Ill L N WNW I 1 W(WWWNW)INHWIHHHII _THS' aaw l . LIBRARY 400/, Michigan State University This is to certify that the dissertation entitled Fibrinolytic Adaptations to a Phase II Cardiac Rehabilitation Program presented by Paul Robert Nagelkirk has been accepted towards fulfillment of the requirements for the PhD degree in Department of Kinesiology w j t/WZ MMafbr Profés’sor’ 5 Signature 6// MC? Date 1 MSU is an Affirmative Action/Equal Opportunity Institution PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE SE P 2 '9'; 3.102% FIBRINOLYTIC ADAPTATIONS TO A PHASE II CARDIAC REHABILITATION PROGRAM By Paul Robert Nagelkirk A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Kinesiology 2005 ABSTRACT F IBRINOLYTIC ADAPTATIONS TO A PHASE II CARDIAC REHABILITATION PROGRAM By Paul Robert Nagelkirk F ibrinolysis, the process of dissolving a fibrin blood clot, plays a pivotal role in the development of vascular disease. Occlusive blood clots are responsible for most acute cardiovascular events, and patients with coronary artery disease (CAD) typically exhibit a blunted fibrinolytic capacity. Initiation of fibrinolysis involves the conversion of plasminogen to plasmin, which is primarily catalyzed by tissue plasminogen activator (tPA). Depressed tPA and elevations of its primary inhibitor, plasminogen activator inhibitor-1 (PAI-l) are associated with morbidity, mortality, and are independent risk factors for various cardiovascular outcomes. Exercise training promotes enhanced fibrinolytic potential in healthy individuals, and individuals with CAD who undergo 12 or more weeks of regular exercise as part of a cardiac rehabilitation program demonstrate improvements in tPA and PAL]. Modern day third—party reimbursement practices often necessitate fewer than 12 weeks of exercise training in cardiac rehabilitation programs. It is unclear if training regimens shorter than 12 weeks will elicit fibrinolytic improvements. The purpose of the present study was to evaluate changes in plasma concentrations of tPA and PAI-l , as well as changes in expression of the tPA and PAI-l genes in whole blood afier three and six weeks of participation in a phase H cardiac rehabilitation program. Fourteen CAD patients (12 male, 2 female) trained three days/week for six weeks. Exercise sessions adhered to American College of Sports Medicine (ACSM) guidelines for intensity and duration. Blood samples were taken at baseline (BL), after three weeks (3W), and afier six weeks of training (6W) in a cardiac rehabilitation program and analyzed for tPA activity and antigen, PAI-l activity and antigen, and relative quantification of tPA and PAL] RNA. Linear regression revealed no confounding influences on any outcome variable. Data were then analyzed using repeated measures analysis of variance. Six weeks of training resulted in significant decreases in submaximal exercise heart rate and systolic blood pressure (SBP), and resting SBP (p<0.05). No significant changes in plasma concentrations of tPA activity (BL=O.69 d: 0.44, 3W=O.94 d: 0.62, 6W=0.77 i 0.49 ng/ml, mean :I: SD, p=0.391) or antigen (BL=13.1 i 3.9, 3W=12.4 :t 3.7, 6W=11.8 i 3.8, mean :I: SD, p=0.59) were observed. No change was observed in plasma PAI-l activity (BL=17.0 d: 16.8, 3W=14.8 i 22.5, 6W=17.9 i 18.8 IU/ml, mean 1: SD, p=0.29) or antigen (BL=28.3 d: 15.5, 3W=24.2 :i: 20.2, 6W=22.4 d: 16.1 ng/ml, mean 2!: SD, p=0.15). No change in tPA (p=0.45) or PAl-l (p=0.44) gene expression was observed during six weeks of exercise training. The six-week cardiac rehabilitation program yielded significant hemodynamic improvements, but did not alter fibrinolytic capacity. Based on the results of the present study and evidence in the literature, it is recommended that traditional cardiac rehabilitation programs that subscribe to ACSM guidelines include at least 12 weeks of regular exercise. ACKNOWLEDGEMENTS This dissertation required a great deal of time and effort from numerous individuals. Barry A. Franklin, Ph.D., director of Cardiac Rehabilitation at William Beaumont Hospital, enthusiastically supported this project and volunteered his staff and facilities. He was instrumental in procuring funding, and has proven to a valuable partner. I also wish to specifically thank the individual members of my dissertation committee. Drs. Suzy Hassouna, Greg Fink, Jim Pivarnik, and Chris Womack were extremely gracious, and only their flexibility and understanding allowed me to proceed quickly through this process so that I could assume the duties of my new life and career. Each time we met, Suzy Hassouna never failed to teach me something new about a topic I was naive enough to think I thoroughly understood. Greg Fink is the only one I know who could address some challenging statistical issues that arose, and is one of those rare individuals who genuinely understands and appreciates the work done outside of his own department. I consider Jim Pivarnik a co-mentor, and cannot count the ways he contributed to my professional development. A source of occasional consternation, Pivarnik’s input regarding my research always strengthened my work. There are few people I value more than J .P. as an ally, advisor, and colleague. Chris Womack has been much more to me than a mentor. Professionally, he provided me with opportunities to conduct original research projects, and publish and present our findings. He is the example after which I model my own teaching practices, and he showed me ways to keep education exciting both for me and my students. The training I received under his tutelage is singularly the most important factor in my career preparation. Furthermore, iv C] W showed me how to remain a committed husband and father while keeping up with the rigors of academia. Most importantly, he is a living example of how one’s faith can and should permeate all aspects of one’s life. My advisor, brother, and fiiend, Chris has my undying gratitude. My friends in the I-[ERL kept me sane through my four years at MSU. We have a good team, and I will miss each of them. In particular, I wish to thank J 0 Ann Janes, who so willingly helped me with countless tasks, and indulged my sarcasm with great patience. I also cannot ignore Adam Coughlin, with whom I have shared notes, research ideas, an office and, at times, a brain. I am extremely grateful for his collaboration on this and many other projects, not to mention his friendship. Finally, and most importantly, I must thank my wife, Stephanie. She deserves more than this, given the sacrifices she made on my behalf. She willingly learned boring physiology concepts just to better understand my interests, and lefi more than one fulfilling career to move across the country to allow for the pursuit of my education. Stephanie endured financial strain, job changes, distance from friends and family simply to follow my dreams. I could not have completed an advanced degree without her support. She and our son, Jack, gave a lot for the sake of this PhD. I love and appreciate them more than I can say. TABLE OF CONTENTS LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii CHAPTER 1 - INTRODUCTION ...................................................................................... 