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Cyclosporine A improves coronary artery function in rat cardiac allografts Moien-Afshari, Farzad 2002

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CYCLOSPORINE A IMPROVES CORONARY ARTERY FUNCTION IN RAT CARDIAC ALLOGRAFTS by F A R Z A D MOIEN-AFSHARI M.D., Mashad University of Medical Sciences, 1996  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENT FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Pharmacology and Therapeutics)  We accept this thesis as conforming to the^required standard  THE UNIVERSITY OF BRITISH C O L U M B I A JUNE 2002 © Farzad Moien-Afshari, 2002  UBC  Rare Books and Special Collections - Thesis Authorisation Form  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t t h e U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  DEPARTMENT OF PHARMACOLOGY & THERAPEUTICS  Department o f  Faculty of Medicine The University of British Columbia 2176 Health Sciences Mall Vancouver, BG g g n a d q V61 MS  The U n i v e r s i t y o f B r i t i s h Columbia Vancouver, Canada Date  AMU <T 1, 2002  ABSTRACT  B A C K G R O U N D : A marked decline in the vascular myogenic response during the course of allograft rejection was previously shown in a rat model of heart transplantation. Two important contributory features are an iNOS-catalyzed, NO-mediated vasodilation and a loss of smooth muscle function. In this study, the effect  of cyclosporine A  immunosuppressive therapy on the alleviation of myogenic and endothelial cell dysfunction of coronary resistance arteries in allograft was examined using pressure myography. We hypothesized that cyclosporine therapy will improve coronary artery function in rat cardiac allografts.  M E T H O D S : Coronary septal arteries (internal diameter 200LUTI) were dissected from heterotopic abdominal heart isografts (Lewis-Lewis) and allografts (Fisher-Lewis) at days 4 and 21 post-transplantation and mounted on a pressure myograph. Pressureinduced vasoconstriction was measured in the absence and presence of an iNOS blocker (aminoguanidine [AG], 100 LiM). Endothelium based (ACh induced) and endothelium independent (SNP induced) vasorelaxation was also tested in each group and compared.  RESULTS: Myogenic response was reduced in allograft coronary arteries at day 21 posttransplantation in comparison with matched isografts (p<0.05). A G potentiated myogenic tone in allograft arteries but had no effect on untreated day-21 isografts indicating the presence of iNOS based relaxation only in allograft vessels. At day 4, however, myogenic tone was potentiated by A G in all CsA-treated groups suggesting that CsA might induce  ii  iNOS in these vessels. Depolarization-induced vasoconstriction was lower in day-21 allografts in comparison to isografts whereas at day 4, KC1 induced tone was similar in both groups. CsA therapy also improved depolarization-induced constriction in day-21 allografts as compared to the other untreated groups (p<0.05). Furthermore, CsA therapy improved endothelium based and endothelium independent vasorelaxation in allograft arteries at day-21 post-transplantation.  CONCLUSIONS: The results of this study suggest that CsA immunosuppressive therapy has a significant effect in the alleviation of early endothelial and smooth muscle cell dysfunction in coronary allograft vascular disease. However, the results reflect a dual and counterbalancing effect of CsA, both increasing the activity of iNOS (reducing tone) at an early time point, while attenuating the extent of smooth muscle destruction at a later time point (augmenting tone).  iii  T A B L E OF CONTENTS  ABSTRACT T A B L E OF CONTENTS LIST OF T A B L E S LIST OF FIGURES LIST OF A B B R E V I A T I O N S ACKNOWLEDGEMENT  ii iv vii viii x xii  CHAPTER ONE CYCLOSPORINS A AND CORONARY ALLOGRAFT  VASCULAR DISEASE  1.1 INTRODUCTION  1  1.2 C A R D I A C A L L O G R A F T V A S C U L A R DISEASE 1.2.1 P A T H O L O G I C A L CHARACTERISTICS 1.2.2 PATHOGENESIS OF C A V  2 2 3  1.3 C Y C L O S P O R I N E A 1.3.1 B A C K G R O U N D 1.3.2 P H A R M A C O K I N E T I C S ABSORPTION DISTRIBUTION V O L U M E OF DISTRIBUTION B L O O D DISTRIBUTION M E T A B O L I S M ELIMINATION 1.3.3 C S A P H A R M A C O D Y N A M I C S M E C H A N I S M OF ACTION A D V E R S E V A S C U L A R EFFECTS OF C S A T O X I C O L O G Y OF CSA B A C K G R O U N D TOXICITY IN RATS TOXICITY IN M O N K E Y S CSA C A R C I N O G E N I C I T Y / M U T A G E N I C I T Y C S A A N D REPRODUCTION AND/OR E M B R Y O T O X I C I T Y V A S C U L A R EFFECTS OF CREMOPHOR, V E H I C L E FOR C S A 1.3.4 C S A A N D T R A N S P L A N T V A S C U L A R DISEASE 1.3.5 CSA VERSUS OTHER IMMUNOSUPPRESSANTS FK506 (TACROLIMUS®) R A P A M Y C I N  iv  8 8 8 8 9 9 9 10 11 11 11 12 17 17 18 19 19 20 20 20 21 21 22 15-DEOXYSPERGUALIN (DSG) M Y C O P H E N O L A T E MOFETIL (MMF) E N D O T H E L I N B L O C K E R S 1.3.6 CSA IN C O M B I N A T I O N WITH OTHER DRUGS  1.4 OTHER DRUGS USED IN THE T R E A T M E N T OF C A V 1.4.1 ANGIOPEPTIN 1.4.2 FK409 1.4.3 H Y D R O X Y M E T H Y L G L U T A R Y L - C O A R E D U C T A S E INHIBITORS 1.4.4 ANGIOTENSIN C O N V E R T I N G E N Z Y M E (ACE) INHIBITORS 1.4.5 FUSION PROTEIN CTLA4IG  23 23 24 24  25 25 25 26 26 27  CHAPTER TWO CYCLOSPORINE A THERAPY AND VASCULAR FUNCTION IN RAT CARDIAC TRANSPLANTS 2.1 INTRODUCTION 2.2 HYPOTHESIS A N D SPECIFIC AIMS 2.3 M A T E R I A L S A N D METHODS 2.3.1 A N I M A L GROUPS 2.3.2 TISSUE PREPARATION A N D D I A M E T E R M E A S U R E M E N T 2.3.3 M Y O G E N I C A N D DEPOLARIZATION-INDUCED TONE 2.3.4 A C H A N D SNP INDUCED V A S O D I L A T A T I O N 2.3.5 M O R P H O L O G I C A L A N A L Y S I S 2.3.6 QUANTITATION OF IN SITU T U N E L 2.3.7 C H E M I C A L S A N D BUFFERS 2.3.8 STATISTICAL A N A L Y S I S A N D C A L C U L A T I O N S 2.4 RESULTS A N D DISCUSSION 2.4.1 V A S C U L A R DIAMETER A N D DISTENSIBILITY 2.4.2 D A Y 21 POST-TRANSPLANTATION RESULTS M E D I A L THICKNESS T U N E L STAINING EFFECT OF CSA T R E A T M E N T O N M Y O G E N I C TONE CYCLOSPORINE A T H E R A P Y IMPROVES D E P O L A R I Z A T I O N INDUCED TONE B Y POTASSIUM CHLORIDE SOLUTION EFFECT OF CSA ON NON-ENDOTHELIUM AND  ENDOTHELIUM-DEPENDENT VASORELAXATION  28 30 30 31 31 32 33 33 34 34 34 35 36 36 36 36 37 38  38  2.4.3 D A Y 21 POST-TRANSPLANTATION DISCUSSION M Y O G E N I C A N D DEPOLARIZATION INDUCED T O N E IMPROVEMENT B Y CSA V A S O R E L A X A T O R Y RESPONSE I M P R O V E M E N T B Y C S A  39  2.4.4 DAY 4 POST-TRANSPLANTATION RESULTS  48  v  40 42 E F F E C T O F C S A T R E A T M E N T O N M Y O G E N I C T O N E T H E R A P Y D O E S N O T C H A N G E D E P O L A R I Z A T I O N INDUCED TONE B Y K C L SOLUTION N O N - E N D O T H E L I U M A N D E N D O T H E L I U M B A S E D R E L A X A T I O N A N D CSA T H E R A P Y 2.4.5 D A Y 4 P O S T - T R A N S P L A N T A T I O N DISCUSSION REFERENCES  48 48 48 49 72  vi  LIST OF TABLES  CHAPTER 1 1.1  A COMPARISON B E T W E E N HISTOPATHOLOGICAL FINDINGS IN CARDIAC ALLOGRAFT VASCULOPATHY AND NATIVE CORONARY A R T E R Y DISEASE  52  1.2  A D V E R S E V A S C U L A R EFFECTS OF CYCLOSPORINE A  53  1.3  CYCLOSPORINE A A C U T E TOXICITY IN M O U S E , R A B B I T A N D R A T  54  CHAPTER 2 2.1  P E R C E N T A G E OF T U N E L POSITIVE C E L L S FOUND A T D A Y 21 POST TRANSPLANTATION  vii  55  LIST OF FIGURES  CHAPTER 1 1.1  DIRECT A N D INDIRECT P A T H W A Y S OF A L L O G R A F T DESTRUCTION A N D SITE IF CYCLOSPORINE A ACTION CHAPTER 2 2.1  2.2  PASSIVE DISTENSIBILITY OF A L L O - A N D ISOGRAFT C O R O N A R Y ARTERIES A C O M P A R I S O N IN M E D I A L THICKNESS A M O N G S T C O R O N A R Y ARTERIES (A &B)  56  57  58,59  2.3  T U N E L STAINING OF T R A N S P L A N T ARTERIES  60  2.4  PRESSURE-DIAMETER TRACINGS F R O M C O R O N A R Y ARTERIES A T DAY-21 POST - T R A N S P L A N T A T I O N  61  M Y O G E N I C TONE IN C O R O N A R Y ARTERIES A T D A Y - 2 1 POSTTRANSPLANTATION  62  DEPOLARIZATION-INDUCED TONE A T DAY-21 POSTTRANSPLANTATION  63  E N D O T H E L I U M DEPENDENT A R T E R I A L R E L A X A T I O N A T D A Y - 2 1 POST-TRANSPLANTATION  64  E N D O T H E L I U M INDEPENDENT A R T E R I A L R E L A X A T I O N A T DAY-21 POST-TRANSPLANTATION  65  M Y O G E N I C TONE IN C O R O N A R Y ARTERIES A T D A Y - 4 POSTTRANSPLANTATION  66  PRESSURE-DIAMETER TRACINGS F R O M C O R O N A R Y ARTERIES A T D A Y - 4 POST-TRANSPLANTATION  67  M Y O G E N I C TONE IN C O R O N A R Y ARTERIES F R O M D A Y - 4 CYCLOSPORINE-A TREATED AND UNTREATED CONTROL  68  DEPOLARIZATION-INDUCED TONE A T D A Y - 2 1 POSTTRANSPLANTATION  69  2.5  2.6  2.7  2.8  2.9  2.10  2.11  2.12  2.13  E N D O T H E L I U M DEPENDENT A R T E R I A L R E L A X A T I O N A T DAY-21  viii  2.14  POST-TRANSPLANTATION.  70  E N D O T H E L I U M INDEPENDENT A R T E R I A L R E L A X A T I O N A T DAY-21 POST-TRANSPLANTATION  71  ix  LIST OF ABBREVIATIONS  ACE ACh AG ANOVA Arg AVP BUN CAD CAV CdA CFR cGMP CsA CsG CTLA4Ig DNA DSG EEL EGF eNOS ET-1 ETC FGF FKBP GM-CSF HBGF HDL HMGCoA ICAM-1 IEL IGF-I IL INF-y iNOS i.v. LD LDL MHC MMF NA NFAT NO NOS 5 0  angiotensin converting enzyme acetylcholine aminoguanidine analysis of variance arginine arginine vasopressin blood urea nitrogen coronary artery disease cardiac allograft vasculopathy chlorodeoxyadenosine coronary Flow Reserve cyclic guanosine monophosphate cyclosporine A cyclosporine G cytotoxic T lymphocyte antigen-4 immunoglobulin deoxyribonucleic acid 15-Deoxyspergualin external elastic lamina epidermal growth factor endothelial nitric oxide synthase endothelin-1 endothelial cells fibroblast growth factor FK506 binding protein granulocye-monocyte colony stimulating factor heparin-binding growth factor high-density lipoprotein hydroxy methyl glutaryl coenzyme A intercellular adhesion molecule-1 internal elastic lamina insulin like growth factor-I interleukin interferon-y inducible nitric oxide synthase intravenous lethal dose 50 low-density lipoprotein major histocompatibility antigen mycophenolate mofetil noradrenaline nuclear factor of activated T-cells nitric oxide nitric oxide synthase  x  PDGF RNA RT-PCR SGPT SMC SNAP SNP TBST TdT TGFb  TGF-p  TUNEL VLDL VSMC  platelet derived growth factor ribonucleic acid reverse transcriptase polymerase chain reaction serum glutamic pyruvic transaminase smooth muscle cell S-Nitroso-N-acetylpenicillamine sodium nitroprusside Tris-Buffered Saline Tween-20 terminal deoxynucleotidyl transferase transforming growth factor beta tumor growth factor betaterminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling very low density lipoprotein vascular smooth muscle cell  xi  ACKNOWLEDGEMENTS  I am particularly grateful to Dr. Ismail Laher, my supervisor, for his scientific and thoughtful guidance and his kind support during my studies and during the process of my application to the program. In fact, he always treated me as a friend, which I really appreciate. I am also grateful to the British Columbia Heart and Stroke Foundation for providing funding to support my study. I also express my appreciation to my research committee members, Drs. Casey van Breemen, Bruce McManus and Ed Moore, who provided excellent guidance and scientific feedback during the course of this study. I would also like to thank my dear friend Kasra, who helped me before my arrival and at every stage since then. He is my childhood friend who will be my friend in my old age. I also appreciate the help and support I received from Holman Chan, Jonathan Choy, Anie Min. I am also very grateful to Nathalie Guardeault, Guy Lagaud, Eugene Lam, and Amy Lui teaching me so much of their technical skills and intellectual ideas. This thesis is dedicated to my parents who always stood by me at difficult stages of life and provided a peaceful environment for their children to grow, and taught them how to live their lives loving others.  xii  CHAPTER ONE CARDIAC ALLOGRAFT VASCULOPATHY AND CYCLOSPORINE A  1.1 INTRODUCTION Cardiac allograft vasculopathy (CAV) is a rapidly progressive and unique form of atherosclerosis, which occurs in about 30 to 60% of transplant recipients within the first 5 years following surgery (Wahlers et al., 1994). Recent studies using intravascular coronary ultrasound techniques show that intimal thickening occurs in 75% of cardiac transplant arteries by the end of the first year post-transplantation. C A V is unique because it affects both intramyocardial as well as epicardial coronary arteries and veins. The disease even involves the aorta of the allograft but does not spread to other arteries. Cardiac allograft vasculopathy is characterized in its early stages by intimal proliferation and in its later phase by luminal stenosis of epicardial branches, occlusion of smaller arteries and myocardial infarction (Billingham, 1992). C A V , or more specifically this form of coronary atherosclerosis, is the main cause of death in cardiac transplant longterm survivors and is a limiting factor for long-term success of cardiac transplantation (Hosenpud et al., 1998). Important in the disease is that myocardial ischemia and/or infarction occurring secondary to C A V in these patients is usually silent due to lack of innervation. Therefore, instead of chest pain, more serious events such as congestive heart failure, ventricular arrhythmias, and sudden death are commonly the first clinical manifestations (Weis and von Scheidt, 1997). CsA is an immunosuppressive drug widely used in transplant medicine to prevent rejection, however it has not changed the outcome of C A V . Two mechanisms are suggested to be responsible for ineffectiveness of CsA in  1  long-term prevention of C A V : 1) involvement of nonimmunologic mechanisms in C A V and 2) CsA itself may have some adverse vascular effects.  