THE ROLE OF HUMAN LEUKOCYTE ANTIGEN-G IN CARDIAC ALLOGRAFT VASCULOPATHY

Transcription

1 THE ROLE OF HUMAN LEUKOCYTE ANTIGEN-G IN CARDIAC ALLOGRAFT VASCULOPATHY By Amelia Georgiana Mociornita, BScN (Hons) A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of the Institute of Medical Science University of Toronto Copyright by Amelia Georgiana Mociornita (2013)

2 THE ROLE OF HUMAN LEUKOCYTE ANTIGEN-G IN CARDIAC ALLOGRAFT VASCULOPATHY Amelia Georgiana Mociornita, BScN (Hons) Master of Science Institute of Medical Sciences, University of Toronto 2013 ABSTRACT Human leukocyte antigen-g (HLA-G), a non-classical MHC I protein, plays an essential role in immune tolerance and is associated with a lower incidence of graft rejection and cardiac allograft vasculopathy (CAV). To examine the pattern of HLA-G expression post-transplantation we determined that HLA-G can be up-regulated in smooth muscle cells (SMCs) following exposure to everolimus. We also determined that HLA-G at 500 and 1000 ng/ml reduces SMC proliferation. In further studies, treatment with HLA-G inhibited TNFα-stimulated neutrophil adhesion to endothelial cells (ECs) at all concentrations tested (0.1-1 ng/ml), suggesting a role in inflammation. The expression of HLA-G is influenced by a polymorphism in the HLA-G gene. We sought to determine if the 14bp insertion/deletion polymorphism can predict the development of CAV. There was no association between this polymorphism and CAV; however, this study had a small number of patients; therefore further investigations are needed to confirm these findings. II

3 ACKNOWLEDGEMENTS Completion of this thesis would not have been possible without the help of many wonderful people I worked with during my years of study. I am very grateful to my supervisor, Dr. Vivek Rao for his guidance, helpful criticism and support. I am indebted to him for the opportunity he gave me to pursue my graduate degree in the field of basic science. I am also deeply thankful for the support of my cosupervisor, Dr. Ren-ke Li and for his countless ideas throughout my project. I wish to thank my committee member, Dr. Gary Newton for his critical comments and valuable suggestions along the way. I would like to address special thanks to Dr. Diego Delgado who gave me the opportunity to add my contributions to his HLA-G research project. Under his supervision I greatly expanded my knowledge about the world of science. I am forever grateful for his valuable friendship, for his mentorship and constant scientific and personal advice, and for guiding me towards a successful career path. It was an unparalleled learning experience and one that I will not forget. I am enormously grateful and forever indebted to Laura Tumiati, the manager of Rao laboratory. Her ongoing support and encouragement had a profound effect on my professional and personal life. I am grateful for her patience while teaching me all the laboratory techniques I know, and for taking time to help me find solutions to endless technical problems. Thank you Laura for your true friendship and for all the much needed valuable talks and advice; I could have not done it without your ongoing help. I would like to express my gratitude to Dr. Heather Ross who provided valuable insights to my overall work and thoughtful contributions to my scientific writing. The time spent as a student was memorable and enjoyable. I had the chance to meet and work with great people who contributed to my learning experience in various ways. These are: Peter Papageorgiou, Jemy Joseph, Liza Grosman-Rimon, Carolina Alba, Arash Ghashghai, Andrzej Chruscinski, and Lisa Garrard. Thank you all for your help and warm friendship. I would like to thank my parents, Roxana and George, my sister Simona, and my brother Catalin for their unconditional love and support throughout my life and in pursuit of my graduate degree. Your continuous help and warm thoughts were felt even from thousands of kilometers away. I thank my son Marco for being the ultimate reason to successfully complete my research project and for keeping me motivated during the final months as a student. III

4 Finally, I would like to acknowledge the Canadian Institutes of Health Research (CIHR), the UHN Research and Education Fund, the University of Toronto, Department of Medicine, and Astellas Pharma for providing me with funding in support of this work. IV

5 Table of Contents ABSTRACT... II ACKNOWLEDGEMENTS... III TABLE OF CONTENTS... V LIST OF ABBREVIATIONS AND SYMBOLS... X LIST OF FIGURES... XII LIST OF TABLES... XIV LIST OF APPENDICES... XV Chapter 1 INTRODUCTION OVERVIEW BACKGROUND Heart Transplantation Cardiac Allograft Vasculopathy Human Coronary Artery Smooth Muscle Cells and CAV Human Coronary Artery Endothelial Cells and CAV The role of everolimus in CAV Human Leukocyte Antigen-G The structure of HLA-G HLA-G inhibitory Receptors Functions of HLA-G a. Direct inhibition of immune cell functions b. Indirect immune suppression through regulatory cell production c. Other functions of HLA-G The Role of HLA-G in Pregnancy The Role of HLA-G in Cancer The Role of HLA-G in Inflammatory diseases The Role of HLA-G in Solid Organ Transplantation The 14-bp insertion/deletion polymorphism in solid organ transplantation V

6 Chapter 2 RATIONALE AND HYPOTHESES The effect of everolimus on HLA-G expression in human coronary artery smooth muscle cells Rationale Hypothesis The effect of HLA-G on human coronary artery smooth muscle cell proliferation Rationale Hypothesis The effect of HLA-G on neutrophil adhesion Rationale Hypothesis The expression of ILT2, ILT4, KIR2DL4 and CD Rationale Hypothesis The 14bp polymorphism and CAV Rationale Hypothesis Chapter 3 METHODS In vitro studies General Methods Cell culture The effect of everolimus on HLA-G expression in human coronary artery smooth muscle cells Cell culture Treatment of SMCs with everolimus Protein extraction Protein determination Western Immunoblotting analysis Statistical analysis VI

7 The effect of HLA-G on human coronary artery smoth muscle cell proliferation Cell culture Treatment of SMCs with HLA-G Statistical analysis Neutrophil adhesion studies The effect of calcineurin inhibitors on neutrophil adhesion to endothelial cells Cell culture Neutrophil isolation Treatment of EC with cyclosporine and tacrolimus Neutrophil adhesion assay Statistical analysis The effect of HLA-G neutrophil adhesion to endothelial cells injured by cytokine exposure Treatment of EC with HLA-G and TNF-α Statistical analysis Expression of ILT2, ILT4, KIR2DL4 and CD160 inhibitory receptors in SMCs and ECs Analysis of ILT2, ILT4, KIR2DL4, and CD160 inhibitory receptors expression on SMCs and ECs via Western Immunoblotting Cell culture Treatment of SMCs and ECs with TNF-α Protein extraction Protein determination Western immunoblot analysis Analysis of ILT2, ILT4, and KIR2DL4 inhibitory receptors expression on SMC via flow cytometry Flow cytometry VII

8 3.2 Clinical study HLA-G polymorphism and cardiac allograft vasculopathy Patients Blood collection DNA extraction bp Insertion/Deletion polymorphism genotyping Coronary angiography Statistical analysis Chapter 4 RESULTS The effect of everolimus on HLA-G expression in SMCs HLA-G expression The effect of HLA-G on human coronary artery smooth muscle cell proliferation Proliferation study using JEG-3 conditioned media as a source of HLA-G Proliferation study using purified HLA-G Neutrophil adhesion studies The effect of calcineurin inhibitors on neutrophil adhesion to endothelial cells The effect of HLA-G on neutrophil adhesion to human coronary artery endothelial cells injured by cytokines TNF- α dose response study Neutrophil adhesion study Expression of ILT2, ILT4, KIR2DL4, AND CD160 inhibitory receptors in SMCs and ECs Analysis of ILT2, ILT4, KIR2DL4 and CD 160 inhibitory receptor expression on SMCs and ECs via Western immunoblotting Analysis of ILT2, ILT4, and KIR2DL4 inhibitory receptors expression via flow cytometry HLA-G polymorphism and cardiac allograft vasculopathy Clinical characteristics HLA-G 14 bp insertion/deletion polymorphism HLA-G 14-bp polymorphism in relation to cardiac allograft vasculopathy VIII

9 Chapter 5 DISCUSSION, CONCLUSION AND FUTURE DIRECTIONS Everolimus induces HLA-G expression in human coronary artery smooth muscle cells HLA-G inhibits human coronary artery smooth muscle cell proliferation in vitro The effect of HLA-G on neutrophil adhesion to human coronary artery endothelial cells injured by cytokines Expression of HLA-G receptors within the human coronary artery smooth muscle cells and human coronary artery endothelial cells HLA-G polymorphism and cardiac allograft vasculopathy Summary and conclusion Future perspectives Chapter 6 REFERENCES Chapter 7 APPENDICES IX

10 LIST OF ABBREVIATIONS AND SYMBOLS 14bp 14bp -/- 14bp +/+ 14bp -/+ ACR ANOVA APC BSA β 2 M CAV CD CMJ CMS CNI CNS DC DMEM DMSO EC ECL ECM EDTA EGM-2 FBS g HLA- HLA-G HLA-G - HLA-G + HRP HUVEC ICAM-1 IFN-γ IL- ILT- ISHLT kda KIR2DL4 mab ml mtor M MHC 14 base pair 14 base pair deletion/deletion 14 base pair insertion/deletion 14 base pair deletion/insertion Acute cellular rejection Analysis of variance Antigen-presenting cell Bovine serum albumin Beta-2-microglobulin Cardiac allograft vasculopathy Cluster of differentiation Conditioned medium from JEG 3 cells Conditioned medium from smooth muscle cells Calcineurin inhibitor Central nervous system Dendritic cells Dulbecco s modified Eagle medium Dimethyl Sulfoxide Human coronary artery endothelial cell Electrochemiluminescence Extracellular matrix Ethylene diamine tetra-acetic acid Endothelial cell growth medium-2 Fetal bovine serum Gram Human leukocyte antigen Human leukocyte antigen-g Human leukocyte antigen-g negative Human leukocyte antigen-g positive Horseradish peroxidase Human umbilical vein endothelial cell Intracellular adhesion molecule-1 Interferon-γ Interleukin- Immunoglobulin-like transcript International Society for Heart and Lung Transplantation Kilodalton Killer cell immunoglobulin-like receptor 2DL4 Monoclonal antibody Millilitre Mammalian target of rapamycin Moles per litre; molar Major histocompatibility complex X

11 MHC-I Major histocompatibility complex class I MHC-II Major histocompatibility complex class II MLR Mixed lymphocyte reaction MMF Mycophenolate mofetil MS Multiple sclerosis NK Natural killer O 2 Oxygen PBMC Peripheral blood mononuclear cell PBS Phosphate buffer saline PE Preeclumpsia ph Negative logarithm of hydrogen ion concentration PMSF Phenylmethylsulphonylfluoride PVDF Polyvinylidene REG Regular medium RT Room temperature SDS Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SEM Standard error of the mean shla-g Soluble human leukocyte antigen-g TBS TRIS-buffered saline TGF-β Transforming growth factor-β TNF-α Tumor necrosis factor-α T reg Regulatory T TRIS Trishydroxymethylaminomethane TTBS TBS + 1% Tween 20 VCAM-1 Vascular cell adhesion molecule-1 µ- Micro- (10-6 ) o C Degrees Celsius XI

12 LIST OF FIGURES Figure 1: Figure 2: Figure 3: Figure 4: Figure 5: Figure 6: Figure 7: Figure 8: Mechanism of CAV - Inflammatory response. Adapted from Abbas and Lichtman Mechanism of CAV - SMC migration and proliferation. Adapted from Mitchell Structures of HLA-G primary transcript and HLA-G mrna transcripts. Adapted from Donadi et al. and Gonzales et al. HLA-G protein isoforms. Adapted from Donadi et al. and Gonzales et al. Location of the 14bp deletion/insertion polymorphism in exon 8 of the 3 UTR. Adapted from Rousseau et al. Immunomodulatory functions of HLA-G. Adapted from Pistoia et al. Assessment of HLA-G expression in smooth muscle cell cultures following everolimus treatment. Assessment of HLA-G expression in smooth muscle cell cultures following everolimus treatment. Western Immunoblot representation. Figures 9(A), (B), (C): Representative microphotographs (4X) of SMCs proliferation at 24, 48, and 120 hours. Figure 10: Figure 11: Assessment of SMC proliferation inhibition in response to HLA-G released by JEG-3 cells. Assessment of SMC proliferation inhibition in response to soluble HLA-G treatment. Figure 12(A), (B), (C): Representative microphotographs (4X) of confluent ECs under different conditions. Figure 13: Figure 14: Figure 15: Assessment of neutrophil adhesion to EC as a result of various degrees of EC injury caused by exposure to tacrolimus and cyclosporine. Assessment of neutrophil adhesion to EC after treatment with tacrolimus, cyclosporine, and TNF-α at various concentrations. Assessment of neutrophil adhesion to EC as a result of various degrees of EC injury caused by exposure to different concentrations of TNF-α XII

13 Figure 16: Figure 17: Figure 18: Figure 19: Figure 20: Figure 21: Figure 22: Figure 23: Figure 24: Figure 25: Figure 26: Figure 27: Assessment of neutrophil adhesion in response to TNF- α with or without HLA-G. A representative Western blot showing no ILT2 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours. A representative Western blot showing no ILT4 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours. A representative Western blot showing no KIR2DL4 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours. A representative Western blot showing no CD160 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours. A representative Western blot showing no ILT2 protein expression in endothelial cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours. A representative Western blot showing no ILT4 protein expression in endothelial cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours. A representative Western blot showing no KIR2DL4 protein expression in endothelial cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours. A representative Western blot showing no CD160 protein expression in endothelial cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours. Flow cytometric analysis of ILT2 expression in cultured smooth muscle cells. Flow cytometric analysis of ILT4 expression in cultured smooth muscle cells. Flow cytometric analysis of KIRDL42 expression in cultured smooth muscle cells XIII

14 LIST OF TABLES Table 1: Table 2: Table 3: Table 4: Clinical characteristics of heart transplant patients HLA-G 14 bp alleles and genotypes frequencies HLA-G 14-bp polymorphism in relation to CAV at 1 year HLA-G 14-BP Polymorphism in Relation to CAV at 5 years XIV

15 LIST OF APPENDICES Mociornita AG, Lim-Shon J, Joseph JM, Twito T, Rao V, Ross HJ, Delgado DH. Can HLA-G polymorphisms predict the development of cardiac allograft vasculopathy? Journal of Human Immunology 2013; 74/4: XV

16 Chapter 1 INTRODUCTION 1

17 1.1 OVERVIEW In humans, the major histocompatibility complex (MHC) is a region of the genome that encodes a series of proteins. The most important of these are the human leukocyte antigens (HLA), which serve to present antigenic peptides onto the cell surface. They are subsequently recognized and bound by the receptors of cells involved in the immune response, such as T lymphocytes and natural killer (NK) cells, which represents the primary mechanism of allograft rejection following transplantation [1]. Human Leukocyte Antigen-G (HLA-G) is a non-classical MHC class I molecule, primarily expressed in the trophoblast cells of the placenta and has been reported to play a crucial role in mediating maternal tolerance of the fetal semi-allograft [2,3]. HLA- G expression in the placenta is known to have a protective effect towards the fetus by inhibiting the cytotoxic activity of T lymphocytes and NK cells [4-6]. This phenomenon is widely believed to be one explanation why mothers are able to accommodate their fetuses even though they do not share the same HLA. Given its immunosuppressive properties in pregnancy, HLA-G has been studied in the context of organ transplantation. Patients with HLA-G expression in serum, plasma, or biopsies post-transplantation were found to have fewer acute rejection episodes and a lower incidence cardiac allograft vasculopathy (CAV) [7]. To gain an understanding of the genetic component of HLA-G in the disease milieu, HLA-G polymorphisms have been recently studied in heart transplantation, with a significant correlation between the 14 bp deletion/deletion (-/-) genotype and acute cellular rejection. This thesis explores 2

18 the functions of HLA-G molecule in the context of heart transplantation and the role of HLA-G polymorphism in predicting the development of cardiac allograft vasculopathy. The in vitro studies presented in this thesis were performed by me with the exception of soluble HLA-G measurement in conditioned medium from JEG-3 cells. The blood collection, DNA analysis and genotyping for the clinical study were performed previously by other members of our laboratory; the coronary angiography data collection and subsequent data analysis were performed by me. 1.2 BACKGROUND Heart Transplantation Heart transplantation (HTx) is the accepted surgical therapy for patients with end-stage heart disease failing medical therapy. With advanced surgical techniques, highly specialized postoperative care and modern immunosuppressive treatments, the 1 year survival rate of 90% has been reached [8]. The long term survival on the other hand still constitutes a limitation of successful transplantation, with the 5-year survival rate of approximately 75%, and the 10-year survival rate of 56% [8]. Heart transplantation becomes the only treatment option when heart failure is advanced and no other pharmacologic or surgical therapies are available. Morbidity and mortality rates in patients with heart failure remain high, despite the advances in drug therapies and innovative device treatments. In patients with symptomatic heart failure, the 5-year life expectancy is 50% and for those with end-stage heart failure mortality rates can reach 80% in the first year of diagnosis [9]. Over the past four decades more than 80,000 3

19 heart transplants have been performed worldwide [10]. Although the cardiovascular advances have led to high success rates with heart transplantation, the scarcity of suitable donor organs remains a constant problem. With improvements in life sustaining therapies and more efficient pharmacologic treatments across the spectrum of the majority of health conditions, now more patients meet the eligibility criteria for transplantation, leading to longer waiting times and higher mortality rates before an organ becomes available [11]. Post heart transplantation, a series of early- and late-onset complications hinders the overall survival of the patient. Early morbidity is predominantly the result of infections and primary graft dysfunction attributable to ischemic injury sustained at the time of organ procurement. Acute cellular rejection is another important limiting factor during the first year following transplantation [12]. The risk of rejection remains high despite the decrease in acute cellular rejection rates in the past decade, due to improvement of immunosuppressive therapies such as calcineurin inhibitors. The first year post transplantation continues to represent a period of high mortality risk. After the first year post transplantation, malignancies and CAV are the most common causes of morbidity and mortality [8,13-15]. Although beneficial in the context of transplantation, prolonged or intensive immunosuppressive treatment is known to increase the risk of secondary malignancies post cardiac transplantation, particularly those of the lymphoid tissue and skin, accounting for 50-80% of malignancies following the transplantation of solid organs [16,17]. The cause of secondary malignancies 4

20 appears to be the chronic suppression of the immune system, as well as the direct carcinogenic effects of immunosuppressive agents. Patients with a history of cancer are at a particularly increased risk of neoplastic transformations following heart transplantation. Compared to other solid organ transplants, heart transplantation seems to place recipients at increased risk of oncologic neoplasms, mostly due to the high complexity and duration of immune suppressive regimen [18]. As a result, the compromised immune system becomes more susceptible to carcinogens and oncologic viruses [19]. Allograft vasculopathy, another long-term complication following heart transplantation, is defined as an accelerated form of atherosclerosis, characterized by concentric intimal thickening of the allograft vascular bed, eventually leading to obstructive lesions [15]. The main cause of CAV is the immunologic vascular injury, although non-immune pathways are major contributors as well. An important consequence is that any cause of vascular damage (e.g., immunologic, infectious, toxic) leads to intimal accumulations of smooth muscle cells (SMC) and extracellular matrix (ECM) [15]. Allograft vasculopathy impacts graft survival, and the pharmacologic or invasive techniques to prevent it or treat it have not had high rates of success. As CAV constitutes a major limitation to long term survival post-transplantation, a substantial area of research is now focusing on acquiring a more in depth knowledge about its pathophysiology, as well as on inhibiting the intimal proliferation associated with CAV. 5

21 Generating allograft tolerance represents the ultimate goal of medical research in the area of solid organ transplantation. Ideally, complete allograft acceptance would eliminate the need for immunosuppressive therapy, decreasing the risk of posttransplant malignancies and protecting against the immune response directed towards the allograft endothelium which leads to vasculopathy [15] Cardiac Allograft Vasculopathy Cardiac allograft vasculopathy is a major diagnostic and therapeutic challenge, occurring as soon as 1 year after transplantation [13,20-22] It is a persistently progressive condition that often presents as sudden cardiac death or heart failure [15,23]. The prevalence of CAV at 3 years post-transplantation reaches 20%, at 5 years 30%, and by the 8 th year, 45% of patients are diagnosed with CAV [24]. CAV affects the entire arterial vasculature of the allograft, with concentric narrowing of the vessels, progressing to obstructive lesions which compromise perfusion, eventually leading to graft ischemia and failure [12,25,26]. At present, the only definitive treatment option for CAV is re-transplantation. Due to its invasive nature, affecting the entire allograft vasculature, any attempt to invasively open the narrowed arteries is of limited benefit, as no current intervention is designed to penetrate the small vessels of the myocardial muscle [12]. The precise pathogenesis of CAV is not well understood and it is usually described as a healing response to vessel injury. Although in the context of transplantation the immunologic response is mainly at fault for the vascular damage, non-immunologic or traditional causes such as cardiovascular risk factors, infections and toxic agents, also contribute [15,26]. Some authors have reported acute rejection 6