1 CHAPTER 2 - REVIEW OF LITERATURE ..................................................................... 5 Fibrinolysis mechanisms - overview ...................................................................... 5 Cardiovascular disease and fibrinolysis .................................................................. 7 Exercise training and fibrinolysis ........................................................................... 8 Exercise training and cardiovascular disease ........................................................ 12 Cardiac rehabilitation and fibrinolysis .................................................................. 15 Summary: .............................................................................................................. 19 CHAPTER 3 - RESEARCH DESIGN / METHODS ....................................................... 21 Subjects ..................................................................................... 21 Training: ................................................................................................................ 22 Laboratory Measures: ........................................................................................... 22 Assays: .................................................................................................................. 23 Statistical Analysis: ............................................................................................... 23 CHAPTER 4 - RESULTS ................................................................................................. 26 CHAPTER 5 - DISCUSSION .......................................................................................... 32 APPENDD( A - CONSENT FORM, MICHIGAN STATE UNIVERSITY .................... 38 APPENDIX B - CONSENT FORM, WILLIAM BEAUMONT HOSPITAL ................. 42 APPENDIX C - HUMAN SUBJECTS APPROVAL, MICHIGAN STATE UNIVERSITY ..................................................... 47 APPENDIX D - HUMAN SUBJECTS APPROVAL, WILLIAM BEAUMONT HOSPITAL .................................................. 49 WORKS CITED .............................................................................................................. 51 vi LIST OF TABLES Table 1. Subject Anthropometric Characteristics ...................................... 28 Table 2. Resting and Submaximal Exercise Hemodynamics ........................ 28 vii Figure 1. Figure 2. Figure 3. LIST OF FIGURES Individual values plus means at SE for tPA activity and antigen at baseline (BL), after three weeks (3W) and after six weeks (6W) of exercise training ................................................................. 29 Individual values plus means :L- SE for PAL] activity and antigen at baseline (BL), after three weeks (3W) and afier six weeks (6W) of exercise training ................................................................. 30 tPA and PAI-l mRNA expression at baseline (BL), after three weeks (3W), and after six weeks (6W) of exercise training. Data are expressed as means :1: SE ............................................. 31 viii Chapter 1 - INTRODUCTION Hemostasis is defined as the cessation of bleeding. Following injury, blood loss is stemmed by the interaction of the severed blood vessel, platelets and soluble coagulation factors to form an insoluble fibrin clot. Fibrin clots are the result of a series of enzymatic reactions that ultimately cause the release of thrombin from prothrombin. A fibrin clot, or thrombus, may impair blood flow if not eliminated. F ibrinolysis is the process by which an insoluble fibrin clot is degraded into fibrin dimer proteins, which are quickly cleared from circulation by the liver. Plasmin, the active form of the zymogen plasminogen, may cause proteolytic digestion of many coagulation proteins and is singularly responsible for fibrin dissolution. Thus, the key to stimulation of fibrinolysis is the conversion of plasminogen to plasmin. The most abundant and rapid plasminogen activator in the blood is tissue plasminogen activator (tPA). tPA is synthesized and released from the endothelium, and other tissues such as leukocytes (153) and sympathetic neurons (71, 72). The primary inhibitor of tPA is plasminogen activator inhibitor-1 (PAI-l), which is produced by endothelial cells, smooth muscle cells, adipocytes, spleen cells, liver cells (4), and leukocytes (6, 16, 152). PAI-l inhibits tPA by forming an inactive bimolecular complex. Plasma concentrations of tPA and PAI-l are widely accepted markers of fibrinolytic activity and correlate with fibrinolysis as assessed by euglobulin clot-lysis time. Coagulation and fibrinolysis play pivotal roles in the development of vascular disease. Vascular injury may be initiated by atherosclerotic plaque, leading to clot formation in an intact arterial wall. Ischemic coronary syndromes such as myocardial infarction (MI), sudden death, and unstable angina share a common pathophysiological course that includes thrombus formation in or around ruptured coronary plaque (26, 50). Atheromatous lesions contain an abundance of pro-thrombotic elements as well as PAI-l, fibrinogen, fibrin, and fibrin degradations products (112, 147). In addition to the plaque itself, atherogenesis is characterized by chronic inflammation (63, 79), which, in turn, induces a procoagulant state. Furthermore, there is evidence of altered fibrinolytic status among patients with advanced CVD (22). Both PAI-l and tPA are considered independent risk factors for cardiovascular disease (CVD). More specifically, decreased tPA and increased PAI-l are associated with CVD (27, 76, 123, 131, 146), coronary artery disease (104, 122), ischemic events (24, 25, 56, 60, 142), stroke (73, 85), and morbidity and mortality (69, 90, 98, 141). Furthermore, impaired fibrinolysis is associated with other CVD risk factors (7, 43, 68, 75, 85, 92, 131). Thus, fibrinolysis is of considerable clinical significance and represents a viable target for therapeutic intervention in CVD. Enhanced fibrinolysis is one of many cardioprotective adaptations associated with endurance exercise training. Athletes and individuals who report high levels of physical activity exhibit lower PAI-l activity, lower tPA antigen (which is indicative of less tPA bound with PAH) and reduced tPA/PAI-l complex formation compared to matched sedentary controls (30, 32, 81, 134). Longitudinal data confirm that aerobic training decreases tPA antigen and PAL] activity (37, 145). A clear mechanism for the observed training-related changes in fibrinolysis has not been posited. Changes in plasma concentrations of tPA and PAI-l may be the result of altered release rates of the stored proteins or a change in hepatic clearance. However, plasma concentrations of tPA and PAI-l are largely genetically determined. Classic twin studies have produced heritability estimates ranging from 42%—71% (l 7, 67) for PAI- land 30-60% for tPA (29, 48, 133). Expression of the tPA and PAI—l genes in response to exercise training has not been studied, but mRNA levels have been reported to be modulated by elements that are known to be influenced by exercise. Experimental models of augmented aerobic metabolism results in