1.2 CARDIAC A L L O G R A F T VASCULAR DISEASE 1.2.1 P A T H O L O G I C A L CHARACTERISTICS Although cardiac allograft vasculopathy resembles atherosclerosis, there are several differences between this phenomenon and the traditional form of atherosclerosis (Table 1-1). In C A V the lesions are diffuse, concentric and involve all portions of coronary vessels. These lesions ultimately affect the entire length of the artery. Calcification is uncommon in these lesions, the internal elastic lamina is mildly damaged, a low-grade vasculitis is sometimes distinguishable, and the disease develops rather abruptly. This is in contrast to traditional atherosclerosis in which lesions are focal, eccentric, and located in the proximal portion of major epicardial coronary arteries. Calcium precipitation is present in these lesions, the internal elastic lamina is often severely disrupted, and it takes years for them to develop (Billingham, 1992). Although in C A V lesions are diffuse, occasionally focal atherosclerotic plaques with traditional characteristics can also be present and may represent the superimposed native atherosclerotic process (Johnson et al., 1989).  Frank atherosclerotic changes in cardiac allograft vasculopathy occur beginning several months after transplantation. During the early stages, cardiac allograft vasculopathy involves circumferential insudation of lipids into the intima, proliferation of smooth  2  muscle cells, and infiltration of T lymphocytes and macrophages (mild vasculitis). Lipid insudation, cellular infiltration, and matrix development results in intimal thickening that ultimately causes decreased lumen size and distal vessel occlusion. Despite the presence of prominent intimal thickening, the media of these vessels rarely shows proliferative changes and occasionally the media in these arteries become thinner than in normal arteries (Hamano et al., 1998). This unique type of vascular lesion is not limited to cardiac transplant vasculature and many occur almost with the same characteristics in renal allograft arteries (Busch et al., 1971;Porter et al., 1963) and in arteries of other solid organ allografts (Liu, Butany, et al. 1993; Radio, Wood, et al. 1996).  1.2.2 PATHOGENESIS OF C A V So far, the detailed pathogenesis of C A V is unknown but the results of previous studies suggest that immunologic mechanisms, which themselves are under modification of nonimmunologic factors, are the major cause. The evidence suggesting a primary immunologic basis of C A V is as follows. First, proliferative vascular disease is limited to allograft arteries and does not include the host's vessels. Second, the nature of allograft vascular involvement is usually diffuse. Third, in animal models, C A V develops more severely and is accelerated in allograft histocompatibility mismatch. Fourth, it is rare to observe C A V in isografts (Weis and von Scheidt, 1997).  The nonimmunologic risk  factors that may contribute to the development of C A V include age, sex, obesity, hypertension, hyperlipidemia, smoking, diabetes, and donor characteristics (Gao et al., 1997;Grady et al., 1996;Johnson, 1992). Peritransplant ischemia also appears to contribute (Tilney, Paz, et al. 2001).  3  Regardless of the vagueness of our understanding of C A V , it is clear that endothelial cell dysfunction and damage is an initial key point in the development of C A V . Therefore, an early initiating event of C A V is probably a subclinical graft coronary endothelial injury. Functional studies show that endothelial cell function to A C h is compromised and therefore  loss of A C h dilation is a useful marker of C A V .  (Andriambeloson et al., 2001a;Hollenberg et al., 2001a;Marti et al., 2001). A n intact endothelium is required for the normal regulation of vascular wall function, including inhibition of leukocyte adhesion, thrombus formation, vascular smooth muscle cell proliferation, and regulation of vasomotor function (Aranda, Jr. and Hill, 2000). Thus, endothelial damage/loss could alter any or all of these functions and lead to lymphocyte and macrophage adhesion to vascular wall and induction of inflammation, thrombosis, smooth muscle proliferation, and vasoconstriction (Treasure and Alexander, 1995). There are several factors involved in endothelial cell damage during the course of allograft rejection. First, ischemia and reperfusion injury during the initial transplantation process activates the endothelium of small vessels to produce oxygen free radicals, which will then activate host leukocytes passage through the vessel. Activated leukocytes release mediators that cause acute inflammation and lead to endothelial dysfunction (Day et al., 1995). Second, at the later stages of transplantation, the host's immune response is the major source of endothelial dysfunction. The host's T-lymphocytes interaction with graft endothelial and antigen presenting cells starts and sustains the inflammatory response against the graft tissue (Libby et al., 1992).  4  The immune response to allograft tissue is mounted by two principal means (direct and indirect) (Figure 1-1). 1) The direct pathway has two mechanisms. First, the recipient's CD8+ lymphocytes differentiate due to recognition of, and reacting to, foreign M H C class-I along with B7 molecules present on the surface of the donor's antigen presenting cells. These antigen-presenting cells are the dendritic cells that present the foreign antigen to the host immune system either within the organ or after migrating to the regional lymph nodes (Libby et al., 1992). Once mature cytototoxic T lymphocytes are formed, they lyse allograft cells. CD8+ differentiation also depends on IL-2 secreted from CD4+ cells that can be blocked by CsA. A second component of the direct pathway is one that is induced by CD4+ lymphocyte activation in response to foreign M H C class-II and B7 molecules also present on the surface of donor's dendritic cells. Upon activation, CD4+ lymphocytes produce IL-2, IL-4&5 and other cytokines. IL-2 induces CD8+ differentiation and activates CD4+ lymphocytes, IL-4&5 activate B-cells, and other cytokines increase vascular permeability and regional accumulation of mononuclear cells. One of the cytokines secreted by activated CD4+ lymphocytes is INFy which potentiates the former component of the direct pathway by the upregulation of M H C type-I expression on graft artery endothelial cells (Libby et al., 1992;Salomon et al., 1991). 2) By indirect pathway the host's antigen presenting cells introduce alloantigens along with native M H C type-II to the host's immune system. These alloantigens are molecules derived form M H C molecules and other alloantigens shed from graft cells that are taken up and processed by recipient's dendritic cells, which are later introduced along with native MHC-II to the host's immune system. Either the direct or indirect pathway can then activate the recipients' humoral immunity through T-helper 2 cells leading to the  5  formation of antibodies by host's plasma cells directed against transplanted endothelial antigens (Ciubotariu et al., 1998;Shoskes and Wood, 1994). These antibodies, either alone or in combination with their antigens, precipitate on graft endothelial cells and can evoke cell mediated immunity or complement dependent cytotoxicity, finally destroying graft endothelial cells (Cotran et al., 1999). Upon direct or indirect activation, CD4+ lymphocytes will produce and secrete cytokines such as INF-Y, which then induce production of intercellular adhesion molecules (ICAM) such as ICAM-1. These adhesion molecules play an essential role in regulating the interaction between the inflammatory cells such as lymphocytes, monocytes and neutrophils and the transplant vascular wall cells; the adhesion of leukocytes is the first step of their transvascular migration (Hayry et al., 1989). Expression of adhesion molecules on vascular endothelial and smooth muscle cells occurs in human heart transplantation (Ardehali et al., 1995). Moreover, activated macrophages, T-lymphocytes, and vascular endothelial and smooth muscle cells produce other cytokines (IL-1, IL-2, IL-6 and tumor necrosis factor-a) or growth factors (PDGF, IGF-I, FGF, HBGF, EGF, GM-CSF, and TGF-p) via their close interactions. These mediators  are involved in the induction and maintenance  of chronic allograft  inflammatory lesions (Duquesnoy and Demetris, 1995;Hachida et al., 1999). This chronic inflammation will lead to a healing process with over-production of connective tissue matrix and also migration and proliferation of medial smooth muscle cells. Finally, this process will lead to vascular luminal narrowing (Rabinovitch et al., 1995).  Since the standard immunosuppressive medication directed toward T-cells, such as CsA, does not overcome C A V , it is logical to assume that other mechanisms are also involved  6  in C A V pathogenesis. Apoptosis, a genetically programmed cell death, is suggested to be involved in the pathogenesis of C A V (Szabolcs et al., 1996). For example, cytolytic lymphocytes mediate their destructive effect in the pathogenesis of C A V through a Fasbased apoptotic pathway (Dong et al., 1996). In addition, TGFb, a cytokine secreted by damaged endothelial cells within the allograft arteries, induces apoptosis and enhances extracellular matrix production (Gibbons and Dzau, 1994). Moreover, nitric oxide (NO) produced in allograft tissue can increase the rate of apoptosis (Busch, Galvanek, and Reynolds, Jr., 1971). Evidence in support of N O mediating apoptosis includes the following: First, iNOS levels and activity are increased in both cardiac myocytes and infiltrating macrophages during allograft rejection (Billingham, 1992;Szabolcs et al., 1996;Weis and von Scheidt, 1997), as measured by RT-PCR (iNOS mRNA), Western blot and immunostaining (iNOS protein) and production of N02-sup from L-arginine (enzyme activity). This induction may be in response to cytokines released during the immune response against transplanted tissue (Szabolcs et al., 1996). Second, the time course and intensity of iNOS induction in cardiac allograft tissue follows the time course and severity of apoptosis of cardiac myocytes. This finding suggests that excessive N O may trigger apoptosis but it does not necessarily prove it (Szabolcs et al., 1996). Third, an N O releasing compound (SNAP) triggers apoptosis of adult rat cardiac myocytes in vitro. Adding reduced hemoglobin, an N O scavenger, to the culture medium eliminated the apoptosis of cardiomyocytes (Russell et al., 1995a). Fourth, in a comparative study between the iNOS (+/+) and iNOS (-/-) mice, cardiac allograft survival was significantly improved in the latter group. At the same time, inflammatory infiltrate, rejection score, and total number of apoptotic nuclei and apoptotic cardiomyocytes were significantly  7  reduced in the iNOS (-/-) mice (Szabolcs et al., 2001). Finally, in a mouse cardiac allograft model treated with A G (400 mg/kg/day), serum levels of NO, and the number of apoptotic cells (using D N A fragmentation detection assay T U N E L , and D N A laddering) were evaluated. There was a significant prolongation of graft survival in A G treated allograft recipients compared to allografts from untreated mice. Serum N O levels were also significantly decreased in the AG-treated mice. Treatment with A G decreased the total number of apoptotic cells and lowered the ratio of the apoptotic cardiac myocytes to the apoptotic infiltrating cells. D N A laddering was present in untreated allografts but was insignificant in AG-treated allografts (Takahashi et al., 2000).  1.3 CYCLOSPORINE A 1.3.1 B A C K G R O U N D CsA is a cyclic polypeptide consisting of 11 amino acids and is a metabolite of the fungus Beauveria Nivea (Borel et al., 1976). Since CsA is highly hydrophobic, Cremophor® is used to solubilize CsA for clinical/in vivo administration (Mankad et al., 1992a). The advantage of CsA over other immunosuppressants, such as cytotoxic drugs, is that it acts specifically on the immune system, particularly T-lymphocytes and therefore it will not destroy rapidly proliferating cells. Bone marrow cells could be affected causing aplastic anemia (Bach, 1975), however such is not typically observed at correct therapeutic doses.  