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Investigating the mechanisms of bronchiolitis obliterans in a murine tracheal transplant model Cheah, Stefanie 2009

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INVESTIGATING THE MECHANISMS OF BRONCHIOLITIS OBLIERANS IN A MURINE TRACHEAL TRANSPLANT MODEL  by   Stefanie Cheah  B.Sc., McGill University, 2005     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF   MASTER OF SCIENCE  in  The Faculty of Graduate Studies  (Experimental Medicine)                THE UNIVERISTY OF BRITISH COLUMBIA (Vancouver)  July 2009  © Stefanie Cheah, 2009  ii ABSTRACT Post-lung transplant bronchiolitis obliterans (BO) is the leading cause of morbidity and mortality in lung transplant patients and is the major limitation to long-term transplant survival. BO is believed to be primarily the result of repetitive immune-mediated injury to the airway epithelium resulting in an aberrant repair phase of fibro-proliferation that occludes the bronchiole lumen. The first aim of this thesis was to investigate the use of gene therapy to prevent airway epithelial cell (AEC) apoptosis and the development of obliterative airway disease (OAD) using the murine heterotopic tracheal transplant model of BO. Using lentivirus-mediated transduction of the anti-apoptotic protein, Bcl-2, an improvement in epithelial integrity was observed in 14 day tracheal allografts.  However, in 28 day allografts, the airway epithelium was lost and the lumens of the tracheae were occluded with fibrotic material in all treatment conditions. These results were caused by difficulties in achieving efficient transduction of the airway epithelium and highlighted the need for futures studies using new mechanisms of gene transfer. Secondly, the role of latent-TGF-β1 activation in the fibrotic processes of OAD was also examined. A synthetic peptide of the thrombospondin-1 (TSP-1) receptor, CD36, was used to block activation of latent-TGF-β1 and inhibit OAD. In a comparison of transplanted tracheae treated with the peptide or PBS alone, allografts that received the peptide demonstrated a lower mean luminal occlusion score than the PBS control allografts. In summary, results from this thesis demonstrated the complexity of gene transfer to the airway epithelium and the need for further exploration in order to achieve ideal transgene expression of anti- apoptotic proteins to prevent the loss of the AECs and the development of OAD. In addition, the preliminary findings on the role of latent-TGF-β1 activation in the fibrotic  iii processes of OAD provide an interesting area of study for future research and a potential therapeutic target for the inhibition of BO after lung transplantation.                       iv TABLE OF CONTENTS ABSTRACT................................................................................................................... ii TABLE OF CONTENTS ............................................................................................. iv  LIST OF TABLES ....................................................................................................... vi  LIST OF FIGURES ....................................................................................................vii  LIST OF ABBREVIATIONS ....................................................................................viii  ACKNOWLEDGEMENTS .......................................................................................... x  DEDICATION.............................................................................................................. xi  CO-AUTHORSHIP STATEMENT............................................................................xii  1. INTRODUCTION1 1.1. Post-lung transplant bronchiolitis obliterans ............................................................. 1 1.2. Murine heterotopic tracheal transplant model ........................................................... 9 1.3. The airway epithelium............................................................................................ 12 1.4. Apoptosis ............................................................................................................... 16 1.5. Transforming growth factor (TGF)-β...................................................................... 23 1.6. Gene therapy in transplantation .............................................................................. 35 1.7. Thesis hypotheses................................................................................................... 37 1.8. Thesis objectives .................................................................................................... 38  2. MATERIALS AND METHODS39 2.1. Mice....................................................................................................................... 39 2.2. Cells....................................................................................................................... 39 2.3. CD36 synthetic peptide .......................................................................................... 39 2.4. Adenoviral and lentivirus vectors ........................................................................... 40 2.5. Lentivirus production ............................................................................................. 41 2.6. Lentiviral and adenoviral transduction of cell lines ................................................. 42 2.7. In vitro induction of apoptosis ................................................................................ 42 2.8. Western blotting ..................................................................................................... 43 2.9. Immunofluorescence .............................................................................................. 44 2.10. Tracheal excision and treatment conditions........................................................... 44 2.11. Murine heterotopic tracheal transplantation .......................................................... 46 2.12. Histological evaluation ......................................................................................... 47 2.13. Immunohistochemistry ......................................................................................... 49 2.14. Statistical analysis ................................................................................................ 50  3. RESULTS ................................................................................................................ 51 3.1. In vitro lentivirus-mediated transduction of HAE cells ........................................... 51  v 3.2. Inhibition of apoptosis by lentivirus-mediated gene transfer of Bcl-2 in HAE cells...................................................................................................................... 53 3.3. Assessment of gene transfer after ex vivo lentivirus-mediated transduction............. 55 3.4. Assessment of lentivirus mediated gene transfer in 3 day isografts ......................... 58 3.5. Development of OAD after heterotopic tracheal transplantation ............................. 60 3.6. Effect of lentivirus-mediated gene transfer of Bcl-2 on 14 day allografts ................ 62 3.7. Inflammatory infiltration remains uniform between transduced and untransduced 14 day allografts............................................................................................................ 66 3.8. Effect of lentivirus-mediated gene transfer Bcl-2 on 28 day allografts .................... 68 3.9. In vitro adenovirus-mediated transduction of HeLa cells......................................... 71 3.10. Assessment of gene transfer after ex vivo adenovirus-mediated transduction................................................................................................................... 73 3.11. Assessment of adenovirus-mediated gene transfer in 3 day isografts..................... 75 3.12. Effect of adenovirus-mediated gene transfer of XIAP on 28 day allografts ........... 77 3.13. Potential inhibition of luminal occlusion and fibrosis with a CD36 synthetic peptide .......................................................................................................................... 80  4. DISCUSSION .......................................................................................................... 84  5. REFERENCES........................................................................................................ 96                            vi LIST OF TABLES  Table 1.1. Classification of bronchiolitis obliterans syndrome ......................................... 3  Table 2.1. Experimental transplant groups..................................................................... 47                                           vii LIST OF FIGURES  Figure 1.1. The hypothesized pathogenesis of BO ........................................................... 8  Figure 1.2. The Bcl-2 family of proteins........................................................................ 20  Figure 1.3. The intrinsic and extrinsic apoptosis pathways............................................. 22  Figure 1.4. Activation of latent-TGF-β1 by thrombospondin-1 and inhibition by a CD36 synthetic peptide ................................................................................................. 26  Figure 1.5. The TGF-β1–Smad signaling pathway......................................................... 28  Figure 3.1. Detection of Bcl-2 in transduced HAE cells................................................. 52  Figure 3.2. Inhibtion of apoptosis activity in Bcl-2 transduced cells .............................. 54  Figure 3.3. Histological and immunohistochemical analysis of ex vivo lentivirus transduced tracheae ....................................................................................................... 58  Figure 3.4. Histological and immunohistochemical analysis of lentivirus transduced 3 day isografts............................................................................................................... 59  Figure 3.5. Histological analysis of isografts and allografts in the HTT model............... 61  Figure 3.6. Tracheal histology of 14 day allografts and wild type tracheae .................... 64  Figure 3.7. Epithelial integrity of 14 day allografts and wild type tracheae .................... 65  Figure 3.8. Immunohistochemical staining for inflammatory infiltrate in 14 day allografts ....................................................................................................................... 67  Figure 3.9. Tracheal histology of lentivirus transduced 28 day allografts ....................... 69  Figure 3.10. Analysis of adenovirus-mediated transduction of HeLa cells ..................... 72  Figure 3.11. Histological and immunohistochemical analysis of ex vivo adenovirus transduction of tracheae................................................................................................. 74  Figure 3.12. Histological and immunohistochemical analysis of adenovirus transduced 3 day isografts ............................................................................................. 76  Figure 3.13. Histological analysis of adenovirus transduced 28 day allografts ............... 78  Figure 3.14. Histological analysis of CD36 synthetic peptide treated 28 day allografts .. 82   viii LIST OF ABBREVIATIONS  Ad adenovirus AEC airway epithelial cell Apaf-1 apoptotic protease activating factor-1 BAL bronchialalveolar lavage Bcl-2 B-cell lymphoma-2 BEC         bronchial epithelial cell BH Bcl-2 homology BIR baculoviral IAP repeat BO  bronchiolitis obliterans BOS  bronchiolitis obliterans syndrome CAD  caspase-activated DNase CMV   cytomegalovirus CTE  constitutive transport element CTGF  connective tissue growth factor DED  death effector domain DISC  death-inducing signaling complex ECM  extracellular matrix EGF  epidermal growth factor EMT  epithelial mesenchymal transition ERK  extracellular signal-regulated kinase FADD  Fas-associating protein with death domain FasL  Fas ligand FEF25-75 mid-expiratory flow rate FEV1  forced expiratory volume in one second FGF  fibroblast growth factor Foxp3  forkhead/winged helix transcription factor 3 GERD  gastroesophageal reflux disease GFP  green fluorescent protein HLA  human leukocyte antigen  ix HTT  heterotopic tracheal transplant IAP  inhibitor of apoptosis IFN  interferon IGF  insulin growth factor IL  interleukin ISHLT  International Society for Heart and Lung Transplantation JNK  cJun NH2 terminal kinase LAP  latency-associated peptide LLC  large latent complex LV  lentivirus MAPK  mitogen-activated protein kinase MCP-1 monocyte chemoattractant protein-1 MHC  major histocompatibility complex MMP  matrix metalloproteinase OAD  obliterative airway disease PARP  poly (ADP-ribose) polymerase PDGF   platelet derived growth factor PGD  primary graft dysfunction PGK  phosphoglycerate kinase SARA  Smad anchor for receptor activation Smurf  Smad ubiquitin regulatory factor STS  staurosporine TBB  transbronchial biopsy TGF-β  transforming growth factor-beta TGF-βR TGF-β Receptor TIMP  tissue inhibitor of matrix metalloproteinases TNFR  tumor necrosis factor receptor Treg  T regulatory cell TSP-1  thrombospondin-1 VSV-G vesicular stomatitis virus G glycoprotein XIAP  X-linked inhibitor of apoptosis  x ACKNOWLEDGEMENTS First and foremost, I would like to thank my group of co-supervisors, Dr. Vince Duronio, Dr. Megan Levings and Dr. Nasreen Khalil. I am grateful to have had the chance to work under the guidance of such great people with diverse research interests. I thank Dr. Darryl Knight for being a part of my supervisory committee and providing guidance, as well as reagents and protocols for my thesis project. I would like to thank all the members of the Duronio, Levings, and Khalil lab, in particular Yan Liu, as well as the entire 4th floor of the Jack Bell Research Centre, for not only the technical support but more importantly, for the friendships and memories that we have made and that I will cherish for many years to come. Over the last three years, I feel that I have obtained a unique graduate experience that I will be able to carry with me in my future endeavors.              xi DEDICATION To my parents, Who have provided endless support no matter how long, dull or incomprehensible they have found my Master's thesis.  To Steve, my loving fiancé, Who can still make me smile and laugh on days when it seems like every experiment has gone wrong.                     xii CO-AUTHORSHIP STATEMENT The work described in this thesis was designed and performed by myself along with the guidance of my supervisors except for the following.  Leslie Sanderson contributed to cloning the modified lentivirus vector (LV-GFP-NGFR and LV-GFP-Bcl-2) that was used in the transplantation experiments. Our animal technician, Yan Liu, performed all the technical work and surgeries involving the mice. Mario Amendola constructed the lentivirus vector backbone with bidirectional promoter. Rupinder Dhesi performed the lentivirus production.  1 1. INTRODUCTION 1.1 Post-lung transplantation bronchiolitis obliterans Lung transplantation is the only accepted treatment option for a variety of end-stage pulmonary disorders. Currently, early post-operative survival rates are at their highest with 88% of patients surviving three months and 78% after one year of transplantation (1). The early success of lung transplantation is largely due to improvements in immunosuppression regimen, advancements in surgical technology, and adherence to appropriate patient selection (2). Unfortunately, long-term survival of lung transplant patients are disappointingly low and are the worst among other solid organ transplants (3). While ten-year survival rates for kidney, liver and heart transplants are 77%, 73.4% and 54.1% respectively; only 29.3% of lung transplant patients survive past ten years. Limitations to long-term survival may be due to many factors including graft versus host disease, acute rejection, side effects and complications to immunosuppression and secondary malignancy. However, the predominant cause of lung transplant failure is chronic allograft dysfunction, manifesting as bronchiolitis obliterans (BO) (1).  