increased tPA mRNA and reduced PAI-l mRNA (33). Additionally, TGF-beta, a cytokine known to rise in response to acute physical exertion, upregulates PAI-l (154). This response is attenuated by vascular endothelial growth factor (154), which increases in response to short-term exercise training (52). Thus, gene expression may be a significant contributor to any training- related adaptations in fibrinolysis. Exercise training is a hallmark of traditional cardiac rehabilitation. Aerobic exercise elicits positive alterations at molecular, systemic, and whole body levels. Recent evidence suggests that individuals with the poorest baseline fibrinolytic capacity may realize the greatest improvement through regular exercise (77). Thus, individuals with CVD, who typically exhibit blunted fibrinolysis, may be more likely to experience hemostatic improvements through training compared to healthy men and women. Cardiac rehabilitation programs typically include three days per week of supervised exercise lasting 20-60 minutes per session. Programs usually continue for 6- 12 weeks. Intensity of exercise is titrated to optimize training benefits while minimizing patient risk. To date, six longitudinal studies have been published exploring the influence of cardiac rehabilitation on fibrinolytic parameters. Detailed in the following chapter, most studies utilized training regimens that are atypical of traditional cardiac rehabilitation programs. Many investigators have trained patients for 24-26 weeks (40, 77, 116, 121), and others utilized training intensities that were either far greater (138) or much less demanding (121) than recommended by the American College of Sports Medicine (3). Since studies of healthy individuals indicate that fibrinolytic improvements are highly dependent upon the specific exercise prescription (12, 37), methodological differences make it unclear if a traditional exercise-based cardiac rehabilitation program that adheres to international guidelines will elicit fibrinolytic improvements. This question is particularly important as insurance companies are now reimbursing hospitals and clinics for fewer rehabilitation sessions than in previous years, often compensating for no more than 18 sessions in six weeks (personal communication, Adam deJong, 7/26/2005). Understanding the time course of fibrinolytic adaptations during exercise training may provide insight as to the effectiveness of traditional cardiac rehabilitation programs. The purpose of the present study was two-fold: (1) to assess fibrinolytic adaptations after three and six weeks of participation in an exercise-based cardiac rehabilitation program; and (2) to evaluate changes in expression of the tPA and PAI-l genes after three and six weeks of participation in an exercise-based cardiac rehabilitation program. It was hypothesized that that no modifications would be observed in any measured variable after three weeks of cardiac rehabilitation, and that significant changes would be observed in plasma concentrations and gene expression of tPA and PAI-l afier six weeks of training. Chapter 2 - REVIEW OF LITERATURE Fibrinolysis mechanisms - overview Hemostasis is defined as the cessation of bleeding. Occurring in distinct phases, hemostasis is initiated with platelets interacting with injured blood vessels and other platelets. This phase, known as primary hemostasis, ends with the formation of a clump of platelets. This primary hemostatic plug serves to temporarily arrest bleeding, but is fragile and easily dislodged from the vessel wall. Secondary hemostasis involves the interaction of soluble plasma proteins, or coagulation factors, in a series of complex enzymatic reactions that conclude with the thrombin catalyzed conversion of fibrinogen to insoluble fibrin. Deposition of fibrin strands on the platelet plug stabilizes the clot and allows healing to occur without further loss of blood. If a clot remained intact afier the damaged tissue healed, the vascular bed might become obstructed. Fibrinolysis, sometimes referred to as tertiary hemostasis, regulates the process of dissolving a fibrin clot. Fibrinolysis is activated in response to the initiation of the coagulation cascade. Activation of the fibrinolytic system produces plasmin, a proteolytic enzyme that is the active form of the zymogen plasminogen, which is able to digest fibrin or fibrinogen. The key factors in fibrinolysis are: (1) plasminogen; (2) plasmin; (3) plasminogen activators; and (4) plasminogen activator inhibitors. The latter two elements are the focal points of the present investigation. Tissue plasminogen activator (tPA), a serine protease, is a rapid activator of plasminogen. Derived primarily from the endothelium, tPA can be found in other tissues such as leukocytes (153), sympathetic neurons (71, 72), the heart, kidneys and other organs (89). tPA has an affinity for fibrin with which it forms a bimolecular complex. The catalytic efficiency of tPA for the activation of plasminogen is increased 1,000-fold in the presence of fibrin. Non-bound tPA has a low affinity for plasminogen and is thus not efficient in producing plasmin. Synthesis and release of tPA are stimulated by coagulation factor Xa, thrombin, bradykinin, and protein C. Furthermore, plasma concentrations of tPA may be elevated in response to hypotensive shock, phannacologic stimulators, venous stasis, and physical exertion. tPA is inhibited by a2-macroglobulin, dl-antitrypsin, antithrombin III, a2-antiplasmin, and a family of plasminogen activator inhibitors. Plasminogen activator inhibitor-1 (PAI-l), part of the serine protease inhibitor (serpin) superfarnily, provides rapid, specific inhibition of tPA and is the primary inhibitor of tPA in blood. PAI-l can occur in an active inhibitory form, which inhibits tPA by forming a 1:1 complex with it. This form is unstable and will spontaneously convert to an inactive latent form that does not react with tPA (64). In humans, PAI-l is produced by endothelial cells, smooth muscle cells, adipocytes, spleen cells, liver cells (4), and blood leukocytes (6, 16, 152). PAI-l gene expression is induced by endotoxin, inflammatory cytokines (57, 127), lipoprotein (57), angiotensin II (42), transforming grth factor-B (TGF-B) (124), tumor necrosis factor-(1(TNF-a) (58) and hypoxia (143). The main reservoir of PAI-l in the blood exists in platelets (100-200 11ng in non- pathological conditions) but only 10% of this PAI-l pool occurs in the active conformation (13, 31, 80). Plasma PAI-l represents a much smaller source (5-20ng/ml) but is the most active pool in the blood (89). Blood concentrations of PAH increase exponentially when platelets are activated due to injury or pathology. Cardiovascular disease and fibrinolysis Atherosclerosis is a complex, multifactorial process. However, ischemic coronary syndromes such as myocardial infarction (MI), sudden death, and unstable angina share common a pathophysiological course that includes thrombus formation in or around ruptured coronary plaque (26, 50). Autopsy studies that observed ruptured or cracked coronary atherosclerotic lesions in individuals without evidence of myocardial infarction (93) suggest plaque rupture must