22 as cause for CAV, due to the immune attack and subsequent inflammatory cascade which injures the endothelial layer of the blood vessel [25]. Both the innate immunity and the adaptive immunity play important roles in initiating immune responses targeting the endothelium of the transplanted heart [1] The innate immune system constitutes a powerful first line of defense mechanism able to combat infections before the adaptive immunity takes over. The innate response is immediate and less specific and acts mainly via inflammatory processes [1]. Among the types of cells that become activated during the inflammatory response seen in innate immunity are macrophages, dendritic cells, neutrophils, eosinophils, mast cells, and natural killer cells [27,28]. These cells are not antigen-specific and they differentiate into effector cells, invade the infection site and eradicate it [29]. The inflammatory response initiated during immune activation plays an important role in allograft rejection as well as in the development of CAV, as it triggers subsequent immunologic events leading to organ damage [29]. Tumor necrosis factor α (TNF-α) is a cytokine with an important role in the inflammatory phase caused by immune responses [30]. TNF-α promotes neutrophil adhesion to the endothelium by acting as a powerful chemoattractant [31]. Neutrophils have particular relevance in this context as they are the first leukocytes to arrive at the sites of inflammation, where they infiltrate causing further tissue damage [29] (Figure 1). 7

23 Neutrophil recruitment Nutrophils Rolling Integrin ligand Activation by chemokines Adhesion Migration through the endothelium Selectin ligand Selectin Cytokines (TNFα, IFN-γ) Integrin Chemokines Figure 1. Mechanism of CAV Inflammatory response. Adapted from Abbas and Lichtman [29] In situations when combating infection solely through innate immune responses is impossible, the adaptive immune response takes over the challenge. The exchange of information between the two immune responses takes place through antigen presenting cells such as dendritic cells [27]. Innate immunity responds in the same way when repeatedly activated by the same pathogen, as opposed to adaptive immunity which uses immunologic memory to create highly specialized immune response against repeated encounters with the same pathogens [29]. Although highly unspecialized, innate immunity has been found to contribute to both acute and chronic rejection of allografts [32]. In the context of transplantation, innate immune responses are triggered due to infection, tissue damage, and ischemia-reperfusion injury leading to an inflammatory reaction and to early episodes of allograft rejection [32]. The cells of the innate immune system participate in the rejection either directly, by releasing substances which damage the cells within the allograft, or indirectly, by activating the highly specific adaptive immune alloresponses [32]. 8

24 In CAV, the persistent immunologic injury to the endothelium of the blood vessel triggers a series of cellular events which progressively lead to luminal narrowing. Once activated, endothelial cells trigger the accumulation of adhesion molecules at the site of injury and the up-regulation of chemokines and pro-inflammatory cytokines; vascular growth factors and thrombogenic molecules are released and immune cells are drawn through the endothelial layer and into the intima of the allograft [26]. Interferon-γ (IFN- γ) seems to be the most important cytokine involved in CAV development, as it has a direct influence on inflammatory cell mobilization and smooth muscle cell activation [15]. These events lead to SMC migration from the media to the intima of the artery and proliferation once they reach the intimal layer. Accumulations and depositions of extracellular matrix also take place [15]. These lesions form a concentric intimal thickening, usually involving the entire arterial vasculature of the heart (Figure 2). 9

25 Immunosuppressants Infection Acute rejection Ischemia-reperfusion Donor disease EC injury Metabolic disorders Hypertension Donor age Preservation damage Adhesion molecules Chemokines Proinflammatory cytokines Growth factors Vascular inflammation Leukocytes T cells Macrophages Phenotypic change SMC migration into vascular intima and proliferation CAV Figure 2. Mechanism of CAV. Endothelial wall injury caused by various stressors leads to vascular inflammation and recruitment of immune cells which cause a phenotypic change of SMCs. These SMCs migrate into vascular intima and proliferate, leading to CAV. Adapted from Mitchell [15] 10

26 Human Coronary Artery Smooth Muscle Cells and CAV Pathologically, CAV is known to be caused by an immunological response of the allograft recipient to the transplanted arterial vasculature in which recipient immune cells injure the endothelial wall of the allograft [25]. In response to this damage, endothelial cells release a series of growth factors and mononuclear cells which in turn secrete proinflammatory cytokines, causing SMC activation. Smooth muscle cells have an important role in regulating arterial vessel tone, as well as synthesizing and secreting collagen [1]. They have the ability to switch their phenotype depending on various pathologic stimuli such as vascular injury, hypertension, and atherosclerosis [33]. In a normal, differentiated state, SMCs express a contractile phenotype, with complex receptors and a well-developed contractile ability. In this normal, quiescent state, their proliferation rate is very slow. In a dedifferentiated state triggered by vascular injury, SMCs switch to a synthetic phenotype able to migrate, proliferate, and secrete extracellular matrix [33]. SMC express receptors for a series of molecules capable of influencing the vasculopathic process, such as growth factors and low density lipoproteins. The endothelial cell layer of the blood vessel protects the SMC lining from these proteins, acting as a cellular shield. With endothelial injury the protective wall is disrupted and plasma molecules gain entrance into the exposed medial layer contributing to the phenotypic alteration of the SMC layer [1]. The most important cytokine involved in CAV is interferon-γ (IFN-γ), as it activates macrophages and influences SMC and inflammatory cell recruitment [15]. IFN-γ up-regulates intracellular adhesion molecule-1 (ICAM-1), attracting more lymphocytes and macrophages to the site of injury[34]. The macrophages release cytokines such as interleukin-1 (IL-1), IL-2, 11

27 and IL-6, and growth factors including transforming growth factor β, basic fibroblast growth factor, endothelial growth factor platelet-derived growth factor, and insulin like growth factor, all potent SMC proliferating factors [34]. Co-stimulatory signals also play a major role in CAV by recruiting T cells and inflammatory cells into the allograft. Upregulation of other adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and E- and P-selectins also facilitate a prolific environment for CAV development [28]. Once activated, SMCs transform from a differentiated state into a dedifferentiated cell capable of proliferation, migration and production of cytokines and ECM [25]. The accumulation of SMCs into the intima of the injured vessels is a result of both their migration from the media to the intima, as well as their proliferation upon arrival into the intimal layer of the artery. The concentric intimal thickening caused by SMCs and ECM protein deposition may ultimately lead to occlusion of the arterial vasculature of the transplanted organ (Figure 2). The migration and proliferation of SMCs might represent the most critical cellular event in neointimal thickening associated with CAV. Therefore, inhibition of SMC migration and proliferation represents a core therapeutic target for the prevention and treatment of CAV Human Coronary Artery Endothelial Cells and CAV The endothelial cell (EC) layer has important functions within the blood vessel. It regulates vascular homeostasis by secreting a series of vasodilatory or vasoconstrictory factors depending on the stimulus, maintains a thrombus free surface by inhibiting platelet aggregation and adhesion, acts as a protective barrier for the other layers of the artery, regulates the transport of substances to and from the vessel walls, controls the 12

28 proliferative state of the SMCs, and mediates the adhesion of neutrophils and other immune cells [35]. If endothelial cells become activated as a result of pathogenic stimuli, they initiate a cascade of cellular events leading to activation of the immune system. Post-transplantation, as soon as the heart allograft is reperfused after a period of ischemia, the innate immune system initiates an inflammatory response. With restored circulation through the heart, donor blood components invade the recipient s vasculature and those of the recipient are carried to the allograft [1]. Within minutes, the immune system detects the foreign cells and activates the defense mechanisms. Donor materials are transported to the spleen and lymph nodes where they come in contact with T-cells. T-lymphocytes bind to MHC molecules and become activated, and further activate B-cells [1]. Immune cells then migrate to the vascular wall of the transplanted heart, invading the endothelium with effector T-cells, B-cells, and macrophages [36]. B- cells bind to the donor antigen and undergo further activation and proliferation into antibody-producing plasma cells [36]. Direct injury to the endothelial wall takes place with subsequent endothelial dysfunction and invasion of inflammatory cytokines. Interferon- γ has an important pro-inflammatory role as it activates macrophages and monocytes. It also up-regulates MHC expression on ECs, making them more prone to antigen presentation to immune cells [1]. TNF-α is also a powerful inflammatory protein causing activation of neutrophils and their subsequent adhesion to the endothelial wall. The expression of adhesion molecules by endothelial cells accelerates and promotes further neutrophil, monocyte and lymphocyte adhesion to the vascular wall [37]. Although much progress has been made in sustaining life post-transplantation, and the survival rates have improved from a decade ago, there is still a long way to go to better 13

29 understand and efficiently prevent acute rejection, and to detect and modify the progression of cardiac allograft vasculopathy. New ways of inducing immune suppression might lead to innovative treatments, eventually minimizing the use of immunosuppressive drugs which are known to have numerous adverse effects The role of everolimus in CAV Immunosuppressive drugs have an important role in promoting allograft tolerance and prolonging life. Many immunosuppressants have been studied over the years, in both animals and humans, and some of them proved to be successful enough to be approved for use in clinical practice. These drugs are divided into different categories, depending on their mechanism of action. Steroids act by inhibiting the transcription of genes that lead to expression of cytokines involved in mediating the immune response [38]. Azathioprine and mycophenolate mofetil (MMF) function as anti-proliferative agents by altering the clonal expansion of lymphocytes [38]. A third group is comprised of cyclophosphamides which inhibit B cell response and interfere with lymphocyte proliferation. The last categories are calcineurin inhibitors such as cyclosporine and tacrolimus, and mammalian target of ramapmycin (mtor) inhibitors such as sirolimus and everolimus. Both cyclosporine and tacrolimus bind to intracellular immunophilins and block the calcineurin pathway therefore inhibiting the transcription of cytokines. The immunosuppressant everolimus will be discussed within the next paragraph, as it is relevant for our studies. 14

30 Everolimus, an mtor inhibitor and a proliferation signal inhibitor, binds to the cytoplasmic immunophilin FK-binding protein 12, forming active complexes which inhibit mtor [39]. More specifically, the everolimus-fkbp12 complex blocks the phosphorylation of enzymes with active roles in protein synthesis, thus arresting the cell cycle progression [39]. Everolimus prevents acute allograft rejection by inhibiting T cell proliferation. Its role extends beyond immune suppressive properties, acting also as a vascular smooth muscle cell proliferation inhibitor [40], this way preventing the development of CAV [41]. Studies have proved the efficacy of everolimus in reducing the incidence of CAV when compared to the use of other immunosuppressive drugs. One study compared the effects of everolimus with those of azathioprine, both administered in combination with cyclosporine or corticosteroids. The acute rejection episodes and CAV incidence at 6, 12, and 24 months were significantly lower in the everolimus group when compared with the azathioprine [42,43]. However, higher rates of infection and a persistent high serum creatinine level were found in the everolimus group. Chou et al. showed that everolimus-treated patients had significant less CAV at 12 months as detected by intravascular ultrasound, when compared with patients on MMF therapy [44]. The use of everolimus-eluting stents also revealed increased superiority in reducing late stent thrombosis when compared with paclitaxel stents [45]. Although a successful immunosuppressant and a potent proliferation signal inhibitor, the adverse effects of everolimus such as hyperlipidemia, wound healing complications, and renal failure remain of concern and warrant a careful selection of the patient population to be placed on this drug [46]. 15

31 Human Leukocyte Antigen-G The major histocompatibility complex (MHC) is a region of the genome that encodes for a series of proteins. The most important of these are the human leukocyte antigens (HLA), which serve to present antigenic peptides onto the cell surface [47,48]. They are subsequently recognized by the receptors of cells involved in the immune response, such as T-lymphocytes and NK cells [49]. This allorecognition and binding of immune cells to HLA presenting donor antigens represents the primary mechanism of allograft rejection following transplantation. Human Leukocyte Antigen-G (HLA-G) belongs to MHC class I molecules, however, its functions make it different from the regular molecules in this category, as HLA-G induces immune suppression rather than immune activation [50]. Its role has been initially studied in the context of pregnancy where it has been found to facilitate a tolerogenic environment for the allogeneic fetus [51-53]. HLA-G is known to protect the fetus from the maternal immune system by exerting a series of inhibitory functions altering the immune cells allorecognition and attack [5,54]. HLA-G has a restricted healthy tissue expression and it is also found in thymic epithelial cells [55], pancreatic islets [56], cornea [57], erythroblasts [58], and mesenchymal cells [59]. HLA-G is up-regulated in pathological conditions such as cancer, autoimmune and inflammatory diseases, viral infections and transplantation [60]. 16

32 Due to its inhibitory roles, HLA-G became an attractive molecule to be further explored in the context of heart transplantation. The next chapters offer an insight into the structure and mechanism of action of HLA-G, as well as into its various functions in health and disease The structure of HLA-G Human Leukocyte Antigen-G belongs to the MHC Class I molecules and although they share similar protein structure, they vary considerably in function, distribution, and properties [61]. Due to these differences HLA-G is called a non-classical MHC Class I protein. Unlike classical MHC, HLA-G has a limited polymorphism, a limited tissue expression, immune suppressive properties, limited protein variability, and distinctive molecular structure with a reduced cytoplasmatic tail [61,62]. Although the genetic structure of HLA-G is similar to other MHC antigens, its primary transcript produces seven isoforms generated through alternative splicing of the same mrna: four membrane-bound (HLA-G1, -G2, -G3, and -G4) and three soluble ones (HLA-G5, -G6, and -G7)[63-65]. The HLA-G transcripts produced can be expressed in various cell types or in different pathologic or non-pathologic situations [66]. The general structure of HLA-G1 is very similar with MHC class I molecules, with cytoplasmatic and transmembrane segments, and three domains, (α1, α2, and α3) that are noncovalently connected with beta-2-microglobulin (β 2 M) [61,67,68] (Figure 3). HLA-G1 and the soluble HLA-G5 are complete proteins, while the rest of the isoforms are shorter versions, smaller, simpler in structure, with no binding to β 2 M [60]. HLA-G2 17

33 has no α2 domain at exon 3, HLA-G3 lacks α2 and α3 encoded by exons 3 and 4, and HLA-G4 has no α3 domain at exon 4 [61,69]. The equivalent of HLA-G1 is HLA-G5, a full length isoform which possesses all the three α domains plus a stop codon at exon 4 which marks its ending, therefore no cytoplasmatic tail and transmembrane domain are present [61,66]. HLA-G6 is the counterpart of HLA-G2 with the α2 domain missing, while HLA-G7 has only the α1 domain encoded by exon 2, followed by a stop codon which dramatically shortens the length of this protein [61,66,70] (Figure 3 and Figure 4). Exon 7 is not present within the mrna and exon 8 is not translated because of the stop codon in exon 6 [61]. 18

34 Intron 2 Intron 4 Exon1 Exon2 Exon3 Exon4 Exon5 Exon6 HLA-G Primary Transcript α 1 α 2 α 3 Tm Cyt HLA-G1 Stop codon HLA-G2 HLA-G3 Membrane bound isoforms HLA-G4 HLA-G5 HLA-G6 Soluble isoforms HLA-G7 Figure 3 Structures of HLA-G primary transcript and HLA-G mrna transcripts. The primary HLA-G transcript can produce 7 different isoforms through alternative splicing: 4 membranebound (HLA-G1, -G2, -G3, -G4) and 3 soluble (HLA-G5, -G6, -G7). Exon 1 marks the leader peptide, followed by exon 2, 3, and 4 which encode the α1, α2, and α3 domains, respectively. Exon 5 codifies the transmembrane domains (Tm) and exon 6 which encodes the cytoplasmic domain is shortened due to a stop codon. HLA-G1 to G4 are membrane-bound isoforms as they end in the transmembrane and the cytoplasmic tail of exons 5 and 6, respectively. HLA-G5 and -G6 are soluble isoforms due to the presence of intron 4, terminating in a stop codon which prevents the continuation with the transmembrane and cytoplasmic tail. Similarly, HLA-G7 is shortened by the presence of intron 2 with a stop codon. Adapted from Donadi et al.[61] and Gonzales et al.[71] 19

35 α 1 α 2 α 1 α 1 Β2 α 3 α 3 α 1 α 2 Membrane HLA-G1 HLA-G2 HLA-G3 HLA-G4 α 1 α 2 α 1 Β2 α 3 α 3 α 1 HLA-G5 HLA-G5 HLA-G7 Figure 4. HLA-G protein isoforms. Membrane-bound HLA-G1 to G4 possess the transmembrane domain. HLA-G5 to -G7 are the soluble isoforms which lack the transmembrane domain. HLA-G1 and G5 are full length isoform which possesses all the three α domains and are non-covalently connected to β2m. Adapted from Donadi et al.[61] and Gonzales et al.[71] 20

36 Class I MHC molecules display a broad polymorphism which affects peptide uptake, the variation of peptides, and their ability to be recognized by immune cells. A high degree of polymorphism gives the classic MHC molecules a greater ability to present a wide range of antigenic peptides for recognition by T-lymphocytes. In contrast, non-classical HLA-G, presents very low polymorphism making it unsuitable for peptide binding and antigen presentation [67]. Instead, due to the unique molecular structure and the three α-domains HLA-G has a high affinity for inhibitory receptors located on immune cells [59]. Insertion/deletion polymorphisms represent genetic variations generated by the existence (insertion) or absence (deletion) of nucleotides [72]. Exon 8 of the HLA-G gene contains a 14bp deletion/insertion (+/-) polymorphism in the 3 untranslated region (UTR) [73] (Figure 5). Those HLA-G mrna displaying the insertion polymorphism can be further spliced alternatively [74,75]. The 14bp +/- polymorphism was found to influence the stability of HLA-G transcripts [76] and HLA-G expression [76-79]. HLA-G expression is linked to immune inhibition; therefore the HLA-G polymorphism influencing the degree of HLA-G expression could be linked to the various effects generated by HLA-G s immune suppression. The 14bp polymorphism has also been studied in transplantation. Its role in this context will be discussed within the following chapters. 21

37 Figure 5. Location of the 14bp deletion/insertion polymorphism in exon 8 of the 3 UTR. Adapted from Rousseau et al. [76] HLA-G Inhibitory Receptors HLA-G acts as an immune suppressant via interaction with three inhibitory receptors expressed on immune cells: the immunoglobulin-like transcript 2 (ILT2), Immunoglobulin-like transcript 4 (ILT4), and the killer immunoglobulin-like (KIR2DL4) [80-83]. The immunoglobulin-like transcript 2 receptor is found on lymphoid and myeloid cells such as B-cells and some of the T-cells and NK cells, monocytes, macrophages, and dendritic cells [84]. Immunoglobulin-like transcript 4 receptor expression is restricted to myeloid cells such as monocytes and dendritic cells [60]. The killer immunoglobulin-like receptor is found only on NK cells, particularly CD56 bright [60] and on some CD8 + T-cells [85,86]. This receptor was found to exert both inhibitory as well activatory functions [60,87], and, therefore, its interaction with HLA-G generating immune supressing effects is somewhat controversial. Other MHC Class I molecules can also bind to ILT2 and ILT4, while KIR2DL4 only interacts with HLA-G [60]. Among all HLA Class I, ILT2 and ILT4 have the highest affinity for HLA-G [67,88]. Moreover, ILT4 has an even higher affinity towards binding with HLA-G when compared to ILT2, 22

38 suggesting that the HLA-G-ILT4 interaction might have an important role in influencing the immunological recognition and response of myeloid cells [88]. All the receptors bind HLA-G at different positions within its molecular structure. ILT2 has a preference for the β 2 M chain while ILT4 also binds to the free heavy chains [60]. Following interaction with these receptors, HLA-G has been reported to influence various functions of the immune cells, such as T-lymphocyte proliferation, antigen presenting cell (APC) maturation, and NK cell [89,90] and CD8 + T-cell cytolysis [59]. HLA-G was also found to induce the expression of some regulatory cells, highlighting even more the inhibitory properties of HLA-G [59]. HLA-G was found to act as a signaling molecule, able to induce phenotypical changes in immune cells such as T-cells, NK cells, and APC. The result is an up-regulation of ILT2, ILT4, and KIR2DL4 inhibitory receptors [85] on the surface of immune cells following exposure to soluble or membrane bound HLA-G [85]. This HLA-G-mediated induction of inhibitory receptors might contribute to the suppression of the immune response in tissues that express HLA-G and in those that are threatened by immune cells, like in the context of transplantation. The HLA-G expressed in tissues might upregulate its own receptors on the T-cells and NK cells once the immune attack is initiated, suppressing their function and minimizing the overall intensity of the attack [85]. Moreover, stimulating the APC to overexpress the inhibitory receptors might alter its ability to start the immunologic cascade targeting the allograft. 23

39 Another receptor identified in the literature and which binds HLA-G is CD160. Fons et al. found that HLA-G interacts with CD 160 present on human umbilical vein endothelial cells (HUVEC) and leads to apoptosis and inhibition of angiogenesis [91]. Besides its presence on endothelial cells, CD 160 expression has been reported on the surface of circulating NK and T cells, spleen and intestines [92]. This anti-angiogenic role of HLA- G seems to be highly relevant in the context of pregnancy, contributing to reshaping of the maternal arteries in preparation for high blood flow demands Functions of HLA-G HLA-G is a powerful tolerogenic molecule with three main roles in immune suppression: direct inhibition of immune cell functions, indirect immune suppression through regulatory cell production, and other functions of HLA-G which result in immune response inhibition [71] (Figure 6). 24

40 Figure 6. Immunomodulatory functions of HLA-G. Target cells and the receptors mediating these functions are also mentioned. Adapted from Pistoia et al. [93]. a) Direct inhibition of immune cell functions The tolerogenic property of HLA-G was initially demonstrated in the context of pregnancy. As classical MHC class I and II molecules are not expressed on the trophoblast cells, the mother s T cells cannot recognize the fetus as non-self, therefore 25