BO, or obliterative bronchiolitis, has been referred to as the "Achilles heel" of lung transplantation (4) as it is the leading cause of morbidity and mortality in patients that survive the first year post transplant (1). It is described as chronic rejection manifesting as dense sub-mucosal fibrosis in the respiratory bronchioles resulting in partial or total luminal occlusion (5). Burke and colleagues at Stanford University first reported the histological features of post-transplant BO in 1984; yet the disease pathogenesis still remains poorly understood (6). The distribution of histological abnormalities is  2 heterogeneous, making the diagnosis by random sampling by transbronchial biopsy (TBB) difficult (7). For this reason, the International Society for Heart and Lung Transplantation (ISHLT) sponsored a committee in which a clinical description of BO termed, bronchiolitis obliterans syndrome (BOS) was developed. Diagnosis of BOS does not require histological confirmation, instead, physiological changes in expiratory flow rates measured by the forced expiratory volume in 1 second (FEV1) and mid-expiratory flow rates (FEF25-75) are used (7 , 8).  As of 2002, BOS is classified into five categories as shown in Table 1.1. BOS 0-p represents potential BOS, created in order to alert the physician to early detection by in- depth patient assessment and monitoring of markers of BOS (8). The first stage of BOS (BOS 1) is defined as an asymptomatic 66-80% decrease in FEV1 and/or a FEF25-75 of less than or equal to 75% of baseline measurements (9). Prevalence of BO/BOS is rare within the first year after transplant, however cumulative data of over 10,000 transplants from the ISHLT Registry demonstrated a prevalence of 27% at 2.5 years and 51% at 5.6 years post-transplant (1). The rate of progression is highly variable and can range from a sudden severe drop in lung function to a slow and gradual decline in FEV1 over several years (10, 11). For simplification, in this thesis, "BO" will be used to describe the fibrotic disease following lung transplantation despite the method of diagnosis.      3 Stage 1993 Classification 2002 Classification BOS 0 FEV1 > 80% baseline value FEV1 > 90% of baseline and   FEV25-75 > 75% of baseline BOS 0-p N/A FEV1 81-90% of baseline and/or   FEV25-75 ≤ 75% of baseline BOS 1 FEV1 66-80% of baseline FEV1 66-80% of baseline BOS 2 FEV1 51-65% of baseline FEV1 51-65% of baseline BOS 3 FEV1 ≤ 50%of baseline FEV1 ≤ 50%of baseline BOS, bronchiolitis obliterans syndrome, FEV1 forced expiratory volume in 1 second, FEV25-75 midexpiratory flow rate, BOS 0-p, potential BOS  Table 1.1. Classification of bronchiolitis obliterans syndrome. A comparison of the 1993 and 2002 classification systems. BOS 0-p has been added in the 2003 classification.  Risk Factors Risk factors for BO can be classified into two categories: alloimmune-dependent and - independent factors. Alloimmune-dependent factors include most dominantly 1) acute rejection 2) lymphocytic bronchitis/bronchiolitis 3) human leukocyte antigen (HLA) mismatches and 4) existence of pre-transplant anti-HLA antibodies (12). Several lines of evidence have shown that severity of acute rejection, characterized by perivascular and interstitial mononuclear cell infiltrates (5), is directly correlated with an increased risk of BO (13-15). However, the relationship between BO and acute rejection appears complex, as many patients with acute rejection never develop BO and some patients with BO never experience acute rejection (16, 17). Another alloimmune risk factor for BO is lymphocytic bronchitis/bronchiolitis, a lesion of CD4+ and CD8+ lymphocytes and macrophages in the subepithelial region of the airway, which is believed to be a predictive precursor to BO (18). In recent studies, donor and recipient mismatched HLA antigens have been confirmed to play an important role in the pathogenesis of BO. In a  4 study reviewing over 182 lung transplants, a significant improvement in graft survival was seen in patients with zero or one HLA mismatch (19). The degree of HLA mismatching was also demonstrated to be a predictor of BO severity (20). Lastly, the presence of preformed anti-class I and -class II HLA antibodies from the recipient have been associated with increased frequency of acute rejection episodes (21), lymphocytic bronchitis and incidence of BO (22, 23).  In addition, several alloimmune-independent risk factors for BO have been identified: 1) primary graft dysfunction (PGD) (formerly ischemia-reperfusion injury) (24-26) 2) cytomegalovirus (CMV) infection (27, 28) and 3) gastroesophageal reflux disease (GERD) (29-31). PGD is proposed to contribute to chronic rejection by causing the upregulation of MHC molecules on the allograft, leading to the amplification of the inflammatory response. In an analysis of 127 transplants by Bharat et al., patients with PGD were found to have significantly higher levels of pro-inflammatory mediators and HLA class I and II antibodies than those without (25).  CMV infections have been associated with elevated HLA I and II expression and the upregulation of the inflammatory response (32, 33), while GERD may exacerbate lung injury due to recurrent aspiration (34, 35).  Pathogenesis Although the mechanisms for the pathogenesis of BO are yet to be fully understood, the fact that acute rejection is a significant risk factor for BO provides supporting evidence that BO is primarily an immune-mediated disease. Immune-independent factors most  5 likely contribute to rejection through the activation of the innate immune response and release of inflammatory mediators (16). The pattern of BO pathogenesis has been suggested to be an "injury response", a concept first proposed in other solid organ transplants, linking graft injury to the priming of the immune response and T cell activation (36). There is histopathological evidence that suggests repeated injury to the lung allograft, whether it is through acute rejection episodes or from alloimmune- independent factors, culminates in a fibro-proliferative repair stage ending in the obliteration of the lumen (5).  The involvement of numerous pro-inflammatory cytokines including Th1 cytokines interleukin (IL)-2, IL-6, interferon (IFN)-γ (37), Th2 cytokines IL-4 and IL-10, tumor necrosis factor (TNF)-α, and chemokines RANTES, IL-8, monocyte chemoattractant protein (MCP)-1, have all been identified in human BO patients as well as in animal models of BO (16). The Th1 immune response has generally been associated with acute rejection while the Th2 response may be involved in chronic rejection and fibrosis (38). The T-cell activated pro-inflammatory response causes the migration of activated neutrophils and macrophages that produce additional pro-fibrotic growth factors and promote myofibroblast proliferation. Pro-fibrotic growth factors such as platelet derived growth factor (PDGF) (39), fibroblast growth factor (FGF) (40) and transforming growth factor (TGF)-β (41) may all be responsible for contributing to extracellular matrix (ECM) deposition and smooth muscle cell proliferation that ultimately occludes the airway (12, 16, 42).   6 There is currently no effective treatment method for BO. Current therapy involves augmenting or changing the immunosuppresion drug regimen that may consist of a combination of the following: corticosteroids, antimetabolites, calcineurin inhibitors and macrolides (9, 12, 43). When employed early in the progression of BO these therapies can only stabilize but not reverse lung functionality and deterioration (16, 44). Newer therapies involving induction of chemotherapeutic agents that deplete recipient immune cells after transplant have been shown to decrease incidence of BO (45), but results are inconsistent due to the difficulty in interpreting small retrospective studies and no consensus has been reached (1). Retransplantation may be a final treatment option for BO, although the benefit of this approach is controversial since some studies revealed a higher incidence of BO in the second allograft than the initial transplant (46-48).  Despite the progress made in identifying mediators and risk factors for BO, the mechanisms involved in the disease pathogenesis are complex and much still remains to be elucidated. It is known that alloimmune-dependent factors play an important role in lung transplant rejection, however treatment with immunosuppression does not prevent BO (12, 16). Therefore, other lung specific factors must contribute to BO and would explain the disease heterogeneity observed. The lung's exposure to various inhaled pathogens such as viruses or infectious agents contribute to the lung's susceptibility to injury and infection and is likely to exacerbate the rejection response contributing to the low survival rates of lung transplant recipients (13, 49). There is the need to investigate the various mechanisms involved in the development of BO and lung transplant rejection  7 in order to provide a greater understanding of the disease and allow for the development of better treatment options to improve long-term survival outcomes.             8  Figure 1.1: The hypothesized pathogenesis of BO. The pathogenesis of BO is believed to be primarily initiated by immune-mediated injury to the bronchial epithelium. In response to the injury and apoptosis of the bronchial epithelial cells (BECs), inflammatory cells are recruited to the bronchiole and infiltrate the lumen. The release of cytokines, chemokines and growth factors exaggerate the inflammatory response as well as cause myofibroblasts to proliferate in an effort to repair the damage. It is believed that repeated injury to the BECs results in an aberrant repair process resulting in the deposition of ECM proteins and smooth muscle cells that ultimately occlude the bronchiole lumen.          9 1.2. Murine Heterotopic Tracheal Transplant Model The murine heterotopic tracheal transplant (HTT) model described by Hertz and colleagues provides a simple and reproducible animal model for studying BO (50). The model involves the heterotopic transplantation of an excised trachea from a donor mouse into a subcutaneous pouch on the upper back of a mouse of genetically different strain (50). After an initial ischemic phase where the epithelium sloughs off, re-epithelialization is observed by day 7 post transplant. From day 10 to 14, the epithelium matures into a mixed squamous and partially ciliated epithelium covering up to 85% of the lumen (51). During this phase the graft also experiences alloimmune injury with substantial subepithelial mononuclear infiltration similar to lymphocytic bronchitis in humans (52). Partial to complete loss of the epithelium has been noted by day 14, ending in ECM deposition and luminal occlusion by day 21 to 28 (51, 53).  Chronic rejection in the tracheal allograft with the histological feature of the fibrotic lesion is referred to as obliterative airway disease (OAD) or experimental BO. The donor and recipient mice strains selected need to be completely MHC mismatched to develop OAD. Control isografts performed in parallel have normal histology, including intact epithelium at day 28 (54-56).  The murine HTT model has been widely used and accepted as a means to study BO, however there are key differences compared to the human disease that should be noted. First, the tracheal allograft is not vascularized or functional. Because the trachea is transplanted into the subcutaneous pouch, it is therefore not exposed to the external environmental stimuli as in normal airways, and is prone to more pronounced ischemia  10 while relying on neovascularization to occur (16). In addition, the behavior of the tracheal allograft adjacent to recipient airway tissues is not accounted for. A murine orthotopic tracheal transplant model has been described by Genden et al, which places the tracheal graft in situ and allows the trachea to be functional (57). In this model, allografts demonstrate loss of ciliated epithelium with cellular infiltration peaking at 21 days. This technique is technically demanding and highly invasive for the animals, as some mice develop debilitating audible stridor and may be required to be euthanized (57). Due to the orthotopic location of the allograft adjacent to recipient airway tissues, the epithelium is regenerated with recipient-derived epithelial cells and do not develop OAD as in the heterotopic model (58). Therefore, this model may be more suitable for studying acute rejection or lymphocytic bronchitis after transplantation but does not characterize the fibro-proliferation and airway obliteration that is observed in BO.  More recently, a murine orthotopic lung transplant model has been described, which provides vascularization, exposure to external stimuli, and involves the small airways (59, 60). This model is the most physiological model of lung transplantation in mice and can be used to successfully reproduce human acute rejection.  Allografts in this model experience severe vascular rejection with extensive necrosis of airway structures but maintain an intact airway epithelium and do not develop OAD after 28 days. The use of this model may not be very practical for many studies as it is technically challenging, requiring several months of personnel training, and time consuming, taking ninety minutes to complete the procedure by an experienced technician (60). For this reason the  11 murine HTT model is favoured for its simplicity, and reliability to reproduce disease outcome.                       12 1.3. The airway epithelium The human and mammalian airway epithelia have developed efficient defense mechanisms against a variety of external environmental factors including inhaled infectious agents and pathogens such as microorganisms and viruses. Several cell types make up the epithelium, each functioning to maintain the local immunological homeostasis in the respiratory tract. Ciliated columnar epithelial cells work together with secretory goblet cells to clear inhaled pathogens through mucociliary mechanisms (61, 62). AECs express receptors on their cell surface allowing them to respond to various stimuli and activate the host inflammatory responses through the production of pro- inflammatory mediators. The airway epithelium also has a second line of defense acting as a physical barrier by using regulated tight junctions to prevent the passage of incoming environmental antigens (61, 63). Injury to the epithelium can result in the shedding of the epithelial cells and denudation of the basement membrane leaving only small clusters of basal cells which can give rise to various differentiated epithelial cell types (64). The restoration of the epithelium consists of a series of regulated events. Collective data from in vivo animal models reveal that the regeneration process begins with the spreading and migration of basal cells to the site of injury, re-establishment of tight junctions, epithelial squamous metasplasia, proliferation and redifferentiation of cells to the complete formation of the mucociliary columnar epithelium. The process of epithelial repair is a key event in airway remodeling and dysregulated bronchial epithelial cell repair has been implicated in several respiratory diseases including asthma, chronic obstructive pulmonary disease, cystic fibrosis and BO (63).   13 Aberrant epithelial repair in BO It is believed that the airway epithelium plays an essential role in lung allograft survival and prevention of chronic rejection. Epithelial cell damage, in the forms of necrosis and apoptosis that cause the denudation of the basement membrane, is observed in lung transplant patients with high-grade lymphocytic bronchiolitis preceding BO (5). A model of aberrant epithelial repair suggests that a continuous cycle of epithelial injury and remodeling is the cause of excessive fibro-proliferation and granulation tissue formation characterized in BO (16, 42). This idea has been supported by several in vitro studies as well as in animal models.  The airway epithelium is considered to be an important immunological target responsible for the dysregulated repair and development of BO. This has been supported by the observation of increased expression of class I and class II HLA antigens on BECs in patients with chronic rejection (65). In vitro studies also showed that a BEC line (66) and primary AECs (32) express HLA class I and II antigens on their cell surface in response to cytokine stimulation and are able to elicit an allogeneic response measured by lymphocyte proliferation. Furthermore, the airway epithelium has been shown to be the primary target of allograft rejection in a murine orthotopic re-transplantation model (67). In this model, mice undergo orthotopic tracheal transplant, in which the allograft is repopulated with recipient-derived epithelium and prevents OAD from developing.  