occur in combination with prothrombotic conditions in order for an ischemic event to occur. In vitro studies have shown that fibrin complexes formed in plasma from patients with a previous MI have tighter, more substantial network structures than gels formed in plasma from healthy subjects (10). There is substantial evidence that hypercoaguability and depressed fibrinolytic capacity promote the formation and maintenance of thromboses both systemically and locally at the exposed surface of damaged plaque (86). Prospective studies have demonstrated that plasma concentrations of prothrombotic markers are predictors of subsequent cardiovascular events in healthy subjects (87, 91, 115, 118, 132), individuals with cardiovascular risk factors (74, 115) and evident coronary disease (142). PAH and tPA, the two critical fibrinolytic proteins that are the focus of this study, are considered independent risk factors for cardiovascular disease (CVD). Decreased tPA and increased PAI-l are associated with CVD (27, 76, 123, 131, 146), coronary artery disease (104, 122), ischemic events (24, 25, 56, 60, 142), stroke (73, 85), morbidity and mortality (69, 90, 98, 141) in both men and women. Increased PAI-l levels have been found in atherosclerotic lesions within the vessel wall (117, 128) and it is now understood that PAI-l contributes to atherosclerotic progression in addition to its role in fibrinolysis, including promotion of neointimal formation afier vascular injury (106, 108). The magnitude of impairment in fibrinolytic potential may correspond to the extent of CVD, as tPA activity is higher in PAD patients with mild claudication versus patients with severe claudication (76). Furthermore, impaired fibrinolysis is independently associated with other CVD risk factors, including body composition (43, 75, 137), hyperlipidemia (7, 68, 85, 92, 131), diabetes, BMI, and low-density lipoprotein concentration (75). These data indicate a potential mechanistic link to the risk factors for CVD as well as the disease itself. Exercise training and fibrinolysis Exercise training elicits numerous physiological adaptations that relate to improved cardiovascular health. Among these cardioprotective adaptations is enhanced fibrinolytic capacity. Resting tPA and PAI-l are correlated with maximal oxygen consumption (32), the gold standard indicator of aerobic fitness, and cross-sectional data indicate individuals who are regularly active demonstrate greater fibrinolytic potential than sedentary individuals. Speiser et al. (134) observed lower resting PAI-l activity in younger athletes compared to sedentary controls. Lower tPA antigen, indicative of less tPA bound with PAI-l, has been observed in athletes compared to matched sedentary controls (81, 134). Furthermore, Depaz, et al. (30) observed reduced tPA/PAI-l complex formation in trained versus untrained individuals. These cross-sectional observations have been supported by longitudinal data. Van den burg and associates (145) documented decreased tPA antigen, PAI-l activity and PAL] antigen in a group of young healthy males that participated in twelve weeks of aerobic exercise training. Furthermore, these investigators observed an increase in the tPA activity/tPA antigen ratio. Exercise training may also influence the fibrinolytic response to acute bouts of exercise. Ferguson et a1. (44) reported significantly higher elevations in global fibrinolysis, assessed by clot-lysis time, in trained versus untrained individuals following a maximal exercise test. Speiser et al. (134) observed higher elevations in tPA activity following maximal cycle ergometry in trained compared to untrained subjects. However, it was suggested that this was due to lower resting PAI-l in the trained subjects, as tPA release was similar for both groups during exercise. Szymanski and Pate (139) also observed that active men experienced greater increases in tPA activity than sedentary men during moderate intensity exercise. Similar to the observations of Speiser’s group, the inactive subjects also had higher resting PAI-l activity. Furthermore, van den Burg, et al. (145) reported the relative increase in tPA from a maximal exercise test was not affected by training status. However, these authors did observe a significantly elevated tPA activity to antigen ratio post-exercise in the aerobically trained group. The relative change in this ratio from resting values was not significant between the trained and sedentary subjects. These data suggest baseline fibrinolytic profile may be the biggest influence of post-exercise values rather than differences in the magnitude of response. Fibrinolytic adaptations to regular exercise may depend on training methods. Yamell, et al. (155) reported that neither leisure-time nor work-related physical activity is associated with resting tPA or PAI-l . Thus, physical activity related to leisure or work may not be of sufficient intensity to modulate fibrinolytic activity. De Geus and associates (28) randomized sedentary men into groups that trained for either four or eight months. A trend for decreased PAI-l activity was observed, but these changes failed to reach the assigned level of significance due to large variation within groups. The training regimen included self-selected exercise frequency. Furthermore, the duration of the exercise sessions in the De Geus study differed among subjects by several hours per week, suggesting the variability of the results may have been impacted by inconsistent training regimens. Length of the training phase is also likely to influence magnitude of fibrinolytic adaptations. Bodary, et a1. (12) demonstrated that very short-term training had no effect on resting measures of fibrinolysis. Sixteen apparently healthy men and women engaged in 50 minutes of moderate intensity treadmill exercise for 10 consecutive days. No change in resting tPA or PAL] was observed. The fibrinolytic improvements related to training may also be age-dependent. Aging is associated with a thrombophilic state that may contribute to cardiovascular complications (2, 32), and baseline fibrinolytic profile appears to exert a strong influence on training adaptations (134, 139). Stratton et al. (136) observed significant changes in resting fibrinolytic profile with training in older (60-82 years), but not younger (24-30 years) subjects. Similar training-related modifications are described elsewhere (14) while other investigators failed to demonstrate such changes (144). Methodological differences specifically related to the exercise regimen (e. g. intensity, duration, frequency, and weeks of training) in these studies make it difficult to draw decisive conclusions regarding the effect of age on hemostatic training adaptations. There is clearly a need for further research to elucidate the evaluate relationships between age, exercise intensity, duration, and modality and fibrinolytic adaptations to training. 