41 they cannot initiate an immune response. NK cells on the other hand, exert their cytotoxicity on cells that do not express MHC class I molecules. Therefore, the mechanism of tolerance of the semi-allogeneic fetus seems to be related to the presence of HLA-G on the fetal cells [4]. HLA-G, expressed by the cytotrophoblast cells of the placenta, was found to inhibit the cytolytic function of semi-allogeneic and allogeneic NK cells, thereby protecting the fetal cells from being the target of the immune response generated by the mother s immune system [4]. In subsequent studies it was confirmed that HLA-G blocks NK cell function through its interaction with inhibitory receptors on the NK cell [85]. Due to the low polymorphism of HLA-G expressed by the trophoblast cells, this molecule is able to bind to all inhibitory receptors on NK cells [4]. Rouas-Freiss et al. found that non-hla-g expressing cells transfected with membrane-bound HLA-G1 and HLA-G2 were able to inhibit NK cytolytic activity [4]. The inhibition of NK cells lytic functions has been extensively documented in the literature [4,94,95]. Using the same human cell line as an NK target, it was also demonstrated that soluble HLA-G has the same immunosuppressive properties as its membrane-bound counterpart [96]. Moreover, HLA-G-transfected cells inhibit the lysing action of NK cells isolated from different donors, suggesting the ability of HLA-G to affect different NK cell subpopulations [97]. The NK cell inhibition by HLA-G is also seen in the context of tumor cells and their escape from immune destruction. HLA-G seems to also contribute in facilitating immune tolerance of cancerous cells. As NK cells are specialized in lysing those cells that lack MHC class I proteins, and because tumors do not express these molecules, they are 26

42 able to escape from immune surveillance only by expressing HLA-G [60]. In a recent study, it was shown that tumor expression of HLA-G1 and its interaction with the ILT2 receptor on NK cell blocks the cytoskeleton reorganization at the NK cell immunologic synapse [59]. As a result, the NK cell becomes unable to release cytolytic enzymes directed against cancerous cells. HLA-G can also be found on fetal endothelial cells within the placental blood vessels, as well as within uterine arteries. In order to elucidate a possible mechanism of immune protection, porcine endothelial cells were transfected with HLA-G and exposed to human NK cells in a transmigration assay [98,99]. HLA-G inhibited the transmigration of NK cells across the endothelial layer. In pregnancy, this might explain how NK cells are blocked from entering the placenta with subsequent immune response activation. This mechanism of action might be especially valuable and also applicable in the context of xenotransplantation where NK cells play an important role in invading the allograft [99]. Riteau et al. investigated the inhibitory ability of HLA-G when co-expressed with the classical MCH class I proteins. For this purpose, HLA class I positive cells were transfected with HLA-G1 and exposed to NK cells. HLA-G exerted a powerful inhibitory effect in the presence of HLA-A, -B, -C, and HLA-E, ensuring a strong defense response against the cytolytic effects of NK cells towards target cells [97]. The immunosuppressive properties of HLA-G extend beyond the innate immunity, with direct influence on adaptive immunity, with respect to T cells. It has been well 27

43 documented in the literature that HLA-G has an important role in modulating T cell activity. When transfected into an MHC class I positive cell displaying an antigen specific for CD8 + T cells, HLA-G1 exerted a powerful inhibitory action towards the CD8 + T cells [96,100]. In mixed lymphocyte reaction (MLR) studies, HLA-G-transfected cells inhibited the interactions between peripheral blood mononuclear cells (PBMC) derived from two donors. HLA-G blocked allogeneic proliferation in both CD4 + and CD8 + T cells [96,101]. Although most of the research has focused on the function of the HLA-G1 isoform, the other membrane-bound molecules seem to play an important role in immune inhibition. In one study, MHC class I positive human cell lines were transfected with HLA-G2, -G3, and HLA-G4. The truncated isoforms were expressed on the cell surface and were fully able to inhibit NK cell and T lymphocyte cytotoxic effects [84]. Other in vitro studies have demonstrated that soluble HLA-G5 can inhibit CD4 + T cell alloproliferation when added to MLR [102,103]. HLA-G5 inhibits not only CD4 + T cell, but also CD8 + T cell proliferation, after their receptor activation and initiation of immune response. Bainbridge et al. engaged in research to elucidate the role of membrane-bound HLA-G in MLR, and found that it strongly supresses the proliferation of CD4 + T cells [104]. In another study, HLA-G5 bound to ILT2 inhibitory receptors arrested cell cycle progression [105]. Moreover, HLA-G1-transfected APCs inhibited the CD4 + T cell immune responses [101,106]. This might be representative for the in vivo transplant situation when APCs expressing HLA-G invade the allograft and the rejection response is blocked. 28

44 To further demonstrate the role of HLA-G in altering immune cell functions, Fournel et al. showed that purified soluble HLA-G1 caused activated CD8 + T cell apoptosis [107]. The mechanism of action was initiated through the interaction of HLA-G with CD8 molecules. Specifically, shla-g1, co-incubation with CD8 + cells, up-regulated CD95-L. This HLA-G isoform is known to be an apoptosis mediator for CD8 cells which express CD95 receptors on their surface. Therefore, HLA-G was able to interact with CD8 molecules, up-regulating apoptotic factors, and mediating CD8+ cell apoptosis through the CD95/CD95-L pathway [107]. Antigen presenting cells play a vital role in initiating immune responses. Dendritic cells have a double function, either immunologic or immunosuppressive, depending on their degree of maturation. In vivo, immature DCs block T lymphocyte activation, whereas mature DC activate T cells [108]. HLA-G was found to interact with the ILT4 receptor expressed by the DCs in vitro, resulting in inhibition of their maturation process [108]. ILT4 is highly expressed by DCs and its binding with HLA-G alters the ability of the DCs to induce allogeneic T lymphocyte proliferation [108]. Animal studies involving HLA-G and DCs revealed similar results to those found in vitro. When compared to non-transgenic controls, the HLA-G transgenic mouse production of functionally competent APC was impaired as a result of altered DC maturation. In accordance with these findings, allogeneic skin grafts from non-transgenic controls transplanted to HLA-G transgenic mice resisted longer to immune attacks than those of controls, and showed an impaired cellular immune response mechanism [109]. The use 29

45 of recombinant HLA-G complexes administered to non-transgenic mice via injection was also reported to promote better skin allograft survival [110]. These HLA-G complexes also inhibited murine DC maturation in vitro, via HLA-G/murine paired immunoglobulinlike inhibitory receptor-b, an equivalent of the human ILT4 inhibitory receptor [108]. It has also been demonstrated that the HLA-G/ILT4 interaction impairs immune activation in transgenic mice following treatment with HLA-G complexes [111,112]. In this study, human ILT4 expression by the DCs was induced in transgenic mice. The result was impaired DC maturation and reduced CD4 + and CD8 + T cells activation. These studies have elucidated the role of HLA-G in inhibiting immune responses through their interaction with APCs and altering their maturation. b) Indirect immune suppression through regulatory cell production The immunosuppressive effects of HLA-G mediated through the interaction with inhibitory receptors are short-term, and only last as long as the two molecules are bound to each other. However, it has been found that HLA-G can also have long-term effects on immune suppression indirectly, through regulatory cell production [71]. These suppressor cells are immune cells that act as vital moderators of immune responses and allogeneic tolerance, and can inhibit the activation of other immune cells. They can be APCs, CD4 + or CD8 + T lymphocytes. It was also found that HLA-G1 + APC induces the long term suppression of CD4 + T cells, and facilitates their differentiation into CD4 + regulatory T cells. This is an important finding as these cells no longer proliferate and differentiate into CD4 + T helper cells and, therefore, no further activation of antigen specific CD8 + effector T cells takes place [106]. This suggests a complete inhibition of 30

46 the immune response right from its initial stage. The same group also found that CD4 + T cells that were exposed to HLA-G + APCs for 12 hours had a much lower ability to undergo subsequent activation by new antigen stimulation than the control group. In addition, the longer the exposure, the less the ability to respond to allogeneic stimuli [106]. Another mechanism through which HLA-G generates regulatory cells is trogocytosis. Through this mechanism, cells in proximity with one another acquire membrane material and molecules from each other [113]. It has been demonstrated that via this process NK cells acquire HLA-G from tumor cells and their entire functionality becomes altered. The HLA-G uptake blocks the NK cells ability to proliferate, inhibits their cytolytic function, and causes them to act like suppressor cells, able to diminish other NK cells allogeneic activation [113]. Interestingly, only those NK cells that become activated during the immune response are able to acquire HLA-G through trogocytosis. Going beyond the innate immunity, Le Maoult et al. investigated whether or not trogocytosis also takes place between APCs expressing membrane-bound HLA-G1 and T lymphocytes [114]. After one hour of exposure of HLA-G1 transfected APC to T cells, there was a significant uptake of HLA-G by activated CD4 + and CD8 + T cells. Following this HLA-G acquisition, the CD4+ T cells proliferation was inhibited, as well as their ability to be re-activated by further allostimulation. Furthermore, these CD4 + T cells became regulatory cells through HLA-G1. Interestingly, two days after trogocytosis, these CD4+ T cells maintained their regulatory function despite losing HLA-G surface 31

47 expression. Moreover, these cells differentiated into regulatory CD4+ T cells at 48h following trogocytosis [114]. Antigen presenting cells have a vital role in HLA-G-APC mediated immune tolerance, as they are the main expressers of HLA-G. Soluble HLA-G was found not to block APCs, but to inhibit their chemokine, cytokine and receptor expression [115,116]. IL-12, CD-80 and HLA-DR are all immune activation molecules, and once inhibited, facilitate T cell unresponsiveness to allogeneic stimuli, and up-regulation of suppressor T cells [115]. Ristich et al. demonstrated that dendritic cells that express ILT2 and ILT4 in abundance become tolerogenic when treated with HLA-G and stimulated with T cells, and are able to induce non-responsiveness in T lymphocytes [111]. APCs that express HLA-G on their surface shed HLA-G molecules, generating immune suppressive surroundings [117]. This way, HLA-G not only impairs the immune functions of lymphocytes, but also those of the APCs, rendering them tolerogenic. Therefore, HLA-G + APCs can be considered as regulatory cells able to induce suppressor lymphocytes. Those APCs containing HLA-G1 are a great source of HLA-G membrane uptake by activated effector cells through trogocytosis, transforming lymphocytes from effectors to suppressors [114]. Membrane and associated molecules transfer seems to be the main mechanism of action of HLA-G1 through which it promotes immune suppression in healthy tissue (pregnancy) or pathological tissue (tumors). 32

48 c) Other functions of HLA-G Several other functions have been attributed to HLA-G. One study showed that membrane bound HLA-G stimulates uterine NK cells to secrete IFN-γ in non-pregnant women [118,119]. This response was achieved by HLA-G binding with the KIR2DL4 receptor on NK lymphocytes. It is speculated that increased production of IFN-γ by NK cells contributes to a normal pregnancy in humans, therefore all human uterine cells, from cytotrophoblasts to macrophages and endothelial cells, express IFN-γ receptors early during pregnancy [118]. Fons et al. elucidated the role of HLA-G as an angiogenesis inhibitor through the modulation of uterine endothelial cell apoptosis. Soluble HLA-G1 is produced by trophoblasts that replace the vascular lining of mother s uterine arteries and promote vessel expansion, accommodating the high volumes of blood required in a healthy pregnancy [91]. During the first trimester, shla-g secreted by cytotrophoblasts induces apoptosis of the endothelial cells of the maternal uterine arteries, contributing to this cellular replacement [91]. This apoptotic effect is reached by the HLA-G1 interaction with CD160 receptor expressed by endothelial cells. Further confirming its antiangiogenic effects, shla-g1 was found to block capillary tube formation in human umbilical vein endothelial cells (HUVEC) by exerting inhibitory effects on fibroblast growth factor 2 and vascular endothelial growth factor, two powerful pro-angiogenic molecules [91]. This finding was validated in vivo, using a rabbit model and testing the inhibition of corneal angiogenesis [91]. The anti-angiogenic properties of HLA-G could potentially be relevant in the context of heart transplantation. Intimal angiogenesis is 33

49 prevalent in CAV lesions and is known to contribute to the development of inward remodelling of the arterial vessels, with vascular endothelial growth factor being the most powerful pro-angiogenic factor [120]. A novel HLA-G function has been described by Morandi et al. Soluble HLA-G was found to affect the expression of cytokine and chemokine receptors on CD4+ T cells and CD8+ T cells [92]. Activated T cells are attracted by chemokine molecules, migrating to sites of inflammation. For this migratory process, chemokines must interact with their receptors. Through binding with the ILT2 inhibitory receptor, HLA-G has been found to inhibit the expression of CCR2, CXCR3 and CXCR5 in CD4 + T cells, CXCR3 in CD8 + T cells, which leads to the blocking of chemotaxis of these cells [92]. Similar findings were reported with NK cells. The migration of CD56 bright and D56dim NK cells towards the sites of inflammation across the vascular wall was inhibited by soluble HLA-G via the alteration of cytokine and chemokine secretion [121]. These cell migration models suggest that HLA-G also limits the availability of activated effector cells at the location of inflammation, contributing to the reduced immune response The Role of HLA-G in Pregnancy As initially discovered in the cytotrophoblast cells of the maternal-fetal interface, HLA-G was initially believed to reside and exert its functions only within the context of pregnancy. The immune system is well developed in pregnancy and performs specific functions of identifying and destroying pathogens of viral or bacterial origins. Interestingly, the highly specialized maternal immune system is able to create an 34

50 immune-tolerant environment for the semi-allogeneic fetal cells. This tolerogenic atmosphere, favourable for the fetus to develop and grow, is mainly stimulated by HLA- G molecules expressed by trophoblast cells [51,122]. Trophoblasts are the cells covering the blastocyst, and appear four days after fertilization. They differentiate rapidly into other cells, gradually forming all the components of the placenta. Cytotrophoblasts play a major role in protecting the semi-allogeneic fetus from rejection by the mother s immune system by expressing a series of immune-inhibitory molecules, with the most relevant one being HLA-G [122]. Numerous studies have confirmed the immunosuppressive role of HLA-G during pregnancy. HLA-G in soluble form can also be detected in the serum of expecting mothers, in substantially higher levels when compared with non-pregnant women [123]. HLA-G is able to regulate the remodeling of uterine blood vessels by inhibiting endothelial cell proliferation, migration, and angiogenesis, this way allowing trophoblast cells to take over the cellular lining of the vascular system within the placenta [91]. The expression of HLA-G constitutes now a prerequisite for a normally developing pregnancy [124]. Low levels of HLA-G during pregnancy have been associated with high rates of miscarriages [125]. The necessity of HLA-G presence during pregnancy is also critical in the context of in vitro fertilization. Rizzo et al. provide powerful indications that HLA-G secretion by embryos is necessary for their implantation into the endometrial wall [54,126]. This finding raised the debate whether or not HLA-G expression by embryos can be used as a strategy to select those embryos with greatest chances of successful implantation and healthy development within uterus [71]. 35

51 Low levels of HLA-G are known to be also associated with complications such as preeclampsia (PE) and placental abruption. Yie et al. reported low shla-g serum and placental levels in women who developed PE when compared with healthy pregnant controls [127]. Analyses of serum HLA-G [128] levels yield similar findings early in pregnancy [128]. Low HLA-G plasma levels were also detected in PE patients later in pregnancy [129]. Steinborn et al. also found an association between low levels of plasma HLA-G and the incidence of placental abruption [130]. All these findings reveal the critical role of HLA-G during pregnancy, from implantation to delivery The Role of HLA-G in Cancer With so many immune suppressive roles confirmed so far, the HLA-G expressed by cancer tumors is believed to contribute to tumor cell escape from immune surveillance, promoting their survival [131]. Upon pathogen recognition and activation of the effector cells of the innate and adaptive immune systems, HLA-G expression seems to protect against both responses by inhibiting effector cell functions and by generating suppressor cells [132]. To date, the HLA-G molecule has been detected in almost all types of cancers [131] and can be expressed on the cell surface, secreted, or present within in the tumor tissue itself, more specifically in tumor exosomes [133]. The location of HLA-G seems to be limited to the tumor tissue, with no expression within the healthy tissue in close proximity, suggesting that the presence of HLA-G is correlated with tumor progression. [134]. Biological studies performed in cancer patients demonstrated that high levels of shla-g were associated with advanced cancer staging, and were predictive of poor prognosis [134]. Increased HLA-G plasma levels in patients with 36

52 neuroblastoma were associated with disease exacerbation and recurrence [135]. In hepato-cellular carcinoma, HLA-G expression was strongly correlated with short life expectancy and tumor relapse [136]. Similar results were observed in non-small cell lung cancer, esophageal-squamous cell carcinoma, colorectal and gastric cancer patients [133]. In all these studies HLA-G was an independent prognostic factor. A few types of cancer such as uveal melanoma and laryngeal carcinoma were found to be free of HLA-G expression [137], suggesting that its presence is dependent on the form of cancer. Depending on the type of cancer, HLA-G can undertake a series of tolerogenic roles which prevent tumor immune rejection, such as inhibiting the activity of NK cells, T lymphocytes, and APC [138]. In primary glioma, HLA-G1 protected the tumor from activated effector cells of the innate and adaptive immune system [139]. Soluble HLA-G inhibited the cytolytic abilities of immune cells directed at the neuroblastoma [135]. Trogocytosis is another mechanism of action of HLA-G in the context of malignancy. The acquisition of HLA-G from the membrane of HLA-G + tumor cells can be accomplished not only by the activated immune cells but also by other HLA-G - tumor cells, explaining the mechanism through which tumors expressing HLA-G protect other HLA-G free tumor cells [138]. Due to its tolerogenic properties which facilitate tumor escape from immune responses, HLA-G has substantial relevance in the context of cancer. Depending on the nature of malignancy, HLA-G expression is associated with oncologic transformation, disease 37

53 progression, poor clinical outcome, low life expectancy, and cancer relapse [64]. Future studies will investigate HLA-G as a potential target for antitumor therapy. Based on current knowledge HLA-G can be used as tumor marker, as a cancer staging tool, and as a prognosis marker The Role of HLA-G in Inflammatory Diseases In addition to its expression in malignant transformations, HLA-G expression has also been studied in the context of inflammatory conditions, particularly in autoimmune diseases such as multiple sclerosis (MS), rheumatoid arthritis, and systemic lupus erythematous [71]. Within the inflammatory context, HLA-G expression in MS has been investigated the most. In MS, the white matter of the central nervous system (CNS) displays areas of myelin sheath damage caused by inflammatory cells [71]. HLA-G and its inhibitory receptor ILT2 were detected in the affected area of the white matter, while they were rarely found in non-diseased specimens [140]. Specifically, HLA-G was expressed by macrophages, microglial cells, and endothelial cells within the brain tissue. Interestingly, shla-g was also found in the cerebrospinal fluid and up-regulated on monocytes [140]. Multiple sclerosis patients display significantly higher levels of HLA-G in the blood when compared with other neurological conditions, suggesting the relevance of this molecule in this type of autoimmune disease. T lymphocytes constantly survey the CNS and can cause substantial damage if they recognize antigens presented by APC. In MS, activated T cells cross the blood brain barrier and upon reaching the CNS they launch an immune attack resulting in inflammation and damaging myelin proteins and axons [141]. Maintaining a balanced immune 38

54 environment depends to a high extent on microglial cells which represent the main APC within CNS. Soluble HLA-G is thought to have a regulatory effect on CNS inflammation. This regulatory effect is believed to take place via an inhibition of pro-inflammatory T lymphocytes by shla-g expression on CNS APCs, and blockage of the subsequent IFN-γ release [140]. In its anti-inflammatory role, HLA-G is believed to suppress CD4 + T, CD8 + T and NK cell lytic activity, and these effects seem to be caused by the HLA-G5 isoform [141]. HLA-G expression has been reported in other inflammatory diseases. In atopic dermatitis, HLA-G was present in skin biopsies, and was thought to exert an inhibitory role towards invading T cells, explaining the spontaneous remissions seen during the disease evolution [142]. Skin lesions from psoriatic patients were found to express HLA- G, as well as the ILT2 receptor, while healthy tissue did not. The interaction of these two molecules seems to alter the effect of T-lymphocyte infiltrate in the initial stage of invasion [143]. HLA-G regulatory CD4 + T and CD8 + T cells have been detected in muscle biopsies of patients with different inflammatory conditions [144]. These suppressive cells expressing HLA-G on their surface might inhibit other effector cells, thereby regulating inflammatory responses. High levels of shla-g were also detected in the synovial fibroblasts in patients with osteoarthritis, and it is believed to have a role in reducing inflammatory responses within the synovial joints [62]. 39

55 Overall, HLA-G seems to have a protective role in the context of inflammation by inhibiting the cytolytic action of immune cells and by transforming them into regulatory cells capable in modulating inflammatory responses The Role of HLA-G in Solid Organ Transplantation Due to its role as a powerful tolerogenic molecule within the pregnancy milieu, HLA-G has been studied in the context of transplantation. Lila et al. were the first to determine the expression of HLA-G in solid organ transplantation, as well as its immunosuppressive role [145]. HLA-G expression in endomyocardial biopsies and in the serum of heart transplant patients was initially found to be associated with fewer acute rejection episodes and a lower incidence of cardiac allograft vasculopathy [145]. The same group also conducted a similar retrospective study, with a larger number of subjects and with a longer follow-up, in order to confirm the preliminary findings stated above. Out of the 51 patients who underwent heart transplantation, 18% expressed HLA-G. These HLA-G positive patients presented with significantly fewer acute rejection episodes, and with no incidence of cardiac allograft vasculopathy when compared with HLA-G negative patients [7]. Some of the patients displayed HLA-G5 and HLA-G6 in their blood, which might provide additional immune suppression. In light of these findings, HLA-G has also been investigated as a tool for monitoring episodes of rejection. Soluble HLA-G in the serum of 72 heart transplant patients was correlated with episodes of rejection and cardiac allograft vasculopathy as measured by angiographic studies and echocardiography [146]. The higher the concentration of HLA- G expressed in the blood (> 200 ng/ml), the fewer acute rejection events and the lower 40

