 20 healthy controls (Adamopoulos et al. 2001; Adamopoulos et al. 2002). They

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51 reported a significant decrease in plasma levels of IL-6 (-29%), TNF(-39%), apoptosis inducer sFasL (-28%) (Adamopolous et al. 2002), and a 12% decrease in sVCAM-1 (Adamapolous et al. 2001). They found a significant negative correlation between the decrease in TNFand the increase in peak VO 2 (Adamopolous et al. 2002). Furthermore, Larsen et al. (2001) reported a 12.5% decrease in TNFbut no change in IL-6 in 28 patients with chronic HF. Taken together, these data suggest that exercise training has a modulating effect on inflammatory mediators in patients with cardiovascular disease. However, the effect of exercise training on inflammatory mediators in HTR has not been investigated.

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CHAPTER 3 METHODS The experiments in this proposal were designed to investigate the effects of orthotopic HT on endothelial function of peripheral limb vasculature and arterial stiffness in end-stage HF patients. Additionally, this study investigated the effects of 12 weeks of supervised endurance exercise training on endothelial function of limb conduit and resistance arteries and arterial stiffness in HTR. A total of twenty end-stage HF patients listed for transplantation at Shands Hospital were recruited and studied prospectively. Before HT, ten HTR were randomly assigned to a program consisting of 12 weeks of supervised endurance exercise training after HT, and ten HTR were assigned to a control group consisting of usual post-HT medical care but did not participate in a supervised exercise program. In addition, ten age-matched, healthy control subjects were recruited to compare with HTR. The study was approved by the University of Florida Health Science Center Institutional Review Board and all subjects signed written informed consent to participate in the study. Subjects All HTR were recruited from the Heart Transplantation Program at Shands Hospital at the University of Florida. Patients were enrolled while inpatient at Shands Hospital and listed as status 1B on United Network of Organ Sharing (UNOS) awaiting HT. The selection of subjects was notbased on gender or racial/ethnic status. Inclusion Criteria 1. Age 18 to 65 52

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53 2. Listed status 1B inpatient on UNOS for HT Exclusion Criteria 1. UNOS status 1A in the intensive care unit 2. Major orthopedic problems that would limit exercise 3. Claudication pain from peripheral artery disease 4. Chronic obstructive lung disease 5. Renal failure Group Assignments Twenty patients (n=20) were randomly assigned to 12 weeks of supervised endurance exercise training (n=10) (e.g. treadmill walking or cycling) or a non-exercise control group (n=10) before HT. The control group consisted of HTR who did not perform supervised exercise training but continued to receive their usual post-HT medical care. Ten age-matched, healthy controls (n=10) were also recruited for the study. Exercise Training Protocol Exercise training was performed at the Living Well Center, College of Health and Human Performance, University of Florida, Gainesville, FL. For subjects who did not reside in the Gainesville area, participation in 12 weeks of supervised endurance exercise training occurred in a hospital in their community with an American Association of Cardiovascular and Pulmonary Rehabilitation (AACVPR) certified cardiac rehabilitation program. Exercise prescription guidelines were provided to each program, and progression and compliance updates were sent every 4 weeks and at the end the study to the primary investigator. HTR in the exercise group participated in 12 weeks of supervised endurance exercise training beginning at 8 weeks after HT. Exercise training will began with 30 minutes of continuous treadmill walking or stationary cycling 3 days

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54 per week not including warm-up and cool-down periods, and progressed to 35-40 minutes as tolerated after the initial 4 weeks. Cardiac denervation in HTR prevents heart rate from being used as an accurate measure of exercise intensity (Braith et al. 1998a). Therefore, intensity begin at 50-60% of VO 2 peak determined from a graded exercise test, and the Borg rating of perceived exertion (RPE) scale was used to maintain intensity in the 11 to 13, or moderate to somewhat hard range, in accordance with ACSM guidelines (ACSM 2000). Exercise intensity was progressed to 60-70% VO 2 peak, or RPE in the 12 to 14 Borg scale range as tolerated by each subject. Each session begin with five minute warm-up period with RPE range of 7 to 9 (very light to light), and a five minute cool down in the same range. Exercise sessions were under the direct supervision of an ACSM exercise specialist or registered nurse certified in basic life support (BLS) and advanced life support (ACLS) and overseen by a physician. Blood pressure, symptoms, and ECG rhythm via three-lead telemetry system were monitored throughout each exercise session. Criteria to terminate an exercise session was be based on ACSM guidelines (ACSM 2000). All exercise facilities were equipped with automated external defibrillators and resuscitation equipment and access to emergency medical services (EMS). The control group received standard of medical care for HTR from their transplant physician, but did not participate in supervised endurance exercise training. Specific Measurements Subjects visited the laboratory 3 times for testing. Details of the study protocol are outlined in Figure 3-1.

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55 DATA COLLECTION 3 (T3)FMD, PLETHYS, PWA, BLOOD, GXT Supervised Exercise (n=10)Standard medical care12 weeks, 3 days/wk DATA COLLECTION 3 (T3)FMD, PLETHYS, PWA, BLOOD, GXT No supervised exercise (n=10)Standard medical care12 weeks DATA COLLECTION 2 (T2)8 weeks post-transplantFMD, PLETHYS, PWA, BLOOD, GXT Orthotopic Heart Transplantation DATA COLLECTION 1 (T1)FMD, PLETHYS, PWA, BLOOD Recruit subjects from UNOS status 1B at Shands Hospitalat the University of FloridaObtain Informed Consent (n=20) Figure 3-1. Study design. UNOS=United Network of Organ Sharing; FMD=flow-mediated dilation of brachial artery; PLETHYS= venous occlusion plethysmography blood flow; PWA=pulse wave analysis; BLOOD= venous blood sample; GXT=graded exercise test Arterial Stiffness Testing Measurement of arterial stiffness were made using pulse wave analysis. Subjects will remained quietly supine for 10 minutes and then blood pressure (BP) was determined in brachial artery in non-dominant arm three times by automated non-invasive BP cuff (Omron, Inc.) and mean was taken as BP value. Next, high-fidelity radial artery pressure waveforms was recorded by applanation tonometry of the radial pulse using a pencil-type micro-tip pressure transducer (Millar Instruments, Inc.). Optimal recording of the radial pressure waveform will be obtained by applying perpendicular hold-down force generating a stable baseline for at least 10 seconds. The radial pressure waveform and

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56 the brachial artery BP was entered into a Sphygmocor (AtCor Medical, Inc., Sydney, Australia) PWA system which synthesizes an aortic pressure waveform using a mathematical generalized transfer function which has been validated (Chen et al. 1997; Cameron et al. 1998) and is reproducible (Wilkinson et al. 1998). As shown in Figure 3-2, the aortic pressure wave (P s -P d ) is the sum of a forward traveling wave with amplitude (P i -P d ) generated by left ventricular ejection, and reflected pressure wave with amplitude (P s -P i ) from the periphery arriving at the ascending aorta (Nichols and Singh 2002). The two pressure waves travel along the artery at the same velocity in opposite directions, whereby the reflected traveling pressure wave augments the forward traveling wave. The amplitude of the reflected traveling pressure was estimated by the aortic augmentation index (AI a ), which was obtained from the aortic pressure waveform. AI a is calculated as the ratio of reflected wave amplitude to the pulse pressure expressed as a percentage, (P s -P i )/(P s -P d ) x 100, where P s is the aortic systolic pressure; P i is the inflection point of the beginning of upstroke of reflected pressure wave; P d is minimum diastolic pressure. The roundtrip travel time (t p ) of the forward traveling pressure wave from the ascending aorta to the major reflection site and back was measured from the foot of the forward traveling wave to P i Round trip travel time (t p ) is inversely related to arterial pulse wave velocity and arterial stiffness, and directly related to the distance to reflecting point (Lo). Furthermore, LV ejection duration (LVED) is equal to duration from P d to the incisura notch (closure of aortic valve). Aortic systolic tension time index (TTI), an indicator of LV myocardial oxygen demand, is equal to the area under the LVED x aortic P s curve. Aortic diastolic TTI is equal to

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57 area under the diastolic duration x aortic P d curve, an indirect indicator of diastolic coronary perfusion (Nichols and Singh 2002). Figure 3-2. Ascending aortic pressure waveform. P s = aortic systolic pressure; P d =aortic diastolic pressure; P i = inflection point of reflected wave; t p = duration of reflected wave from the heart to the periphery and back; t r = systolic duration of reflected wave Endothelial Function Testing Brachial artery flow-mediated dilation Brachial artery reactivity testing was performed using high-resolution ultrasound (ATL, Inc.). Brachial artery reactivity tests were performed when the subject was fasted for at least 4 hours, abstained from caffeine for 12 hours and exercise for 24 hours, and the subject was asked to eat a low fat meal on the day of testing (Plotnick et al. 1997; Gudmundsson et al. 2000). After lying quietly for 15 minutes, a 10.5 MHz linear array ultrasound transducer was used to image the right brachial artery longitudinally and record on a super VHS recorder. After baseline artery diameter was obtained, a blood pressure cuff was inflated to 200 mmHg for 5 minutes on the upper arm proximal to the location brachial artery measurement. The transducer was held in the same location for

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58 the duration of the cuff inflation to ensure the same section of the brachial artery was measured before and after cuff inflation. The proximal cuff position elicits a greater increase in blood flow and dilation during reactive hyperemia, compared to distal (e.g., forearm) cuff inflation (Corretti et al. 2002). Upon release of the cuff, brachial artery diameter was imaged and recorded for three minutes during reactive hyperemia blood flow. Reactive hyperemia blood flow results in flow-mediated dilation (FMD) of the brachial artery due to increased shear stress-induced nitric oxide release from the endothelial wall. This EDV of the brachial artery has been reported to peak between 60 to 90 seconds after cuff deflation, and is a valid measure endothelial-mediated arterial reactivity (Corretti et al. 2002). Images of the brachial artery were transferred to computer by a frame grabber (DT-4152, Data Translation, Inc.) and brachial artery diameter was made during end-diastole by measuring the distance between anterior and posteriors wall of the intima using image analysis software (Image Pro, Data Translation, Inc.). Five anterior to posterior point measures within a 3 cm segment were made and the average distance was recorded as diameter. Reliability of brachial artery testing was confirmed by a pilot study of four young healthy adults who had brachial artery FMD performed during three visits separated by one-week which yielded coefficient of variation (CV%) of 14.5%. NO donors, such as nitroglycerin or sodium nitroprusside, are commonly used to test endothelial-independent vasodilation (EIV). NO donors act directly on vascular smooth muscle, resulting in normal vasodilation in subjects with CAD, HF, and HT. EIV using an NO donor was not performed in this study in order to reduce the risk of a hypotensive episode in patients with diminished baroreflex sensitivity.

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59 Forearm and calf flow-mediated vasodilation Forearm blood flow (FBF) and calf blood flow (CBF) responses were determined separately by venous occlusion plethysmography (EC-6, D.E. Hokanson, Inc.) using calibrated mercury strain-gauges as previously described (Hokanson et al. 1975; Wilkinson and Webb 2001). Patients were tested in a quiet, temperature-controlled room approximately 21-22C and relative humidity approximately 40-50%. Strain-gauges were applied to the widest part of the non-dominant forearm (~5 cm below anticubital fossa) or calf (~10 cm below patella). Patients remained quietly supine for 10 minutes with arms or legs elevated above the right atrium n order to achieve stable baseline measurements of FBF and CBF. To measure FBF, an upper arm cuff (EC-20, Hokenson, Inc) was inflated to 40 mmHg for 5 seconds every 15 seconds using a rapid cuff inflator to prevent venous outflow (Wilkinson and Webb 2001). To measure CBF, an upper thigh cuff was inflated to 40 mmHg for 5 seconds every 15 seconds. One minute before each measurement, a wrist or ankle cuff was inflated to pressure 50 mmHg above systolic pressure to occlude hand or ankle circulation respectively, during FBF or CBF measurements. The FBF or CBF output signal was transmitted to NIVP3 software program (Hokanson, Inc) on a laptop PC computer and expressed as milliliters (mL) per minute per 100 mL of forearm tissue (mLmin -1 per 100 mL tissue). Absolute blood flow was determined by the rate of change of limb circumference (e.g., slope) during the fivesecond venous occlusion, which has been validated to correlate highly to arterial blood inflow into the limb (Greenfield et al. 1963; Hokanson et al. 1975). FBF or CBF for one minute is the average of one plethysmographic measurement every 15 seconds. Mean arterial pressure (MAP) was determined by systolic blood pressure (SBP) and diastolic

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60 blood pressure (DBP) measured by an automatic oscillometric cuff (HEM-739, Omron, Inc) and calculated as DBP + [0.33(SBP-DBP)]. Endothelium-dependent FBF was measured during reactive hyperemia blood flow of the forearm following 5 minutes of upper arm occlusion using a BP cuff inflated at 200 mmHg (Wilkinson and Webb 2001). A blood pressure cuff was placed on the upper arm 5 cm above the anticubital fossa. After baseline FBF was confirmed to be stable for 2 minutes and recorded, the cuff was rapidly inflated to 200 mmHg for 5 minutes and then released. FBF was measured every 15 sec for 4 minutes. Peak FBF was recorded as the highest FBF observed immediately following releases of the cuff, and total FBF for three minutes was recorded as the area under the time x blood flow curve after baseline FBF is subtracted using the trapezium rule (Matthews et al. 1990). Peak FBF during reactive hyperemia has been shown to correlate highly with acetylcholine-induced FBF in patients with essential hypertension (Higashi et al. 2001), therefore it is a good non-invasive measurement of EDV of forearm resistance arteries (Wilkinson and Webb 2001; Higashi et al. 2001). Meredith et al. (1996) reported that peak FBF during reactive hyperemia is NO-dependent, where other studies report that peak FBF is not NO-dependent (Tagawa et al. 1994; Engelke et al. 1996). In fact, these studies suggest that vasodilation of resistance vessels of forearm is prostacyclin-dependent (Engelke et al. 1996), and that total area under the time x blood flow curve is NO-dependent (Tagawa et al. 1994). Endothelium-dependent CBF was measured following 5 minutes of upper leg (thigh) arterial occlusion. After baseline CBF was stable for 2 minutes, the cuff was rapidly inflated to 200 mmHg for 5 minutes and then released. Peak CBF during reactive hyperemia was recorded as CBF observed immediately following release of the cuff, and

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61 total CBF was recorded as the area under the time x blood flow curve after baseline CBF is subtracted using the trapezium rule (Matthews et al. 1990). A reliability of peak FBF and CBF using venous occlusion plethysmography was confirmed by a pilot study of 9 young healthy adults who had peak FBF and CBF during reactive hyperemia performed during three visits separated by one week (Pierce et al. 2004). Mean CV% of resting and peak FBF was 17% and 6.6%, respectively, and resting and peak CBF was 15.2% and 8.4%, respectively. Graded Exercise Test All HTR performed a symptom-limited graded exercise test (GXT) at 8 weeks after HT and again after 12 weeks of the exercise training or the control period. The GXT was be performed on a motorized treadmill (Quinton, Inc.) with collection of respiratory gas analysis using a calibrated metabolic cart (Parvomedics, Inc.) for determination of peak oxygen uptake (VO 2 ). Subjects performed a Modified Naughton walking protocol, which begins at 1.2 MPH and 0% grade for two minutes, increases to 2.0 MPH for two minutes, and then increases 3.5% grade every 2 minutes thereafter. HTR were monitored continuously during the GXT with a 12-lead electrocardiogram (Quinton, Inc.), blood pressure, and Borg rating of perceived exertion measured once each stage. Criteria for termination of GXT was based upon guidelines published by ACSM (ACSM 2000). All GXTs were performed in the Clinical Exercise Physiology (CEP) Laboratory in the Center for Exercise Science at the University of Florida and were supervised by a cardiologist and a certified ACSM exercise specialist. The CEP laboratory was equipped with a Lifepak 500 automated external defibrillator (Medtronic, Inc.), supplemental oxygen, emergency crash cart medications, and telephone.

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62 Blood Collection Venous blood samples were collected in tubes containing no additive, allowed to clot at room temperature for 15 minutes, and immediately centrifuged at 3,000 rpm for 15 minutes at 4C. Venous blood for plasma samples were collected in tubes containing EDTA, placed on ice, and centrifuged immediately as noted above. Plasma that was used for measurement of lipid peroxidation was stored with diethylenetriamine pentaacetic acid (DTPA) and butylated hydroxytoluene (BHT) for a final concentration of 0.01 mM to prevent autooxidation during freezing and thawing. All serum and plasma samples were aliquoted into 1.5 ml epindorff tubes and immediately stored at C until analysis at the end of study. Plasma Biochemical Analysis Vasoactive balance Since the vasodilator, NO, is rapidly converted to nitrate and nitrite (NOx) in plasma, NOx will be used to estimate NO production. Plasma NOx has a half-life of 8 hours and can be influenced by dietary nitrate, therefore all subjects will be asked to follow National Institute of Health low nitrate diet guidelines 36 hours prior to each blood draw (Pannala et al. 2003). Plasma NOx was measured using a commercially available kit (Cayman Chemical, Inc.), which converts all nitrate to nitrite using nitrate reductase. Spectrophotometric analysis of total nitrite was performed using Greiss reagent and the absorbance measured at 540 nm. The vasoconstrictor ET-1 was measured using an ELISA kit (Cayman Chemical, Inc.). Lipid peroxidation Oxidative stress-induced lipid peroxidation was assessed by measuring plasma levels of 8-iso-PGF 2 using a enzyme-linked immunoassay (ELISA) (Stressgen, Inc.). 8

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63 iso-PGF 2 in plasma competes for binding with 8-isoprostane covalently attached to alkaline phosphatase The assay plate is then incubated with p-nitrophenyl phosphate and the reaction stopped with the addition of an acid. The plate is read at 405 nm on spectrophotometer and the absorbance is inversely proportional to 8-iso-PGF 2 in the plasma sample. Extracellular antioxidant enzyme activity SOD activity in plasma was measured using coloremtric assay which uses cytochrome c reduction technique (Cayman Chemical, Inc.). This method utilizes the reduction of cytochrome c by superoxide ions produced by the xanthine oxidase reaction which causes a change in absorbance via spectrophotometry at 450nm. One unit of SOD activity is defined as the amount of SOD required for a 50% decrease in cytochrome c reduction rate or absorbance. Inflammatory markers Plasma CRP was measured using a sandwich ELISA (Alpha Diagnostics, Inc.). The ELISA is based on simultaneous binding of human CRP from plasma samples to two antibodies, one immobilized on the microtiter well plates, and the other conjugated to the enzyme horseradish peroxidase. The product is read at 450 nm and represents CRP bound to horseradish peroxidase. Plasma levels of IL-6, TNF-, and sICAM-1 were measured using an ELISA (R&D Systems, Inc) which employ the quantitative sandwich enzyme assay technique. Endogenous NO inhibition Plasma levels of the endogenous eNOS competitive inhibitor, ADMA, was measured using an ELISA (Alpco, Inc).

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64 Blood hemoglobin, hematocrit, serum lipids, glucose, creatinine, white blood cell count, cyclosporine, and cytomegliovirus status Blood hemoglobin, hematocrit, and serum total cholesterol, LDL, HDL cholesterol, triglycerides, glucose, cyclosporine trough levels, and cytomegliovirus (CMV) status were measured by the Clinical Chemistry Laboratory at Shands Hospital at the University of Florida using standard blood lipid chemistry analyzer. Endocardial biopsy rejection history The number of allograft rejection episodes identified from endocardial heart biopsies were obtained during the study period in all subjects from the Shands Hospital Transplant Program database. Statistical Considerations Data is presented in table format as mean standard deviation (SD) for continuous variables and as percent frequencies (%) for categorical variables. Continuous variables were be analyzed by analysis of variance (ANOVA) with repeated measures of brachial FMD, peak and total FBF, peak and total CBF, AI a plasma NOx, ET-1, 8-iso-PGF 2 SOD activity, ADMA, CRP, IL-6, TNF-, sICAM-1, demographics, and serum metabolic parameters before and after HT. ANOVA was performed between the exercise and control HTR groups at baseline before the exercise intervention to analyze for baseline group differences. ANOVA with repeated measures was used to compare the above vascular and blood parameters before and after 12 weeks of exercise training or control period. When a significant group-by-time interaction was observed, within-group comparisons between time points and betweengroup comparisons at each time point were performed using Tukeys post-hoc analysis. Categorical variables were analyzed by

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65 2 analysis. All statistical analysis were performed using Microsoft Excel and SPSS 10.0 (SPSS, Inc.). An alpha level of p0.05 will be required for statistical significance. A power analysis was performed to estimate statistical power related to testing the following hypothesis: 12 weeks of exercise training will result in greater peak FBF and brachial artery FMD when compared to a 12 week control period in HTR. Preliminary data on three HTR (n=3) was a peak FBF of 23.1 4.8 ml/min/100ml (mean SD) two months after HT. Based on the study by Higashi et al. (1999), a 24% increase in peak FBF was conjectured after 12 weeks of exercise training. As such, the statistical power related to testing the hypothesis that peak FBF is greater in the exercise trained HTR compared to the control HTR is 0.80 for a two-tailed test when the group means were estimated to be 23.1 ml/min/100ml in the control group, and 28.6 ml/min/100ml in the exercise group; the standard deviation was assumed to be 4.8 ml/min/100ml; total sample size was 20 patients; and the alpha level was set at 0.05. Preliminary data on four HTR (n=4) was a brachial FMD of 8.8 2.5% (mean SD) two months after HT. Based on the study by Edwards et al. (2004a) a 42% increase in brachial FMD was conjectured after 12 weeks of exercise training. The statistical power related to testing the hypothesis that brachial artery FMD is greater in the exercise trained HTR compared to the control HTR is 0.94 for a two-tailed test when the group means were estimated to be 8.8% in the control group and 12.5% in the exercise group; the standard deviation was assumed to be 2.5%; total sample size was 20 patients; and the alpha level was set at 0.05.

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CHAPTER 4 RESULTS A total of twenty subjects (n=20) were recruited and signed written informed consent for this study. Twelve subjects (n=12) completed all measurements before HT (PREHTX) and after HT (POSTHTX). A total of eight consented subjects (n=8) did not complete the measurements because 24-48 hours from the time of written consent the subject received heart transplantation, increased to status 1A and transferred to intensive care unit, or had left ventricular assist device implanted. Therefore, PREHTX data collection was unobtainable. Seven age-matched healthy controls (n=7) were recruited and completed all measurements. Before transplantation, ten subjects were randomly assigned to the exercise group (TRAINED; n=10) and ten were assigned to the control group (CONTROL; n=10). One subject in the TRAINED group withdrew for a non-cardiac medical problem not related to exercise. Three subjects in the CONTROL group withdrew from the study, one for a non-cardiac medical reason, and the other two were lost to follow up. Therefore, sixteen subjects (n=16) completed the 12-week intervention part of the study of which seven were assigned to the CONTROL group (n=7) and nine to the TRAINED group (n=9). Subject Characteristics before and after Heart Transplantation The characteristics for PREHTX, POSTHTX, and age-matched healthy control subjects are presented in Table 4-1. The PREHTX and POSTHTX groups did not differ with respect to age, weight, body mass index, gender ratio, or number of ischemic etiology of heart failure. PREHTX had more on beta-blocker therapy (12 vs. 0, p<0.01) 66

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67 and nitrate therapy (4 vs. 0, p<0.05) than POSTHTX. All PREHTX and POSTHTX subjects were on ACE inhibitor or angiot ensin receptor blocker therapy (12 vs. 12, p=NS), however, there were more POSTHT X subjects on statin therapy (12 vs. 5, p<0.05), and insulin therapy (5 vs. 1, p<0.05) All subjects after HT were receiving standard triple immunosuppressive ther apy including cyclosporine (Neoral ), myophenolate mofetil (Cellcept ), and prednisone. Age-matched healthy controls did not differ significantly from PREHTX and POSTHTX with respect to age, weight, body mass index (BMI), or male/female ratio. Two healthy controls were on statin and AC E inhibitor therapy, but no healthy controls were on beta-blocker, nitrate, or insulin therapy. Table 4-1: Patient characteristics be fore and after heart transplantation PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) Age (years) 56.8 8.0 57.3 8.0 61.7 8.5 Weight (kg) 85.6 12.6 84.5 11.9 89.0 9.2 Body mass index (kg/m2) 27.5 3.4 27.1 2.7 28.3 2.4 Male, no. (%) 10 (83) 10 (83) 6 (86) Female, no. (%) 2 (17) 2 (17) 1 (14) Ischemic HF etiology, no. (%) 7 (58) 7 (58) N/a Days before transplant 76.9 42.3 N/a N/a Days after transplant N/a 66.4 11.6 N/a IV inotrope therapy, no. (%) 12 (100) N/a N/a ACEI/ARB therapy, no. (%) 12 (100) 7 (58) 2 (29)* Beta-blocker therapy, no. (%) 12 (100) 0* 0* Nitrate therapy, no. (%) 4 (33) 0* 0* Calcium channel blocker therapy, no. (%) 1 (8) 3 (25) 0 Statin therapy, no. (%) 5 (42) 12 (100)* 2 (29) Insulin therapy, no. (%) 1 (8) 5 (42) 0 Cyclosporine therapy, no. (%) N/a 12 N/a Prednisone therapy, no. (%) N/a 12 N/a Mycophenolate mofetil therapy, no. (%) N/a 12 N/a Data are mean SD; *P 0.05 vs. PREHTX; P 0.05 vs. POSTHTX; PREHTX= preheart transplantation; POSTHTX=post-hea rt transplantation; HF=heart failure; IV=intravenous; ACEI=angiot ensin converting enzyme i nhibitor; ARB=angiotensin receptor blocker; N/A=not applicable Table4 1 Subjectcharacteristicsbeforeand afterhearttransplantation

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68 Serum Metabolic Parameters before and after Heart Transplantation Fasting metabolic parameters are presented in Table 4-2. As shown in Table 4-2, hemoglobin (13.1 vs. 11.3 g/L, p<0.05) and hematocrit (38.3 vs. 34.6%, p<0.05) decreased significantly in POSTHTX compared to PREHTX. Serum lipid analysis showed that total cholesterol (164.9 vs. 193.3, p<0.05) and HDL cholesterol (45.8 vs. 64.8 mg/dl, p<0.05) increased significantly in POSTHTX, but there was no change in LDL (84.9 vs. 92.0 mg/dl) or triglycerides (171.0 mg/dl vs. 182.6 mg/dl, p=NS) in POSTHTX compared to PREHTX. However, total cholesterol/HDL cholesterol decreased significantly in POSTHTX (3.94 vs. 3.20, p<0.05). Finally, fasting glucose (95.0 vs. 113.0 mg/dl, p=NS), creatinine (1.43 vs. 1.26 mg/dl, p=NS), and WBC (8.0 vs. 7.0, 1x10 9 p=NS) count did not differ in POSTHTX compared to PREHTX, respectively. Table 4-2 Serum metabolic parameters before and after heart transplantation PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) Hemoglobin (g/L) 13.11.5 11.31.1* N/a Hematocrit (%) 38.34.0 34.62.5* N/a Total cholesterol (mg/dl) 164.929.5 193.326.7* 195.654.5 LDL cholesterol (mg/dl) 84.928.2 92.019.7 117.741.5* HDL cholesterol (mg/dl) 45.817.5 64.819.2* 54.311.2 Total cholesterol/HDL ratio (no.) 3.941.20 3.200.98* 3.630.79 Triglycerides (mg/dl) 171.075.0 182.660.9 117.155.5 Glucose (mg/dl) 113.055.3 95.022.3 103.310.6 Creatinine (mg/dl) 1.260.37 1.430.41 0.970.13 White blood cells, 1x 10 9 (no.) 7.02.0 8.02.1 N/a Data are mean SD. *P0.05 vs. PREHTX; P0.05 vs. POST-HTX; PREHTX=pre-heart transplantation; POSTHTX=post-heart transplantation; LDL=low-density lipoprotein; HDL=high density lipoprotein; N/a=not available Age-matched healthy controls did not differ from PREHTX and POSTHTX subjects with respect to total cholesterol, HDL cholesterol, total cholesterol/HDL ratio, fasting glucose, or white blood cell count. LDL cholesterol was significantly higher in healthy controls compared to PRETX (117.7 vs. 84.9 mg/dl, p=0.05), but not with

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69 POSTHTX (117.7 vs. 92.0 mg/dl, p=NS). Fasting triglycerides were significantly lower in healthy controls than POSTHTX (117.1 vs. 182.6 mg/dl, p<0.05), but not significantly different from PREHTX (117.1 vs. 171.0 mg/dl, p=NS). Healthy controls had significantly lower serum creatinine than POSTHTX (0.97 vs. 1.43 mg/dl, p=0.01), but not different than PREHTX (0.97 vs. 1.26, p=NS). Brachial Artery Endothelial Function before and after Heart Transplantation Brachial artery flow-mediated dilation (FMD) and absolute diameter dilation results in PREHTX, POSTHTX, and healthy controls are presented in Table 4-3, Figure 4-1, and Figure 4-2. Brachial artery FMD (9.63 vs. 6.44%, p<0.05) and the absolute diameter dilation (0.45 vs. 0.32 mm, p<0.05) was significantly increased in POSTHTX compared to PREHTX. However, there was no significant change in resting baseline diameter in POSTHTX compared to PREHTX (4.70 vs. 4.93 mm, p=NS). Table 4-3 Brachial artery flow-mediated dilation before and after heart transplantation PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) Baseline diameter (mm) 4.930.78 4.700.63 4.460.78 Absolute diameter dilation (mm) 0.320.16 0.450.14* 0.300.13 Flow-mediated dilation (%) 6.443.30 9.633.05* 6.812.92 Data are mean SD; *P0.05 vs. PREHTX; P0.05 vs. POSTHTX; PREHTX=pre-heart transplantation; POSTHTX=post-transplantation Resting diameter and brachial artery FMD did not differ between age-matched healthy controls and PREHTX or POSTHTX. There was a trend for brachial artery FMD to be significantly greater in POSTHTX than age-matched controls (9.63 vs. 6.81, p=0.06). Lastly, absolute diameter dilation was significantly less in healthy controls compared with POSTHTX (0.30 vs. 0.45 mm, p<0.05), but not significantly different than PREHTX (0.30 vs. 0.32 mm, p=NS).

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70 Blood Pressure and Pulse Wave Analysis before and after Heart Transplantation Blood pressure components for PREHTX, POSTHTX, and age-matched healthy controls are presented in Table 4-4. POSTHTX had a significant increase in heart rate (95.6 vs. 66.5 b/min, p<0.01), peripheral systolic blood pressure (138.8 vs. 109.8 mmHg, p<0.05) and peripheral diastolic blood pressure (90.8 vs. 70.7 mmHg, p<0.01) compared to PREHTX, but no significant change in peripheral pulse pressure (47.9 vs. 39.1 mmHg, p=NS). There was a significant increase in central systolic blood pressure (122.0 vs. 98.5 mmHg, p<0.01), central diastolic blood pressure (92.2 vs. 71.0 mmHg, p<0.01), but no significant change in central pulse pressure (29.8 vs. 27.5 mmHg, p=NS) in POSTHTX compared to PREHTX. There was a significant increase in mean blood pressure (105.3 vs. 81.3 mmHg, p<0.01). Arterial pulse wave analysis data are presented in Table 4-4 and Figure 4-3, 4-4, and 4-5. There was no significant change in AI a normalized for heart rate at 75 b/min (8.9 vs. 13.5 mmHg, p=NS), or augmentation pressure (3.8 vs. 1.8 mmHg, p=NS). In addition, there was no significant change in time duration of the reflected wave to periphery and back (t p ) in POSTHTX compared to PREHTX (140.6 vs. 146.0 ms, p=NS), but a significant increase in systolic tension time index (A s TTI; 3254.3 vs 1826.1 mmHg/sec/min, p<0.01), an indicator of systolic LV myocardial oxygen demand, in POSTHTX compared to PREHTX. Lastly, there was no significant difference in diastolic pressure tension index (DPTI), an indicator of diastolic coronary perfusion, in POSTHTX vs. PREHTX. Age-matched healthy controls had significantly lower heart rate than POSTHTX (58.3 vs. 95.6 b/min, p<0.01), and had higher peripheral and central systolic, diastolic,

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71 pulse and mean blood pressure than PREHTX but no significant difference from POSTHTX. In addition, healthy controls had significantly higher augmentation blood pressure than PREHTX (10.4 vs. 3.8 mmHg, p<0.01) and POSTHTX (10.4 vs. 1.8 mmHg, p<0.01), however, AI a normalized for heart rate (at 75 b/min) was not significantly different than PREHTX (17.6 vs. 8.9%, p=0.09) or POSTHTX (17.6 vs. 13.5%, p=NS). There was no significant difference in t p between healthy controls and PREHTX or POSTHTX, but healthy controls had a significantly higher A s TTI than PREHTX (2402.3 vs. 1826.1 mmHg/sec/min, p<0.01) and significantly lower A s TTI than POSTHTX (2402.3 vs. 3254.3 mmHg/sec/min, p<0.01). However, there was no significant difference in DTPI between healthy controls, PREHTX, or POSTHTX. Table 4-4 Blood pressure components and pulse wave analysis before and after heart transplantation PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) HR (b/min) 66.516.7 95.611.7* 58.39.0 PSBP (mmHg) 109.89.5 138.819.2* 133.313.6* PDBP (mmHg) 70.77.1 90.812.2* 81.89.0* PPBP (mmHg) 39.18.6 47.914.6 51.410.4* CSBP (mmHg) 98.58.2 122.017.2* 122.913.5* CDBP (mmHg) 71.07.3 92.212.1* 82.68.9* CPBP (mmHg) 27.56.7 29.810.9 40.39.6* MBP (mmHg) 81.37.0 105.313.1* 98.79.3* AgBP (mmHg) 3.82.9 1.85.1 10.46.8* AI a at HR=75 b/min (%) 8.911.6 13.515.9 17.67.4 t p (ms) 146.015.7 140.611.7 147.14.9 A s TTI (mmHg/sec/min) 1826.1392.6 3254.3617.5* 2402.3311.3* DPTI (mmHg/sec/min) 2843.5497.4 3079.7606.1 3719.7442.5 Data are mean SD. *P0.05 vs. PREHTX; P0.05 vs. POSTHTX; PREHTX=pre-heart transplantation; POSTHTX=post-transplantation; HR=heart rate; PSBP=peripheral systolic blood pressure; PDBP=peripheral diastolic blood pressure; PPBP=peripheral pulse pressure; CSBP=central systolic blood pressure; CDBP=central diastolic blood pressure; CPBP=central pulse blood pressure; MBP=mean blood pressure; AGBP=augmentation blood pressure; AI a =aortic augmentation index; t p =round triptravel time of reflected pressure wave from ascending aorta to peripheral reflecting sites and back; A s TTI=aortic systolic tension-time index; DPTI=diastolic perfusion time index