The orthotopic allografts are then extracted and heterotopically transplanted into the subcutaneous pouch of a mouse allogeneic of the original donor mouse. Results of re- transplantation, revealed no evidence of OAD development even after 150 days post  14 transplantation.  When the orthotopic allografts were re-transplanted into their syngeneic heterotopic recipient, development of OAD and complete fibrous obliteration was observed. Taken together, these results demonstrate that the phenotype of the epithelium is able to dictate the rejection outcome and the importance of the epithelium acting as an immunological target in OAD development.  Non-immunological injury to the epithelium is also important in the development of BO. Using a rat HTT model, Adams et al. were able to induce OAD after two weeks of transplant by denuding the epithelium with proteases prior to transplantation. The ability to induce OAD in isografts is significant, as they are normally indistinguishable from wild type tracheae. When the denuded isografts were reseeded with epithelial cells at time of transplant, the fibrotic lesion and luminal occlusion was significantly diminished (68). In a more in depth study using the rat HTT model, the relationship between the loss of the epithelium and luminal occlusion over a time course ranging from 4 days to 8 weeks after transplant was analyzed (69). In allografts, the intensity of inflammatory infiltrate in the tracheal lumen was associated with a more extensive loss of airway epithelial integrity.  Complete disappearance of epithelium by 2 weeks and simultaneous obliteration of the lumen was observed.  The work of Adams et al. was reinforced when it was shown that reseeding of denuded tracheal isografts with viable epithelial cells restored the normal ciliated epithelium and prevented OAD as early as 6 days post- transplant.  From these studies, the importance of the integrity of the epithelium is emphasized and it is clear that the presence of healthy epithelium can adequately suppress airway obliteration.  15 The removal of damaged AECs is believed to occur through apoptotic mechanisms. AECs can undergo apoptosis in response to viral or bacterial infections, to restore homeostasis after hyperplastic changes, and to control inflammatory responses (70). An increase in apoptotic activity was observed with epithelial destruction and preceded luminal occlusion in a swine heterotopic bronchial allograft model (71). In the murine HTT model, apoptosis of the epithelium was measured by TUNEL staining and by electron microscopy to evaluate morphological features of apoptosis.  A higher percentage of apoptotic cells were identified in allografts compared to isografts at day 10, 12 and 14 post-transplant during epithelial cell regeneration (51).  In summary, the proper regeneration and maintenance of the airway epithelium integrity is necessary to prevent airway obliteration. A potential approach for preventing BO could include the protection of the airway epithelium from undergoing apoptosis. Another theory would be to induce proliferation of the AECs to replenish any that are lost by necrosis or apoptosis.          16 1.4. Apoptosis It is hypothesized that injury to the airway epithelium causes them to undergo apoptosis, starting the aberrant repair process associated with BO. Apoptosis is a highly regulated process in which a cell is programmed to undergo cell death. Apoptosis is involved in normal development, maintenance of cell populations, and functions as a means of defense in immune mechanisms or against diseased cells (72).  The other major mechanism of cell death is necrosis, usually occurring in cells damaged by external injury. Unlike necrosis, apoptotic cells can be induced to commit cell suicide by internal and external stimuli and proceed through the intrinsic or extrinsic pathway respectively. Both pathways eventually converge at a common point in which the cell undergoes apoptosis: DNA fragmentation, degradation of nuclear proteins, cross-linking of proteins, formation of apoptotic bodies and finally uptake by phagocytic cells. The initiation of apoptotic processes is dependent on the activation of a group of cysteine proteases called caspases that specifically cleave proteins at aspartic acid residues (73).  Under normal physiological conditions, caspases exist in their inactive proenzyme state. During apoptosis, the pro-caspase is cleaved into its active state of separated large and small subunits (74). The caspases can be divided into two groups: initiator caspases which include caspases-2, -8, -9 and -10 and effector caspases which comprise of caspase-3, -6 and -7 (75). Activated initiator caspases subsequently cleave and activate effector caspases, which then proceed to cleave regulatory proteins such as endonucleases, kinases, cytoskeletal proteins and DNA repair proteins, a process referred to as the caspase cascade (76). Caspase-3 plays a very important role in the final  17 execution of apoptosis by cleaving the inhibitor of caspase-activated DNAse (ICAD) to release CAD. CAD is responsible for degrading chromosomal DNA into fragments and causing chromosomal condensation. Another target of caspase 3 is the DNA repair protein, poly (ADP-ribose) polymerase (PARP), leading to the depletion of cellular ATP and ultimately cell death. The formation of apoptotic bodies and uptake by phagocytic cells such as macrophages is the final step of apoptosis. The externalization of the phospholipid, phosphatidylserine, from the inner to outer leaflet of the lipid bilayer allows for recognition, uptake and removal by phagocytes, without the release of cellular constituents (77).  In the extrinsic pathway, apoptosis is activated by external death signals binding to death receptors at the cell surface. Death receptors contain a death domain (DD) in their cytosolic portion, which is important for recruiting adaptor proteins and for oligomerization. Well known death receptors include Fas, tumor necrosis factor receptor 1 (TNFR1) and DR3. Binding of Fas ligand (FasL) to the Fas receptor induces receptor trimerization, recruitment of adaptor proteins and the activation of initiator caspases (78). Fas-associating protein with death domain (FADD) is an adaptor molecule recruited to the Fas receptor forming a multi-component death-inducing signaling complex (DISC). The death effector domain (DED) of FADD is critical in recruiting and binding to the DEDs in the prodomain of procaspase-8 or -10 (79). Once recruited, procaspase-8 is proteolytically cleaved to its active form allowing it to then cleave and activate downstream effector or executioner caspases such as caspase 3, 6 and 7 (73, 78).   18 Bcl-2 Family Factors such as stress, DNA damage, cytotoxic agents and deficiency in growth factors can lead to the intrinsic apoptotic pathway of a cell. The intrinsic pathway of apoptosis involves regulation of mitochondria membrane integrity by the B-cell lymphoma-2 (Bcl- 2) family of proteins (80). Conserved regions called Bcl-2 homology (BH) domains 1-4 are shared among family members with both pro- and anti-apoptotic properties. There are three subfamilies of the Bcl-2 family based on function and sequence homology: anti- apoptotic proteins, the multidomain pro-apoptotic proteins, and BH3 only proteins. The anti-apoptotic proteins include Bcl-2, Bcl-XL, BCL-W, Mcl-1, Bcl-B and A1, all of which promote cell survival (80). The pro-apoptotic proteins are Bax, Bak and Bok of the multi-domain subfamily, and Bad, Bik, Bid, Hrk, Bim, Bmf, Noxa and Puma of the BH3- only subfamily. The balance of anti-apoptotic and pro-apoptotic proteins, Bcl-2 and Bax, in particular, is important in the progression of apoptosis (76). Although widely studied, the exact mechanism of the Bcl-2 family of proteins has yet to be proven. The pro- apoptotic functions of Bax and Bak are believed to occur following a conformational change and translocation from the cytosol to the mitochondria (75). Homo- oliogomerization of Bax or Bak is most likely required to form pores that induce permeabilization of the outer mitochondrial membrane allowing the release of soluble pro-apoptotic proteins such as cytochrome c and Smac/DIABLO into the cytosol (81, 82). Cyotchrome c binds and activates Apoptotic protease activating factor-1 (Apaf-1) and pro-caspase-9, forming the apoptosome complex that can cleave caspase-3 (83, 84), It is hypothesized that Bcl-2 and other anti-apoptotic family members prevent the release of cytochrome c from the mitochondria by constraining Bax and Bak (85, 86). Binding of  19 the anti-apoptotic proteins to Bax or Bak may or may not take place through Bax and Bak's BH3 domain. The mechanism of action of BH3 proteins is unclear, and two hypotheses exist in which they may promote apoptosis (86). The direct activation model proposes that some of the BH3 only proteins, Bim, tBid and Puma, directly bind Bax and Bak to promote their activation and induce apoptosis (87, 88). Puma also has secondary pro-apoptotic functions by binding and inhibiting the anti-apoptotic proteins Bcl-2 and Bcl-XL, thereby allowing Bax and Bak to become activated (89). The indirect activation model proposes BH3 only proteins function solely by binding to the anti-apoptotic proteins, thereby releasing the inhibition of Bax and Bak and allowing them to proceed with apoptosis (90, 91). Recent evidence has favoured the indirect pathway, as cells deficient in Bid and Bim did not show any impairment in apoptosis (91) suggesting BH3 only proteins are not essential for apoptosis by the direct activation of Bax or Bak (86)  20   Figure 1.2. The Bcl-2 family of proteins. The Bcl-2 family of proteins can be divided into three distinct subsets consist of: the anti- apoptotic proteins including Bcl-2, and the pro-apoptotic proteins of which are the BH3 containing, and the BH3 only proteins. The proteins share sequence homology in regions known as Bcl-2 homology (BH) domains. Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Molecular Cell Biology] (80), copyright (2008).      21 IAP Family The inhibitors of apoptosis (IAP) family is another important protein family capable of suppressing apoptosis by directly inhibiting caspase function. To date, eight human IAPs have been identified, targeting caspases-3, -7 and -9 for inhibition (92). Members of the IAP family share sequence homology in functional conserved zinc-binding baculoviral IAP repeat (BIR) domains. One of the better-known IAPs is the X-linked inhibitor of apoptosis (XIAP), which binds to caspase-9 through its third BIR domain (BIR3) preventing it from forming its active dimeric state (93, 94). XIAP's inhibition of caspase- 3 and -7 occurs through binding the N-terminus linker region of BIR2, to the active site of the caspases, preventing substrate entry and activation by catalysis (95, 96). XIAP also contains a RING domain at the C-terminus, which functions as an E3 ubiquitin ligase, targeting proteins for ubiquitination and subsequent degradation by the proteosome (97). Caspase-3 (97), caspase-9 and Smac/DIABLO are all targeted to the proteosome by XIAP (98, 99).    22   Figure 1.3. The intrinsic and extrinsic apoptosis pathways. Apoptosis may be induced by the extrinsic pathway through ligands binding to their cell surface receptors such as Fas and TNFR1, or by stress, DNA damage and cytotoxic agents in the intrinsic pathway. Bcl-2 is an anti-apoptotic protein that regulates the mitochondrial membrane permeability in the intrinsic pathway, preventing the release of cytochrome C from the mitochondria and the assembly of the apoptosome complex. XIAP (not shown) is another anti-apoptotic protein that can inhibit both intrinsic and extrinsic pathways of apoptosis by binding directly to caspases and inhibiting the downstream apoptotic signaling events. Reprinted by permission from Macmillan Publishers Ltd: [Nature Reviews Molecular Cell Biology] (80), copyright (2008).     23 1.5. Transforming growth factor-β The transforming growth factor-β (TGF-β) isoforms (TGF-β1-3) belong to a family of multifunctional cytokines that are important regulators in wound repair and fibrosis among other cellular processes (100, 101). TGF-β is produced by various pulmonary cell types including BECs, AECs, alveolar macrophages, mesenchymal cells and airway smooth muscle cells (102-104). In particular, abnormalities in the TGF-β1 isoform expression, regulation and signaling have been implicated in a number of acute and chronic lung diseases such as BO (41, 102).  Conversion of latent -TGF-β  1 to active TGF-β1 Initially, all three TGF-β isoforms are synthesized as 75kDa inactive precursor proproteins (105-107). Although all three isoforms are believed to undergo similar mechanisms of activation, the structure and activation of TGF-β1 is studied the most extensively. The pro-TGF-β1 complex consists of TGF-β1 bound to homodimeric proteins at the N-terminus known as latency-associated peptides-1 (LAP-1). Once TGF- β1 is associated with LAP-1, the complex, called latent-TGF-β-1, is blocked from binding to its receptors and cannot induce signal transduction, making latent-TGF-β-1 biologically inert. This complex also binds to the latent-TGF-β-binding protein1 (LTBP1) through specific cysteine disulfide bonds to form the large latent complex (LLC) (105, 108).  LTBP1 consists of a series of epidermal growth factor (EGF)-like repeats and cysteine rich (8-cys) domains (109). The LLC has been suggested to act as a sensor to extracellular signals for TGF-β1 activation (105) and can help in secretion out of the cell (110).  24 Cleavage of LAP-1 from TGF-β1 occurs in the Golgi apparatus by the intracellular endonuclease, furin convertase (111). However, LAP-1 remains non-covalently bound with TGF-β1, and its removal is necessary for complete activation of TGF-β1 (107). Several mechanisms have been identified to activate latent-TGF-β1. Activation through changes in physical conditions such as heat or acidification can denature LAP-1 and release the mature 25kDa TGF-β1 dimer (112). Proteases such as plasmin (113) and matrix metalloprotease-9 (114) are capable of cleaving LAP-1, resulting in the destabilization of the latent-TGF-β1 complex and release of active TGF-β1. The αvβ6 integrin and the matricellular glycoprotein, thrombospondin-1 (TSP-1), activate latent- TGF-β through direct interactions with LAP-1, most likely disrupting the non-covalent bonds between LAP-1 and TGF-β1 (115, 116).  A mechanism of action of TSP-1 in TGF-β1 activation has been elucidated in an animal model of bleomycin-induced lung inflammation and fibrosis. Elevated levels of TGF-β1, plasmin, (103) and TSP-1 (117) were reported after bleomycin-induced injury in alveolar macrophages. Yehualaeshet et al. noted that latent-TGF-β1 is released in complex with TSP-1 and that the TSP-1 component of the complex binds to its natural cell surface receptor, CD36. The binding of the TSP-1/ latent-TGF-β1 to CD36 brings the complex in close approximation to plasmin, a protease that is the cleaved product of plasminogen and is located at the cell surface. Plasmin efficiently cleaves TGF-β1 from its association with LAP-1 at the cell surface, allowing active TGF-β1 to proceed with its signaling events. The external domain of CD36 is critical for the interaction of TSP-1 with CD36. It was discovered that a synthetic CD36 peptide from amino acids 93-110 that mimicked the  25 external domain of CD36, could block TGF-β1 signaling. Blocking TSP-1 binding with the CD36 synthetic peptide lead to the interference of plasmin-mediated lysis of TGF-β1 from LAP-1 and was effective in inhibiting TGF-β1 signaling (117). The activation of latent TGF-β has been a popular target in regulation of TGF-β expression for treatment possibilities. Preventing the removal of the TGF-β1 from LAP-1 would be a strategic method for inhibiting TGF-β1 signaling and an effective therapeutic approach for pulmonary diseases in which TGF-β1 is implicated.             