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The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates.

The active state of plasminogen activator inhibitor type-1 (PAI-1) is prolonged when it forms a complex with vitronectin (VN), a major serum protein. Active PAI-1 in the PAI-1:VN complex serves many functions related to fibrinolysis and cell migration but key to these effects is its extracellular distribution. PAI-1:VN complexes can bind to exposed vimentin (VIM) on activated platelet and platelet microparticles, resulting in the assembly of PAI-1:VN:VIM ternary complexes. However, the manner in which the vimentin cytoskeleton is presented extracellularlyi s not well understood. I hypothesized that PAI-1:VN:VIM ternary complex assembly occurs on cell surfaces when microparticle release leads to exposure of vimentin cytoskeleton which can lead to either assembly of the ternary complex or become involved in an autoimmune response specific for vimentin. To follow the intracellular and extracellular fate of PAI-1, I constructed an expression vector encoding PAI-1-dsRed, a fluorescent form of PAI-1, which would permit live cell tracking of PAI-1 in megakaryocytes and endothelial cells. Secondly, to study how vimentin is expressed on platelets and platelet microparticles, flow cytometry was used to isolate vimentin positive platelets or PMP's and atomic force microscopy was performed to image platelets or PMP's at nanoscale resolution. From these studies, I propose a model of vimentin expression in which the junction of microparticle release results in the exposure of cytoskeletal vimentin on both the cell and the microparticle. This exposed vimentin could potentially induce VN multimerization on the same cell surface leading to incorporation of multiple PAI-1:VN complexes. Finally, I investigated how anti-vimentin antibodies can induce platelet:leukocyte conjugate formation. To achieve this, in vitro tests were performed to determine the binding site of anti-vimentin antibodies (AVA's) and how they induce blood cell activation. Overall, my results suggest that vimentin exposure in our model of microparticle release can lead to ternary complex assembly if suitable quantities of PAI-1 are released during platelet activation. In the setting of transplant vasculopathy with high titres of AVA's, vimentin-positive granulocytes can bind these autoantibodies, which then leads to platelet activation and the formation of platelet:leukocyte conjugates. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

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

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

Long-term survival of cardiac transplant recipients continues to be severely limited by the development of a pathological, chronic rejection process, termed allograft vasculopathy (AV). This remains to be the case despite dramatic improvements in the areas of surgical techniques, pre- and post-operative care, and immunosuppression. To model the clinical setting we used calcineurin inhibitor (CNI) immunosuppression, the cornerstone of post-transplant immunosuppression, in a murine aortic interposition transplant model for our analysis of AV development. This model mimics the presentation of AV in human cardiac transplants through the development of a progressively occlusive neointimal lesion. Our previous work in this model has demonstrated that CD8+, but not CD4+, T cells play a role in neointimal lesion formation. Further investigation also highlighted a specific requirement for either CD8+ T cell-derived IFN-γ or direct cytotoxicity in the development of lesion formation. In the current study we confirmed that CD8+ T cell-derived IFN-γ also leads to the loss of medial smooth muscle cells, an event which inversely correlates with lesion formation. The Fas/FasL direct cytotoxic pathway was also significantly involved in neointimal lesion formation and medial remodeling. This work clarified the pathways utilized by CD8+ T cells in their role as mediators of AV development. Recognizing the threat that CD8+ T cells pose to cardiac transplant recipients in the presence of CNI immunosuppression, and a growing concern with the presence of anti-donor memory T cells in transplant recipients, we next explored the development of memory CD8+ T cells in the presence of CNI immunosuppression. We first established that memory CD8+ T cells could not develop when CNI immunosuppression was initiated immediately post-challenge. Next, we hypothesized that the clinical practice of CNI delay post-transplant would permit the development of de novo memory CD8+ T cells. Immediate and early initiation was sufficient to prevent the development of de novo memory CD8+ T cells. However, later delay to within a clinically practiced timeframe did permit the development of de novo memory CD8+ T cells. Our analysis revealed that this population demonstrated equivalent functionality to de novo memory CD8+ T cells generated in the absence of CNI immunosuppression.