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72 Forearm and Calf Resistance Artery Endothelial Function before and after Heart Transplantation Forearm and calf resistance artery blood flow during reactive hyperemia results are displayed in Table 4-5 and Figure 4-6 and 4-7. A subgroup of five subjects (n=5) completed FBF and CBF before and after HT. POSTHTX compared to PREHTX. However, there was a significant increase in peak CBF in POSTHTX compared to PREHTX (22.4 vs. 17.4 ml/min/100ml, p<0.05), but no significant change in CBF AUC 3min (11.2 vs. 9.3 ml/min/100ml, p=NS) in POSTHTX compared to PREHTX. There was no significant difference in peak FBF (22.9 vs. 20.4 ml/min/100ml, p=NS) and total excess FBF for 3 min (AUC 3min ) (8.1 vs. 7.2 ml/min/100ml, p=NS) in Lastly, there was a trend for, but no significant increase in resting FBF in POSTHTX compared to PREHTX (2.4 vs. 1.7 ml/min/100ml, p=0.06), and no significant change in resting CBF in POSTHTX vs. PREHTX (2.6 vs. 2.8 ml/min/100ml, p=NS). Table 4-5 Forearm and calf flow-mediated vasodilation before and after heart transplantation PREHTX (n=5) POSTHTX (n=5) Healthy Controls (n=7) Resting FBF 1.70.16 2.40.5 2.51.8 Peak FBF 20.44.8 22.94.5 26.46.5* Total FBF AUC 3 min 7.21.1 8.12.6 7.71.5 Resting CBF 2.80.9 2.60.7 3.31.9 Peak CBF 17.40.5 22.44.4* 22.47.0* Total CBF AUC 3min 9.34.1 11.27.3 7.15.4 Values are meanSD; units are ml/min/100 ml tissue; *P0.05 vs. PREHTX; P0.05 vs. POSTHTX; PREHTX=pre-heart transplantation; POSTHTX=post-heart transplantation BF=blood flow; AUC=area under flow x time curve. Peak FBF was significantly greater in healthy controls compared to PREHTX (26.4 vs. 20.4 ml/min/100ml, p=0.05), but not significantly different compared to POSTHTX (26.4 vs. 22.9 ml/min/100ml, p=NS). Peak CBF was significantly greater in the healthy controls compared to PREHTX (22.4 vs. 17.4 ml/min/100ml, p=0.05), but not

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73 significantly different than POSTHTX (22.4 vs. 22.4 ml/min/100ml, p=NS). There was no significant difference between healthy controls, PREHTX, and POSTHTX in resting FBF, resting CBF, total FBF AUC3min, and total CBF AUC3min. Vasoactive Balance before and after Heart Transplantation Plasma NOx and ET-1 are displayed in Table 4-6 and Figures 4-8 and 4-9. Plasma NOx, the product of NO metabolism, was not significantly different in POSTHTX compared to PREHTX (39.7 vs. 55.5 mol/L, p=NS, respectively). The endothelial-derived vasoconstrictor ET-1, was not significantly different in POSTHTX compared to PREHTX (4.9 vs. 4.1 pg/ml, p=NS, respectively). Plasma NOx was significantly lower in age-matched healthy controls compared to PREHTX (24.6 vs. 55.5 mol/L, p<0.05), but not significantly different than POSTHTX (24.6 vs. 39.7 mol/L, p=NS). Additionally, ET-1 was not significantly different in age-matched healthy controls compared to PREHTX (5.0 vs. 4.1 pg/ml, p=0.15) and vs. POSTHTX (5.0 vs. 4.9 pg/ml, p=NS). Table 4-6 Vasoactive balance before and after heart transplantation PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) NOx (mol/L) 55.537.7 39.723.9 24.617.6* ET-1 (pg/ml) 4.12.2 4.93.0 5.04.9 Values are meanSD; *P0.05 vs. PREHTX; PREHTX=pre-heart transplantation; POSTHTX=post-heart transplantation; NOx=nitrate/nitrite; ET-1= endothelin-1 Plasma Lipid Peroxidation, Antioxidant Defense, and Endogenous Nitric Oxide Inhibition before and after Heart Transplantation Plasma 8-iso-PGF 2 SOD activity, and ADMA are presented in Table 4-7 and Figures 4-10, 4-11, and 4-12. There was no significant difference in plasma 8-iso-PGF 2 (1597.4 vs.1474.8 pg/ml, p=NS) or ADMA (0.65 vs. 0.65 mol/L, p=NS) in POSTHTX

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74 vs. PREHTX, but compared to PREHTX, there was a significant decrease in SOD activity in POSTHTX (2.16 vs. 1.79 U/ml, p<0.05, respectively). Plasma 8-iso-PGF 2 was significantly greater in the age-matched healthy controls than PREHTX (2089.5 vs. 1474.8 pg/ml, p<0.05), but not POSTHTX (2089.5 vs. 1597.4 pg/ml, p=NS). SOD activity was not significantly different than PREHTX (2.56 vs. 2.16, p=NS), but was significantly higher in healthy controls compared to POSTHTX (2.56 vs. 1.79 U/ml, p<0.01). Finally, plasma ADMA was not significantly different in healthy controls than PREHTX (0.75 vs. 0.65 mol/L, p=NS) or POSTHTX (0.75 vs. 0.65 mol/L, p=NS). Table 4-7 Lipid peroxidation, antioxidant enzyme activity, and endogenous nitric oxide inhibition before and after heart transplantation PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) 8-iso-PGF 2 (pg/ml) 1474.8564.9 1597.4566.2 2167.7248.2* SOD activity (U/ml) 2.160.54 1.790.34* 2.560.43 ADMA (mol/L) 0.650.18 0.650.23 0.750.12 Values are meanSD; *P0.05 vs. PREHTX; P0.05 vs. POSTHTX; PREHTX=pre-heart transplantation; POSTHTX=post-heart transplantation; PGF 2 =prostaglandin F 2 isoprostane; SOD=superoxide dismutase; ADMA= asymmetric dimethylarginine Inflammatory Markers before and after Heart Transplantation Plasma markers of CRP, logCRP, IL-6, TNF-, and sICAM-1 are displayed in Table 4-8 and Figures 4-13, 4-14, 4-15, 4-16, and 4-17. There was no significant difference in PREHTX vs. POSTHTX in CRP (7.0 vs. 6.0 mg/L, p=NS, respectively) and IL-6 (6.2 vs. 6.6 pg/ml, p=NS, respectively). However, CRP is well known to be non-normally distributed in the population and is skewed to the right (Blake and Ridker 2003), therefore, log transformation of CRP was performed which resulted in a significant decrease in log CRP from PREHTX to POSTHTX (0.75 vs. 0.51 mg/L, p=0.05). Furthermore, there was a significant decrease in TNF(2.6 vs. 2.0 pg/ml,

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75 p<0.05, respectively) and sICAM-1 (363.4 vs. 237.8 ng/ml, p<0.01, respectively) from PREHTX to POSTHTX. Age-matched healthy controls had significantly lower CRP (2.6 vs. 7.0 mg/L, p<0.05), logCRP (0.28 vs. 0.75 mg/L, p=0.01), IL-6 (1.4 vs. 6.2 pg/ml, p<0.05), TNF(1.8 vs. 2.6 pg/ml, p=0.05), and sICAM-1 (249.2 vs. 363.4 ng/ml, p<0.05) than PREHTX, but only IL-6 was lower compared to POSTHTX (1.4 vs. 6.6 pg/ml, p<0.05). Table 4-8 Inflammatory markers before and after heart transplantation PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) CRP (mg/L) 7.024.20 6.005.72 2.632.04* logCRP (mg/L) 0.750.32 0.510.58* 0.280.40* IL-6 (pg/ml) 6.215.08 6.584.22 1.430.60* TNF(pg/ml) 2.630.84 1.970.54* 1.880.49* sICAM-1 (ng/ml) 363.4144.5 237.861.6* 249.240.6* Values are meanSD; *P0.05 vs. PREHTX; P0.05 vs. POSTHTX; PREHTX=pre-heart transplantation; POSTHTX=post-heart transplantation; CRP=c-reactive protein; IL-6= interluekin-6; TNF-=tumor necrosis factor-; sICAM-1=soluble intercellular adhesion molecule-1 Baseline Subject Characteristics before Exercise Training or Control Baseline characteristics of the sixteen transplant subjects who completed the exercise intervention (TRAINED; n=9) or control (CONTROL; n=7) period are displayed in Table 4-9. Subjects in the CONTROL group did not differ significantly from the TRAINED group with respect to age, male/female ratio, ischemic etiology, days after transplant, percentage on immunosuppressive therapy, dose of immunosuppressive therapy, percentage on statin therapy, percentage on ACEI/ARB therapy, percentage on insulin therapy, or number of endocardial biopsy rejection episodes. There was a trend, but no significant difference in body weight for the CONTROL vs. TRAINED at baseline (90.4 vs. 78.6 kg, p=0.08), however, CONTROL had a significantly higher BMI than TRAINED at baseline (28.5 vs. 25.5 kg/m 2 p=0.05).

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76 Table 4-9 Baseline patient characteristics before exercise training or control CONTROL (n=7) TRAINED (n=9) Age (years) 54.39.5 54.413.1 Body weight (kg) 90.411.5 78.612.6 Body mass index (kg/m 2 ) 28.51.4 25.53.6* Male, no. (%) 6 (86) 7 (78) Female, no (%) 1 (14) 2 (22) Ischemic HF etiology, no. (%) 4 (57) 5 (56) Days after transplant 73.630.6 67.311.2 Cyclosporine therapy, no. (%) 6 (86) 7 (78) Cyclosporine dose (mg/day) 379.2123.0 328.677.0 Serum cyclosporine trough level (ng/dl) 353.8125.2 446.7268.7 Tacrolimus therapy, no. (%) 1 (14) 2 (22) Prednisone therapy, no. (%) 7 (100) 9 (100) Prednisone dose (mg/day) 24.316.7 22.86.1 Mycophenolate mofetil therapy, no. (%) 7 (100) 9 (100) Mycophenolate mofetil dose (mg/day) 2714.3393 2800.0632 Statin therapy, no. (%) 7 (100) 9 (100) ACEI/ARB therapy, no. (%) 3 (43) 5 (56) Calcium channel blocker therapy, no. (%) 2 (29) 3 (33) Insulin therapy, no. (%) 2 (29) 5 (56) Endocardial biopsy rejection episodes (no.) 12 14 Grade 1A/B mild (no.) 8 10 Grade 2 mild/moderate (no.) 3 2 Grade 3 moderate/severe (no.) 1 2 Grade 4 severe (no.) 0 0 Values are meanSD. *P<0.05 vs. CONTROL; ACEI=angiotensin converting enzyme inhibitor; ARB=angiotensin receptor blocker Body Weight, Serum Metabolic Parameters, and Endocardial Rejection History after Exercise Training Fasting serum metabolic parameters in the CONTROL and the TRAINED group before and after the 12-week control or exercise intervention period are displayed in Table 4-10. Body weight significantly increased in the CONTROL group (90.4 vs. 96.0 kg, p=0.01), but not in the TRAINED group (78.6 vs. 80.9 kg, p=0.06) after 12 weeks. There was no significant change in hemoglobin, hematocrit, total cholesterol LDL cholesterol, HDL cholesterol, total cholesterol/HDL ratio, triglycerides, glucose, or creatinine in the CONTROL or TRAINED group after 12 weeks. There was a significant decrease in white blood cell count in the TRAINED group (p<0.05) after 12 weeks, but

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77 not in the CONTROL group. One subject in each group tested positive for antibodies for CMV infection at baseline but both were negative at the 12 weeks measurement. Table 4-10 Body weight, serum metabolic parameters, and endocardial rejection episodes at baseline and after exercise training or control CONTROL (n=7) TRAINED (n=9) Baseline 12 weeks Baseline 12 Weeks Body weight (kg) 90.411.5 96.011.0* 78.612.6 80.912.7 Hemoglobin (g/L) 11.51.3 11.81.1 11.11.2 11.41.3 Hematocrit (%) 35.64.2 34.83.1 33.92.6 34.94.8 Total cholesterol (mg/dl) 188.338.8 177.738.4 193.915.1 172.720.0 LDL cholesterol (mg/dl) 81.918.6 88.429.8 97.618.5 86.318.6 HDL cholesterol (mg/dl) 69.021.6 57.017.9 65.620.3 61.818.5 Total cholesterol/HDL ratio 2.840.57 3.240.80 3.231.12 3.030.94 Triglycerides (mg/dl) 188.357.3 161.358.6 153.962.2 122.679.0 Glucose (mg/dl) 104.126.7 114.448.8 87.417.6 103.728.3 Creatinine (mg/dl) 1.260.33 1.540.85 1.440.52 1.320.47 WBC, 1x 10 9 (no.) 8.41.9 6.82.0 7.32.2 5.51.2* CMV positive IgG (no.) 1 0 1 0 Endocardial rejection (no.) 12 3 14 5 Grade 1 very mild (no.) 8 3 10 4 Grade 2 mild (no.) 3 0 2 1 Grade 3 moderate (no.) 1 0 2 0 Grade 4 severe (no.) 0 0 0 0 Data are mean SD. *P0.05 vs. Baseline within-groups; P0.05 vs. CONTROL at same time-point; LDL=low-density lipoprotein; HDL=high-density lipoprotein; CMV=cytomegliovirus; IgG=immunoglobulin G antibody; WBC=white blood cells Brachial Artery Endothelial Function after Exercise Training Brachial artery FMD results in the CONTROL and TRAINED group are displayed in Table 4-11 and Figure 4-18 and 4-19. There was a significant decrease in brachial artery FMD (11.1 vs. 7.9%, p<0.05) and the absolute change in diameter (0.51 vs. 0.39 mm, p<0.05) in the CONTROL group after 12 weeks, but no significant change in brachial artery FMD (10.1 vs. 9.6%, p=NS) or absolute change in diameter (0.48 vs. 0.42 mm, p=NS) in the TRAINED group after 12 weeks of exercise training. Furthermore, there was no significant change in baseline diameter in the CONTROL or TRAINED group after 12 weeks.

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78 Table 4-11 Brachial artery flow-mediated dilation at baseline and after exercise training or control CONTROL (n=7) TRAINED (n=9) Baseline 12 weeks Baseline 12 Weeks Baseline diameter (mm) 4.500.60 4.460.64 4.740.87 4.640.82 Absolute change diameter (mm) 0.510.16 0.390.23* 0.480.22 0.420.24 Flow-mediated dilation (%) 11.12.6 7.95.1* 10.06.1 9.66.2 Data are mean SD. *P0.05 vs. Baseline within-group. Blood Pressure and Pulse Wave Analysis after Exercise Training Blood pressure components and pulse wave analysis results are displayed in Table 4-12 and Figure 4-20, and 4-21. There was no significant change in heart rate or peripheral or central systolic, diastolic, pulse and mean blood pressure in the CONTROL group or TRAINED group after 12 weeks. Pulse wave analysis results showed that there was no significant change in augmentation pressure, AIa corrected for heart rate at 75 b/min, Tp, AsTTI, or DTPI in the CONTROL or TRAINED group after 12 weeks. Table 4-12 Blood pressure components and pulse wave analysis at baseline and after exercise training or control CONTROL (n=7) TRAINED (n=9) Baseline 12 weeks Baseline 12 Weeks HR (b/min) 90.77.4 95.19.7 95.813.7 91.117.4 PSBP (mmHg) 150.618.3 144.916.8 132.9 18.1 129.416.5 PDBP (mmHg) 93.114.1 94.09.9 89.710.3 87.011.3 PPBP (mmHg) 57.48.9 50.99.1 43.515.0 42.412.8 CSBP (mmHg) 132.117.9 127.616.5 116.614.1 114.414.0 CDBP (mmHg) 94.914.1 95.310.0 90.79.7 88.210.9 CPBP (mmHg) 37.38.7 32.37.5 25.98.9 26.27.2 MBP (mmHg) 111.315.1 110.013.1 102.110.6 99.912.1 AgBP (mmHg) 4.35.4 2.93.1 0.32.9 1.63.2 AI @ HR=75 b/min (%) 17.013.3 17.310.0 11.414.5 14.313.4 t p (ms) 141.613.5 143.410.3 142.99.7 145.89.7 A s TTI (mmHg/sec/min) 3389.1598 3296.0533 3134.0524 2925.5444 DPTI (mmHg/sec/min) 3263.7462 3253.6329 3054.4583 3013.9429 Data are mean SD. P0.05 vs. CONTROL at same time-point; HR=heart rate; PSBP=peripheral systolic blood pressure; PDBP=peripheral diastolic blood pressure; PPBP=peripheral pulse pressure; CSBP=central systolic blood pressure; CDBP=central diastolic blood pressure; CPBP=central pulse blood pressure; MBP=mean blood pressure; AGBP=augmentation blood pressure; AI a =augmentation index; t p =round trip travel time of reflected pressure wave from ascending aorta to peripheral reflecting sites and back; A s TTI=aortic systolic tension-time index; DPTI=diastolic perfusion time index

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79 Forearm and Calf Resistance Artery Blood Flow after Exercise Training Forearm and calf resistance artery blood flow during reactive hyperemia in the CONTROL and TRAINED group are displayed in Table 4-13 and Figure 4-22 and 4-23. Peak FBF increased 34% (21.5 vs. 28.8 ml/min/100ml, p<0.05) in the TRAINED group after 12 weeks compared to baseline, but there was no significant increase (+14%) in peak FBF (25.5 vs. 29.2 ml/min/100ml, p=0.08) in the CONTROL group. There was no significant change in resting FBF or total FBF AUC 3 min in the CONTROL or TRAINED group after 12 weeks. There was a significant 17% increase in peak CBF (25.0 vs. 29.3 ml/min/100 ml, p=0.05) in the TRAINED group after 12 weeks compared to baseline, and no significant change (-4%) in the CONTROL group (26.3 vs. 25.2 ml/min/100ml, p=NS). There was no significant increase in resting CBF or total CBF AUC 3 min in the CONTROL and TRAINED group after 12 weeks. Table 4-13 Forearm and calf flow-mediated vasodilation at baseline and after exercise training or control CONTROL (n=5) TRAINED (n=6) Baseline 12 weeks Baseline 12 Weeks Resting FBF 2.40.5 2.60.7 2.91.0 3.31.1 Peak FBF 25.511.6 29.28.1 21.54.6 28.83.7* Total FBF AUC 3min 14.213.0 13.94.4 9.85.2 10.33.0 Resting CBF 3.01.1 3.00.7 3.01.0 3.72.8 Peak CBF 26.35.4 25.25.8 25.06.4 29.37.7* Total CBF AUC 3min 18.37.3 10.93.0 13.510.6 14.812.2 Values are meanSD; Units are ml/min/100 ml tissue; *P0.05 vs. Baseline within-group; P0.05 vs. CONTROL at same time-point; BF=blood flow; AUC=area under flow x time curve Vasoactive Balance after Exercise Training Plasma NOx and ET-1 in the CONTROL and TRAINED group after 12 weeks compared to baseline are displayed in Table 4-14 and Figure 4-24 and 4-25. There was no significant change in plasma NOx from baseline in the CONTROL (30.5 vs. 45.3

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80 mol/L, p=NS) or TRAINED group (42.7 vs. 56.1 mol/L, p=NS). There was a was a trend but no significant change in ET-1 in the TRAINED group (-41.4%) after 12 weeks compared to baseline (3.8 vs. 2.2 pg/ml, p=0.09), and no significant change in ET-1 in the CONTROL group (-36.5%) after 12 weeks compared to baseline (4.4 vs. 2.8 pg/ml, p=0.15). Table 4-14 Vasoactive balance at baseline and after exercise training or control CONTROL (n=7) TRAINED (n=9) Baseline 12 weeks Baseline 12 Weeks NOx (mol/L) 30.541.6 45.314.2 42.7323.7 56.141.2 ET-1 (pg/ml) 4.453.01 2.820.90 3.832.54 2.241.02 Values are meanSD; *P0.05 vs. Baseline within-group; P0.05 vs. CONTROL at same time-point; NOx=nitrate/nitrite; ET-1=endothelin-1. Lipid Peroxidation, Antioxidant Enzyme Activity, and Endogenous Nitric Oxide Inhibition after Exercise Training Plasma levels of 8-isoPGF 2 SOD activity, and ADMA in the CONTROL and TRAINED group compared to baseline are displayed in TABLE 4-15 and Figure 4-26, 4-27, and 4-28. There was no significant change in 8-iso-PGF 2 SOD activity, or ADMA in the CONTROL or TRAINED group after 12 weeks compared to baseline. Table 4-15 Lipid peroxidation, antioxidant enzyme activity, and endogenous nitric oxide inhibition at baseline and after exercise training or control CONTROL (n=7) TRAINED (n=9) Baseline 12 weeks Baseline 12 Weeks 8-iso-PGF 2 (pg/ml) 1815.0516.7 1711.3904.1 1387.8506 1535.0514 SOD activity (U/ml) 1.670.36 1.750.48 1.860.35 1.800.67 ADMA (mol/L) 0.730.26 0.720.25 0.600.17 0.680.23 Values are meanSD; *P0.05 vs. Baseline within-group; P0.05 vs. CONTROL at same time-point; PGF 2 =prostaglandin F 2 isoprostane; SOD=superoxide dismutase; ADMA= asymmetric dimethylarginine Inflammatory Markers after Exercise Training Plasma CRP, IL-6, TNF-, and sICAM-1 in the CONTROL and TRAINED group after 12 weeks compared to baseline are displayed in Table 4-16 and Figures 4-29, 4-30,

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81 4-31, and 4-32. There was a non-significant 19% decrease in plasma CRP in the CONTROL group (5.62 vs. 4.54 mg/L, p=NS) and a non-significant 49% decrease in the TRAINED group (5.03 v. 2.55 mg/L, p=NS) after 12 weeks compared to baseline. Log transformation of CRP did not alter the results in either group. There was no change in IL-6 in the CONTROL group (6.65 vs. 6.16 pg/ml, p=NS) and a non-significant 35% decrease in IL-6 in the TRAINED group (5.02 vs. 3.25 pg/ml, p=NS) after 12 weeks compared to baseline. However, there was a significant increase in TNF(1.56 vs. 2.38 pg/ml, p<0.05) in the CONTROL group, but no significant change in TNFin the TRAINED group (1.83 vs. 1.79 pg/ml, p=NS) after 12 weeks. Lastly, there was no significant change in sICAM-1 in the CONTROL (269.2 vs. 295.6 ng/ml, p=NS) or TRAINED group (216.5 vs. 198.2 ng/ml, p=NS) after 12 weeks compared to baseline. Furthermore, TNF(1.79 vs. 2.38 pg/ml, p<0.05) and sICAM-1 (198.2 vs. 295.6 ng/ml, p<0.05) were significantly lower at 12 weeks in TRAINED compared to CONTROL at 12 weeks. Table 4-16 Inflammatory markers at baseline and after exercise training or control CONTROL (n=7) TRAINED (n=9) Baseline 12 weeks Baseline 12 Weeks CRP (mg/L) 5.624.29 4.543.36 5.036.55 2.552.81 IL-6 (pg/ml) 6.655.03 6.162.72 5.023.15 3.252.55 TNF(pg/ml) 1.560.38 2.380.79* 1.830.59 1.790.50 sICAM-1 (ng/ml) 269.2104.8 295.686.1 216.564.0 198.239.1 Values are meanSD; *P0.05 vs. Baseline within-group; P0.05 vs. CONTROL at same time-point; CRP=c-reactive protein; IL-6=interluekin-6; TNF-=tumor necrosis factor-alpha; sICAM-1=soluble intercellular adhesion molecule-1 Peak Cardiopulmonary Exercise Testing Variables after Exercise Training Peak cardiopulmonary variables during graded exercise testing with respiratory gas analysis in the CONTROL and TRAINED group are displayed in Table 4-17 and Figure 4-33 and Figure 4-34. There was no significant change in peak heart rate, peak systolic

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82 blood pressure, peak diastolic blood pressure in the CONTROL or TRAINED group after 12 weeks compared to baseline. There was no significant change in peak oxygen uptake (VO 2 ) in the CONTROL group (16.2 vs. 16.8 ml/kg/min, p=NS), but there was a significant 26% increase in peak VO 2 in the TRAINED group (15.4 vs. 19.4 ml/kg/min, p<0.01) after 12 weeks compared to baseline. Moreover, there was no significant change in exercise duration in the CONTROL group (561.5 vs. 554.5 sec, p=NS), but there was a significant 44.5% increase in exercise duration in the TRAINED group (518.9 vs. 750.0 sec, p<0.01) after 12 weeks compared to baseline. Lastly, there was no significant difference in peak respiratory exchange ratio (RER) and rating perceived exertion (RPE) in the CONTROL or TRAINED group after 12 weeks compared to baseline. Table 4-17 Peak cardiopulmonary graded exercise testing variables at baseline and after exercise training or control CONTROL (n=7) TRAINED (n=9) Baseline 12 weeks Baseline 12 Weeks Peak HR (b/min) 123.710.2 133.014.0 124.316.6 124.942.7 Peak systolic BP (mmHg) 173.721.0 178.318.1 147.123.7 167.225.0 Peak diastolic BP (mmHg) 88.75.9 90.37.1 84.016.9 83.617.9 Peak VO 2 (ml/kgBW/min) 16.25.2 16.82.8 15.44.3 19.45.5* Peak RER 1.050.07 1.010.08 1.050.08 1.080.06 Peak RPE 16.21.6 16.31.2 15.41.6 15.31.5 Peak exercise duration (sec) 561.5202.8 554.5110.9 518.9198.2 750.0274.4* Data are mean SD. *P0.05 vs. Baseline within-groups; P0.05 vs. CONTROL at same time-point; HR=heart rate; BP=blood pressure; VO 2 =rate of oxygen consumption; BW=body weight; RER=respiratory exchange ratio; RPE=rating of perceived exertion (Borg scale)

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83 0.0 2.5 5.0 7.5 10.0 12.5PREHTX (n=12) POSTHTX (n=12) *Healthy Controls (n=7) Flow-mediated dilation (%) Figure 4-1. Brachial artery flow-mediated dilation before and after heart transplantation. *P0.05 vs. PREHTX. 0.0 0.1 0.2 0.3 0.4 0.5PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) *#Diameter dilation (mm) Figure 4-2. Brachial artery flow-mediated diameter dilation before and after heart transplantation. *P0.05 vs. PREHTX; #P0.05 vs. POSTHTX. 0 5 10 15 20 25PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) AIa (%) @ HR 75 b/min Figure 4-3. Aortic augmentation index (AI a ) corrected for heart rate=75 b/min before and after heart transplantation.

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84 0 25 50 75 100 125 150 175 200PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) tp (ms) Figure 4-4. Roundtrip travel duration of reflected wave (t p ) before and after heart transplantation. 0 500 1000 1500 2000 2500 3000 3500PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) **#AsTTI (mmHg/min/sec) Figure 4-5. Aortic systolic tension-time index (A s TTI) before and after heart transplantation. *P0.05 vs. PREHTX; #P0.05 vs. POSTHTX. Rest FBF Peak FBF AUC FBF 0 10 20 30PREHTX (n=5) POSTHTX (n=5) Healthy Controls (n=7) FBF (ml/min/100ml) Figure 4-6. Forearm blood flow (FBF) before and after heart transplantation. *P0.05 vs. PREHTX; AUC=area under blood flow x time curve for 3 min

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85 Rest CBF Peak CBF AUC CBF 0 5 10 15 20 25 30PREHTX (n=5) POSTHTX (n=5) Healthy Controls (n=7) **CBF(ml/min/100ml) Figure 4-7. Calf blood flow (CBF) before and after heart transplantation. *P0.05 vs. PREHTX; AUC=area under blood flow x time curve for 3 min. 0 10 20 30 40 50 60 70PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) *NOx (mol/L) Figure 4-8. Nitrate/nitrite (NOx) before and after heart transplantation. *P0.05 vs. PREHTX. 0 1 2 3 4 5 6 7PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) *ET-1 (pg/ml) Figure 4-9. Endothelin-1 (ET-1) before and after heart transplantation.

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86 0 500 1000 1500 2000 2500PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) *#8-iso-PGF2 (pg/ml) Figure 4-10. Eight (8)-iso-prostanglandin-F2 (PGF2) before and after heart transplantation. *P0.05 vs. PREHTX; #P0.05 vs. POSTHTX. 0.0 0.5 1.0 1.5 2.0 2.5 3.0PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) *#SOD activity (U/ml) Figure 4-11. Superoxide dismutase (SOD) activity before and after heart transplantation. *P0.05 vs. PREHTX; #P0.05 vs. POSTHTX. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) ADMA (mol/L) Figure 4-12. Asymmetric dimethylarginine (ADMA) before and after heart transplantation.

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87 0 1 2 3 4 5 6 7 8 9PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) *CRP (mg/L) Figure 4-13. C-reactive protein (CRP) before and after heart transplantation. *P0.05 vs. PREHTX; #P0.05 vs. POSTHTX. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) **logCRP Figure 4-14. Log-transformed C-reactive protein (logCRP) before and after heart transplantation. *P0.05 vs. PREHTX. 0 1 2 3 4 5 6 7 8PREHTX (n=12) POSTHTX (n=12) Healthy Controls (n=7) *#IL-6 (pg/ml) Figure 4-15. Interluekin-6 (IL-6) before and after heart transplantation. *P0.05 vs. PREHTX; #P0.05 vs. POSTHTX.

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88 0.0 0.5 1.0 1.5 2.0 2.5 3.0PREHTX (n=12) POSTHTX (n=12) *Healthy Controls (n=7) *TNF(pg/ml) Figure 4-16. Tumor-necrosis factor-alpha (TNF-) before and after heart transplantation. *P0.05 vs. PREHTX; #P0.05 vs. POSTHTX. 0 50 100 150 200 250 300 350 400 450PREHTX (n=12) POSTHTX (n=12) *Healthy Controls (n=7) *sICAM-1 (ng/ml) Figure 4-17. Soluble intercellular adhesion molecule-1 (sICAM-1) before and after heart transplantation. *P0.05 vs. PREHTX. CONTROL (n=7) TRAINED (n=9) 0.0 2.5 5.0 7.5 10.0 12.5BASELINE 12 WEEKS *Brachial artery FMD (%) Figure 4-18. Brachial artery flow-mediated dilation (FMD) at baseline and after 12 weeks of exercise training or control. *P0.05 vs. BASELINE within-groups.

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89 CONTROL (n=7) TRAINED (n=9) 0.0 0.1 0.2 0.3 0.4 0.5 0.6BASELINE 12 WEEKS Absolute dilation (mm) Figure 4-19. Brachial artery absolute diameter dilation at baseline and after 12 weeks of exercise training or control. *P0.05 vs. BASELINE within-groups. CONTROL (n=7) TRAINED (n=9) 0 5 10 15 20 25BASELINE 12 WEEKS AIa (%) @ HR=75 b/min Figure 4-20. Aortic augmentation index (AI a ) normalized for heart rate at 75 b/min at baseline and after 12 weeks of exercise training or control. HR=heart rate. CONTROL ( n=7 ) TRAINED ( n=9 ) 0 25 50 75 100 125 150 175 200BASELINE 12 WEEKS tp (ms) Figure 4-21. Roundtrip travel time of reflected wave (t p ) at baseline and after 12 weeks of exercise training or control.

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90 CONTROL TRAINED CONTROL TRAINED 0 5 10 15 20 25 30 35BASELINE 12 WEEKS *(n=5)(n=6)(n=5)(n=6)Peak FBF A UC FBFFBF (ml/min/100ml) Figure 4-22. Peak and total area under curve (AUC) forearm blood flow (FBF) at baseline and after 12 weeks of exercise training or control. *P0.05 vs. BASELINE within-groups. CONTROL TRAINED CONTROL TRAINED 0 5 10 15 20 25 30 35 BASELINE 12 WEEKS Peak CBF A UC CBF*(n=5)(n=6)(n=5)(n=6)CBF (ml/min/100ml) Figure 4-23. Peak and total area under curve (AUC) calf blood flow (CBF) at baseline and after 12 weeks of exercise training or control. *P0.05 vs. BASELINE within-groups.

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91 CONTROL (n=7) TRAINED (n=9) 0 10 20 30 40 50 60 70BASELINE 12 WEEKS NOx (mol/L) Figure 4-24. Nitrate/nitrite (NOx) at baseline and after 12 weeks of exercise training or control. CONTROL (n=7) TRAINED (n=9) 0 1 2 3 4 5 6BASELINE 12 WEEKS ET-1 (pg/ml) Figure 4-25. Endothelin-1 (ET-1) at baseline and after 12 weeks of exercise training or control. *P0.05 vs. BASELINE within-group. CONTROL (n=7) TRAINED (n=9) 0 500 1000 1500 2000 2500BASELINE 12 WEEKS 8-iso-PGF2 (pg/ml) Figure 4-26. Eight (8)-iso-prostaglandin-F 2 (PGF 2 ) at baseline and after 12 weeks of exercise training or control.

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92 CONTROL (n=7) TRAINED (n=9) 0.0 0.5 1.0 1.5 2.0 2. 5 BASELINE 12 WEEKS SOD activity (U/ml) Figure 4-27. Superoxide dismutase (SOD) activity at baseline and after 12 weeks of exercise training or control. CONTROL (n=7) TRAINED (n=9) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9BASELINE 12 WEEKS ADMA (mol/L) Figure 4-28. Asymmetric dimethylarginine (ADMA) at baseline and after 12 weeks of exercise training or control. CONTROL (n=7) TRAINED (n=9) 0 1 2 3 4 5 6 7 8 9BASELINE 12 WEEKS CRP (mg/L) Figure 4-29. C-reactive protein (CRP) at baseline and after 12 weeks of exercise training or control.

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93 CONTROL (n=7) TRAINED (n=9) 0 1 2 3 4 5 6 7 8 9BASELINE 12 WEEKS IL-6 (pg/ml) Figure 4-30. Interleukin-6 (IL-6) at baseline and after 12 weeks of exercise training or control. CONTROL (n=7) TRAINED (n=9) 0.0 0.5 1.0 1.5 2.0 2.5 3.0BASELINE 12 WEEKS *#TNF(pg/ml) Figure 4-31. Tumor necrosis factor-alpha (TNF-) at baseline and after 12 weeks of exercise training or control. *P0.05 vs. BASELINE within-groups; #P0.05 vs. CONTROL at same time-point. CONTROL ( n=7 ) TRAINED ( n=9 ) 0 50 100 150 200 250 300 350BASELINE 12 WEEKS #sICAM-1 (ng/ml) Figure 4-32. Soluble intercellular adhesion molecule-1 (sICAM-1) at baseline and after 12 weeks of exercise training or control. #P0.05 vs. CONTROL at same time-point.

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94 CONTROL (n=7) TRAINED (n=9) 0 5 10 15 20 25BASELINE 12 WEEKS *VO2 (ml/kg/min) Figure 4-33. Peak exercise oxygen uptake (VO 2 ) on graded exercise test at baseline and after 12 weeks of exercise training or control. *P0.05 vs. BASELINE within-groups. CONTROL (n=7) TRAINED (n=9) 0 100 200 300 400 500 600 700 800 900BASELINE 12 WEEKS *Exercise duration (sec) Figure 4-34. Peak exercise duration on graded exercise test at baseline and after 12 weeks of exercise training or control. *P0.05 vs. BASELINE within-groups.