1.3.2 P H A R M A C H O K I N E T I C S ABSORPTION  8  Following oral administration of radio labeled CsA ( H-CsA) to rats, it was observed that 3  absorption was slow and incomplete; the vehicle did not affect absorption. A n average of 30% of the radioactive dose was absorbed (Wagner et al., 1987), consistent with the oral bioavailability of CsA in rats observed by other investigators (Ueda et al., 1984). Approximately 70% of the radioactivity was excreted in feces and 15% in urine. Bile elimination accounts for 10% of the oral and 60% of the intravenous doses(Wagner st al., 1987). DISTRIBUTION: Volume of distribution: CsA is a lipophilic agent and can cross most biological membranes and it is also heavily bound to tissue and blood components. Therefore its volume of distribution at a steady state in human adults is several hundred liters. Even though CsA is highly lipophilic, it crosses the blood-brain barrier poorly. However, CsA does have central nervous system side effects, possibly due to accumulation of minor levels within the microcirculation of the brain (Fahr, 1993). Blood distribution: At 25°C, 58% of CsA (total concentration 500ug/l) is in erythrocytes, 9% in leukocytes, 4% in plasma water, while 21 % is bound to lipoproteins (34% L D L , 34% H D L , 10% V L D L , 22% other lipoproteins (Gurecki et al., 1985)) and 8% bound to other plasma proteins (Lemaire and Tillement, 1982). Thus erythrocytes carry the highest percentage of CsA in blood and therefore may act as a fast and readily accessible reservoir of CsA. This important source itself is therefore dependent on the  9  hematocrit, which can vary after transplantation (Rosano, 1985;van den Berg et al., 1985). M E T A B O L I S M After oral consumption, CsA metabolism starts in the gastrointestinal tract. The rate of metabolism however is slower than what is expected from the peptidic structure of this drug due to the presence of 7 N-methylated amino acids in its structure (Tjia et al., 1991). The main site of metabolism for CsA is the liver by the cytochrome P450-dependent mono-oxygenase (CYP3A isoform) system (Combalbert et al., 1989). Therefore, any other drug metabolized by this system will interfere with CsA metabolism. Metabolism of CsA occurs by mono- and dihydroxylation, and N-demethylation (Fahr, 1993). The prominent metabolism of CsA by cytochrome P-450 is unusual because peptides are uncommon substrates for this enzyme, and peptides containing nitrogen are very unusual substrates for its reaction (Bruke et al., 1989). In humans and rats, CsA is extensively metabolized, with metabolites accounting for 50-70% of all CsA derived material in whole blood measured within a few hours after oral administration (Wagner et al., 1987).  Up to 27 metabolites of CsA are found in humans (Wallemacq et al., 1989) and in some studies the metabolites are predicted to be more than 60 (Wenger, 1990). M l and M l 7 were the most prominent metabolites found in rat and human blood, respectively, and the ratio of CsA to total metabolites is much higher in rats (3:1) than in human (1:0.5). This ratio is important because results of previous studies indicate that unchanged CsA is more toxic than its metabolites (Gunson et al., 1988;Ryffel et al., 1988). Also, rodent drug  10  metabolism is markedly affected by strain and gender as well as the route of administration. Therefore, it is important to consider species-specific CsA metabolism differences when evaluating CsA toxicology and pharmacology in animals, especially since some of the metabolites may have more toxic or immunosuppressive activity than others (Fahr, 1993). ELIMINATION Only 1 % of an oral dose is excreted unchanged in urine or bile. Therefore, in the case of CsA total clearance and hepatic clearance are almost the same (Burckart et al., 1986;Wagneretal., 1988).  1.3.3 C S A P H A R M A C O D Y N A M I C S M E C H A N I S M OF A C T I O N CsA suppresses the immune response by inhibiting the signal transduction pathway responsible for the activation of B- and T-lymphocytes. CsA immunosuppression occurs as follows (Ho et al., 1996a)(Figure 1-1):  1- Binding to the CsA receptor "immunophilin" within T-cell cytoplasm. 2- Production of intracytoplasmic CsA/cyclophilin complex. 3- The complex will act as a composite surface and block the phosphatase activity of calcineurin. Calcineurin is a key compound for T-call activation. 4- Calcineurin inhibition will lead to a full blockade of translocation of the N F A T from the cytoplasm to the nucleus.  11  5- Failure to activate the genes regulated by the N F A T transcription factor in T-cells, such as those required for B-lymphocytes recruitment (e.g. CD40 ligand) as well as those necessary for T-helper and T-cytotoxic lymphocyte proliferation, e.g. IL2.  CsA may also allow the expansion of the T-suppressors population (Cohen et al., 1984) A D V E R S E V A S C U L A R EFFECTS OF C S A CsA therapy can cause several adverse effects including hypertension and nephrotoxicity by affecting the recipient's renal and systemic vasculature. Some of these adverse effects of CsA on vascular system are as follows (Table 1-2): 1- A characteristic endothelialitis occurs with CsA therapy in rat aortic grafts. This endothelialitis may lead to accelerated arteriosclerosis in allograft arteries. In fact, in allografts treated with CsA there was an early inflammatory lesion in the intimal subendothelial space. This inflammation may lead to secondary events such as rapid accumulation of proliferative cells in this space and arteriosclerosis  development  (Mennander et al., 1992). 2- CsA may not prevent allograft arterial vasculitis. In a rat cardiac transplant model, CsA treated allografts had a modified pattern of rejection in which M H C class II antigen was over expressed in the vascular bed endothelium and to a lesser extent in myocardium. Unmodified rejection, which develops rapidly, is associated with dense  12  M H C class I rather than class II antigen expression on both myocytes and endothelium. In the setting of CsA modified rejection, the expression of M H C class II antigens within the arterial bed produces a delayed-type hypersensitivity response directed toward either the endothelium and/or nearby M H C class II expressing myocytes. This prolonged periarterial and intraluminal inflammatory reaction may then induce vasculitis and compromise the survival of the allograft (Herskowitz et al., 1989).  3- CsA induces upregulation of the N O system in transplanted patients by increasing the mRNA of endothelial cells NOS (eNOS) and NOS activity (Calo et al., 2000). Similar results are observed in other in vivo (Stroes et al., 1997) or in vitro studies. In cultured endothelial cells, an increased expression of eNOS occurs after incubation of cells with CsA (Lopez-Ongil et al., 1996).  4- CsA at therapeutic doses causes hypertension in animals and human. For example in a human clinical trial, mean arterial blood pressure that was 113 ± 5 mmHg (mean ± SEM) during CsA administration, came down to 94 ± 4 mmHg after washout for three months (Forstermann et al., 1989). In a rat model, systolic blood pressure increased from 144 ± 1.4 up to 160 ± 2.5 with CsA therapy (Gonzalez-Santiago et al., 2000). In addition, in spontaneously hypertensive rats, CsA administration significantly increased systolic blood pressure as compared to control animals (245 ± 6 vs 208 ± 9 mmHg, respectively) (Mervaala et al., 1999). Several mechanisms are possibly responsible for CsA induced hypertension:  13  a) CsA induces superoxide ( O 2 ) production, which will then react with N O to produce peroxynitrite that will break down to  NO27NO3".  This N O destruction by CsA can lead to  vasoconstriction and hypertension (Calo, Semplicini, Davis, Bonvicini, Cantaro, Rigotti, D'Angelo, Livi, and Antonello, 2000). Nguyen et al (Nguyen et al., 1999) reported that CsA can produce reactive oxygen species, most likely superoxide and hydroxyl radicals, after cytochrome P-450 mediated metabolization in rat aortic vascular smooth muscle cells; reactive oxygen species inactivate NO.  b) Free radicals can degrade tetrahydrobiopterin (BH4), a necessary co-factor for NOS activity. At the same time, CsA produces free radicals (Calo, Semplicini, Davis, Bonvicini, Cantaro, Rigotti, D'Angelo, Livi, and Antonello, 2000). Therefore, it seems reasonable that CsA can decrease the production of N O through degrading BH4. However, this effect is controversial because some studies report that CsA increases the synthesis of BH4 (Hattori and Nakanishi, 1995).  c) CsA and its metabolites increase ET-1 production from endothelial/mesangial cells(Kon et al., 1990) and decrease prostacyclin (PGI2) production from these cells in vitro (Rosenthal et al., 1989;Voss et al., 1988). Since endothelin-1 is a potent vasoconstrictor and PGI2 a dilator, the net effect of CsA is to cause vasoconstriction. CsA and its metabolites also significantly reduce prostacycline production in kidney mesangial cells. Moreover, the dihydroxylated metabolites of CsA such as A M 19 and A M l c 9 significantly increase endothelin-1 production by these cells. These effects can be contributing factors to decreased renal blood flow, which in turn will increase arterial  14  blood pressure by activating the renhin-angiotensin pathway (Copeland and Yatscoff, 1992). Both CsA and CsG induce a significant increase in the release of ET-1 by rabbit endothelial cells but not mesangial cells (CsG is an analogue of CsA in which norvaline is substituted for the a-amino butyric acid residue in position two of the molecule). However, neither parent drug nor their metabolites result in a significant decrease in prostacyclin release from either cell type (Langman and Yatscoff, 1994). The latter finding is in contrast with other studies that indicate CsA decreases the level of prostacyclin (Rosenthal, Chukwuogo, Ocasio, and Kahng, 1989). Finally, CsA also increases the expression of ET-1 receptors on rat endothelial cells (Nambi et al., 1990).  d) Pretreatment of rat mesenteric arteries with CsA (10" M) potentiates vascular 6  vasoconstriction by noradrenaline and Arg-vasopressin (AVP), possibly by amplifying the calcium response to vasoconstrictor hormones in vascular smooth muscle cells(Lo et al., 1996; Lo et al., 1997a; Lo et al., 1997b). Magnification of the calcium response is neither due to augmented calcium loading of the cells nor to calcium efflux from intracellular pools. CsA may interact with signal transduction pathways activated by vasoconstrictors. This effect is separate from the immunosuppressive effect of CsA occurring through cyclophilins and calcineurin, since some CsA metabolites do not bind to these elements and therefore do not have an immunosuppressive effect, despite increasing the calcium response to vasoconstrictors (Lo, Passaquin, Andre, Skutella, and Ruegg, 1996). Other experiments using [ H]-AVP indicate that CsA increases the 3  expression of A V P receptors on the surface of vascular smooth muscle cells by two fold without affecting their affinity. This will potentiate inositol phosphate formation and  15  increase calcium responsiveness to A V P in these cells. This effect of CsA can also occur with other vasoconstrictor hormone receptors such as angiotensin-II receptors and be an essential part of CsA induced vasoconstriction and hypertension (Avdonin et al., 1999;Lo, Passaquin, and Ruegg, 1997a). Lastly, CsA-induced hypertension is related to increases in plasma rennin substrate and activity (Perico et al., 1986).  e) C s A significantly increases renal and lumbar sympathetic nerve activity when administered intravenously (5 mg/kg)(Morgan et al., 1991). This increase in sympathetic nerve activity was progressive during 60 minutes of CsA administration and was followed by increases in proportional resistance of the femoral and renal vasculature. Clonidine, a central sympatholytic, and ganglionic blockers attenuate this response confirming the sympathetic stimulatory nature of vascular resistance increase by CsA (Morgan, Lyson, Scherrer, and Victor, 1991). This sympathetic tone dependent increase in blood pressure by CsA was also indicated in other studies (Avdonin, Cottet-Maire, Afanasjeva, Loktionova, Lhote, and Ruegg, 1999;Chiu et al., 1992). The mechanism of sympathetic activation by CsA is through binding with calcineurin. The evidence for this hypothesis is as follows: First, rapamycin which is structurally similar to CsA but does not bind to calcineurin, is unable to increase sympathetic activity, whereas, FK506 which is structurally different from CsA but also binds calcineurin, has the same effect as C s A on the sympathetic nervous system. Second, both CsA and FK506 induced sympathetic activation can be reduced by changes in their molecular structure that limit their ability to inhibit the phosphatase activity of brain calcineurin and calcineurin mediated T-cell signaling (Lyson et al., 1993). Calcineurin may act as a C a  16  ++  sensitive step of a negative  feedback pathway that decreases the possibility of neurotransmitter vesicle release in response to presynaptic [Ca ] increase during nerve terminal depolarization. This finding ++  may further explain the reason for induction of central nervous system excitation and neurogenic hypertension as side effects of immunosuppression therapy with calcineurin inhibitors, such as CsA and FK506 (Victor et al., 1995).  