56 incidence of cardiac allograft vasculopathy was reported. On the other hand, those patients with low levels of HLA-G (< 6 ng/ml) or not expressing it at all, sustained significantly more rejection and vasculopathy [146]. These findings were in accordance with Luque et al. who reported an inverse correlation between HLA-G expression in serum and severe rejection, suggesting that HLA-G protects the allograft against immune responses [147]. Luque et al. also found that shla-g concentration was increased during the first month post-transplantation, suggesting that highly administrated immunosuppressive agents at the time of transplant might have a role in up-regulating HLA-G. A study was conducted to evaluate the fluctuations of shla-g in the serum over 12h, as a result of immunosuppressant administration [147]. In these investigations, 42% of patients showed a significant rise in shla-g 4h post immunosuppressant drug administration, compared to baseline serum concentrations, and going down to baseline at 10h after dose administration. Therefore, an association between immunosuppressant administration and shla-g level cannot be excluded. Our group investigated the expression of HLA-G in myocardial biopsies of 76 heart transplant recipients [148]. Only 11% of patients with recurrent moderate to severe rejection showed HLA-G expression in biopsy specimens while 86% of patients who never experienced acute cellular rejection were HLA-G-positive. Fifteen out of the 25 (60%) HLA-G positive patients, who had no rejection episodes, had at least two HLA-G positive biopsies, suggesting a stable expression. In accordance with results from previous studies, none of the patients in the non-rejecting group experienced any moderate or severe rejection episodes up to 10 years post-transplant [148]. Our group 41

57 also investigated the relationship between HLA-G expression and antibody-mediated rejection reflected by C4d staining in 50 heart transplant patients. Results showed a negative association between shla-g levels and C4d staining in heart transplant patients, with only 13% of HLA-G positive patients displaying C4d deposition in their biopsies, compared with 38% in the HLA-G negative group, suggesting inhibition of the humoral response [149]. To further investigate the assumption that HLA-G is up-regulated by immunosuppressants, Sheshgiri et al. compared plasma HLAA-G levels in 17 heart transplant recipients receiving two different anti-rejection drugs: everolimus and mycophenolate mofetil (MMF). Seventy eight percent of patients treated with everolimus were found to express HLA-G, versus only 25% of patients receiving MMF, suggesting an association between everolimus and HLA-G expression [150]. As the investigations in the heart transplantation context revealed strong arguments suggesting improved allograft tolerance with HLA-G expression, research in the area of HLA-G expanded towards other solid organ transplants. Qiu et al. investigated the role of shla-g in 65 patients who underwent kidney transplantation. Prior to surgery, none of the patients had detectable HLA-G concentrations in the serum. However, posttransplantation, out of the 26 patients with satisfactory kidney function, 13 patients expressed HLA-G, compared with only 8 out of 39 patients with renal allograft rejection [151]. In the same study, high concentrations of HLA-G in the serum were also correlated with a low generation of HLA IgG antibodies, which play an important role in 42

58 acute and chronic rejection. Investigations of kidney biopsies post-transplantation also reveal important information regarding the protective action of HLA-G against allograft rejection. Crispim et al. found that out of 73 kidney transplant recipients, 40 patients expressed HLA-G and this expression was seen primarily in tubule epithelial cells. Only 4 of these HLA-G positive patients had episodes of acute rejection or chronic allograft nephropathy [152]. Of the remaining 33 patients who suffered from rejection and nephropathy, only 4 were HLA-G positive. Those patients treated with tacrolimus showed higher levels of HLA-G expression, further supporting the idea that immunosuppressants might play a role in HLA-G up-regulation. The results from this study support the protective role of HLA-G in the area of transplantation, contributing to freedom from rejection. However, this study has limitations, such as the lack of routinely performed biopsies, as well as the use of data collected from only one biopsy, with no further follow up to assess the stability of HLA-G expression. Further investigations have also examined the expression and role of HLA-G in lung transplantation. Brugiere et al. detected HLA-G presence in the transbronchial biopsies of 30 patients of the 64 included in their study. Twenty-two out of 34 clinically stable patients at the time of biopsies expressed higher levels of HLA-G in bronchial epithelial cells, had fewer acute rejection episodes, and a lower incidence of bronchiolitis obliterans syndrome. Of the 40 patients with moderate or severe acute rejection, and high incidence of bronchiolitis obliterans syndrome, only 8 were HLA-G positive [153]. This data highlights the importance of HLA-G in assessing graft tolerance. 43

59 In combined kidney-liver transplantation Creput et al. carried out a study involving 40 patients. The expression of HLA-G was detected in 55% of kidney biopsies and 35% of liver biopsies [154]. In the liver, HLA-G presence was seen in biliary epithelial cells during periods of acceptance, as well as in infiltrating mononuclear cells during acute rejection. In the kidney, HLA-G was detected in tubular epithelial and endothelial cells. HLA-G expression in biliary epithelial cells was associated with graft acceptance in liver transplant, suggesting that HLA-G may block the immune response. No episode of acute or chronic rejection was seen in kidney allografts when the patients expressed HLA-G in the liver [154]. This suggests a potential protective effect by the HLA-G positive transplanted liver towards the kidney graft tissue. In another study, high levels of shla-g detected in the serum of kidney-liver transplant recipients were also associated with better graft acceptance and could be used to monitor allograft function and to assess the need for immunosuppressive therapy post-transplantation [155]. Naji et al. reported that the two main HLA-G isoforms, the membrane-bound HLA-G1 and its soluble counterpart HLA-G5, were able to induce regulatory T cells in liver and kidney-liver transplant patients [156]. These CD3 + CD4 low and CD3 + CD8 low suppressor T cells might represent important contributors to HLA-G-related allograft acceptance of the transplanted organ. The expression of HLA-G in biopsies, serum and plasma samples of heart, liver, lung, and kidney transplant recipients is associated with decreased graft rejection. Although the pattern of HLA-G expression in the period surrounding the transplantation is not fully 44

60 elucidated, it appears that immunosuppressive therapy plays a role in its presence. The findings resulting from transplantation research illustrate that HLA-G is an attractive molecule, with important tolerogenic functions, which might potentially be used in promoting organ acceptance The 14-bp insertion/deletion polymorphism in solid organ transplantation The expression of the HLA-G gene is co-dominant [157,158]. A 14-bp insertion/deletion polymorphism located in exon 8 in the three prime untranslated region (3' UTR) of the gene (rs16375) was found to influence mrna stability and splicing, and to play an important role in HLA-G expression [76, ]. The importance of HLA-G polymorphisms extends in many areas of transplantation. In kidney transplantation, Crispim et al. found no association between the presence of the 14bp -/+ polymorphisms in either transplant patients or healthy controls [152]. However, the same group found an association between the homozygous 14-bp insertion genotype and acute rejection. This is in agreement with previous studies reporting a decreased HLA-G production in this genotype [77]. In bone marrow transplantation, the 14bp -/- genotype was associated with increased risk of developing severe graft versus host disease (GvHD), while the homozygous 14bp insertion and the heterozygous genotypes predicted low risk of GvHD [162]. 45

61 In heart transplantation, Torres et al reported in a small retrospective study that the 14bp polymorphism influenced the expression of soluble HLA-G and accordingly resulted in low rejection rates [163]. Our research group has recently investigated the association of the 14bp polymorphism and cellular rejection in a larger population of heart transplant recipients [164]. We found a significant association between the 14bp - /- polymorphism and serum HLA-G levels. We also observed a significant correlation between the 14bp -/- genotype and decreased acute cellular rejection [165]. The 14bp insertion/deletion polymorphism has been studied in various organ transplantations and according to the research done in our lab seems to represent a potential marker for cardiac allograft acute rejection. Other roles of this polymorphism may be determined, such as its ability to serve as a tool to identify patients at risk for CAV development. In conclusion, despite the pharmacological advances in the area of immunosuppressive therapy and the innovative techniques of organ preservation, cardiac allograft vasculopathy continues to limit the long-term success of heart transplantation and contributes to persistently high incidences of morbidity and mortality posttransplantation [166,167]. At present, the only optimal therapy for CAV is retransplantation, which is largely limited by the availability of donor organs. HLA-G, a molecule that has been studied in context of various pathological and non-pathological conditions, seems to play an important immune suppressive role within the body. At the genetic level, the HLA-G 14bp genotype represents a potential marker to identify 46

62 patients at risk for organ rejection. The understanding of HLA-G s expression and its role in heart transplantation is still in the early stages. Similarly, the HLA-G polymorphism gains more and more interest as its functions are gradually being discovered. Future studies are needed to fully elucidate whether or not HLA-G exerts beneficial functions within the coronary artery, modulating cardiac allograft vasculopathy formation. Previous studies suggesting HLA-G-mediated immune suppressive effects in pregnancy, inflammatory diseases, cancer, and heart transplantation, make HLA-G a truly attractive candidate for further study. As such, in keeping with the reports that HLA- G has anti-inflammatory and anti-proliferative properties, our group is interested in examining whether or not these effects can also be exerted in the context of cardiac allograft vasculopathy post heart transplantation. We also aim to elucidate if the 14bp HLA-G polymorphism is associated with CAV development with the future possibility for use as a clinical tool to identify patients at risk. 47

63 Chapter 2 RATIONALE AND HYPOTHESES 48

64 2.1 The effect of everolimus on HLA-G expression in human coronary artery smooth muscle cells Rationale Immunosuppressive therapy has been found to up-regulate HLA-G expression in heart transplant recipients [147,152,168]. Sheshgiri et al. found that shla-g was detected in significantly more patients receiving everolimus (78%) than in patients receiving MMF (25%) [150]. Everolimus, a powerful SMC proliferation signal inhibitor, is known to reduce CAV development post heart transplantation [46]. HLA-G has been previously associated with decreased CAV incidence after heart transplantation [7]. As SMCs have a core role in CAV development, we aimed at determining if HLA-G can be up-regulated in this type of cells. Therefore, in our studies we examined the ability of everolimus to up-regulate HLA-G. In this way, a potential mechanism of HLA-G expression posttransplant might be elucidated. The choice of using only everolimus as cell treatment in our experiments and no other immunosuppressive treatments is based on the previous clinical study conducted by Sheshgiri Hypothesis We hypothesize that everolimus induces HLA-G expression in SMC The effect of HLA-G on human coronary artery smooth muscle cell proliferation Rationale The intimal proliferation associated with CAV, leading to impaired organ perfusion, represents the main cause of graft failure. Vascular smooth muscle cells have a pivotal 49

65 role in CAV development, as they are the main generators of vascular compromise. The activation and proliferation of SMCs represents the most important causative factor of CAV [166]. Inhibition of SMCs activation and proliferation represents an important research focus, with the ultimate goal of generating effective therapeutic interventions meant to alter these critical cellular processes and prevent CAV development. As the main cause of chronic endothelial injury, which initiates CAV, is immunologic, allograft rejection is considered an important risk factor for CAV; therefore, with no efficient therapies for reversing CAV, the best current practice is to focus on prevention through the use immunosuppressive therapies. Although newer and more advanced immunosuppressive drugs have become available on the market, and their target of action extends beyond immune response alteration into reducing SMCs proliferation, these treatments still do not ensure long-lasting graft function [15]. Moreover, chronic use of immunosuppressants has been linked with high incidence of malignant transformations post-transplant, making malignancy the main cause of morbidity and mortality after heart transplantation [46]. Other side effects of immunosuppressive therapy such as nephrotoxicity, impaired wound healing, and edema, also limit the success of solid organ transplantation [46]. In light of this knowledge, the need for new strategies of directly targeting the inhibition of SMCs proliferation associated with CAV is increasing. Le Bouteiller et al. conducted a series of in-vitro and in-vivo experiments and demonstrated that soluble HLA-G molecules affected endothelial cell activity. HLA- G was found to inhibit human umbilical vein endothelial cell proliferation, migration, and capillary tubule formation [169]. Given these interesting findings it is reasonable to look 50

66 into a similar effect of the HLA-G molecule on SMC, as their proliferation represents the main factor leading to allograft vasculopathy. The aim of our investigations was to determine the antiproliferative role of the soluble HLA-G molecule on smooth muscle cell activity in the post-transplant setting as it pertains to the subsequent development of CAV Hypothesis We hypothesize that HLA-G inhibits SMC proliferation The effect of HLA-G on neutrophil adhesion Rationale As previously mentioned, immune responses targeting the allograft constitute a risk factor for the development of CAV [25]. Following cardiac transplantation, both innate and adaptive immune systems take action, contributing to early and late rejection. Innate immune responses are activated following ischemia/reperfusion, tissue injury, inflammation, and infection; events commonly present at the time of transplantation [32]. Neutrophils have been previously shown to contribute to donor tissue damage and rejection post-solid organ transplantation [32]. They are the first cells to arrive at the site of injury and, due to selectins produced by activated endothelial cells they begin rolling on the endothelium. Integrins and adhesion molecules facilitate neutrophil adhesion to the endothelial wall where they infiltrate, causing further inflammation and tissue damage [29]. 51

67 Blocking the action of neutrophils by inhibiting their adhesion to the endothelium might prove highly useful in reducing the effects of inflammatory responses associated with organ rejection. HLA-G has been shown to inhibit various immune cell functions through binding to inhibitory receptors expressed on their surface. Neutrophils have been shown to express ILT4 receptors [170], making them suitable for HLA-G inhibitory action. Our studies were therefore meant to determine whether or not the HLA-G molecule is able to exert an inhibitory effect on neutrophil adhesion to EC Hypothesis We hypothesize that HLA-G inhibits neutrophil adhesion to EC injured by cytokine exposure The expression if ILT2, ILT4, KIR2DL4 and CD Rationale As mentioned previously, HLA-G has been found to exert its inhibitory actions via interaction with four inhibitory receptors (ILT2, ILT4, KIR2DL4, and CD160) expressed on the membrane of immune cells [88,171]. The presence of inhibitory receptors in tissues which express HLA-G and which are targeted by immune cells might represent an escape mechanism from immune attack. While in the context of viral and cancer conditions the inhibition of the immune response has a detrimental outcome, in heart transplantation, the presence of inhibitory receptors might prove beneficial. The expression of the ILT2, ILT4, andkir2dl4 inhibitory receptors within human coronary artery might facilitate a decrease in immune responses directed against the allograft. The presence of CD160 and its binding with HLA-G might elicit an antiproliferative 52

68 effect. Gaining knowledge about the type of cells within the coronary artery expressing these receptors might offer new perspectives regarding which cells to be used as targets for future treatments with HLA-G. The aim of our study was determine whether or not ILT2, ILT4, KIR2DL4, and CD160 receptors are expressed within the SMCs and ECs Hypothesis We hypothesize that ILT2, ILT4, KIR2DL4, and CD160 receptors are expressed by the SMCs and ECs of the human coronary artery The 14 bp polymorphism and CAV Rationale Only a few HLA-G genetic studies have been conducted in the area of transplantation. The association between HLA-G and CAV post transplantation remains unclear. Only one retrospective study suggested a significant association between serum HLA-G and CAV assessed by coronary angiography [7]. Our research group has recently investigated the association of the 14bp polymorphism and cellular rejection in a larger population of heart transplant recipients and found a significant association between the 14bp -/- polymorphism and serum HLA-G levels and a significant correlation between the 14bp -/- genotype and decreased acute cellular rejection [165]. No investigations have been conducted to determine the association between HLA-G polymorphisms and CAV. Understanding the association between the 14bp genotype and CAV may generate valuable tools for disease monitoring and prognosis. We intend to determine if the HLA-G polymorphism can predict the development of CAV post transplantation in 53

69 heart transplant recipients. For this, we followed a group of heart transplant recipients up to five years post transplantation [165] Hypothesis We hypothesize that there is an association between the 14bp insertion/deletion polymorphism and CAV as evaluated by coronary angiography, in patients who underwent heart transplantation. The knowledge gained from our studies will provide insights into the role of HLA-G in transplantation rejection and cardiac allograft vasculopathy, and will potentially lead to innovative pharmacologic therapies that will facilitate a better control of these pathological processes. The results from these investigations will provide essential data to support the importance of HLA-G within the heart transplant milieu, and its potential future use as a tool to improve both the short-term and the long-term outcomes of heart transplantation. 54

70 Chapter 3 METHODS 55

71 3.1. In vitro studies General Methods Cell Culture Commercially available primary human coronary artery smooth muscle cells (SMC, Cell Applications, Inc, San Diego, California) and human coronary artery endothelial cells (EC, PromoCell GmbH, Heidelberg, Germany) were cultured and grown to full confluence in a sterile, humidified tissue incubator (Model MCO-18AIC, Sanyo Electric Co., Ltd, Moriguchi, Japan) at 37 C and 5% CO for all experiments. SMCs were cultured in Smooth Muscle Cell Growth Medium (Cell Applications, Inc, San Diego, California, 5% FBS) optimized for smooth muscle cells, while ECs were cultured in MesoEndo Cell Growth Medium (Cell Applications Inc, San Diego, California, 5% FBS). From the primary culture, cells were passaged once and cryopreserved in a solution consisting of culture medium, fetal bovine serum (FBS, Life technologies, Grand Island, NY) and dimethyl sulfoxide (DMSO, Sigma, St. Louis, Missouri), until further experimental use. Cells used for these experiments had been cryopreserved no more than 6 months and were subsequently passaged 2 to 4 times, and used for studies between 7 and 21 days from the first culture post cryopreservation. All cells were initially grown in 10 cm polystyrene culture dishes (Corning Incorporated, Corning, NY) with 10 ml medium for expansion, then either used for Western immunobloting analysis, or transferred to 24-well plates (Corning Incorporated, Corning, NY) for the proliferation or neutrophil adhesion assays. 56

72 To culture the cells, the cryopreserved tube was thawed and the cells were placed in 10 cm culture dishes with 10 ml culture medium, for 24 h to allow cell attachment. The culture medium was then aspirated and replaced with fresh medium, to wash off all remaining DMSO from the initial cryopreservation. Once grown to full confluence, the medium was again discarded and the cells were washed with 10 ml of warm phosphatebuffered saline (PBS). Cells were detached by adding 2 ml 0.25% trypsin solution with ethylene diamine tetra-acetic acid (EDTA, Life technologies, Grand Island, NY) and incubated for 2 minutes at room temperature. A volume of 4 ml of medium was then added to the culture dishes to block the enzymatic activity of trypsin. Cells were resuspended by repeated pipetting and transferred into new culture dishes. Medium was then added to achieve the full desired volume of 10 ml. The cells were incubated for 48h to allow adequate time for attachment, and then the culture medium was replaced to allow the cells being used for experimentation to grow to full confluence and to prevent contamination. This cell culture method was used for all experiments The effect of everolimus on HLA-G expression in human coronary artery smooth muscle cells Cell Culture SMCs were initially cultured and grown in 10 cm dishes with 10 ml medium, as previously described. Once reaching full confluence they were subjected to various concentrations of everolimus (LC Laboratories, Woburn, Massachusetts). 57

73 Treatment of SMCs with everolimus To determine whether or not human coronary artery smooth muscle cells grown in culture could be induced into expressing HLA-G, they were treated with everolimus, an immunosuppressant with anti-proliferative properties and which was found to be associated with increased HLA-G levels in the plasma of heart transplant patients. We only chose everolimus as the treatment due to the previous clinical study showing higher HLA-G expression in patients treated with this immunosuppressant [150]. Cells previously grown in 10 cm dishes were subjected to everolimus for 24h, at a range of 0.1 to 1000ng/ml. The vehicle for our treatment was DMSO. Cells used as controls were treated with vehicle alone. Following treatment, cultures were assessed for HLA-G expression by Western immunoblotting Protein extraction Following the treatment period, the SMC were washed twice with cold PBS to ensure disposal of any remaining culture medium and non-adherent cells. Cells were then harvested by scraping the dishes with cell lifters (Fisher Scientific, Fair Lawn, New Jersey) and immediately transferred into 1.5 ml microcentrifuge tubes with 1 ml of cold trishydroxymethylaminomethane (TRIS)-buffered saline (TBS) (10mM TRIS, 0.1M NaCl, ph 7.5) (Fisher Scientific, Fair Lawn, New Jersey), containing a protease inhibitors cocktail (AEBSF, aprotinin, bestatin, E-64, leupeptin, pepstatin and phenylmethylsulphonylfluoride (PMSF)) (Sigma, St. Louis, Missouri.) at a concentration of 0.5µl/ml. Harvested cells were then centrifuged at 163 g for 10 minutes at 4 C. Following centrifugation, the cells were re-suspended in TBS containing 1% Triton X- 58