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CHAPTER 5 DISCUSSION This is the first prospective study to evaluate the effects of HT on peripheral endothelial function, arterial stiffness, plasma vasoactive balance, oxidative stress, antioxidant enzyme activity, inflammation, and NO inhibition in end-stage HF patients. The first major finding is that upper limb conduit artery endothelial function (brachial FMD) is improved after HT. Second, peak vasodilatory capacity of resistance arteries of lower limb (peak calf BF) is increased after HT and comparable to values recorded in age-matched healthy controls, but peak vasodilatory capacity of upper limb (peak forearm BF) is not significantly changed. Third, peripheral and central systolic, diastolic, mean, and pulse blood pressures are increased after HT, but indices of arterial stiffness are not significantly altered. Fourth, plasma levels of inflammatory markers CRP, IL-6, TNF-, and sICAM-1 are significantly elevated in end-stage HF subjects before HT compared to age-matched healthy control subjects. More importantly, CRP, TNF-, and sICAM-1 are significantly decreased following HT. Fifth, there is a significant decrease in plasma SOD activity after HT to levels significantly lower than age-matched healthy controls, but there is no change in plasma 8-iso-PGF 2 Sixth, there is no significant change in plasma NOx or ET-1 after HT. Lastly, there is no significant change in plasma levels of the endogenous NO inhibitor, ADMA in HF patients after HT. This is also the first prospective, controlled study to investigate the effects of supervised endurance exercise training on peripheral endothelial function, arterial stiffness, plasma vasoactive balance, oxidative stress, antioxidant enzyme activity, 95

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96 inflammation, and endogenous NO inhibition in HTR. The major findings of this study are that 12 weeks of supervised endurance exercise training (walking) attenuates a progressive decline in upper limb conduit artery endothelial function (brachial FMD) in HTR. Second, 12 weeks of supervised endurance exercise training improves peak vasodilatory capacity of resistance arteries of upper (forearm) and lower (calf) peak blood flow in HTR, but peak limb vasodilatory capacity is unaltered in control HTR who do not participate in supervised exercise training. Third, 12 weeks of supervised exercise training results in a significant increase in peak exercise VO 2 and exercise duration, but remain unchanged in HTR who do not participate in supervised exercise training. Fourth, 12 weeks of supervised exercise training attenuates a progressive increase in the major inflammatory cytokine TNFin HTR, but does not alter the inflammatory proteins CRP, IL-6, or sICAM-1. Fourth, exercise training does not alter plasma levels of 8-iso-PGF 2 SOD activity, NOx, or ET-1 in HTR. Fifth, exercise training does not appear to alter peripheral or central systolic, diastolic, or pulse blood pressure, and does not alter arterial stiffness (AI a ) in HTR. Sixth, plasma levels of the endogenous competitive inhibitor of nitric oxide synthase, ADMA, is not altered following 12 weeks of supervised exercise training in HTR. Peripheral Conduit Artery Endothelial Function and Heart Transplantation There is conflicting data on whether peripheral endothelial function of upper limb conduit arteries improves after HT. One cross-sectional study reported that endothelial function in the brachial artery does not improve in HTR with antecedent ischemic HF etiology, but that brachial artery endothelial function does improve in HTR with non-ischemic HF etiology (Patel et al. 2001). In contrast, other studies report reduced

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97 brachial artery FMD in HTR compared to healthy controls irrespective of etiology of HF (Saxonhouse et al. 2000; Lim et al. 2002; Schmidt et al. 2002; Cuppoletti et al. 2003). However, there have been no prospective, longitudinal studies of brachial artery endothelial function in the same cohort of HF subjects before and after HT compared to values in age-matched, healthy controls. The present study demonstrates that brachial artery FMD in end-stage HF subjects is not significantly different than age-matched healthy controls (6.44 vs. 6.81%, p=NS), but that brachial artery FMD improves significantly after HT (6.44 vs. 9.63%, p<0.05). In addition, there was a trend (p=0.06) in brachial artery FMD to be greater in HTR than age-matched healthy controls (9.63 vs. 6.81%). These data suggest that brachial artery FMD is improved early after HT to levels greater than age-matched healthy controls. Several cross-sectional studies report that brachial artery FMD is impaired in HTR compared to healthy controls (Saxonhouse et al. 2000; Patel et al. 2001; Lim et al. 2002; Schmidt et al. 2002; Cuppoletti et al. 2003). Saxonhouse et al (2000) reported that brachial artery FMD in HTR, one to seven years post-transplant, was similar to stable class IV HF patients (4.4 vs. 3.3%, p=NS), but was decreased compared to age-matched healthy controls (4.4 vs. 9.8%, p<0.01). Patel et al. (2001) compared brachial FMD of ischemic vs. non-ischemic HF patients, to two groups of HTR with antecedent ischemic and non-ischemic HF etiology. HTR with ischemic HF etiology had significantly decreased brachial FMD compared to non-ischemic HTR (5.5% vs. 13.0%, p=0.002). FMD in non-ischemic HTR did not differ from healthy, age-matched controls (13.0% vs. 13.9%, p=NS). Lim et al. (2002) reported reduced brachial artery FMD in14 young HTR (mean age 18 years) with non-ischemic HF etiology, compared to ageand gender

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98 matched healthy controls (3.0% vs. 15.5%, p<0.05). Lastly, Schmidt et al. (2002) reported a reduced brachial FMD (1.4% vs. 8.4%, p<0.05) in sedentary HTR (age 60 6 years) six years post-transplant, compared to age-matched sedentary, healthy controls. The major reason for these discordant FMD results is likely due to the differences in study design and time after transplant that the measurements were obtained. The present study was a prospective, longitudinal design and brachial artery FMD measurements were obtained at approximately 8 weeks after HT in all subjects. In contrast, the above studies were cross-sectional designs and the time after HT that the measurements were obtained ranged from 1 to 6 years. Thus, selection bias and the heterogeneous length of time after HT likely affected the results. It is possible that early after HT brachial artery FMD is improved, but that brachial artery FMD progressively declines months to years after HT. The present study demonstrates that although FMD was improved after HT in all patients, the HTR who did not participate in exercise training, experienced significant declines in brachial artery FMD over a period of two to five months after HT (11.1 vs. 7.9%, p<0.05). Taken together, these data suggest that brachial artery FMD is improved early after HT, but progressively declines in the first year and persists indefinitely in sedentary HTR. The potential mechanisms for this phenomenon will be discussed in the sections that follow. Peripheral Resistance Artery Endothelial Function and Heart Transplantation Several studies have evaluated flow-mediated dilation of limb resistance arteries in HF subjects before and after HT using venous occlusion strain-gauge plethysmography. In a longitudinal study, Sinoway et al. (1988) reported that resting and peak hyperemic forearm blood flow did not increase immediately after transplant (21 vs.

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99 25 ml/min/100ml, p=NS), but increased significantly at four months (21 vs. 43 ml/min/100ml,p <0.05). Kubo et al. (1993) measured forearm BF after methacholine and reactive hyperemia in the same six patients before and 4 months after HT. Resting forearm BF was similar before and after HT (3.3 vs. 3.7 ml/min/100ml), but peak FBF increased significantly at four months after HTX at each dose of methacholine and peak forearm BF during reactive hyperemia increased significantly after HTX from 19.0 to 44.8 ml/min/100 ml. These studies suggests that impaired forearm BF is not directly related to normalizing cardiac output and mean arterial pressure, but that it is increased after several months possibly as a result of resumption of daily physical activities. In addition, these data suggest that both agonist-mediated (methacholine) and flow-mediated (reactive hyperemia) forearm vasodilation of resistance vasculature is increased after HT. In contrast, Cavero et al. (1994) reported that peak FBF during reactive hyperemia was increased significantly 24-36 hours after HT before cyclosporine therapy started (11.2 vs. 21.2 ml/min/100 ml, p<0.05), and despite initiation of cyclosporine therapy peak FBF was similar 6-8 days after HT (22.3 ml/min/100ml), and after 6 weeks post-HT (22.7 ml/min/100ml). One possible explanation of these discordant results is that the peak forearm BF of the pre-HT subjects in the study by Cavero and colleagues. (1994) were extremely low, therefore subjects may have had more peripheral edema, lower cardiac index, or have been more severely deconditioned than the subjects in the former studies. Furthermore, because there was no age-matched healthy controls in these studies, it was unknown whether the forearm vasodilatory capacity returns to normal. In the present study, we found that peak forearm BF in our small cohort of HTR was

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100 significantly lower in end-stage HF subjects before HT, compared to healthy-age-matched control subjects, but did not increase significantly after HT. Peak CBF was significantly lower in end-stage HF subjects compared to age-matched healthy controls. However, peak CBF significantly increased after HT. This is the first report of vasodilatory capacity of resistance arteries in the calf before and after HT. Thus, it appears that the calf resistance vasculature may be more responsive to the effects of normalized cardiac output and arterial pressure after HT, or affected by the resumption of daily physical activity. Although it must be emphasized that the cohort is small, it appears that forearm and calf resistance artery function returns to levels similar to age-matched-healthy controls. Pulse Wave Analysis and Heart Transplantation This was the first investigation that studied the effects of HT on systemic arterial stiffness in end-stage HF subjects. Our results demonstrate that arterial stiffness does not change significantly following HT. AI a a measure of stiffness in central elastic and peripheral muscular arteries, was not significant changed after HT (8.9 vs. 13.5%, p=NS). Additionally, roundtrip travel time of the reflected wave to the periphery and back to the aorta (t p ), an inverse of pulse wave velocity, was not significantly altered after HT (146 vs. 140 ms, p=NS). This positive outcome occurs in the setting of a significant increase in peripheral (brachial) and central (aorta) systolic, diastolic, and mean blood pressure after HT, but without a change in peripheral and central pulse pressure. Thus, restoration of cardiac output results in significant increase in mean arterial blood pressure without significantly altering arterial reflected pulse wave properties (AI a and central pulse pressure) after HT. However, the increased aortic systolic blood pressure is accompanied

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101 by increased aortic systolic tension time index (AsTTI), an indicator of LV myocardial oxygen demand during systole, after HT (1826.1 vs. 3254.3 mmHg/sec/min, p<0.01), without any change in diastolic tension time index (DTPI), an indicator of diastolic perfusion (2843.5 vs. 3079.7 mmHg/sec/min). The study also demonstrates that augmentation pressure, pressure generated by reflected wave, is significantly higher in age-matched healthy controls than in end-stage HF subjects before HT (10.4 vs. 3.8 mmHg, p<0.05) and after HT (10.4 vs. 1.8 mmHg, p<0.05). However, there was no significant difference in AI a normalized for heart rate, between age-matched healthy controls vs. before HT (17.6 vs. 8.9%, p=0.09), and vs. after HT (17.6 vs. 13.5%, p=NS). These data suggest that systemic arterial stiffness in age-matched healthy controls is not significantly different than end-stage HF and HTR subjects. It is possible that poor cardiac function and the combination of antihypertensive medications in HF subjects accounted for the lower mean blood pressure and lack of difference in arterial stiffness between HF and age-matched healthy controls. This is because muscular artery vasodilators such as ARBs, decrease pulse wave velocity and wave reflection amplitude resulting in reduced AI a and central and systolic pulse pressure (Nichols 2005). Furthermore, antihypertensive medications which alter arterial pulse wave properties likely account for the lack of difference in AI a between HTR and age-matched healthy controls. Endothelial-Derived Vasoactive Balance and Heart Transplantation In the present study, there was a 40% non-significant reduction in plasma NOx in end-stage HF subjects after HT (55.5 vs. 39.7 uM, p=NS). This finding did not support our hypothesis that plasma NOx would increase following HT reflecting improved endothelial function. Paradoxically, an improvement in conduit and resistance artery

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102 flow-mediation vasodilation after HT occurred despite the 40% reduction in NOx. We suspect that these puzzling findings can be attributed to the multiple stimuli for NO. For example, the source of NO in end-stage HF subjects before HT is influenced by sources in addition to eNOS. It is well established that inflammatory cytokines, such as TNFand IL-6, both elevated in HF (Levine et al. 1990), stimulate expression of iNOS in vascular smooth muscle (Chester et al. 1998) and the failing myocardium (Drexler 1998) resulting in overproduction of NO. In this scenario, increased NO production has a negative inotropic effect on the myocardium (Habib et al. 1996; Drexler 1998) and impairs EDV (Kessler et al. 1997). Indeed, in the present study plasma NOx levels significantly higher in HF subjects than age-matched healthy controls. Although TNFis decreased after HT levels, plasma NOx did not change significantly after HT. However, NOx levels may be confounded by several factors in HTR. Urinary NOx is elevated during acute endomyocardial rejection in animal models (Mugge et al. 1996), and also has been reported to increase before any clinical signs of rejection are evident (Winlaw et al. 1994). In contrast, the administration of the immunosuppressive agent cyclosporine inhibits induction of cytokine stimulated iNOS mRNA, and reduces the accumulation of NOx (Marumo et al. 1995). Thus, when HTR are not in acute clinical rejection, NOx levels likely reflect the competition between cytokine induced iNOS expression, subclinical rejection, and iNOS inhibition by cyclosporine. Moreover, we speculate that in healthy controls, where cytokine induction of iNOS is minimal, plasma levels of NOx more accurately reflect NO derived from the eNOS pathway in the vascular endothelium.

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103 In the present study, plasma levels of ET-1 levels did not significantly change after HT, and ET-1 in end-stage HF and HTR were not significantly different than age-matched healthy controls. Cyclosporine has been demonstrated to increase ET-1 in vitro (Marsen et al. 2003), and acute oral dose of cyclosporine in transplant subjects results in an acute increase in plasma ET-1 levels by six hours. As such, Haas et al. (1993) reported that plasma ET-1 was elevated in HTR 2 years after HT (range 9 days to 3 years post-HT) compared to normal healthy controls (5.2 vs. 1.9 pg/ml). In contrast, Piquard et al. (2001) reported in a prospective study, that ET-1 increased significantly within the first 2 weeks after surgery, but normalized by 2 months post-HT to levels similar to healthy controls (2.8 vs. 1.4 pg/ml). These discordant results may be explained by the cross-sectional design and the heterogenous time after transplant which the samples were obtained by Haas and colleagues (1993). Although cyclosporine therapy may be a primary contributor to elevated ET-1 in some HTR, the data of Piquard et al. (2001) and the present study suggest that ET-1 levels may, in fact, be similar to healthy controls by two months after HT. Lipid Peroxidation, Antioxidant Enzyme Activity, Endogenous Nitric Oxide Inhibition and Heart Transplantation Administration of vitamin C, a known scavenger of superoxide anion radical, reverses impaired radial artery EDV in HF, suggesting that superoxide plays a significant role in endothelial dysfunction in HF patients (Hornig et al. 1999). In addition, lipid peroxidation products such as lipid peroxides (LPO), malonylaldehyde (MDA), and 8-iso-PGF 2 are elevated in chronic HF subjects (Keith et al. 1998; Polidori et al. 2004). In the present study, there was no significant difference in 8-iso-PGF 2 before and after HT. Surprisingly, 8-iso-PGF 2 was significantly greater in age-matched healthy controls than

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104 in both end-stage HF subjects (2167.7 vs. 1474.8, p<0.01) and HTR (2167.7 vs. 1597.4 pg/ml, p<0.05). These data are in contrast to the study by Polidori et al. (2004) who reported significantly higher plasma F 2 isoprostanes in chronic HF than in healthy controls, and Keith et. al. (1998) who reported higher plasma MDA and LPO in chronic HF subjects. Although the precise mechanism for these discordant results are unclear from the present study, we propose that the HF and HTR subjects may have lower levels of lipid peroxidation due their pharmacological regimen which includes ACE inhibitors/ARBs and HMG-CoA reductase inhibitors (statins). Infusion of ANG II into pigs (Haas et al. 1999) and humans (Murphey et al. 2003) increases levels of lipid peroxidation in plasma, suggesting that ANG II is a significant contributor to plasma levels of oxidative stress. In the present study, all HF and HTR subjects were on ACE inhibitors or ARBs, both of which are known to inhibit ANG II stimulated production of superoxide by activation of NADPH oxidase via the AT-1 receptor in the vascular wall (Nickenbig and Harrison 2002). In addition to their lipid-lowering effects, statins also have a well documented antioxidant effect via upregulation of eNOS and decreased NADPH oxidase activity in vitro (Mason et al. 2004). In humans, Desideri et al. (2003) reported that statin therapy lowers urinary and plasma levels of 8-iso-PGF 2 and in the present study five subjects before HT and all subjects after HT were on statins. Therefore, these studies suggest that ACEI/ARB and statin pharmacological regimens may have influenced our measurement of lipid peroxidation in the HF and HTR in this study. The present study also observed that plasma SOD activity is decreased after HT (2.16 vs. 1.79 U/ml, p<0.05), and is significantly lower than age-matched healthy

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105 controls (1.79 vs. 2.56 U/ml, p<0.01). These data are consistent with animal studies that report that cyclosporine decreases plasma and tissue total antioxidant status, SOD activity, glutathione peroxidase (GPX) activity, and catalase activity in rats (Mun 2000). In humans, Perez et al. (2002) reported reduced plasma SOD activity in HTR compared to class III HF patients, but no difference in GPX or catalase activity (Perez et al. 2002). Thus, in the present study, the toxic effects of cyclosporine likely account, in part, for the decrease in plasma SOD activity after HT. ADMA also did not change in end-stage HF subjects after HT. We hypothesized that ADMA would be elevated due to oxidative inactivation of DDAH, and therefore would result in accumulation of ADMA. Although our hypothesis was not supported, plasma lipid peroxidation did not increase after HT and therefore the lack of ADMA elevation was consistent with this finding. Inflammatory Markers and Heart Transplantation CRP is an acute phase protein released by liver cells in response to inflammatory cytokines IL-6 and TNF-. CRP has been reported to be a strong independent predictor of coronary artery vasculopathy, cardiac allograft failure, and mortality in HTR (Eisenberg et al. 2000; Labarrare et al. 2002). In addition to being a marker of future cardiovascular risk, several in vitro studies suggest that CRP is directly involved in the development of endothelial dysfunction because endothelial cells incubated with CRP demonstrate decreased expression of eNOS mRNA, eNOS protein, and eNOS bioactivity (Venupogal et al. 2002), as well as increased expression of vascular adhesion molecules VCAM-1 and ICAM-1 (Pasceri et al. 2000). Fichtlscherer et al. (2000) reported that elevated CRP correlated inversely with impaired EDV in a cohort of CAD patients, and a

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106 reduction in CRP after 3 months was associated with improved EDV. In the present, study CRP and IL-6 were significantly higher in end-stage HF subjects than in age-matched healthy controls. Following HT, IL-6 remained higher than age-matched controls, but CRP (log transformed) decreased significantly after HT to levels comparable to healthy controls. To our knowledge, this is the first report of plasma IL-6 and CRP in same cohort of subjects before and after HT which clearly illustrates the hyperinflammatory state of end-stage HF subjects compared to normal healthy controls. TNFis an inflammatory cytokine produced from activated macrophages, T-lymphocytes (Blake and Ridker 2003), and adipose tissue (Kern et al. 2001). In end-stage HF subjects, TNFis expressed in the failing myocardium (Torre-Amione et al. 1996b) which can be shed into the circulation and increase plasma levels of TNFas severity of HF increases (Torre-Amione et al. 1996a). TNFhas a negative inotropic effect on myocardium and increases expression of iNOS in myocardium (Habib et al. 1996; Wildhirt et al. 2001a) and vascular smooth muscle (Chester et al. 1998). Importantly, in vitro studies demonstrate that TNFhas a direct effect on endothelial function by causing post-transcriptional modification of eNOS mRNA leading to early mRNA degradation (Yoshizumi et al. 1993). In chronic HF subjects, Katz et al. (1994) found TNFlevels to be highly correlated with impaired forearm EDV in response to acetylcholine. Moreover, Weis et al. (2001) reported that treatment with simvastatin lowered IL-6 and TNFand was associated with improved acetylcholine-induced coronary EDV. In the present study, TNFwas significantly elevated in end-stage HF subjects compared to age-matched healthy controls, and was significantly lower after HT. Thus, it is possible that the reduction in TNFafter HT is one mechanism that

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107 contributed to the improvement in conduit and resistance artery endothelial function after HT. Expression of sICAM-1 and VCAM-1 on the endothelial wall in response to activation by ROS, CRP, TNF-, and decreased NO, is a critical step in the development and progression of atherosclerosis (Hope and Meredith 2003). sICAM-1 is the circulating form of the endothelial adhesion molecule ICAM-1 which has been shed from the activated endothelium. When the endothelium is activated, ICAM-1 and VCAM-1 expression on the endothelial surface are involved in the adherence and transendothelial migration of circulating leukocytes into the subintimal space at sites of inflammation, including developing atheroslerotic plaques (Hope and Meredith 2003). The present study demonstrates that plasma sICAM-1 is elevated in end-stage HF subjects compared to age-matched healthy controls, and is significantly reduced after HT. The mechanism for the decreases in CRP, TNF-, and sICAM-1 and not IL-6 may be due to several factors. First, a major source of TNF-, during end-stage HF is the failing myocardium (Torre-Amione et al. 1996b). Therefore, transplantation removes this source and therefore is likely a major contributor to the reduction in TNFafter HT. Second, the calcineurin inhibitor cyclosporine inhibits IL-2 gene expression in T-lymphocytes, which prevents allograft rejection by inhibiting T-lymphocyte proliferation and stimulation of specific cytokines during allograft rejection. Cyclosporine suppresses the post-surgical increase in CRP and IL-6 usually observed after cardiac surgery (Van Lente et al. 1985) and decreases cytokine induced ICAM-1 and VCAM-1 expression on endothelial cells in vitro (Markovic et al. 2002). Therefore, chronic cyclosporine therapy in HTR may account for the decrease in CRP, TNF-, and sICAM-1 after HT.

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108 Peripheral Conduit Artery Endothelial Function and Exercise Training The present study demonstrates that 12 weeks of supervised endurance exercise training in HTR preserves but does not improve brachial artery endothelial function (FMD: 10.0 vs. 9.6%, p=NS). However, HTR who do not participate in supervised exercise training show a progressive decline in endothelial function of the brachial artery (FMD: 11.1% vs. 7.9%, p<0.05). Two studies have reported that that brachial artery FMD is improved in CAD patients following supervised exercise training (Walsh et al. 2003; Edwards et al. 2004). Walsh et al. (2003) recently reported that 8 weeks of cross-training (aerobic/resistance training) in CAD patients resulted in improved brachial artery FMD (3.0% to 5.7%, p<0.05). Edwards et al. (2004a) found that 12 weeks of treadmill walking as part of a cardiac rehabilitation program resulted in an improvement in brachial artery FMD (7.9 vs. 11.2%, p<0.05). In contrast, Gokce et al. (2002) reported that 12 weeks of lower body endurance exercise training resulted in a significant increase in FMD of the posterior tibialis artery of the lower leg (7.9 vs. 11.2%, p<0.05), but only a non-significant trend in improvement in brachial artery FMD (6.4% vs. 8.3%, p>0.05) in CAD patients. Our results are consistent with Gokce et al. and suggest that 12 weeks of endurance training may not be sufficient to improve upper limb endothelial function. However, the finding of a progressive decrease in brachial artery FMD after 12 weeks in HTR who did not participate in supervised exercise training is a novel finding. One cross-sectional study evaluated brachial artery FMD in trained and untrained HTR six years after HT (Schmidt et al. 2002). Brachial artery FMD was significantly higher in HTR who participated in 6 months of cycling for 40 minutes 2 to 3 times per week compared to sedentary HTR (7.1% vs. 1.4%, p<0.05). Our prospective study support the results of Schmidt and colleagues (2002) and suggest that exercise training may have, in

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109 fact, attenuated a progressive decline in peripheral endothelial function of upper limb conduit arteries that occurs early after HT. Taken together, these data demonstrate that supervised exercise training has a beneficial effect on the peripheral vasculature in HTR early after HT by attenuating a progressive decline in upper limb endothelial dysfunction demonstrated by a decrease in brachial artery FMD. Peripheral Resistance Artery Endothelial Function and Exercise Training Our study demonstrates that welve weeks of endurance exercise training improved the maximal flow-mediated vasodilatory capacity of forearm and the calf resistance arteries in a subgroup of HTR. Peak FBF during reactive hyperemia increased 34% (21.5 vs. 28.8 ml/min/100 ml, p<0.05) in HTR who performed supervised exercise training, but there was a non-significant 14% increase in peak FBF (25.5 vs. 29.2 ml/min/100ml, p=NS) in the control group who did not perform supervised training. Peak CBF increased 17% in HTR following 12 weeks of exercise training (25.0 vs. 29.3 ml/min100 ml, p=0.05), but there was no change in peak CBF in control HTR (26.3 vs. 25.2 ml/min/100ml, p=NS) who did not participate in supervised training. These data support the hypothesis that maximal vasodilatory capacity of the forearm and calf resistance arteries are improved following 12 weeks of lower body endurance exercise training in HTR. Two prospective studies in subjects with essential hypertension reported improvements in maximal vasodilatory capacity of forearm resistance arteries during reactive hyperemia. In 20 subjects with essential hypertension, Higashi et al. (1999) reported a 23% increase in peak FBF during reactive hyperemia using plethysmography after a 12-week exercise training intervention. In subjects who showed an improvement in peak FBF, the increased FBF was abolished by the NO inhibitor, L-NMMA,

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110 suggesting that the increase in peak FBF during reactive hyperemia of forearm resistance arteries was NO mediated. Tanaka et al. (1998) reported a 17% increase in peak VO 2 a 17.5% increase in peak FBF, and 18.9% increase in peak CBF during reactive hyperemia in older, hypertensive subjects after exercise training (walking) for 6 months 3-4 days per week. Two studies have reported conflicting results on forearm and calf resistance artery vasodilatory capacity in subjects with chronic HF. Testa et al. (2000) reported a 50% increase in peak CBF during reactive hyperemia along with a 23% increase in peak VO 2 following 12 weeks of supervised exercise training in chronic HF subjects. In contrast, Dziekan and colleagues (1998) reported a decrease in peak forearm BF and an increase in peak calf BF in both trained and control HF subjects. The investigators suggest that that exercise training had no additional benefit in forearm or calf despite a 25% increase in peak VO 2 in the trained HF subjects. In aggregate, our blood flow data suggest that lower body dynamic endurance exercise training results in a systemic improvement in maximal flow-mediated vasodilatory capacity of limb resistance arteries likely due to in part to improvements in endothelial function in HTR. The possible mechanisms for this improvement in both forearm and calf resistance artery endothelial function will be discussed in the sections to follow. Pulse Wave Analysis, Blood Pressure, and Exercise Training Twelve weeks of supervised exercise training resulted in no significant change in arterial stiffness in HTR in the present study. Moreover, exercise training resulted in no change in AI a or the round trip travel time of the reflected pressure wave (t p ). These findings were surprising due to the recent reports of exercise-induced improvements in

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111 arterial stiffness in healthy sedentary, young men (Cameron et al. 1994), healthy, sedentary middleaged and older men (Tanaka et al. 2000), and men with coronary artery disease (Edwards et al. 2004). The study by Edwards et al. (2004) reported a significant decrease AI a and an increase in t p with no change in systolic or mean blood pressure. Their results suggest that endurance exercise training improved systemic arterial stiffness independent of blood pressure in men with CAD. Consistent with our results, however, Parnell et al. (2002) reported that 8 weeks of endurance exercise training in chronic HF patients resulted in no change in aortic pulse pressure, aortic AI a and aortic pulse wave velocity after the exercise intervention. It is possible antihypertensive medications confounded the results of the present study (Table 4-9). Angiotensin receptor blockers and calcium channel blockers decrease the amplitude and timing of the reflected wave returning to the aorta, and thus decrease AI a pulse wave velocity, aortic systolic and pulse blood pressure (Nichols 2005). Although the number of the antihypertensive medication was not different between TRAINED and CONTROL group at baseline and after 12 weeks, individual variability in response to medication may have confounded the results. Twelve weeks of supervised endurance exercise training also resulted in no change in peripheral and central blood pressure components in HTR early after HT. Specifically, 12 weeks of exercise training resulted in no change in peripheral or central systolic, diastolic, pulse, or mean blood pressure in HTR. These results are consistent with the randomized, controlled study Kobashigawa et al. (1999) who randomized twenty-seven HTR two weeks after HT to an exercise or control group. Subjects performed supervised endurance exercise training as part of a cardiac rehabilitation

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112 program for six-months. Resting brachial systolic blood pressure did not significantly change in the exercise trained group (126 vs. 121 mmHg, p=NS) or the control group (130 vs. 114 mmHg, p=NS) after 6 months of endurance exercise training beginning early after HT. As in the pulse wave analysis, these findings may be due to the confounding effects of antihypertensive medications being taken by all HTR in the study. An additional confounding factor could be the effects of cyclosporine on blood pressure (Ventura et al. 1997; Scherrer et al. 1990). Although the dose and serum trough levels of cyclosporine were not significantly different at baseline and at 12 weeks, the individual effects of cyclosporine on blood pressure at the time of each measurement is unknown. Alternatively, it is possible that the exercise training stimulus (frequency, intensity, or duration) was not sufficient to elicit changes in peripheral vascular resistance and thus alter mean or systolic blood pressure. Endothelial-Derived Vasoactive Balance and Exercise Training It has been well documented in animals (Sessa et al. 1994; Fukai et al. 2000; Spier et al. 2004) and humans (Hambrecht et al. 2003) that chronic exercise training increases eNOS mRNA and eNOS protein leading to increased synthesis of NO. Previous studies have reported that endurance exercise training results in an increase in plasma NOx in both healthy (Jungersten et al. 1997) and CAD subjects (Edwards et al. 2004), reflecting the increase in vascular NO production from the endothelial wall. In the present study, twelve weeks of exercise training resulted in a non-significant 31% increase in resting plasma nitrate/nitrite (NOx) levels in HTR. In addition, plasma NOx levels increased 48.5% over 12 weeks in the HTR who did not perform exercise training, but this change was not statistically different. Schaefer et al. (2001) found an acute increase in plasma NOx following a 30 minute bout of cycle exercise in healthy humans, but no change in

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113 plasma NOx following exercise in HTR. The authors suggested that this was due to endothelial dysfunction and the inability of HTR to increase shear-stress mediated endothelial-derived NO synthesis. In support of this hypothesis, they noted that exercise capacity was significantly correlated to NOx in the healthy subjects (r=0.95, p<0.01), but not in the HTR. The lack of change in exercise-induced plasma NOx in the study by Schaefer (2001) and the large non-significant increase in NOx in both groups in the present study, suggests that an additional source of NO, in addition to endothelial-derived NO, contributes to plasma NOx levels in HTR. In light of the fact that resistance artery endothelial function increased after 12 weeks of exercise training, but the NO-mediated brachial artery FMD did not, it is possible that a non-NO mediated mechanism may have accounted for this increase in forearm and calf resistance artery endothelial function. Although several studies confirm that limb resistance artery vasodilation during reactive hyperemia is NO-dependent (Higashi et al. 1999; Meredith et al. 1996), indeed several studies suggest that peak vasodilation of limb resistance vasculature during reactive hyperemia is prostacyclin-dependent (Engelke et al. 1996; Tagawa et al. 1994). As stated in a previous section, urinary NOx has been reported to be elevated during acute allograft rejection in HTR (Mugge et al. 1996). In the present study no HTR were in active rejection on endomyocardial biopsy at the time the blood sample was obtained. However, Winlaw et al. (1994) reported that an increase in urinary NOx precedes clinical evidence of allograft rejection in rats. Also, Wildhirt et al. (2001a) reported that 26% of HTR at 1 month post-transplantation had increased endomyocaridal iNOS expression which was associated with impaired coronary endothelial function and increased transcardiac NOx release. In a separate study, Wildhirt et al. (2001b) reported

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114 that transcardiac TNFlevels correlated to NOx levels at one month (r=0.081, p=0.001) and 12 months (r=0.62, p=0.04) post-HT suggesting that an inflammatory process is involved in coronary microvasculature dysfunction. Thus, increased NOx may be an early indicator of coronary artery vasculopathy and contribute to plasma NOx levels in HTR. Therefore, we conclude that in the present study it is possible that NO production from smooth muscle of coronary microvasculature iNOS expression and non-clinical allograft rejection contributed to the variable NOx levels and temporal trend for NOx to increase over 12 weeks in both trained and control HTR. In the present study, plasma ET-1 levels did not significantly change after 12 weeks of exercise training or control period. However, there was a trend for a decrease (-41%) in ET-1 (p=0.09) and a non-significant decrease (-36%) in the control group. Endurance exercise training has been reported to reduced plasma ET-1 levels in young, healthy subjects (Maeda et al. 2001), and older women (Maeda et al. 2003). However, Callaerts-Vegh et al. (1998) reported that 12 weeks of exercise training did not alter ET-1 in chronic HF patients. It has been suggested that NO produced from the endothelium during chronic exercise training decreases ET-1 production, and that plasma NOx correlates inversely with plasma ET-1 in healthy young subjects (Maeda et al. 2001). Therefore, we hypothesized that exercise training would decrease ET-1 levels in HTR due to an increase in NO synthesis or bioavailablilty. Thus, our study demonstrates that endurance exercise training does not significantly alter plasma levels of ET-1 in HTR. Lipid Peroxidation, Antioxidant Enzyme Activity, Endogenous Nitric Oxide Inhibition and Exercise Training F 2 -isoprostanes are a family of prostaglandin products of free-radical oxidation of polyunsaturated fatty acids which circulate esterified to lipid membranes in plasma and in

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115 free plasma form. Using the F 2 -isoprostane isomer, 8-iso-PGF 2 as a marker of in vivo lipid peroxidation, we hypothesized that 12 weeks of supervised endurance exercise training would reduce plasma lipid peroxidation primarily by an increase in activity of the extracellular antioxidant enzyme SOD. SOD is the primary SOD isoform found in the vascular wall and is the principal regulator of superoxide radical production and the primary mechanism for preservation of NO from superoxide-induced degradation (Fukai et al. 2002). As mentioned, chronic exercise training has been reported in animals and humans to enhance NO production by increased eNOS protein production. In addition, Fukai et al. (2000) reported that three weeks of exercise training in eNOS wildtype (+/+) mice resulted in a 3-fold increase in aortic eNOS protein which was paralleled by a 3-fold increase in aortic ecSOD. However, this increased ecSOD protein expression was not observed in aortas from eNOS knockout (-/-) mice suggesting that NO production modulates ecSOD expression in the vascular wall via a feed-forward mechanism, therefore increasing its own biological effects. Therefore, we hypothesized that exercise training would enhanced ecSOD activity and subsequently lower levels of free-radical induced lipid peroxidation and therefore preserve NO. However, twelve weeks of supervised endurance exercise training did not alter basal levels of plasma 8-iso-PGF 2 or ecSOD activity in HTR. The reasons for the lack of change in NOx data were explained in the previous section, however, the lack of change in SOD activity are surprising in light a recent report that exercise training enhanced SOD activity and lowered lipid peroxidation in humans (Edwards et al. 2004). The lack of improvement in brachial artery FMD in HTR who participated in supervised exercise training is consistent with the lack of change in SOD activity and 8-iso-PGF 2 However, we would have expected

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116 that a decrease in SOD activity and an increase in 8-iso-PGF 2 would have paralleled the decrease in brachial artery FMD in the control HTR after 12 weeks. However, we speculate that additional factors may have contributed to the decrease in brachial artery FMD in the control HTR. For example, sympathetic activation increases in HTR due to chronic cyclosporine therapy. Thus, one hypothesis is that exercise training attenuated a progressive increase in sympathetic activation in HTR because endurance training decreases muscle sympathetic nerve activity (MSNA) in chronic heart failure subjects (Roveda et al. 2003). Although we did not measure MSNA in this study, studies show that elevated sympathetic activation in end-stage HF subjects normalizes immediately after HT (Rundqvist et al. 1996), but chronic cyclosporine therapy contributes to enhanced sympathetic activation months to years after HT (Scherrer et al. 1990). Indeed, Hijmering et al. (2002) reported that acute enhanced sympathetic activation results in a decreased brachial artery FMD, but has no effect in peak forearm vasodilatory capacity or resistance arteries during reactive hyperemia (Hijmering et al. 2002). Hence, we speculate that this could explain the decline in brachial artery FMD in control HTR, but not in the peak forearm vasodilatory BF and calf BF. Lastly, the endogenous NO inhibitor, ADMA, was not altered by 12 weeks of exercise training in HTR. This is not surprising because we hypothesized that oxidative stress following exercise training would decrease ADMA by decreasing oxidant inactivation of DDAH, the enzyme that degrades ADMA and the primary pathway of ADMA regulation (Sydow and Munzel 2003). Thus, the lack of change in plasma 8-iso-PGF 2 and SOD activity suggest that oxidative stress was not altered following HTR and support the lack of change in ADMA.