4) Chronic treatment (21 days) with CsA in rats facilitates Ca -induced Ca  release by  the sarcoplasmic reticulum in isolated myocardial trabeculae such that the same force develops in the trabeculae at lower exteracellular calcium concentrations and the chance for occurrence of spontaneous contractions increases(Banijamali et al., 1993). T O X I C O L O G Y OF CSA B A C K G R O U N D At therapeutic doses, CsA does not have systemic side effects in animal models (rats, monkeys, and dogs), however CsA toxicity occurs in animals at higher doses and has three distinguished patterns: acute, subacute and chronic. Acute toxicity (up to 4 weeks) of CsA does not affect specific end organs and is evaluated as LD50 (Table 1-2). Subacute (4-13 weeks duration) CsA toxicity mainly affects kidney, liver, lymphoid tissue, gingiva (atrophy), and in rats treated with very high doses it may suppress their erythropoietic system. Chronic (52-104 weeks duration) CsA (45mg/kg/day) toxicity in dogs appears as hypertrophic gingivitis and atypical cutaneous papillomatosis. LD50 of CsA in various species of laboratory animals is different probably due to species-specific bioavailability or activity of cytochrome P-450, CsA metabolizing enzymes. Table 1-3 includes  17  examples of intravenous or oral LD o for acute toxicity of CsA in animals (Ryffel et al., 5  1983). TOXICITY IN RATS 1- Toxicity of CsA after 4-week intravenous (i.v.) therapy at doses of 6, 24 and 48 mg/kg/day was dose related. Mortality was zero at 6mg/kg; however, it increased at 24 and 48 mg/kg (3 and 9 out of 20, respectively). At the highest dose, hepatic pathology was not observed, but the kidneys contained morphological signs of toxicity such as degenerative lesions in the proximal part of the nephron, and renal dysfunction was apparent from serum and urine biochemistry (increased blood urea nitrogen, decreased serum sodium and chloride, low specific gravity of urine and glucosuria) (Ryffel, Donatsch, Madorin, Matter, Ruttimann, Schon, Stoll, and Wilson, 1983).  2- Rats receiving oral CsA for 13 weeks at doses of 14 and 45 mg/kg/day were observed to have severe toxic effects on kidney and liver at the highest dose. Hepatotoxicity first appeared as elevated liver enzymes (SGPT), and later there was fatty infiltration, degeneration and single cell death of hepatocytes. Nephrotoxicity manifested as vacuolation, necrosis, and regeneration of the epithelium of the proximal tubules. In fact, the liver and kidneys were the organs with major toxic effects of CsA. Even at the highest doses, CsA did not have toxic effects on bone marrow, and bone marrow morphology remained normal. At the lower dose (14mg/kg/day), which is considered a high immunosuppressive dose of CsA in rats, there were no toxic effects (Ryffel, Donatsch, Madorin, Matter, Ruttimann, Schon, Stoll, and Wilson, 1983). Lack of bone marrow  18  toxicity  is  a  great  advantage  for  CsA therapy  as  compared  to cytotoxic  immunosuppressive drugs which induce severe bone marrow suppression that may lead to aplastic anemia (den Ottolander et al., 1982; Hogge et al., 1982). TOXICITY IN M O N K E Y S CsA toxicity in monkeys was evaluated after 4 weeks of i.v. therapy on cynomolgus monkeys and after 13 weeks of oral therapy in rhesus monkeys.  1- Cynomolgus monkeys were treated with CsA at doses 5, 25, 45 mg/kg/day/iv for four weeks. No animals died at the lower doses, whereas three out of eight animals that received 45mg/kg/day of CsA died due to the drug's toxic effects on the liver and kidney. Hepatotoxicity appeared as cholestasis, lipidosis, and single cell death of liver cells and nephrotoxicity manifested as vacuolation, degeneration, and regeneration of proximal tubular cells with focal inflammatory cell infiltration.  2- Rhesus monkeys were treated with 20, 60, and 200 mg/kg/day/oral for 13 weeks and the dose was increased up to 300mg/kg/day for the last 4 weeks of the study. The side effects in this experiment were trivial and considered nonsignificant. This study suggests that CsA toxicity is quite variable in different animals and even within the same species, with rhesus monkeys tolerating high doses of CsA better than cynomolgus monkeys. C S A CARCINOGENECITY/ M U T A G E N E C I T Y  19  In comparison to controls, CsA did not change the type, frequency of occurrence, or appearance of hyperplasic or neoplasic growth in mice that were treated for 78 weeks with 1, 4, and 16 mg/kg/day of the drug. Moreover, CsA did not change the frequency of tumors between the control animals and rats treated with the drug for two years. No mutagenic effect was observed in different in vivo and in vitro experiments (Matter et al., 1982). C S A A N D REPRODUCTION AND/OR E M B R Y O T O X I C I T Y Treatment of rats with an immunosuppressive dose CsA (up to 15-17 mg/kg/day) that is below its toxic dose, neither affected fertility nor induced embryotoxicity. This is unlike the effects of cytotoxic immunosuppressants, wherein impaired fertility and congenital malformations are common (Janssen and Genta, 2000). V A S C U L A R EFFECTS OF CREMOPHOR, THE V E H I C L E FOR C S A Cremophor (poly-oxy-ethylated caster oil) at a low dose (50ng/ml) decreases the basal flow rate in perfused hearts in vitro by inducing vasoconstriction (Mankad, Spatenka, Slavik, O'Neil, Chester, and Yacoub, 1992a ;Tatou et al., 1996); however, at higher doses (lOOOng/ml) it increases flow in a dose dependent manner by inducing coronary vasodilation (Mankad, Spatenka, Slavik, O'Neil, Chester, and Yacoub, 1992a).  1.3.4 C S A A N D T R A N S P L A N T V A S C U L A R DISEASE CsA improves 1-year survival in cardiac transplant recipients, however long-term survival or the incidence of allograft coronary artery disease is not changed(Gao et al.,  20  1989; Olivari et al., 1989; Uretsky et al., 1987). There is data from heterotopic heart transplantation in rats showing that CsA administration can prevent the development of intimal thickening. Even when it is administered with 40 days delay after transplantation, CsA can reverse established changes of chronic rejection in allograft arteries. The amount of CsA administered (CsA dose) may have a significant effect on the development and progression of coronary intimal thickening in heart transplant recipients.  1.3.5 C S A V E R S U S OTHER IMMUNOSUPPRESSANTS FK506 (TACROLIMUS ®) 1- Mechanism of action and adverse effects: FK506, like CsA, binds to calcineurin as cyclophilin-CsA and F K binding protein (FKBP)-FK505 complexes (Avdonin, CottetMaire, Afanasjeva, Loktionova, Lhote, and Ruegg, 1999), (Schreiber, 1991). In terms of adverse effects, FK506 increases sympathetic activity much like CsA, and such may be related to the hypertension induced by this drug (Lyson, Ermel, Belshaw, Alberg, Schreiber, and Victor, 1993)  2- A comparison between the final outcome of allograft treatment with CsA or FK506: There is some controversy on this issue. Results of some studies will be reviewed as examples of this uncertainty. 1) In a study performed by Arai et al in 1992, FK506 at a dose of 0.32 mg/kg/day intramuscularly was associated with a more progressive graft C A V than CsA (lOmg/kg/day). Meiser et al (Meiser et al., 1991), found similar effects in a study of rat cardiac allografts. This more severe C A V in FK506 treated rats is not an expression of the generalized vascular adverse effects of this compound, as FK506 did  21  not have any adverse effect on the native heart or on the hearts of control rats that were only treated with FK506. The proportion of CD8 positive lymphocytes to other Tlymphocytes subsets infiltrating allograft tissue is higher in FK506 treated rats as compared to CsA treated animals (Arai et a l , 1992). 2- In contrast, there are studies of rat and human that do not find any significant differences in the severity of C A V between the groups that were treated with CsA or FK506 (Hisatomi et al., 1995) (Pham et al., 1996). Moreover, studies on endothelium dependent and independent coronary flow reserve (CFR) indicate that neither of these two functions was different in cardiac allograft recipient patients treated with CsA or FK506. Coronary flow reserve is an important parameter for the functional assessment of micro vessel disease (Rieber et al., 1998). 3- In human subjects, neither of these calcineurin inhibitors (FK506 or CsA) change the outcome of C A V (Gao, Schroeder, Alderman, Hunt, Valantine, Wiederhold, and Stinson, 1989). R A P A M Y C I N The rapamycin molecule is similar to FK506 and binds to FKBP but this complex does not bind to calcineurin due to small structural differences  between  the  two  immunosuppressants (Schreiber, 1991). Rapamycin interferes with signal transduction by cytokines and growth factors, and is thus able to interrupt cell proliferation in fibroblasts, smooth muscle cells, and also B cells, thereby preventing C A V (Schmid et al., 1995; Xiao et al., 1995). Rapamycin also prevents intimal thickening produced by mechanical injury (Gregory et al., 1995) and vascular lesion formation after balloon-catheter-induced vascular injury due probably to inhibition of cell cycle progression (Marx et al., 1995).  22  In long term cardiac allograft follow up, continuous immunosuppression with rapamycin almost completely inhibited the proliferation of intima and myocytes (Cao et al., 1995; Morris et al., 1995; Schmid et al., 1995). Recent studies also indicate that in comparison to an equipotent dose of CsA, allograft recipients treated with rapamycin have a more significant effect in reducing C A V . Furthermore, rapamycin blocks the formation of antiM H C (RT1 in rat), a molecule which has an important role in C A V pathogenesis (Poston etal., 1999). 15-DEOXYSPERGUALIN DSG particularly suppresses macrophage function in a direct manner. In a heterotopic rat cardiac transplant model, this agent appeared to be more effective than CsA in preventing C A V in the process of chronic rejection (Nagamine et al., 1994). Also, in a rat aortic allograft model, this agent significantly improved the ominous signs of chronic rejection in comparison to control animals (Raisanen-Sokolowski et al., 1994). A recent study of rat heterotopic cardiac allograft, reports that D S G is more effective in the treatment of C A V than CsA (Hachida, Zhang, Lu, Hoshi, and Koyanagi, 1999). M Y C O P H E N O L A T E MOFETIL M M F is an antimetabolite derived from mycophenolic acid. In rodent and primate models of cardiac allograft and also in human subjects M M F induces donor-specific tolerance, prolongs cardiac allograft survival, and also reverses progressive acute cellular rejection (Kobashigawa et al., 1998; Nair and Morris, 1995). It was later shown that this compound retards the development of C A V by direct inhibition of vascular smooth muscle cell  23  proliferation. In a rat model of balloon-induced injury, M M F reverses the arterial thickening and smooth muscle cell substitution of endothelial cells (Gregory, Huang, Pratt, Dzau, Shorthouse, Billingham, and Morris, 1995). In a swine model of cardiac allograft, a short course of M M F administration induces longer allograft survival with less severity and later onset of C A V in comparison to a CsA treated group (Schwarze et al.,2001). E N D O T H E L I N B L O C K E R S In heart transplant recipients, endothelin levels are elevated and may be related to the pathogenesis of allograft coronary vasculopathy (Okada et al., 1998; Ravalli et al., 1996). In fact, some data indicates that blocking endothelin receptors with bosentan or with endothelin converting enzyme inhibitors to diminish endothelin synthesis decreases the incidence of transplant C A V (Okada, Nishida, Murakami, Sugimoto, Kosaka, Morita, Yamashita, and Okada, 1998).  1.3.6 CSA IN C O M B I N A T I O N WITH OTHER DRUGS 1- Co-administration of CsA and 2-CdA, an anticancer agent, in heterotopic rat cardiac transplant model inhibits the development of transplant arteriosclerosis more effectively than CsA monotherapy (Cramer et al., 1997). 2-CdA causes both T- and B-lymphocytes depletion and inhibits the proliferative changes occurring in transplant arteriopathy, especially of the vascular intima. 2- In a rat heterotopic cardiac transplant model, pre-operative co-administration of a single dose of rapamycin with a short course of CsA significantly reduces the prevalence  24  of C A V in different sized coronary vessels as compared to vessels from allografts in CsA treated recipient animals using the same schedule (Goggins et al., 1996).  1.4 OTHER DRUGS USED IN T H E TREATMENT OF CAV 1.4.1 ANGIOPEPTIN Angiopeptin, an octapeptidic somatostatin analogue, inhibits myointimal proliferation by more than 50% in a rabbit heart transplant model (Wagner, Schreier, Heitz, and Maurer, 1987). Angiopeptin in vitro inhibits vascular smooth muscle cell migration through a Gprotein-mediated  pathway  (Mooradian  et  al.,  1995).  Angiopeptin  also  has  immunosuppressive properties, probably through the inhibition of IGF-I, T lymphocytes, macrophages, and ICAM-1 and by reducing the expression of M H C class II (Weis and von Scheidt, 1997). However, some studies do not indicate a significant beneficial effect from angiopeptin therapy; for example, Wahlers et al. (Wahlers, et al., 1994), indicate that in transplant patients, adding angiopeptin to the CsA immunosuppressive regimen did not significantly decrease the incidence of C A V (examined by angiography) after one year of treatment.  