74 100 (Fisher Scientific Inc.; Pittsburgh, PA) a non-ionic surfactant detergent, and then sonicated on ice to disrupt cell membranes and release cellular contents (Sonic Dismembrator Model 500, Fisher Scientific Inc.; Pittsburgh, PA). The cells were then exposed to three cycles of vortexing and 10 minute incubations on ice. Lysates were then centrifuged once more at 163 g for 10 minutes at 4 C. Supernatants containing soluble proteins were then divided into two aliquots (one for protein determination, the other for Western immunoblotting analysis). Aliquots were flash frozen in liquid nitrogen and stored at -80 C Protein Determination Total protein concentrations in each cell lysate sample were determined with the Bio- Rad DC protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Working reagent A was prepared by combining 2% Reagent S with Reagent A. Protein standards of 0, 0.05, 0.1, 0.25, 0.5 and 0.77 mg/ml of BSA in the extraction buffer containing the protease inhibitors, was also made. Ten microliters of each sample and standard were combined with 50 µl of Working Reagent A and 400 µl of Reagent B. Then 200 µl of the samples and standards were transferred to a 96-well polystyrene plate and incubated for approximately 15 minutes at room temperature. The plate was read by a spectrophotometer (µquant Universal Microplate Spectrophometer, BioTek Instruments, Inc., Winooski, VT) at an absorbance wavelength of 750 nm. Absorbance readings from blank wells were subtracted from the values of the samples and standards. Total protein concentrations of samples were calculated by plotting the corrected absorbances on the linear protein standard curve. 59

75 Western Immunoblot Analysis Twenty micrograms of protein samples were loaded and separated using 10% running and 4% stacking TRIS-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was achieved over 60 minutes at 150 V, at RT. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp.; Bedford, MA) at 40V for 16h at 4 C. The blots were then dried and stored at -20 C until further use. On the day of blocking, dry blots were soaked in 100% methanol and washed with TBS and 1% Tween 20 (Fisher Scientific Inc.; Pittsburgh, PA) polysorbate surfactant detergent (TTBS) 2 times for 15 minutes at RT. Blocking was performed in a 5% skim milk solution with TTBS for 1 hour at room temperature. The expression of HLA-G was determined with a protein specific monoclonal antibody (Exbio Praha a.s., Vestec, Czech Republic). Blots were stained with mouse monoclonal IgG at a dilution of 1:3000 overnight at 4 C then washed with 2 times with TTBS for 15 minutes at RT. Incubation with secondary antibody was done for 1 hour at RT using a goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Santa Cruz Biotechnology Inc.; Santa Cruz, CA) at a dilution of 1:7500. Enhanced chemilumininescence using the ECL Plus Western Blotting reagent System (GE Healthcare Canada; Baie d Urfe, QC) and subsequent autoradiography using a medical film processor (Konica Minolta Model SRX-101A, Konica Minolta Medical Imaging USA, Inc.; Wayne NJ) was employed to visualize the proteins. Blots were then washed in TTBS as previously described, reprobed, and visualized in a similar manner for the protein β-actin, with both primary 60

76 mouse monoclonal IgG (Abcam Ltd.; Cambridge, MA) at a dilution of 1:22,000 and secondary goat anti-mouse IgG-HRP at a dilution of 1:20,000. HLA-G protein expression was determined in all experimental SMC culture samples. Comparisons between groups were made using computerized densitometric analysis with a commercially available software program (GS800 Calibrated Densitometer and Quantity One 1D-Analysis Software version 4.6.1, Bio-Rad Laboratories Canada, Ltd.; Mississauga, ON) Statistical Analysis Parametric statistical comparisons of all-pairwise values were performed as appropriate using the one way analysis of variance (ANOVA) using Student-Newman-Keuls post hoc test. A value of p<0.05 was required for statistical significance The effect of HLA-G on human coronary artery smooth muscle cell proliferation Cell Culture For the proliferation experiment, the SMCs were initially grown in 10 cm dishes with 10 ml medium, as previously described. Cells were washed with 10 ml PBS, detached with 2 ml 0.25% trypsin solution and diluted with 4 ml of growth medium. The cells were centrifuged at 2500 rpm for 5 minutes. The supernatant was discarded and the cells were re-suspended in the appropriate volume of medium. Cell counting was performed with an automated cell counter (Invitrogen Countess, Life technologies, Grand Island, 61

77 NY) to determine the number of cells per 1 ml of medium. The appropriate volume of cell suspension to yield 50,000 cells was transferred to each well of a 24-well culture plate. SMC culture medium was added for a final total volume of 1.5 ml/well. The cells were then incubated for 24h at 37 C to ensure attachment, and then the medium was discarded and replaced with various treatments Treatment of SMCs with HLA-G To determine the anti-proliferative effects of HLA-G, human coronary artery smooth muscle cells grown in culture were exposed to HLA-G for various periods of time. Smooth muscle cell proliferation was assessed via automated cell counting at different time points. For the initial proliferation studies, JEG-3 cells, a choriocarcinoma cell line, were used as the source of HLA-G as these cells are known to release substantial amounts of HLA-G [172]. JEG-3 cell line was a kind gift from Dr. Librach, Women s College Hospital, Toronto, ON) and the cells were cultured in 10 cm dishes in RPMI medium (Life Technologies, Grand Island, NY) containing FBS and antibiotics (100 U/mL streptomycin and 100 µg/ml penicillin) and maintained at 37 C in a sterile humid environment under 5% CO 2. Every 48h the medium was collected, filtered (0.22 µm, Millipore, Carrigtwahill, Co. Cork, Ireland) and stored at 4 C for subsequent use in the proliferation studies as conditioned medium from JEG-3 cells (CMJ). The concentration of soluble HLA-G in the CMJ was measured with Elisa methodology as previously described [172,173], yielding 75U/ml, the equivalent of approximately 30ng/ml 62

78 according to the manufacturer. In parallel, to minimize the effect of other variables, the medium from cultured SMCs (CMS) was also collected in a similar fashion. In preparation for experimental use, the medium was filtered in a sterile environment to eliminate any cell debris. Two groups of cells were created for each time point: (a) cells cultured in 1ml CMS plus 0.5ml regular, non-conditioned SMC medium (R), (b) cells cultured in CMJ, supplemented with 0.5ml R medium. The addition of R medium was done to ensure that a subsequent inhibition of SMC proliferation would not be due to the potential lack of nutrients within the CMS or CMJ, rather than the effect of HLA-G. All the cells were subjected to 24, 72, and 120 hours of treatment. The groups cultured for 120 hours had the medium changed at 72 hours, with the appropriate medium combination outlined above, to promote growth and prevent contamination. Following each treatment time point, the cultures were assessed for SMC proliferation via automated cell counting. Specifically, on the day of counting, the medium was discarded and each well was washed with 2 ml of warm PBS. A volume of 500 µl of trypsin was added to each well and incubated at room temperature for 2 minutes to allow time for cell detachment. One ml of medium was then added to each well. The total volume of cell suspension from each well was then transferred to 1.5 ml micro tubes and centrifuged at 130g for 5 minutes at RT. The supernatant was discarded and the cell pellet was re-suspended in 200 µl of culture medium in preparation for counting. The 63

79 cells were counted with the automated cell counter set to detect particles of between 2 and 10 microns in diameter. When the soluble purified recombinant HLA-G became available on the market, the study was repeated using recombinant HLA-G in place of that derived from JEG-3 cells. SMCs were cultured as previously mentioned, and subjected to recombinant HLA-G (OriGene Technologies, Rockville, MD, USA) (100, 500, and 1000ng/ml) for 24, 72, and 120 hours. These concentrations were chosen to allow assessment of HLA-G effects at a wide series of doses, ranging from low to high. The vehicle for the treatments was the HLA-G buffer (10% Glycerol, 100mM Glycine, 25mM Tris-HCl, ph 7.3). Cells used as controls were treated with vehicle alone. The medium of cells treated for 120 hours was replaced with fresh medium, containing vehicle or HLA-G in the appropriate concentrations at 72 hours. Following each treatment time point, the cultures were assessed for SMC proliferation in a non-blinded fashion, using an automated cell counter set to detect particles of 2-10 microns in diameter Statistical Analysis Parametric statistical comparisons of all-pairwise values for the study using JEG-3 conditioned medium were performed as appropriate using the one way analysis of variance (ANOVA) using Student-Newman-Keuls post hoc test. A value of p<0.05 was required for statistical significance. 64

80 Parametric statistical comparisons of all-pairwise values for the study using purified HLA-G were performed as appropriate using the two-way analysis of variance (ANOVA) using Student-Newman-Keuls post hoc test Neutrophil adhesion studies HLA-G is known to have a regulatory role in inflammatory conditions [140]. We tested whether or not HLA-G decreases the inflammatory response to endothelial injury The effect of calcineurin inhibitors on neutrophil adhesion to endothelial cells It has been previously reported that exposure to calcineurin inhibitors (CNI) directly influences neutrophil adhesion to coronary artery endothelial cells [174]. The role of HLA-G in the context of endothelial activation in response to CNI injury has not been studied. In order to establish an injury model, we tested whether or not exposure to CNIs would up-regulate neutrophil adhesion to cultured ECs Cell Culture ECs were initially grown in 10 cm dishes with 10 ml medium, as previously described. Once confluent, cells were further washed with 10 ml PBS, detached with 2 ml 0.25% trypsin solution for 2 minutes, and diluted with 4 ml growth medium. The cells were centrifuged at 1428 g for 5 minutes. The supernatant was discarded and the cells were re-suspended in the appropriate volume of medium. Cell counting was performed with an automated cell counter as previously described, to determine the number of cells per 1 ml of medium. The appropriate volume of cell suspension to yield 50,000 cells was 65

81 transferred to each well of a 24-well culture plate. EC culture medium was added for a final total volume of 1.5 ml/well. The cells were incubated for 24 h to achieve full confluence Neutrophil Isolation Neutrophils were isolated from the peripheral blood of healthy volunteers [145]. Peripheral blood was drawn into 8 ml Vacutainer CPT tubes that contained sodium citrate (VWR International LLC, Mississauga, ON) and inverted three times to mix. The tubes were centrifuged for 25 minutes at 1700g at RT. The plasma and the peripheral blood mononuclear cells were aspirated and discarded and the gel lock was washed twice with 5 ml of ice cold PBS. The red blood cells and neutrophil mix, below the gel lock, was collected by aspiration into a 3 ml syringe attached to an 18 gauge 1.5 inch needle. The cell mix was then deposited into a 50 ml conical tube and washed with 5 ml of ice cold PBS containing 2% FBS. This process was followed by 10 minutes of centrifugation at 404 g at 4 C. The supernatant was discarded and the cell pellet was resuspended in 16 ml of red blood cell (RBC) lysing buffer (10 mmol/l KHCO3, 150 mmol/l NH4Cl, 0.1 mmol/l EDTA, ph 8.0) and incubated at RT for 10 minutes with intermittent vortexing. After centrifugation for 10 min at 4 C, the supernatant was discarded and the cells were washed with 5 ml PBS with 2% FBS as described above. After centrifugation for 10 min at 4 C, the supernatant was again discarded and the cells were subjected to 5 ml of RBC lysing buffer for 10 min at RT with intermittent vortexing, followed by 10 min of centrifugation as described above. The remaining cell pellet was washed once more with PBS with 2% FBS and after centrifugation for 10 min at 404 g 66

82 at 4 C, the cells were resuspended in RPMI 1640 medium supplemented with 10% FBS, 2% L-glutamine, and 1% antibiotic-antimycotic solution. The number of cells/ml of medium was calculated via automated cell counting Treatment of ECs with cyclosporine and tacrolimus Cyclosporine and tacrolimus were previously found to increase neutrophil adhesion to endothelial cells [174]. In baseline experiments we examined the effect of CNI on neutrophil adhesion to treated ECs. The cells were exposed to 48 hours of treatment with cyclosporine (CyA) (LC Laboratories, Woburn, Massachusetts ) at concentrations of 10 ng/ml and 100ng/ml, and with tacrolimus (Tac) (LC Laboratories, Woburn, Massachusetts) at concentrations of 100ng/ml and 500 ng/ml. DMSO (0.01%) was used as vehicle control for both CyA and Tac. Recombinant TNF-α (10ng/ml) (RnD Systems, Minneapolis, MN) was used as positive control. Cells were exposed to TNF-α for 4 hours prior to adhesion assay. In a subsequent study we examined the effect of Tac at a lower concentration, on EC. The cells were exposed to 48 hours of treatment with Tac at 10 ng/ml and 100 ng/ml, and Cya at 10 ng/ml. TNF-α 10 ng/ml was again used as positive control. A concentration of TNF-α of 1 ng/ml was added to the study to test its ability to cause injury to ECs and subsequent neutrophil adhesion. 67

83 Neutrophil Adhesion Assay Neutrophil adhesion to the human coronary artery endothelial cell cultures subjected to TNF-α treatment with or without HLA-G was assessed with a non-static adhesion assay as follows. The neutrophil adhesion assay was started within one hour post neutrophil isolation. Isolated neutrophils were re-suspended in RPMI medium and the cell count was determined. The dishes containing confluent EC were discarded of medium and to each well was added 300 µl of neutrophil solution to yield a total of 500,000 cells/well. The same solution was used to create a standard curve. A 24-well plate was also used for the standards, and appropriate volumes of cell suspension and RPMI medium were added in order to reflect a wide range of percentage of adhesion (10 to 100%). All plates were incubated for 1 hour at 37 C under rotating conditions at 62 rpm [175]. All wells containing EC and neutrophils were then gently washed with 250 µl of RPMI medium to eliminate any non-adherent cells. They were then resupplied with 300 µl RPMI medium and all the wells, including the standards, were added 300 µl Myeloperoxidase assay buffer containing 67mM Na 2 HPO 4, 35mM Citric acid, 0.1% Triton X-100, with 5.5 mmol/l o-phenylenediamine and 4 mmol/l H2O2. All cell dishes were incubated at RT for 4 minutes and the reaction was stopped by the addition of 300 µl of 1M H 2 SO 4. The optical density of absorbance was read spectrophometrically at 492nm with a microplate reader (μquant Universal Microplate Spectrophotometer, BioTek Instruments, Inc., Winooski, VT). 68

84 Statistical Analysis Parametric statistical comparisons of all-pairwise values were performed as appropriate using the one way analysis of variance (ANOVA) using Dunnett`s method. A value of p<0.05 was required for statistical significance The effect of HLA-G on neutrophil adhesion to endothelial cells injured by cytokine exposure Treatment of EC with HLA-G and TNF- α To assess the degree and severity of EC injury caused by TNF-α, and to determine the optimal concentrations which elicit a response of increased neutrophil adhesion, a dose response study was employed. ECs cultured as mentioned above, were subjected to TNF-α exposure for 4 hours, at concentrations varying from 0.01ng/ml to1ng/ml. For the final study, confluent EC were exposed to purified recombinant HLA-G (1000 ng/ml) for 48 hours. This concentration was chosen based on our previous study results, where SMC proliferation was inhibited following exposure to 1000ng/ml of HLA- G. The vehicle for our treatment in the current study was the HLA-G buffer. Cells used as controls were treated with vehicle alone. On the day of the experiment the cells were exposed to various doses of TNF-α (0.1 1 ng/ml) for 4 h with or without the presence of HLA-G. 69

85 Statistical Analysis Parametric statistical comparisons of all-pairwise values for the TNF-α preliminary study were performed as appropriate using the one way analysis of variance (ANOVA) using Student-Newman-Keuls post hoc test. A value of p<0.05 was required for statistical significance. Parametric statistical comparisons of all-pairwise values for the neutrophil adhesion study using HLA-G and TNF-α were performed as appropriate using the two way analysis of variance (ANOVA) using Student-Newman-Keuls post hoc test. A value of p<0.05 was required for statistical significance Expression of ILT2, ILT4, KIR2DL4 and CD160 inhibitory receptors in SMCs and ECs Human leucocyte antigen G exerts its inhibitory actions via interaction with ILT2, ILT4, KIR2DL4 and CD160 inhibitory receptors currently detected only on immune cells and HUVEC, respectively. Our objective was to determine if the cells used in our experiments, SMCs and ECs, express these receptors. 70

86 Analysis of ILT2, ILT4, KIR2DL4, and CD160 inhibitory receptors expression on SMCs and ECs via Western Immunoblotting Cell Culture SMCs and ECs were initially cultured and grown in 10 cm dishes with 10 ml medium, as previously described. Once reaching full confluence they were subjected to TNF-α Treatment of SMCs and ECs with TNF-α To determine whether or not human coronary artery smooth muscle cells and human coronary artery endothelial cells grown in culture could be induced into expressing the ILT2, ILT4, KIR2DL4, and CD160 receptors following cytokine induced injury, they were treated with TNF-α for 4 and 24h, at a concentration of 10ng/ml. The vehicle for our treatment was PBS. Cells used as controls were treated with vehicle alone. Following treatment, cultures were assessed for ILT2, ILT4, KIR2DL4, and CD160 expression by Western immunoblotting Protein extraction Following the treatment period, the SMCs and ECs were collected and used for protein extraction as previously described Protein Determination Total protein concentrations in each cell lysate sample were determined with the Bio- Rad DC protein assay as described above. 71

87 Western Immunobloting Analysis Twenty micrograms of protein samples were loaded and separated using 10% running and 4% stacking TRIS-glycine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis was achieved over 60 minutes at 150 V, at RT. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp.; Bedford, MA) at 40V for 16h at 4 C. The blots were then dried and subsequently soaked in 100% methanol. They were then washed with TTBS twice for 15 minutes at RT. Blocking was performed in a 5% skim milk solution with TTBS for 1 hour at room temperature. For both SMCs and ECs, the expression of ILT2 was determined with protein specific polyclonal rabbit antibody (Biorbyt, San Francisco, CA) at a dilution of 1:500, over night at 4 C. Recombinant human ILT2 (RnD Systems, Minneapolis, MN) was used as positive control. The presence of ILT4 was determined with protein specific polyclonal goat antibody (RnD Systems, Minneapolis, MN) at a concentration of 1:1,000. Recombinant human ILT4 (RnD Systems, Minneapolis, MN) was used as positive control. For detection of KIR2DL4 expression a polyclonal rabbit antibody (Abgent, San Diego, CA) at a concentration of 1:5,000 was used. Recombinant human KIR2DL4 was used as positive control (RnD Systems, Minneapolis, MN). Detection of CD160 was achieved using a polyclonal sheep antibody (RnD Systems, Minneapolis, MN) at a concentration of 1:500. Recombinant human CD 160 was used as positive control (RnD Systems, Minneapolis, MN). Blots were subsequently washed 2 times with TTBS for 15 minutes at RT. Incubation with secondary antibody was done for 1 hour at RT using a 72

88 goat anti-rabbit IgG (Santa Cruz Biotechnology Inc.; Santa Cruz, CA) at a dilution of 1:5000 for ILT2, with a donkey anti-goat IgG antibody (RnD Systems, Minneapolis, MN) at a dilution of 1:10,000 for ILT4, with goat anti-rabbit IgG HRP at a dilution of 1:30,000 for KIR2DL4, and with a polyclonal donkey anti-sheep IgG (RnD Systems, Minneapolis, MN) at a dilution of 1:20,000 for CD 160. Enhanced chemilumininescence using the ECL Plus Western Blotting reagent System (GE Healthcare Canada; Baie d Urfe, QC) and subsequent autoradiography using a medical film processor (Konica Minolta Model SRX-101A, Konica Minolta Medical Imaging USA, Inc.; Wayne NJ) was employed to visualize the proteins. Blots were then washed in TTBS as previously described, re-probed, and visualized in a similar manner for the protein β-actin, with primary mouse monoclonal IgG (Abcam Ltd.; Cambridge, MA) at a dilution of 1:22,000 and secondary goat anti-mouse IgG-HRP dilutions of 1:40,000 for ILT2 and ILT4, and primary antibody dilution of 1:25,000 and secondary antibody dilution of 1:60,000 for KIR2DL4 and CD160. ILT2, ILT4, KIR2DL4 and CD 160 protein expression was determined in all experimental SMC and EC culture samples. Analysis of samples was made using computerized densitometric analysis with commercially available Bio-Rad Quantity One software. 73

89 Analysis of ILT2, ILT4, and KIR2DL4 inhibitory receptors expression on SMC via Flow Cytometry Flow cytometry Smooth muscle cells To assess cell surface receptor expression, cells cultured in 10 cm dishes were washed with 6 ml PBS and detached with 2 ml Accutase cell detachment medium (ebioscience, Inc., San Diego, CA, USA) at 37 ºC for 5 minutes. Each dish was added 3 ml of cold flow buffer and the cells were re-suspended and centrifuged at 914 g at 4ºC for 5 minutes. The supernatant was discarded and the cells were re-suspended in 3 ml of flow buffer. Cell count was performed with the automated cell counter and 500,000 cells were transferred into polypropylene round bottom tubes (BD Biosciences, San Jose, CA, USA), to be assessed for protein expression or to be used as isotype controls. Upon cell counting and transfer of SMCs into tubes, 2 ml of flow buffer was added to each sample tube which was then centrifuged as above. The supernatant was discarded. To minimize the nonspecific binding of antibodies, FCR blocking agent (Miltenyi Biotec, Cambridge, MA) was used. To each tube was added 10 µl of FCR and the suspension was incubated at 4 ºC for 10 minutes. To test the ability of the antibodies to detect positive and negative signals, beads (BD CompBead Anti-mouse Ig, BD Bioscience, San Jose, CA, USA) were used. An individual tube was prepared for each antibody to be tested. One drop of positive beads and one drop of negative beads was added to each tube, followed by the addition of 100 µl of flow buffer. All tubes 74