26   Figure 1.4. Activation of latent-TGF-β1 by thrombospondin-1 and inhibition by a CD36 synthetic peptide. (A) The latent form of TGF-β1 consists of the dimeric latency-associated protein-1(LAP- 1) bound to the N-terminus of TGF-β1. During pulmonary inflammation and fibrosis, latent-TGF-β1 is associated with TSP-1. TSP-1 binds to the cell surface receptor CD36 bringing latent-TGF-β1 in close proximity to plasmin. Plasmin cleaves LAP-1 releasing the active form of TGF-β1 that can then go on to bind its receptors and initiate signaling events. (B) A CD36 synthetic peptide of amino acids 93-110 blocks the binding of the TSP-1/latent-TGF-β1 complex. As TGF-β1 is not present at the cell surface, plasmin cannot cleave LAP-1 from TGF-β1 and downstream signaling events are inhibited.   27 TGF-β  Signaling The predominant signaling pathway for TGF-β1 involves the phosphorylation of Smad proteins (118, 119). Activated TGF-β binds to a serine/threonine receptor kinase complex consisting of two type II (TGF-βRII) and two type I receptors (TGF-βRI) (118, 120). The third receptor TGF-βRIII or betaglycan, is not directly involved in TGF-β signaling, but allows TGF-β to bind to TGF-βRII with high affinity (121, 122). Once TGF-β binding occurs, the constitutively active TGF-βRII initiates its kinase activity and subsequently phosphorylates TGF-βRI. TGF-βRI is then capable of phosphorylating downstream receptor-associated Smad (R-Smad) proteins. The R-Smads consist of Smad1, Smad2, Smad3, Smad5 and Smad8, several of which are recruited to TGF-βRI by the Smad anchor for receptor activation protein (SARA) (119, 123). R-Smads then become associated with the co-Smad, Smad4, forming a complex that can translocate to the nucleus, associate with additional transcription factors and bind to the Smad binding element (SBE) of target genes (100, 124, 125).  Signaling is negatively regulated by members of the inhibitory Smads (I-Smads): Smad6 and Smad7. I-Smads can inhibit signaling by a few mechanisms. First, I-Smads competitively bind to TGF-βRI, thus preventing the phosphorylation of R-Smads. Second, they can terminate signaling by recruiting ubiquitin ligases called Smad ubiquitin regulatory factors (Smurfs) to the activated TGF-βRI to target the receptor for degradation (120). Lastly, they may recruit phosphatases such as phosphatase-1 to the receptor complex to dephosphorylate and inactivate TGF-βRI (126).   28   Figure 1.5. The TGF-β1-Smad signaling pathway. TGF-βRIII or betaglycan, acts as an accessory receptor, bringing TGF-β1 to the constitutively active TGF-β1 type II receptor (TGF-βRII). TGF-βRII phosphorylates TGF-βRI on specific serine and threonine residues. Activated TGF-βRI phosphorylates the downstream R-Smads, Smad2 and Smad3, which may be recruited to TGF-βRI by as SARA. Smad2 and Smad3 can associate with the co-Smad, Smad4, and form heterotrimeric or dimeric complexes that can translocate to the nucleus and regulate gene transcription. Inhibitory Smads, Smad6 and Smad7, negatively regulate TGF-β1 signaling and can recruit Smurfs that target TGF-βRI for ubiquitination and degradation. Reprinted from Trends in Biochemical Sciences, May;29 (5),  ten Dijke P, Hill CS., New insights into TGF-beta-Smad signalling, 265-73, Copyright (2004), with permission from Elsevier (119).   29 Another signaling pathway regulated by TGF-β is the mitogen-activated protein kinases (MAPKs) in cell proliferation. The MAPKs dictate the proliferation of various cell types and consist of extracellular signal-regulated kinase (ERK), p38 and cJun NH2 terminal kinase (JNK) (127). The MAPK pathway of TGF-β1 signaling may also be important in fibrosis as the induced proliferation of phosphorylated MAPK proteins has been associated with airway smooth muscle proliferation (128).  TGF-β  and Fibrosis in BO Tissue injury normally results in an inflammatory response followed by a repair phase involving wound healing that restores tissue integrity (129). TGF-β1 has been implicated in the wound healing process; recruiting inflammatory mediators to the site of injury and promoting the proliferation of myofibroblasts to synthesize ECM proteins important for normal repair (100, 130). In some instances, resolution of injury may not occur and fibroblast and myofibroblast proliferation result in excessive fibrosis taking place (131). In a number of animal models and human diseases characterized by excessive fibrosis, it has been shown that continuous expression of TGF-β1 contributes to the pathogenesis of fibrosis. In particular, aberrant expression and release of biologically active TGF-β1, but not TGF-β2 or TGF-β3 has been associated with animal models of pulmonary fibrosis (102, 132-134). TGF-β1 contributes to fibrosis by stimulating the production of various proteins involved in cytoskeletal organization, matrix formation and remodeling in addition to its own transcription in the latent form in a positive feedback loop (100) (130). In an informative review by Branton and Kopp, a compilation of extracellular matrix proteins regulated by all of the TGF-β isoforms included several collagens,  30 fibronectin and elastin (130). It was also noted that TGF-β is associated with the decreased protein expression of the matrix metalloproteinases (MMPs), which breakdown matrix proteins, and the increased production of their inhibitors, the tissue inhibitors of MMPs (TIMPs). Therefore, prolonged expression of TGF-β leads to an imbalance between collagen production and degradation resulting in connective tissue accumulation and fibrosis (135). The effect of the TGF-β1 isoform on inducing fibrosis was also noted even in the absence of any inflammatory infiltrate or injury in rat lung explants (136). Alveolar epithelial cells in sections of normal rat lung free of inflammation were transfected with cDNA of active TGF-β1. Analysis of the lung sections 14 days after transfection revealed remodeling of the lung and increased fibrosis. These observations demonstrated TGF-β1 controlled fibrosis is able to occur in settings where no injury repair or inflammatory response has taken place.  TGF-β1 has also been implicated in the pathogenesis of various fibro-proliferative diseases related to transplant rejection including chronic rejection of heart, kidney and lung transplants (130). The analysis of 380 TBBs from 91 lung transplants by El-Gamel and colleagues revealed elevated expression levels of TGF-β1 among patients with BO than transplant recipients without BO (41). In addition, immunohistochemical detection of TGF-β1 was directly correlated with severity of BO and preceded BO diagnosis by six to eighteen months (137). Another study found increased levels of TGF-β mRNA expression in BAL fluid of BO patients compared to stable patients, although this observation was not confirmed at protein levels (138). TGF-β1 is considered one of the  31 major inducers of an abnormal fibrotic response that causes morbidity and mortality in several lung diseases, particularly BO (41, 133).  Using the rat HTT model, it was observed that adenoviral-mediated topical transfer of soluble TGF-βRIII contributed to the inhibition of luminal occlusion in allografts (139). TGF-βRIII was administered at the site of allograft on day 5 after transplantation. It was observed that the presence of soluble TGF-βRIII could abrogate the effect of TGF-β by acting as an antagonist and competitively binding to TGF-β receptors. In the murine HTT model of BO, the upregulation of the TGF-β1-Smad3 signaling has been associated with increased levels of the pro-fibrotic growth factors PDGF and connective tissue growth factor (CTGF) as well as ECM components, fibronectin and collagen (140). Ramirez and colleagues demonstrated that TGF-β1-induced myofibroblast transdifferentiation was dependent on Smad3 (141) and the use of a Smad3 deficient mouse in the murine HTT model was capable of ameliorating luminal occlusion and fibrosis in allografts (142). In a large animal porcine bronchial model of BO, upregulation of PDGF, PDGF receptors and TGF-β1 was observed in allografts compared to isografts (143). Collectively, these studies confirm the importance of TGF-β in fibrosis in chronic allograft rejection and blocking the effect TGF-β could be beneficial in preventing the fibrotic processes in BO.  A novel mechanism by which TGF-β1 is involved in the progression of BO has been recently described. The effect of TGF-β1 on the ability of the airway epithelium to undergo epithelial-mesenchymal transition (EMT) has been studied in lung transplant patients with BO (144, 145). EMT is the process in which epithelial cells lose epithelial  32 properties and gain a mesenchymal cell phenotype such as the production of ECM proteins (146, 147). Primary BECs obtained from stable lung transplant patients were treated with TGF-β1 and overall showed characteristics of EMT including change in cell morphology, the down regulation of epithelial proteins, upregulation of mesenchymal proteins, ECM secretion and invasive and migratory capacity (145). Flow cytometry analysis of large airway brushings from BO patients showed an increased expression of mesenchymal proteins including α-smooth muscle actin. Surprisingly, in this study there was no change in expression levels of TGF-β1 among patients with BO and without, although only 33 bronchoscopies were analyzed, 6 of which were BO positive (144). The recent findings from these two studies suggest a novel role for TGF-β1 by the induction of EMT in the pathogenesis of BO.  TGF-β  and T regulatory Cells In what appears to be contradictory to its role as a pro-fibrotic cytokine in the pathogenesis of BO, overexpression of TGF-β has been reported to be beneficial in experimental organ transplantation. In animal models of heart (148, 149) and lung (150, 151) transplant, expression of TGF-β has lead to a reduction in acute rejection and inflammation and the prolongation of graft survival. This is explained by the fact that in addition to its effects as a pro-fibrotic cytokine, TGF-β also has potent immunosuppressive functions. TGF-β has been shown to regulate immune responses by controlling T cell proliferation, differentiation and survival (152). Over two decades ago, in vitro studies first documented anti-proliferative effects of TGF-β on T cells (153).  It is now known that TGF-β inhibits T cell proliferation through multiple pathways including  33 the inhibition of IL-2, the T cell activation cytokine, and through regulation of cell cycle proteins (152). Another important mechanism by which TGF-β inhibits immune responses is through the generation of regulatory T cells (Tregs) (154-156) that express CD4, CD25 and the forkhead/winged helix transcription factor (Foxp3) (157).  Tregs are capable of modulating allograft rejection by suppressing the immunological response after transplantation (158, 159). In several experimental transplantation models, CD4+CD25+Tregs have been implicated in the maintenance of tolerance to donor antigens (160, 161) and have been found at higher percentages in mice with stable allografts than those that have experienced rejection episodes (162). The adoptive transfer of Tregs has also been used to establish antigen-specific dominant tolerance to allogeneic transplants in animal models (163, 164). It is believed that TGF-β promotes the generation of Tregs from CD4+CD25- naive T cells by the induction of Foxp3 expression, a process that has been observed in vitro (156, 165, 166). TGF-β may also have an essential role in maintaining naturally occurring CD4+CD25+Tregs numbers and their suppressive function (154, 167).  It is unknown whether the immune regulatory functions of TGF-β are observed in lung transplantation. Mamessier et al. observed that the TGF-β1 isoform was expressed in lung transplant patients with acute rejection and during BO progression, but was not overexpressed in patients that had a stable form of BO (168). This observation and the fact that Treg cells were dramatically higher in patients with stable BO than evolving BO  34 or healthy recipients, has led to the speculation that TGF-β1 is unlikely to be produced by the Tregs in this setting.  TGF-β1 has multiple diverse functions that may be contradictory in some settings. In the context of lung transplantation, the overexpression of TGF-β1 is observed with its major function believed to be in mediating wound repair and fibrotic processes.  Larger cohort studies are necessary to fully understand the role of TGF-β1 in Treg generation and function and whether this role is observed during lung transplantation.                 35 1.6. Gene Therapy in Transplantation Gene therapy can improve transplantation outcomes by a number of approaches. The focus of this thesis is to investigate the ability of gene therapy to improve graft survival by preventing damage to the graft at the time of transplantation (169). Primary graft dysfunction and subsequent inflammation are causes of graft loss in several organs. The overexpression of anti-apoptotic genes in transplanted tissue can potentially overcome graft injury and prevent chronic rejection.   The development of viral vectors for gene therapy is widely used for experimental transplantation models with success. Common viral vectors used for treatment of lung diseases are the adenovirus and lentivirus (170). Each vector system offers its own advantages and disadvantages, and each requiring efficient gene transfer for an effective outcome. Adenoviruses have been used in clinical trials for treatment of cystic fibrosis; however problems with efficient gene transfer and activation of inflammatory responses have arisen, and studies are continually being conducted. The use of a lentivirus vector has the advantage of being able to transduce non-dividing cells and to provide stable transgene expression. However, Vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus vectors also have difficulty with efficient gene transfer to the airway epithelium as their receptors are located on the basolateral surface of the epithelium (171).  Adenovirus has been used to deliver anti-apoptotic proteins Bcl-2 (172, 173) and XIAP (174) to prolong graft survival in experimental animal liver and islet transplantations. The  36 prevention of PGD, ischemic injury and epithelial cell apoptosis in lung transplantation using virus-mediated delivery of anti-apoptotic proteins would therefore be an interesting area of study. Both Bcl-2 and XIAP would make ideal candidate anti-apoptotic genes for the inhibition of AECs apoptosis in donor tracheal tissue. Expression of Bcl-2 has already been detected to be upregulated in the airway epithelium of the vascularized mouse whole lung transplant model, in which the epithelium is intact and OAD is prevented (59). XIAP would also be effective for gene therapy as well since it is a downstream apoptotic protein and can inhibit the final common pathway of effector caspase activation. In this thesis, both lentivirus and adenovirus will be used as mechanisms for gene transfer, and the Bcl-2 and XIAP genes will be used to inhibit apoptosis of AECs in donor tracheal tissue.              37 1.7. Thesis hypotheses BO remains the leading cause of morbidity and mortality in lung transplant patients, contributing to the lowest patient survival rates among solid organ transplants. It is evident that understanding the multifactorial process and mechanism of BO is required for developing effective treatment approaches. Combined data from animal and human studies suggest that injury to the airway epithelium is a crucial initiating factor in aberrant tissue remodeling and the progression of BO. The injury and apoptosis of BECs in lung allografts may occur by alloimmune or non-alloimmune mechanisms, causing a massive inflammatory and pro-fibrotic response leading to myofibroblasts and fibroblasts depositing ECM and granulation tissue in the bronchiole lumen (16). Therefore the initial injury and apoptosis of the BECs, and the fibro-proliferative stage of the disease are two ideal targets in preventing the chronic rejection process.  1. The inhibition of AEC apoptosis in donor tracheal tissue will prevent the subsequent progression of murine OAD. 2. TGF-β1 is a major contributor to the fibro-proliferative response in the airways and inhibition of latent TGF-β1 activation will reduce luminal fibrosis in OAD.        38 1.8. Thesis objectives 1. Inhibit AECs apoptosis in donor tracheal tissue to prevent murine OAD. The first objective was to inhibit AEC apoptosis using gene therapy and ectopic expression of anti-apoptotic proteins to prevent the progression of OAD.  