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117 Inflammatory Markers and Exercise Training As mentioned, it is now known that CRP has a direct pro-inflammatory effect on the endothelium in addition to its clinical value as a predictor of future cardiovascular disease. Therefore pharmacological or non-pharmacological interventions which modify levels of CRP may have important clinical implications. There have only been two prospective studies on effects of exercise training on CRP in patients with CAD (Milani et al. 2004; Edwards et al. 2004). Milani et al. (2004) reported a 41% decrease in CRP in 277 CAD patients after 12 weeks of exercise training and Edwards et al. (2003) reported that 12 weeks of endurance exercise training lowered CRP by 45% in CAD patients. However, there have been no prospective studies on the effects of exercise training on CRP in HTR. In the present study, 12 weeks of exercise training resulted in a non-significant 49% decrease in plasma CRP, and a non-significant 19% decrease in CRP in the control HTR. The relative change in CRP is similar to that reported by Milani et al. (2004) and Edwards et al. (2003), but the lack of a statistical significant change is likely due to the large inter-individual variation in CRP within each group and the small number of subjects. However, the decrease in CRP in the trained HTR in the present study may be clinically significant because Eisenberg et al. (2000) reported that for every 2 fold increase in CRP increased the risk of allograft failure by 32%. Furthermore, Pethig et al. (2000) reported that HTR with significant coronary artery vasulopathy had significantly higher CRP (4.1 vs. 1.8 mg/L) than HTR with no coronary vasculopathy. Thus, exercise training did not significantly alter CRP in this small cohort of HTR, but more research is needed with larger cohort to confirm these findings. There have been two prospective studies on the effects of exercise training on inflammatory cytokines in chronic HF patients (Adamopoulos et al. 2002; Adamopoulos

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118 et al. 2001). Adamopoulos and colleagues conducted a 12-week, randomized, controlled, cross-over design study in 24 chronic HF patients and 20 healthy controls. They reported a significant 29% decrease in plasma levels of IL-6 and 39% decrease in TNF(Adamopolous et al. 2002). Second, Larsen et al. (2001) reported a 12.5% decrease in TNFbut no change in IL-6 in 28 patients with chronic HF. In CAD patients, Edwards et al. (2003) reported that 12 weeks of endurance exercise training lowered IL-6 by 32%. In the present study, the effect of 12 weeks exercise training in HTR resulted in a non-significant 35% decrease in IL-6 (5.02 vs. 3.25 pg/ml) and no significant change in TNF-. However, in the control HTR, there was a significant 53% increase in plasma TNF(1.56 vs. 2.38 pg/ml, p<0.05) after 12 weeks. These data suggest that endurance exercise training attenuates a progressive increase in TNFover 12 weeks early after HT. The mechanism for this increase in the control HTR is unknown, but a possible explanation is that the greater increase in body weight in the control HTR than in trained HTR contributed to this finding, since TNFis correlated modestly to body weight and body fat (Kern et al. 2001). As mentioned in a previous section, in vitro studies demonstrate that TNFhas a direct effect on endothelial function by causing post-transcriptional modification of eNOS mRNA leading to early mRNA degradation (Yoshizumi et al. 1993), and in humans, TNFlevels are highly correlated with impaired forearm EDV in response to acetylcholine in HF patients (Katz et al. 1994). Therefore, in the present study we propose that this increase in circulating TNFover 12 weeks in the control HTR, may have contributed to the progressive decrease in brachial artery endothelial function during the control period.

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119 In HTR, the development of coronary artery vasculopathy is preceded by expression of ICAM-1 on the endothelium (Labarrere et al. 2000). Also, serum levels of sICAM-1 are a significant predictor of coronary vasculopathy, post-transplant ischemic events, and graft failure in HTR (Labarrere et al. 2000; Labarrere et al. 2002). For example, HTR with sICAM-2 of 308 ng/ml or greater had a 2.6 fold increase risk of CAV, and 3.6 fold increased risk of graft failure (Labarrere et al. 2000). Thus, a pharmacological or non-pharmacological intervention which lowers sICAM-1 may be indicator of improved endothelial function or decreased CV risk in HTR. Two studies on the effects of exercise on plasma sICAM-1 levels have been reported in chronic HF patients (Adamapolous et al. 2001; Niebauer et al. 2005). Adamapolous et al. (2001) reported a 14.4% decrease in sICAM-1 (367 vs. 314 ng/ml, p<0.01) after 12 weeks of endurance exercise training in chronic HF patients and Niebauer et al. (2005) reported no change in basal plasma levels of sICAM-1 after 8 weeks of a home-based endurance cycling program in chronic HF. Prior to the present study, the effects of exercise training on plasma sICAM-1 in HTR were unknown. The present study demonstrates that twelve weeks of endurance exercise training in HTR does not significantly alter plasma levels of sICAM-1 (216.5 vs. 198.2 ng/ml, p=NS). Additionally, there was no significant change in the control HTR (269.2 vs. 295.6 ng/ml, p=NS) after 12 weeks. These results are consistent with the study by Niebauer (2005) but not with Adamopoulos et al. (2001). It is possible that since TNFand the measure of brachial artery endothelial function did not change with training, that endothelial activation was not altered significantly and therefore did not change circulating sICAM-1 levels. However, sICAM did not increase in the control group where TNFincreased, however, ICAM-1 expression is regulated

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120 by several factors in addition to TNFwhich may have influenced plasma sICAM-1 levels. Exercise Capacity and Exercise Training There has been only one randomized, controlled study of the effect of endurance exercise training on exercise capacity in HTR. Kobashigawa et al. (1999) randomized twenty-seven HTR two weeks after HT to an exercise or control group. They reported that 24 weeks of supervised exercise training as part of a cardiac rehabilitation program increased peak VO 2 by 49% and exercise duration 59%, compared to 18% and 18% in the non-supervised control HTR, respectively. In the present study, HTR who performed 12 weeks of endurance exercise training increased peak VO 2 by 26%, whereas there was no significant change in peak VO 2 in the HTR who did not perform supervised exercise training. Furthermore, exercise duration on the graded treadmill test increased 45% in the trained HTR, but there was no change in exercise duration in the control HTR. Although Kobashigawa and colleagues (1999) reported greater changes in peak VO 2 and exercise duration, these differences are likely due to the longer duration of their exercise training program (24 weeks) than the program in our study (12 weeks). Conclusions HT results in an improvement in brachial artery FMD and forearm and calf vasodilatory capacity. The mechanisms responsible for these post-HT are not clear from the present study, however attenuation of the hyper-inflammatory state after HT present in end-stage HF subjects may be a significant contributor. Although, it is possible that NO mediated the improvement in endothelial function, the confounding influences of HT pharmacology and low-grade non-clinical allograft rejection on plasma NOx levels prevent such conclusions. Additionally, although lipid peroxidation was unchanged after

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121 HT, but is suppressed in HTR compared to normal healthy controls possibly due to pharmacological therapy which attenuates oxidative stress. In contrast, SOD activity is significantly depressed after HT to levels below healthy controls likely due to chronic cyclosporine therapy. Although peripheral and central blood pressure components increase after HT due to normalization of cardiac function, reflected wave properties and arterial stiffness does not appear to change significantly. This study was the first study to report that 12 weeks of exercise training attenuates a progressive decline in brachial artery endothelial function in HTR. Although the mechanism for this finding is unclear, it may be due a progressive increase in the inflammatory mediator TNFin HTR who do not perform endurance exercise training. This study also demonstrates for the first time the 12 weeks of exercise training improved upper and lower limb resistance artery vasodilation. The mechanism for this finding is unclear because we could not confirm whether increases in NOx were due to endothelial-derived NO or non-endothelial sources of NO. It is possible that the increase is a non-NO-mediated mechanism, because several studies suggest that limb resistance vasculature during reactive hyperemia is prostacyclin-dependent. However, we cannot confirm this in the present study. Lastly, HTR who participated in 12 weeks of supervised exercise training experienced a 26% increase in peak VO 2 and 44% increase in exercise duration, but there was no significant changes in peak VO 2 or exercise duration in the control group. This may be the most important clinical finding because it suggests that the HTR who are instructed to initiate self-monitored, rather than supervised, exercise training programs, likely adopt a sedentary lifestyle despite their recent life-saving surgery.

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122 Limitations and Future Research The present study was designed to investigate the effects of HT on endothelial function and arterial stiffness in end-stage HF patients. In addition, we sought to investigate the effects of endurance exercise training on endothelial function and arterial stiffness in HTR. The study is limited by the small sample size. Also, it is possible that our findings are a result of selection bias or confounding variable such as dietary influences, or antihypertensive medications. However, we believe that a larger sample size would have supported the present results, and perhaps teased out some of the additional mechanisms that may be involved in changes in endothelial function following exercise training. Future research should include more invasive studies including infusion of pharmacological agonists and inhibitors of endothelial-derived substances via brachial artery catheterization to better pharmacodissect the mechanisms of endothelial function after exercise training in HTR. Furthermore, novel techniques such as human endothelial cell biopsies would allow superior isolation of changes of protein expression in endothelial cells than measurement by traditional blood plasma samples.

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140 Winlaw DS, Schyvens CG, Smythe GA, Du Z, Rainer SP, Keogh AM, Mundy JA, Lord RS, Spratt PM, MacDonald PS. Urinary nitrate excretion is a non-invasive indicator of acute cardiac allograft rejection and nitric oxide production in the rat. Transplantation 1994;58:1031-1036. Woodman CR, Muller JM, Laughlin MH, Price EM. Induction of nitric oxide synthase mRNA in coronary resistance arteries isolated from exercise-trained pigs. Am J Physiol 1997;273:H2575-2579. Woodman CR, Muller JM, Rush JW, Laughlin MH, Price EM. Flow regulation of ecNOS and Cu/Zn SOD mRNA expression in porcine coronary arterioles. Am J Physiol 1999;276:H1058-1063. Yoshizumi M, Perrella MA, Burnett JC, Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res 1993; 73 :205. Yousufuddin M, Yamani MH. The renin-angiotensin hypothesis for the pathogenesis of cardiac allograft vasculopathy. Int J Cardiol 2004;95:123-127.

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BIOGRAPHICAL SKETCH Gary Leon Pierce was born October 9, 1971 in Fitchburg, MA and graduated from Fitchburg High School in 1989. He attended Worcester State College in Worcester, MA and graduated in 1994 with a B.S. in biology. He then received an M.S. in clinical exercise physiology in 1997 from Northeastern University, and worked from 1997 to 2001 as the supervising clinical exercise physiologist in the Exercise Testing Laboratory in the Division of Cardiovascular Medicine, Brigham and Womens Hospital in Boston. He was also an adjunct faculty in the Department of Exercise Physiology at Lasell College from 1999-2001, and in the Sargent College of Health Sciences at Boston University in 2000. In 2001, he enrolled in the doctoral program in Applied Physiology and Kinesiology in the College of Health and Human Performance (HHP) at the University of Florida (UF) in Gainesville, FL and received the Jane Edmonds Predoctoral Fellowship. He was a graduate teaching assistant and adjunct instructor in the Department of Applied Physiology and Kinesiology at UF from 2001-04, and adjunct faculty in the Department of Health Sciences at Santa Fe Community College in Gainesville, FL in 2000. He received a Predoctoral Research Fellowship from the American Heart Association Florida/Puerto Rico Affiliate from 2003-05, and earned a Ph.D. in exercise physiology from UF in 2005. In summer 2005, he began work as a post-doctoral research associate in the Department of Integrative Physiology at the University of Colorado in Boulder, 141

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142 CO. He is married to Catherine (King) Pierce of Rochester, MN, and they have a daughter, Carolyn Louise Pierce, born Sept. 27, 2004.


Permanent Link: http://ufdc.ufl.edu/UFE0011375/00001

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Title: Endothelial Dysfunction and Arterial Stiffness in Heart Transplant Recipients
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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Permanent Link: http://ufdc.ufl.edu/UFE0011375/00001

Material Information

Title: Endothelial Dysfunction and Arterial Stiffness in Heart Transplant Recipients
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
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ENDOTHELIAL DYSFUNCTION AND ARTERIAL STIFFNESS IN HEART
TRANSPLANT RECIPIENTS
















By

GARY L. PIERCE


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2005






























Copyright 2005

by

Gary L. Pierce




























This dissertation is dedicated to my wife Cathy. Her unwavering support, patience, and
unconditional love for the past 4 years has made this accomplishment the most rewarding
of my life. I am happy I could share it with the woman I love. I want to thank her for
being a remarkable friend, spouse, career woman, and caring and dedicated mother to our
daughter Carolyn.















ACKNOWLEDGMENTS

No project this size can be completed without help from many people. I first thank

my supervisory committee chair and mentor (Randy Braith, Ph.D.) who gave me

invaluable support and guidance through this 3-year project. I am also indebted to Dr.

Braith for giving me the freedom to explore my research ideas, encouraging my teaching

style, and being an advocate for my career development. I look forward to continued

future collaboration and friendship with him.

I would also like to thank my committee members (Scott Powers, Ph.D., Christiaan

Leeuwenburgh, Ph.D., and Wilmer Nichols, Ph.D.). They offered valuable

encouragement and advice during this project, and were instrumental in instilling in me

the enthusiasm for scientific research.

I would like to thank several individuals in the Heart Transplant Program in the

Division of Cardiology at the University of Florida College of Medicine. In particular,

Rich Schofield, M.D. helped me recruit patients for the study, supervised the graded

exercise tests, and offered valuable advice on clinical issues involving heart-transplant

recipients. I also thank James Hill, M.D. (the director of the Heart Transplantation

program) for his full support of this project from its conception. Randy Harris, CVT,

unselfishly offered his time and knowledge and taught me about high-resolution vascular

ultrasound. I thank the heart transplant nurse practitioners at Shands Hospital

(particularly Suzanne Conrad, Suzy Holder, Tim Cleeton, Tracy Walker, and Alex Price)

for graciously responding to my frequent calls and emails.









I would like to thank several individuals in our laboratory. My colleagues, David

Edwards, Ph.D. and Peter Magyari, Ph.D. offered me valuable advice and friendship

during my first year at the University of Florida. I also thank Darren Casey, M.S. and

Scott Hamlin, M.S. for their camaraderie in the laboratory during the last 2 years and for

their help in supervising the graded exercise tests, processing blood samples, and with

biochemistry assays. I also thank Louise Perras and Kim Hatch (Center for Exercise

Science) for their endless administrative help for the last 4 years.

I would like to thank my parents Al and Rita Pierce; my brother Mark and his

family; and my sister Margie and her family for their encouragement and support to

pursue my professional goals. I thank my in-laws, Dr. and Mrs. John and Judy King for

supporting my academic pursuits and for their love and support of me and Cathy during

these past 4 years.

I need to thank several colleagues and mentors in Boston who encouraged me to

pursue doctoral training and a career in academic research: in particular, L. Howard

Hartley, M.D.; Kyle McInnis, Sc.D; Avery Faigenbaum, Ed.D.; Gary Balady, M.D.;

William Gillespie, Ed.D.; and Joe Libonati, Ph.D.

Finally, I thank the heart-transplant recipients who participated in this project.

Many of them inspired me with their "will for life," positive attitude, and unselfish

willingness to contribute to scientific research. I am grateful for the trust they placed in

me.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ....................................................... ............ ....... ....... ix

LIST OF FIGURES ......... ......................... ...... ........ ............ xi

ABSTRACT ........ .............. ............. ...... ...................... xiv

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

Rationale for the Study ................................. ... ....... ................. .6
Specific A im s and H ypotheses .......................................................... ............. 8

2 REVIEW OF LITERATURE ........................................................ .............. 14

N orm al Endothelial Function .................................. .....................................15
N itric O xide ............................................................................................... 15
Nitric Oxide Synthase .................. ............................ .... .... ............... 15
M echanism of N O R elease...................................................................... .. .... 16
B asal release of N O ....................................... ................ .. ................16
Agonist-mediated release of NO ............... ......... ................16
Shear stress-m ediated release of N O .................... .............................17
Pleiotropic Actions of NO .................................. .....................................17
O their Endothelial V asodilators ........................................ ....................... 18
E ndothelial V asoconstrictors..................................... ......................... .. ......... 19
E ndothelin- 1 ............................... .............. .......... .......... ...............19
A ngiotensin II.................................................. ......... 19
V asoconstrictor prostaglandins ....................................... ............... 20
V ascular Endothelial D dysfunction ........ ................ ................... .....................20
Decreased N O Synthesis by eN OS.................................... ...................... 21
NO Degradation by Reactive Oxygen Species.............................................. 23
Vascular Endothelial Dysfunction before Heart Transplantation .....................24
Vascular Endothelial Dysfunction after Heart Transplantation ........................26
Cyclosporine and vascular endothelial dysfunction in heart transplant
recipients ........................................................... .... ...... 31









Inflammation and vascular endothelial dysfunction in heart transplant
recipients ........................................... ....... ............ .... ......... 32
Asymmetric dimethylarginine and vascular endothelial dysfunction in
heart transplant recipients ..... ............. ........ ....................... 33
A rterial Stiffness .......... ...................... ..... .......... ..................... 33
Arterial Stiffness and Cardiovascular Risk ............................... ............... 37
Arterial Stiffness before Heart Transplantation ..............................................37
Arterial Stiffness and Hypertension after Heart Transplantation........................39
R ole of Exercise Training in H TR .............................. ..... ........................ ........... 41
Exercise Training and Functional Capacity ................................ ............... 41
Exercise Training and Endothelial Dysfunction......................................42
Exercise Training and Arterial Stiffness .................................. ............... 44
Exercise Training and Nitric Oxide Synthesis.............................................. 46
Exercise Training and Oxidative Stress ................................... .................48
Exercise Training and Vasoconstrictors........................................................49
Exercise Training and Inflammation .........................................................50

3 M E T H O D S ........................................................................................................... 5 2

S u b j e c ts ................................................................................................................. 5 2
Inclusion C criteria ................................................ ........ .. ............ 52
E x clu sio n C riteria .......... .............................................................. .. .... .. .. .. .. 5 3
G roup A ssignm ents ......................... .. .................... .. ...... ........... 53
E x ercise T raining P rotocol .............................................................. .....................53
Specific M easurem ents ............................................................. ............. ............... 54
A rterial Stiffness T testing .......................................................... ............... 55
Endothelial Function Testing ........................................... ....................... 57
Brachial artery flow-m ediated dilation ................................ ............... 57
Forearm and calf flow-mediated vasodilation................... ....... ...........59
G raded E exercise T est........... ...... .................................................. ............. 6 1
B lood C collection ......... ................................................................. .. .... .... .. 62
Plasm a B iochem ical A analysis ........................................ ......... ............... 62
Vasoactive balance ............ ..... ........... ...... .... .... .. ................62
L ipid peroxidation .......... .... ..... ........ ...... ............ ............ .. .......... 62
Extracellular antioxidant enzyme activity ................................................63
Inflammatory markers ....................................................63
Endogenous NO inhibition................ ... .... ...............63
Blood hemoglobin, hematocrit, serum lipids, glucose, creatinine, white
blood cell count, cyclosporine, and cytomegliovirus status ...................64
Endocardial biopsy rejection history.........................................................64
Statistical C on sideration s......................................... .............................................64

4 R E S U L T S .......................................................................... 6 6

Subject Characteristics before and after Heart Transplantation ...............................66
Serum Metabolic Parameters before and after Heart Transplantation......................68
Brachial Artery Endothelial Function before and after Heart Transplantation ..........69









Blood Pressure and Pulse Wave Analysis before and after Heart Transplantation....70
Forearm and Calf Resistance Artery Endothelial Function before and after Heart
Transplantation............... ....... .......... ........ ...... ....................................72
Vasoactive Balance before and after Heart Transplantation ...................................73
Plasma Lipid Peroxidation, Antioxidant Defense, and Endogenous Nitric Oxide
Inhibition before and after Heart Transplantation.................. ...............73
Inflammatory Markers before and after Heart Transplantation.............................. 74
Baseline Subject Characteristics before Exercise Training or Control .................75
Body Weight, Serum Metabolic Parameters, and Endocardial Rejection History
after Exercise Training ......................... .......... ........... ........ .............. 76
Brachial Artery Endothelial Function after Exercise Training..............................77
Blood Pressure and Pulse Wave Analysis after Exercise Training ..........................78
Forearm and Calf Resistance Artery Blood Flow after Exercise Training .............79
Vasoactive Balance after Exercise Training.......................................... ......... ......79
Lipid Peroxidation, Antioxidant Enzyme Activity, and Endogenous Nitric Oxide
Inhibition after Exercise Training ..........................................80
Inflammatory M arkers after Exercise Training .................................. ...................80
Peak Cardiopulmonary Exercise Testing Variables after Exercise Training ............81

5 D ISCU SSIO N ............................................................... ... .... ......... 95

Peripheral Conduit Artery Endothelial Function and Heart Transplantation ............96
Peripheral Resistance Artery Endothelial Function and Heart Transplantation.........98
Pulse Wave Analysis and Heart Transplantation......................... ............... 100
Endothelial-Derived Vasoactive Balance and Heart Transplantation ....................101
Lipid Peroxidation, Antioxidant Enzyme Activity, Endogenous Nitric Oxide
Inhibition and Heart Transplantation ............................................................. 103
Inflammatory Markers and Heart Transplantation .............................. ..................105
Peripheral Conduit Artery Endothelial Function and Exercise Training................08
Peripheral Resistance Artery Endothelial Function and Exercise Training ............109
Pulse Wave Analysis, Blood Pressure, and Exercise Training .............................110
Endothelial-Derived Vasoactive Balance and Exercise Training.............................112
Lipid Peroxidation, Antioxidant Enzyme Activity, Endogenous Nitric Oxide
Inhibition and Exercise Training................................... ..................................... 114
Inflammatory M arkers and Exercise Training.............. ................. ....................117
Exercise Capacity and Exercise Training ............... ............................................. 120
Conclusions.............. ....................................... .120
Lim stations and Future R research .................................... ........................... ......... 122

L IST O F R E FE R E N C E S ........................................................................ ................... 123

BIOGRAPHICAL SKETCH ............................................................. ............... 141
















LIST OF TABLES


Table page

4-1 Patient characteristics before and after heart transplantation.............................. 67

4-2 Serum metabolic parameters before and after heart transplantation........................68

4-3 Brachial artery flow-mediated dilation before and after heart transplantation ........69

4-4 Blood pressure components and pulse wave analysis before and after heart
tran plantation .........................................................................7 1

4-5 Forearm and calf flow-mediated vasodilation before and after heart
tran plantation .........................................................................72

4-6 Vasoactive balance before and after heart transplantation ........... ...............73

4-7 Lipid peroxidation, antioxidant enzyme activity, and endogenous nitric oxide
inhibition before and after heart transplantation ............................................... 74

4-8 Inflammatory markers before and after heart transplantation..............................75

4-9 Baseline patient characteristics before exercise training or control.........................76

4-10 Body weight, serum metabolic parameters, and endocardial rejection episodes at
baseline and after exercise training or control ............................... ............... .77

4-11 Brachial artery flow-mediated dilation at baseline and after exercise training or
control ............... ....... ............................................... ....... ......... ....... 78

4-12 Blood pressure components and pulse wave analysis at baseline and after
exercise training or control .............. ........... ..... ............... 78

4-13 Forearm and calf flow-mediated vasodilation at baseline and after exercise
training or control ............ ........ ....... ............... ..... ............ 79

4-14 Vasoactive balance at baseline and after exercise training or control....................80

4-15 Lipid peroxidation, antioxidant enzyme activity, and endogenous nitric oxide
inhibition at baseline and after exercise training or control ...................................80









4-16 Inflammatory markers at baseline and after exercise training or control ...............81

4-17 Peak cardiopulmonary graded exercise testing variables at baseline and after
exercise training or control .................................................. .......... ............... 82
















LIST OF FIGURES


Figure page

3-1 Study design. ...................................................................55

3-2 Ascending aortic pressure waveform ............................................ ............... 57

4-1 Brachial artery flow-mediated dilation before and after heart transplantation. .......83

4-2 Brachial artery flow-mediated diameter dilation before and after heart
tran plantation ..................................................... ................. 83

4-3 Aortic augmentation index (Ala) corrected for heart rate=75 b/min before and
after heart transplantation .......................................................................... ......... 83

4-4 Roundtrip travel duration of reflected wave (Atp) before and after heart
transplantation. .........................................................................84

4-5 Aortic systolic tension-time index (AsTTI) before and after heart transplantation..84

4-6 Forearm blood flow (FBF) before and after heart transplantation.........................84

4-7 Calf blood flow (CBF) before and after heart transplantation ..............................85

4-8 Nitrate/nitrite (NOx) before and after heart transplantation ....................................85

4-9 Endothelin-1 (ET-1) before and after heart transplantation ....................................85

4-10 Eight (8)-iso-prostanglandin-F2a (PGF2a) before and after heart transplantation....86

4-11 Superoxide dismutase (SOD) activity before and after heart transplantation..........86

4-12 Asymmetric dimethylarginine (ADMA) before and after heart transplantation......86

4-13 C-reactive protein (CRP) before and after heart transplantation ...........................87

4-14 Log-transformed C-reactive protein (logCRP) before and after heart
transplantation ..................................................... ................. 87

4-15 Interluekin-6 (IL-6) before and after heart transplantation ............. ..................87

4-16 Tumor-necrosis factor-alpha (TNF-ac) before and after heart transplantation.........88









4-17 Soluble intercellular adhesion molecule-1 (sICAM-1) before and after heart
transplantation ..................................................... ................. 88

4-18 Brachial artery flow-mediated dilation (FMD) at baseline and after 12 weeks of
ex ercise training or control......................................... ........................................ 88

4-19 Brachial artery absolute diameter dilation at baseline and after 12 weeks of
ex ercise training or control......................................... ........................................ 89

4-20 Aortic augmentation index (Ala) normalized for heart rate at 75 b/min at
baseline and after 12 weeks of exercise training or control................................89

4-21 Roundtrip travel time of reflected wave (Atp) at baseline and after 12 weeks of
exercise training or control. .......................................................... .....................89

4-22 Peak and total area under curve (AUC) forearm blood flow (FBF) at baseline
and after 12 weeks of exercise training or control. .............................................90

4-23 Peak and total area under curve (AUC) calf blood flow (CBF) at baseline and
after 12 weeks of exercise training or control. ................... ........................ 90

4-24 Nitrate/nitrite (NOx) at baseline and after 12 weeks of exercise training or
c o n tro l ...................................... ................................................... 9 1

4-25 Endothelin-1 (ET-1) at baseline and after 12 weeks of exercise training or
control .................................................................... .......... ...... 91

4-26 Eight (8)-iso-prostaglandin-F2a (PGF2a) at baseline and after 12 weeks of
exercise training or control............................................... ............. ............... 91

4-27 Superoxide dismutase (SOD) activity at baseline and after 12 weeks of exercise
training or control ................................... ......................... ......... 92

4-28 Asymmetric dimethylarginine (ADMA) at baseline and after 12 weeks of
exercise training or control............................................... ............................ 92

4-29 C-reactive protein (CRP) at baseline and after 12 weeks of exercise training or
c o n tro l .......... .................... ............................................................... 9 2

4-30 Interleukin-6 (IL-6) at baseline and after 12 weeks of exercise training or
c o n tro l ............ ............ .. .......... ...... .................................................. 9 3

4-31 Tumor necrosis factor-alpha (TNF-ac) at baseline and after 12 weeks of exercise
train in g o r co n tro l...................................................................................... 9 3

4-32 Soluble intercellular adhesion molecule-1 (sICAM-1) at baseline and after 12
weeks of exercise training or control. ........................................... ............... 93









4-33 Peak exercise oxygen uptake (V02) on graded exercise test at baseline and after
12 weeks of exercise training or control. ..................................... ............... 94

4-34 Peak exercise duration on graded exercise test at baseline and after 12 weeks of
exercise training or control............................................... ............. ............... 94















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ENDOTHELIAL DYSFUNCTION AND ARTERIAL STIFFNESS IN HEART
TRANSPLANT RECIPIENTS

By

Gary L. Pierce

August 2005

Chair: Randy W. Braith
Major Department: Applied Physiology and Kinesiology

Heart transplantation (HT) has become a life-extending intervention for patients

with end-stage heart failure (HF). However, frequent complications after HT, such as

hypertension and coronary artery vasculopathy (CAV), have been linked to vascular

endothelial dysfunction (VED) and arterial stiffness (AS), and jeopardize the long-term

survival of heart transplant recipients (HTR). Recent studies suggest that endurance

exercise training modifies VED and AS in chronic HF and hypertensive individuals.

Therefore, the purpose of this study was to investigate the effects of HT on VED and AS

in end-stage HF patients, and the effects of endurance exercise training early after HT.

Twelve (n=12) end-stage HF patients awaiting orthotopic HT at Shands Hospital

at the University of Florida were recruited in the observational part of the study.

Peripheral endothelial function, AS, and plasma vasoactive balance, lipid peroxidation,

antioxidant enzyme activity, inflammation and nitric oxide (NO) inhibition were

measured before and 8 weeks after HT. HTR were randomly assigned to an exercise









training group (TRAINED; n=9) who performed 12 weeks of supervised endurance

exercise training beginning at 8 weeks post-HT, or to a non-exercise control group

(CONTROL; n=7). All vascular and plasma measurements were performed before and

after 12 weeks.

Brachial artery flow-mediated dilation (+49.5%) and calf (+34.8%) hyperemic

blood flow (BF) improved after HT. Superoxide dismutase activity (SOD), C-reactive

protein (CRP), tumor necrosis factor-alpha (TNF-a), and soluble intercellular adhesion

molecule (sICAM-1) decreased significantly (p<0.05) 17, 23, 63, and 35% after HT,

respectively. In the exercise training study, brachial artery FMD did not change in

TRAINED, however there was a significant decrease in FMD (-29%) in CONTROL after

12 weeks. Peak forearm (+34%) and calf (+17%) hyperemic blood flow increased

significantly in TRAINED, but did not change in CONTROL. Furthermore, there was a

significant increase in TNF-a (+53%) in CONTROL, but no change in TRAINED after

12 weeks.

We found that HT improved peripheral endothelial function and partially reduced

the hyperinflammatory state in end-stage HF subjects. Furthermore, endurance exercise

training attenuated a progressive decline in brachial artery endothelial function, improved

endothelial function of limb resistance vasculature, and attenuated a progressive increase

in TNF-a in HTR.














CHAPTER 1
INTRODUCTION

Heart transplantation (HT) has become a life-extending intervention for patients

with end-stage heart failure (HF). Although HT reverses many of the primary

symptomatic and physiological derangements of the chronic HF syndrome, lifelong

immunosuppressive therapy and cardiac denervation result in de novo physiological and

clinical sequelae that jeopardize long-term survival of the heart-transplant recipient

(HTR). Post-transplant hypertension and coronary artery vasculopathy (CAV) may be

the most frequent complications in HTR surviving greater than one year (Ventura et al.

1997; Davis et al. 1996; Hollenberg et al. 2001), and have been linked to vascular

endothelial dysfunction (VED) and arterial stiffness (Davis et al 1996; Hollenberg et al.

2001; Schofield et al. 2003).

One of the earliest events in the pathophysiology of cardiovascular disease is VED.

VED is associated with traditional risk factors for cardiovascular disease before

manifestations of clinical atherosclerosis, and is a key contributor in the progression of

advanced symptomatic disease. VED refers to a pathological phenotype of the vascular

endothelium including pro-inflammatory and thrombotic properties, enhanced platelet

aggregation, impaired inhibition of vascular smooth-muscle growth, and impaired

endothelial-dependent vasodilation (EDV) (Cai and Harrison 2000). Clinically, impaired

EDV of the coronary (Halcox et al. 2002; Suwaidi et al. 2000) and peripheral (Gokce et

al. 2003; Heitzer et al. 2001) circulation are independent predictors of adverse

cardiovascular events in individuals with (or at risk for) cardiovascular disease.









Physiologically, impaired EDV of coronary arteries may contribute to impaired

myocardial perfusion and myocardial ischemia (Halcox et al. 2002), whereas impaired

EDV of peripheral conduit and resistance arteries may contribute to increased vascular

resistance (Hambrecht et al. 2000), large-conduit artery stiffness (Wilkinson et al. 2004),

and reduced exercise muscle blood flow during exercise (Kao 1994). Thus,

pharmacological or non-pharmacological interventions that preserve endothelial function

should be a major therapeutic goal for individuals at risk for cardiovascular disease.

EDV occurs from the release of endothelial-derived vasodilators including nitric

oxide (NO), prostacyclin (PGI2), and endothelial-derived hyperpolarizing factor (EDHF)

(Mombouli and Vanhoutte 1999). Of the three vasodilators, endothelial-derived NO has

been the most widely studied because impaired synthesis or enhanced degradation of NO

plays a key role in VED. NO is synthesized by a 5-electron oxidation of the amino acid

L-arginine by the endothelial isoform of nitric oxide synthase (eNOS) enzyme. NO

induces EDV via agonist stimulation of a muscarinic receptor on the endothelial

membrane, or by a mechanical shear stress-mediated mechanism from increased laminar

blood flow along the endothelial wall. One mechanism responsible for impaired EDV is

decreased bioavailability of endothelial-derived NO (Vallance and Chan 2001). Cai and

Harrison (2000) hypothesized that reduced bioavailability of endothelial-derived NO

occurs due to several potential mechanisms. First, decreased synthesis of NO may be due

to decreased eNOS gene transcription, increased post-transcriptional degradation of

eNOS mRNA, or post-translational modification eNOS enzyme activity. Moreover,

increased asymmetric dimethylarginine (ADMA), an endogenous intracellular









competitive inhibitor of eNOS, may also contribute to decreased NO synthesis (Tran et

al. 2003).

A second potential mechanism of impaired EDV is increased inactivation of NO by

reactive oxygen species (ROS), such as superoxide anion (Cai and Harrison 2000). In

states of vascular homeostasis, superoxide is rapidly dismutated to hydrogen peroxide

(H202) and water by intracellular Cu/Zn superoxide dismutase (SOD) in the endothelial

cytosol (Cai and Harrison 2000). In the vascular wall, superoxide produced is quickly

dismutated by the primary extracellular isoform of SOD (ecSOD), which is strategically

located on the endothelial membrane between the endothelial and smooth muscle layer

(Fukai et al. 2002). However, when excess superoxide overwhelms the primary

antioxidant defenses, superoxide reacts quickly with NO to generate the potent oxidant,

peroxynitrate (ONOO-), and the metabolite nitrate, both of which have minimal

vasodilating properties (Cai and Harrison 2000). In addition, ROS (such as superoxide

and ONOO-), inactivate dimethylarginine dimethylaminohydrolase (DDAH), the enzyme

that degrades intracellular ADMA, thus increasing ADMA and further inhibiting NO

synthesis (Sydow and Munzel 2003).

A third mechanism that contributes to VED and impaired EDV is increased

vasoconstrictor peptides endothelin (ET-1) and angiotensin II (ANG-II), which oppose

the vasodilator action of NO and promote vasoconstriction (Nickenbig and Harrison

2002). ET-1 is produced by the endothelial cells and other tissues when exposed to

cytokines such as TNF-ac and the immunosuppressive agent cyclosporine (Bunchman and

Brookshire 1991). ANG II is increased in plasma and vascular tissues in diseased









conditions such as acute and chronic HF due to overactivation of the renin-angiotensin

system and sympathetic activity to the kidney (Nickenbig and Harrison 2002).

In HTR receiving immunosuppressive therapy, EDV is impaired in the coronary

arteries early after HT (Fish et al. 1988; Mills et al. 1992; Davis et al. 1996; Hollenberg et

al. 2001), and is an independent predictor of the development of CAV (Davis et al. 1996;

Hollenberg et al. 2001) and cardiac death (Hollenberg et al. 2001). In the peripheral

vasculature, impaired EDV is present in conduit brachial artery of HTR (Patel et al. 2002;

Lim et al. 2003; Saxonhouse et al. 2000), particularly in HTR with antecedant ischemic

HF etiology (Patel et al. 2001). EDV of forearm resistance arteries is improved after HT

in end-stage HF patients (Sinoway et al. 1988; Kubo et al. 1993; Cavero et al. 1994),

however it is currently unknown whether it returns to that of age-matched controls.

Physiologically, impaired EDV contributes to increased vascular resistance (Kao et al.