90 received the appropriate antibodies and isotype controls. To assess ILT2 expression we used a primary allophycocyanin (APC) conjugated mouse monoclonal anti-human ILT2 antibody (RnD Systems, Minneapolis, MN) at 10 µg/ml. For the detection of ILT4, a primary carboxyfluorescein (CFS)-conjugated mouse monoclonal anti-human ILT4 antibody (RnD Systems, Minneapolis, MN) was used at 10 µg/ml. A primary phycoerythrin (PE)-conjugated mouse monoclonal anti-human KIR2DL4 antibody (RnD Systems, Minneapolis, MN) was used for KIR2DL4 detection at a concentration of 10 µg/ml. Cells serving as negative controls were incubated with isotype-matched mouse IgG (RnD Systems, Minneapolis, MN) at the same antibody concentration in similar fashion. All tubes were vortexed and incubated for 25 minutes on ice. One ml of flow buffer was added to each tube followed by vortexing and centrifugation as above. The supernatant was discarded and the samples were fixed as follows. Each tube received 900 µl of cold PBS followed by vortexing and the subsequent addition of 100 µl of 16% Paraformaldehyde with vortexing. The samples were incubated for 20 minutes on ice. One ml of cold flow buffer was added to each tube followed by vortexing and centrifugation as above. The supernatant was discarded and 500 µl of cold flow buffer was added to each tube. The ILT2, ILT4 and KIR2DL4 receptor expression was assessed using a BD LSR II flow cytometer (BD Bioscience). IsoFlow sheath fluid (Beckman Coulter) was used to top up sample volumes in the round-bottom tubes. 75

91 3.2. Clinical study HLA-G polymorphism and cardiac allograft vasculopathy This retrospective study was approved by institutional ethics research board and included 81 heart transplant recipients who underwent transplantation at Toronto General Hospital between 1995 and 2008 [165]. Each patient underwent blood testing for DNA analysis. Coronary angiography testing was done routinely at 1 year and 5 years post transplantation. We aimed to identify if there is an association between HLA- G insertion/deletion polymorphism and CAV post heart transplantation Patients All 81 patients received induction therapy with rabbit antithymocyte serum, basiliximab or anti-thymocyte rabbit globulin. The immunosuppression treatment consisted of cyclosporine or tacrolimus, azathioprine (prior to 1998) or mycophenolate mofetil (since 1998) and prednisone. Patients who developed renal dysfunction after transplantation were placed on sirolimus (n=14) and the calcineurin inhibitors were minimized or withdrawn. Those treated with everolimus were part of a clinical trial. In this study, only those patients taking a stable immunosuppressive regime for the first year post transplant were selected Blood collection Each patient had one blood sample drawn for DNA analysis. For this purpose, 8ml of venous blood was collected in EDTA-coated BD vacutainer collection tubes. Samples were then centrifuged at 4 C for 20 minutes and the serum was transferred in 76

92 microtubes, flash frozen in liquid nitrogen and stored at -80 C for subsequent DNA analysis [165] DNA extraction DNA extraction was done previously (161). Genomic DNAs were extracted from serum samples using the QiaAmp kit (QIAGEN, Grand Island, NY) and according to the manufacturer's recommendations. DNA concentration and purity (A260/A280 ratio) was determined by the NanoDrop Spectrophotometer (Thermofisher) [165] BP Insertion/Deletion Polymorphism Genotyping The samples were assayed previously for polymorphism (161). Briefly, genotyping for the 14 bp insertion/deletion polymorphism was performed by polymerase chain reaction (PCR) amplification of exon 8 (rs16375), using a forward primer conjugated to a fluorescent dye FAM: 5'- /FAM/ TGATGGGCTGTTTAAAGTGTCACC-3' (Integrated DNA Technologies, Inc), and the reverse primer: 5'-GGA AGGAATGCAGTTCAGCATGA-3'. PCR amplifications were performed with about 20 ng genomic DNA as described previously [164] Coronary angiography Coronary angiography was routinely performed at 1 year (n=60) and at 5 years (n=56) post-transplant. We excluded from the angiographic studies those patients who developed complications such as renal failure. Cardiac allograft vasculopathy was diagnosed according to the International Society for Heart and Lung Transplantation 77

93 (ISHLT) classification system [21]. The severity of the disease was analyzed by one independent cardiologist blinded to the polymorphism classification of these patients. Patients were classified into two groups, within the two time points (1 year and 5 years), based on their CAV profile: Group 1, CAV negative, included patients with no detectable angiographic lesions (ISHLT CAV 0 ) and Group 2, CAV positive, included patients with mild, moderate, or severe angiographic lesions (ISHLT CAV 1, ISHLT CAV 2, and ISHLT CAV 3 respectively). The increase in severity of angiographic lesions from mild to moderate or moderate to severe was considered as CAV progression [165] Statistical analysis All statistical analyses were carried out using the SAS software (version 10). To compare differences between groups, we performed Fisher s exact test. HLA-G allele frequencies were tested for Hardy Weinberg equilibrium by Chi-Square test. In all analysis a p value of 0.05 was considered statistically significant [165]. 78

94 CHAPTER 4 RESULTS 79

95 4.1. The effect of everolimus on HLA-G expression in SMCs It has been previously suggested in clinical studies that HLA-G might be up-regulated by immunosuppressants [147] such as tacrolimus [152] and everolimus [150]. We examined whether or not HLA-G can be modulated by everolimus in SMCs, one of the main contributors to CAV development HLA-G Expression To determine HLA-G expression in SMC cultures, the cells were treated with various doses (1 1000ng/ml) of everolimus and protein expression was assessed via Western Immunoblotting (Figure 7). BActin was used as a loading control and there were no differences between groups (Figure 8). Protein content is expressed as the ratio of HLA- G to BActin. SMC cultures expressed HLA-G at baseline (0.27±0.06). Expression of HLA-G was detected following treatment with everolimus. At 0.1ng/ml of everolimus, the HLA-G:BActin ratio was 0.22±0.04, at 10ng/ml the ratio was 0.33±0.06, at 100ng/ml the ratio was 0.35±0.03, and at 1000ng/ml the ratio was 0.54±0.06. Total protein expression increased significantly in response to everolimus treatment at a concentration of 1000ng/ml (p<.014) compared to control, as detected by Western immunoblotting analysis. 80

96 Density (HLA-G/B-actin) * Everolimus treatment (ng/ml) Figure 7. Assessment of HLA-G expression in smooth muscle cell cultures following everolimus treatment. SMC were incubated with everolimus ( ng/ml) for 24 h. Significantly higher HLA-G expression was induced by the highest concentration of everolimus (1000ng/ml) when compared with control and with the other lower doses of everolimus, as determined by Western blot. Individual significant values of everolimus treatment at 1000 ng/ml versus 0 ng/ml (*p=0.014); vs. 0.1 ng/ml (*p=0.003); vs. 10 ng/ml (*p=0.024); vs. 100 ng/ml (*p=0.014). 81

97 39 kda Whole SMC lysates HLA-G Control E 0.1 E 10 E 100 E 1000 Β-actin Figure 8. A representative Western blot demonstrating higher HLA-G expression in smooth muscle cell cultures following treatment with everolimus at 1000ng/ml. BActin is used as a loading control. 82

98 4.2. The effect of HLA-G on human coronary artery smooth muscle cell proliferation Smooth muscle cells have an important role in the development of CAV. By inhibiting their proliferation, the progression of CAV could be altered with subsequent reduction of intimal thickening. We examined if HLA-G has an inhibitory effect in SMC proliferation. A B C Figure 9. Representative microphotographs (4X) of SMCs proliferation at 24 hours with a cell count of 0.5 x 10 5 /ml (A), at 48 hours with a cell count of 1.5 x 10 5 /ml (B), and 120 hours with a cell count of 4.5 x 10 5 /ml (C), under no treatment conditions. A time dependent increase in proliferation is observed. 83

99 Proliferation study using JEG-3 conditioned media as a source of HLA-G Commercially available primary SMC (n=8) were seeded for 24h, and then exposed to conditioned medium from JEG-3 cells (CMJ). Groups: (a) cells cultured in conditioned medium from SMC (CMS) (1ml) plus 0.5ml regular, non-conditioned SMC medium (R) (b) cells cultured in CMJ (1ml), supplemented with 0.5ml R. Proliferation was measured at various time points ( hours) post-treatment by automated cell counting. The concentration of HLA-G in the CMJ was approximately 30ng/ml which is comparable with the concentration found in patients plasma. There was no significant difference in proliferation between the cells treated with HLA-G conditioned media (CMJ) and the control group, within the 3 different time points (p>.05) (Figure 9). The lack of purified HLA-G was a major limitation of this study. Other factors released by JEG-3 in the medium might have had greater effects on SMC than the potential inhibitory effects of HLA-G. In this study, the JEG-3 conditioned media did not prove to be efficient for use as a substitution for purified HLA-G protein. Therefore the next study was done using the purified HLA-G. 84

100 1.5 CMS/R CMJ/R Cell count (#cells x 10 5 /ml) Time (hours) Figure 9. Assessment of SMC proliferation inhibition in response to HLA-G released by JEG-3 cells. There was no significant difference in proliferation between the cells treated with HLA-G conditioned media (CMJ) and the control group, within the 3 different time points (p>0.05). 85

101 Proliferation study using purified HLA-G In the subsequent study, commercially available primary SMC (n=8) were seeded for 24h, and then exposed to recombinant human HLA-G at various concentrations ( ng/ml). Proliferation was measured at various time points ( hours) posttreatment by automated cell counting (Figure 10). SMCs in the control group proliferated as expected, and their cell count (# cells/ml) was 0.60±0.08x10 5 at 24 hours, 1.3±0.04 x10 5 at 48 hours, and 4.59±0.29 x10 5 at 120 hours. The increase in proliferation was significant at all-time points (p<0.001). HLA-G at 100ng/ml was not statistically significant compared to control (p>0.05). At 24 and 48 hours of treatment there was no significant difference in proliferation between the groups. At 120 hours, the proliferation of each one of the concentration groups was significantly higher than the corresponding concentration group at 24 ( # p<0.001) and 48 hours ( # p<0.001). At 120 hours post-treatment, the proliferation of cells treated with HLA-G at 500 and 1000ng/ml was significantly lower (*p=0.041 and *p=0.004, respectively) than that of the untreated, control group. At 120 hours there was also a significant decrease in proliferation of the cells treated with 1000ng/ml when compared to the group exposed to 100ng/ml (*p=0.020). No significant differences were seen between the cells treated with 100 ng/ml (p=0.454) of HLA-G and control, at any of the 3 time points. The viability of the cells was similar at all time-points. 86

102 Cell count (#cells x 10 5 /ml) Control HLA-G 100 ng/ml HLA-G 500 ng/ml HLA-G 1000 ng/ml * # # # # Time (hours) Figure 10. Assessment of SMC proliferation inhibition in response to soluble HLA-G treatment. There was a significant increase in proliferation over time (by two-way ANOVA, time effect F=954, # p<0.001). HLA-G demonstrated an inhibitory effect (HLA-G effect F=, *p=0.03). There was no interaction between time and HLA-G exposure (time and HLA-G effect F=1.35, p=0.2). Student-Newman-Kleus multiple comparison method specified a decrease in proliferation of cells treated with 1000ng/ml of HLA-G when compared to control and with 100ng/ml dose (*p<0.05), and also a difference between groups treated with 500ng/ml and control (*p<0.05). 87

103 4.3. Neutrophil adhesion study Neutrophil adhesion to the sites of injury as part of the inflammatory response triggered at the time of transplantation, has been shown to generate further tissue damage and to contribute in time to CAV development. We examined whether or not HLA-G inhibits neutrophil adhesion to ECs. A B C Figure 12. Representative microphotographs (4X) of confluent ECs under different conditions. Microphotograph A shows confluent ECs with no treatment and no neutrophils added (0% adhesion); microphotograph B shows confluent ECs with no treatment and with added neutrophils (4% adhesion); microphotograph C shows confluent ECs treated with TNFα at 1ng/ml and with neutrophils added post treatment (33% adhesion). 88

104 The effect of calcineurin inhibitors on neutrophil adhesion to endothelial cells Commercially available EC were exposed to tacrolimus (100 ng/ml and 500 ng/ml) and cyclosporine A (10 ng/ml and 100 ng/ml) for 48 hours. The cells in the positive control group were incubated with TNF-α (10ng/ml) for 4 hours. The vehicles for our treatments were DMSO. Neutrophils were isolated from the blood of healthy volunteers and added to EC under nonstatic conditions. The adhesion of neutrophils to EC was measured spectrophotometrically at 492nm. Incubation with tacrolimus at 100 ng/ml and 500 ng/ml had no significant effect on neutrophil adhesion when compared with control (p>0.05). Similarly, the cells treated with cyclosporine A at 10 ng/ml and 100 ng/ml did not show an increase in neutrophil adhesion (p>0.05). As expected, exposure of the EC to 4 hours of treatment with TNF-α at a concentration of 10 ng/ml increased neutrophil adhesion (*p<0.001) compared to control (Figure 13). 89

105 Figure 13. Assessment of neutrophil adhesion to EC as a result of various degrees of EC injury caused by exposure to tacrolimus (TAC) and cyclosporine A (CyA). Tacrolimus at a concentration of 100 ng/ml and 500 ng/ml, as well as cyclosporine A at 10ng/ml and 100 ng/ml did not cause increase in neutrophil adhesion when compared to untreated cells. TNF-α at 10ng/ml was used as a positive control and caused significantly higher adhesion (*p<0.05) than the control group. 90

106 In a subsequent study we tested whether or not a lower dose of tacrolimus would yield a stronger effect on the ECs. We also tested whether or not a lower concentration of TNFα would elicit similar effects of increased neutrophil adhesion to EC. For this experiment, the confluent ECs were exposed to tacrolimus at concentrations of 10 ng/ml and 100 ng/ml for 48 hours. Cyclosporin A was used again at 10 ng/ml for the same duration of time. The cells were also exposed to TNF-α at a concentration of 1 ng/ml, as well as the 10 ng/ml dose, for 4 hours. Incubation with tacrolimus at 10 ng/ml and 100 ng/ml had no significant effect on neutrophil adhesion when compared with control (p>0.05). Similarly, the cells treated with cyclosporine A at 10 ng/ml did not show an increase in neutrophil adhesion (p>0.05). The exposure of ECs to 4 hours of treatment with TNF-α at both concentrations of 1 ng/ml and 10 ng/ml increased neutrophil adhesion (*p<0.005) compared to control (Figure 14). 91

107 Figure 14. Assessment of neutrophil adhesion to EC after treatment with tacrolimus (TAC), cyclosporine A (CyA), and TNF-α at various concentrations. Tacrolimus at a concentration of 10 ng/ml and 100 ng/ml, as well as cyclosporine A at 10ng/ml did not cause increase in neutrophil adhesion when compared to untreated cells. TNF-α at 1ng/ml and 10 ng/ml caused significantly higher adhesion (*p<0.05) than the control group. 92

108 The effect of HLA-G on neutrophil adhesion to human coronary artery endothelial cells injured by cytokines TNF- α dose response study As our previous studies with immunosuppressants failed to cause a relevant increase in neutrophil adhesion, we decided to use TNF- α as our treatment to cause endothelial cell injury. In order to determine which concentrations of TNF- α to be used to create EC injury and subsequent neutrophil adhesion, a dose response study was implemented. EC were incubated for 4 h with various concentrations (0.01, 0.1, 0.5, 1, 10 ng/ml) of TNF-α. Neutrophils were isolated from the blood of healthy volunteers and added to the EC under nonstatic conditions. The adhesion of neutrophils to the EC was measured spectrophotometrically at 492nm. The study revealed a dose dependent increase in neutrophil adhesion, when compared to control. The 0.1, 0.5, 1, and 10 ng/ml concentrations caused significantly higher neutrophil adhesion than control (*p<0.05). Based on these results, three concentrations were chosen to be used as treatment for the subsequent study (0.1, 0.5, and 1ng/ml). The control was 3.98±0.23% of adhesion, 0.1ng/ml was 5.32±0.23% of adhesion, the 0.5ng/ml was 7.44±0.12% of adhesion, and 1ng/ml was 8.87±0.07% of adhesion. The highest concentration of 10ng/ml was excluded as its strong effect was thought to overwhelm the inhibitory action of HLA-G (Figure 15). 93

109 Adhesion (%) * * * * C TNF 0.01 TNF 0.1 TNF 0.5 TNF 1 TNF 10 Figure 15. Assessment of neutrophil adhesion to EC as a result of various degrees of EC injury caused by exposure to different concentrations of TNF-α. A dose dependent increase in neutrophil adhesion was observed. The TNF-α concentrations of 0.1, 0.5, 1, and 10ng/ml caused significantly higher adhesion (*p<0.05) than the control group. 94

110 Neutrophil adhesion study Commercially available EC were exposed to recombinant human HLA-G (1000ng/ml) for 48 hours. The cells were then co-incubated with various doses of TNF-α (0.1-1ng/ml) for 4 hours. Neutrophils were isolated from the blood of healthy volunteers and added to the EC under nonstatic conditions. The adhesion of neutrophils to the EC was measured spectrophotometrically at 492nm. Incubation with HLA-G at 1000ng/ml alone had no significant effect on neutrophil adhesion compared to control (4.84±0.25 vs. 3.73±0.22, p>0.05, respectively). As expected, exposure of the EC to 4 hours of treatment with TNF-α at concentrations of 0.1, 0.5, and 1ng/ml increased neutrophil adhesion to 9.98±0.31, 26.97±0.50, and 31.98±0.51, respectively (*p<0.001) compared to control 3.73±0.22. Incubation of the EC with HLA-G in combination with TNF-α, significantly decreased TNF-α-stimulated neutrophil adhesion. The adhesion decreased from 9.98% to 7.36±0.45% ( # p<0.001) at 0.1ng/ml of TNF-α, from 26.97% to 24.20±0.83% ( # p<0.001) at 0.5ng/ml TNF-α, and from 31.98% to 29.41±0.91% ( # p=0.005) at 1ng/ml TNF-α (Figure 16). 95

111 Adhesion (%) Control HLA-G TNF-α TNF-α + HLA-G * # * # Figure. * # 0 Figure 16. Assessment of neutrophil adhesion in response to TNF- α with or without HLA-G. Higher neutrophil adhesion was observed as the TNF-α concentration increased (by two way ANOVA, TNF-α effect F=687, *p<0.001). The addition of 1000ng/ml of HLA-G significantly reduced neutrophil adhesion (HLA-G effect F=27, # p<0.001). There was no interaction between TNF-α and HLA-G exposure (TNF and HLA-G effect F=0.01, p=0.9). Student-Newman-Kleus multiple comparison method specified significant differences between HLA-G and non-hla-g treated groups at all TNF-α concentrations tested (p<0.001, p<0.001, p=0.005). 96

112 4.4. Expression of ILT2, ILT4, KIR2DL4, AND CD160 inhibitory receptors in SMCs and ECs HLA-G has been shown to exert its inhibitory functions via membrane receptors expressed by immune cells and endothelial cells. We examined if the ILT2, ILT4, KIR2DL4 and CD160 receptors for HLA-G are expressed by SMCs and ECs Analysis of ILT2, ILT4, KIR2DL4 and CD 160 inhibitory receptor expression on SMCs and ECs via Western immunoblotting Human coronary artery smooth muscle cells and ECs were exposed to 10 ng/ml of TNF-α for 4 and 24 hours. The cell lysates were then used to determine the protein concentration. ILT2, ILT4, KIR2DL4 and CD160 protein expression was determined via Western immunoblotting. Figure 17 shows a representative image of Western blot demonstrating that ILT2 was not detected in SMCs following treatment with TNF-α at 10ng/ml for 4 hours or 24 hours. At the same treatment conditions, ILT4, KIR2DL4, and CD160 were also not detected in SMCs (Figures 18, 19 and 20, respectively). Similarly, ILT2, ILT4, KIR2Dl4 and CD160 were not detected in ECs after treatment with TNF-α at 10ng/ml for 4 hours or 24 hours via Western Immunoblotting (Figures 21, 22, 23 and 24 respectively). Based on our studies, SMCs and ECs do not express the four HLA-G inhibitory receptors. 97

113 Figure 17. A representative Western blot showing no ILT2 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours BActin is used as a loading control. Figure 18. A representative Western blot showing no ILT4 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours BActin is used as a loading control. 98

114 Figure 19. A representative Western blot showing no KIR2DL4 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours BActin is used as a loading control. Figure 20. A representative Western blot showing no CD160 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours BActin is used as a loading control. 99

115 Figure 21. A representative Western blot showing no ILT2 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours BActin is used as a loading control. Figure 22. A representative Western blot showing no ILT4 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours BActin is used as a loading control. 100

116 Figure 23. A representative Western blot showing no KIR2DL4 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours BActin is used as a loading control. Figure 24. A representative Western blot showing no CD160 protein expression in smooth muscle cell cultures following treatment with TNF-α at10ng/ml for 4 hours or 24 hours BActin is used as a loading control. 101

117 Analysis of ILT2, ILT4, and KIR2DL4 inhibitory receptors expression via flow cytometry We used flow cytometry analysis to determine ILT2, ILT4, and KIR2DL4 receptor expression in untreated SMCs cultures. Microbeads were used as positive control (Figures 23A, 24A, 25A). SMCs did not express ILT2 at baseline (Fig 23B). Similarly, SMCs did not express ILT4 and KIR2DL4, as represented in figure 24B and 25B, respectively. In conclusion, SMCs do not express ILT2, ILT4 and KIR2DL4 inhibitory receptors. A Beads + Beads - B ILT2 IgG Figure 25. Flow cytometric analysis of ILT2 expression in cultured smooth muscle cells. Negative and positive microbeads were used as controls (A). Cell surface expression of ILT2 was not detected in SMCs (B). 102