Using the murine HTT model, lentivirus-mediated gene transfer of Bcl-2 or adenovirus-mediated gene transfer of XIAP was utilized to deliver these gene products to the AECs in donor trachea prior to transplantation.  2. Determine importance of TGF-β1 in the fibroproliferative process of OAD by inhibiting latent- TGF-β1 activation. The donor tracheae from the murine HTT model were treated with a synthetic CD36 peptide to block the cleavage of LAP-1 from TGF-β1. The effect of the inhibition of TGF-β1 activation on the development of fibrosis in OAD was then examined.            39 2. MATERIALS AND METHODS 2.1. Mice Pathogen-free BALB/c and C57 BL/6 male mice were purchased from the UBC Animal Care Centre. Mice were between 8 - 12 weeks of age at time of experimentation. All experiments were performed in compliance to the University of British Columbia Animal Care Committee protocols.  In this thesis, BALB/c mice refer to the wild type (WT) mouse.  2.2. Cells The human embryonic kidney cell line, HEK 293T, the transformed human airway epithelial cell line, HAE, and the human cancer cell line, HeLa, were used. All cells were cultured in complete media consisting of Dulbecco's Modified Eagle Media (DMEM) (Invitrogen Corp.) supplemented with 10% fetal bovine serum (Invitrogen Corp.), 10mM L-glutamine (Invitrogen Corp.), 10mM sodium pyruvate (Invitrogen Corp.) and 10units/mL penicillin-streptomycin (Invitrogen Corp.). Cell culture conditions consisted of cells incubated at 37°C with 5% CO2.  2.3. CD36 synthetic peptide A synthetic peptide of the TSP-1 cell surface receptor, CD36, YRVRFLAKENVTQDAEDNC (from amino acids 93-100) was previously synthesized using an Applied Biosystems model 431A peptide synthesizer (117) and was provided by Dr. Nasreen Khalil. The peptide was dissolved in PBS to required concentrations.   40 2.4. Adenoviral and lentivirus vectors The adenovirus vectors used were kindly provided by Dr. Bruce Verchere  (Child and Family Research Institute, BC) and were prepared in the laboratory of Dr. Bob Korneluk (University of Ottawa, ON). The production of the adenoviral vectors are described elsewhere (175). Adenovirus encoding the human XIAP protein (AdXIAP) and adenovirus encoding the green fluorescent protein (AdGFP) were used. Lentivirus vectors constructed were: 1) Lentivirus-encoding GFP and the truncated nerve growth factor receptor (ΔNGFR) (LV-CTE-GFP-NGFR) 2) Lentivirus-encoding GFP and Bcl-2 (LV-CTE-GFP-Bcl-2) 3) A modified lentivirus with the constitutive transport element removed, encoding GFP and ΔNGFR (LV-GFP-NGFR) and 4) Modified lentivirus encoding GFP and Bcl-2 (LV-GFP-Bcl-2). The original lentivirus vector (LV-CTE) was used for some in vitro experiments and the new modified lentivirus (LV) used for all transplant experiments.  Lentiviral vector construction To clone the Bcl-2 gene into the lentivirus vector, complementary DNA for murine Bcl-2 was generated by reverse transcriptase-PCR using RNA isolated from mouse spleen. Sense and anti-sense oligonucleotide primers used were 5' GGAAGG ATG GCG CAA GCC GGG AGA ACA G 3' and 5' GAC CCG GTG TTC ACT CCAGCTGTTTGG 3', inserting SmaI and SalI restriction sites for cloning purposes. The 768bp SmaI/SalI Bcl-2 gene product was verified by DNA sequencing and gel purified. The lentivirus transfer vector was constructed from the plasmid pCCL.sin.cPPT.GFP.WPRE that contained a synthetic bidirectional promoter, MA1, developed by Amendola et al. (176). MA1  41 consists of a minimal core cytomegalovirus (mCMV) promoter driving expression of GFP and the human phosphoglycerate kinase (PGK) promoter driving expression of the second transgene, truncated nerve growth factor receptor (ΔNGFR). To optimize lentivirus-mediated gene delivery, two expression cassettes were inserted flanking the bidirectional promoter. The upstream cassette contains a constitutive transport element (CTE) from the Mason-Pfizer virus and polyadenylation site from the Simian Virus 40. The downstream cassette contains the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and the self inactivating (SIN) HIV-1 LTR polyadenylation site. For simplification, we name this transfer vector LV-CTE. A modified version of this transfer vector was also used in which the CTE was found unnecessary and removed from the upstream cassette. This vector was used for all transplant experiments and is referred to as LV. For both the LV-CTE and LV lentiviral transfer vectors, Bcl-2 was cloned into the MA1 driven plasmid as the second transgene replacing ΔNGFR.  2.5. Lentivirus production Third-generation HIV-1 derived lentivirus pseudotyped with vesicular stomatitus virus (VSV)-G was produced by four-plasmid co-transfection of HEK 293T cells with each of the transfer plasmids, LV-CTE-GFP-NGFR, LV-CTE-GFP-Bcl-2, LV-GFP-NGFR, LV- GFP-Bcl-2 and the complementing packaging (pMDLg/pRRE), expression (pRSV/Rev) and envelope (pMD.G) plasmids. Virus titer was determined by adding serial dilutions of virus to HEK 293T cells and expression of GFP and/or ΔNGFR was read by flow cytometry on day 4 or 5. Normal virus titers achieved ranged from 3 x 108 transduction  42 units (TU)/mL to 1.2 x 109 TU/mL. Aliquots of concentrated virus were stored at -80°C until use.  2.6. Lentiviral and adenoviral transduction of cell lines HAE cells Approximately 2.5 x 105 HAE cells were plated per well in a 6-well plate and allowed to adhere for 4 hours. Cells were infected at a MOI of 5 for each LV-CTE-GFP-NGFR, LV- CTE-GFP-Bcl-2, LV-GFP-NGFR and LV-GFP-Bcl-2. Cells were cultured for four days producing stable populations of lentivirus transduced cells. HAEs were subjected to apoptosis studies, western blotting and immunofluorescence staining.  HeLa cells Approximately 5 x 105 HeLa cells were plated per well in a 6-well plate and allowed to adhere for 4 hours. Cells were infected at multiplicity of infections (MOI) of 1 pfu/cell or 10pfu/cell of each AdGFP and AdXIAP adenoviral vectors. Cells were left for 48 hours to allow for gene expression before western blotting.  2.7. In vitro induction of apoptosis Staurosporine (STS) (Sigma-Aldrich Co.) at a concentration of 10µm was used to induce apoptosis in untransduced (UT) HAEs, and HAEs stably transduced with LV-CTE-GFP- NGFR or LV-CTE-GFP-Bcl-2 . Cells were treated with STS for 6 hours followed by fixing with 4% paraformaldehyde. Cells were lysed with a urea-containing buffer (6M urea, 62.5 mM Tris/HCl; 10% glycerol, 5% b-mercaptoethanol, 2% SDS; 0.00125%  43 bromophenol blue; pH 6.8), followed by heating at 65°C for 15 minutes and sonication before western blotting.  2.8. Western blotting Approximately 1 x 106 cells were obtained and lysed in ice-cold solubilization buffer (20mM Tris HCl, pH 8.0, 1% NP40, 10% glycerol, 137mM NaCl and 10mM NaF) with protease inhibitor cocktail (Sigma-Aldrich Co.). Cells were centrifuged for 10min at 32,000 x g and supernatant was collected. Protein concentration was determined following a BCA protein assay and 50µg of protein was loaded onto a 12% SDS- polyacrylamide gel and run by gel electrophoresis. After electrophoresis, the proteins were transferred to nitrocellulose membranes and blocked with 5% skim milk for 30 minutes at room temperature. Membranes were probed with the following antibodies, mouse anti-PARP at 1:500 (BD Biosciences), polyclonal rabbit anti-Bcl-2 at 1:500 (Abcam Inc.), monoclonal mouse anti-XIAP at 1:500 (BD Biosciences), polyclonal rabbit anti-GFP at 1:1000 (Abcam Inc.), polyclonal goat anti-β-actin at 1:1000 (Santa Cruz Biotechnology Inc.) and mouse anti-vinculin at 1:500 (Sigma-Aldrich Co.). All primary antibodies were incubated with the membrane for 2 hours at room temperature or overnight at 4°C. Appropriate secondary antibodies, goat anti-mouse, goat anti-rabbit and rabbit anti-goat (Dako Ltd.) conjugated to horseradish peroxidase were used at 1:5000 and proteins were detected using an enhanced chemiluminescence (ECL) system (GE Healthcare Co.).    44 2.9. Immunofluorescence Approximately 2.5 x 105 HAE cells were plated per chamber well and allowed to adhere overnight. Cells were then washed in PBS and fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature.  Cells were blocked with TBS containing 1% Triton-X for 10 minutes at room temperature followed by incubation with the primary antibody, rabbit anti-Bcl-2 at a 1:50 dilution (Abcam Inc.), in blocking buffer for 1 hour at room temperature. Bound antibody was detected using goat anti-rabbit secondary antibody conjugated to AlexaFluor594, a red fluorescent dye. Cells were also briefly stained with Hoescht33342 nuclear stain (Invitrogen Corp.) at 1:10,000 dilution in PBS before mounting media was added and glass coverslips were mounted. Expression of GFP and Bcl-2 were visualized using a Zeiss Axioplan 2 imaging microscope.  2.10. Tracheal excision and treatment conditions Donor BALB/c mice were sacrificed by CO2 inhalation and an incision was made on the anterior of the mouse exposing the trachea. The trachea was carefully separated from surrounding tissues and excised by transection below the larynx and above the carina bifurcation. Tracheae were placed immediately in complete DMEM on ice and subjected to different treatments described below. Three different parameters were investigated in studying the development of murine OAD, the effect of lentivirus-mediated gene transfer of Bcl-2 (Group 1), adenovirus-mediated gene transfer of XIAP (Group 2), and treatment with a CD36 synthetic peptide (Group 3).    45 Group 1: Ex vivo lentivirus-mediated transduction of Bcl-2 Excised donor tracheae were placed in individual wells of a 24-well plate and infected with 3x106 transduction units (TU) of LV-GFP-Bcl-2 virus or control virus LV-GFP- NGFR in 0.5mL of complete DMEM. Untransduced tracheae incubated with complete DMEM alone also served as additional controls. Viral infection was allowed to take place over 16 hours at 37°C with 5% CO2. All tracheae were subsequently washed in fresh complete DMEM and returned to the incubator for an additional 48 hours in 1mL of media to allow gene expression. Tracheae were either histologically examined after the ex vivo 48-hour incubation, or used for transplantation experiments.  Group 2: Ex vivo adenovirus-mediated transduction of XIAP Adenovirus gene transfer to excised tracheae was performed in similar fashion as with the lentivirus. Tracheae were incubated with 1 x 105pfu of AdGFP or AdXIAP in 0.5mL of  complete DMEM for 16 hours overnight followed by washing and additional incubation for 48h. Trachea were either histologically examined after the ex vivo 48-hour incubation or used for transplantation experiments.  Group 3: Administration of CD36 synthetic peptide To investigate the role of TGF-β1 in the fibrotic process of BO, a synthetic CD36 peptide developed and provided by Dr. Nasreen Khalil, was used to block activation of latent TGF-β1. Excised donor tracheae were initially incubated ex vivo with 80µg of CD36 peptide in 320ul of PBS for 3 hours at 37°C and 5% CO2. Following treatment, tracheae were then heterotopically transplanted into the subcutaneous pouch of the recipient C57  46 BL/6 mouse and given a dose of 16µg of peptide in 150µl of PBS in the pouch. In vivo administration of the CD36 synthetic peptide was performed at 7, 14 and 21 days post- transplant by carefully injecting 16µg in 150µl of PBS with a 32½ gauge needle into the pouch. Ex vivo and in vivo administration of PBS served as control.  2.11. Murine heterotopic tracheal transplantation Allografts performed used BALB/c mice as donors and C57 BL/6 mice as recipients. Isografts consisted of C57 BL/6 tracheae transplanted into C57 BL/6 recipient mice. Recipient mice were anesthetized by isoflurane inhalation (initial concentration of 2.5- 3% for induction followed by 1-1.5% for maintenance) and an incision was made on the dorsal neck of the recipient mouse to create a small subcutaneous pouch and the donor trachea was heterotopically transplanted. The pouch was closed using 3-0 nylon sutures. In some of the CD36 peptide experiments, two BALB/c tracheae were transplanted per recipient mouse, one on each side of the recipient mouse's dorsal back. All mice were given pain medication of 1mgm/kg buprenorphine following transplantation, housed individually and monitored daily for signs of distress. No immunosuppresion regimen was given to any of the animals. Transplants for each experimental group are outlined in the Table 2.1.        47 Group 1 n Donor Strain Recipient Strain Untransduced Isograft, 4 weeks 2 C57 BL/6 C57BL/6 Untransduced Allograft, 2 weeks 8 BALB/c C57BL/6 Untransduced Allograft, 4 weeks 8 BALB/c C57BL/6 LV-GFP-NGFR Allograft, 2 weeks 8 BALB/c C57BL/6 LV-GFP-NGFR Allograft, 4 weeks 5 BALB/c C57BL/6 LV-GFP-Bcl-2 Allograft, 2 weeks 10 BALB/c C57BL/6 LV-GFP-Bcl-2 Allograft, 4 weeks 7 BALB/c C57BL/6 Group 2 AdGFP Allograft, 4 weeks 7 BALB/c C57BL/6 AdXIAP Allograft, 4 weeks 8 BALB/c C57BL/6 Group 3 PBS Allograft, 4 weeks 4 BALB/c C57BL/6 CD36 peptide Allograft, 4 weeks 12 BALB/c C57BL/6  Table 2.1. Experimental transplant groups.  2.12. Histological evaluation Tracheae were examined following 48 hour ex vivo incubation or at specified time points after transplantation.  Transplanted recipient mice were sacrificed by CO2 inhalation and tracheae were harvested from the subcutaneous pouch.  All tracheae were briefly washed in fresh DMEM before being embedded in Tissue Tek O.C.T. compound (Sakura Finetek USA Inc.), submerged in ice-cold methylbutane and snap frozen in liquid nitrogen. Frozen tissue blocks were stored at -80°C until use. Blocks were cut into 5µm sections and serial sections were adhered onto Superfrost plus gold coating glass slides (Fisher Scientific Inc.). Slides were left for approximately 30 minutes to dry by air and then fixed in ice-cold acetone for 10 minutes and stored at -20°C until use. For histological evaluation, slides were stained with hematoxylin and eosin (H&E).     48 Computer-aided morphological analysis Images of tissue sections stained by H&E were captured with a Nikon Eclipse E600 photomicroscope and SpotFlex V4.6. imaging software. Images were analyzed using ImageProPlus Software 4.0. (Media Cybernetics Inc.). The percentage of luminal occlusion was calculated by tracing the outer circumference of the trachea (including smooth muscle surrounding the cartilage rings) and the inner surface of the residual lumen, following the basement membrane when present.  Luminal occlusion was then calculated using the following formula: Area of Outer Trachea - Area or Residual Lumen x 100% Area of Outer Trachea   To determine the amount epithelial damage and apoptosis after transplantation, the perimeter of the residual lumen was used. Epithelium integrity was then scored based on three categories of integrity: healthy intact epithelium, flattened or damaged epithelium, and absent epithelium. For each category, the cursor was used to trace the epithelium coverage.  Epithelial integrity for each category was then calculated using the following formula:  Distance of epithelial coverage   x 100%   Perimeter of residual lumen   Computer-aided morphological analysis was performed on 5 serial sections taken from the graft and mean values were determined.    49 2.13. Immunohistochemistry Serial sections of frozen mouse tracheal tissue were analyzed for transgene expression by immunostaining for GFP, Bcl-2 and XIAP. Inflammatory infiltrate was monitored by immunostaining with antibodies for CD45 and CD11b.  All tissue sections were serially washed in three changes of PBS and blocked for endogenous peroxidase with 3% H2O2 in PBS for 10 minutes at room temperature. Sections were then blocked with 5% goat serum in PBS + 0.1% Trition-X for 30 minutes at room temperature.  Blocking buffer was gently tipped off the slides and primary antibody in blocking buffer was applied. Dilutions of primary antibodies used were as follows: rabbit anti-GFP at 1:500 (Abcam Inc.), rabbit anti-Bcl-2 at 1:200 (Abcam Inc.), rabbit anti-RIAP3 at 1:500 (a XIAP antibody generously provided by Dr. Bob Korneluk), rat anti-CD45 at 1:50 (BD Biosciences,), rat anti-CD11b  at 1:50 (BD Biosciences).  