10 The potential molecular mechanisms of fibrinolytic adaptations to regular exercise are poorly understood. Catecholamines are associated with release of tPA from endothelial cells (18, 113), and B-adrenergic blockade attenuates the normal fibrinolytic response to acute exercise (39). However, it is unlikely that catecholamines influence the fibrinolytic response, because tPA release occurs before an increase in epinephrine during and acute bout of exercise (38). Training-induced reductions in plasma catecholamines may be related to enhanced fibrinolytic capacity, but this relationship is unclear. Increased blood flow due to repetitive bouts of physical exertion may exert multiple effects on the fibrinolytic system (110). Vascular shear stress may cause damage to the arterial intimal layer, particularly in regions disturbed by atherosclerotic plaque. Platelets and coagulation factors, notably thrombin, become activated at the site of injury, leading to increased release of both tPA and PAI-l (110, 120). Elevated blood flow and vascular shear stress also enhance the basal formation of nitric oxide (NO) (126). NO inhibits platelet adhesion and aggregation and facilitates the dissolution of small platelet granules. Furthermore, NO regulates the release of both tPA and PAI-l (126). It has been suggested that atherosclerosis-related impairment in NO synthesis and release may be mediated by the renin-angiotensin system, which is also involved in the regulation of fibrinolysis. In this system, angiotensinogen is converted to angiotensin I by the renal protease renin. Cleavage of angiotensin I by the angiotensin converting enzyme (ACE) yields angiotensin II, which is a potent vasoconstrictor and primary tool in the regulation of blood pressure. ACE may either promote increased levels of angiotensin I or bradykinin, which in turn induce the expression of PAH and t-PA, respectively (15, 114). Studies of in vivo infusion of angiotensin II demonstrate a direct effect of 11 angiotensin on fibrinolysis (114). Training effects on the renin-angiotensin system may provide a link between regular exercise and fibrinolysis, but this remains speculative. Recent findings related to atherosclerosis indicate a key role of inflammation in the disease process, and coronary disease is often characterized by high levels of circulating pro-inflammatory cytokines. Two such cytokines, TNF-a and IL-1 increase PAI-l synthesis and/or release from endothelial cells and also decrease tPA synthesis (82). Regular exercise suppresses the activity of pro-inflammatory cytokines (107), though the extent to which this translates to fibrinolytic adaptations is unclear. Exercise training and cardiovascular disease Cardiac rehabilitation is a coordinated collection of interventions designed to improve physical, psychological, and social conditions so that patients with cardiovascular disease may preserve or resume optimal functioning and slow or reverse the progression of disease. This complex intervention may involve any of a variety of therapies including risk factor education, psycho-social counseling, drug therapy, nutritional and smoking cessation input. Nonetheless, the central element of cardiac rehabilitation is exercise therapy (45, 135, 149). A large body of evidence overwhelmingly suggests exercise-based interventions produce significant overall benefits compared to usual medical care, including increased physical performance, improved angina threshold and myocardial perfusion (36, 129). Various meta-analyses of the effects of exercise training among patients with CVD demonstrate numerous improvements in modifiable risk factors such as hypertension, lipid profile, smoking habit, body composition, and glucose tolerance, as well as improvements in health-related 12 quality of life as assessed by a range of outcome measures (140). Moreover, estimated reductions in total and cardiac mortality range from 20-32% (11, 100, 101, 140). A primary goal of exercise training of an individual with cardiovascular disease is improved cardiac health, which necessitates improved myocardial perfusion. Exercise training attenuates ST-segrnent depression during exercise (36) and decreases perfusion defects on thallium scanning (129), indicating an increase in myocardial perfusion. Regional myocardial hypoperfusion may result from vascular stenosis, microvascular dysfunction (105), and microrheology (53). Each of these basic pathogenic components may be affected by exercise training, and several mechanisms may contribute to the exercise training-related improvements in cardiovascular health. First, exercise training may arrest the progression of atherosclerosis, or even result in a net regression of coronary stenosis. A study of lifestyle changes including stress management, dietary modifications, and 3 hours of exercise training per week resulted in a significant regression of coronary stenoses compared to a non-exercising control group (102). This was associated with a 2.5-fold risk reduction in cardiac events after a 5-yr follow-up period (103). The Stanford Coronary Risk Intervention Project (62) did not observe atherosclerotic plaque regression in a group of patients undergoing exercise therapy, but the rate of change in minimal diameter per patient in this risk- reduction group was 47% less than for a non-exercising usual-care group. Similarly, the Heidelberg Regression Study used exercise and a low-fat diet to halt the progression of atherosclerosis, evidenced by an unchanged luminal diameter while the non-exercising control group experienced decreased luminal diameter (96). 13 Second, exercise training may improve myocardial circulation through the creation of collateral blood vessels. Animal studies suggest that long-term, intensive training increases coronary collateralization (20, 94, 125). Human data are more equivocal. Belardinelli observed significant increases in collateralization (9) while Niebauer did not (95). The lack of agreement between these studies is perplexing considering the exercise regimens employed were similar in intensity and frequency, the subject pools were comparable, and Niebauer’s study trained its patients for a longer period of time than the Belardinelli project (12 months versus 8 weeks). One explanation for the negative findings reported by Niebauer is that the protracted training regimen resulted in net regression of atherosclerotic plaque, thus reducing the need for additional blood supply distal to the stenosis. It has also been suggested that angiography, the typical method for assessment of collateralization in humans, may not be sensitive enough to detect the formation of blood vessels smaller than 100 um, especially in patients without previous MI (53, 95). Finally, regular exercise may improve myocardial blood flow through enhanced dilation of microvasculature. Atherosclerosis is associated with progressive impairment of coronary endothelial function, which decreases nitric oxide (N 0) release from endothelial cells. Since endothelium-derived nitric oxide is thought to be necessary to maintain an adequate vascular response to increased blood-flow demands during exertion, correction of endothelial dysfunction has become a rehabilitation goal of paramount importance. A recent study of 54 men and women with a recent MI documented significant improvement in endothelium-dependent vasodilation following three months of aerobic training, theorized to involve a chronic increase in