1.4.2 FK409 The local presence of nitric oxide donors has a therapeutic benefit in inhibiting the proliferartion of smooth muscle cells. For example, FK409, which is a spontaneous nitric oxide releaser, attenuates transplant vascular disease in rat aortic allografts. Aortic allografts in rats treated with FK409 manifested a dose-dependent (l-10mg/kg) reduction in the neointimal thickness and the drug also significantly decreased D N A synthesis and  25  upregulated Fas expression in smooth muscle cells of the vascular neointima (Fukada et al., 2000). Administration of L-arginine, the substrate for NOS, could improve the maintenance of normal endothelial function in coronary allograft recipients (Drexler et al., 1994). L-arginine feeding of rabbits undergoing cardiac allograft transplantation decreased myointimal hyperplasia in graft arteries by decreasing the mitogenic effect of IGF-I and IL-6 on arterial cells (Lou et al., 1996).  1.4.3 H Y D R O X Y M E T H Y L G L U T A R Y L C O A R E D U C T A S E INHIBITORS Hydroxymethyl glutaryl coenzyme A (HMGCoA) reductase inhibitors are administered to organ transplant patients to control hyperlipidemia. In addition to this effect, these agents (pravastatin, simvastatin) also reduce arterial intimal thickening in heart transplant patients (Escobar et al., 1994;Jaeger et al., 1997;Kobashigawa et al., 1995). These agents appear to have immunosuppressive effects independent of their cholestrol lowering property (Cutts et al., 1989;Cutts and Bankhurst, 1990;Kobashigawa et al., 1995). In vitro, H M G C o A reductase inhibitors have a suppressive effect on natural killer cell activity, cytotoxic lymphocyte activity, T-cell proliferation and monocyte chemotaxis (Katznelson and Kobashigawa, 1995). A possible mechanism for the immunosuppressive action of H M G C o A reductase inhibitors may be a reduction in the formation of farnesylated proteins, particularly Ras. Ras activation of the cell membrane is part of the intracellular signaling pathway common to both growth factor and cytokine cell stimulation (O'Donnell et al., 1995).  1.4.4 ANGIOTENSIN C O N V E R T I N G E N Z Y M E INHIBITORS  26  a) In a Lewis-to-Fisher rat heterotopic cardiac transplant model, recipient animals treated with captopril for three months had less intimal and smooth muscle cell proliferation (Kobayashi et al., 1993). b) Cardiac transplant recipient patients treated with A C E inhibitors during the first year after transplantation had a lower grade intimal proliferation in comparison to the control group (Mehra et al., 1995). Furthermore, these compounds postpone diabetic nephropathy by attenuating vascular remodeling in glomerular microvasculature (Lewis et al., 1993). The mechanism of these actions by A C E inhibitors may be by the inhibition of angiotensin-II formation, thus preventing its proliferative effect on vascular cells as well reducing its potent vasoconstrictor effect (Yang et al., 1993).  1.4.5-FUSION PROTEIN CTLA4IG Fusion protein CTLA4Ig: cardiac allografts in recipient rats treated with fusion protein for 120 days manifested a significantly lower frequency and severity of C A V in comparison to CsA treated rats (Russell et al., 1996).  27  CHAPTER TWO C Y C L O S P O R I N E - A T H E R A P Y A N D V A S C U L A R F U N C T I O N IN R A T CARDIAC TRANSPLANTS  2.1 INTRODUCTION More than two thousand North Americans benefit from cardiac transplantation yearly (Canada's National Organ and Tissue Information Site; Transplant Patient DataSource); however, long-term allograft survival is limited by transplant vascular disease (TVD), a severe and progressive form of atherosclerosis (Young, 1999). Early T V D manifests as a malfunction of endothelial and arterial S M C , so that changes in arterial function are apparent (Hollenberg et al., 2001; Skarsgard et al., 2000) and can be monitored to assess the progression of T V D .  It is assumed that these early arterial malfunctions are  secondary to immune mechanisms and cytotoxic materials released by immune cells infiltrating the allograft tissue (Andriambeloson et al., 2001b). The therapeutic goals for immunosuppressive therapy are to inhibit these secondary events of T V D mediated by immune cells.  The mediators released by immune cells during the course of allograft rejection have a number of effects including increasing the inducible isoform of nitric oxide synthase (iNOS) in V S M C s (Russell et al., 1995b;Yang et al., 1994). The activity of this subtype of NOS is independent of intracellular calcium levels and is directly related to the amount of enzyme present in the cytoplasm (Loscalzo and Welch, 1995). Therefore, once induced, iNOS can produce sufficient amounts of N O to cause sustained vasodilation of resistance arteries and increased coronary flow, which can lead to deterioration of  28  coronary circulation homeostasis and cause interstitial edema. The resulting edema decreases cardiac muscle compliance and contractile ability, sufficiently enough to cause ventricular failure (Szabo et al., 2001). Increased levels of N O in arteries can also cause contractile dysfunction indirectly by decreasing the number of viable SMCs through induction of apoptosis (Szabolcs, Michler, Yang, Aji, Roy, Athan, Sciacca, Minanov, and Cannon, 1996). The host's immune system also damages SMCs in allograft tissue directly. For example, neutrophils and T-cytotoxic lymphocytes in vitro can lyse myocytes or alter their function. Similarly, macrophages and T-helper lymphocytes infiltrating allograft interstitial space can produce cytokines that hamper  SMCs  contractility (Barry, 1994).  Cyclosporine-A (CsA) is an immunosuppressive agent currently used for prevention of allograft rejection. CsA inhibits the production of interleukin-2 and other lymphokines by T-helper lymphocytes. If not reduced, these lymphokines act further to activate Tcytotoxic and B-lymphocyes and macrophages and together they destroy allograft tissue (Ho, Clipstone, Timmermann, Northrop, Graef, Fiorentino, Nourse, and Crabtree, 1996a). Furthermore, lymphokines are strong iNOS inducers, thus preventing their production decreases excessive N O production in transplant tissue (Marumo et al., 1995).  CsA therapy, however, has some side effects. For example, CsA induces upregulation of the N O system in transplanted patients by increasing the mRNA of eNOS and NOS activity (Calo, Semplicini, Davis, Bonvicini, Cantaro, Rigotti, D'Angelo, Livi, and Antonello, 2000). The same result is observed in other in vivo (Stroes, Luscher, de Groot,  29  Koomans, and Rabelink, 1997) or in vitro studies. For example, in an in vitro study on cultured endothelial cells, Lopez et al. indicated an increased expression of eNOS after incubation of cells with CsA (Lopez-Ongil, Saura, Rodriguez-Puyol, Rodriguez-Puyol, and Lamas, 1996). Furthermore, CsA has some other side effects such as hypertension in vivo, and apoptosis of endothelial cells in vitro. The latter effect may happen through iNOS induction (Amore et al., 1995).  2.2 HYPOTHESIS AND SPECIFIC AIMS Based on the above-mentioned evidence, I hypothesized that direct adverse effects of CsA therapy will occur in transplant and control arteries. Also, I proposed that treating heart allograft recipient rats with CsA would prevent early transplant vascular dysfunction by preventing tissue damage caused by host cell-mediated immunity. The following data indicates that CsA (5mg/Kg) at day 21 post-transplantation reduces the effect of immune-mediated and iNOS-induced injury, and possibly other damaging mechanisms on cardiac allograft vasculature in rats. At day 4 post-transplantation, however, an iNOS inducing effect of CsA is indicated. The latter is perhaps an acute temporary effect of this drug that does not last until day-21 post-transplantation. Therefore, the net result of CsA therapy is preservation of vascular smooth muscle and endothelial cell function, which may in turn contribute to graft vascular integrity, thereby limiting longterm graft injury due to C A V .  2.3 MATERIALS AND METHODS  30  2.3.1 A N I M A L GROUPS Protocols were all designed according to the University of British Columbia Animal Care Committee Guidelines. Male rats, body weight 200-250g at the time of surgery, were used for transplantation. Isograft (Lewis to Lewis) and allograft (Lewis to Fisher) hearts were heterotopically transplanted by suturing the ascending aorta of donor hearts to the abdominal aorta and the pulmonary artery to the inferior vena cava of the recipient animals. Venous connections to the donor atria were ligated (Ono and Lindsey, 1969). Both isograft and allograft groups were then divided into two subgroups according to the duration of transplantation and treatment schedule. Animals were sacrificed either at day 4 (early time point) or at day 21 (late time point) postsurgery. Within each group, recipients were divided into three subsets receiving either CsA or its solvent/vehicle cremophor or no treatment. CsA was administered subcutaneously (SC) at a dose of 5mg/kg and the vehicle treated group received the same volume of solvent as the C s A treated group. The non-treated group was injected with distilled water (SC). Rats received heparin (50 units/kg, i.p.) and were sacrificed by an overdose of sodium pentobarbital (40mg/kg, i.p.).  2.3.2 TISSUE P R E P A R A T I O N A N D D I A M E T E R M E A S U R E M E N T After animals were sacrificed, donor hearts were isolated and dissected while maintained in ice-cold physiological salt solution (PSS: see Chemicals and Buffers). Coronary septal arteries were located through a right ventricular wall opening and dissected and cleaned of adherent cardiac muscle tissue. For all functional studies, a 0.8-1.2 mm segment of the artery (inner diameter ~ 200 ixm) at the level of the superior papillary muscle was excised  31  and mounted at both ends onto glass canulae in a pressure myograph chamber (Danish Myo Technology, Aarhus, Denmark). Both ends of the artery were tied using a single strand teased from a 4-0 surgical silk thread and the chamber was placed on an inverted microscope stage to measure the arterial diameter. One of the canulae was close ended and the other connected through a pressure transducer to a peristaltic feedback pump to maintain constant pressure and also to monitor transmural pressure. The vessels were continuously supervised with bubbled (95%0 , 5%C0 ) PSS (pH 7.35-7.4) at 37°C. 2  2  Transmural pressure was then gradually increased to induce myogenic tone.  2.3.3 M Y O G E N I C A N D DEPOLARIZATION-INDUCED TONE Pressure-constriction curves were determined in all artery segments. Intravascular pressure was gradually increased in a stepwise fashion to 80mmHg. Vessels that did not develop a leak were equilibrated for one hour at this pressure to develop myogenic tone (vessels that did not develop spontaneous constriction were excluded). Following the development of pressure-induced constriction, transmural pressure was then reduced to 10 mmHg and increased in a stepwise manner at 5-minute intervals to 120 mmHg. The internal diameter was recorded at each step and then compared with diameters occurring in a calcium free PSS at the end of the experiment to determine the degree of myogenic tone at each pressure. The artery was incubated for an hour with A G (lOOuM) and the protocol was repeated to determine the effect of Ca independent production of N O on the myogenic response. Potassium chloride depolarization-induced constriction was also measured in arteries incubated with A G . These experiments were made at a low pressure (20mmHg) to  32  eliminate pressure-induced vasoconstriction. A series of increasing concentrations of KC1 solutions were prepared by mixing PSS with KC1 (127mM) solution (KC1: see Chemicals and Buffers). Finally, the vessel chamber was perfused with calcium free PSS and vessel diameters were measured with stepwise increases in pressure to determine the passive diameter at each pressure. Myogenic and depolarization induced tones were then calculated (see Statistical Analysis and Calculations).  2.3.4 A C H A N D SNP INDUCED V A S O D I L A T A T I O N A separate segment of septal artery was used to determine A C h or SNP concentration response curves. After developing maximum myogenic tone (80mm Hg), cumulative concentrations of A C h (lOnM-lOuM) or SNP (lOnM-luM) were added and the new steady-state internal diameters of the artery were registered.  2.3.5 M O R P H O L O G I C A L A N A L Y S I S Transverse sections of allograft and isograft hearts, untreated or treated with CsA, were fixed in formalin and embedded in paraffin, and stained with Verhoff s elastin to visualize the internal and external arterial elastic laminae. Digital photographs of the arteries with diameter between 50-200fim were taken using a Spot digital camera. The external (EEL) and internal elastic laminae (IEL) were outlined and their lengths measured using the image analysis software ImageProPlus® (IPP). Medial thickness was measured by the following formula: EEL/IEL-1. Since an increase in medial thickness would result in a greater difference in the length of the E E L and IEL, this ratio provides an accurate assessment of the medial thickness.  