1994), arterial stiffness (Schofield et al. 2002), decreased exercise capacity (Kao et al.

1994), and decreased exercise muscle blood flow in HTR (Kao et al. 1994). Clinically,

impaired peripheral EDV may partly contribute to the development of de-novo

hypertension after HT, which jeopardizes the long-term success of the cardiac allograft

(Lim et al. 2002; Caveo et al. 1994).

Impaired peripheral EDV may also contribute to increased stiffness (reduced

compliance) of large elastic and muscular conduit arteries (Wilkinson et al. 2004).

Increased arterial stiffness of large elastic and muscular conduit arteries increases the

amplitude of the forward (incident) pressure wave during LV ejection, and increases

pulse wave velocity of reflected pressure waves returning to the ascending aorta. This

change in amplitude and timing of forward and reflected pressure waves causes the









reflected waves to return early to the ascending aorta during systole instead of diastole,

augmenting aortic systolic pressure. Thus, augmented aortic systolic and pulse pressure

due to arterial stiffness of large conduit arteries contributes to increased LV afterload,

myocardial oxygen demand, and wasted energy by the heart (Nichols and Singh 2002).

Large-artery stiffness has been reported in primary aging (Tanaka et al. 2000),

hypertension (Nichols and Singh 2002), renal failure (Laurent et al. 2003), and chronic

HF (Nichols and Pepine 1992; Lage et al. 1994; Mitchell et al. 2001) population. Arterial

stiffness is also an independent predictor of adverse cardiovascular events in patients with

renal failure (Laurent et al. 2003) and coronary artery disease (Weber et al. 2003). To

date, only one cross-sectional study has evaluated arterial stiffness in HTR. Schofield et

al. (2002) reported that that 82% of 53 HTR had an elevated aortic augmentation index,

elevated aortic pulse pressure, and a decreased time of reflected wave (inverse of pulse

wave velocity), compared to age-matched controls, despite normal brachial systolic and

mean blood pressure controlled by antihypertensive medications. Although there are

currently no data on the prognostic implications of increased arterial stiffness in HTR,

Schofield and colleagues show that HTR may be at increased cardiovascular risk despite

having optimally managed brachial blood pressure.

Chronic cyclosporine therapy in HTR results in increased ROS in the vascular wall

(Dietrich et al. 1994) and elevated circulating ET-1 and ANG II (Lerman et al. 1992;

Haas et al. 1993;Grief et al.1993; Perez-Villa et al.2004). ET-1 has been implicated in

the development of post-transplant hypertension and vascular remodeling of smooth

muscle. ANG II is elevated secondary to chronic cardiac denervation (Braith et al. 1996;

Perez-Villa et al 2004) and cyclosporine-induced overactivation of the renin-angiotensin









system (Julien et al. 1993). ANG II is a potent vasoconstrictor and stimulates production

of ROS in the vascular wall via activation of membrane-bound NADPH oxidase. As

such, ANG II contributes to impaired EDV and hypertension in HTR (Nickenbig and

Harrison 2002), and has been implicated in the development of coronary artery

vasculopathy in HTR (Yousufuddinn and Yamani 2004). Thus, interventions that reduce

ANG II and ET-1 levels may have important physiological and clinical benefit in HTR.

Cardiovascular disease has recently gained widespread acceptance as an

inflammatory disease. As such, inflammatory mediators C-reactive protein (CRP) and

soluble intercellular adhesion molecule (sICAM-1) have been reported to be elevated in

HTR (Laberrere et al. 2000) and are strong predictors of coronary artery vasculopathy

(Pethig et al. 2000), allograft failure (Eisenburg et al. 2000), and mortality in HTR

(Laberrere et al. 2002). Besides being a marker of future cardiovascular outcomes in

HTR, in vitro studies have recently confirmed that elevated CRP actively contributes to

VED by activating expression of the endothelial adhesion molecule sICAM-1 (Pasceri et

al. 2000), and decreasing eNOS mRNA, protein, and bioactivity (Venupogal et al. 2002).

Proinflammatory cytokines IL-6 and TNF-ac (known hepatic stimulants of CRP) are also

elevated in HTR and may be involved in VED in HTR (Katz et al. 1994; Holm et al.

2000; Weis et al. 2001). Thus, interventions that attenuate basal levels of inflammatory

mediators in HTR may improve endothelial function.

Rationale for the Study

Alterations in endothelial function in humans were first described by Ludmer et al.

(1986) who observed paradoxical vasoconstriction of the left coronary artery in response

to acetylcholine in patients with CAD. Hambrecht et al. (2000b) reported that lower









body-dynamic endurance-exercise training improved this paradoxical vasoconstriction of

the coronary arteries in response to acetylcholine in patients with CAD, suggesting that

lower body exercise training can alter endothelial function systemically. In addition,

other studies report that lower body exercise training improves peripheral VED in the

upper limbs in individuals with essential hypertension (Higashi et al. 1999), CAD

(Edwards 2004a et al.; Walsh et al. 2003), and chronic HF (Linke et al. 2001). This

supports the theory of a systemic improvement in endothelial function, since lower body

exercise improved upper-body limb vasculature. This theory was further supported by

Anderson et al. (1995) who reported that brachial artery flow-mediated EDV is correlated

with acetlycholine-induced coronary EDV in patients with CAD (r=0.36). Furthermore,

Takase et al. (1998) reported a strong positive correlation between brachial artery flow-

mediated EDV and coronary artery flow-mediated EDV (r=0.79) in CAD patients. Such

studies suggest that endothelial function testing in the peripheral vasculature of the upper

limbs may be a good adjunct method to detect changes in systemic endothelial function in

response to a therapeutic intervention.

As mentioned, impaired coronary artery EDV is a strong predictor of coronary

artery vasculopathy (Davis et al. 1996; Hollenberg et al. 2001) and cardiac death in HTR

(Hollenberg et al. 2001). Therefore, restorating endothelial function (such as with

exercise training) should be a major therapeutic goal to potentially decrease long-term

cardiovascular risk in HTR. However, there have been no prospective reports in the

literature on the modulating effects of an endurance-exercise training intervention on

endothelial function in HTR.









Specific Aims and Hypotheses

Our study is the first prospective, randomized, controlled study to investigate the

effects of endurance exercise training on endothelial function, arterial stiffness, oxidative

stress, and inflammation in HTR. Our study is also the first longitudinal, prospective

study on the effects of HT on endothelial function and arterial stiffness in end-stage HF

patients, and comparing them to an age-matched healthy control group. Thus, our

experiments are novel and will further the understanding of the mechanisms contributing

to VED in HTR.

Specific Aim 1: To measure EDV of limb conduit and resistance arteries, systemic

arterial stiffness, plasma vasoactive balance, oxidative stress, antioxidant enzyme

activity, and endogenous NO inhibition before and 2 months after HT in patients with

end-stage HF, and in an age-matched healthy control group.

Hypothesis 1: Brachial artery flow-mediated dilation (FMD), forearm/calf flow

mediated vasodilation (peak FBF/CBF), aortic augmentation index (Ala), plasma

nitrate/nitrite (NOx), ET-1, 8-iso-prostaglandin-F2a, (PGF2a), ecSOD activity,

inflammatory cytokines, and ADMA will improve 2 months after HT, but will remain

abnormal compared to age-matched healthy controls.

Rationale: Although it is accepted that coronary artery VED exists in HTR (Fish et

al. 1988; Mills et al. 1992), data are conflicting on whether endothelial function of

peripheral limb vasculature improves in end-stage HF patients after HT. Several studies

suggest that endothelial function of brachial artery does not improve after HT in HTR

patients of ischemic HF etiology, and that it does improve in those HTR of non-ischemic

HF etiology (Patel et al. 2001). Other studies report reduced brachial artery FMD in









HTR compared to controls irrespective of etiology of HF (Saxonhouse et al. 2000; Lim et

al. 2002; Schmidt et al. 2002; Cuppoletti et al. 2003). However, there have been no

longitudinal studies of brachial artery FMD before and after HT in the same cohort

compared to age-matched, healthy controls.

Three studies have evaluated endothelial function of forearm resistance arteries

before and after HT (Sinoway et al. 1988; Kubo et al. 1993; Cavero et al. 1994). In a

prospective study before and after HT, Sinoway et al. (1988) reported that forearm EDV

in response to reactive hyperemia did not improve within several weeks of HT, but

improved by 4 months. Kubo et al. (1993) reported that EDV in response to reactive

hyperemia in forearm resistance arteries improved 4 months after HT. Cavero et al.

(1994) reported an increase in forearm EDV 24-36 hours after HT, and then no change 1

week and 6 weeks after starting cyclosporine therapy. However, these studies did not

have an age-matched, healthy control group for comparison, so it is unclear whether

endothelial function in peripheral resistance arteries HTR was restored to normal.

Furthermore, no studies have investigated the effects of HT on systemic arterial stiffness

in end-stage HF patients, or on the potential mechanisms involved in VED such as

oxidative stress, enzymatic antioxidant capacity, NO inhibition, and inflammation.

Specific Aim 2: To measure EDV of limb conduit and resistance arteries, systemic

arterial stiffness, plasma vasoactive balance, oxidative stress, antioxidant enzyme

activity, and endogenous NO inhibition in HTR before, and after 12 weeks of supervised

endurance-exercise training or a 12-week control period.









Hypothesis 2: In HTR, 12 weeks of supervised endurance exercise training will

increase brachial-artery FMD, peak forearm and calf BF, plasma NOx, and ecSOD

activity; and decrease aortic AI, plasma ET-1, 8-iso-PGF2a, and ADMA.

Rationale: Several studies have reported that exercise training improves EDV in

both conduit (Hambrect et al. 1998; Linke et al. 2001) and resistance arteries (Katz et al.

1997) in chronic HF patients, and that the improvement is NO-mediated. In a design

similar to ours, subjects with essential hypertension, Higashi et al. (1999) reported a 22%

increase in peak FBF during reactive hyperemia using plethysmography, after a 12 week

exercise training intervention. The increase in FBF was abolished by the NO inhibitor, L-

NMMA, suggesting that the increase in EDV of forearm resistance arteries was NO

mediated. In HTR, one cross-sectional study has investigated the effects of exercise on

endothelial function. Schmidt et al. (2002) reported higher brachial artery FMD in

exercise-trained HTR than sedentary HTR. No prospective studies have yet reported the

effects of endurance-exercise training on either peripheral-conduit or resistance-artery

endothelial function in HTR.

Cross-sectional studies suggest that exercise capacity is positively associated with

increased arterial compliance of large arteries in healthy older individuals (Vaitkevicius

et al. 1993) and persons with dilated cardiomyopathy (Bonapace et al. 2003). Several

prospective studies report that exercise training decreased arterial stiffness in sedentary

young (Cameron et al. 1994), sedentary aged (Tanaka et al. 2000), individuals with CAD

(Edwards et al. 2004), and chronic HF (Pamell et al. 2002). However, no prospective

studies have reported the effects of endurance-exercise training on arterial stiffness in

HTR.









Endurance-exercise training increased aortic expression of eNOS mRNA and

eNOS protein in both animal (Sessa et al. 1994, Fukai et al. 2000; Woodman et al. 1999)

and human models (Hambrecht et al. 2003). Concominant increases in agonist-mediated

EDV suggest an increased NO synthesis after chronic exercise training. Plasma NOx, the

stable end product of NO metabolism in plasma, also is increases after 8 weeks of

exercise training in healthy humans (Jungersten et al. 1997; Maeda et al. 2001) and in

CAD patients (Edwards et al. 2004a). Together, these data support the hypothesis of

increased systemic NO synthesis after chronic exercise training.

ET-1 is elevated in HTR due to chronic exposure of the endothelium to

cyclosporine and may contribute to hypertension and impaired EDV in HTR (Haas et al.

1993; Greiff et al. 1993). Endurance exercise training reduces reduce plasma ET-1 levels

in young, healthy subjects (Maeda et al. 2001), and in older women (Maeda et al. 2003),

but not in chronic HF patients (Callaerts-Vegh et al. 1998). Plasma ET-1 levels are also

inversely correlated to increased plasma NOx after exercise training in young healthy

subjects, suggesting that NOx has a modulating effect on ET-1 levels (Maeda et al.

2001). However, the effects of endurance exercise training on circulating ET-1 levels in

HTR has not been investigated.

The F2 isoprostane isomer, 8-iso-prostaglandin-F2a (PGF2a), a stable and specific

marker for in vivo oxidative stress-induced lipid peroxidation, can be measured in plasma

or as its metabolite in urine (Roberts and Morrow 2000). Plasma or urinary levels of

8-iso-PGF2a), are elevated in patients with cardiovascular risk factors such as smoking,

hypercholesterolemia, diabetes (Pratico 1999; Patrono and Fitzgerald 1997), coronary

artery disease (Vassalle et al. 2003), and chronic HF (Polidori et al. 2004). Also, 8-iso-









PGF2a has biological vasoconstrictor action (Roberts and Morrow 2000) and is an

independent predictor for development of cardiovascular disease (Schwedhelm et al.

2004). Furthermore, Edwards et al. (2004a) found that 12 weeks of endurance exercise

training reduced plasma levels of 8-iso-PGF2a, and increased plasma NOx and ecSOD

activity in patients with CAD. However, no prospective study has yet reported the effects

of endurance exercise training on plasma levels of 8-iso-PGF2a, NOx, and ecSOD

activity in HTR. Moreover, no human studies have tested the effects of endurance

exercise training on ADMA. We hypothesize that by attenuating production of ROS and

preventing inactivation of DDAH, exercise training may lower ADMA levels and thus

preserve NO synthesis.

Specific Aim 3: To measure plasma levels of inflammatory cytokines in HTR

before and after 12 weeks of supervised endurance-exercise training or 12 week control

period.

Hypothesis 3: In HTR, 12 weeks of supervised endurance-exercise training will

decrease plasma levels of CRP, TNF-c, IL-6, and sICAM-1.

Rationale: In addition to being markers of future cardiovascular risk, experimental

evidence shows that CRP, TNF-c, and sICAM-1 actively contribute to VED (Blake and

Ridker 2003). Several prospective exercise-training studies have tested the effects of

endurance-exercise training on inflammatory mediators in patients with CAD (Milani et

al. 2004; Edwards 2002) and chronic HF (Larsen et al. 2001; Adamopoulos et al. 2003).

Milani et al. (2004) reported a 41% decrease in CRP in a cohort of 277 CAD patients

who completed 12 weeks of exercise training as part of cardiac rehabilitation. Edwards

et al. (2002) reported that 12 weeks of exercise training as part of cardiac rehabilitation






13


(in patients with CAD) lowered CRP by 45% and IL-6 by 32%. In 28 patients with

chronic HF, Larsen et al. (2001) reported a 12.5% decrease in TNF-a but no change in

IL-6. Furthermore, in 24 chronic HF patients, Adamopoulos et al. (2003) reported that 12

weeks of endurance exercise training significantly lowered IL-6, TNF-a, sVCAM-1 and

sICAM-1. However, no prospective studies have tested the effects of endurance-exercise

training on inflammatory mediators in HTR.














CHAPTER 2
REVIEW OF LITERATURE

Before the 1980s, the endothelial layer of the vasculature was believed to be a

physiological inert layer of epithelial cells acting as a barrier between the blood and

medial layer of the vascular wall. This changed with Furchgott and Zawadzki's (1980)

discovery that vasorelaxation of vascular smooth muscle cells in response to

acetylcholine is dependent on an endothelial-derived relaxing factor released from

endothelial cells. They reported that if the endothelial layer was removed from rabbit

aorta, the vessel vasoconstricted in response to acetylcholine, but its vasodilatory

response to nitrates was preserved. In the late 1980s, this endothelial-relaxing compound

was discovered to be the free radical gas, NO (Palmer et al. 1987; Ignarro et al. 1987).

Furthermore, over the last decade it has been discovered that the endothelial layer is not

inactive, but is intimately involved in regulating vascular tone and homeostasis.

NO has proven to be a critical component of vascular health such that the decreased

bioavailability of NO results in the phenomenon of ED (Cai and Harrison 2000). ED is

present in individuals with primary cardiovascular risk factors such as

hypercholesterolemia, hypertension, diabetes, and obesity; and with primary aging and in

those with documented cardiovascular disease (Drexler 1997). As mentioned, VED

develops years before clinical evidence of atherosclerosis develops and remains evident

in individuals with occult cardiovascular disease (Drexler 1997). In particular, VED is

evident in individuals with both ischemic and non-ischemic HF (Kubo et al. 1991; Patel

et al. 2001), and is believed to partly contribute to increased peripheral vascular









resistance (Hambrecht et al. 2000), arterial stiffness (Arnold et al. 1991), and impaired

muscle-blood flow and exercise capacity in HF patients (Hambrecht et al. 1998; Linke et

al. 2001). Furthermore, both coronary and peripheral ED persists in end-stage HF

patients who undergo orthotopic HT (Fish et al. 1988; Mills et al. 1992; Patel et al. 2001;

Schmidt et al. 2003), however, the mechanisms have not been elucidated.

Normal Endothelial Function

Nitric Oxide

Endothelial-derived NO is synthesized from the amino acid L-arginine which

undergoes a five-electron oxidation to NO and L-citrulline by the endothelial isoform of

the nitric oxide synthase (eNOS) enzyme (Moncada and Higgs 1993). Since NO has a

short biological half-life (3-10 seconds) at physiological pH, and is rapidly oxidized to

nitrate (NO2-) and then nitrite (NO3-) by oxygenated hemoglobin (Moncada and Higgs

1993), the primary biological signaling activity of endothelial NO occurs a short

diffusion distance across the endothelial wall into the smooth muscle layer. NO binds to

the heme moiety of the enzyme guanylate cyclase activating it to catalyze the conversion

of GTP to the second messenger cyclic guanosine 3', 5-monophosphate (cGMP). cGMP

mediates vascular smooth muscle relaxation via increase Ca+2 extrusion from the smooth

muscle cells (Moncada and Higgs 1993).

Nitric Oxide Synthase

There are three isoforms of nitric oxide synthase (NOS): constitutively expressed

neuronal NOS (nNOS) and endothelial NOS (eNOS), and the inducible NOS isoform

(iNOS). nNOS and eNOS activation are Ca+2-dependent and are located in neurons and

vascular endothelial cells, respectively (Mayer and Hemmens 1997). Endothelial cells

constitutively express eNOS which is an NADPH-dependent oxygenase that requires the









cofactors tetrahydrobiopterin (BH4), FAD, and FMN (Mayer and Hemmens 1997). In

endothelial cells, eNOS, is located in special invaginations in the cell membrane called

caveolae, and is associated with a specialized protein, caveolin, which interacts with

signaling proteins, such as eNOS, and inhibits its activity (Feron et al. 1998). Stimulation

of endothelial cells by agonists such as acetylcholine or bradykinin, dissociates the

caveolin/NOS complex and allows Ca+2/calmdulin complex to bind to NOS and activate

NO synthesis (Feron et al. 1998). This compartmentalization of eNOS allows rapid

conversion of L-arginine to NO since the y+ transporter for L-arginine is also located in

the cell membrane near the caveolae (Harrison 1997).

Mechanism of NO Release

Basal release of NO

There is a continuous basal release of NO from vascular endothelium to maintain

resting vascular tone. The first evidence of this was in 1989 by Vallance et al. (1989)

who demonstrated that by infusing an inhibitor of eNOS, NG-monomethyl-L-arginine (L-

NMMA), into the brachial artery of the human forearm, there was a dose-dependent

reduction in resting blood flow. L-NMMA is a methylated analogue of L-arginine which

prevents the synthesis of NO and when systemically infused into experimental animals

and humans (Vallance et al. 1989), results in an increase in mean arterial pressure. Thus,

these data demonstrate the importance of NO in maintaining tonic peripheral arterial

vasodilation and blood pressure in vivo.

Agonist-mediated release of NO

Several substances can stimulate muscarinic receptors on the endothelial

membrane and activate eNOS to increase NO synthesis via a Ca+2-dependent mechanism.

In particular, acetylcholine, bradykinin, and substance P, can stimulate EDV of resistance









and conduit arteries in humans, which can be partially inhibited by L-NMMA. In

addition, other endothelial vasodilators, such as prostacyclin and EDHF may also be

involved in EDV but to a lesser extent.

Shear stress-mediated release of NO

Laminar pulsatile flow of blood along the endothelial wall causes a mechanical

shear stress which provides the stimulus for both short-term and long-term regulation of

eNOS and NO synthesis. In vitro studies suggest that specific potassium ion channels

respond immediately to increase shear stress and induce increase intracellular calcium

within one minute and increase short-term eNOS activity and NO synthesis (Cooke et al.

1991). After one hour exposure of endothelial cells to increased shear stress, serine-

threonine protein kinase B (Akt) phosphorylates eNOS activating the enzyme and

increasing NO synthesis six-fold independent of increase in intracellular calcium

(Dimmeler et al. 1999). Prolonged exposure of increased shear stress for 24 hours,

induces increased eNOS mRNA expression in a dose dependent manner in bovine and

human endothelial cells (Uematso et al. 1995), and in isolated soleus feed arteries from

rats exposed to increased luminal shear stress (Woodman et al. 2004). Furthermore,

human studies suggest that flow-mediated dilation of forearm brachial (Lieberman et al.

1996) and resistance arteries (Meredith et al. 1996) is partially attenuated by the eNOS

inhibitor, L-NMMA, suggesting that shear-stress mediated vasodilation is NO dependent.

Pleiotropic Actions of NO

Endothelial-derived NO not only modulates vascular tone, but also has

antiatherosclerotic, antithrombotic, and anti-inflammatory functions on the endothelial

wall (Vallance and Chan 2001). Specifically, NO suppresses platelet aggregation,

leukocyte migration and adhesion to the endothelial wall, and prevents vascular smooth









muscle migration and proliferation into the subendothelial space (Mombouli and

Vanhoutte 1999). Thus, the decrease in bioavailability of NO not only promotes an

endothelial phenotype of vasoconstriction, but also of platelet aggregation, leukocyte

migration and adhesion to the endothelial layer, and smooth muscle migration and

proliferation into the subendothelial layer (Mombouli and Vanhoutte 1999).

Other Endothelial Vasodilators

The endothelium also was discovered to release several other vasodilating

compounds including prostacyclin and an unknown endothelial derived hyperpolarizing

factor (EDHF). The production of prostaglandins is regulated by the availability of

membrane-bound arachadonic acid (AA) and the activity of the enzyme cyclooxygenase

(COX). AA is derived from the phospholipid membrane which is enzymatically

released via action of the enzyme phospholipase A2 and is converted to prostaglandin H2

by COX and peroxidase (Savidge 2001). Prostacyclin is the major endothelial metabolite

derived from arachadonic acid (AA) and diffuses into vascular smooth muscle and

activates the enzyme adneylate cyclase. Adenylate cyclase converts ATP to 3', 5 cyclic

adenosine monophosphate (cAMP) and induces vasorelaxation of vascular smooth

muscle (Savidge 2001).

Endothelial hyperpolarizing factor (EDHF) is less well characterized but is believed

to act through activation of calcium-activated K+ channels on the smooth muscle

membrane resulting in hyperpolarization and vasorelaxation (Mombouli and Vanhoutte

1999; Triggle et al. 2004). Although identification of EDHF is still unclear, possible

EDHF's include hydrogen peroxide, isoprostanes, potassium, or the AA metabolite,

epoxyeicosatrienoic acid (Triggle et al. 2004)









Endothelial Vasoconstrictors

The endothelial wall also secretes vasoconstrictors ET-1, ANG II, and

vasoconstrictor prostaglandins which compete with NO for the vasoactive balance.

Endothelin-1

ET-1, the major endothelin isoform, is produced by the endothelium and is derived

from the precursor big ET-1 by the enzyme endothelin converting enzyme (ECE). ET-1

can act in a paracrine or autocrine manner via ET type A or ET type B receptors on

adjacent endothelial and smooth muscle cells. ETA and ETB receptors are on smooth

muscle cells and both mediate vasoconstriction, cell proliferation, and hypertrophy. ETB

exist on endothelial cells as well, and mediate vasodilation via release of NO and

prostacyclin (Taddei et al. 2001). Although ET-1 is generally considered to be a potent

vasoconstrictor, this activity can be attenuated by the increased expression or activity of

ETB receptor mediated release of NO by the endothelium (Taddei et al. 2001). However,

systemic infusion of ET-1 into animals results in a decrease in glomerlular filtration rate,

renal blood flow, and an increase in mean arterial blood pressure (Goetz et al. 1988). In

humans, infusion of an ET-1 receptor antagonist significantly decreased peripheral

vascular resistance and blood pressure (Haynes et al. 1996). Taken together, these data

support the idea that ET-1's vasoconstrictor activity on the smooth muscle predominate

and strongly contributes to basal vascular tone and blood pressure.

Angiotensin II

ANG II is a strong vasoconstrictor peptide that also has direct and indirect salt and

water regulatory actions on the kidney (Nickenig and Harrison 2002). ANG II is formed

in the circulation when angiotensinogen production is increased via increased adrenergic

stimulation release of renin from juxtaglomerular cells in the afferent arteriole of the









kidney. Angiotensinogen is converted to angiotensin I, which is quickly converted to

ANG II by angiotensin converting enzyme (ACE). Besides being a potent

vasoconstrictor, ANG II stimulates kidney tubule absorption of salt and water directly, as

well as indirectly via stimulation of aldosterone from the adrenal cortex. In addition, the

endothelial and smooth muscle wall contain a vascular form of ACE which is responsible

for production of ANG II in the vascular wall (Nickenig and Harrison 2002).

Furthermore, ANG II is major stimulus of NADPH oxidase production of superoxide

anion in the vascular wall (Nickenig and Harrison 2002; Cai and Harrison 2000), thus, in

conditions of elevated levels of ANG II such as chronic HF, ANG II may be partially

responsible for increased vascular oxidative stress.

Vasoconstrictor prostaglandins

As mentioned earlier, the production of prostaglandins is regulated by the

availability of AA and the activity of the enzyme COX (Savidge 2001). Under certain

pathophysiological conditions, increased thromboxane A2 is formed from its precursor

prostaglandin H2, both of which bind to endoperoxide/thromboxane receptors on vascular

smooth muscle and induce vasoconstriction (Mombouli and Vanhoutte 1999). However,

in states of vascular homeostasis, NO, EDHF, and prostacyclin, override any influence of

endothelial vasoconstrictors, promote vasodilation and an antithrombotic phenotype of

the endothelium. (Mombouliand Vanohoutte 1999). However, when the endothelial-NO

pathway is disrupted, the vasoconstrictor, pro-thromobotic, and proinflammatory

phenotype of the endothelium prevails.

Vascular Endothelial Dysfunction

Several mechanisms have been implicated in VED including: 1) decreased NO

synthesis due to decreased expression or activity of nitric oxide synthase (eNOS); 2)









decreased NO synthesis due to increased competitive inhibition by ADMA; 3) post-

translational inactivation of NO by ROS such as superoxide radical; and 4) enhanced

production of vasoconstrictor substances such as ET-1, ANG II, and vasoconstricting

prostaglandins, which oppose vasodilatory effects of NO.

Decreased NO Synthesis by eNOS

Decreased synthesis of NO can occur via several distinct mechanisms.

Pathophysiological factors such as TNF-ca, hypoxia, oxidized LDL (Harrison 1999), and

reduced blood flow and shear-stress in heart failure (Smith et al. 1996), have been shown

to decrease eNOS levels through both transcriptional regulation and post-transcriptional

modifications in half-life of eNOS mRNA (Harrison 1999). In contrast, shear stress

increases gene transcription of eNOS mostly by transcriptional regulation (Uematsu et al.

1995; Harrison 1999). Thus, although eNOS is constitutively expressed, eNOS

undergoes various degrees of expression under different physiological and

pathophysiological conditions (Harrison 1999).

NO synthesis can be also can be decreased when eNOS becomes uncoupled due to

reduction of the essential eNOS cofactor, BH4 (Cai and Harrison 2000). Decreased BH4

results in eNOS transferring electrons to molecular oxygen instead of L-arginine,

resulting in increased production of superoxide radical. As such, pathophysiological

conditions such as insulin resistance, cigarette smoking, and hypercholesterolemia can

cause impaired EDV due to BH4 depletion, and supplementation with BH4 restores EDV

in these clinical conditions (Vallance and Chan 2001).

Lastly, ADMA, an endogenous inhibitor of eNOS, can also decrease NO synthesis.

ADMA is derived from methylation of the side chain nitrogen of arginine residues in









nuclear proteins by enzymes called protein-arginine methyltransferases (PRMTs) (Tran et

al. 2003). Specific PRMTs methylate L-arginine residues on intracellular RNA binding

proteins in endothelial cells, which are released into the cytoplasm upon normal cellular

protein turnover (Tran et al. 2003). DDAH, the enzyme responsible for degradation of

ADMA into L-citrulline and dimethylamine (Tran et al. 2003), is responsible for 85-90%

of ADMA degradation with only a small amount of ADMA excreted in the urine (Tran et

al. 2003). As such, impairment of this DDAH may be a key mechanism for ADMA

accumulation under certain pathophysiological conditions (Tran et al. 2003).

Vallance et al. (1992) first described the in vivo effects of ADMA on EDV in

healthy humans. They infused ADMA into brachial artery of healthy subjects and caused

a dose-dependent vasoconstriction that was completely reversed with infusion of L-

arginine. In the same study, ADMA was reported to be elevated in chronic renal failure

patients which was attributed to reduced renal excretion. However, ADMA has been

reported to be elevated in clinical populations with normal renal function including,

CAD, peripheral artery disease (PAD), hyperhomocysteinemia, hypercholesterolemia,

primary aging (Tran et al. 2003), chronic HF (Usui et al. 1998), and HT (Fearon et al.

2004), suggesting that elevated ADMA must be due to some other mechanism than poor

renal function. As such, in vitro studies report that DDAH contains a reactive cysteine

residue in the active site which can be reversibly inhibited by s-nitrosylation by NO-

derived oxidants or via oxidation by superoxide radical (Sydow and Munzel 2003).

Additionally, there is some evidence that suggests that ADMA may be a direct mediator

of oxidative stress by causing uncoupling of eNOS from oxidation of the eNOS cofactor,

BH4 (Sydow and Munzel 2003). Hence, ROS may contribute to decreased NO









bioavailibility indirectly by inhibiting DDAH activity, thus promoting the accumulation

of ADMA, or directly, by enhanced degradation of synthesized NO (Sydow and Munzel

2003).

NO Degradation by Reactive Oxygen Species

Vascular redox homeostasis involving superoxide levels are normally controlled by

intracellular superoxide dismutase (SOD) isoforms copper/zinc (Cu/Zn) SOD and

manganese (Mn) SOD; and the extracellular isoform of SOD (ecSOD), which is located

on the endothelial membrane and in extracellular space between endothelial and smooth

muscle cells (Fukai et al. 2002). However, during states of increased production of

superoxide, excess superoxide reacts rapidly with NO because this reaction has a rate

constant three times faster than with SOD. This results in loss of bioactivity and forming

of peroxynitrite anion (ONOO-) which is a potent oxidant and has minimal vasodilating

properties (Cai and Harrison 2000).

There are several potential sources of superoxide production in the vascular wall.

Membrane-bound NADH/NADPH oxidase is postulated to be the major source of ROS

in endothelial and smooth muscle vasculature (Fukai et al. 2002; Cai and Harrison 2000).

NADH/NADPH oxidase uses NADH and NADPH as substrates for electron transfer to

molecular oxygen. Experimental evidence shows that NADH/NAPDH oxidase activation

can occur via stimulation by ANG II and TNF-ca, both of which are elevated in HF and

HTR's. In vitro studies show that ANG II is a primary stimulus of NADH/NADPH

oxidase activity, and in vivo studies report that chronic infusion of ANG II in rats results

in increased superoxide production and impaired EDV (Harrison 1999).









Another potential enzymatic source of superoxide production in the vascular wall is

xanthine oxidase, which catalyzes conversion of hypoxanthine to xanthine in endothelial

and smooth muscle cells (Cai and Harrison 2000). Xanthine oxidase is synthesized as

xanthine dehydrogenase and under normal conditions uses NAD+ as an electron acceptor.

However, under pathophysiological conditions such as exposure to TNF-ac or ONOO- in

endothelial cells, xanthine dehydrogenase is converted to xanthine oxidase (Landmesser

et al. 2002). Thus, increased xanthine oxidase activity transfers electrons to molecular

oxygen instead of NAD+, resulting in excess superoxide production and impaired EDV

(Harrison 1999). Moreover, supplementation with the xanthine oxidase inhibitor,

allopurinol, results in improved EDV in smokers (Guthikonda et al. 2003), diabetics

(Butler et al. 2000), and chronic HF patients (Farquharson et al. 2002), suggesting that

xanthine oxidase contributes at least, in part, to impaired EDV in these conditions.

Vascular Endothelial Dysfunction before Heart Transplantation

Impaired EDV of peripheral conduit and resistance arteries exists before HT in

chronic HF patients (Kubo et al. 1991; Katz et al. 1992; Homig et al. 1996; Hambrecht et

al. 1998; Linke et al. 2001). Kubo et al. (1991) was the first to demonstrate that EDV of

forearm resistance vasculature was attenuated in patients with chronic HF in response to

a muscarinic agonist, methacholine, compared to healthy controls. The mean increase in

forearm blood flow to three dosages of methylcholine using strain gauge

plethysmography was significantly attenuated in HF patients. Katz et al (1992) then

demonstrated that endothelium-dependent blood flow velocity of conduit femoral artery

was attenuated in HF patients compared to healthy controls in response to infusion of









acetylcholine. Taken together, these early studies confirmed that EDV was impaired in

both resistance and conduit arteries of HF patients.

The mechanisms for impaired EDV in HF likely include several pathophysiological

mechanisms. First, Smith et al. (1996) reported a significant decreased expression of

eNOS and COX-1 mRNA and a 70% reduction in eNOS protein in aortas of dogs after 1

month left ventricular pacing-induced HF suggesting regulation at the transcriptional or

post-transcriptional level. Second, HF is associated with increased levels of circulating

TNF-a (Levine et al. 1990) which, in vitro, post-transcriptionally degrades eNOS mRNA

(Yoshizumi et al. 1993). In vivo evidence to support this, Katz et al. (1994) found that

elevated TNF-a levels to be highly correlated with impaired forearm EDV in response to

acetylcholine. Thirdly, in humans, administration of vitamin C, a known scavenger of

superoxide anion radical, reverses impaired radial artery EDV in HF, suggesting that

superoxide plays a significant role in endothelial dysfunction in HF patients (Hornig et al.

1999). Excess superoxide production may be due to hyperactivity of the RAAS in HF,

which results in increased levels of ANG II via the enzyme angiotensin-converting

enzyme (ACE) (Nickenbig and Harrison 2002). However, not only does ANG II and

TNF-a stimulate NADPH oxidase production of superoxide anion, but vascular ACE

also degrades bradykinin, which stimulates release of NO and endothelial-derived

hyperpolarizing factor (EDHF) from the endothelium (Drexler 1997). TNF-a also

upregulates ET-1 production, which is also elevated in HF, and therefore competes with

endogenous vasodilators and promote systemic vasoconstriction and impairesd EDV.

Indeed, ETA receptor blockade improves EDV of the brachial artery in chronic HF

patients (Berger et al. 2001). Taken together, decreased eNOS and COX-1 gene









expression, increased ANG II-stimulated superoxide degradation of NO, and increased

vasoconstrictors ET-1 and ANG II likely all contribute to impaired EDV in chronic HF.

ADMA levels are also elevated in chronic HF patients (Usui et al. 1998). Usui et

al. (1998) found that chronic HF patients had elevated plasma levels of ADMA and NOx

compared to healthy controls. In addition, ADMA and NOx were significantly associated

with NYHA functional HF class and NOx inversely associated with ejection fraction (r=-

0.33, p=0.004) (Usui et al. 1998). Moreover, there was a significant positive relationship

between plasma ADMA and NOx in only the moderate and severe HF patients (r=0.41,

p=0.01). Thus, the authors suggested that increased NOx may be due to inflammatory

cytokine-induced excessive NO production in severe HF, which may have negative

inotropic effects on the myocardium. Therefore, increased ADMA may be a

compensatory mechanism against hyperactive systemic or myocardial NO synthase

activity and NO production.