118 A Beads + Beads - B ILT4 IgG Figure 26. Flow cytometric analysis of ILT4 expression in cultured smooth muscle cells. Microbeads were used as the positive control (A). Cell surface expression of ILT4 was not detected in in SMC (B). A Beads + Beads - B KIR2DL4 IgG Figure 27. Flow cytometric analysis of KIRDL42 expression in cultured smooth muscle cells. Microbeads were used as the positive control (A). Cell surface expression of KIR2DL4 was not detected in in SMC (B). 103

119 4.5. HLA-G polymorphism and cardiac allograft vasculopathy The 14bp HLA-G polymorphism has been previously studied in the context of heart transplantation and the deletion/deletion genotype was found to be associated with increased HLA-G expression and fewer episodes of acute cellular rejection. We intended to determine if the 14bp polymorphism is also associated with CAV Clinical characteristics Table 1 illustrates the clinical characteristics of the patients who underwent heart transplantation and who were recruited for participation in the study [165]. Of the 81 study patients, 63 were male and 18 female, with a mean age of 46 ± 13 years at the time of transplant. The indication for transplant was idiopathic cardiomyopathy (43%), ischemic cardiomyopathy (31%) and other heart disease (21%). At 1 year posttransplant 60 patients underwent coronary angiography. 50 patients had no detectable angiographic lesions and were therefore included in Group 1 (CAV negative), and 10 patients were found to have CAV and were included in Group 2 (CAV positive). Out of the 10 patients with CAV, 2 patients had a normal angiographic test at one month post transplantation and 2 other patients donor hearts had normal coronary angiographies. Donor hearts of the other 6 patients did not undergo coronary angiography testing because the donors were young, with no cardiovascular conditions or risk factors. At 5 years post-transplant 56 patients had coronary angiography and 22 patients were found to have CAV. There were no significant differences in mean age, gender, indication for transplant, or immunosuppressive therapy between the two groups [161]. 104

120 Table 1. Clinical characteristics of heart transplant patients Baseline Characteristics All patients n Demographics Sex (male/female) 63/18 Mean Age 46±13 Reason For Transplantation Idiopathic Cardiomyopathy 39 Ischemic Cardiomyopathy 25 Other 17 Immunosuppressive Treatment Cyclosporine 48 Tacrolimus 18 Mycophenolate Mofetil 46 Sirolimus 14 Everolimus

121 4.5.2 HLA-G 14 bp insertion/deletion polymorphism The genotyping analysis of the 81 patients is shown in table 2. HLA-G genotype frequencies were in Hardy Weinberg equilibrium. Frequencies of the genotypes +14 bp/+14 bp, +14 bp/ 14 bp and 14 bp / 14 bp were 13.5%, 53% and 33.2%, respectively. Frequencies of the alleles +14 bp and 14 bp were 40.1% and 59.8%, respectively; the latter was the dominant allele in our 81 cardiac transplant patients. The 14 bp polymorphism frequencies of male and female subjects are shown in Table 2 [165]. Table 2. HLA-G 14 bp alleles and genotypes frequencies All patients No (%) Males No (%) Females No (%) Genotype +14 bp/+14 bp 11(13.5) 7(11.1) 4(26.6) +14 bp/ 14 bp 43(53) 38(60.3) 5(33.3) 14 bp/ 14 bp 27(33.3) 18(28.5) 9(60) Total(n) 81(100) 63(100) 18(100) Allele +14 bp 65(40.1) 52(41.2) 13(36.1) 14 bp 97(59.8) 74(58.7) 23(63.8) Total Hardy-Weinberg equilibrium Chi-Square p-value* *Probability is based on Chi-square test (Pearson test) 106

122 HLA-G 14-bp polymorphism in relation to cardiac allograft vasculopathy Patients were further divided into three groups, according to the HLA-G genotype (-14 bp/-14, +14 bp/-14 bp, and +14 bp/+14 bp), and analyzed in relation to the CAV profile (Table 3). There were no statistically significant differences in the presence of the three HLA-G genotypes between patients without CAV and patients with CAV, at 1 year post transplant (p=0.61). Similarly, no statistically significant differences were observed between patients without CAV and patients with CAV and the HLA-G genotypes (-14 bp/-14bp, +14 bp/-14 bp, +14 bp/+14 bp) at 5 years post-transplant (p=0.55) (Table4). Out of 81 patients included in the study 36 patients had angiography at both 1 and 5 years post-transplant. At 1 year 6 patients were found to have CAV and at 5 years post transplantation 8 patients had CAV. We found no correlation between HLA-G polymorphisms and CAV progression from baseline to 5 years post-transplant (p=0.76). With 60 patients at 1 year, the power calculated was 0.12 or 12% chance of detecting a significant difference in the two groups. With 56 patients at 5 years, the power calculated was 0.09 or 9% chance of detecting a difference [165]. 107

123 Table 3. HLA-G 14-bp polymorphism in relation to CAV at 1 year No CAV (n=50) CAV (n=10) HLA-G polymorphism +14 bp/+14 bp 6 (12%) 2 (20%) +14 bp/ 14 bp 27 (54%) 6 (60%) 14 bp/ 14 bp 17 (34%) 2 (20%) *Probability is based on Fisher s exact test (p=0.61) Table 4. HLA-G 14-BP Polymorphism in Relation to CAV at 5 years No CAV (n=34) CAV (n=22) HLA-G polymorphism +14 bp/+14 bp 4 (12%) 4 (18%) +14 bp/ 14 bp 18 (53%) 10 (46%) 14 bp/ 14 bp 12 (35%) 8 (36%) *Probability is based on Fisher s exact test (p=0.76) 108

124 Chapter 5 DISCUSSION, CONCLUSION AND FUTURE DIRECTIONS 109

125 The purpose of this thesis was to address important gaps in our understanding of the role of HLA-G in heart transplantation, in particular its effect in altering CAV development following transplantation. Our experiments reveal several novel functions of HLA-G in the context of heart transplantation, portraying HLA-G as an antiinflammatory and anti-proliferative agent Everolimus induces HLA-G expression in human coronary artery smooth muscle cells The factors which regulate HLA-G protein expression have been extensively studied and a few endogenous and exogenous elements have been identified. In lung transplantation, cytokines such as IFN-γ and TNF-α have been identified [153]. Progesterone has been found to up-regulate HLA-G in pregnancy, cancer, and heart transplantation [59]. Interleukins such as IL-10, IL-4, IL-5, IL-6, and growth factors like TGFβ are some of the HLA-G regulators in pregnancy [176]. Increased attention has been focused on the exogenous factors that influence HLA-G expression. In this regard, immunosuppressants have been shown to modulate the expression of soluble HLA-G. In kidney transplantation, therapy with betalacept, a co-stimulation blocker which inhibits T cell activation [177], was associated with increased HLA-G plasma levels [178]. Cyclosporine and tacrolimus were also correlated with high plasma HLA-G levels in heart transplant patients [179]. Prednisolone and cyclosporine up-regulated HLA-G post liver transplantation [180]. Previous investigations carried out by our laboratory compared shla-g expression in heart transplant patients treated with two different immunosuppressants, everolimus and MMF. It was found that patients treated with 110

126 everolimus had higher HLA-G plasma levels [150]. These results influenced our rationale for using everolimus in our experiments. Other immunosuppressive agents such as cyclosporine and tacrolimus could have been included in the study; however, they have been previously studied and found to be associated with HLA-G expression in plasma; everolimus in the context of HLA-G up-regulator has not been studied before. Our objective was to assess whether or not vascular smooth muscle cells were capable of expressing HLA-G in vitro as a result of exposure to everolimus. Our experiment illustrates for the first time in vitro HLA-G expression in SMC following treatment with this anti-proliferative, immunosuppressive agent. Previously, HLA-G had been found to be expressed in endothelial cells of chorionic fetal vessels during embryonic development; however, as the vessels matured, HLA-G was no longer expressed [181,182]. Investigations done in our laboratory revealed increasing HLA-G levels in human coronary artery endothelial cells, human aortic endothelial cells, and human coronary artery smooth muscle cells, following treatment with progesterone [183]. These findings were in accordance with other studies that found an up-regulation of HLA-G in JEG-3 choriocarcinoma cells and first trimester trophoblasts [172]. In vitro experiments with macrophage cell lines, as well as blood monocytes exposed to IFN-γ, have been shown to modulate mrna levels of HLA-G, causing an up-regulation of its expression inside the cells and on their surface [ ]. The influence of IFN-γ on HLA-G induction is expected as IFN-γ expression is intense during the first trimester of pregnancy and IFN-γ receptors are present throughout the pregnancy [187]. In another study, IL-10 was found to modulate HLA-G gene expression in trophoblasts and to up- 111

127 regulate HLA-G cell surface expression in human peripheral blood monocytes [188]. It was proposed by the same authors that IL-10 secretion by trophoblasts with subsequent HLA-G up-regulation functions as a mechanism which regulates the immune response, protecting the fetus from rejection. Although HLA-G was initially thought to be expressed solely by the trophoblasts cells of the placenta [2,3], further extensive research confirmed its ability to be induced in a series of cell types and pathologic conditions. Our finding that HLA-G is up-regulated in vascular cells by everolimus can be attributed various meanings. It can be speculated that HLA-G expression constitutes a mechanism of action of everolimus. Everolimus is known to be a powerful proliferation signal inhibitor exerting its effects on the SMCs of human coronary arteries, reducing intimal thickening following endothelial damage [46]. According to our findings everolimus is also able to induce SMCs to express HLA-G. One retrospective clinical study showed an association between HLA-G expression and freedom from CAV post heart transplantation [7]. Soluble HLA-G was detected in serum of transplant patients and all these HLA-G positive patients had no CAV up to 15 years post transplantation [7]. With these results in mind we can therefore speculate that everolimus and HLA-G have synergistic anti-proliferative and immune-suppressive roles within the coronary artery. Nonetheless, the clinical relevance and accuracy of these suppositions remain unclear and need to be studied in depth in order to reach a definite conclusion. However, induction of HLA-G expression in SMCs is of core importance as they are the main cells to migrate and proliferate, leading to the intimal thickening of CAV. 112

128 The relevance of our findings needs to be tested in vivo situations. According to Kovarik et al. [189], the recommended everolimus therapeutic range is 3 to 8 ng/ml in transplant patients. In our in vitro studies, the dose that had an inhibitory effect on SMCs was the highest one, of 1000ng/ml. Everolimus at 10 ng/ml, the closest concentration to that used in clinical practice, did not have a significant effect in SMCs. Therefore, according to our findings, everolimus at therapeutic doses would probably not be able to up-regulate HLA-G in vivo. The expression of HLA-G following solid organ transplantation is not fully elucidated. Some patients express HLA-G following heart transplantation, while others do not. Our finding that a drug is able to induce HLA-G expression post heart transplantation supports the previous hypothesis sustaining that the presence of HLA-G can be influenced by immunosuppressive drugs. Our study is in accordance with previous clinical findings in our laboratory, where patients treated with everolimus had higher shla-g levels in the plasma [150]. Whether or not HLA-G has true protective effects remains to be determined. In summary, previous studies have found that HLA-G can be up-regulated by cytokines [153], interleukins [176,188], hormones [59], growth factors [188] and immunosuppressive agents [168,177]. We have shown for the first time that everolimus can induce HLA-G expression in human coronary artery smooth muscle cells. Whether or not this expression represents a mechanism of action of everolimus needs to be determined in future studies exploring the anti-proliferative actions of HLA-G, if any. A 113

129 potential synergistic anti-proliferative effect of everolimus and HLA-G in SMCs, leading to decreased intimal proliferation and preventing coronary occlusion, would have a significant clinical impact in heart transplantation. Using exogenous HLA-G in combination with everolimus as therapy against CAV could allow a decrease in the dose of the latter, minimizing its use and its side effects, while achieving the same antiproliferative effects HLA-G inhibits human coronary artery smooth muscle cell proliferation in vitro Currently, cardiac allograft vasculopathy is the second leading cause of morbidity and mortality, after malignancy in heart transplant recipients [26]. Due to the progressive nature of CAV and the lack of efficient diagnostic methods, CAV can evolve undetected and can ultimately manifest as heart failure due to general ischemia as a result of coronary vessel obstruction and impaired perfusion [26]. Revascularization interventions such as angiography and bypass grafting proved to be beneficial for the short term but in the long term they have limited success due to a high incidence of restenosis [190]. The diffuse and aggressive nature of CAV, involving the entire coronary vasculature of the transplanted heart, makes revascularization difficult [26]. Drug therapies to alleviate CAV progression, such as immunosuppressants, need to be used early in order to be efficient, and even so, the increased risk of infection and malignancy associated with their use makes them unappealing for use as a strategy for CAV prevention [166]. So far, the only definitive treatment for CAV is re-transplantation, 114

130 which brings to discussion numerous other issues such as limited organ availability and lower survival rate compared to the first transplant [26]. In reality, only patients who fit a strict profile and eligibility criteria are considered good candidates for a second transplantation [26]. Providing efficient preventative therapies and treatments for CAV constitutes challenges both in research as well as in the medical field. Current research is focusing on the inhibition of cellular mechanisms of injury, inflammation, and smooth muscle cell proliferation, and on achieving graft tolerance. The vascular smooth muscle cells phenotypic changes and their migration and proliferation represent important components of the rapidly progressing vasculopathic process. This cytokine-induced SMC activation with subsequent migration, proliferation and production of cytokines and extracellular matrix, is the cause of arterial thickening and obstructive lesions seen in CAV [166]; therefore, therapeutic strategies aimed at inhibiting these processes are key elements to alter the development of CAV. We have previously found that everolimus, a powerful immunosuppressant and antiproliferative agent up-regulates HLA-G expression in SMCs. It has been demonstrated that HLA-G exerts immunosuppressive functions in the context of pregnancy and in pathological conditions. Based on clinical studies, in transplantation, HLA-G confers allograft immune protection in a variety of solid organ transplants such as heart, liver, lung, and kidney [180,191,192]. As everolimus and HLA-G seem to share similar properties, and because HLA-G is up-regulated in the presence of everolimus 115

131 potentially enhancing its immune suppressive effects, we were interested to determine if HLA-G exerts similar anti-proliferative properties. Our investigations were undertaken to determine the role of HLA-G as a SMC proliferation inhibitor. In our baseline study we showed that soluble HLA-G released by JEG-3 cells within the culture medium exerts no effect on SMC proliferation. Multiple variables might have played a role in our findings. The concentration of soluble HLA-G in each ml of medium was approximately 30ng/ml. This amount may be too low and may not be sufficient to alter the SMC functions; therefore it may not elicit the expected effect of inhibition of proliferation. Also, because the HLA-G protein was not isolated from other soluble factors released by the JEG-3 cells, its potential inhibitory effect might have been altered or masked due to possible interactions with other soluble molecular elements also released into the medium. Finally, the other soluble factors released into the culture medium may have far stronger effects on SMC than HLA-G, thereby diminishing the action of HLA-G. The use of HLA-G conditioned medium was the main limitation of this study, not only due to the presence of a variety of soluble factors that might have had an effect on SMC function, but also because of the alteration of the actual nutritive value of the medium. As the JEG-3 cells were exposed to the medium for 48h, it is reasonable to believe that some of the nutrients present in the fresh medium were lost in this process. This variable was well controlled in our study. The two groups were treated with a combination of conditioned medium (CMJ and CMS, respectively) and regular medium (R), to account for any loss of nutrients which might have caused an eventual decrease in proliferation. However, no inhibition 116

132 of proliferation was found between the cells treated with CMJ and those treated with CMS, suggesting that soluble HLA-G released by JEG-3 cells has no inhibitory effect on SMC proliferation. To effectively assess whether or not HLA-G has inhibitory effects on SMC, purified recombinant HLA-G was used as soon as it became available on the market. Our experiments are the first to demonstrate the ability of HLA-G to inhibit SMC proliferation. Interestingly, significantly higher inhibition of proliferation was detected following treatment with a high HLA-G concentration, compared to control and to the lowest doses of HLA-G, suggesting that high doses of HLA-G are needed to elicit an inhibitory effect. After 24 and 48h of treatment, minimal and relatively similar proliferation was seen across the groups. At 120h post treatment, the group treated with 1000ng/ml of HLA-G showed a significant inhibition of proliferation when compared to the control group and with the 100ng/ml group. Similarly, at the same time point, the group treated with 500ng/ml HLA-G showed a significant decrease in proliferation when compared to the control group. These findings suggest that a longer exposure of the SMC to HLA-G is needed in order to alter the proliferative ability of the cells. The high HLA-G concentrations which had an inhibitory effect might not have significance in clinical practice as in our heart transplant population the maximum concentration of HLA-G detected in plasma is 214ng/ml. However, in future studies, exogeously administered HLA-G might prove to be an efficient way of enhancing its expression and maximizing its beneficial inhibitory effects. 117

133 This study is the first to examine the inhibitory role of HLA-G in vascular smooth muscle cells. Previously, one other study demonstrated that HLA-G can exert powerful inhibitory functions in non-immune cells. Fons et al. studied the effect of soluble HLA- G1 on human umbilical vein endothelial cells proliferation and found that HLA-G is able inhibit their proliferation [91]. Interestingly, the mechanism through which this effect was obtained was apoptosis. HLA-G was shown to bind to the CD160 receptor on HUVEC and induce apoptotic effects. This function of HLA-G is believed to be highly important in the context of pregnancy, as it seems to facilitate the replacement of HUVEC with endovascular trophoblasts necessary to remodel the maternal blood vessels. This remodelling allows their expansion and adaptation to the high volumes of blood required during pregnancy. In the current study, although HLA-G has an inhibitory effect on SMC, it is not mediated through apoptosis. Cell viability was measured at each time point, with consistent high scores across time points and HLA-G concentrations. The main limitation of this study was the lack of sample blinding; however, the cell counting was accomplished with an automated cell counter, and the sample dilutions and preparation for counting were kept consistent at all times, with all samples. The mechanism through which HLA-G exerts its inhibitory effects remains to be determined. In other cell types HLA-G employs inhibitory roles by binding with the ILT2, ILT4, KIR2DL4 and CD160 inhibitory receptors; however, we weren t able to detect these molecules in SMCs. The fact that HLA-G expression in biopsies and serum and/or plasma of heart transplant patients is associated with reduced episodes of rejection and CAV [7,145,193] suggests that HLA-G might play a 118

134 role within the coronary artery. A similar inverse association between HLA-G expression and graft failure has been reported across other solid organ transplants. In kidney and/or liver transplantation, soluble HLA-G levels have been inversely correlated with acute rejection and organ failure [180,192,194]; however, no research has been conducted to determine the protective effect of HLA-G within the vasculature of these allografts. As previously mentioned, HLA-G exerts inhibitory effects on immune cells, more specifically NK cells and T cells. The acquisition of membrane-bound HLA-G from HLA- G + cells via trogocytosis is another way to block the proliferative ability of NK cells [113]. Moreover, HLA-G inhibits CD4 and CD8 T cell allo-proliferation. It has been shown that HLA-G acts as a proliferation signal inhibitor via its interaction with ILT2, ILT4, and KIR2DL4 inhibitory receptors expressed by the cells of both innate and adaptive immunity [195]. Although not previously studied in SMC, the expression of other inhibitory receptors might elucidate the mechanism of action of HLA-G in these cells. Future studies need to be conducted to determine how the proliferation of SMC is inhibited as a result of soluble HLA-G treatment. The precise cellular mechanisms of action remain to be determined. Nevertheless, our findings might have a favorable impact in the context of heart transplantation; more specifically, they can represent a starting point towards discovering new roles of HLA-G, roles which may eventually be used as strategies to prevent the development of allograft vasculopathy. A considerable area of research is currently focused on detecting efficient SMC anti-proliferative methods to slow the progression of CAV post-transplantation. Although various 119

135 relatively new and improved immunosuppressants such as everolimus and sirolimus act as SMC proliferation signal inhibitors, their adverse effects are still substantially detrimental. The use of the HLA-G protein, whether exogenously administered or endogenously stimulated, as treatment or prevention therapy, alone or in combination with other immunosuppressants, might be a more efficient approach, and might eventually allow a decrease in immunosuppressant doses, leading to fewer side effects and long-term complications associated with their use. One of the limitations of our study was the cellular environment. In the case of CAV, SMC proliferation takes place as a response to injury to the EC layer, which causes a release and activation of a series of molecular factors which alter the SMC behaviour, causing them to migrate and proliferate. In our study, the SMC growth was stimulated under normal conditions, using growth medium, therefore it can be called a natural proliferation not a pathological one. Another limitation of our study was the absence of a second experimental technique to confirm the present findings. However, the validity of this study can be argued, as consistent experimental methodology was maintained across all samples, at all-time points. The time-dependent increase in proliferation observed in all control groups was the expected result, suggesting the cells were behaving as anticipated, eliminating any doubts regarding their ability to grow. 120