NO production (148). NO 14 concentrations are influenced by various factors, each of which is susceptible to modifications through exercise. L-arginine is the precursor to NO, and must be present at the active site of endothelial NO synthase (eNOS) for NO production. During exercise, augmented vascular shear stress increases the velocity of the endothelial high afi'mity/low-capacity transport system for L-arginine (109), which ensures substrate availability for eNOS. Furthermore, eNOS activity and expression are both enhanced in response to elevated shear stress both in vitro (23, 34, 49, 99) and in vivo (130, 151). Reactive oxygen species (ROS) accelerate the degradation of NO, and are associated with atherosclerosis and endothelial dysfunction (111). Exercise training increases total oxygen uptake as well as production of ROS (70). However, regular exercise also improves endothelial function (55, 59). These seemingly contradictory facts may be explained by the fact that exercise training increases both eNOS and extracellular superoxide dismutase, a potent antioxidant. Through this mechanism, exercise training may attenuate the deleterious effects of ROS on NO. Exercise training further affects endothelium-mediated vasomotion of coronary arteries by attenuating the paradoxical vasoconsuictive response to acetylcholinein patients with CVD (55, 59), thus improving peak flow velocity in larger conduit arteries, and increasing sensitivity and responsiveness to adenosine in smaller resistance vessels (5 9). Cardiac rehabilitation and fibrinolysis As described above, there is evidence that regular aerobic exercise elicits enhanced fibrinolytic capacity in healthy individuals. Exercise training is also effective 15 in improving rheological variables among individuals with confirmed diagnosis of or risk factors for CVD. Although post-menopausal women typically have an impaired fibrinolytic profile, DeSouza et al. (32) observed that trained post-menopausal women exhibit fibrinolytic profiles similar to pre-menopausal active women. Additionally, Lindahl et al. (84) observed significant decreases in tPA antigen and PAI-l activity in patients with non-insulin dependent diabetes mellitus (NIDDM) in response to chronic aerobic exercise training combined with a nutritional intervention. Gardner (51) reported that patients with peripheral arterial disease (PAD) who expended fewer than 175 calories through physical activity were particularly susceptible to experiencing a prothrombotic state. In this study of 106 PAD patients subjects in the low physical activity group, as determined by monitoring with an accelerometer, exhibited lower tPA activity and higher PAI-l activity than the moderate and high physical activity groups (p<0.05). It has been hypothesized that training-related fibrinolytic improvements in a CVD population may be related to changes in body mass (144), suggesting the link between exercise and fibrinolysis may be circuitous with body composition mediating the association. However, Lindahl et al. (83) observed that a 5-6 kg reduction in body weight in obese individuals failed to have a significant effect on PAI-l, supporting a more direct relationship between exercise and hemostasis. Similar to studies involving apparently healthy subjects, fibrinolytic improvements related to exercise training of a CVD population may depend on duration and/or intensity of training. Estelles, et al. (40) studied a group of post-MI patients, that entered an exercise training program and another group of patients that did not. The non- exercise control group experienced a significant decline in fibrinolytic potential over 6 l6 months, as evidenced by decreased tPA activity and increased PAI-l activity. Fibrinolytic capacity did not specifically improve in response to training (i.e. nonsignificant increase in tPA activity) but the exercise group did not experience this decrement in fibrinolysis. Six months of aerobic training produced significant fibrinolytic changes in a recent study by Killewich, et a1. (77). Twenty-one men with intermittent claudication underwent 6 months of treadmill exercise training and were compared to a group with intermittent claudication who did not train. Significant increases in tPA activity and decreases in PAI-l activity were observed in the exercise group, while no fibrinolytic changes were observed in the non-exercising group. It was noted that patients with the highest baseline PAI-l experienced the greatest decline, suggesting that those with the greatest fibrinolytic impairment may benefit most from regular exercise. Pararno, et al. (104) studied 30 survivors of a first MI and 30 healthy controls who underwent 9 months of cardiac rehabilitation training. Patients had higher tPA antigen and PAI-l activity and antigen at baseline than healthy control subjects. Three months of training elicited a significant decrease in PAI-l activity (p<0.01), and 9 months of exercise produced a decrease in PAI-l antigen (p<0.05). Not all published studies showed a fibrinolytic benefit of participating in at least 3 months of exercise-based cardiac rehabilitation. Rigla, et al. (116) evaluated 27 diabetic patients and 11 healthy controls before and after a 3 month training program. The primary finding of this study was a decrease in thrombomodulin after training, indicating improved endothelial integrity. Regarding fibrinolysis, type I diabetics experienced a significant increase in PAI-l activity (p<0.05). Type II diabetics demonstrated a similar, 17 though not statistically significant, response to training. No change was observed in tPA activity. Since healthy controls exhibited similar results, the authors disregard this unusual finding as biologically unimportant. It is not clear fi'om the description of the methods used why such a finding was observed. One explanation is that 10 of the enrolled subjects were current smokers, which has substantial effects on fibrinolytic parameters. A more recent investigation (121) randomized 29 male patients with congestive heart failure (CHF) to training or control groups. The training program included 26 weeks of combined strength and endurance exercise 4 times per week, 2 supervised and 2 “at home,” unsupervised, sessions. Home training sessions lasted 11 minutes and included 4 exercises for relaxation, flexibility and strength, and one endurance exercise. Supervised exercise sessions included these same 5 “home” exercises plus an interval workout on a cycle ergometer designed for CHF patients. The cycle ergometer workout alternated 30-second work phases with 60-second recovery phases a total of 10 times. Work phases were at an intensity associated with 50% of the individual’s maximum exercise capacity. Training improved exercise tolerance but none of the endothelial- derived variables under examination, including tPA and PAI-l. It is probable that the exercise stimulus, which included approximately 5-10 minutes of moderate-intensity aerobic activity, may not have been adequate to elicit the molecular adaptations necessary to produce significant hemostatic improvements. Only one published investigation of the fibrinolytic adaptations during cardiac rehabilitation used an exercise regimen of less than 3 months. Suzuki and associates (138) studied 56 post-MI patients before and after one month of exercise training. 