Mechanisms for increased ADMA in HF are unclear, but may include decreased

renal excretion of ADMA due to renal failure, since renal plasma flow and excretion

decline as HF progresses. However, Usui et al. (1998) excluded all HF patients with

renal dysfunction making this hypothesis unlikely. Finally, as mentioned earlier, it is

postulated that increased ROS in endothelial cells in severe HF may contribute to

increased ADMA by oxidatively inactivating DDAH, the enzyme that degrades

intracellular ADMA. However, the effects of ROS on ADMA in HF patients has not

been investigated.

Vascular Endothelial Dysfunction after Heart Transplantation

Fish et al. (1988) were the first to demonstrate that HTR had impaired coronary

EDV early after HT. They demonstrated paradoxical vasoconstriction to acetylcholine in









12 of 13 HTR who were 12 months post-HT. Using intravascular ultrasound, Mills et al.

(1992) also described vasoconstriction of conduit coronary arteries in response to

acetylcholine in HTR who had no evidence of coronary vasculopathy one year after HT.

Davis et al. (1996) reported that impaired coronary artery EDV predicts development of

coronary allograft arteriosclerosis by one year. Furthermore, Hollenburg et al. (2002) not

only reported that impaired coronary EDV is an independent predictor of development of

coronary allograft arteriosclerosis but also of cardiac death in HTR. Thus, coronary ED

develops early after HT, and provides valuable prognostic information on long-term risk

of the cardiac allograft in HTR.

In contrast, similar prognostic data using peripheral endothelial function testing in

HTR is not available at this time. However, Anderson et al. (1995) reported that

peripheral endothelial function and coronary endothelial function correlate, albeit

modestly, and therefore may provide some valuable clinical information in HTR if

coronary endothelial function testing is not available. As such, peripheral endothelial

dysfunction is present after HT and persists indefinitely (Saxonhouse et al. 2000; Patel et

al. 2001; Lim et al. 2002; Schmidt et al. 2002; Cuppoletti et al. 2003). Several cross-

sectional studies report that brachial artery FMD is impaired in HTR compared to healthy

controls (Saxonhouse et al. 2000; Patel et al. 2001; Lim et al. 2002; Schmidt et al. 2002;

Cuppoletti et al. 2003). Saxonhouse et al. (2000) reported that brachial artery FMD in

HTR one to seven years post-transplant, was similar to stable class IV HF patients, and

was decreased compared to age-matched healthy controls. Patel et al. (2001) compared

brachial FMD in a group of ischemic vs. non-ischemic HF patients, to two groups of

HTR with antecedent ischemic and non-ischemic HF etiology. Ischemic and non-









ischemic HF patients did not differ in ejection fraction, duration of heart failure illness,

total cholesterol, or ACE inhibitor use. Brachial artery FMD was the same in both HF

groups (3.6% vs. 5.1%, p=NS), but significantly less than controls (13.9%, p<0.001).

Ischemic vs. non-ischemic HTR did not differ in time since transplant, duration of pre-

transplant heart failure, ejection fraction, cyclosporine levels, lipid lowering therapy, and

cardiac risk factors. HTR with ischemic HF etiology had significantly decreased brachial

FMD compared to non-ischemic HTR (5.5% vs. 13.0%, p=0.002). FMD in non-ischemic

HTR did not differ from healthy, age-matched controls (13.0% vs. 13.9%, p=NS). Thus,

this data suggests that EDV of conduit brachial artery is restored after HT in non-

ischemic HTR, but not in HTR with ischemic etiology. Although this was not a

prospective follow-up of the same patients before and after transplant, this study

illustrates that etiology of heart failure can influence endothelial function after HT.

Lim et al. (2002) reported reduced brachial artery FMD inl4 young HTR (mean

age 18 years) with non-ischemic HF, etiology compared to age- and gender-matched

healthy controls (3.0% vs. 15.5%, p<0.05). Interestingly, there was no relationship in

HTR between impairment in brachial artery FMD and gender, time since transplantation,

number of rejection episodes, cyclosporine levels, or presence of hypertension. Thus,

this suggests that other factors may contribute to impairment of brachial FMD, however,

the small size of the study limits the generalizability of the results.

Schmidt et al. (2002) reported a reduced brachial FMD in sedentary HTR (age 60 +

6 years) six years post-transplant compared to age-matched sedentary, healthy control

group (1.4% vs. 8.4%, p<0.05). Lastly, Cuppoletti et al. (2003) measured brachial FMD

at one and six months after heart transplant in the same 12 HTR. They reported that 10









of HTR (83%) had a brachial FMD < 4% at one month (0.4% vs. 9.9%, p=0.01), and at

six months, brachial FMD remained < 4% in all 10 HTR and as well as the remaining

two HTR suggesting that VED persists early after HT.

In a longitudinal study, Sinoway et al. (1988) measured EDV of resistance arteries

of forearm using strain-gauge plethysmography during reactive hyperemia following 5

minutes of upper arm occlusion with a blood pressure cuff. They measured EDV before,

18 days after HT, and four months after HT in 10 HTR who had severe HF. Basal

forearm blood flow did not significantly increase immediately after transplantation, but

increased four months after-transplant. Similarly, peak hyperemic forearm blood flow

following arterial occlusion did not increase immediately after transplant, but increased

significantly at four months. This suggests that impaired forearm blood flow is not

directly related to normalizing cardiac output, but that it is increased after several months

probably as a result of resumption of daily physical activities. However, because there

was no age- and weight-matched healthy group, it was unknown whether the four month

peak blood flow was completely normalized.

In a cross-sectional study and longitudinal design, Kubo and associates (1993)

investigated EDV of forearm resistance arteries using measurement of forearm blood

flow by strain gauge plethysmography. In the cross-sectional design, forearm blood flow

was measured during infusion of the muscarinic agonist metacholine in three doses in a

group of HF and HTR. Forearm blood flow at three doses of methacholine was higher in

HTR than CHF patients. In addition, they measured EDV in the forearm during reactive

hyperemia in both groups as well. Reactive hyperemia forearm blood flow following

upper arm occlusion was not statistically different in HTR than HF patients. This data









suggests that the EDV following brief ischemia of forearm resistance vasculature is

similar, but agonist-mediated vasodilation is impaired in HF compared to HTR.

Furthermore, endothelium-independent vasodilation (EIV) using an NO donor,

nitroprusside, showed that smooth muscle vasculature was not different between groups.

In the longitudinal design, forearm blood flow was measured after methacholine

and reactive hyperemia in the same six patients before and 4 months after HT. Patient

characteristics before and after HT had similar resting forearm blood flow (3.3 1.1 vs.

3.7 1.5 ml/min/100 ml) and forearm vascular resistance (30.5 11 vs. 37 16 U).

After HT, HTR had higher blood pressure, cholesterol, and cyclosporine levels, but a

lower pulmonary wedge pressure and norepinphrine. Forearm blood flow increased

significantly at four months after HTX at each doses of methacholine. Also, reactive

hyperemia forearm blood flow increased significantly after HTX from 19.0 3.7 to 44.8+

6.4 ml/min/100 ml. This data suggests that both agonist-mediated methacholinee) and

flow-mediated (reactive hyperemia) forearm vasodilation is increased after HT.

However, subjects did not have nitroprusside EIV mediated forearm blood flow

measured, so it is not known if this increase in blood flow after transplant was partially

mediated by improvement in forearm vascular smooth muscle. Furthermore, it is not

known whether this increase in blood flow is normalized since there was no age-matched

healthy control group. The discordant results in forearm EDV to reactive hyperemia may

be due to the inherent limitations in cross-sectional study and small number of subjects

studied.

In a longitudinal study, Cavero et al. (1994) investigated the effects cyclosporine

on peripheral vascular EDV in end-stage HF patients before and after HT. Peak FBF









during reactive hyperemia was measured in HF patients 1) before HT; 2) 24-36 hours

after HT but before initiation of cyclosporine therapy; 3) 6-8 days after HT in presence of

therapeutic cyclosporine levels; and 4) 4-6 weeks post-transplantation on cyclosporine.

Forearm blood flow to reactive hyperemia after 10 minutes of upper arm cuff occlusion,

increased significantly after HT (11.2 vs. 21.2 ml/min/100 ml, p<0.05), but did not

change significantly after 6-8 days (22.3 ml/min/100ml), or after 6 weeks (22.7

ml/min/100ml) on cyclosporine therapy. Taken together, this suggests that HT results in

an immediate increase in peak forearm blood flow during reactive hyperemia which does

not change in the ensuing several weeks. However, there was no age-matched healthy

control group to compare to so it is unknown if peak FBF is completely normalized.

Cyclosporine and vascular endothelial dysfunction in heart transplant recipients

Cultured endothelial cells exposed to cyclosporine increase expression of eNOS

mRNA (Navarro-Antolin et al. 2000), but also increase production of ET-1 (Bunchman et

al. 1991) and superoxide anion levels (Navarro-Antolin et al. 2001). Diederich et al.

(1994) reported that pretreatment of mesenteric arteries with SOD, normalized

acetylcholine-induced impaired vasodilation in cyclosporine treated rats, suggesting that

superoxide was a contributing mechanism for impaired NO mediated vasodilation.

Sudhir et al. (1994) showed attenuated acetylcholine-induced vasodilation of epicardial

and conductance coronary arteries of dogs treated with cyclosporine. Vasoconstriction

induced with L-NAME (NO inhibitor) was exacerbated by cyclosporine suggesting that

cyclosporine attenuates release of, or increases degradation of, NO in coronary arteries.

Furthermore, human studies show that cyclosporine is associated with increased

production of ET-1 (Grief et al. 1993; Lerman et al. 1992), and sympathetic nervous

hyperactivity and hypertension in HTR (Scherrer et al. 1990). Thus, taken together these









data suggest that despite increased eNOS expression in endothelial cells, cyclosporine

therapy may partly contribute to impaired EDV via increased superoxide anion release,

ET-1 production, and increased sympathetic hyperactivity in HTR.

Inflammation and vascular endothelial dysfunction in heart transplant recipients

CRP is an acute phase protein released by liver cells in response to inflammatory

cytokines IL-6 and TNF-c. CRP is a strong predictor of cardiovascular events in

previously healthy men and women, and in individuals with existing cardiovascular

disease (Blake and Ridker 2003). However, recently inflammatory proteins have been

suggested as a contributing mechanism to VED by in vitro studies which suggest that

CRP is directly involved in the development of VED (Pasceri et al. 2000; Venupogal et

al. 2002). Venupogal et al. (2002) demonstrated that endothelial cells incubated with

CRP decrease expression of eNOS mRNA, eNOS protein, and eNOS bioactivity, and

increase expression of vascular adhesion molecules VCAM-1, ICAM-1, and P-selectin

(Pasceri et al. 2000). Decreased NO and increased ROS also activate expression of

endothelial VCAM-1 and sICAM-1 which initiates an inflammatory response and

activate T-lymphocytes, monocytes, and macrophages into the endothelial wall to release

proinflammatory cytokines such as IL-6 and TNF-a (Blake and Ridker 2003).

Furthermore, Fichtlscherer et al. (2000) reported that elevated CRP correlated inversely

with impaired EDV in a cohort of CAD patients, and a reduction in CRP after 3 months

was associated with improved EDV.

In HTR, CRP and has been reported to be a strong independent predictor of

coronary artery vasculopathy, cardiac allograft failure, and mortality (Pethig et al. 2000;

Eisenberg et al. 2000; Labarrare et al. 2002). Holm et al. (2001) found that elevated









levels of IL-6 and TNF-a correlated negatively with acetylcholine-induced peripheral

EDV. Moreover, Weis et al. (2001) reported that treatment with simvastatin lowered IL-

6 and TNF-a and was associated with improved acetylcholine-induced coronary EDV.

Taken together, these in vitro and clinical data suggest that CRP may not solely a marker

of future cardiovascular risk in HTR, but may be intimately involved in the development

of VED.

Asymmetric dimethylarginine and vascular endothelial dysfunction in heart
transplant recipients

Lastly, recent accumulating evidence suggests that a contributing mechanism for

VED in HTR may due to elevated intracellular and plasma ADMA. A recent study

reported a 200% increase in plasma ADMA levels in HTR compared to healthy controls

(Weis et al. 2004) and was slightly more elevated in CMV-positive HTR's (Weis et al.

2004). In addition, in vitro, human endothelial cells infected with CMV, had decreased

DDAH activity and produced more ADMA (Weis et al. 2004). Furthermore, a recent

study demonstrated that the degree of impaired EDV of coronary arteries of HTR was

more profound in those with elevated ADMA levels (Fearon et al. 2004). Therefore,

ADMA may also be a key contributor of decreased NO bioavailability and impaired EDV

in HTR, particularly in those who are CMV-positive.

Arterial Stiffness

The arterial system can be divided into three anatomic regions with distinct

physiological functions. Large elastic arteries of the central circulation, such as the aorta

and carotids, act as a bufferingg" or "cushioning" function to absorb pressure and flow

pulsations from LV ejection. This "Windkessel" effect, allows blood (and potential

energy) to be stored in the large arteries during systole, and then expelled to the









peripheral circulation during diastole (Nichols and Singh 2002). This allows for

continuous blood flow in the capillaries throughout the cardiac cycle, and dampening of

pressure oscillations from intermittent ejection from the LV (Safar et al. 2003). Second,

large muscular conduit arteries, such as femoral, brachial, or radial, have a thicker layer

of smooth muscle (Wilkinson et al. 2004) and are about twice as long as the elastic

arteries (Nichols and Singh 2002). The muscular conduit arteries can alter smooth

muscle tone and therefore, modify the speed of forward and reflected pressure waves,

also known as pulse wave velocity. Third, the small arterioles, or resistance vessels,

control blood flow into tissues and can affect mean arterial pressure by altering their

diameter. Additionally, the state of arteriolar tone can affect the distance of reflecting

sites, whereby increased tone can result in reflecting sites "closer" to the ascending aorta

and an early return of reflected waves from the periphery to the heart (Nichols and Singh

2002).

Arterial compliance (inverse of stiffness) is a function of structural elements of the

vessel wall (elastin/collagen/smooth muscle) and distending pressure or mean arterial

pressure (Wilkinson et al. 2004). During aging, decreased arterial compliance (increased

arterial stiffness) in large elastic central arteries can occur due to 'passive" alterations in

elastin/collagen matrix resulting in the inability to absorb pulsations from LV ejection

and increased pulse wave velocity of forward and reflected traveling pressure waves.

Increased arterial stiffness in the central elastic arteries results in increased central

systolic pressure and pulse pressure because they are dependent on LV stroke volume and

compliance of the proximal ascending aorta. In contrast, "active" increases in peripheral

muscular conduit artery and arteriolar tone, contribute to arterial stiffness by increasing









pulse wave velocity of forward and reflected traveling pressure waves, and by decreasing

the distance to peripheral reflecting sites, respectively (Nichols and Singh 2002).

Increased pulse wave velocity results in early return of reflected pressure waves to the

ascending aorta during systole instead of diastole. Reflected waves merge with forward

(incident) pressure waves resulting in augmentation of systolic pressure. This alteration

in arterial reflected wave properties creates an undesirable "mismatch" between the LV

and the arterial system (ventricular/vascular coupling), thus increasing LV afterload and

myocardial oxygen demand. Moreover, increased LV afterload or 'wasted energy,"

increases the risk of LV hypertrophy and myocardial ischemia (Nichols and Singh 2002).

In 1980, Murgo et al. published a detailed description of invasively recorded

ascending aortic pressure and flow waves in humans. Murgo et al. (1980) characterized

the aortic pressure and flow wave reflection in young and older individuals. In young

individuals, they reported that the aortic pressure augmentation occurs in diastole after

LV ejection and closing of the aortic valve. This aortic pressure waveform was

designated a "type c" wave, in which augmentation occurs in late systole or diastole after

peak pressure and flow ejection. Therefore, augmentation is negative and the augmented

pressure wave corresponded to the peak flow wave. In middle-aged individuals,

augmented pressure occurred earlier in the cardiac cycle during mid-late systole resulting

in a "type b' wave. Type b wave had an augmentation between 0 and 12% of pulse

pressure, and also did not affect aortic flow. Lastly, older individuals had a "type a"

wave which showed augmented pressure in early systole, resulting in augmentation >12%

of pulse pressure (O'Rourke and Pauca 2004). Calculation of augmentation, also known

as AI, is the ratio of the reflected wave amplitude or augmented pressure divided by pulse









pressure. Thus, AI is a measure of amplitude and timing of reflected pressure waves

arriving at the large central arteries, therefore, is an indirect measure of pulse wave

velocity and arterial stiffness of the entire arterial tree (Nichols and Singh 2002).

Furthermore, AI of the carotid or aorta can be measured noninvasively using a high

fidelity transducer (e.g., applanation tonometry) to record the pressure waveform of the

carotid directly, or the aorta indirectly by recording the radial pressure wave and using a

generalized transfer function to obtain an aortic pressure waveform (O'Rourke et al.

2001).

A primary mechanism for a change in arterial stiffness in both large elastic and

muscular arteries is acute changes in distending or mean arterial pressure (Wilkinson et

al. 2004). However, smooth muscle tone of muscular conduit arteries, are also influenced

by circulating and local vasoactive substances, and sympathetic nervous tone. In

particular, endothelial-derived NO can influence arterial stiffness through its vasodilatory

properties. Systemic studies to stimulate or inhibit release of NO can be confounded by

changes in mean arterial pressure (Wilkinson et al. 2004). However, recent in vivo studies

in sheep and in humans, show that NO released by local intraarterial infusion of

acetylcholine, indicate that NO release decreases arterial stiffness (Wilkinson et al. 2004).

Furthermore, exogenous NO donors (e.g., nitroglycerin) and phosphodiesterase inhibitors

(e.g., sildenifil), reduce arterial reflected pressure waves and arterial stiffness,

independent of any change in mean arterial pressure (Wilkinson et al. 2004). Thus,

arterial stiffness may be present in clinical conditions in which endothelial dysfunction

and reduced NO bioavailability are present.









Arterial Stiffness and Cardiovascular Risk

Several recent studies investigated the effects of arterial stiffness on cardiovascular

risk. Weber et al. (2003) recently reported that aortic AI was an independent predictor

for developing premature coronary artery disease in men undergoing coronary

angiography. London et al (2001) reported that increased carotid AI to be an

independent predictor of all cause and cardiovascular mortality in renal failure patients.

Furthermore, Laurent et al. (2001) reported that aortic pulse wave velocity was an

independent predictor of all-cause and cardiovascular mortality in patients with essential

hypertension. Thus, measures of arterial stiffness may provide important prognostic in

populations at risk for cardiovascular disease. However, the long-term prognostic

implication of arterial stiffness in HTR has not been determined.

Arterial Stiffness before Heart Transplantation

Systemic measures of arterial stiffness such as aortic input impedance are increased

are increased in chronic HF patients (Nichols and Pepine 1992; Mitchell et al. 2001).

Regional measures of arterial stiffness, such as in the carotid (Lage et al. 1994), iliac

(Ramsey et al. 1995), and brachial (Arnold et al. 1991; Ramsey et al. 1995; Nakamura et

al. 2004) conduit arteries are also elevated in HF patients (Nichols and Pepine 1992;

Ramsey et al. 1995; Nakamura et al. 2004). Using invasive simultaneous measures of

ascending aortic pressure and flow, Nichols and Pepine (1992) reported an increased

aortic impedance and resistance, the pulsatile and nonpulsatile component of LV

afterload, respectively, in HF patients compared to age- and pressure- matched controls.

Elevation of aortic input impedance, consisting of aortic elastance (stiffness) and wave

reflection at various oscillatory frequencies, suggests that arterial stiffness of the arterial

tree is increased in HF patients (Nichols and Pepine 1992).









Ramsey et al. (1995) found decreased pulse wave velocity in the common iliac

artery in response to intraarterial infusion of acetylcholine in healthy subjects but not

patients with HF. This suggests that distensibility of the iliac artery in healthy subjects is

influenced by stimulated release of NO, and that decreased release of NO may be

contributing to increased stiffness of large muscular conduit artery in HF patients. This

was further supported by noninvasive measures of brachial artery flow-mediated dilation

and distensibility during reactive hyperemia, which both were significantly decreased in

HF patients compared to healthy controls. Using high resolution ultrasound, Nakamura

et al. (2004) and Arnold et al. (1991) also found decreased compliance of the brachial

artery in HF patients, and Lage et al. (1994) reported reduced carotid artery compliance

and increased wall thickness in non-ischemic HF patients. Taken together, these data

support the hypothesis that regional muscular conduit artery stiffness is present in chronic

HF patients.

However, systemic noninvasive measures of arterial stiffness such as aortic-femoral

pulse wave velocity, total arterial compliance, and AI have yielded conflicting results

(Mitchell et al. 2001). Using noninvasive applanation tonometry and high-resolution

ultrasound, Mitchell et al. (2001) reported that pulse wave velocity and total arterial

compliance was not different than age-matched controls who had coronary artery disease

or risk factors. However, they reported a lower carotid AI in the HF patients (8 vs. 21%,

p=0.001) than in controls, but a higher central pulse pressure, characteristic impedance,

and lower proximal aortic compliance. This discordant results do suggest that proximal

aortic stiffness is elevated, but that systemic measures of AI and pulse wave velocity may

not be able to detect these changes. As such, during chronic HF, reduced cardiac output









and reduced left ventricular ejection duration lead to reduced mean arterial pressure,

pulse pressure of incident pressure wave, and decreased perfusion pressure of organs

(kidney, skeletal muscle, etc). Thus, return of reflected waves to the ascending aorta

from the periphery reduces the aortic blood flow wave during deceleration phase, rather

than add to second systolic peak of the aortic pressure wave (Nichols and O'Rourke

1998). Moreover, augmented pressure and AI may appear normal in HF patients, but

ascending aortic flow is reduced due to the increased arterial stiffness.

Arterial Stiffness and Hypertension after Heart Transplantation

Post-transplant hypertension is a common complication in HTR occurring in 72%

of HTR by one year, and 95% HTR by five years (Hertz et al. 2002). Post-transplant

hypertension jeopardizes the long-term survival of the allograft by increasing left LV

afterload and LV mass, and increases the risk of the of coronary artery vasculopathy, the

leading cause of death in HTR surviving one year (Hertz et al. 2002). Mechanisms

proposed for this de novo hypertension in HTR include 1) cyclosporine-induced

sympathetic system hyperactivity and nephrotoxicity (Scherrer et al. 1990); 2) failure of

the renin-angiotensin-aldosterone axis to reflexly suppress volume expansion-induced

hypertension due to cardiac denervation (Braith et al. 1996); and 3) arterial stiffness due

to structural changes in large elastic arteries and increased peripheral conduit arterial tone

due to endothelial dysfunction. However, it is currently unknown the relative

contribution of each of the above mechanisms on post-transplant hypertension.

Schofield et al. (2002) recently reported that 82% of 53 HTR had elevated aortic AI

and decreased time of the reflected wave (ATp), despite being on optimal hypertensive

therapy as indicated by brachial blood pressure measured by standard cuff









sphygmomanometry. In addition, when HTR were stratified by Murgo aortic wave types,

this revealed significantly different aortic pulse pressure, reflected wave amplitude, and

aortic AI between the three groups, despite having similar mean arterial blood pressure.

This data suggests that a subgroup of HTR have increased aortic augmented systolic and

pulse pressure and arterial stiffness which cannot be identified by standard brachial

sphygmomanomtry assessment. Although it is currently unknown whether elevated

aortic augmented pressure and AI is a predictor of future cardiovascular risk in HTR, it is

conceivable that any intervention that can attenuate these physiological parameters of

arterial stiffness may have beneficial prognostic implications.

There may be several mechanisms for increased arterial stiffness in HTR. Structural

changes in the large proximal elastic arteries (e.g. aorta, carotids) during chronic HF prior

to HT may occur due to chronic neurohormonal and sympathetic hyperactivity.

Increased chronic salt and water retention in the vascular wall, and the hypertrophic

effects of elevated ANG II and ET-1 on the vascular smooth muscle layer may contribute

to increased proximal arterial stiffness. As such, increased stroke volume, cardiac output,

and mean arterial pressure from HT in this setting, may lead to increased pulse pressure

and amplitude of the forward traveling pressure wave and increased pulse wave velocity

(Pierce et al. 2004).

ED and cyclosporine-induced sympathetic hyperactivity (Scherrer et al. 1990) may

also contribute to increase stiffness in HTR due to elevated vascular resistance via

increased tone of peripheral muscular conduit and resistance arteries. Increased vascular

tone leads to increased pulse wave velocity of forward and reflected pressure waves,

while increased tone of small resistance vessels decreases distance to reflecting sites.









The cumulative effect contributes to increased timing and amplitude of forward and

reflected waves to the peripheral reflecting sites and back to the ascending aorta. Thus,

increased LV afterload due to increased vascular resistance and AI due to alterations in

wave reflections, may lead to increased myocardial oxygen demand and long-term

deleterious effects on the cardiac allograft.

Role of Exercise Training in HTR

Exercise Training and Functional Capacity

Exercise capacity and peak heart rate increase in the first year after transplantation,

but remain approximately 60-70% of age-matched normals for the subsequent five years

(Givertz et al. 1997). The reasons for this persistent subnormal exercise capacity in HTR

is related to several mechanisms. First, cardiac output may be limited during peak

exercise due to impaired chronotropic reserve and mild allograft diastolic dysfunction

(Kao et al. 1994). Chronic cardiac denervation limits heart rate during submaximal and

peak exercise and mild diastolic dysfunction of the cardiac allograft reduces end-diastolic

volume and stroke volume during peak exercise (Kao et al. 1988). Thus, at low

workloads submaximal cardiac output is maintained by augmenting stroke volume in

plasma volume expanded HTR via the Frank Starling mechanism, however peak heart

rate and stroke volume contribute to a reduced cardiac output at peak exercise compared

to age-matched controls (Braith et al. 1998a). Second, myopathy of peripheral skeletal

muscle due to chronic deconditioning and glucocorticoid therapy may also contribute to

reduced exercise capacity (Braith et al. 1998b). Reduced muscle girth, muscle strength,

and metabolic enzyme activity of muscle (Braith et al. 2005), contribute to decreased

oxygen utilization and aerobic ATP production during exercise (Kao et al. 1994). Lastly,

impaired EDV of peripheral vasculature persists after transplantation in HTR, and may









contribute to reduced muscle blood flow and a-VO2 difference during peak exercise (Kao

et al. 1994).

There has been only one randomized, controlled study of the effect of endurance

exercise training in HTR. Kobashigawa et al. (1999) randomized twenty-seven HTR two

weeks after HT to an exercise or control group. They reported that 24 weeks of

supervised exercise training as part of a cardiac rehabilitation program increased peak

VO2 by 49% and exercise duration 59%, compared to 18% and 18% in the non-

supervised control HTR, respectively. To date there is no evidence that exercise training

improves central hemodynamic parameters such as LV ejection fraction or stroke volume

at rest or during exercise in HTR, so it reasons that improvement in exercise capacity in

HTR following chronic exercise training likely involves peripheral mechanisms. As such,

peripheral improvements in metabolic capacity of muscle or increased muscle blood flow

due to improvement in shear-stress mediated EDV of peripheral conduit and resistance

arteries, likely play a significant role improved exercise capacity in HTR.

Exercise Training and Endothelial Dysfunction

Several recent randomized, controlled studies in chronic HF patients report that

chronic lower-body endurance training improves EDV of peripheral conduit arteries

(Hambrecht et al.1998; Linke et al. 2001). Linke et al. (2001) reported that 4 weeks of

lower body cycle training improved radial artery EDV in response to acetylcholine. In

addition, they reported that change in EDV of the radial artery after the exercise

intervention correlated positively with the change in peak V02 (r=0.63, p<0.05).

Hambrecht et al. (1998) also reported a high positive correlation (r=0.64) between the

increase in EDV of conduit femoral artery blood flow velocity and the increase in peak

oxygen uptake (V02) following an exercise training program in HF patients. The results









of these studies suggest that lower body exercise training results in a systemic

improvement in endothelial function in chronic HF, since EDV was improved in both an

upper limb conduit artery (Linke et al. 2001), and lower body conduit artery (Hambrecht

et al. 2000). Moreover, the studies suggest that peripheral endothelial dysfunction

contributes significantly to impaired exercise capacity in HF because improvement in

EDV correlated highly with improvement in peak VO2.

Brachial artery FMD has become an accepted non-invasive test of EDV of upper

limb conduit artery function (Corretti et al. 2002). Edwards et al. (2004a) showed that 12

weeks of treadmill walking as part of a cardiac rehabilitation program resulted in an

improvement in brachial artery FMD (7.9 vs. 11.2%, p<0.05). In contrast, Goyce et al.

(2002) showed a trend but no statistical difference in brachial FMD after 12 weeks of

cardiac rehabilitation in patients with coronary artery disease (6.4% vs. 8.3%, p>0.05),

but they did report a significant increase in posterior tibial artery FMD after the training

period (9.7% vs. 11.7%, P<0.05). Furthermore, Walsh et al. (2003) recently reported that

8 weeks of cross-training (aerobic/resistance training) in CAD patients resulted in

improved brachial artery FMD (3.0% to 5.7%, p<0.05).

In a similar design to our study, Higashi et al. (1999) reported a 24% increase in

peak FBF during reactive hyperemia using plethysmography in 20 patients with essential

hypertension after a 12-week exercise training intervention. In eight of the 20 exercise

patients who showed an improvement in peak FBF, the increased FBF was abolished by

the NO inhibitor, L-NMMA, suggesting that the increase in EDV during reactive

hyperemia of forearm resistance arteries was NO mediated.









In HTR, there is a paucity of data on the effects of exercise training on endothelial

function. Only, one cross-sectional study has evaluated brachial artery FMD in trained

and untrained HTR six years after HT (Schmidt et al. 2002). Trained HTR participated in

6 months of cycling for 40 minutes 2 to 3 times per week. Brachial artery FMD was

significantly higher in trained HTR compared to sedentary HTR (7.1% vs. 1.4%, p<0.05).

Patel et al. (2003) reported that brachial artery FMD was significantly correlated to

maximal exercise treadmill time and to duration of exercise after ventilatory threshold,

but not with time to threshold in HTR four years after transplantation. Thus, these

observational data suggest that peripheral artery conduit function may be a valuable

therapeutic target for improving exercise capacity in HTR. However, there have been no

prospective, controlled studies on the effects of lower body dynamic exercise training

(e.g., walking, cycling) on brachial artery FMD or resistance vessel EDV in HTR.

Exercise Training and Arterial Stiffness

Cross-sectional studies indicate that elevated large artery stiffness is associated

with reduced exercise capacity in healthy, sedentary individuals (Vaitkevicius et al.

1993), middle-aged athletes (Kingwell et al. 1995), and individuals with chronic HF

(Bonapace et al. 2003). In a large cohort of sedentary, healthy individuals (mean age 55

years) in the Baltimore Longitudinal Study of Aging, Vaitkevisius et al. (1993) reported

that aortic AI (men: r=-0.34; women: r=-0.49) and aortic pulse wave velocity (men: r=-

0.54; women: r=-0.74) were inversely correlated to peak V02 even after controlling for

age. In another study, Kingwell et al. (1995) found an inverse correlation between aortic

P-stiffness index and peak V02 (r=-0.44) in aerobically trained middle-aged athletes (age

30-59). Lastly, in a study of 78 patients with stable, chronic HF and dilated









cardiomyopathy, Bonepace et al. (2003) reported an inverse correlation between peak

VO2 and aortic pulse wave velocity (r=-0.39). Thus, these data suggest that large artery

stiffness, at least partially, affects exercise capacity in various populations.

Several prospective, controlled exercise training studies have reported an improved

arterial stiffness in healthy sedentary, young men (Cameron and Dart 1994), healthy,

sedentary middle- aged and older men (Tanaka et al. 2000), men with coronary artery

disease (Edwards et al. 2004), and chronic HF patients (Pamell et al. 2002). Cameron

and Dart (1994) reported increased systemic arterial compliance and aortic P-stiffness

index in previously, healthy young men after 30 minutes of cycling 3 days per week for 4

weeks. Tanaka et al. (2000) studied twenty middle-aged and older men who exercise

trained for three months of walking 3 to 4 days per week at 60% of maximal heart rate,

and progressed to 4 to 6 days per week at 70-75% of maximal heart rate. They reported a

5% increase peak VO2 and a 25% increase in dynamic carotid arterial compliance and

20% decrease in carotid P-stiffness index after the exercise intervention. Lastly, Edwards

et al. (2004) investigated the effects of 12 weeks of endurance exercise training (walking)

in men with CAD. Twenty patients with previous myocardial infarction or documented

CAD via coronary angiography were assigned to supervised exercise as part of cardiac

rehabilitation program or a non-exercise control group. The 12-week training

intervention resulted in a decrease in aortic AI and an increase in duration of the reflected

wave (inverse of pulse wave velocity) in the exercise group, but with no change in

brachial systolic or mean arterial blood pressure. Furthermore, there was no change in

the time-control group, thus suggesting that regular exercise training decreased wave









reflection and systemic arterial stiffness of large arteries in patients with CAD and

reduced the dynamic components of LV afterload.

In a prospective, controlled study using simultaneous noninvasive measures of

aortic blood flow velocity and right carotid arterial pressure, Parnell et al. (2002) reported

that 8 weeks of endurance exercise training in chronic HF patients improved systemic

arterial compliance. However, other indices of arterial stiffness, such as aortic pulse

pressure, aortic AI, and aortic pulse wave velocity, did not change following the exercise

intervention. The authors explained the discordant results by suggesting that the total

arterial compliance measurement was specific for changes in the ascending aorta

independent of any changes in pulse wave reflection, and thus explaining the lack of

change in pulse wave velocity or AI. Taken together, these studies indicate that regular

endurance exercise training may be a valuable adjunct to attenuate arterial stiffness and

thus may be one mechanism by which exercise reduces long-term cardiovascular risk.

Furthermore, the mechanism for improved large artery stiffness with regular exercise

training are currently unknown, but may include reduced vascular smooth muscle

hypertrophy, reduced connective tissue cross linking, reduced sympathetic nervous tone,

or improvement in endothelial function (Joyner 2000).

Exercise Training and Nitric Oxide Synthesis

Regular endurance exercise training results in increased aortic expression of eNOS

and EDV of aorta (Sessa et al. 1994; Fukai et al. 2000), coronary (Woodman et al. 1997),

and peripheral resistance vessels in animal models (Spier et al. 2004), and in human

models using the left internal mammary coronary artery (LIMA) (Hambrecht et al. 2003).

Sessa et al. (1994) reported increased eNOS mRNA and eNOS protein content in aortas

of dogs after 10 days of treadmill training. Woodman et al. (1997) reported that 6 weeks









of exercise training in miniature swine resulted in increased eNOS mRNA in coronary

resistance arteries. Fukai et al. (2000) reported a 3-fold increase in aortic eNOS protein

expression in mice after 3 weeks of treadmill exercise. Furthermore, Spier et al. (2004)

recently reported increased eNOS mRNA and eNOS protein were increased rat skeletal

muscle soleus arterioles after 10-12 weeks of treadmill exercise in aged rats. Thus, in

animals, there is clear evidence in the aorta, coronary arteries, and the peripheral

resistance arteries, that chronic exercise training increases expression of eNOS, the

enzyme responsible for synthesis of vascular NO.