33  2.3.6 Q U A N T I T A T I O N OF IN SITU T U N E L T U N E L staining was performed, using the suggested protocol by the manufacturer, with some minor modifications (Intergen; Purchase, N Y , USA) to detect D N A fragmentation suggestive o f apoptosis.  Formalin-fixed,  paraffin-embedded  sections were de-  paraffinized in xylene and rehydrated in a series of ethanol washes. Sections were then incubated with 20 Ltg/mL proteinase K for 20 min., TdT enzyme for l h , and alkaline phosphatase conjugated anti-digoxigenin for 30 min. Chromagen Vector Red (Vector Laboratories; Burlingame, C A , USA) was used to visualize the staining and hematoxylin was used for counterstaining. At the end of alternate steps, washings with TBST were performed. Then IPP was used which is a color segmentation file that recognizes either TUNEL-positivity or nuclear staining with hematoxylin on the basis of hue, saturation, and intensity. TUNEL-positive cells and total nuclei were then counted automatically in the medial layer by IPP, and results were expressed as the percentage of apoptotic cells in the media of the studied arteries.  2.3.7 C H E M I C A L S A N D BUFFERS A l l buffer reagents were purchased from B D H and drugs were purchased from Sigma (St. Louis, MO). The composition of the PSS (mM) was: NaCl (119), KC1 (4.7), K H P 0 2  (1.18), M g S 0  4  4  (1.17), N a H C 0 (24.9), E D T A (0.023), C a C l (1.6), dextrose (11.1). 3  2  Isotonic substitutions (replacement of Na with equimolar concentrations of K ) were used when using PSS solutions with increased K concentrations.  2.3.8 STATISTICAL A N A L Y S I S A N D C A L C U L A T I O N S  34  Results of all calculations are expressed as mean ± SEM. Data was analyzed with either A N O V A and/ or repeated measurement A N O V A with multiple comparisons using a Bonferroni test where appropriate. The results of statistical tests were considered significant if p-values were < 0.05. '  Myogenic tone was calculated as percentage of arterial constriction at each pressure step using the formula: % Constriction = 100 x (Dc free,P - D )/ D c free,P where D c free,P is a  p  a  a  the arterial diameter at pressure P in calcium free PSS and D is the diameter in PSS at p  pressure P.  Depolarization induced constriction was measured as: 100 x (Dc free,20 - D[KCI])/ D c fr , a  a  ee  20 where D c fr ,20 is arterial diameter in calcium free PSS at pressure 20 mmHg and a  D[KCI]  ee  is the vessel diameter at any concentration of potassium chloride  Percentage of relaxation/dilation of the arteries in response to Ach and SNP was calculated as: 100 x  (D[Dru ], g  80 - Dgo)/(Dc free,80 - Dgo) where D[Dru ],80 is the diameter a  g  in PSS in the presence of a particular concentration of drug (ACh or SNP) at pressure 80mmHg, and Dso is the diameter at PSS and pressure 80, and D c fr , 80 is the passive a  ee  diameter of vessel in calcium free PSS at pressure 80.  2.4 RESULTS AND DISCUSSION In all of the following experimental results, coronary arterial function of transplanted heart in vehicle treated recipient rats was the same as in untreated animals (data not included).  35  2.4.1 V A S C U L A R D I A M E T E R A N D DISTENSIBILITY The mean diameter for septal arteries was 218 ± 2.7ixM (lOmmHg in calcium free PSS, n=45). Pressure-diameter data in calcium-free PSS, indicating vascular compliance, was similar in all CsA treated and untreated isograft and allograft arteries (Figure 2-1).  2.4.2 D A Y 21 POST-TRANSPLANTATION RESULTS M E D I A L THICKNESS There were marked morphological differences in the media of arteries from non-treated allograft hearts in comparison to allograft arteries from CsA-treated recipients and isograft hearts. The media of vessels from allograft hearts were thinner than their isograft counterparts (Figure 2-2A). Treatment of recipient rats with CsA completely preserved medial thickness in allograft arteries similar to the isograft controls, suggesting that the observed morphological changes were the result of the activated immune response (Figure 2-2A). Histologically, there was marked perivascular immune cell accumulation and infiltration of these cells into the medial layer. VSMCs appeared disorganized within the media layer. Finally, treatment with CsA prevented these histological changes in allograft coronary arteries (Figure 2-2B). T U N E L STAINING There were a limited number of TUNEL-positive cells in coronary arteries of untreated allograft hearts and no T U N E L staining in vessels from isograft and CsA-treated allograft hearts. TUNEL-staining was localized to cellular nuclei, and the number of T U N E L positive cells in the media was very small. As assessed by quantitation of T U N E L -  36  positivity by IPP, there were significantly more TUNEL-positive cells in the medial layer of vessels from non-treated allograft hearts (n=12) as compared to their isograft (n=7) counterparts and animals treated only with CsA (n=13) (Table 2-l)(Figure 2-3). EFFECT OF CSA T R E A T M E N T O N M Y O G E N I C TONE The tracings in Figure 2-4 demonstrate the relationship between arterial diameters in response to increasing transmural pressure in an artery from a 21-day post-transplant isograft (N21S) heart in comparison to an artery from a CsA treated allograft (C21A) and untreated recipient rat (N21A). Pressure-induced myogenic tone was lower in coronary arteries from untreated allograft recipient rats when compared to tone obtained in nontreated isograft tissue (p<0.05, n=8-9; Figure 2-5A). CsA immunosuppressive therapy in graft recipients markedly improved myogenic tone in allograft vessels in comparison to untreated animals (p<0.05, n=6-8; Fig. 2-5 A & B right). As can be seen in Figure 2-5 B , CsA abolished the difference in myogenic tone between arteries from allograft and isograft cardiac transplant rats.  Coronary septal arteries of allografts in both CsA treated and untreated recipients tended to have an increased myogenic tone in the presence of A G . This difference was statistically significant only in the latter group. In fact, vascular tone of untreated allograft recipients increased after incubation with A G , indicating that iNOS produced N O may be responsible for part of the loss of tone in these arteries (Figure 2-5 A & B ) .  37 C S A T H E R A P Y IMPROVES DEPOLARIZATION-INDUCED T O N E B Y P O T A S S I U M CHLORIDE SOLUTION In untreated allograft recipients, there was a decreased constriction of coronary septal arteries to K as compared to responses in isograft hearts (p< 0.05, different at [KC1] > +  66mM, n=8-9; Figure 2-6). CsA therapy of allograft recipient rats restored arterial constrictor responses to K to values similar to those in isograft rat hearts (n=6-8, Figure +  2-6). Treatment with CsA thus abolished the differences in response to K of arteries +  from isografts and allografts. Treatment of isograft animals did not change the constrictor response of coronary arteries to K (Figure 2-6). +  The reduced response to K  +  (a non-receptor mediated constriction) and diminished  myogenic constriction to pressure suggests a possible decrease in the number of viable or functionally active smooth muscle cells, or that there may be a defect in the common portion of the smooth muscle contraction pathway (Skarsgard et al., 2000). It is likely that CsA therapy improves cardiac function through increasing the smooth muscle viability. EFFECT OF C S A O N E N D O T H E L I U M B A S E D A N D E N D O T H E L I U M INDEPENDENT V A S O R E L A X A T I O N Vasodilatation to A C h occurs through endothelial production of NO. The function of endothelial cells was evaluated by comparing the effect of A C h in CsA treated vs. untreated allograft arteries. The dilation to A C h was then compared with that of SNP (a  38  direct N O releasing agent) to investigate whether there is also a smooth muscle component for the weaker response to ACh. The vasodilation induced by A C h was significantly greater in isograft- than in allograftarteries (p<0,01) resulting in maximal relaxations (at 5uM ACh) of 83.4 ± 5.2% vs. 27.0 ± 4.9%, respectively. Treating allograft recipients with CsA markedly improved vasodilation to A C h (P<0.01, n=6-8) and the maximum response was increased to 80 ± 4.3%. CsA treatment did not alter the responses to A C h in isograft arteries (Figure 2-7).  The magnitude of SNP induced relaxation was significantly lower in non-treated allografts in comparison to responses in isografts (p< 0.01, n=8-9). The maximal response to SNP (5uM) in non-treated allografts was 41.3 ± 5.5% (n=8) compared to 75.7 ± 4.4% (n=9) in isografts (p<0.05). Treatment with CsA improved vasorelaxation to SNP in allograft arteries (p< 0.01, n=6-8) so that the maximum was increased to 67.6 ± 8.2% (n=6). The responses to SNP were unaffected by CsA treatment of isograft rats (Figure 28).  2.4.3 D A Y 21 POST-TRANSPLANTATION DISCUSSION In this study of 21 day old cardiac transplant recipients, the findings were as follows: 1) There was a profound inhibition of myogenic and depolarization induced tone in allograft septal coronary arteries in comparison with age- and size- matched isograft vessels. CsA therapy prevented loss of tone in allograft arteries. 2) There was a significant decline in both endothelium dependent and endothelium independent vasodilation in arteries of  39  untreated allograft hearts. Immunosuppression with CsA restored vasorelaxation in allograft arteries. M Y O G E N I C A N D DEPOLARIZATION INDUCED TONE I M P R O V E M E N T B Y CSA There was a significant decline in myogenic tone in allograft arteries in comparison with isograft vessels. Reduced myogenic tone in allograft arteries was significantly increased by blocking iNOS with A G , however, the magnitude of tone in these arteries in the presence of A G was still less than their isograft counterparts.  These two findings  together indicate that there is another reason, other than the presence of excessive amount of NO, for the weaker myogenic tone in arteries of rejected allografts. Other experiments indicated that depolarization-induced constriction by KC1 solution was also significantly lower in coronary arteries of allografts in comparison to isografts. This weaker response to KC1 could not be due to the effect of increased level of iNOS-produced N O in arterial wall, since these experiments were performed after iNOS blockade. Taking these two findings into account, it can be deduced that the defect is not limited to specific mechanisms responsible for initiation of myogenic tone but it has also affected the common pathway involved in smooth muscle constriction. Alternatively, the population of viable and functional smooth muscle cells is lower in rejected allograft arteries. The lower number of viable cells in arteries of rejected allograft can be either the result of cell destruction by host cellular and humoral immunity (Barry, 1994) or the effect of cytokine-induced NO-mediated apoptosis (Szabolcs, Michler, Yang, A j i , Roy, Athan, ' Sciacca, Minanov, and Cannon, 1996). The latter mechanism is again related to the host  40  immune system but indirectly through the upregulation of iNOS by cytokines released from the attacking immune cells (Goren et al., 1998).  This study indicated for the first time that when allograft recipients were treated with CsA (5mg/Kg), myogenic tone observed in graft arteries at day 21 post-transplantation was well preserved and was the same as isograft vessels. Moreover, it was shown that incubation with A G did not significantly increase the magnitude of myogenic tone in these arteries.  Therefore, treating cardiac transplant recipients with CsA (5mg/Kg)  prevented N O induction and, as a result, fewer apoptotic VSMCs are expected to exist in this tissue.  Poor myogenic tone in allograft resistant arteries, as was indicated, increases coronary blood flow and hydrostatic pressure surpasses the oncotic pressure in end-arterioles. The net result of this pressure inequilibrium is the formation of cardiac interstitial edema. Furthermore, endothelial cell damage, which happens in allograft arteries, makes these cells directly penetrable to plasma proteins and serves as another mechanism for further progression of edema (Skarsgard et al., 2000). This generalized edema will decrease ventricular compliance and will lead to heart failure in the course of acute immune rejection of the graft (Lewis et al., 1996). CsA, by preventing myogenic tone decline, as this study indicated, and preserving endothelial cells integrity, as was shown in an aortic allograft model before (Andriambeloson, Pally, Hengerer, Cannet, Nikolova, Bruns, Zerwes, and Bigaud, 2001b), may prevent interstitial edema formation and, as a result, can slow down acute transplant heart failure.  