18 Compared to a non-exercising control group of MI patients, coagulation activity was suppressed following training as evidenced by numerous markers including F VIII activity, vWF antigen, VH activity, and thrombin-antithrombin. In a subset of 20 patients who underwent physical training, tPA antigen and PAI-l activity decreased (p<0.05). This was an atypical cardiac rehabilitation program in that it involved two 40-minute sessions per day of treadmill walking and cycle ergometry for six days per week. The extreme duration and frequency of exercise explains the rapid fibrinolytic improvements that were not apparent in other studies until 3-9 months of training. Historically, phase H cardiac rehabilitation programs included 3 days per week of supervised exercise lasting 20-60 minutes for 3-6 months. In recent years, insurance companies reduced the number of exercise sessions for which they would reimburse hospitals and clinics. Today, most programs are 6-12 weeks in duration. The literature suggests three months of training is sufficient to elicit positive fibrinolytic changes, but it is unclear if fewer than 12 weeks of traditional cardiac rehabilitation is beneficial with regard to hemostasis. Summary: In summary, fibrinolysis is clinically significant due to its regulation of clot dissolution, particularly in regard to thrombosis in or around atherosclerotic plaque. Two of the primary elements of the fibrinolytic process, tPA and PAL], are strong predictors of many outcomes related to CVD. Low plasma concentrations of tPA and high concentrations of PAI-l are considered independent risk factors for CVD. Exercise training produces numerous cardiovascular benefits, which may include enhanced fibrinolytic capacity and, thus, reduced risk of acute cardiovascular events. Individuals 19 with CVD often exhibit reduced fibrinolytic capability and may experience profound hemostatic improvements following a regular training regimen. Exercise training is the cornerstone of modern cardiac rehabilitation, and previous studies have demonstrated that fibrinolytic improvements may be realized through typical rehabilitation programs lasting three to nine months. Exercise regimens of more than three months that adhered to exercise guidelines provided by the American College of Sports Medicine (3) were effective in producing fibrinolytic improvements (40, 77), and one study demonstrated that as few as three months of training improved fibrinolytic potential (104). Only one study examined fibrinolysis in patients with CVD after fewer than three months of training, but the exercise regimen involved very high-intensity and high-frequency activity that would not be part of a traditional cardiac rehabilitation program (138). The present study is the first to assess fibrinolytic adaptations during a cardiac rehabilitation program of fewer than 12 weeks that utilized an exercise prescription as recommended by the American College of Sports Medicine. 20 Chapter 3 - RESEARCH DESIGN / METHODS All methods described herein have been approved by Michigan State University’s Committee on Research Involving Human Subjects and William Beaumont Hospital’s Human Investigation Committee. Each participant had the study explained in full, and provided written informed consent prior to enrollment in the study. Subjects: Individuals referred for Phase II (monitored) cardiac rehabilitation at William Beaumont Hospital in Royal Oak, M1 were recruited to participate in this study. This cardiac rehabilitation program enrolls approximately 300 patients in its Phase 11 program annually, and has been shown to produce numerous beneficial physiological adaptations through six to eight weeks of physical training (47). To determine effect sizes for training adaptations in fibrinolysis, data were collected from patients with coronary artery disease preparing to begin Phase H cardiac rehabilitation (n = 4) and those who recently completed six weeks of cardiac rehabilitation (n = 3). It was estimated that 10 subjects would be sufficient to detect statistically significant training-induced increases in plasma tPA and decreases in PAI-l at an alpha level of P<0.05 with power of 0.8. Tobacco users and those with diagnosed liver disease were excluded from the study due to the possible effects on the variables under examination. Subjects were encouraged not to modify their course of pharmacological treatments during this study unless otherwise directed by their personal physicians. Individuals who discontinued or began taking a medication likely to influence fibrinolytic variables (i.e. fibrates, statins, B-blockers, ACE inhibitors) during participation in the study were excluded from data analysis. Twenty-two volunteers qualified and agreed to participate in the study. Three subjects withdrew from the study 21 due to prolonged illness, four provided unusable baseline blood samples, and one was excluded for discontinuing a significant medication. Thus, 14 patients (12 male, 2 female) completed the study and were included in final analyses. Training: Subjects participated in 18 sessions (3 sessions/week) of structured exercise therapy as part of the Phase II cardiac rehab program at Beaumont Hospital. Supervised exercise sessions consisting of bouts of treadmill walking, cycling, or combined arm/leg ergometry were conducted three days per week for approximately one hour per session. Exercise intensity corresponded to 50-70% of heart rate reserve. Subjects were monitored continuously during exercise via ECG by hospital personnel. Data from subjects who participated in fewer than 80% of the scheduled exercise sessions or who required more than eight weeks to complete 18 sessions were not included in the final analyses. Laboratory Measures: Each participant’s height, weight, hip circumference, waist circumference, resting systolic and diastolic blood pressure was measured at program entry and exit by Beaumont Hospital personnel. To determine changes in submaximal exercise responses as a result of the rehabilitation program, 13 participants completed constant-load, treadmill exercise tests during their 2“d and 18th exercise sessions. Intensity for these tests was set at the workload prescribed at the beginning of the phase 11 program. Steady- state heart rate and blood pressure were collected during these exercise tests. Blood samples were taken at three time points: prior to enrollment in Phase II cardiac rehabilitation, after nine sessions (approximately three weeks), and after 18 22 sessions (approximately six weeks). Following a 12 hour overnight fast, subjects reported to the Beaumont rehabilitation center where blood was drawn from an antecubital vein. All blood samples were acquired between 6 and 10 AM to control for diurnal variations in fibrinolysis (5, 66). Blood samples were collected in tubes containing 1:10 0.45M sodium citrate, pH 4.3 (Biopool Stabilytem) and platelet-poor plasma was isolated by centrifugation at 11,200 g for 20 min at 4°C. Plasma aliquots were frozen and stored at -80°C