Sections were incubated with primary antibody for 1-2 hours at room temperature followed by appropriate secondary antibody for 30 minutes. Biotinylated goat anti-rabbit antibody at 1:300 (Dako Ltd.) and biotinylated goat anti-rat at 1:50 (BD Biosciences) were used. Unbound secondary antibody was washed in serial dilutions of PBS. Sections were incubated with Streptavidin-Horseradish peroxidase (BD Biosciences) for 30 minutes at room temperature to label biotinylated secondary antibodies. 3,3'-diaminobenzidine (DAB) (BD Biosciences) was used to visualize protein signals and counterstained with hematoxylin. Sections were then placed through increasing successions of ethanol, dehydrated in xylene and mounted with glass coverslips.    50 2.14. Statistical analysis Analysis of Variance (General Linear Model) and Least Significant Difference (LSD) with Bonferroni's corrections were used to compare the mean values of the luminal occlusion and epithelial integrity scores in this study.                             51 3. RESULTS 3.1. In vitro lentivirus-mediated transduction of HAE cells. The murine Bcl-2 gene was cloned into a VSV-G pseudotyped bidirectional lentivirus vector to allow coordinate expression of both GFP and Bcl-2. It was demonstrated that HAEs stably infected with the virus had dual overexpression of GFP and Bcl-2 that could be detected by western blotting and immunofluorescence staining. Overexpression of Bcl-2 was detected in both the original LV-CTE and modified LV lentiviral vectors by western blotting (Figure 3.1A). The 26kDa Bcl-2 protein was detected in both LV-CTE- GFP-Bcl-2 and LV-GFP-Bcl-2 transduced cell lysates, but not in the control vectors, LV- CTE-GFP-NGFR, LV-GFP-NGFR or untransduced (UT) cells.  β-actin served as loading control for the samples. Expression of GFP was detected by immunofluorescence imaging in both LV-CTE (Figure 3.1 C and D) and LV transduced cells (Figure 3.1 F and G). Bcl-2 could also be detected by immunofluorescence after incubation with rabbit anti-Bcl-2 antibody and goat anti-rabbit AlexaFluor594 (red) secondary antibody (Figure 3.1 D and G).  In both control vectors, LV-CTE-GFP-NGFR and LV-GFP-NGFR, only GFP could be detected (Figure 3.1 C and F). Untransduced cells were negative for GFP and Bcl-2 staining (Figure 3.1B and E). These results show that the Bcl-2 gene was properly cloned into the lentivirus vector and Bcl-2 overexpression was detectable in transduced cells.  52  Figure 3.1. Detection of Bcl-2 in transduced HAE cells. (A) Western blot analysis of HAE lysates of untransduced (UT) and stably transduced cells with the original LV-CTE vector and modified lentivirus, LV. Overexpression of Bcl-2 is detected as the 26kDa band in LV-CTE-GFP-Bcl-2 and LV-GFP-Bcl-2 transduced cells only. β-actin served as loading control. (B-G) Immunofluorescence staining for GFP and Bcl-2. (B and E) Untransduced HAEs stain positive for Hoescht33342 nuclear stain only. (C) HAEs transduced with LV-CTE- GFP-NGFR are GFP positive but Bcl-2 negative. (D) HAEs transduced with LV-CTE- GFP-Bcl-2 demonstrate GFP and AlexFluor594 tagged (Red) Bcl-2 positive staining. (F) HAEs transduced with modified LV-GFP-NGFR are positive for GFP expression only. (G) HAEs transduced with modified LV-GFP-Bcl-2 coordinately express both GFP and Bcl-2.  53 3.2. Inhibition of apoptosis by lentivirus-mediated gene transfer of Bcl-2 in HAE cells. To confirm that the lentivirus construct containing Bcl-2 was capable of inhibiting apoptosis, an in vitro apoptosis study was performed.  Untransduced and stably transduced LV-CTE-GFP-NGFR and LV-CTE-GFP-Bcl-2 HAE cell populations were treated with STS for 6 hours. STS is a non-selective protein kinase inhibitor that has been demonstrated to activate apoptosis pathways and thus increasing proteolytic caspase activity (177, 178). PARP is an important DNA repair protein and is also a target of caspases during apoptosis (179). The presence of cleaved PARP by western blotting is used as a hallmark for apoptosis in in vitro assays. In Figure 3.2., we observe full length 113kDa PARP detected in all three populations of HAE cells that were not induced to undergo apoptosis. Upon 6-hour treatment with STS (6H STS), the expression of the 85kDa cleaved PARP protein was detected in untransduced (UT) and LV-CTE-GFP- NGFR transduced cells. However, the presence of the cleaved PARP product was not detected in the Bcl-2 transduced cell population even after apoptosis induction with STS. The lack of the 85kDa cleaved PARP indicated that apoptosis and caspase activity had been inhibited by overexpression of Bcl-2. Therefore, the lentivirus construct, LV-CTE- GFP-Bcl-2, was confirmed to be functional and was sufficient to inhibit apoptosis in STS treated cells.      54  Figure 3.2. Inhibition of apoptosis activity in Bcl-2 transduced cells. The detection of full length PARP and cleaved PARP was determined in cell lysates from untreated and STS treated untransduced (UT), LV-CTE-GFP-NGFR (NGFR) stably transduced, and LV-CTE-GFP-Bcl-2 (Bcl-2) stably transduced HAE cells. Full length PARP is detected in all samples. Presence of cleaved PARP, an indicator of apoptosis and caspase activity, is detected after 6 hours of STS treatment in untransduced and NGFR stably transduced HAEs and not Bcl-2 transduced cells.                  55 3.3 Assessment of gene transfer after ex vivo lentivirus-mediated transduction  The ability of the lentivirus construct to efficiently transduce the mouse tracheal epithelium ex vivo was tested. Donor tracheae were incubated with LV-GFP-Bcl-2 for 16 hours ex vivo, washed and followed by another 48 hour incubation to allow gene expression.  Untransduced tracheae, and LV-GFP-NGFR transduced tracheae served as controls. After the ex vivo incubation, tracheae were frozen and cut into 5 µm thick sections for histological and immunohistochemical analysis. The appearance of the airway epithelium appeared abnormal and flattened with some epithelial loss in all tracheae (Figure 3.3. A and B). No ciliated epithelial cells were observed, with the remaining epithelium being composed of what appeared to be single layered basal cells. The detection of GFP (Figure 3.3. C) and Bcl-2 (Figure 3.3. D) by immunohistochemical staining proved to be difficult with the abnormal epithelium of the tracheae. Although putative GFP positive staining was detected in the flattened epithelial cells of tissue transduced with LV-GFP-NGFR and LV-GFP-Bcl-2, there was also faint false positive GFP staining in the untransduced trachea. Difficulties with detecting Bcl-2 expression were also encountered: LV-GFP-NGFR transduced trachea and the untransduced trachea both demonstrated staining for Bcl-2. Whether this staining was low levels of endogenous Bcl-2 present in the epithelium of all tracheae or if it was background staining was not known. The difficulties experienced with immunohistochemical staining was most likely due to the ex vivo incubation of the tracheae that exacerbated the ischemic damage to the tracheal epithelium. It was expected that ectopic expression of Bcl-2 in the airway epithelium would be able to inhibit apoptosis by ischemic damage as well as immunological damage to the epithelium after transplantation. However, it  56 appears the ischemic damage may have occurred prior to the expression of Bcl-2 could take place and therefore transduction may not be detectable at this point after ex vivo incubation.  57     58 Figure 3.3. Histological and immunohistochemical analysis of ex vivo lentivirus transduced tracheae. Original magnification 4X (A) and 40X (B-D). (A and B) Histology of tracheae were examined after ex vivo incubation. Untransduced, LV-GFP-NGFR and LV-GFP-Bcl-2 transduced tracheae showed abnormal epithelium, with a flattened single layer of epithelial cells with some epithelial loss. (C) Immunohistochemical staining for GFP revealed false positive background staining in untransduced trachea similar to the staining observed in the LV-GFP-NGFR and LV- GFP-Bcl-2 transduced tracheae. (D) Bcl-2 staining was also difficult to confirm due to the positive staining present in the untransduced trachea and LV-GFP-NGFR transduced trachea (n = 2).   3.4. Assessment of lentivirus mediated gene transfer in 3 day isografts Due to the epithelial cell damage observed after ex vivo incubation, the ability of the lentivirus to transduce the basal progenitor cells in order to regenerate epithelium that would express the genes of interest was assessed. To overcome the difficulties with immunohistochemical staining of the abnormal epithelium of ex vivo transduced tracheae, C57 BL/6 to C57 BL/6 isografts were performed to ensure healthy epithelium after 3 days of transplantation. The airway epithelium of isografts after 3 days of transplantation appeared regenerated with improved histology (Figure 3.4A and B) in comparison to ex vivo transduced tracheae. Immunohistochemical staining of GFP in isografts appeared to be stronger in LV-GFP-NGFR and LV-GFP-Bcl-2 transduced tracheae than the untransduced control. However, this was not as obvious as expected (Figure 3.4C). There were no positive labeled cells for Bcl-2 in any of the isografts (Figure 3.4D) indicating the lentivirus transduction levels of the epithelial progenitor cells were too low to be detected by immunohistochemistry.    59      60 Figure 3.4. Histological and immunohistochemical analysis of lentivirus transduced 3 day isografts. Original magnification 4X (A) and 40X (B-D). (A and B) Histological examination of 3 day C57 BL/6 to C57 BL/6 isografts demonstrate an intact epithelium with minimal epithelial cells loss. (C) Immunohistochemical staining for GFP appeared to be slightly stronger in both LV-GFP- NGFR and LV-GFP-Bcl-2 transduced tracheae, however untransduced trachea also showed some background staining. (D) Bcl-2 staining was not detected in the LV-GFP- Bcl-2 transduced trachea as was expected, demonstrating the difficulties in efficiently transducing the airway epithelium.    3.5. Development of OAD after heterotopic tracheal transplantation The HTT model was used to study the development of OAD in mice. BALB/c to C57 BL/6 allografts and C57 BL/6 to C57 BL/6 isografts were first performed without any treatment to confirm the model outcome. C57 BL/6 to C57 BL/6 isografts were harvested at 28 days and BALB/c to C57 BL/6 allografts were harvested at 14 and 28 days. In this model, both isografts and allografts lose their AECs during an early ischemic phase before neovascularization of the pouch occurs. Isografts and allografts begin a re- epithelialization phase around day 7, however allografts experience alloimmune- mediated injury during an inflammatory phase between days 10 to 14 and the AECs undergo apoptosis (51).  These observations are confirmed upon the histological analysis of 14 day allografts that demonstrated extensive inflammatory infiltrate and epithelial cell loss with areas of basement membrane denudation (Figure 3.5E). After the inflammatory infiltration subsides, fibroproliferation and ECM deposition leading to luminal occlusion is observed by day 28 (Figure 3.5C and D). As OAD is primarily an immune-mediated rejection disease, the complete regeneration of the airway epithelium is allowed in isografts (Figure 3.5D) and the lumen remains free of fibrosis even after 28 days of transplantation (Figure 3.5 A).  61  Figure 3.5. Histological analysis of isografts and allografts in the HTT model. Original magnification 4X (A-C) and 40X (D-F). (A and D) Isografts 28 days after transplantation maintain a normal histology with intact airway epithelium and no evidence of fibrosis. (B and F) 14 day allografts demonstrate inflammatory infiltrate and thickening of submucosal tissue. Basement membrane is denuded of epithelial cells. (C and F) 28 day allografts are characterized by severe fibrosis and total obliteration of the airway lumen. No epithelium is present.                 62  3.6. Effect of lentivirus-mediated gene transfer of Bcl-2 on 14 day allografts.  The effect of lentivirus-mediated gene transfer of Bcl-2 on the development of OAD was investigated in the HTT model. Although Bcl-2 levels could not be detected in 3 day isografts, gene expression in the regenerated epithelium may have been delayed, or minimal expression of Bcl-2 could be sufficient for inhibiting AECs loss. As before, BALB/c donor tracheae were subjected to ex vivo incubation and left untransduced in media alone, or incubated with LV-GFP-NGFR or LV-GFP-Bcl-2 for 16 hours overnight. Excess virus was washed the following day and tracheae were incubated for an additional 48 hours in fresh complete media to allow gene expression to take place. All tracheae were then heterotopically transplanted into the subcutaneous pouch of the recipient C57 BL/6 mouse. Allografts were harvested at 14 days, frozen down and sectioned, and scores for luminal occlusion and epithelial integrity were determined by computer-aided morphological analysis. The luminal occlusion of the 14 day allografts appeared histologically similar (Figure 3.5A-C). Mean luminal occlusion scores for untransduced, LV-GFP-NGFR and LV-GFP-Bcl-2 14 day allografts were 70.07%, 69.94% and 71.84% respectively (Figure 3.6). Neither LV-GFP-NGFR nor LV-GFP-Bcl-2 showed any effect on the percentage of luminal occlusion compared to the untransduced donor trachea. As expected, all allografts showed an increase in luminal occlusion when compared to a normal wild-type trachea (Figure 3.6D). While luminal occlusion scores were similar between each allograft, there were some differences noted in epithelial integrity (Figure 3.7A-C). The epithelial integrity of all 14 day allografts ranged from healthy and ciliated, flattened and squamous, to completely absent. Wild type trachea demonstrated the highest percentages of healthy epithelium with no absent epithelium.  The lowest  63 percentage of healthy epithelium and accordingly the greatest percentage of absent epithelium were observed in the untransduced trachea (Figure 3.7A). Both lentivirus transduced tracheae had similar percentages of absent epithelium, but the mean percentage of healthy epithelium was higher in the LV-GFP-Bcl-2 treated trachea (Figure 3.7C). It is important to note that the range of epithelial integrity scores between all the transplant experiments was considerable, depicted by the large error bars (Figure 3.6E), and so the differences between the LV-GFP-Bcl-2 transduced allografts and the controls were considered statistically insignificant. These results indicate that efficient transduction of the AECs is rather variable but in some transplants, successful transduction of epithelium may have occurred, as the Bcl-2 transduced tracheae were able to maintain the highest percentage of healthy epithelium among allografts.     64    Figure 3.6. Tracheal histology of 14 day allografts and wild type tracheae. Original magnification 4X. (A) Untransduced 14 day allograft (B) 14 day allograft treated with lentivirus-GFP- NGFR (C) 14 day allograft treated with lentivirus-GFP-Bcl-2. (D) Balb/C wildtype trachea, no transplantation. (E) Computer morphometric analysis of luminal occlusion. Values are represented as mean ± SEM (n = 8-10/ group for allografts, n = 2 for WT).    65  Figure 3.7. Epithelial integrity of 14 day allografts and wild type tracheae. Original magnification 40X. (A) Untransduced trachea demonstrated the lowest percentage of healthy epithelium and highest percentage of absent epithelium. Arrow points to areas of epithelial cell loss. (B) LV-GFP-NGFR treated trachea had the highest percentage of damaged epithelium. Arrow points to flattened abnormal epithelium. (C) LV-GFP-Bcl-2 treated trachea had the highest percentage of healthy epithelium of the allografts. Arrow points to healthy ciliated epithelium. Asterisk shows slight protrusion of the sub-mucosal layer despite the intact epithelium. (D) Wild type trachea has a majority of healthy epithelium without any absent epithelial cells. Values are represented as mean ± SEM (n = 8-10/ group for allografts, n = 2 for WT).       66 3.7. Inflammatory infiltration remains uniform between transduced and untransduced 14 day allografts. The presence of inflammatory infiltration in the tracheal lumen and sub-mucosal layers in 14 day allografts was detected by immunohistochemical staining of lymphocytes with anti-CD45 (Figure 3.7A-C) and macrophages granulocytes, dendritic cells and natural killer cells with anti-CD11b (Figure 3.7D-F) antibodies. Positive staining for inflammatory cells was observed in the airway wall as well as the lumen of all tracheal allografts. In allografts with comparable rejection or luminal occlusion, inflammatory infiltration was also uniform between untransduced and transduced tracheae.  