41 V A S O R E L A X A T O R Y RESPONSE I M P R O V E M E N T B Y C S A The results of this study indicate that the vasorelaxatory response to A C h was significantly lower in allograft coronary arteries in untreated recipients at day-21 posttransplantation in comparison to matched isografts. This finding was in accordance with the results of previous studies indicating the existence of endothelial cell malfunction in allograft arteries that appears as an attenuated vasorelaxatory response to A C h (Andriambeloson, Pally, Hengerer, Cannet, Nikolova, Bruns, Zerwes, and Bigaud, 2001b),(Hollenberg, Klein, Parrillo, Scherer, Burns, Tamburro, Oberoi, Johnson, and Costanzo, 2001b). In fact, endothelial cell malfunction is the first event in the development of transplant vasculopathy and therefore can be used to predict graft vasculopathy before the clinical endpoint (Andriambeloson, Pally, Hengerer, Cannet, Nikolova, Bruns, Zerwes, and Bigaud, 2001b;Hollenberg, Klein, Parrillo, Scherer, Burns, Tamburro, Oberoi, Johnson, and Costanzo, 2001b), (Marti, Romeo, Aymat, Garcia, Guiteras, Ballester, Aminian, Caralps, and Auge, 2001). Whereas, allograft arteries from CsA treated recipient rats were able to relax in response to A C h with the same magnitude as vessels in isograft hearts. It was also found that arteries from CsA treated animals that did not undergo any surgery dilated in response to A C h with the same magnitude as untreated control animals. This finding seems different from the results of some previous studies regarding endothelium dependent vasodilation after CsA therapy. For example, in 1994, Gallego et al. discovered in a series of in vitro experiments that after C s A administration (25mg/kg/day) to Wistar rats for 15 days A C h induced vasorelaxation was markedly inhibited in femoral arteries (Gallego et al., 1994). The same finding was indicated in other studies with a CsA dose range of 25-50 mg/kg/day in different types of  42  arteries (Diederich et al., 1992), (Roullet et al., 1994), (Stephan et al., 1995), (Rego et al., 1990). The common finding in all these studies was that CsA attenuated ACh-induced dilation when it was used at a much higher dose (>25 mg/kg/day) than was used in the present study. However, in accordance with the finding of this study, when CsA is used with a dose (5-15 mg/kg/day) close to what we used (5mg/kg/day) it does not affect endothelium-based relaxation (Chan et al., 1992),(Diederich, Yang, and Luscher, 1992), (Rego, Vargas, Wroblewska, Foegh, and Ramwell, 1990). Thus, it can be deduced that CsA at doses >25mg/kg/day will negatively affect endothelial cells function. At lower doses (5-10 mg/kg/day), however, it does not inhibit endothelial cell N O production but can still act effectively as an immunosuppressor as indicated.  A C h vasorelaxation happens through an upregulation of eNOS (Moncada and Higgs, 1995). Endothelium released N O will then relax SMCs by either increasing their intracellular cGMP content or opening potassium channels. Moreover, there is an N O independent relaxatory mechanism for A C h in resistance arteries, which acts through releasing endothelium derived hyperpolarizing factor (Woodman et al., 2000). Therefore, in this study, the weaker A C h induced vasorelaxtion in allograft arteries can be due to a defect in endothelial cells or SMCs of these vessels jeopardizing one or more of the above-mentioned mechanisms involved in A C h induced vasorelaxation. Another reason for poor response to A C h in allograft arteries may exist. It has been proven that A C h can induce vasoconstriction by direct stimulation of receptors on vascular SMCs (Nasa et al., 1997;Tsuji and Cook, 1995). In allograft tissue, A C h has the opportunity to directly affect subendothelial smooth muscle cell and cause constriction as evidenced by electron-  43  micrographs and silver staining methods indicating some degrees of endothelial cell denudation at day 20 post-transplantation (Andriambeloson, Pally, Hengerer, Cannet, Nikolova, Bruns, Zerwes, and Bigaud, 2001b), (Gohra et al., 1995). Acting in this way, the constrictive effect of A C h in allograft arteries counterbalances its dilatory effect via released N O from the remaining functional endothelial cells and may lead to a poor vasorelaxatory response. The results of this study also indicated that when allograft arteries were subjected to cumulative doses of sodium nitroprusside (SNP), vasorelaxation was again significantly lower than in matched vessels from isograft hearts. However, this defective response was significantly improved by CsA treatment, so that no difference in relaxatory response was observed between coronary arteries of CsA treated allograft- and untreated isograftrecipients. The mechanism of action of SNP occurs by release of N O after its direct metabolization by arterial S M C (Bates et al., 1991).  N O will then cause vascular  relaxation through increasing cGMP concentration within V S M C s (Murad, 1986). Since both A C h and SNP use the common NO-induced cGMP-mediated vasorelaxation pathway, the weak relaxatory response to both compounds in allograft heart arteries suggests the presence of a defect in the cGMP-mediated pathway of smooth muscle relaxation. However, it is hard to say i f there is also a specific defect in endothelial cell production of N O in allograft arteries since the magnitude of endothelium based vasorelaxation (ACh induced) was not significantly lower than endothelium independent (SNP induced) dilation in these vessels. It was also indicated that the vasodilatory response in CsA (5mg/kg) treated arteries was not different from the response in untreated control animals, therefore CsA at 5mg/kg/day did not affect endothelium  44  independent vasorelaxation. This finding is in accordance with previous studies that used the same dose of CsA with almost the same treatment length (Chan, Kern, Flanagan, Kron, and Tribble, 1992),(Diederich, Yang, and Luscher, 1992), (Rego, Vargas, Wroblewska, Foegh, and Ramwell, 1990).  Whereas, CsA therapy at higher doses  (>25mg/kg/day) suppressed SNP induced vasorelaxation (Diederich, Yang, and Luscher, 1992), (Rego, Vargas, Wroblewska, Foegh, and Ramwell, 1990), (Roullet, Xue, McCarron, Holcomb, and Bennett, 1994).  Based on the results, CsA affects  neither endothelium-based  nor endothelium-  independent vasorelaxation i f administered at a low dose (5mg/kg/day). However, at higher doses (>25mg/kg/day), it attenuates both mechanisms of vasorelaxation. The lower vasorelaxation can be due to CsA inhibition of cGMP formation (Busch, Galvanek, and Reynolds, Jr., 1971) or an increase in endothelium-released  vasoconstrictor  cyclooxygenase products (Hosenpud, Bennett, Keck, Fiol, Boucek, and Novick, 1998). This effect has been recognized as a possible explanation for CsA induced hypertension.  It was also found that arteries of allograft hearts had an infiltration of immune cells in the perivascular space in their media as well as a thinner media in comparison to isografts (not indicated). This medial thickness loss in allograft arteries was a direct result of immune-mediated damage because CsA therapy could prevent it as it also prevented medial perivasular immune cell infiltration. In contrast to the results of the present study, Hamano et al. indicated in 1998 that in allograft arteries despite CsA therapy (lOmg/kg/day) intimal thickness increases by V S M C proliferation whereas medial  45  thickness decreases due to V S M C and collagen atrophy at day 30 and 60 posttransplantation follow up (Hamano et al., 1998). This contrast is probably attributable to examining the vessels at a later time point during post-transplant follow up. In another study on a dog model of arterial transplantation, it was indicated that after 3 months of implantation, medial thickness was well preserved in carotid allografts in recipients treated with 25mg/kg/day CsA in. comparison to lOmg/kg/day or control group. Taking into account the above-mentioned data, it can be deduced that C s A at low doses (5mg/kg/day) can preserve allograft arteries wall thickness in short term  after  transplantation (Vischjager et al., 1996). However, over time (>8 weeks) even lOmg/kg/day of CsA is not effective in prevention of graft arterial changes and higher doses of CsA (e.g. 25mg/kg/day) are needed to prevent arterial medial changes.  Finally, in this study it was indicated that the number of apoptotic cells in the medial layer of allograft arteries was significantly higher in comparison with isograft arteries. CsA treatment in allograft recipients could significantly inhibit programmed cell death in the graft arteries. However, the grafts were only followed for 21 days and therefore longterm results of CsA therapy on apoptosis in allograft arteries can be different. For example, in a previous related study it was indicated that CsA therapy only prevented apoptosis at an early time point (< 14 days) post-transplantation, however, at a later time point (> 28 days) the number of T U N E L positive cells were similar in coronary arteries of cardiac allograft in CsA treated and untreated recipients (Dong et al., 1999). As was indicated, CsA can preserve V S M C s and endothelium in allograft arteries. The mechanism of allograft preservation by CsA is suppression of T cell-mediated immunity  46  through inhibition of the activation of cytokine genes. These genes include those involved in B-cell activation such as IL-4, as well as those necessary for T-cell proliferation, by shortening the G l phase of the cell cycle in these cells (Cristillo et al., 1997), such as IL-2 (Ho et al., 1996b). CsA mediates this effect by inhibiting calcineurin which is a phosphatase enzyme required for activation of genes encoding IL-2 and other lymphokines.  Another finding of this study was that arteries from Cremophor® (vehicle for CsA) treated control rats acted the same as distilled water treated (no treatment) animals in response to pressure, KC1, A C h and SNP. Other studies have also addressed the effect of cremophor on coronary artery function. For example, Mankad P. et al. (1992), by using isolated and perfused rat hearts, showed that at intravenous doses lower than 50ng/ml, cremophor had no effect on vascular function. However, at higher doses cremophor induced dose-dependent vasodilation (Mankad et al., 1992b). It is possible that the cremophor content of the CsA dose in this in vivo study is comparable with the in vitro cremophor dose used by Mankad P. et al. and therefore, it had no effect on vascular function.  In conclusion, this study indicated that CsA treatment in allograft recipients can preserve endothelial and smooth muscle cells function and, as a result, is expected to prevent ventricular failure secondary to myocardial edema and to slow down the development of TVD.  47  2.4.4 D A Y 4 POST-TRANSPLANTATION RESULTS EFFECT OF CSA T R E A T M E N T O N M Y O G E N I C TONE A n induction of iNOS occurred in all CsA treated rats regardless of the type of graft (allograft or isograft) they received. These arteries from treated animals first developed a poor myogenic tone but in repeated experiments after incubation with A G , a specific iNOS blocker, this depression of tone was significantly improved (p<0.05) (Figure 2-9). Control rats that did not undergo surgery but received CsA treatment for 4 days also had A G sensitive myogenic tone (Figure 2-10). These data indicate an induction of iNOS by CsA at day 4 post-treatment (Figure 2-11). C S A T H E R A P Y A N D DEPOLARIZATION INDUCED TONE To eliminate the effect of iNOS, KC1 experiments were performed after treating the vessels with A G . The extent of KC1 induced tone was similar in untreated and CsA treated rats for isograft, allograft, and control groups (Figure 2-12). N O N - E N D O T H E L I U M A N D E N D O T H E L I U M - B A S E D R E L A X A T I O N A N D CSA THERAPY There was no significant difference between the allograft and the isograft arteries in relaxatory response to either A C h (endothelium based) or SNP (non-endothelium based). There was also no difference in dilation in CsA treated and untreated rats in either the isograft or allograft group (Figure 2-13 & 2-14).  48  In control experiments, treatment with CsA vehicle (cremophor) did not cause any change in myogenic and KC1 induced tone or in SNP or A C h relaxatory responses (no difference from untreated/control rats).  2.4.5 D A Y 4 POST-TRANSPLANTATION DISCUSSION The main finding of day-4 posttreatment is that CsA itself may induce iNOS in arteries of transplant and control hearts at this early time point after administration. This conclusion was made because myogenic tone reduction in CsA treated arteries was compensatable by treating the vessels with iNOS blocker A G . Thus, it can be suggested that the extra amount of N O produced by an over-expression of iNOS suppresses myogenic tone in these arteries. This possible iNOS induction is the specific effect of CsA treatment and is not attributable to an immune reaction or the effect of surgery because it does not occur in untreated allografts and was present even in CsA treated control animals that did not undergo any surgery. In a previous study in 2000, Amore et al. indicated the in vitro induction of iNOS by CsA (Amore et al., 2000). In this study, four different cultured cell types were used including murine endothelial cells and human mesangeal, renal cortical tubular epithelial, and umbilical vein cells. Incubation of these cells with CsA induced apoptosis via inducing iNOS and increasing levels of p53 (a proapoptotic tumor suppression protein). Adding L - N A M E to the culture medium prevented apoptosis and iNOS m R N A was elevated as detected with Northern blot analysis (Amore, Emancipator, Cirina, Conti, Ricotti, Bagheri, and Coppo, 2000). Later studies indicated that the addition of A C E inhibitors and angiotensin-I receptor antagonists to the culture medium  49  of the cells attenuated apoptosis possibly by inhibiting the activation of nuclear factor K B (Conti etal., 2001).  