BACKGROUND. Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a risk factor for coronary artery disease (CAD) in nontransplant patients. We evaluated the association between Lp-PLA2, cardiac allograft vasculopathy (CAV) assessed by 3D intravascular ultrasound, and incidence of cardiac adverse events in heart transplant recipients. MATERIALS AND METHODS. Fasting blood samples were obtained and stored from a cross-section of 112 cardiac transplant recipients attending the Mayo cardiac transplant clinic in 2000 to 2001, mean of 4.7 years after transplant. Lp-PLA2 was measured in plasma aliquots using an enzyme-linked immunoassay. Fifty-six of these patients subsequently underwent two 3D intravascular ultrasound studies in 2004 to 2006 12 months apart. Cardiovascular (CV) events included percutaneous coronary intervention, coronary artery bypass grafting (CABG), reduction in left ventricular ejection fraction (LVEF) ≤45% secondary to CAV and CV death. RESULTS. High Lp-PLA2 level was associated with increase in plaque volume (r=0.43, P=0.0026) and percent plaque volume (r=0.45, P=0.0004). The association remained significant after adjusting for clinical and lipid variables. During follow-up of 5.1±1.6 years, 24 CV adverse events occurred in 15 of 112 (13%) heart transplant patients. Lp-PLA2 level>236 ng/mL (higher tertile) identified a subgroup of patients having a 2.4-fold increase of relative risk for combined endpoint of CV events (percutaneous coronary intervention, CABG, LVEF<45%, and CV death; 95% CI 1.16-5.19, P=0.012) compared with patients with Lp-PLA2≤236 ng/mL. CONCLUSIONS. Lp-PLA2 is independently associated with progression of CAV and predicts a higher incidence of CV events and CV death in transplant patients. This finding supports the concept that systemic inflammation is an important mediator of CAV. Lp-PLA2 may be a useful marker for risk of CAV and a therapeutic target in posttransplant patients.

Près de la moitié des patients atteints de sclérodermie systémique (ScS) présentent des troubles de la pigmentation cutanée. La mélanogenèse est sous la dépendance, outre les facteurs épidermiques, de facteurs dermiques produits par les fibroblastes et les cellules endothéliales. Parmi ces facteurs se trouve NOV (CCN3) (protéine appartenant à la même famille que CTGF (CCN2), protéine pro-fibrotique augmentée dans la ScS) qui aurait un rôle anti-fibrotique. Nous souhaitons ainsi disséquer le lien entre la composante fibrotique dermique et la pigmentation épidermique. Pour cela, des biopsies cutanées ont été réalisées chez 21 patients ScS, en zone lésionnelle (scléreuse avec ou sans trouble de pigmentation) et non lésionnelle pour 12 d’entre eux. Ces biopsies ont été séparées en 3 fragments qui ont été soit fixés pour analyses histologiques et immunohistochimiques, soit congelés à -80°C pour analyse protéomique ou transcriptomique, soit dissociés pour isoler les cellules cutanées afin de les analyser en protéomique ou transcriptomique. Des témoins de peau et des cellules saines sont utilisées en contrôle. Nous avons dégagé deux types de troubles pigmentaires : une hyperpigmentation s’apparentant à un photo-vieillissement, et une dépigmentation péri-folliculaire apparaissant de manière précoce. Les niveaux d’expression de CCN2 et CCN3 varient chez les patients en fonction des données cliniques, aussi bien au niveau protéique qu’au niveau immunohistochimique. S’il existe des différences en fonction de l’atteinte pigmentaire, il semble que dans les fibroblastes sclérodermiques, la balance CCN2/CCN3 soit déséquilibrée tant au niveau de la zone lésionnelle que cliniquement non lésionnelle, suggérant que CCN2 et 3 pourraient être dérégulés intrinsèquement ou à des stades précoces. Ce travail pourrait aboutir à l’identification d’un phénotype précoce basé sur l’atteinte pigmentaire, et au développement d’une thérapie basée sur la rééquilibration du ratio CCN2/CCN3. / Systemic Sclerosis (SSc) is a rare but potentially deadly connective tissue disease. Its pathophysiology remains partly unknown but combines auto-immunity, small and large vessels involvement and fibrosis of the connective tissue, affecting all organs. Skin features are considered as diagnostic and prognosis markers and include for some patients the presence of pigmentary disorders. In this work, we looked into pigmentary disorders in SSc and their relationship with the pathophysiology of the disease. First, we analyzed the presence of pigmentary disorders among a local cohort of 239 patients as well as their association with systemic involvement in the disease. We have found that diffuse hyperpigmentation was associated with an increased risk of vascular involvement in SSc, particularly digital ulcers. Then, we investigated the molecular basis behind this association. Proteins of the CCN (CYR61 / CTGF / NOV) family are multimodular proteins secreted in the extra-cellular matrix, where they take part in numerous biological processes, such as cell proliferation, adhesion, collagen secretion. Within this family, CCN3 (also called NOV) is a promising candidate, being implicated both in angiogenesis and epidermal homeostasis. We studied CCN3 expression in the skin of SSc patients presenting or not pigmentary disorders, as compared to healthy controls. We found that CCN3 expression was particularly decreased in the dermal vessels in situ, as well as in endothelial cells in vitro. CCN3 inhibition in endothelial cells resulted in altered angiogenesis in vitro, through a decrease in cell migration. We also studied CCN3 expression in SSc epidermis. SSc patients presenting hyperpigmentation exhibited decreased CCN3 in their melanocytes as well as increased CCN3 in their keratinocytes, compared to patients without pigmentary disorders. Overall, CCN3 represents a promising therapeutic lead for SSc patients with vascular involvement, which could bespotted early thanks to the presence of hyperpigmentation.