These results indicated that treatment of tracheae with LV-GFP-Bcl-2 did not prevent the pro- inflammatory response and the infiltration of inflammatory cells into the lumen in 14 day allografts.    67  Figure 3.8. Immunohistochemical staining for inflammatory infiltrate in 14 day allografts. Original magnification 40X. (A-C) Immunohistochemical staining with an anti-CD45 antibody demonstrate presence of lymphocytes. (D-F) Immunohistochemical staining with an anti-CD11b antibody positively identifies granulocytes, macrophages, dendritic cells and natural killer cells. No difference in inflammatory infiltration was observed between differentially treated allografts.           68 3.8. Effect of lentivirus-mediated gene transfer Bcl-2 on 28 day allografts. The effect of lentivirus-mediated transduction of Bcl-2 on the development of fibrosis and OAD was also examined 28 days post-transplantation. The lumen of untransduced, LV-GFP-NGFR and LV-GFP-Bcl-2 transduced tracheae were completely obliterated with fibrotic tissue after transplantation (Figure 3.9A-F). The mean luminal occlusion scores for untransduced, LV-GFP-NGFR and LV-GFP-Bcl-2 treated allografts were 90.38%, 90.32% and 83.95% respectively (Figure 3.9G). The AECs of all the allografts were also completely absent  (Figure 3.9D-F and H). These findings suggest that transduction of the donor tracheae  with Bcl-2 prior to heterotopic transplantation was not capable of preventing long term epithelial cell loss or progression of OAD.       69  Figure 3.9. Tracheal histology of lentivirus transduced 28 day allografts. Original magnification 4X (A-C) and 40X (D-F). (A and D) Untreated trachea 28 day allograft demonstrated complete loss of epithelium and complete luminal obliteration. (B and E) LV-GFP-NGFR does not show any effect on prevention of luminal occlusion or preservation of epithelial integrity. (D and F) LV- GFP-Bcl-2 was not able to inhibit luminal occlusion or preserve epithelial integrity after 28 days of transplantation.       70   Figure 3.9. (G) Mean scores of luminal occlusion for 28 day allografts by computer- aided morphological analysis. All allografts had high levels of luminal occlusion. (H) Average epithelial integrity scores for 28 day allografts by computer morphological analysis. All allografts had no healthy epithelium and very high percentages of absent epithelium. Values are represented as mean ± SEM (n = 5-8/ group).           71 3.9. In vitro adenovirus-mediated transduction of HeLa cells  In the second group of experiments, a different mechanism of gene transfer and different anti-apoptotic protein were investigated. Adenovirus encoding the anti-apoptotic gene, XIAP, was provided by Dr. Bruce Verchere and used in similar methods as the lentivirus to inhibit AEC apoptosis. In vitro overexpression of XIAP was determined by western blotting of HeLa cell lysates transduced  with the adenovirus construct. HeLa cells were left untransduced (UT), or transduced with AdGFP or AdXIAP at either 1pfu/cell (1) or 10 pfu/cell (10) for 48 hours (Figure 3.10). HeLa cells transduced with AdXIAP at both concentrations demonstrated an overexpression of XIAP as detected by the 55kDa band. Untransduced and AdGFP treated cells showed basal levels of XIAP. The 117kDa protein, vinculin, served as loading control. Therefore, the AdXIAP construct was confirmed to be functional and allowed overexpression of XIAP in transduced cells.          72 Figure 3.10. Adenovirus transduction of HeLa cells. Cell lysates from Untreated HeLa cells (UT), AdGFP treated with 1pfu/cell (1) or 10pfu/cell (10) and AdXIAP treated with 1 or 10 pfu/cell. Overexpression of XIAP is observed as the 55kDa band in AdXIAP transduced cells only at both 1 and 10 pfu/cell concentrations compared to untransduced and the AdGFP control. Vinculin served as loading control.                 73 3.10. Assessment of gene transfer after ex vivo adenovirus-mediated transduction Ex vivo treatment of excised trachea with AdGFP and AdXIAP was performed in similar manner as with the lentivirus previously. Tracheae were subjected to 16 hours of overnight incubation with the adenovirus, an additional 48 hour incubation in fresh media, and then frozen and sectioned for assessment of GFP and XIAP overexpression. As with the ex vivo lentivirus transduction results, the airway epithelium of both the AdGFP and AdXIAP transduced tracheae was damaged, flattened and abnormal (Figure 3.11A and B). Immunohistochemical staining for GFP and XIAP was again difficult as with the lentivirus transduced tissues. The AECs appeared to have background staining at similar levels for both GFP in AdGFP and AdXIAP transduced tracheae (Figure 3.11C). Immunohistochemical staining for XIAP had similar results to the GFP staining, as both AdGFP and AdXIAP transduced tracheae showed similar levels of staining (Figure 3.11D). These results again demonstrate the difficulty in performing immunohistochemical staining on damaged or abnormal epithelium as observed by the high levels of background staining. Treatment of tracheae with AdXIAP was not sufficient for inhibiting AEC apoptosis caused by ischemia during the ex vivo incubation period, however it is still possible that epithelial progenitor cells may have been transduced.   74   75 Figure 3.11. Histological and immunohistochemical analysis of ex vivo adenovirus transduced tracheae. Original magnification 4X (A) and 40X (B-D). (A and B) Both AdGFP and AdXIAP transduced tracheae have flattened and damaged airway epithelium after ex vivo incubation. (C) AdGFP and AdXIAP tranduced tracheae stain positive for GFP. GFP staining of transduced tracheae could not be confirmed due to background staining present in AdXIAP transduced trachea. (D) AdGFP and AdXIAP demonstrate positive staining for XIAP. Expression of XIAP was also not confirmed due to presence in both the control AdGFP and AdXIAP treated trachea (n = 2).   3.11. Assessment of adenovirus-mediated gene transfer in 3 day isografts The ability of the adenovirus constructs to transduce the airway epithelial progenitor cells and regenerate epithelial cells expressing GFP or XIAP was investigated. To allow full regeneration of the airway epithelium, isografts were performed and heterotopically transplanted for 3 days. Histological analysis of the 3 day isografts revealed a regenerated epithelium, however, the epithelial integrity was still varied as flattened epithelium was still present in both AdGFP and AdXIAP transduced tracheae (Figure 3.12A and B). Immunohistochemical staining for GFP appeared to be prominent in the AECs of the AdGFP transduced trachea. However, background staining of GFP was still present in the AdXIAP transduced trachea (Figure 3.12C). Detection of XIAP expression was not improved in the 3 day isografts, as immunohistochemical staining of the AECs for XIAP appeared to be negative for both AdGFP and AdXIAP (Figure 3.12D). These findings suggest that the ex vivo transduction of the tracheal epithelium with adenovirus could not produce significant gene transfer of GFP or XIAP to the epithelial progenitor cells, and transgene expression could not be easily detected by immunohistochemical staining.  76   77 Figure 3.12. Histological and immunohistochemical analysis of adenovirus transduced 3 day isografts. Original magnification 4X (A) and 40X (B-D). (A and B) Histological assessment of AdGFP and AdXIAP transduced isografts 3 days post-transplant. Both tracheae show some epithelial regeneration, but still have some epithelial abnormalities including shedding and flattened epithelium. (C) Analysis of GFP expression by immunohistochemical staining. AdGFP transduced isografts show positively transduced epithelial cells, however AdXIAP transduced isografts also show GFP staining, which is a false positive. (D) Weak background staining for XIAP is observed in the AECs for both AdGFP and AdXIAP transduced tracheae (n = 2).  3.12. Effect of adenovirus-mediated gene transfer of XIAP on 28 day allografts Despite the lack of positive staining for XIAP in the 3 day isografts, the transduced tracheae were heterotopically transplanted to investigate whether transgene expression may be delayed or minimal expression of XIAP was capable of inhibiting OAD progression. Allografts were harvested after 28 days of transplantation and histological analysis of the tracheae was performed. AdGFP and AdXIAP transduced allografts were harvested after 28 days and assessed for luminal occlusion and epithelial integrity. Although the mean luminal occlusion score for AdXIAP of 80.57%, was lower than the mean of 90.93% in AdGFP transduced allografts, the difference was determined not to be significant. The 28 day allografts from both adenovirus treated trachea showed similar histological appearance to each other (Figure 3.13A and B) and to control untransduced allografts previously described (Figure 3.9A and D). The airway epithelium was completely absent in both of the adenovirus transduced allografts, as the lumen was already obliterated (Figure 3.13C and D). These findings indicated that adenovirus- mediated transduction of XIAP, was not effective in preventing the apoptosis of the airway epithelium or the development of OAD in the murine HTT model.    78  Figure 3.13. Histological analysis of adenovirus transduced 28 day allografts. Original magnification 4X (A and B), 40X (C and D). (A and C) AdGFP treated tracheae demonstrated on average a high percentage of luminal occlusion. Granulation tissue and fibrosis is observed in the lumen. (B and D) AdXIAP treated tracheae had similar results as the control adenovirus, AdGFP, and luminal occlusion was not significantly reduced. Both AdGFP and AdXIAP transduced allografts demonstrated complete loss of their airway epithelium.   79  Figure 3.13. (E) Mean values of luminal occlusion in adenovirus transduced allografts 28 days post-transplant. There was no significant difference observed between AdGFP or AdXIAP treated tracheae. Values are represented as mean ± SEM (n = 7-8/ group).                          80 3.13. Potential inhibition of luminal occlusion and fibrosis with a CD36 synthetic peptide  In the third group of experiments, the inhibition of latent TGF-β1 by a CD36 synthetic peptide on the development of OAD and inhibition of fibrosis in murine tracheal allografts was investigated. Excised donor tracheae were initially incubated with 80µg of the CD36 peptide for 4 hours prior to transplantation. Additional 16µg administrations of the peptide were given at the site of the graft, at the time of the transplantation, and at 7, 14 and 21 days post-transplant. Tracheae were harvested at 28 days and frozen for histological assessment. The results showed that treatment with the synthetic CD36 peptide was capable of decreasing the luminal occlusion and severity of OAD in 28 day tracheal allografts (Figure 3.14B and D) compared to PBS controls (Figure 3.14A and C), thus supporting the initial hypothesis. The average luminal occlusion score for CD36 peptide treated allografts was 78.36% compared to the control allografts treated with PBS of 94.74% (Figure 3.14E). However, upon statistical analysis, these results were determined to be not significant. This was likely due to the inconsistency of the luminal occlusion scores amongst the transplant experiments performed. While some transplants demonstrated what appeared to be a significant reduction in luminal occlusion, many transplants developed OAD and had completely obliterated lumens. As control allografts consistently demonstrate obliterated lumens after 28 days of transplantation, any reduction in the luminal occlusion score is an exciting observation. Although the consistent prevention of luminal obliteration was not observed by the use of the CD36 peptide, it appears it may have some capability to decrease the severity of the fibro- proliferative processes of OAD. As may be expected, the CD36 synthetic peptide did not  81 appear to have an effect on the preservation of epithelial integrity in allografts. These transplantation experiments suggest that the inhibition of latent-TGF-β1 activation may be a desirable route for manipulation in inhibiting fibrosis and the development of OAD.        82  Figure 3.14. Histological analysis of CD36 peptide treated 28 day allografts. Original magnification 4X (A and B) and 40X (C and D). (A and C) Allografts treated with PBS did not show any reduction in luminal occlusion after 28 days of transplantation. (B and D) Administration of the synthetic CD36 peptide showed a small decrease in luminal occlusion and fibrosis in allografts but was determined to be statistically insignificant.      83  Figure 3.14. (E) Allografts treated with the CD36 peptide had on average a lower percentage of luminal occlusion. Values are represented as mean ± SEM (n = 4-12/ group).                  84 DISCUSSION  The long-term success of lung transplantation is hindered due to patients developing chronic allograft rejection manifesting as BO (1). Small rodent models of BO involving tracheal transplants develop what is called OAD and have revealed the importance of the airway epithelium as a primary immunological target and that the loss of the epithelium is sufficient for the development of OAD (67-69). The ineffective repair and regeneration of the epithelium is believed to initiate a cycle of injury and repair leading to fibroproliferative processes that cause the obliteration of the lumen (16, 42). While these studies have presented compelling evidence that the maintenance of the airway epithelium is important in OAD, there has been a lack of studies on the prevention of OAD by inhibiting the loss of the airway epithelium. The first objective of this thesis project sought to investigate this theory. Using gene therapy, it was hypothesized that ectopic expression of anti-apoptotic proteins by viral-mediated delivery would be able to regulate the survival of the AECs and therefore inhibit the progression of the disease. The murine HTT model was chosen due to its simplicity and reproducibility of the fibrotic OAD lesion.  In the past decade, gene therapy has become an increasingly desirable approach in numerous settings including the prevention of ischemia induced graft loss in transplantation, as well as in delivering necessary gene products in severe respiratory diseases. Lentivirus vectors derived from HIV have become popular gene delivery vehicles due to their ability to transduce non-dividing cells at high efficiencies (180-182). Modifications to the third generation lentivirus vectors provided a significant  85 improvement to the virus biosafety and gene transfer efficiency. Lentiviruses were pseudotyped with the envelope protein glycoprotein G from the unrelated vesicular stomatitus virus (VSV-G), which allowed the deletion of accessory HIV genes and altered the virus' mode of entry from fusion at the plasma membrane, to the endocytic pathway. The VSV-G envelope also broadened the range of cell types the lentivirus could target and allowed production of higher titers and multiplicity of infections (183, 184).  Another vector that has been extensively studied for use in gene therapy is the adenovirus. Like the lentivirus, the adenovirus offers high gene transfer efficiency and has the ability to transduce a wide range of target cells (185, 186). While the lentivirus is capable of stable integration into its host's genome, the adenovirus provides transient gene expression lasting two to four weeks (187). As adenoviruses were common causes of respiratory tract infections in humans, they were therefore proposed for use in lung- directed gene therapy (170, 188). For these reasons, both the lentivirus and adenovirus were chosen to determine which mode of gene transfer would be most effective in transducing the airway epithelium of tracheal explants.  