until assayed. Blood to be used for assessment of mRNA was collected in commercially available tubes containing a lysing and a stabilizing buffer (Qiagen, Inc., Valencia CA) and stored at -20°C until assayed. Assays: Plasma concentrations of tPA and PAI-l were measured using enzyme-linked immunosorbancy assays (ELISA). All blood assays were performed in Michigan State University’s F ibrinolysis Research and Genetics Laboratory in the Department of Kinesiology. Samples were measured in duplicate, and blood assays were batched so that all data points for a given subject were run using the same kit. Intra-assay coefficients of variation in the Fibrinolysis Research and Genetics Laboratory are consistently < 5% for these measurements when this protocol is used (personal communication, Christopher Womack, Ph.D.). RNA was isolated from whole blood using commercially available kits (Qiagen, Inc., Valencia CA) and amplified using real time PCR. As endogenous control to correct for potential variation in RNA loading and quantification, RNA Pol II was used. RNA was treated with DNA-Free (Ambion part no: 1906), according to manufacturer's protocol, to remove any genomic DNA contamination. 0.5ug of RNA was converted into 23 cDNA using Taqman Reverse Transcription reagents (Applied Biosystems no. N808- 0234), according to ABI directions. Relative levels of expression for PAI-l , tPA and RNA Pol II were determined using Taqman Gene Expression Assays for RNA Pol II (no.Hs00172187_m1), tPA (no.Hs00263492_m1), and PAI-1(no. Hs00167155_m1) and Taqman mastermix (ABI no.430443 7) according to manufacturer’s standard protocol (10min 95C initial denaturation, followed by 40 cycles of 95C 155 and 60C 608). Levels of PAH and tPA expression were normalized to RNA pol H, and fold changes of gene expression are relative to baseline. Gene expression assays were done in Michigan State University’s Genomics Technology Support Facility. Statistical Analysis: Statistical calculations included means, SD, and SE. Student’s t test for paired samples was used to assess changes from baseline to program conclusion for the following variables: weight, waist circumference, hip circumference, waist/hip ratio, body mass index (BMI), resting blood pressure, heart rate, submaximal exercise heart rate and blood pressure. All paired comparisons were two-tailed. Linear regression was used to assess the relationship between the outcome variables (plasma concentrations of tPA activity, tPA antigen, PAI-l activity, PAI-l antigen, and tPA and PAI-l RNA) and any potential confounders, including comorbid diagnoses, medication use, waistzhip ratio, body mass index (BMI), weight change, month of enrollment in the program, and elapsed time since coronary event. When no confounding influences were observed, change in plasma concentrations and gene expression of tPA and PAI-l from baseline (BL) to three weeks (3W) and six weeks of training (6W) were assessed using repeated measures AN OVA. Tukey’s post-hoe test was used to elucidate the differences in the event 24 statistical significance was observed. Statistical significance for all analyses was set at alpha = 0.05. P5010 was considered a nonsignificant trend. Unless otherwise stated, all values are displayed as means :t SD. 25 Chapter 4 - RESULTS Subject anthropometric characteristics are displayed in Table 1. Twelve males and two females, aged 67.4 i 10.5 yrs, completed the study. Time elapsed from the most recent cardiac event or surgery to enrollment in the cardiac rehabilitation program was 77.7 i 14.5 days. No significant changes in weight, body mass index (BMI), waist circumference, hip circumference or waist:hip ratio were observed following the six- week training regimen (P>0.05). As expected, baseline values for plasma tPA and PAI-l indicated a blunted fibrinolytic profile. Unpublished data from our laboratory show that comparably aged, sedentary healthy individuals exhibit higher tPA activity (0.92 d: 0.35 vs. 0.65 :I: 0.43 IU/ml), lower tPA antigen (5.8 i 1.8 vs. 13.1 :1: 3.9 ng/ml) and lower PAI- 1 activity (8.1 i 12.1 vs. 17.0 :I: 16.8 IU/ml) than participants in the present study. Resting and submaximal exercise HR, SBP and DBP are displayed in Table 2. Six weeks of participation in the cardiac rehabilitation program produced significant decreases in resting HR, as well as resting and exercise SBP (P<0.05). A non-significant trend was observed for decreased resting DBP (P=0.075) and increased resting HR fiom baseline to 6 weeks. Linear regression was used to assess potential confounding influences on the outcome variables in question. Prevalence of comorbid diagnoses that may influence fibrinolytic parameters are as follows: 21% diabetes mellitus, 67% hypertension, and 56% hypercholesterolemia. None of these conditions were predictive of the change in tPA activity, tPA antigen, PAI-l activity or PAI-l antigen (p>0.05) during the six weeks of training. Medications such as ACE inhibitors, anticoagulants, aspirin, nitrates, platelet inhibitors, calcium channel blockers, and diuretics were likewise unrelated to tPA 26 and PAI-l changes (p>0.05). All participants were taking beta-blockers and statins, so these classes of medication were not included in regression analyses. Waistzhip ratio and weight change during the six-week cardiac rehabilitation program also demonstrated no relationship to changes in tPA and PAI-l (p>0.05). Repeated measures AN OVA showed no significant changes in tPA or PAI-l following six weeks of training. Plasma concentrations of tPA activity (BL=O.69 i 0.44, 3W=O.94 i 0.62, 6W=0.77 :I: 0.49 ng/ml, mean :1: SD, p=0.391) and antigen (BL=13.1 :l: 3.9, 3W=12.4 :t 3.7, 6W=11.8 :i: 3.8, mean :1: SD, p=0.59) are shown in figure 1. Figure 2 illustrates the changes in PAI-l activity (BL=17.0 d: 16.8, 3W=14.8 :I: 22.5, 6W=17.9 :i: 18.8 IU/ml, mean 1 SD, p=0.29) and antigen BL=28.3 :t 15.5, 3W=24.2 :1: 20.2, 6W=22.4 d: 16.1 ng/ml, mean 1 SD, p=0.15). Gene expression responses are displayed in Figure 3. RNA data were normalized to the housekeeping gene RNA Pol II and displayed as fold changes relative to baseline. Repeated measures ANOVA showed no changes in tPA (p=0.45) or PAI-l (p=0.44) gene expression during six weeks of exercise training. 27 Table 1: Subject Anthropometric Characteristics (N=14) Baseline 6 Weeks Height (cm) 174.9 i 7.0 - Weight (kg) 92.2 :t 14.7 92.4 :I: 13.1 Waist circumference (cm) 106.5 :t 12.4 102.9 :t 12.4 Hip circumference (cm) 110.7 d: 12.0 110.0 d: 11.3 Waist/Hip Ratio 0.96 :l: 0.07 0.94 :t 0.10 BMI 30.3 i 5.2 30.3 :t 5.3 Table 2: Resting and Submaximal Exercise Hemodynamics (N=13) Baseline 6 Weeks Resting HR (bpm) 61.4 :1: 8.8 68.7 :t 12.2 T Resting SBP (mmHg) 129.7 d: 20.7 120.0 i: 17.9 * Resting DBP (mmHg) 73.8 :t 10.5 70.5 i 7.4 T Submaximal HR (bpm) 94.1 :I: 16.8 87.9 i 17.0 * Submaximal SBP (mmHg) 137.5 :i: 16.2 126.2 i 18.5 * Submaximal DBP (mmHg) 67.8 :I: 13.0 66.5 :l: 9.7 * P<0.05 compared to baseline 1' P5010 compared to baseline 28 Figure l. tPA Activity (lU/ml) tPA Antigen (ng/ml) 2.25 2.0% 1.8; 1.6- 1.43 [-v-MEAN13E -nAA ......... 22- 20: 18- 16- 14S 12: 10$ 3: +MEAN 1: SE 29 Figure 2. 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