In humans, a recent elegant study by Hambrecht et al. (2003), stable coronary

artery disease patients undergoing elective coronary artery bypass surgery were randomly

assigned to a 4 week exercise training program or control period. EDV and average peak

velocity in response to acetylcholine (agonist-mediated EDV) and adenosine (flow-

mediated EDV) were measured invasively before and after the 4-week period in both

groups. Additionally, during bypass surgery, part of the LIMA tissue was harvested and

measured eNOS mRNA, eNOS protein, and phosphorlayed eNOS at serine 1177. After

the exercise intervention, LIMA average peak velocity and EDV in response to

acetylcholine and adenosine was significantly increased. Additionally, the trained group

had 96% higher eNOS mRNA, 200% higher eNOS protein expression, and 300% higher

phosphorlated eNOS in the explanted LIMA compared to controls. Furthermore,

phosporylated eNOS levels was significantly correlated to change in LIMA average peak

velocity. Thus, this was the first study in humans to demonstrate increased eNOS

mRNA, eNOS protein content, and phosphorylated eNOS and its relationship to impaired

agonist-mediated EDV after exercise training in humans.









Finally, several studies in humans have reported an increase in plasma nitrate, the

major metabolite of NO in vivo (Jungersten et al. 1996), in response to acute exercise and

after chronic exercise training (Jungersten et al. 1997; Edwards et al. 2004). Jungersten

(1997) reported higher resting plasma nitrate in athletes compared to sedentary controls

(45 vs. 34 uM, p<0.01), and an 18% and 16% increase in plasma nitrate following two

hours of cycling in athletes and controls, respectively. Edwards et al. (2004) found a

22% increase in plasma nitrate (28.2 vs. 34.4 uM, p<0.05) after 12 weeks of endurance

exercise training (walking) in patients with CAD. Together, these animal and human

studies suggest that acute and chronic exercise result in increased NO availability in part

due to increased NO synthesis.

Exercise Training and Oxidative Stress

CuZn SOD is the major intracellular isoform of SOD in the cytosol that responds to

increased shear stress and likely plays an important role in preventing intracellular

superoxide accumulation and NO degredation. As such, laminar shear stress in cultured

endothelial cells increased Cu/Zn SOD mRNA in a time and dose-dependent manner, and

increased Cu/Zn SOD protein and enzyme activity as well (Inoue et al. 1996). In isolated

soleus feed arteries, exposure to intraluminal shear stress resulted in an increase Cu/Zn

SOD protein, Cu/Zn SOD mRNA, and Cu/Zn SOD activity in coronary arterioles of pigs.

(Woodman et al. 1999).

However, the major isoform of SOD in the vessel wall is the extracellular form

(ecSOD). ecSOD is believed to be the principal regulator of endothelial-derived NO

bioactivity in the vascular wall (Fukai et al. 2002). ecSOD is produced by smooth

muscle and is bound to heparin sulfate proteoglycans on the endothelial wall between

endothelial and smooth muscle, and is in equilibrium with plasma ecSOD (Faraci et al.









2004). Fukai et al. (2000) reported that three weeks of exercise training in mice

increased ecNOS protein in aortas, but aortic Cu/Zn SOD was not changed. Additionally,

they studied the effects of exercise training in eNOS knockout (eNOS -/-) mice, and its

potential role in modulating ecSOD expression. At baseline, ecSOD expression was

decreased in eNOS -/- mice, suggesting that basal NO modulates ecSOD. Interestingly,

in control eNOS +/+ mice, aortic eNOS protein was increased 3 fold after exercise

training, which was paralleled by a 3-fold increase in aortic ecSOD. However, this

increased ecSOD protein expression was not observed in aortas from eNOS -/- mice.

Thus, this study strongly suggest that endogenous NO production modulates ecSOD

expression in the vascular wall, both under basal conditions and in response to exercise

training. The authors speculated that enhanced NO formation serves as a feed-forward

mechanism by increasing ecSOD expression, therefore increasing its own biological

effects.

To date there has been only one human study that has investigated the effects of

exercise training on ecSOD activity in plasma. Edwards et al. (2004) observed an 8.3%

increased in ecSOD activity in CAD patients who completed 12 weeks of endurance

training. In addition, they found an increase in plasma nitrate, a reduction in lipid

peroxidation, and an improvement in brachial artery FMD, but no change in non-exercise

controls. This data, along with the studies by Fukai et al. (2000), suggests that ecSOD

may be an important modulator of oxidative stress in vivo, however more studies in

humans are needed to confirm this hypothesis.

Exercise Training and Vasoconstrictors

Several studies have evaluated the effects of exercise training on ET-1 and ANG II.

Endurance exercise training has been reported to reduced plasma ET-1 levels in young,









healthy subjects (Maeda et al. 2001), and older women (Maeda et al. 2003). However,

Callaerts-Vegh et al. (1998) reported that 12 weeks of exercise training did not alter ET-1

in chronic HF patients. Vanhees et al. (1984) reported that 3 months of endurance

exercise training reduced plasma renin activity, but did not change ANG II levels in

patients with ischemic heart disease. In chronic HF patients, Braith et al. (1998) reported

that 16 weeks of exercise training lowered basal levels of ANG II and aldosterone levels,

suggesting that exercise training can modify renin-angiotensin-aldosterone activation in

HF patients. However, the effects of endurance exercise training on circulating ET-1 and

ANG II levels in HTR has not been investigated.

Exercise Training and Inflammation

Several cross-sectional studies report that higher exercise capacity is associated

with lower CRP levels in men and women at risk for cardiovascular disease (LaMonte et

al. 2002; Church et al. 2003). In addition, there have only been two prospective studies

on effects of exercise training on CRP in patients with CAD (Milani et al. 2004;Edwards

et al. 2004). Milani et al. (2004) reported a 41% decrease in CRP in a cohort of 277 CAD

patients who completed 12 weeks of exercise training as part of cardiac rehabilitation.

Additionally, Edwards et al. (2003) reported that 12 weeks of cardiac rehab lowered CRP

by 45% and IL-6 by 32% in CAD patients. Thus, these studies suggest that endurance

exercise training has a modulating effect on CRP levels in patients with documented

CAD.

There have been two prospective studies on the effects of exercise training on

inflammatory cytokines in chronic HF patients. Adamopoulos and colleagues conducted

two 12-week, randomized, controlled, cross-over design studies in 24 chronic HF patients

and 20 healthy controls (Adamopoulos et al. 2001; Adamopoulos et al. 2002). They









reported a significant decrease in plasma levels of IL-6 (-29%), TNF-a (-39%), apoptosis

inducer sFasL (-28%) (Adamopolous et al. 2002), and a 12% decrease in sVCAM-1

(Adamapolous et al. 2001). They found a significant negative correlation between the

decrease in TNF-a and the increase in peak V02 (Adamopolous et al. 2002).

Furthermore, Larsen et al. (2001) reported a 12.5% decrease in TNF-a but no change in

IL-6 in 28 patients with chronic HF. Taken together, these data suggest that exercise

training has a modulating effect on inflammatory mediators in patients with

cardiovascular disease. However, the effect of exercise training on inflammatory

mediators in HTR has not been investigated.














CHAPTER 3
METHODS

The experiments in this proposal were designed to investigate the effects of

orthotopic HT on endothelial function of peripheral limb vasculature and arterial stiffness

in end-stage HF patients. Additionally, this study investigated the effects of 12 weeks of

supervised endurance exercise training on endothelial function of limb conduit and

resistance arteries and arterial stiffness in HTR. A total of twenty end-stage HF patients

listed for transplantation at Shands Hospital were recruited and studied prospectively.

Before HT, ten HTR were randomly assigned to a program consisting of 12 weeks of

supervised endurance exercise training after HT, and ten HTR were assigned to a control

group consisting of usual post-HT medical care but did not participate in a supervised

exercise program. In addition, ten age-matched, healthy control subjects were recruited

to compare with HTR. The study was approved by the University of Florida Health

Science Center Institutional Review Board and all subjects signed written informed

consent to participate in the study.

Subjects

All HTR were recruited from the Heart Transplantation Program at Shands

Hospital at the University of Florida. Patients were enrolled while inpatient at Shands

Hospital and listed as status 1B on United Network of Organ Sharing (UNOS) awaiting

HT. The selection of subjects was notbased on gender or racial/ethnic status.

Inclusion Criteria

1. Age 18 to 65









2. Listed status 1B inpatient on UNOS for HT

Exclusion Criteria

1. UNOS status 1A in the intensive care unit

2. Major orthopedic problems that would limit exercise

3. Claudication pain from peripheral artery disease

4. Chronic obstructive lung disease

5. Renal failure

Group Assignments

Twenty patients (n=20) were randomly assigned to 12 weeks of supervised

endurance exercise training (n=10) (e.g. treadmill walking or cycling) or a non-exercise

control group (n=10) before HT. The control group consisted of HTR who did not

perform supervised exercise training but continued to receive their usual post-HT medical

care. Ten age-matched, healthy controls (n=10) were also recruited for the study.

Exercise Training Protocol

Exercise training was performed at the "Living Well Center," College of Health

and Human Performance, University of Florida, Gainesville, FL. For subjects who did

not reside in the Gainesville area, participation in 12 weeks of supervised endurance

exercise training occurred in a hospital in their community with an American Association

of Cardiovascular and Pulmonary Rehabilitation (AACVPR) certified cardiac

rehabilitation program. Exercise prescription guidelines were provided to each program,

and progression and compliance updates were sent every 4 weeks and at the end the study

to the primary investigator. HTR in the exercise group participated in 12 weeks of

supervised endurance exercise training beginning at 8 weeks after HT. Exercise training

will began with 30 minutes of continuous treadmill walking or stationary cycling 3 days









per week not including warm-up and cool-down periods, and progressed to 35-40

minutes as tolerated after the initial 4 weeks.

Cardiac denervation in HTR prevents heart rate from being used as an accurate

measure of exercise intensity (Braith et al. 1998a). Therefore, intensity begin at 50-60%

of V02 peak determined from a graded exercise test, and the "Borg" rating of perceived

exertion (RPE) scale was used to maintain intensity in the 11 to 13, or "moderate" to

"somewhat hard" range, in accordance with ACSM guidelines (ACSM 2000). Exercise

intensity was progressed to 60-70% V02 peak, or RPE in the 12 to 14 Borg scale range

"as tolerated" by each subject. Each session begin with five minute warm-up period with

RPE range of 7 to 9 ("very light to light"), and a five minute cool down in the same

range. Exercise sessions were under the direct supervision of an ACSM exercise

specialist or registered nurse certified in basic life support (BLS) and advanced life

support (ACLS) and overseen by a physician. Blood pressure, symptoms, and ECG

rhythm via three-lead telemetry system were monitored throughout each exercise session.

Criteria to terminate an exercise session was be based on ACSM guidelines (ACSM

2000). All exercise facilities were equipped with automated external defibrillators and

resuscitation equipment and access to emergency medical services (EMS). The control

group received standard of medical care for HTR from their transplant physician, but did

not participate in supervised endurance exercise training.

Specific Measurements

Subjects visited the laboratory 3 times for testing. Details of the study protocol are

outlined in Figure 3-1.










Recruit subjects from UNOS status 1B at Shands Hospital
at the University of Florida
Obtain Informed Consent (n=20)


DATA COLLECTION 1 (T1)
FMD, PLETHYS, PWA, BLOOD


Orthotopic Heart Transplantation


DATA COLLECTION 2 (T2)
8 weeks post-transplant
FMD, PLETHYS, PWA, BLOOD, GXT
SI I

Supervised Exercise (n=10) No supervised exercise (n=10)
Standard medical care Standard medical care
12 weeks, 3 days/wk 12 weeks


DATA COLLECTION 3 (T3) DATA COLLECTION 3 (T3)
FMD, PLETHYS, PWA, BLOOD, GXT FMD, PLETHYS, PWA, BLOOD, GXT
Figure 3-1. Study design. UNOS=United Network of Organ Sharing; FMD=flow-
mediated dilation of brachial artery; PLETHYS= venous occlusion
plethysmography blood flow; PWA=pulse wave analysis; BLOOD= venous
blood sample; GXT=graded exercise test

Arterial Stiffness Testing

Measurement of arterial stiffness were made using pulse wave analysis. Subjects

will remained quietly supine for 10 minutes and then blood pressure (BP) was determined

in brachial artery in non-dominant arm three times by automated non-invasive BP cuff

(Omron, Inc.) and mean was taken as BP value. Next, high-fidelity radial artery pressure

waveforms was recorded by applanation tonometry of the radial pulse using a "pencil-

type" micro-tip pressure transducer (Millar Instruments, Inc.). Optimal recording of the

radial pressure waveform will be obtained by applying perpendicular hold-down force

generating a stable baseline for at least 10 seconds. The radial pressure waveform and









the brachial artery BP was entered into a SphygmocorTM (AtCor Medical, Inc., Sydney,

Australia) PWA system which synthesizes an aortic pressure waveform using a

mathematical generalized transfer function which has been validated (Chen et al. 1997;

Cameron et al. 1998) and is reproducible (Wilkinson et al. 1998).

As shown in Figure 3-2, the aortic pressure wave (Ps-Pd) is the sum of a forward

traveling wave with amplitude (Pi-Pd) generated by left ventricular ejection, and reflected

pressure wave with amplitude (Ps-Pi) from the periphery arriving at the ascending aorta

(Nichols and Singh 2002). The two pressure waves travel along the artery at the same

velocity in opposite directions, whereby the reflected traveling pressure wave augments

the forward traveling wave. The amplitude of the reflected traveling pressure was

estimated by the aortic augmentation index (Ala), which was obtained from the aortic

pressure waveform. Ala is calculated as the ratio of reflected wave amplitude to the pulse

pressure expressed as a percentage, (Ps-Pi)/(Ps-Pd) x 100, where Ps is the aortic systolic

pressure; Pi is the inflection point of the beginning of upstroke of reflected pressure

wave; Pd is minimum diastolic pressure. The roundtrip travel time (Atp) of the forward

traveling pressure wave from the ascending aorta to the major reflection site and back

was measured from the foot of the forward traveling wave to Pi. Round trip travel time

(Atp) is inversely related to arterial pulse wave velocity and arterial stiffness, and directly

related to the distance to reflecting point (Lo). Furthermore, LV ejection duration

(LVED) is equal to duration from Pd to the incisura notch (closure of aortic valve).

Aortic systolic tension time index (TTI), an indicator of LV myocardial oxygen demand,

is equal to the area under the LVED x aortic Ps curve. Aortic diastolic TTI is equal to









area under the diastolic duration x aortic Pd curve, an indirect indicator of diastolic

coronary perfusion (Nichols and Singh 2002).






Pressure




Flow


i At


Figure 3-2. Ascending aortic pressure waveform. Ps= aortic systolic pressure; Pd=aortic
diastolic pressure; Pi= inflection point of reflected wave; Atp= duration of
reflected wave from the heart to the periphery and back; Atr= systolic duration
of reflected wave

Endothelial Function Testing

Brachial artery flow-mediated dilation

Brachial artery reactivity testing was performed using high-resolution ultrasound

(ATL, Inc.). Brachial artery reactivity tests were performed when the subject was fasted

for at least 4 hours, abstained from caffeine for 12 hours and exercise for 24 hours, and

the subject was asked to eat a low fat meal on the day of testing (Plotnick et al. 1997;

Gudmundsson et al. 2000). After lying quietly for 15 minutes, a 10.5 MHz linear array

ultrasound transducer was used to image the right brachial artery longitudinally and

record on a super VHS recorder. After baseline artery diameter was obtained, a blood

pressure cuff was inflated to 200 mmHg for 5 minutes on the upper arm proximal to the

location brachial artery measurement. The transducer was held in the same location for









the duration of the cuff inflation to ensure the same section of the brachial artery was

measured before and after cuff inflation. The proximal cuff position elicits a greater

increase in blood flow and dilation during reactive hyperemia, compared to distal (e.g.,

forearm) cuff inflation (Corretti et al. 2002). Upon release of the cuff, brachial artery

diameter was imaged and recorded for three minutes during reactive hyperemia blood

flow. Reactive hyperemia blood flow results in flow-mediated dilation (FMD) of the

brachial artery due to increased shear stress-induced nitric oxide release from the

endothelial wall. This EDV of the brachial artery has been reported to peak between 60

to 90 seconds after cuff deflation, and is a valid measure endothelial-mediated arterial

reactivity (Corretti et al. 2002). Images of the brachial artery were transferred to

computer by a frame grabber (DT-4152, Data Translation, Inc.) and brachial artery

diameter was made during end-diastole by measuring the distance between anterior and

posteriors wall of the intima using image analysis software (Image Pro, Data Translation,

Inc.). Five anterior to posterior point measures within a 3 cm segment were made and the

average distance was recorded as diameter. Reliability of brachial artery testing was

confirmed by a pilot study of four young healthy adults who had brachial artery FMD

performed during three visits separated by one-week which yielded coefficient of

variation (CV%) of 14.5%.

NO donors, such as nitroglycerin or sodium nitroprusside, are commonly used to

test endothelial-independent vasodilation (EIV). NO donors act directly on vascular

smooth muscle, resulting in normal vasodilation in subjects with CAD, HF, and HT. EIV

using an NO donor was not performed in this study in order to reduce the risk of a

hypotensive episode in patients with diminished baroreflex sensitivity.









Forearm and calf flow-mediated vasodilation

Forearm blood flow (FBF) and calf blood flow (CBF) responses were determined

separately by venous occlusion plethysmography (EC-6, D.E. Hokanson, Inc.) using

calibrated mercury strain-gauges as previously described (Hokanson et al. 1975;

Wilkinson and Webb 2001). Patients were tested in a quiet, temperature-controlled

room approximately 21-220C and relative humidity approximately 40-50%. Strain-

gauges were applied to the widest part of the non-dominant forearm (-5 cm below

anticubital fossa) or calf (-10 cm below patella). Patients remained quietly supine for 10

minutes with arms or legs elevated above the right atrium n order to achieve stable

baseline measurements of FBF and CBF. To measure FBF, an upper arm cuff (EC-20,

Hokenson, Inc) was inflated to 40 mmHg for 5 seconds every 15 seconds using a rapid

cuff inflator to prevent venous outflow (Wilkinson and Webb 2001). To measure CBF,

an upper thigh cuff was inflated to 40 mmHg for 5 seconds every 15 seconds. One

minute before each measurement, a wrist or ankle cuff was inflated to pressure 50 mmHg

above systolic pressure to occlude hand or ankle circulation respectively, during FBF or

CBF measurements. The FBF or CBF output signal was transmitted to NIVP3 software

program (Hokanson, Inc) on a laptop PC computer and expressed as milliliters (mL) per

minute per 100 mL of forearm tissue (mLmin-1 per 100 mL tissue). Absolute blood flow

was determined by the rate of change of limb circumference (e.g., slope) during the five-

second venous occlusion, which has been validated to correlate highly to arterial blood

inflow into the limb (Greenfield et al. 1963; Hokanson et al. 1975). FBF or CBF for one

minute is the average of one plethysmographic measurement every 15 seconds. Mean

arterial pressure (MAP) was determined by systolic blood pressure (SBP) and diastolic









blood pressure (DBP) measured by an automatic oscillometric cuff (HEM-739, Omron,

Inc) and calculated as DBP + [0.33(SBP-DBP)].

Endothelium-dependent FBF was measured during reactive hyperemia blood flow

of the forearm following 5 minutes of upper arm occlusion using a BP cuff inflated at 200

mmHg (Wilkinson and Webb 2001). A blood pressure cuff was placed on the upper arm

5 cm above the anticubital fossa. After baseline FBF was confirmed to be stable for 2

minutes and recorded, the cuff was rapidly inflated to 200 mmHg for 5 minutes and then

released. FBF was measured every 15 sec for 4 minutes. Peak FBF was recorded as the

highest FBF observed immediately following releases of the cuff, and total FBF for three

minutes was recorded as the area under the time x blood flow curve after baseline FBF is

subtracted using the trapezium rule (Matthews et al. 1990). Peak FBF during reactive

hyperemia has been shown to correlate highly with acetylcholine-induced FBF in patients

with essential hypertension (Higashi et al. 2001), therefore it is a good non-invasive

measurement of EDV of forearm resistance arteries (Wilkinson and Webb 2001; Higashi

et al. 2001). Meredith et al. (1996) reported that peak FBF during reactive hyperemia is

NO-dependent, where other studies report that peak FBF is not NO-dependent (Tagawa

et al. 1994; Engelke et al. 1996). In fact, these studies suggest that vasodilation of

resistance vessels of forearm is prostacyclin-dependent (Engelke et al. 1996), and that

total area under the time x blood flow curve is NO-dependent (Tagawa et al. 1994).

Endothelium-dependent CBF was measured following 5 minutes of upper leg

(thigh) arterial occlusion. After baseline CBF was stable for 2 minutes, the cuff was

rapidly inflated to 200 mmHg for 5 minutes and then released. Peak CBF during reactive

hyperemia was recorded as CBF observed immediately following release of the cuff, and









total CBF was recorded as the area under the time x blood flow curve after baseline CBF

is subtracted using the trapezium rule (Matthews et al. 1990). A reliability of peak FBF

and CBF using venous occlusion plethysmography was confirmed by a pilot study of 9

young healthy adults who had peak FBF and CBF during reactive hyperemia performed

during three visits separated by one week (Pierce et al. 2004). Mean CV% of resting and

peak FBF was 17% and 6.6%, respectively, and resting and peak CBF was 15.2% and

8.4%, respectively.

Graded Exercise Test

All HTR performed a symptom-limited graded exercise test (GXT) at 8 weeks after

HT and again after 12 weeks of the exercise training or the control period. The GXT was

be performed on a motorized treadmill (Quinton, Inc.) with collection of respiratory gas

analysis using a calibrated metabolic cart (Parvomedics, Inc.) for determination of peak

oxygen uptake (VO2). Subjects performed a Modified Naughton walking protocol, which

begins at 1.2 MPH and 0% grade for two minutes, increases to 2.0 MPH for two minutes,

and then increases 3.5% grade every 2 minutes thereafter. HTR were monitored

continuously during the GXT with a 12-lead electrocardiogram (Quinton, Inc.), blood

pressure, and Borg rating of perceived exertion measured once each stage. Criteria for

termination of GXT was based upon guidelines published by ACSM (ACSM 2000). All

GXT's were performed in the Clinical Exercise Physiology (CEP) Laboratory in the

Center for Exercise Science at the University of Florida and were supervised by a

cardiologist and a certified ACSM exercise specialist. The CEP laboratory was equipped

with a Lifepak 500 automated external defibrillator (Medtronic, Inc.), supplemental

oxygen, emergency "crash cart" medications, and telephone.









Blood Collection

Venous blood samples were collected in tubes containing no additive, allowed to

clot at room temperature for 15 minutes, and immediately centrifuged at 3,000 rpm for 15

minutes at 40C. Venous blood for plasma samples were collected in tubes containing

EDTA, placed on ice, and centrifuged immediately as noted above. Plasma that was used

for measurement of lipid peroxidation was stored with diethylenetriamine pentaacetic

acid (DTPA) and butylated hydroxytoluene (BHT) for a final concentration of 0.01 mM

to prevent autooxidation during freezing and thawing. All serum and plasma samples

were aliquoted into 1.5 ml epindorff tubes and immediately stored at -800C until analysis

at the end of study.

Plasma Biochemical Analysis

Vasoactive balance

Since the vasodilator, NO, is rapidly converted to nitrate and nitrite (NOx) in

plasma, NOx will be used to estimate NO production. Plasma NOx has a half-life of 8

hours and can be influenced by dietary nitrate, therefore all subjects will be asked to

follow National Institute of Health low nitrate diet guidelines 36 hours prior to each

blood draw (Pannala et al. 2003). Plasma NOx was measured using a commercially

available kit (Cayman Chemical, Inc.), which converts all nitrate to nitrite using nitrate

reductase. Spectrophotometric analysis of total nitrite was performed using Greiss

reagent and the absorbance measured at 540 nm. The vasoconstrictor ET-1 was measured

using an ELISA kit (Cayman Chemical, Inc.).

Lipid peroxidation

Oxidative stress-induced lipid peroxidation was assessed by measuring plasma

levels of 8-iso-PGF2 using a enzyme-linked immunoassay (ELISA) (Stressgen, Inc.). 8-









iso-PGF2a in plasma competes for binding with 8-isoprostane covalently attached to

alkaline phosphatase. The assay plate is then incubated with p-nitrophenyl phosphate and

the reaction stopped with the addition of an acid. The plate is read at 405 nm on

spectrophotometer and the absorbance is inversely proportional to 8-iso-PGF2 in the

plasma sample.

Extracellular antioxidant enzyme activity

SOD activity in plasma was measured using coloremtric assay which uses

cytochrome c reduction technique (Cayman Chemical, Inc.). This method utilizes the

reduction of cytochrome c by superoxide ions produced by the xanthine oxidase reaction

which causes a change in absorbance via spectrophotometry at 450nm. "One unit of

SOD activity" is defined as the amount of SOD required for a 50% decrease in

cytochrome c reduction rate or absorbance.

Inflammatory markers

Plasma CRP was measured using a sandwich ELISA (Alpha Diagnostics, Inc.). The

ELISA is based on simultaneous binding of human CRP from plasma samples to two

antibodies, one immobilized on the microtiter well plates, and the other conjugated to the

enzyme horseradish peroxidase. The product is read at 450 nm and represents CRP

bound to horseradish peroxidase. Plasma levels of IL-6, TNF-a, and sICAM-1 were

measured using an ELISA (R&D Systems, Inc) which employ the quantitative sandwich

enzyme assay technique.

Endogenous NO inhibition

Plasma levels of the endogenous eNOS competitive inhibitor, ADMA, was measured

using an ELISA (Alpco, Inc).









Blood hemoglobin, hematocrit, serum lipids, glucose, creatinine, white blood cell
count, cyclosporine, and cytomegliovirus status

Blood hemoglobin, hematocrit, and serum total cholesterol, LDL, HDL cholesterol,

triglycerides, glucose, cyclosporine trough levels, and cytomegliovirus (CMV) status

were measured by the Clinical Chemistry Laboratory at Shands Hospital at the University

of Florida using standard blood lipid chemistry analyzer.

Endocardial biopsy rejection history

The number of allograft rejection episodes identified from endocardial heart

biopsies were obtained during the study period in all subjects from the Shands Hospital

Transplant Program database.

Statistical Considerations

Data is presented in table format as mean + standard deviation (SD) for continuous

variables and as percent frequencies (%) for categorical variables. Continuous variables

were be analyzed by analysis of variance (ANOVA) with repeated measures of brachial

FMD, peak and total FBF, peak and total CBF, Ala, plasma NOx, ET-1, 8-iso-PGF2a,

SOD activity, ADMA, CRP, IL-6, TNF-a, sICAM-1, demographics, and serum

metabolic parameters before and after HT. ANOVA was performed between the exercise

and control HTR groups at baseline before the exercise intervention to analyze for

baseline group differences. ANOVA with repeated measures was used to compare the

above vascular and blood parameters before and after 12 weeks of exercise training or

control period. When a significant group-by-time interaction was observed, within-group

comparisons between time points and between- group comparisons at each time point

were performed using Tukey's post-hoc analysis. Categorical variables were analyzed by









X2 analysis. All statistical analysis were performed using Microsoft Excel and SPSS

10.0 (SPSS, Inc.). An alpha level of p<0.05 will be required for statistical significance.

A power analysis was performed to estimate statistical power related to testing the

following hypothesis: 12 weeks of exercise training will result in greater peak FBF and

brachial artery FMD when compared to a 12 week control period in HTR. Preliminary

data on three HTR (n=3) was a peak FBF of 23.1 4.8 ml/min/100ml (mean SD) two

months after HT. Based on the study by Higashi et al. (1999), a 24% increase in peak

FBF was conjectured after 12 weeks of exercise training. As such, the statistical power

related to testing the hypothesis that peak FBF is greater in the exercise trained HTR

compared to the control HTR is 0.80 for a two-tailed test when the group means were

estimated to be 23.1 ml/min/100ml in the control group, and 28.6 ml/min/100ml in the

exercise group; the standard deviation was assumed to be 4.8 ml/min/100ml; total sample

size was 20 patients; and the alpha level was set at 0.05. Preliminary data on four HTR

(n=4) was a brachial FMD of 8.8 2.5% (mean SD) two months after HT. Based on

the study by Edwards et al. (2004a) a 42% increase in brachial FMD was conjectured

after 12 weeks of exercise training. The statistical power related to testing the hypothesis

that brachial artery FMD is greater in the exercise trained HTR compared to the control

HTR is 0.94 for a two-tailed test when the group means were estimated to be 8.8% in the

control group and 12.5% in the exercise group; the standard deviation was assumed to be

2.5%; total sample size was 20 patients; and the alpha level was set at 0.05.














CHAPTER 4
RESULTS

A total of twenty subjects (n=20) were recruited and signed written informed

consent for this study. Twelve subjects (n=12) completed all measurements before HT

(PREHTX) and after HT (POSTHTX). A total of eight consented subjects (n=8) did not

complete the measurements because 24-48 hours from the time of written consent the

subject received heart transplantation, increased to status 1A and transferred to intensive

care unit, or had left ventricular assist device implanted. Therefore, PREHTX data

collection was unobtainable. Seven age-matched healthy controls (n=7) were recruited

and completed all measurements.

Before transplantation, ten subjects were randomly assigned to the exercise group

(TRAINED; n=10) and ten were assigned to the control group (CONTROL; n=10). One

subject in the TRAINED group withdrew for a non-cardiac medical problem not related

to exercise. Three subjects in the CONTROL group withdrew from the study, one for a

non-cardiac medical reason, and the other two were lost to follow up. Therefore, sixteen

subjects (n=16) completed the 12-week intervention part of the study of which seven

were assigned to the CONTROL group (n=7) and nine to the TRAINED group (n=9).

Subject Characteristics before and after Heart Transplantation

The characteristics for PREHTX, POSTHTX, and age-matched healthy control

subjects are presented in Table 4-1. The PREHTX and POSTHTX groups did not differ

with respect to age, weight, body mass index, gender ratio, or number of ischemic

etiology of heart failure. PREHTX had more on beta-blocker therapy (12 vs. 0, p<0.01)









and nitrate therapy (4 vs. 0, p<0.05) than POSTHTX. All PREHTX and POSTHTX

subjects were on ACE inhibitor or angiotensin receptor blocker therapy (12 vs. 12,

p=NS), however, there were more POSTHTX subjects on station therapy (12 vs. 5,

p<0.05), and insulin therapy (5 vs. 1, p<0.05). All subjects after HT were receiving

standard triple immunosuppressive therapy including cyclosporine (Neoral),

myophenolate mofetil (Cellcept), and prednisone.

Age-matched healthy controls did not differ significantly from PREHTX and

POSTHTX with respect to age, weight, body mass index (BMI), or male/female ratio.

Two healthy controls were on station and ACE inhibitor therapy, but no healthy controls

were on beta-blocker, nitrate, or insulin therapy.

Table 4-1 Subiect characteristics before and after heart transplantation
PREHTX POSTHTX Healthy
(n= 12) (n= 12) Controls
(n=7)
Age (years) 56.88.0 57.38.0 61.78.5
Weight (kg) 85.612.6 84.511.9 89.09.2
Body mass index (kg/m2) 27.53.4 27.12.7 28.32.4
Male, no. (%) 10 (83) 10 (83) 6 (86)
Female, no. (%) 2(17) 2(17) 1(14)
Ischemic HF etiology, no. (%) 7 (58) 7 (58) N/a
Days before transplant 76.942.3 N/a N/a
Days after transplant N/a 66.411.6 N/a
IV inotrope therapy, no. (%) 12 (100) N/a N/a
ACEI/ARB therapy, no. (%) 12 (100) 7 (58) 2 (29)*
Beta-blocker therapy, no. (%) 12 (100) 0* 0*
Nitrate therapy, no. (%) 4 (33) 0* 0*
Calcium channel blocker therapy, no. (%) 1 (8) 3 (25) Of
Statin therapy, no. (%) 5 (42) 12 (100)* 2 (29)t
Insulin therapy, no. (%) 1 (8) 5 (42) 0
Cyclosporine therapy, no. (%) N/a 12 N/a
Prednisone therapy, no. (%) N/a 12 N/a
Mycophenolate mofetil therapy, no. (%) N/a 12 N/a
Data are mean + SD; *P<0.05 vs. PREHTX; tP<0.05 vs. POSTHTX; PREHTX= pre-
heart transplantation; POSTHTX=post-heart transplantation; HF=heart failure;
IV=intravenous; ACEI=angiotensin converting enzyme inhibitor; ARB=angiotensin
receptor blocker; N/A=not applicable









Serum Metabolic Parameters before and after Heart Transplantation

Fasting metabolic parameters are presented in Table 4-2. As shown in Table 4-2,

hemoglobin (13.1 vs. 11.3 g/L, p<0.05) and hematocrit (38.3 vs. 34.6%, p<0.05)

decreased significantly in POSTHTX compared to PREHTX. Serum lipid analysis

showed that total cholesterol (164.9 vs. 193.3, p<0.05) and HDL cholesterol (45.8 vs.

64.8 mg/dl, p<0.05) increased significantly in POSTHTX, but there was no change in

LDL (84.9 vs. 92.0 mg/dl) or triglycerides (171.0 mg/dl vs. 182.6 mg/dl, p=NS) in

POSTHTX compared to PREHTX. However, total cholesterol/HDL cholesterol

decreased significantly in POSTHTX (3.94 vs. 3.20, p<0.05). Finally, fasting glucose

(95.0 vs. 113.0 mg/dl, p=NS), creatinine (1.43 vs. 1.26 mg/dl, p=NS), and WBC (8.0 vs.

7.0, lx109, p=NS) count did not differ in POSTHTX compared to PREHTX, respectively.

Table 4-2 Serum metabolic parameters before and after heart transplantation
PREHTX POSTHTX Healthy
(n=12) (n=12) Controls
(n=7)
Hemoglobin (g/L) 13.11.5 11.31.1* N/a
Hematocrit (%) 38.34.0 34.62.5* N/a
Total cholesterol (mg/dl) 164.929.5 193.326.7* 195.654.5
LDL cholesterol (mg/dl) 84.928.2 92.019.7 117.741.5*
HDL cholesterol (mg/dl) 45.817.5 64.819.2* 54.311.2
Total cholesterol/HDL ratio (no.) 3.941.20 3.200.98* 3.630.79
Triglycerides (mg/dl) 171.075.0 182.660.9 117.1+55.5t
Glucose (mg/dl) 113.055.3 95.022.3 103.310.6
Creatinine (mg/dl) 1.260.37 1.430.41 0.970.13t
White blood cells, lx 109 (no.) 7.02.0 8.02.1 N/a
Data are mean + SD. *P<0.05 vs. PREHTX; tP<0.05 vs. POST-HTX; PREHTX=pre-
heart transplantation; POSTHTX=post-heart transplantation; LDL=low-density
lipoprotein; HDL=high density lipoprotein; N/a=not available

Age-matched healthy controls did not differ from PREHTX and POSTHTX

subjects with respect to total cholesterol, HDL cholesterol, total cholesterol/HDL ratio,

fasting glucose, or white blood cell count. LDL cholesterol was significantly higher in

healthy controls compared to PRETX (117.7 vs. 84.9 mg/dl, p=0.05), but not with









POSTHTX (117.7 vs. 92.0 mg/dl, p=NS). Fasting triglycerides were significantly lower

in healthy controls than POSTHTX (117.1 vs. 182.6 mg/dl, p<0.05), but not significantly

different from PREHTX (117.1 vs. 171.0 mg/dl, p=NS). Healthy controls had

significantly lower serum creatinine than POSTHTX (0.97 vs. 1.43 mg/dl, p=0.01), but

not different than PREHTX (0.97 vs. 1.26, p=NS).