In animal models, the overexpression of the anti-apoptotic genes to promote survival of transplanted tissues has already been demonstrated in pancreatic islets (174, 189) and hepatocytes (172, 173, 190). In experimental OAD, successful gene therapy with anti- apoptotic genes should inhibit AECs apoptosis caused by primary graft dysfunction, as well as other non-immune and immune-mediated graft injury (169, 191). The use of the murine HTT model in this thesis project also presented the opportunity for ex vivo  86 transduction, avoiding the many obstacles involved in in vivo clinical situations (191) and thus providing a realistic and simple approach for transplantation gene therapy.  The foundation for successful gene therapy requires long lasting efficient gene transfer. This aspect has proven to be very difficult regarding the transduction of the airway epithelium and is the major challenge to overcome in continuing studies. Although it was shown that the LV-GFP-Bcl-2 and AdXIAP vectors could exhibit overexpression of their respective anti-apoptotic genes in vitro in cell lines, transgene expression could not be detected in ex vivo transductions. When donor tracheae were analyzed after the initial overnight virus infection and the following 48-hour incubation, the integrity of the airway epithelium was much lower than anticipated. The histology of untransduced, LV-GFP- NGFR, LV-GFP-Bcl-2, AdGFP and AdXIAP treated tracheae revealed epithelial abnormalities and significant epithelial cell loss. The most likely cause of damage was from ischemia related apoptosis. Ischemia is also a major cause of damage to the airway epithelium in isografts and allografts in the murine HTT model, as the subcutaneous pouch is not vascularized in the first few days following transplantation (51). It is not surprising that the removal of vascularization to the trachea for several days would lead to epithelial damage.  It is also important to note that the ex vivo incubation involving the complete submerging of the tracheal tissue into the liquid media was not optimal and is different than the normal air-liquid interface that the trachea is accustomed to in vivo. Ex vivo tracheal culture systems have been developed in which the air-liquid interface is preserved and  87 organ culture takes place under a sealed pressure jar (192). However, due to limited resources and the objective of infecting tracheae by incubation with the virus vectors, this system was not feasible.  It was expected that the expression of the anti-apoptotic genes in the AECs would aid in the prevention of the ischemic damage to the epithelium. Instead, the histological analysis of the ex vivo transduced tracheae revealed a dilemma in which the incubation time that was necessary to allow gene expression in the AECs actually exacerbated the amount of ischemic damage to the epithelium. Due to the damaged and apoptotic epithelium, proper immunohistochemical staining of positively transduced AECs with GFP, Bcl-2 or XIAP, was not possible. It is known that the tracheal epithelium has a great capacity for regeneration by basal progenitor cells (193) (194) and so it was still possible that the immune-mediated damage to the AECs after transplantation could be prevented. If efficient transduction of the basal progenitor cells with Bcl-2 or XIAP was achieved, the regenerated epithelium would also express Bcl-2 or XIAP and could potentially be protected from undergoing apoptosis and stop the progression of OAD.  Subsequent transplantation experiments were performed with ex vivo transduced tracheae and analyzed at 14 and 28 day time points after transplant. In the 14-day allografts transduced with LV-GFP-Bcl-2 or LV-GFP-NGFR, the mean luminal occlusion scores were not significantly lower than the untransduced allografts and ranged from 70-72%. Wild type BALB/c tracheae that were not transplanted provided a mean baseline luminal occlusion score of approximately 42%. The results for luminal occlusion in 14-day  88 allografts were consistent with previous studies using the murine HTT model, in which allografts experienced thickening of the tracheal wall due to submucosal inflammatory infiltration (142, 195). Immunohistochemical staining of CD45 and CD11b identified the infiltration inflammatory cells as leukocytes, granulocytes and macrophages. Lentivirus transduction did not show any effect on inflammatory infiltration compared to untransduced allografts.  Upon analysis of the epithelial integrity of the 14-day allografts, it appeared LV-GFP- Bcl-2 and LV-GFP-NGFR transduced tracheae had reduced epithelial cell loss as noted by the mean percentages of absent epithelium compared to untransduced allografts. Untransduced tracheal allografts had the highest percentage of absent epithelium of 76% and accordingly lowest mean percentage of healthy epithelium at 10%. The highest mean percentage of healthy epithelium was observed in the LV-GFP-Bcl-2 transduced allografts. This result suggested that although transgene expression could not be detected, some minimal expression of Bcl-2 may have promoted the survival of the AECs and therefore increased epithelial integrity. However, after performing statistical analysis, these observations were determined to be insignificant. The large range in the histological assessment scores between transplant experiments suggests that transduction of the Bcl-2 transgene was not consistent and successful inhibition of AEC apoptosis could also not be consistently achieved. Another unexpected observation was that allografts treated with the LV-GFP-NGFR control vector appeared to have overall better epithelial integrity than the untransduced allografts, although this was also shown to be statistically insignificant. It is unknown whether the lentivirus itself can inhibit apoptosis, but this property would  89 be advantageous for the survival of the virus during infection. The observation that the Bcl-2 transduced tracheae did maintain the highest percentage of healthy epithelium may be taken as a preliminary result, and suggests that better mechanisms for AEC transduction and expression should be considered for the future.  Both lentivirus and adenovirus-mediated transduction of Bcl-2 and XIAP failed to promote survival of the AECs and inhibit the progression of OAD in 28-day allografts. As expected, untransduced allografts experienced complete loss of the epithelium and developed the OAD fibrotic lesion occluding the lumen. Control virus vectors, LV-GFP- NGFR and AdGFP also showed similar results of luminal occlusion. These observations suggest that either the transduction of Bcl-2 or XIAP was not sufficient to protect the epithelium from immunological injury, or insufficient gene transfer and expression of Bcl-2 or XIAP took place. As detection of the transgene expression was still difficult even in areas of intact epithelium in allografts, the efficiency of the gene transfer was questioned.  Effective lentivirus and adenovirus-mediated gene delivery to the mature airway epithelium is challenging (196). Despite initial results demonstrating high levels of gene transfer by adenoviruses (197-199), several limitations were experienced in subsequent trials. For example, diminished transgene expression was observed due to the activation of host inflammatory responses and the generation of neutralizing anti-adenoviral antibodies and cytotoxic T lymphocytes that eliminate transduced cells (170, 188, 200, 201). Additionally, the ability of the lentivirus and adenovirus to transduce non-dividing  90 cells in other applications, relatively limited gene transfer efficiency has been achieved in the differentiated respiratory epithelium (196, 202, 203). Unlike other easily transducible cells types, the airway epithelium has a discriminating defense mechanism to protect the organism from pollutants, irritants, viruses and bacteria found in the environment (61, 62). Mucociliary activity and epithelial tight junctions act as physical barriers, preventing viral vectors from being able to infect the epithelium. Lastly, and perhaps most importantly, the preferred route of entry for VSV-G pseudotyped lentiviruses and adenoviruses is through receptors located on basolateral surface of the epithelial cell (202, 204).  Therefore, it is not surprising that lentivirus and adenovirus vectors do not efficiently transduce AECs.  Much effort has been made to achieve efficient gene delivery in the airway epithelium, particularly for the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene as a gene therapy approach for cystic fibrosis.  Recent studies investigated the in vivo transduction of fetal developing airways with CFTR as a means to overcome the low levels of transduction in mature airways previously described (205, 206). Another approach is to pre-treat the airways to transiently disrupt epithelial tight junctions and allow the viral vectors to reach their basolateral receptors. Agents such as EGTA (207), sodium caprate (208, 209) and the detergent lysophosphatidylcholine (LPC) (171, 210) have all been used to modify the tight junction barrier to allow vector entry and increase transduction efficiency. LPC has also been shown to have other side effects that benefit transduction efficiency such as acting as a mucolytic agent to dissolve mucus and decrease cilia beat, both of which increase residence time of the virus (171). Another  91 alternative is to use lentiviruses pseudotyped with apically targeted viral envelopes like the baculovirus GP64 envelope or Ebola Zaire filovirus envelope (171, 211, 212). In a direct comparison between VSV-G pseudotyped lentivirus with LPC pre-treatment and an apically targeted pseudotyped lentivirus without pre-treatment, higher persisting levels of transduction were observed in pre-treated VSV-G lentivirus transduced cells. Pre- treatment with LPC may be the best approach for gene delivery to the airway epithelium; however, absolute transduction is still relatively low with approximately only 2.5% of the total epithelium receiving the gene of interest (171). Thus, gene delivery to the airway epithelium to treat pulmonary diseases such as cystic fibrosis or OAD is not a simple and straightforward process.  In future studies, in vivo transduction of the tracheal epithelium following pre-treatment should be investigated to achieve higher gene transfer efficiencies. In vivo transduction would also minimize the epithelial damage caused by ischemia during ex vivo incubations. Another option that would completely bypass any difficulties in transduction efficiency would be to obtain the trachea from transgenic mice overexpressing Bcl-2 or XIAP and perform transplant experiments. By using the transgenic mice as donors, all cells including the AECs lining the tracheal lumen should express the anti-apoptotic gene of interest at significant levels without requiring any ex vivo incubations. The transplanted transgenic tracheae should have an intact epithelium; and the hypothesis that the survival of the AECs will prevent the luminal obliteration in OAD, can then be verified. Although the use of transgenic mice as donors in the murine HTT model is not a  92 realistic therapeutic option, the findings from these experiments could support the theory of inhibiting AECs apoptosis as an approach to inhibit OAD.  In the second objective of this study, the prevention of the fibroproliferative phase of OAD by the inhibition of latent TGF-β1 activation was examined. TGF-β1 has already been implicated in numerous pulmonary fibrotic diseases including the development of BO (41, 135, 137, 143, 213). Other studies have demonstrated that deficiency of the downstream TGF-β1 signal transducer, Smad3, could significantly inhibit ECM deposition (142) and decrease fibroblast to myofibroblast trans-differentiation (141) in murine tracheal allografts. This strategy however, fails to account for other pathways of TGF-β1 signaling, such as through MAPKs.  Therefore, the early activation of latent- TGF-β1 is a favourable therapeutic target for intervention.  A model has been described for latent-TGF-β1 activation in pulmonary fibrosis that involves latent-TGF-β1 forming a complex with TSP-1, and the association of latent- TGF-β1/TSP-1 complex with the TSP-1 cell surface receptor, CD36 (117).  The recruitment of latent-TGF-β1 to the cell surface places it in proximity to plasmin, which can then cleave LAP-1 and release the active form of TGF-β1. A synthetic peptide of the receptor CD36 of amino acids 93-110 has been previously described to successfully inhibit bleomycin-induced pulmonary inflammation and fibrosis in a rat model (214). CD36 is consistently found on monocytes and macrophages (215, 216), both of which have been identified in the OAD model (55). It is therefore hypothesized that the model  93 of plasmin-mediated cleavage of TSP-1/latent-TGF-β1 bound to the CD36 receptor may occur in the progression of OAD.  In findings from this thesis project, it was observed that pre-treatment of tracheae with the CD36 peptide and weekly readministrations were able to reduce luminal occlusion scores in 28-day allografts compared to control mice that received PBS. Although the results were shown to be statistically insignificant due to the wide range in luminal occlusion scores observed between transplants, these results can still be considered valuable in understanding the role of latent-TGF-β1 activation in OAD as the CD36 peptide is still undergoing modifications for improved function. The inhibition of fibrosis with the CD36 peptide demonstrates for the first time that TSP-1 binding to CD36 is an important step in the fibrotic processes in the murine HTT model. These findings also provide supportive data for previous work on the importance of TGF-β1 in promoting fibrosis in OAD. Based on these observations, strategies targeting the function of TGF-β1 would be ideal for inhibiting the fibrotic processes of OAD. Interestingly, some studies have shown that TGF-β1 is beneficial in organ rejection due to its potent immunosuppresive functions that can prevent acute rejection and inflammation (149, 151). In this study, the preliminary results suggest that the pro-fibrotic effects of TGF-β1 may be the more dominant function in the context of murine OAD, as tracheae treated with the CD36 peptide did show a trend in reduced luminal occlusion.  Recently, it is suggested the mechanism in which TGF-β1 contributes to fibrosis in BO is by promoting EMT (144, 145). An interesting aspect to investigate for additional studies  94 would be the effect of CD36 peptide treatment on the presence of EMT in tracheal allografts. Suppression of TGF-β1 activation should decrease the occurrence of EMT and cells should maintain their epithelial cell phenotype expressing junction associated proteins, cell adhesion molecules like E-cadherin, cytokeratins and the apical actin- binding transmembrane protein-1 (146). Accordingly, mesenchymal cell morphology, expression of mesenchymal markers and an invasive phenotype should be more prominent in the untreated allografts versus allografts receiving doses of the peptide. Future studies examining the long-term effects of inhibiting TGF-β1 activation on fibrosis in OAD will be important for determining a functional approach for preventing disease outcome. Results of this study have provided a solid framework for future investigators to design approaches that will suppress TGF-β1 activity and allow for further elucidation of the mechanism in which TGF-β1 acts in lung transplants and the development of BO.  In conclusion, the first thesis objective to transduce the airway epithelium in tracheal allografts proved to be unexpectedly difficult and other mechanisms of gene delivery should be investigated to achieve sufficient transgene expression. While an improvement in the epithelial integrity was observed in Bcl-2 transduced allografts after 14 days of transplantation, there were no significant changes in the luminal occlusion scores after 28 days. Alternative methods that would increase the level of transgene expression should be considered for future studies such as in vivo transduction with pre-treatment of the epithelial tight junctions, the use of apically targeted virus vectors, or the use of transgenic animals overexpressing the anti-apoptotic genes of interest. These methods  95 would also avoid epithelial cell damage experienced during ischemic ex vivo incubations. In the second thesis objective, inhibition of TGF-β1 activation by a CD36 synthetic peptide was capable of suppressing fibrosis observed by the decrease in luminal occlusion and inhibition of the development of OAD in tracheal allografts. 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