The degree of vasoconstriction induced by depolarization, using KC1 solution, was the same in all three groups (iso- and allograft recipients as well as control animals). Taking into account that KC1 dose-response experiments were performed after blocking iNOS with A G it can be deduced that four-day exposure to extra amounts of N O in coronary arteries of CsA treated animal does not permanently affect the contractile function of vascular smooth muscle cells.  ACh-induced endothelium dependent vasorelaxation was similar in CsA treated (5mg/kg/day) and untreated allografts and isograft heart septal arteries. This finding indicates that early signs of rejection such as leukocyte infiltration occurs in CsA untreated allografts at day 4 post transplantation at the microscopic level (Dong, Granville, Tuffhel, Kenyon, English, Wilson, and McManus, 1999) and that endothelial cells and smooth muscle cells are normal at this stage. Furthermore, it can be deduced that CsA itself does not have an adverse effect on endothelial production of N O or smooth muscle relaxation in response to NO. This finding is in accordance with previous studies indicating that low doses of CsA does not affect endothelium dependent vasodilation (Chan, Kern, Flanagan, Kron, and Tribble, 1992;Diederich, Yang, and Luscher, 1992;Rego, Vargas, Wroblewska, Foegh, and Ramwell, 1990).  50  Endothelium independent vasorelaxation was the same in all three groups, which further supports the conclusion that the ability of smooth muscle to dilate in response to N O is not affected at day 4 by the rejection process or CsA. In conclusion, CsA at a low dose (5mg/kg/day) may induce iNOS in arteries of allograft, isograft and control hearts. However, CsA does not affect endothelium dependent or independent vasorelaxation and V S M C contractile function permanently.  51  Histopathology  CAV  CAD  Development period Vascular localization Lipid  Months Diffuse Prominent, early & late Concentric Never until very late Occasionally Mildly disrupted  Years Focal and proximal Prominent, generally later Eccentric Common Absent Disrupted & frequently reduplicated Macrophages / Fas negative oncosis  Intimal proliferation Calcium deposit Vasculitis Intrernal elastic lamina Main Inflammatory cell involved / mechanism of death  CD4 T cells / Fas signaling apoptosis  Table 1-1: A comparison between histopathological findings in cardiac allograft vasculopathy (CAV) and coronary artery disease (CAD)(Billingham 1992; Dong et al. 1996; Personal communication, McManuc 2002).  52  Adverse  Concentration  effect  Model  Reference  /Dose Endothelialitis  5 mg/kg/day  Rat  ( M e n n a n d e r et a l . 1992)  c N O S upregulation  1 nM-1 u M  Human  ( L o p e z - O n g i l e t a l . 1996)  Hypertension induction ( C a l o et a l . 2 0 0 0 )  B l o o d level Human  - T Superoxide production  ( N g u y e n e t a l . 1999)  150-200 ng/ml a n d N O degradation - T E T 1 production  20 mg/kg i.v.  - -1 P r o s t a c y c l i n e  Culture  Rabbit  ( K o n , S u g i u r a , e t a l . 1990; ( L a n g m a n et a l . 1994)  Cultured  ( R o s e n t h a l e t a l . 1989)  Concentration HUVEC 0.5-5 u M  - T R e s p o n s e to A V P  - 1 R e s p o n s e to n o r e p i n e p h r i n  Cultured  ( L o e t a l . 1996; L o e t a l . 1997;  concentration  Human  ( A v d o n i n e t a l . 1999)  0.1-10 u M .  ETC  Bath concentration  Rats  (Loetal.  25 m g / k g / d a y  Rats  ( P e r i c o e t a l . 1986)  5 mg/kg/day  Rats  ( M o r g a n et a l . 1991; A v d o n i n et a l .  15 m g / k g / d a y  Rats  ( B a n i j a m a l i et a l . 1 9 9 3 )  Culture  1996)  1-10 u M - T P l a s m a rennin substrate activity  - 1 Sympathetic activity 1999; C h i u e t a l . 1992) Facilitation o f C a i n d u c e d 2 +  Ca  2 +  release  Table 1-2: Adverse vascular effects of CsA  53  LD o (±95% confidence interval) (mg/kg) 5  Animal species  Intravenous therapy  Oral therapy  Mouse Rabbit Rat  107 > 10 25 (22-29)  2329(1848-3020) > 1000 1480(1105-1997)  Table 1-3: CsA acute toxicity in mouse, rabbit, and rat.  54  Section  N21A (n=12)  C21A (n=13)  N21S (n=7)  C21S (n=10)  TUNELpositivity  1.6% ± 0 . 5 % *  0.0% ± 0.0%  0.0% ± 0.0%  0.0% ± 0.0%  Table 2-1: Percentage of T U N E L positive cells found at day 21 posttransplantation in untreated and CsA treated alio- and isograft coronary arteries (*p< 0.01). N : untreated, C: CsA treated, A : allograft, S: isograft, 21: day 21 posttransplantation.  55  Dendritic Cells  Cytotoxic T cell Host T-Cells  y  CsA  Figure 1-1: Schematic representation of the direct and indirect pathways of the events that lead to the destruction of allograft and site of CsA action. Donor class I and class II M H C antigens along with B7 molecules on the surface of donor's dendritic cells are recognized by host's CD8+ cytotoxic T cells and CD4+ helper T cells respectively. In response to M H C class II antigens, CD4+ cells proliferate and differentiate to T H 1 and T H 2 cells. TH1 releases IL-2 and which further augments the proliferation of CD4+ cells and also provides signals for differentiation of CD8+ cytotoxic cells. T H 1 cells also secrete INFy and other cytokines that potentiate the expression of M H C molecules on graft endothelial cells and will increase vascular permeability respectively. T H 1 cells are also responsible for the induction of a local delayed hypersensitivity reaction. Activation of TH 2-type CD4+ cells generates IL-4 and IL-5 that promote B-cell differentiation. Eventually the following mechanisms come together to destroy allograft: 1) lysis of cells bearing M H C I by CD8+ cytotoxic T cells, 2) antigraft antibodies produced by plasma cells, 3) nonspecific damage induced by macrophages and other leukocytes that accumulate as a result of the delayed hypersensitivity reaction. M H C : major histocompatibility antigen, IL: interleukin, INF: interferon, A g : antigen, Ab: antibody, E T C : endothelial cells, C: complement, CsA: cyclosporine A .  56  Figure 2-1: Passive distensibility in day 4 and day 21 CsA treated or untreated allograft and isograft coronary arteries. Distensibility is presented according to the passive diameter at pressure 80mmHg in order to increase the sensitivity for detecting the possible differences. Diameter at pressure 80 was selected because at this pressure there was no difference in passive diameters. C:CsA treated, N : no treatment, 2 l:day-21 post-transplantation, 4: day-4 post-transplantation.  57  A 0.4  n  0.35 -  N21A  N21S  C21A  C21S  Figure 2-2: A comparison in medial thickness at day-21 posttransplantation amongst the coronary arteries of untreated or C s A treated alio- and isograft hearts. A - Coronary artery medial thickness df allografts in untreated recipients is significantly lower than the other three groups (*p<0.01) indicating the destruction of V S M C s in the media of these arteries. B- Verhoff s elastin stain indicating the arterial internal and external elastic laminae (next page). N : no treatment, C: CsA treated, A : allograft, S: isograft, 21: day 21 post-transplantation.  58  C21A  C21S  Figure 2-2B: (see page 58 for description)  59  Figure 2-3: T U N E L staining o f transplant arteries. A comparison in the number o f T U N E L positive cells at day-21 post-transplantation amongst the coronary arteries o f untreated or C s A treated hosts receiving alio- or isograft hearts. The arrow represents an apoptotic cell in the media o f an allograft artery in an untreated recipient. N : no treatment, C : C s A treated, A : allograft, S: isograft, 21: day 21 post-transplantation.  60  120 100  TRANSMURAL PRESSURE  Figure 2-4: Day 21 post-transplantation, pressure-diameter tracings from coronary arteries harvested from CsA treated and untreated alio- or isograft recipients, There is a profound inhibition of myogenic tone in untreated allograft in comparison with untreated isografts that develop a strong constriction in response to pressure. This tone reduction in allografts is reflected in large diameter increase as pressure increases. CsA treatment preserves the tone in allograft arteries. N:no treatment, C: CsA treated, A : allograft, S: isograft, 21: day 21 post-transplantation.  61  A  TRANSMURAL P R E S S U R E (mmHg)  TRANSMURAL P R E S S U R E (mmHg)  Figure 2-5: Day 21 post-transplantation, myogenic tone in CsA treated or untreated allografts and isografts in the presence or absence of AG. A- In the absence of AG, there is a significant decline in myogenic tone in untreated allograft arteries in comparison to isograft vessels (°p<0.05, n=8-9 repeated measurement ANOVA). Treating allograft vessels with A G has improved the tone (*p<0.05 repeated measurement ANOVA) indicating the presence of iNOS based NO. B- CsA treatment in graft recipient has no effect in isograft arteries but improved the myogenic tone reduction in allografts arteries in comparison to their untreated counterpart (**p< 0.05, n=6 repeated measure ANOVA) so that the response is not different from the isograft tissue. In both CsA treated isograft and allograft arteries incubation with AG potentiates the tone but not significantly. C: CsA treated, N: no treatment, 21: day 21 posttransplantation, AG: aminoguanidine.  62  Figure 2-6: Day 21 post-transplantation, KC1 depolarization induced tone- there is a significant inhibition in KC1 induced tone in untreated allograft arteries in comparison to isograft vessels (**p<0.05, n=8-9, repeated measure A N O V A ) . CsA therapy markedly improves this tone reduction (*p<0.05, repeated measure A N O V A , n=6-8). N : no treatment, C: CsA treated, 21: day 21 poat-transplantation.  63  Figure 2-7: Day 21 post-transplantation, A C h induced endotheliumdependent arterial relaxation. The response in allograft arteries of untreated recipient rats is markedly lower in comparison to isografts (**p<0.01 repeated measure A N O V A , n=6-8). CsA therapy of the graft recipient has no effect in isograft tissue but significantly improves the response in the arteries of the allograft hearts (*p<0.01). N : no treatment, C: CsA treated, 21: day 21 post-transplantation.  64  Figure 2-8: Day 21 post-transplantation, SNP induced endotheliumindependent vascular relaxation. The response of the arteries in allograft hearts was significantly lower than isograft vessels (**p<0.05, n=8-9, repeated measure A N O V A ) . The graphs also indicate that CsA therapy in allograft recipients has an enormous effect in improving vascular relaxatory response to SNP in comparison to untreated animals (*p<0.05, n=8-9, repeated measure A N O V A ) . N : no treatment, C: CsA treated, 21 : day 21 post-transplantation.  65  TRANSMURAL PRESSURE (mmHg)  TRANSMURAL PRESSURE (mmHg)  Figure 2-9: Day 4 post-transplantation. A - Myogenic tone in untreated isograft and allograft arteries before and after iNOS inhibition with A G . There is no tone inhibition in untreated graft arteries and incubation with A G does not increase the tone. B Myogenic tone in CsA treated alio- and isograft arteries in the presence or absence of A G . There is an apparent tone reduction in CsA treated allografts, which is compensatable by A G (*p<0.05, n=6, repeated measure A N O V A ) . Isograft arteries indicated a similar pattern. N : no treatment, C: CsA treated, 4: day 4 poattransplantation, A G : aminoguanidine.  66  N4-Control  Figure 2-10: Pressure-diameter tracings from coronary septal arteries harvested from day-4 CsA treated and untreated control rats. Arteries of untreated animals develop a stronger myogenic tone in response to pressure increase in comparison with arteries from CsA treated rats. N : no treatment, C: CsA treated, Control: no surgery, 4: day 4 after treatment.  67  TRANSMURAL PRESSUE (mmHg)  Figure 2-11: Pressure-percent constriction in day-4 untreated (NControl, left) and CsA treated (C-Control, right) control animals. Myogenic tone is lower in arteries of CsA treated animals in comparison with untreated ones. Incubating the vessel with A G compensates for tone weakness in C-control arteries. N : no treatment, C: CsA treated, 4: day 4 post-transplantation, Control: no surgery, A G : aminoguanidine.  68  ALLOGRAFT  ISOGRAFT  [KCQ mM  Figure 2-12: Day 4 post-transplantation. KC1 depolarization induced constriction in CsA treated and untreated isograft and allograft arteries. The amount of induced tone in both types of graft regardless of the type of treatment is the same. N : no treatment, C: CsA treated, 4: day 4 post-transplantation.  69  Figure 2-13: Day 4 post-transplantation. 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