Brachial Artery Endothelial Function before and after Heart Transplantation

Brachial artery flow-mediated dilation (FMD) and absolute diameter dilation

results in PREHTX, POSTHTX, and healthy controls are presented in Table 4-3, Figure

4-1, and Figure 4-2. Brachial artery FMD (9.63 vs. 6.44%, p<0.05) and the absolute

diameter dilation (0.45 vs. 0.32 mm, p<0.05) was significantly increased in POSTHTX

compared to PREHTX. However, there was no significant change in resting baseline

diameter in POSTHTX compared to PREHTX (4.70 vs. 4.93 mm, p=NS).

Table 4-3 Brachial artery flow-mediated dilation before and after heart transplantation
PREHTX POSTHTX Healthy
(n=12) (n=12) Controls
(n=7)
Baseline diameter (mm) 4.930.78 4.700.63 4.460.78
Absolute diameter dilation (mm) 0.320.16 0.450.14* 0.300.13t
Flow-mediated dilation (%) 6.443.30 9.633.05* 6.812.92
Data are mean + SD; *P<0.05 vs. PREHTX; tP<0.05 vs. POSTHTX; PREHTX=pre-
heart transplantation; POSTHTX=post-transplantation

Resting diameter and brachial artery FMD did not differ between age-matched

healthy controls and PREHTX or POSTHTX. There was a trend for brachial artery FMD

to be significantly greater in POSTHTX than age-matched controls (9.63 vs. 6.81,

p=0.06). Lastly, absolute diameter dilation was significantly less in healthy controls

compared with POSTHTX (0.30 vs. 0.45 mm, p<0.05), but not significantly different

than PREHTX (0.30 vs. 0.32 mm, p=NS).









Blood Pressure and Pulse Wave Analysis before and after Heart Transplantation

Blood pressure components for PREHTX, POSTHTX, and age-matched healthy

controls are presented in Table 4-4. POSTHTX had a significant increase in heart rate

(95.6 vs. 66.5 b/min, p<0.01), peripheral systolic blood pressure (138.8 vs. 109.8 mmHg,

p<0.05) and peripheral diastolic blood pressure (90.8 vs. 70.7 mmHg, p<0.01) compared

to PREHTX, but no significant change in peripheral pulse pressure (47.9 vs. 39.1 mmHg,

p=NS). There was a significant increase in central systolic blood pressure (122.0 vs. 98.5

mmHg, p<0.01), central diastolic blood pressure (92.2 vs. 71.0 mmHg, p<0.01), but no

significant change in central pulse pressure (29.8 vs. 27.5 mmHg, p=NS) in POSTHTX

compared to PREHTX. There was a significant increase in mean blood pressure (105.3

vs. 81.3 mmHg, p<0.01).

Arterial pulse wave analysis data are presented in Table 4-4 and Figure 4-3, 4-4,

and 4-5. There was no significant change in Ala normalized for heart rate at 75 b/min

(8.9 vs. 13.5 mmHg, p=NS), or augmentation pressure (3.8 vs. 1.8 mmHg, p=NS). In

addition, there was no significant change in time duration of the reflected wave to

periphery and back (Atp) in POSTHTX compared to PREHTX (140.6 vs. 146.0 ms,

p=NS), but a significant increase in systolic tension time index (AsTTI; 3254.3 vs 1826.1

mmHg/sec/min, p<0.01), an indicator of systolic LV myocardial oxygen demand, in

POSTHTX compared to PREHTX. Lastly, there was no significant difference in

diastolic pressure tension index (DPTI), an indicator of diastolic coronary perfusion, in

POSTHTX vs. PREHTX.

Age-matched healthy controls had significantly lower heart rate than POSTHTX

(58.3 vs. 95.6 b/min, p<0.01), and had higher peripheral and central systolic, diastolic,










pulse and mean blood pressure than PREHTX but no significant difference from

POSTHTX. In addition, healthy controls had significantly higher augmentation blood

pressure than PREHTX (10.4 vs. 3.8 mmHg, p<0.01) and POSTHTX (10.4 vs. 1.8

mmHg, p<0.01), however, Ala normalized for heart rate (at 75 b/min) was not

significantly different than PREHTX (17.6 vs. 8.9%, p=0.09) or POSTHTX (17.6 vs.

13.5%, p=NS). There was no significant difference in Atp between healthy controls and

PREHTX or POSTHTX, but healthy controls had a significantly higher AsTTI than

PREHTX (2402.3 vs. 1826.1 mmHg/sec/min, p<0.01) and significantly lower AsTTI than

POSTHTX (2402.3 vs. 3254.3 mmHg/sec/min, p<0.01). However, there was no

significant difference in DTPI between healthy controls, PREHTX, or POSTHTX.

Table 4-4 Blood pressure components and pulse wave analysis before and after heart
transplantation


HR (b/min)
PSBP (mmHg)
PDBP (mmHg)
PPBP (mmHg)
CSBP (mmHg)
CDBP (mmHg)
CPBP (mmHg)
MBP (mmHg)
AgBP (mmHg)
Ala at HR=75 b/min (%)
Atp (ms)
AsTTI (mmHg/sec/min)
DPTI (mmHg/sec/min)


PREHTX
(n=12)
66.516.7
109.8+9.5
70.77.1
39.1+8.6
98.58.2
71.07.3
27.56.7
81.37.0
3.82.9
8.911.6
146.015.7
1826.1392.6
2843.5497.4


POSTHTX
(n=12)
95.611.7*
138.8+19.2*
90.8+12.2*
47.914.6
122.017.2*
92.212.1*
29.8+10.9
105.313.1*
1.85.1
13.515.9
140.611.7
3254.3617.5*
3079.7606.1


Healthy Controls
(n=7)
58.39.0t
133.313.6*
81.89.0*
51.410.4*
122.913.5*
82.68.9*
40.39.6*
98.79.3*
10.46.8*t
17.6+7.4
147.1+4.9
2402.3+311.3*t
3719.7442.5


Data are mean + SD. *P<0.05 vs. PREHTX; tP<0.05 vs. POSTHTX; PREHTX=pre-
heart transplantation; POSTHTX=post-transplantation; HR=heart rate; PSBP=peripheral
systolic blood pressure; PDBP=peripheral diastolic blood pressure; PPBP=peripheral
pulse pressure; CSBP=central systolic blood pressure; CDBP=central diastolic blood
pressure; CPBP=central pulse blood pressure; MBP=mean blood pressure;
AGBP=augmentation blood pressure; AIa=aortic augmentation index; Atp=round
triptravel time of reflected pressure wave from ascending aorta to peripheral reflecting
sites and back; AsTTI=aortic systolic tension-time index; DPTI=diastolic perfusion time
index









Forearm and Calf Resistance Artery Endothelial Function before and after Heart
Transplantation

Forearm and calf resistance artery blood flow during reactive hyperemia results are

displayed in Table 4-5 and Figure 4-6 and 4-7. A subgroup of five subjects (n=5)

completed FBF and CBF before and after HT. POSTHTX compared to PREHTX.

However, there was a significant increase in peak CBF in POSTHTX compared to

PREHTX (22.4 vs. 17.4 ml/min/100ml, p<0.05), but no significant change in CBF

AUC3min (11.2 vs. 9.3 ml/min/l00ml, p=NS) in POSTHTX compared to PREHTX.

There was no significant difference in peak FBF (22.9 vs. 20.4 ml/min/100ml,

p=NS) and total excess FBF for 3 min (AUC3min) (8.1 vs. 7.2 ml/min/100ml, p=NS) in

Lastly, there was a trend for, but no significant increase in resting FBF in POSTHTX

compared to PREHTX (2.4 vs. 1.7 ml/min/100ml, p=0.06), and no significant change in

resting CBF in POSTHTX vs. PREHTX (2.6 vs. 2.8 ml/min/100ml, p=NS).

Table 4-5 Forearm and calf flow-mediated vasodilation before and after heart
transplantation
PREHTX POSTHTX Healthy
(n=5) (n=5) Controls
(n=7)
Resting FBF 1.70.16 2.40.5 2.51.8
Peak FBF 20.44.8 22.94.5 26.46.5*
Total FBF AUC3 mn 7.21.1 8.12.6 7.71.5
Resting CBF 2.80.9 2.60.7 3.31.9
Peak CBF 17.40.5 22.44.4* 22.47.0*
Total CBF AUC3mn 9.34.1 11.27.3 7.15.4
Values are mean+SD; units are ml/min/100 ml tissue; *P<0.05 vs. PREHTX; tP<0.05 vs.
POSTHTX; PREHTX=pre-heart transplantation; POSTHTX=post-heart transplantation
BF=blood flow; AUC=area under flow x time curve.

Peak FBF was significantly greater in healthy controls compared to PREHTX (26.4

vs. 20.4 ml/min/100ml, p=0.05), but not significantly different compared to POSTHTX

(26.4 vs. 22.9 ml/min/100ml, p=NS). Peak CBF was significantly greater in the healthy

controls compared to PREHTX (22.4 vs. 17.4 ml/min/100ml, p=0.05), but not









significantly different than POSTHTX (22.4 vs. 22.4 ml/min/100ml, p=NS). There was

no significant difference between healthy controls, PREHTX, and POSTHTX in resting

FBF, resting CBF, total FBF AUC3min, and total CBF AUC3min.

Vasoactive Balance before and after Heart Transplantation

Plasma NOx and ET-1 are displayed in Table 4-6 and Figures 4-8 and 4-9. Plasma

NOx, the product of NO metabolism, was not significantly different in POSTHTX

compared to PREHTX (39.7 vs. 55.5 |jmol/L, p=NS, respectively). The endothelial-

derived vasoconstrictor ET-1, was not significantly different in POSTHTX compared to

PREHTX (4.9 vs. 4.1 pg/ml, p=NS, respectively).

Plasma NOx was significantly lower in age-matched healthy controls compared to

PREHTX (24.6 vs. 55.5 [lmol/L, p<0.05), but not significantly different than POSTHTX

(24.6 vs. 39.7 [Lmol/L, p=NS). Additionally, ET-1 was not significantly different in age-

matched healthy controls compared to PREHTX (5.0 vs. 4.1 pg/ml, p=0.15) and vs.

POSTHTX (5.0 vs. 4.9 pg/ml, p=NS).

Table 4-6 Vasoactive balance before and after heart transplantation
PREHTX POSTHTX Healthy
(n=12) (n=12) Controls
(n=7)
NOx (|umol/L) 55.537.7 39.723.9 24.617.6*
ET-1 (pg/ml) 4.12.2 4.93.0 5.04.9
Values are mean+SD; *P<0.05 vs. PREHTX; PREHTX=pre-heart transplantation;
POSTHTX=post-heart transplantation; NOx=nitrate/nitrite; ET-1= endothelin-1

Plasma Lipid Peroxidation, Antioxidant Defense, and Endogenous Nitric Oxide
Inhibition before and after Heart Transplantation

Plasma 8-iso-PGF2a, SOD activity, and ADMA are presented in Table 4-7 and

Figures 4-10, 4-11, and 4-12. There was no significant difference in plasma 8-iso-PGF2a

(1597.4 vs.1474.8 pg/ml, p=NS) or ADMA (0.65 vs. 0.65 [tmol/L, p=NS) in POSTHTX









vs. PREHTX, but compared to PREHTX, there was a significant decrease in SOD

activity in POSTHTX (2.16 vs. 1.79 U/ml, p<0.05, respectively).

Plasma 8-iso-PGF2a was significantly greater in the age-matched healthy controls

than PREHTX (2089.5 vs. 1474.8 pg/ml, p<0.05), but not POSTHTX (2089.5 vs. 1597.4

pg/ml, p=NS). SOD activity was not significantly different than PREHTX (2.56 vs. 2.16,

p=NS), but was significantly higher in healthy controls compared to POSTHTX (2.56 vs.

1.79 U/ml, p<0.01). Finally, plasma ADMA was not significantly different in healthy

controls than PREHTX (0.75 vs. 0.65 [lmol/L, p=NS) or POSTHTX (0.75 vs. 0.65

[lmol/L, p=NS).

Table 4-7 Lipid peroxidation, antioxidant enzyme activity, and endogenous nitric oxide
inhibition before and after heart transplantation
PREHTX POSTHTX Healthy Controls
(n=12) (n=12) (n=7)
8-iso-PGF2z (pg/ml) 1474.8564.9 1597.4566.2 2167.7248.2*"
SOD activity (U/ml) 2.160.54 1.790.34* 2.560.43t
ADMA (imol/L) 0.650.18 0.650.23 0.750.12
Values are mean+SD; *P<0.05 vs. PREHTX; tP<0.05 vs. POSTHTX; PREHTX=pre-
heart transplantation; POSTHTX=post-heart transplantation; PGF2a=prostaglandin F2
isoprostane; SOD=superoxide dismutase; ADMA= asymmetric dimethylarginine

Inflammatory Markers before and after Heart Transplantation

Plasma markers of CRP, logCRP, IL-6, TNF-a, and sICAM-1 are displayed in

Table 4-8 and Figures 4-13, 4-14, 4-15, 4-16, and 4-17. There was no significant

difference in PREHTX vs. POSTHTX in CRP (7.0 vs. 6.0 mg/L, p=NS, respectively) and

IL-6 (6.2 vs. 6.6 pg/ml, p=NS, respectively). However, CRP is well known to be non-

normally distributed in the population and is skewed to the right (Blake and Ridker

2003), therefore, log transformation of CRP was performed which resulted in a

significant decrease in log CRP from PREHTX to POSTHTX (0.75 vs. 0.51 mg/L,

p=0.05). Furthermore, there was a significant decrease in TNF-a (2.6 vs. 2.0 pg/ml,









p<0.05, respectively) and sICAM-1 (363.4 vs. 237.8 ng/ml, p<0.01, respectively) from

PREHTX to POSTHTX.

Age-matched healthy controls had significantly lower CRP (2.6 vs. 7.0 mg/L,

p<0.05), logCRP (0.28 vs. 0.75 mg/L, p=0.01), IL-6 (1.4 vs. 6.2 pg/ml, p<0.05), TNF-a

(1.8 vs. 2.6 pg/ml, p=0.05), and sICAM-1 (249.2 vs. 363.4 ng/ml, p<0.05) than

PREHTX, but only IL-6 was lower compared to POSTHTX (1.4 vs. 6.6 pg/ml, p<0.05).

Table 4-8 Inflammatory markers before and after heart transplantation
PREHTX POSTHTX Healthy Controls
(n=12) (n=12) (n=7)
CRP (mg/L) 7.024.20 6.005.72 2.632.04*
logCRP (mg/L) 0.750.32 0.51+0.58* 0.280.40*
IL-6 (pg/ml) 6.215.08 6.584.22 1.430.60*t
TNF-a (pg/ml) 2.630.84 1.970.54* 1.880.49*
sICAM-1 (ng/ml) 363.4144.5 237.861.6* 249.240.6*
Values are mean+SD; *P<0.05 vs. PREHTX; tP<0.05 vs. POSTHTX; PREHTX=pre-
heart transplantation; POSTHTX=post-heart transplantation; CRP=c-reactive protein; IL-
6= interluekin-6; TNF-a-tumor necrosis factor-a; sICAM-l=soluble intercellular adhesion
molecule-1

Baseline Subject Characteristics before Exercise Training or Control

Baseline characteristics of the sixteen transplant subjects who completed the

exercise intervention (TRAINED; n=9) or control (CONTROL; n=7) period are

displayed in Table 4-9. Subjects in the CONTROL group did not differ significantly

from the TRAINED group with respect to age, male/female ratio, ischemic etiology, days

after transplant, percentage on immunosuppressive therapy, dose of immunosuppressive

therapy, percentage on station therapy, percentage on ACEI/ARB therapy, percentage on

insulin therapy, or number of endocardial biopsy rejection episodes. There was a trend,

but no significant difference in body weight for the CONTROL vs. TRAINED at baseline

(90.4 vs. 78.6 kg, p=0.08), however, CONTROL had a significantly higher BMI than

TRAINED at baseline (28.5 vs. 25.5 kg/m2, p=0.05).









Table 4-9 Baseline patient characteristics before exercise training or control
CONTROL TRAINED
(n=7) (n=9)
Age (years) 54.39.5 54.413.1
Body weight (kg) 90.4+11.5 78.612.6
Body mass index (kg/m2) 28.51.4 25.53.6*
Male, no. (%) 6 (86) 7 (78)
Female, no (%) 1(14) 2(22)
Ischemic HF etiology, no. (%) 4 (57) 5 (56)
Days after transplant 73.6+30.6 67.3+11.2
Cyclosporine therapy, no. (%) 6 (86) 7 (78)
Cyclosporine dose (mg/day) 379.2123.0 328.677.0
Serum cyclosporine trough level (ng/dl) 353.8125.2 446.7268.7
Tacrolimus therapy, no. (%) 1 (14) 2 (22)
Prednisone therapy, no. (%) 7 (100) 9(100)
Prednisone dose (mg/day) 24.316.7 22.86.1
Mycophenolate mofetil therapy, no. (%) 7 (100) 9(100)
Mycophenolate mofetil dose (mg/day) 2714.3+393 2800.0632
Statin therapy, no. (%) 7(100) 9(100)
ACEI/ARB therapy, no. (%) 3 (43) 5 (56)
Calcium channel blocker therapy, no. (%) 2 (29) 3 (33)
Insulin therapy, no. (%) 2 (29) 5 (56)
Endocardial biopsy rejection episodes (no.) 12 14
Grade 1A/B mild (no.) 8 10
Grade 2 mild/moderate (no.) 3 2
Grade 3 moderate/severe (no.) 1 2
Grade 4 severe (no.) 0 0
Values are mean+SD. *P<0.05 vs. CONTROL; ACEI=angiotensin converting enzyme
inhibitor; ARB=angiotensin receptor blocker

Body Weight, Serum Metabolic Parameters, and Endocardial Rejection History
after Exercise Training

Fasting serum metabolic parameters in the CONTROL and the TRAINED group

before and after the 12-week control or exercise intervention period are displayed in

Table 4-10. Body weight significantly increased in the CONTROL group (90.4 vs. 96.0

kg, p=0.01), but not in the TRAINED group (78.6 vs. 80.9 kg, p=0.06) after 12 weeks.

There was no significant change in hemoglobin, hematocrit, total cholesterol LDL

cholesterol, HDL cholesterol, total cholesterol/HDL ratio, triglycerides, glucose, or

creatinine in the CONTROL or TRAINED group after 12 weeks. There was a significant

decrease in white blood cell count in the TRAINED group (p<0.05) after 12 weeks, but









not in the CONTROL group. One subject in each group tested positive for antibodies for

CMV infection at baseline but both were negative at the 12 weeks measurement.

Table 4-10 Body weight, serum metabolic parameters, and endocardial rejection
episodes at baseline and after exercise training or control


CONTROL (n=7)


Baseline


12 weeks


Body weight (kg) 90.411.5 96.0+11.0*
Hemoglobin (g/L) 11.51.3 11.8+1.1
Hematocrit (%) 35.64.2 34.83.1
Total cholesterol (mg/dl) 188.338.8 177.738.4
LDL cholesterol (mg/dl) 81.918.6 88.429.8
HDL cholesterol (mg/dl) 69.021.6 57.017.9
Total cholesterol/HDL ratio 2.840.57 3.240.80
Triglycerides (mg/dl) 188.357.3 161.358.6
Glucose (mg/dl) 104.126.7 114.448.8
Creatinine (mg/dl) 1.260.33 1.540.85
WBC, lx 109 (no.) 8.41.9 6.82.0
CMV positive IgG (no.) 1 0
Endocardial rejection (no.) 12 3
Grade 1 very mild (no.) 8 3
Grade 2 mild (no.) 3 0
Grade 3 moderate (no.) 1 0
Grade 4 severe (no.) 0 0
Data are mean + SD. *P<0.05 vs. Baseline within-groups;


TRAINED (n=9)
Baseline 12 Weeks
78.612.6 80.912.7t
11.11.2 11.41.3
33.92.6 34.94.8
193.915.1 172.720.0
97.618.5 86.318.6
65.620.3 61.818.5
3.231.12 3.030.94
153.962.2 122.679.0
87.417.6 103.728.3
1.440.52 1.320.47
7.32.2 5.51.2*
1 0
14 5
10 4
2 1
2 0
0 0
tP<0.05 vs. CONTROL at


same time-point; LDL=low-density lipoprotein; HDL=high-density lipoprotein;
CMV=cytomegliovirus; IgG=immunoglobulin G antibody; WBC=white blood cells

Brachial Artery Endothelial Function after Exercise Training

Brachial artery FMD results in the CONTROL and TRAINED group are displayed

in Table 4-11 and Figure 4-18 and 4-19. There was a significant decrease in brachial

artery FMD (11.1 vs. 7.9%, p<0.05) and the absolute change in diameter (0.51 vs. 0.39

mm, p<0.05) in the CONTROL group after 12 weeks, but no significant change in

brachial artery FMD (10.1 vs. 9.6%, p=NS) or absolute change in diameter (0.48 vs. 0.42

mm, p=NS) in the TRAINED group after 12 weeks of exercise training. Furthermore,

there was no significant change in baseline diameter in the CONTROL or TRAINED


group after 12 weeks.










Table 4-11 Brachial artery flow-mediated dilation at baseline and after exercise training
or control


CONTROL (n=7)
Baseline 12 weeks
Baseline diameter (mm) 4.500.60 4.460.64
Absolute change diameter (mm) 0.51+0.16 0.390.23*
Flow-mediated dilation (%) 11.1+2.6 7.95.1*
Data are mean SD. *P<0.05 vs. Baseline within-group.


TRAINED (n=9)
Baseline 12 Weeks
4.740.87 4.640.82
0.480.22 0.420.24
10.06.1 9.66.2


Blood Pressure and Pulse Wave Analysis after Exercise Training

Blood pressure components and pulse wave analysis results are displayed in Table

4-12 and Figure 4-20, and 4-21. There was no significant change in heart rate or

peripheral or central systolic, diastolic, pulse and mean blood pressure in the CONTROL

group or TRAINED group after 12 weeks. Pulse wave analysis results showed that there

was no significant change in augmentation pressure, Ala corrected for heart rate at 75

b/min, ATp, AsTTI, or DTPI in the CONTROL or TRAINED group after 12 weeks.

Table 4-12 Blood pressure components and pulse wave analysis at baseline and after
exercise training or control


HR (b/min)
PSBP (mmHg)
PDBP (mmHg)
PPBP (mmHg)
CSBP (mmHg)
CDBP (mmHg)
CPBP (mmHg)
MBP (mmHg)
AgBP (mmHg)
AI @ HR=75 b/min (%)
Atp (ms)
AsTTI (mmHg/sec/min)
DPTI (mmHg/sec/min)


CONTROL (n=7)


12 weeks
95.19.7
144.916.8
94.09.9
50.99.1
127.616.5
95.310.0
32.37.5
110.013.1
2.93.1
17.310.0
143.410.3
3296.0533
3253.6329


Baseline
90.77.4
150.618.3
93.114.1
57.48.9
132.117.9
94.914.1
37.38.7
111.315.1
4.35.4
17.013.3
141.613.5
3389.1598
3263.7462


TRAINED (n=9)
Baseline 12 Weeks
95.813.7 91.1+17.4
132.9 18.1 129.416.5
89.710.3 87.011.3
43.515.0t 42.412.8
116.614.1 114.414.0
90.79.7 88.210.9
25.98.9t 26.27.2
102.110.6 99.912.1
0.32.9 1.63.2
11.414.5 14.313.4
142.99.7 145.89.7
3134.0524 2925.5444
3054.4583 3013.9429


Data are mean SD. tfP<0.05 vs. CONTROL at same time-point; HR=heart rate;
PSBP=peripheral systolic blood pressure; PDBP=peripheral diastolic blood pressure;
PPBP=peripheral pulse pressure; CSBP=central systolic blood pressure; CDBP=central
diastolic blood pressure; CPBP=central pulse blood pressure; MBP=mean blood pressure;
AGBP=augmentation blood pressure; Ala=augmentation index; Atp=round trip travel time
of reflected pressure wave from ascending aorta to peripheral reflecting sites and back;
AsTTI=aortic systolic tension-time index; DPTI=diastolic perfusion time index









Forearm and Calf Resistance Artery Blood Flow after Exercise Training

Forearm and calf resistance artery blood flow during reactive hyperemia in the

CONTROL and TRAINED group are displayed in Table 4-13 and Figure 4-22 and 4-23.

Peak FBF increased 34% (21.5 vs. 28.8 ml/min/100ml, p<0.05) in the TRAINED group

after 12 weeks compared to baseline, but there was no significant increase (+14%) in

peak FBF (25.5 vs. 29.2 ml/min/100ml, p=0.08) in the CONTROL group. There was no

significant change in resting FBF or total FBF AUC3 min in the CONTROL or TRAINED

group after 12 weeks.

There was a significant 17% increase in peak CBF (25.0 vs. 29.3 ml/min/100 ml,

p=0.05) in the TRAINED group after 12 weeks compared to baseline, and no significant

change (-4%) in the CONTROL group (26.3 vs. 25.2 ml/min/100ml, p=NS). There was

no significant increase in resting CBF or total CBF AUC3 min in the CONTROL and

TRAINED group after 12 weeks.

Table 4-13 Forearm and calf flow-mediated vasodilation at baseline and after exercise
training or control
CONTROL (n=5) TRAINED (n=6)
Baseline 12 weeks Baseline 12 Weeks
Resting FBF 2.40.5 2.60.7 2.91.0 3.31.1
Peak FBF 25.511.6 29.28.1 21.54.6 28.83.7*
Total FBF AUC3mn, 14.213.0 13.94.4 9.85.2 10.33.0
Resting CBF 3.01.1 3.00.7 3.01.0 3.72.8
Peak CBF 26.35.4 25.25.8 25.06.4 29.37.7*
Total CBF AUC3mn 18.37.3 10.93.0 13.510.6 14.812.2
Values are mean+SD; Units are ml/min/100 ml tissue; *P<0.05 vs. Baseline within-
group; tP<0.05 vs. CONTROL at same time-point; BF=blood flow; AUC=area under
flow x time curve

Vasoactive Balance after Exercise Training

Plasma NOx and ET-1 in the CONTROL and TRAINED group after 12 weeks

compared to baseline are displayed in Table 4-14 and Figure 4-24 and 4-25. There was

no significant change in plasma NOx from baseline in the CONTROL (30.5 vs. 45.3









pmol/L, p=NS) or TRAINED group (42.7 vs. 56.1 |jmol/L, p=NS). There was a was a

trend but no significant change in ET-1 in the TRAINED group (-41.4%) after 12 weeks

compared to baseline (3.8 vs. 2.2 pg/ml, p=0.09), and no significant change in ET-1 in

the CONTROL group (-36.5%) after 12 weeks compared to baseline (4.4 vs. 2.8 pg/ml,

p=0.15).

Table 4-14 Vasoactive balance at baseline and after exercise training or control
CONTROL (n=7) TRAINED (n=9)
Baseline 12 weeks Baseline 12 Weeks
NOx (imol/L) 30.541.6 45.314.2 42.7323.7 56.141.2
ET-1 (pg/ml) 4.453.01 2.820.90 3.832.54 2.241.02
Values are meanSD; *P<0.05 vs. Baseline within-group; tP<0.05 vs. CONTROL at
same time-point; NOx=nitrate/nitrite; ET-1=endothelin-1.

Lipid Peroxidation, Antioxidant Enzyme Activity, and Endogenous Nitric Oxide
Inhibition after Exercise Training

Plasma levels of 8-iso- PGF2a, SOD activity, and ADMA in the CONTROL and

TRAINED group compared to baseline are displayed in TABLE 4-15 and Figure 4-26, 4-

27, and 4-28. There was no significant change in 8-iso-PGF2a, SOD activity, or ADMA

in the CONTROL or TRAINED group after 12 weeks compared to baseline.

Table 4-15 Lipid peroxidation, antioxidant enzyme activity, and endogenous nitric oxide
inhibition at baseline and after exercise training or control
CONTROL (n=7) TRAINED (n=9)
Baseline 12 weeks Baseline 12 Weeks
8-iso-PGFz2 (pg/ml) 1815.0516.7 1711.3904.1 1387.8+506 1535.0514
SOD activity (U/ml) 1.670.36 1.750.48 1.860.35 1.800.67
ADMA (timol/L) 0.730.26 0.720.25 0.600.17 0.680.23
Values are meanSD; *P<0.05 vs. Baseline within-group; tP<0.05 vs. CONTROL at
same time-point; PGF2a=prostaglandin F2a isoprostane; SOD=superoxide dismutase;
ADMA= asymmetric dimethylarginine

Inflammatory Markers after Exercise Training

Plasma CRP, IL-6, TNF-ac, and sICAM-1 in the CONTROL and TRAINED group

after 12 weeks compared to baseline are displayed in Table 4-16 and Figures 4-29, 4-30,









4-31, and 4-32. There was a non-significant 19% decrease in plasma CRP in the

CONTROL group (5.62 vs. 4.54 mg/L, p=NS) and a non-significant 49% decrease in the

TRAINED group (5.03 v. 2.55 mg/L, p=NS) after 12 weeks compared to baseline. Log

transformation of CRP did not alter the results in either group. There was no change in

IL-6 in the CONTROL group (6.65 vs. 6.16 pg/ml, p=NS) and a non-significant 35%

decrease in IL-6 in the TRAINED group (5.02 vs. 3.25 pg/ml, p=NS) after 12 weeks

compared to baseline. However, there was a significant increase in TNF-a (1.56 vs. 2.38

pg/ml, p<0.05) in the CONTROL group, but no significant change in TNF-a in the

TRAINED group (1.83 vs. 1.79 pg/ml, p=NS) after 12 weeks. Lastly, there was no

significant change in sICAM-1 in the CONTROL (269.2 vs. 295.6 ng/ml, p=NS) or

TRAINED group (216.5 vs. 198.2 ng/ml, p=NS) after 12 weeks compared to baseline.

Furthermore, TNF-a (1.79 vs. 2.38 pg/ml, p<0.05) and sICAM-1 (198.2 vs. 295.6 ng/ml,

p<0.05) were significantly lower at 12 weeks in TRAINED compared to CONTROL at

12 weeks.

Table 4-16 Inflammatory markers at baseline and after exercise training or control
CONTROL (n=7) TRAINED (n=9)
Baseline 12 weeks Baseline 12 Weeks
CRP (mg/L) 5.624.29 4.543.36 5.036.55 2.552.81
IL-6 (pg/ml) 6.655.03 6.162.72 5.023.15 3.252.55
TNF-a (pg/ml) 1.560.38 2.380.79* 1.830.59 1.790.50t
sICAM-1 (ng/ml) 269.2104.8 295.686.1 216.564.0 198.239.1t
Values are mean+SD; *P<0.05 vs. Baseline within-group; tP<0.05 vs. CONTROL at
same time-point; CRP=c-reactive protein; IL-6=interluekin-6; TNF-a-tumor necrosis
factor-alpha; sICAM-1=soluble intercellular adhesion molecule-1

Peak Cardiopulmonary Exercise Testing Variables after Exercise Training

Peak cardiopulmonary variables during graded exercise testing with respiratory gas

analysis in the CONTROL and TRAINED group are displayed in Table 4-17 and Figure

4-33 and Figure 4-34. There was no significant change in peak heart rate, peak systolic









blood pressure, peak diastolic blood pressure in the CONTROL or TRAINED group after

12 weeks compared to baseline. There was no significant change in peak oxygen uptake

(V02) in the CONTROL group (16.2 vs. 16.8 ml/kg/min, p=NS), but there was a

significant 26% increase in peak VO2 in the TRAINED group (15.4 vs. 19.4 ml/kg/min,

p<0.01) after 12 weeks compared to baseline. Moreover, there was no significant change

in exercise duration in the CONTROL group (561.5 vs. 554.5 sec, p=NS), but there was a

significant 44.5% increase in exercise duration in the TRAINED group (518.9 vs. 750.0

sec, p<0.01) after 12 weeks compared to baseline. Lastly, there was no significant

difference in peak respiratory exchange ratio (RER) and rating perceived exertion (RPE)

in the CONTROL or TRAINED group after 12 weeks compared to baseline.

Table 4-17 Peak cardiopulmonary graded exercise testing variables at baseline and after
exercise training or control
CONTROL (n=7) TRAINED (n=9)
Baseline 12 weeks Baseline 12 Weeks
Peak HR (b/min) 123.710.2 133.014.0 124.316.6 124.942.7
Peak systolic BP (mmHg) 173.721.0 178.318.1 147.123.7t 167.225.0
Peak diastolic BP (mmHg) 88.75.9 90.37.1 84.016.9 83.617.9
Peak V02 (ml/kgBW/min) 16.25.2 16.82.8 15.44.3 19.45.5*
Peak RER 1.050.07 1.010.08 1.050.08 1.080.06t
Peak RPE 16.21.6 16.31.2 15.41.6 15.31.5
Peak exercise duration (sec) 561.5202.8 554.5110.9 518.9198.2 750.0274.4*
Data are mean + SD. *P<0.05 vs. Baseline within-groups; tP<0.05 vs. CONTROL at
same time-point; HR=heart rate; BP=blood pressure; VO2=rate of oxygen consumption;
BW=body weight; RER=respiratory exchange ratio; RPE=rating of perceived exertion
(Borg scale)






83



^ 12.5-
2. l PREHTX (n=12)
10.0- I POSTHTX (n=12)
3 Healthy Controls (n=7)
7.5-

5.0

E 2.5
0
E 0.0


Figure 4-1. Brachial artery flow-mediated dilation before and after heart transplantation.
*P<0.05 vs. PREHTX.

*
0.5-
E PREHTX (n=12)
E 0.4- POSTHTX (n=12)
o" 03 Healthy Controls (n=7)


S0.2

E 0.1

0.0
Figure 4-2. Brachial artery flow-mediated diameter dilation before and after heart
transplantation. *P_<0.05 vs. PREHTX; #P<0.05 vs. POSTHTX.
25-
.c PREHTX (n=12)
20- M POSTHTX (n=12)
LO Healthy Controls (n=7)
15-
10,


157

0
Figure 4-3. Aortic augmentation index (Ala) corrected for heart rate=75 b/min before and
after heart transplantation.









200-
S17PREHTX (n=12)
175-
1POSTHTX (n=12)


S100-
< 750- I Healthy Controls (n=7)


0.
75
50-
25-
0
Figure 4-4. Roundtrip travel duration of reflected wave (Atp) before and after heart
transplantation.


3500-
-G= PREHTX (n=12)
S3000' *# POSTHTX (n=12)
2500- = Healthy Controls (n=7)
2000-
E 1500-
E
1000-
I-
500
0

Figure 4-5. Aortic systolic tension-time index (AsTTI) before and after heart
transplantation. *P<0.05 vs. PREHTX; #P<0.05 vs. POSTHTX.


= PREHTX (n=5)
SPOSTHTX (n=5)
= Healthy Controls (n=7)


LU
M
L..
U 0

Rest FBF Peak FBF AUC FBF
Figure 4-6. Forearm blood flow (FBF) before and after heart transplantation. *P<0.05 vs.
PREHTX; AUC=area under blood flow x time curve for 3 min











25-
o
S20-

E 15-
E 10-
L.
) 5-
0o--

Figure 4-7.


70-
60-
50-

E 40-
30-
x
z 20


* *


1 PREHTX (n=5)
POSTHTX (n=5)
E Healthy Controls (n=7)


Rest CBF Peak CBF AUC CBF
Calf blood flow (CBF) before and after heart transplantation. *P<0.05 vs.
PREHTX; AUC=area under blood flow x time curve for 3 min.


TL


l PREHTX (n=12)
POSTHTX (n=12)
- Healthy Controls (n=7)


-71


Figure 4-8. Nitrate/nitrite (NOx) before and after heart transplantation. *P<0.05 vs.
PREHTX.


=* 5
E
) 4-
- 3-
LU 2-
1.


-I


= PREHTX (n=12)
POSTHTX (n=12)
= Healthy Controls (n=7)


ON I I I
Figure 4-9. Endothelin-1 (ET-1) before and after heart transplantation.