136 In summary, our investigation illustrates a new role of HLA-G within the heart transplantation field. HLA-G as a SMC proliferation inhibitor offers new insights into the ability of this molecule to not only to protect against rejection, but also to potentially prevent CAV formation. While other investigations determined the inhibitory role of HLA- G in immune cell populations via inhibitory receptor activation, or in HUVEC via apoptotic mechanisms, we demonstrated that the anti-proliferative action of HLA-G is not restricted to immune cells and also, it is not achieved through apoptotic pathways as is the case with HUVEC. Our results demonstrate that HLA-G is a SMC proliferation inhibitor. Further studies need to be conducted to fully elucidate its mechanism of action, and its ability to exert the same inhibitory effects in pathologic conditions. Our findings might serve as a stepping stone towards new areas of transplantation research, where HLA-G can be further explored and eventually used as a prevention therapy against CAV The effect of HLA-G on neutrophil adhesion to human coronary artery endothelial cells injured by cytokines Allograft rejection is known to significantly increase the risk of mortality during the first year post transplantation. The persistent immunologic-mediated injury directed against the allograft not only harms the transplanted organ in the short term, but also represents a risk factor for the development of CAV [196]. It has been found that immunological events taking place during the early phase post-transplantation cause inflammatory reactions within the endothelial wall of the allograft vasculature [197]. Sustained inflammatory responses are also known to contribute to CAV pathophysiology [198]. 121

137 The correlation between organ rejection and cardiac allograft vasculopathy has been frequently addressed in the literature with numerous findings revealing a significant association between episodes of acute rejection and the development of CAV [197,199,200]. As a link between acute rejection and CAV, it has been proposed that inflammatory responses and vascular cellular damage, following episodes of rejection, cause injury to the endothelium, initiating CAV formation [198]. The interaction of neutrophils with the endothelial wall plays an important role during the inflammatory process [198]. With this in mind, we tested whether soluble HLA-G is able to exert an inhibitory role on adhering neutrophils. To create an injury to the endothelial cell layer we used TNF-α, one of the cytokines of allograft rejection, which is highly cytotoxic, and able to induce the expression of adhesion molecules which contribute to the adherence of circulating leukocytes to the endothelium [198]. We determined that exposure to TNF-α increases neutrophil adhesion to EC. Neutrophils are the first leukocytes to arrive at the site of injury and are thought to play a key role in contributing to further tissue damage and in maintaining the arteriopathy which develops with sustained episodes of rejection [174]. Our results revealed decreased neutrophil adhesion in response to shla-g with each of the different concentrations of TNF-α. The inhibition of neutrophil adhesion to the endothelium is of particular interest as this mechanism plays an important role in the initial phase of acute rejection, with subsequent repercussions on CAV formation. If a significant number of neutrophils are stopped from adhering to endothelial cells, then fewer are infiltrating into the vascular endothelial layer to cause further tissue damage 122

138 and inflammation. The exact mechanism through which HLA-G exerts this effect has not yet been determined. We can propose that prolonged EC exposure to shla-g confers protection from cytokine-induced injury, or inhibits EC activation post injury, thereby inhibiting the release of pro-inflammatory cytokines which perpetuate the inflammatory response. Also, in response to endothelial damage and inflammatory molecules, the activated vascular endothelial layer expresses on its surface a series of cell adhesion molecules such as ICAM-1, VCAM-1, and selectins [201,202]. All these molecules interact with surveying neutrophils and facilitate their rolling and their adhesion to the endothelium [202,203]. Another potential mechanism of action of HLA-G is the inhibition of the expression of adhesion molecules by endothelial cells. With less stimulation from factors which cause adhesion, fewer neutrophils adhere to the vessel wall and transmigrate into the tissue, leading to decreased overall detrimental effects. Another potential explanation for the effects seen is the fact that neutrophils are known to express the ILT4 inhibitory receptor [170]. The shla-g remnants, once EC culture medium is discarded, could bind to ILT4 receptor on neutrophils and exert inhibitory effects. The ILT4 receptor has been shown to have a much stronger preference for binding with HLA-G than any other MHC class I, when compared to the ILT2 receptor [88]. This finding suggests that neutrophils can indeed be the target of the inhibitory effects of HLA-G due to the presence of ILT4 on their surface. Although the role of HLA-G as an adhesion inhibitor has not yet been extensively studied, an investigation of Forte et al. revealed the ability of HLA-G to block human NK 123

139 rolling adhesion on porcine EC [204]. In this study, porcine EC were transfected with HLA-G. The expression of HLA-G on the cell surface inhibited the adhesion of NK cells to the endothelium. This finding is of particular interest since the rolling adhesion of NK represents the main mechanism of their recruitment to the site of injury where they adhere tightly and migrate through the endothelium causing cytotoxic effects [204]. A limitation of our neutrophil adhesion study was the absence of multiple HLA-G doses. The current concentration of 1000ng/ml was chosen based on the proliferation study, this concentration having a more dramatic effect on SMCs. Also, as TNF-α is a powerful cytokine with strong cytolytic effects even at low doses, we felt that a smaller concentration of HLA-G would not be able to overcome the effects of the TNF-α. Even at 1000ng/ml, HLA-G was only able to partially inhibit the adhesion of neutrophils. Based on our findings HLA-G is able to diminish the TNF-α-induced inflammatory response directed against the vessel wall by preventing the neutrophils from infiltrating through the endothelium. This role of HLA-G indicates its ability to influence innate immune responses thereby potentially exerting protective effects towards the allograft endothelium. By modulating these cellular events which take place within the vasculature of the transplanted organ as a result of immunologic responses during episodes of rejection, HLA-G may contribute to preventing CAV formation. Further investigations are required to determine the precise role of HLA-G in modulating inflammatory responses and altering the development of CAV. 124

140 5.4. Expression of HLA-G receptors within the human coronary artery smooth muscle cells and human coronary artery endothelial cells According to our studies, HLA-G plays a role in inhibiting proliferation of SMCs and neutrophil adhesion to ECs. It has been reported previously that HLA-G exerts its effects on immune cells via three inhibitory receptors: ILT2, ILT4 and KIR2DL4 [88,171]. On HUVEC, receptor CD160 is bound by HLA-G triggering an inhibition of their proliferation by an apoptotic pathway [169]. We sought to determine if the receptors, ILT2, ILT4, KIR2DL4, and CD160 are also present on SMCs and ECs. To-date these are the only receptors found to bind HLA-G and enable its inhibitory functions. Previously the ILT2, ILT4 and KIR2DL4 receptors have been exclusively found on immune cells. As HLA-G functions primarily to inhibit immune responses, these receptors have been of primary focus in the research field so far. The new roles of HLA- G, such as anti-proliferative and anti-inflammatory, might be facilitated by other receptors. We performed cellular experiments to determine whether or not ILT2, ILT4, KIR2DL4, and CD160 can be detected in SMCs and ECs. We analyzed the expression of ILT2, ILT4, KIR2DL4, and CD160 in SMCs via Flow cytometry. Our results showed no membrane bound receptor expression on these cells. Positive and negative beads, able confirm the ability of the antibodies to detect the proteins of interest, were used as controls. Moreover, the antibodies chosen for this study have been previously tested for flow cytometry assays and confirmed to detect the specific protein they are designed 125

141 for. In these studies the cells used were not subjected to any treatments. Our objective was to determine if the receptors are expressed at baseline, in the normal state. One limitation of this study was the use of beads as positive controls, instead of a biological control. However, the beads are designed to bind to the antibody of interest and confirm its ability to detect a specific protein. Another limitation was the lack of initial studies to confirm the type of cells used in the experiments. However, the SMCs used were bought as primary lines, and they were previously grown and studied under the microscope and it was confirmed by their appearance that they were indeed SMCs. A second method used to detect the expression of ILT2, ILT4, and KIR2DL4 in SMCs was via Western Immunoblotting. Besides these three receptors, we also looked at CD160 expression. We also tested the expression of all four receptors in ECs. For all experiments we used the appropriate purified human recombinant protein as a positive control for each receptor of interest. SMCs and ECs were stimulated with TNFα for 4 and 24 hours. Untreated cells were used as positive control. According to our results, both SMCs and ECs stained negative for all receptors. In these experiments we used not only various detection techniques, but also various categories of cells. Both SMCs and ECs used were healthy cells from purchased, primary cell lines. Our studies demonstrated that SMCs and ECs do not express ILT2, ILT4 KIR2DL4, and CD160 receptors at baseline, in the non-diseased state. We have also shown that SMCs and ECs do not express any of the receptors when stimulated with TNFα for 4 and 24 hours. Future studies need to be conducted to elucidate whether or not SMCs 126

142 and ECs express other inhibitory receptors on their surface, other than those investigated in our studies HLA-G polymorphism and cardiac allograft vasculopathy The importance of HLA-G polymorphisms is relevant to many areas in transplantation. In kidney transplantation, Crispim et al. found no association between the three genotypes (-14 bp/-14bp, +14 bp/-14 bp, +14 bp/+14 bp) and either transplant patients or healthy controls [152]. However a positive association between the homozygous 14- bp insertion genotype and acute rejection was reported. This is in accordance with previous results reporting decreased HLA-G production in this genotype [77], therefore potentially increasing the risk of rejection. In bone marrow transplantation the 14-bp deletion genotype was associated with increased risk of developing severe graft versus host disease (GvHD), while the homozygous 14-bp insertion and the heterozygous genotypes predicted low risk of GvHD [162]. In heart transplantation, Torres et al. reported a strong association between the homozygous 14-bp deletion genotype and the bioavailability of cyclosporine [163]. The same -14/-14-bp genotype was also linked with increased soluble HLA-G levels. Our research group has recently investigated the genetic component influencing HLA-G expression in heart transplant patients and found a positive association between the 14 bp/ 14 bp genotype and shla-g expression [164]. We have also found a significant correlation between the -14bp/-14bp genotype and acute cellular rejection in a population of heart transplant recipients. 127

143 Considering these previous findings highlighting the importance of HLA-G polymorphism in identifying patients at risk for acute rejection, we investigated whether the HLA-G 14-bp polymorphism was associated with the development of CAV [161]. In a previous study, Lila et al reported that HLA-G expression is not only associated with fewer episodes of acute organ rejection in the first year post transplant, but also with the absence of graft vasculopathy, as assessed by coronary angiography [7]. However, this was a retrospective study, with a small number of subjects. To the best of our knowledge this is the first study to evaluate a potential association between HLA-G polymorphism and cardiac allograft vasculopathy. We observed no association between the +14 bp/+14 bp, +14 bp/ 14 bp and 14 bp / 14 bp and the presence of CAV at one and five years post heart transplant. Also, the HLA-G polymorphism was not correlated with the progression of CAV from baseline to 5 years following transplant [161]. One of the limitations of this study was the sample size of 81 patients. Out of these, only 60 patients were angiographycally tested at 1 year and 56 at 5 years, with a calculated power of 0.12 and 0.09, respectively. For a power of 0.8 we would need to enrol 586 patients in the study. Furthermore, only 36 of patients had coronary angiography testing at both time points of 1 year and 5 years, making the CAV progression analysis from baseline (1 year) to 5 years even more under powered. 128

144 Another limitation of our study is the use of coronary angiography as a diagnosis tool for CAV. A more sensitive alternative detection tool is the use of intravascular ultrasound (IVUS) which can increase the accuracy of CAV diagnosis; however, the high cost of this technique makes it less accessible for use. Extraneous factors, such as other polymorphisms in the HLA-G gene or in other genes, may also play a role in our findings. Other factors, including immunosuppressive therapy and co-morbidities such as metabolic syndrome (markers of dyslipidemia, hypertension and diabetes) may play a role in the development of CAV, accelerating its progression in such ways that overcome the potential inhibitory effects of HLA-G [161]. In our study we did not find an association between the various 14bp genotypes and CAV or CAV progression from 1 to 5 years post transplantation, however, the study was heavily underpowered. Larger studies are needed to fully elucidate if the 14bp polymorphism can predict CAV [165] Summary and conclusion The present work analyzed the role of HLA-G in the context of transplant vasculopathy with four in vitro studies which determined the up-regulation of HLA-G by everolimus, the anti-proliferative role of HLA-G in the SMC, the inhibitory function of HLA-G in neutrophil adhesion to EC, and absence of HLA-G receptors within the cells of coronary arteries, and one clinical study which determined that the genetic component of HLA-G, the 14bp polymorphism, is not associated with CAV. 129

145 The exact pattern of HLA-G expression in patients undergoing transplantation is not fully elucidated. As immunosuppressants administered during the post transplantation period were previously shown to be associated with increased HLA-G expression, we examined whether or not everolimus induces HLA-G in SMCs. Those cells treated with the highest concentration of everolimus revealed a significant up-regulation of HLA-G. Our results are consistent with clinical studies which revealed associations between everolimus treatment and the presence of HLA-G in serum or plasma of transplant patients. These observations potentially elucidate another mechanism of action of everolimus, and, based on previous findings, open the possibility for a synergistic effect of everolimus and HLA-G, as both seem to share anti-proliferative properties. The inhibitory effect of shla-g on the proliferation of SMCs of the coronary arteries has particular relevance within the CAV milieu. The activation, phenotypic change, and migration of SMCs culminate with their pathologic proliferation, leading to intimal thickening and subsequent arterial narrowing, eventually resulting in occlusion. After five days of treatment with HLA-G, SMC proliferation it was significantly inhibited. Our study is the first to attribute an anti-proliferative role of HLA-G in human coronary artery smooth muscle cells. This finding is of particular relevance as a considerable area of research is currently aimed at determining ways to modulate SMC proliferation with the ultimate goal of preventing or treating CAV development. So far the reduction in SMC proliferation is achieved solely through pharmacologic means which come with 130

146 undesirable adverse effects. Further exploration of the anti-proliferative functions of HLA-G is required, potentially leading to effective treatments for CAV prevention. Organ rejection represents a risk factor for CAV development [25]. Inflammatory processes initiated during immunologic insults promote further tissue damage and the development of CAV. Within minutes of vessel injury, neutrophils invade the allograft vasculature. Their interaction with adhesion molecules expressed by EC, causes them to adhere tightly to the endothelium where they transmigrate, causing more inflammation. We tested whether treatment with shla-g confers protection to the endothelium by inhibiting neutrophil adhesion post cytokine-induced injury. The exposure of EC to shla-g significantly inhibited the adhesion of neutrophils to endothelial cells following all degrees of injuries caused by the various concentrations of TNF-α. We demonstrated for the first time the anti-adhesive role of HLA-G in neutrophils. The reduction of the inflammatory response at the time of immunologic injury decreases the overall effect of acute rejection, as well as the risk of CAV development. In previous studies it has been demonstrated that HLA-G exerts its immunosuppressive and anti-proliferative roles via four inhibitory receptors: ILT2, ILT4, KIR2DL4 and CD 160, respectively. Given this knowledge, we sought to determine if these receptors are expressed by the SMCs and ECs. Using various experimental techniques we found that ILT2, ILT4, KIR2DL4 and CD 160 are not expressed on the surface of SMCs and ECs. This finding suggests that HLA-G might exert effects on these cells via other receptors 131

147 or cellular processes. However, other studies must be done to confirm these suppositions. Previous studies determined that HLA-G expression and implicitly its effect is influenced by both genetic and non-genetic factors. Given the importance of HLA-G within the context of heart transplantation, we sought to determine any associations between the genetic component of HLA-G and CAV development in a population of heart transplant recipients. We did not identify any associations between the 14bp polymorphism and CAV at 1 and 5 years follow-up or CAV progression within this time frame. HLA-G polymorphism appears to play an important role as a genetic indicator for cellular rejection post-transplant; however, further research with larger sample size is recommended to fully elucidate if the HLA-G polymorphism might predict the development of CAV post-transplant Future perspectives Transplantation represents the ultimate option for life prolongation for patients with advanced heart disease. Innovative discoveries in both clinical and basic science research have allowed for wider eligibility criteria for heart transplantation, with the inclusion of high risk patients who are being provided a highly specialized follow-up care after the surgery. More and more efficient techniques of donor organ preservation are becoming available, allowing the use of less than optimal hearts for transplantation with highly successful outcomes. Despite these positive aspects of current practices in transplantation, the reality of short- and long-term complications limits their overall success. Current research is highly focused on finding ways to promote immunologic 132

148 tolerance both immediately after transplantation, as well as in the long term. HLA-G expression has been negatively associated with acute rejection, as well as with chronic rejection in the form of CAV [7,145,146,205]. Based on these findings it is reasonable to consider HLA-G a promising molecule with potentially useful immunosuppressive properties which could eventually be used as a tool for preventing organ rejection and CAV, or even as a marker for identifying patients at risk for developing these complications post heart transplantation. We have shown that everolimus induces HLA-G expression in SMCs. Everolimus is known to be a proliferation signal inhibitor. We showed that HLA-G also alters the proliferation of SMCs. Used in combination they might elicit synergistic effects in SMC cultures. Subsequent studies need to explore this hypothesis and examine in depth the cellular processes involved. These findings would prove particularly important as they may potentially allow for a therapeutical use of HLA-G, eventually minimizing the concentration of everolimus required, therefore greatly reducing the adverse effects associated with its use. Our in vitro investigations revealed the ability of HLA-G to exert anti-proliferative effects on SMCs. More in depth studies need to be conducted to fully elucidate the role of HLA- G as an anti-proliferative agent. As in our study the SMC proliferation effect was seen under normal conditions, future studies may determine if HLA-G has similar effects when proliferation is achieved under pathologic conditions. This can be done in vivo, using animal studies, and using different types of immunologic injuries. Higher 133

149 concentrations of HLA-G might be used in future experiments to determine if there is a further decrease in proliferation. In order to confirm the direct effect of HLA-G on SMC proliferation, an HLA-G sirna could be used to block HLA-G and reverse the antiproliferative effects. Furthermore, it is particularly relevant to elucidate the mechanism through which HLA-G blocks proliferation. Numerous studies have confirmed that in immune cells HLA-G inhibits alloproliferation by binding with ILT2, ILT4, and KIR2DL4 receptors expressed by the cells of innate or adaptive immunity. To date our studies are the only ones to determine whether or not SMCs and ECs express these inhibitory receptors at baseline or when stimulated with TNFα. It would be interesting to see if the receptors can be detected under other conditions, such as SMCs and ECs treatment with other stressors such as hypoxia/reoxygenation or immunosuppressants. For a potential synergistic, anti-proliferative effect on SMCs, immunosuppresants such as everolimus can be used in combination with HLA-G. Within the context of inflammation, HLA-G seems to protect the ECs from neutrophil adhesion to their surface following cytokine-induced injury. It remains to be determined whether or not this effect is achieved as a result of HLA-G interaction with ILT4 inhibitory receptor expressed on neutrophils. An ILT4 blocking antibody or an ILT4 sirna used to reverse the anti-adhesive effects of HLA-G would potentially elucidate the involvement of ILT4 as a mediator of these effects. Another mechanism warranting further exploration is the possible interaction of neutrophils with adhesion molecules translocated to the membrane of ECs upon their activation. In this case HLA-G could either block their activation/function, or inhibit their actual expression by ECs. Similar 134

150 neutrophil adhesion experiments might be carried out using other methods of injury relevant in the context of heart transplant. IFN-γ is a known cytokine implicated in organ rejection, which has strong cytolytic properties [198]. The ischemia-reperfusion insult is highly relevant in heart transplantation [198]. In vitro hypoxia-reoxygenation studies simulating this condition, with a wider range of exposure times than those used in our experiments, would reveal important data regarding the protective properties of HLA-G in this context. Injury from immunosuppressive drugs has been documented in the literature as being a cause for inflammation, as well as an important risk factor for the development of CAV [198]. Evaluating the ability of HLA-G to confer protection on ECs exposed to these stressors, would provide useful and innovative data, with implications for future anti-rejection therapies. Looking further into the tolerance induction, co-culture experiments might be done in vitro to assess whether HLA-G treated ECs are protected from T cell and NK cell immune responses. Also, future in vivo studies need to determine if HLA-G has the same inhibitory role in the actual post-transplantation situation and if the degree of inhibition is significant enough to delay or ameliorate CAV development. The expression of ILT2, KIR2DL4, and CD160 receptors in SMCs and ECs was not detected in our studies. This could be due to the fact that cell surface proteins are often lost due to passaging. A future use of cells during early passages might be helpful in assessing if this is the reason for the results seen. Another way of improving the accuracy of results might be the isolation of SMCs and ECs from fresh coronary artery 135

151 tissue procured from heart transplant patients during routine biopsies followed by cell culture and then used to determine the expression of receptors. In order to adequately assess if there are any association between the 14bp polymorphism and CAV, a larger number of subjects should be recruited and enrolled in the study, with coronary angiography testing at both time points. For an even more accurate diagnosis, IVUS should be used as diagnosing method. Detecting other HLA- G polymorphisms and exploring their potential association with CAV can potentially reveal new roles of HLA-G in the field of genetics. Prior to considering HLA-G for therapeutic use in heart transplant patients, a number of issues need to be resolved. These include whether to use an exogenously administrated recombinant protein, or endogenously induced HLA-G in target cells. Moreover, its adverse effects need to be extensively examined. Considering the role of HLA-G in cancer, namely facilitating tumor escape from the immune surveillance [137,138], and taking into account the risk of malignancies which is the leading longterm complication following heart transplantation [206], careful analysis of these issues must be pursued. To date, HLA-G has not been shown to play a role in oncologic transformations. Also, no HLA-G expression has been associated with any onset of new malignancy. Moreover, a high risk of post-transplantation malignancies is associated with the chronic use of immunosuppressants, not with HLA-G expression. 136

152 As demonstrated by our findings, as well as by other investigations within the literature, HLA-G exerts numerous functions, ranging from immune suppression, to antiproliferative and anti-adhesive properties within the vascular wall. These roles make HLA-G highly relevant in the context of CAV prevention. The negative associations between HLA-G and short- and long-term complications support the importance of HLA- G in heart transplantation, and its potential use as prevention therapy or as a marker to identify patients at risk for developing allograft vasculopathy. 137

153 CHAPTER 6 References 138

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171 Chapter 7 APPENDICES 156

172 157

173 158

174 159