Chronic rejection is a major obstacle to long-term survival of organ transplants. PPAR-γ agonist rosiglitazone has been shown to reduce graft rejection but the underlying mechanisms remain unclear. Combined treatment of rosiglitazone and anti-IL-5 antibody prevented MHC class II histoincompatiblecardiac graft rejection with a reduction of cellular infiltration, vasculopathy and interstitial fibrosis in a heterotopic heart transplantation model. In particularly, rosiglitazone decreased CD8 T cells infiltration and luminal occlusion, while anti-IL-5 antibody reduced eosinophil infiltration and collagen deposition. Adiponectin gene (APN) is a PPAR-γ target gene, and the expression of APN receptor AdipoRII in grafts, dendritic cells (DCs) and T cells are increased by rosiglitazone. These findings prompted me to further examine the immunomodulatory role of APN in graft rejection. APN is an anti-inflammatory adipocytokine, and has been shown to inhibitimmunostimulatory function of monocytes and macrophages. Rosiglitazone suppresses DCs maturation, activation and proliferation;hence, it is possible that APN could protect graft rejection through immunoregulation of DCs. Here, using in vitro culture systems, I found that APN has only moderate effect on the differentiation of bone marrow derived DCs but itcould alter DC phenotypes. APN-treated DCs showed an increased expression of PD-L1, which is consistent with the increased PD-L1 expression in rosiglitazone treated cardiac allografts. APN-treated DCs led to a decreased proliferation and reduction of IL-2production of T cell. Moreover, APN-treated DCs increased the expansion of Tregs (regulatory T cells) which could be inhibited by the blockage of PD-1/PD-L1 pathway, suggesting that PD-1/PD-L1 pathway and expansion of Tregs played important roles in APN-treated DCs mediated immunomodulation. Further, I employed APN-/-mice for functional and mechanistic studies, and found that cardiac allografts were not rejected by APN-/-recipient mice even after 120 days post-transplantation. Histological analyses revealed very little eosinophils, CD4 and CD8 T cells infiltration; no collagen deposit and no vessel occlusion in the cardiac allografts. Furthermore, Th2 cytokines such as IL-4 and IL-5 were lower in cardiac allografts and in the serum of APN-/-recipient. Inhibition of AMPK signaling, a major APN mediated pathway, reduced the eosinophils infiltration in wild type recipient. In contrast, AMPK activation increased eosinophils infiltration in APN-null recipient. APN enhanced T cell proliferation. AMPK and P38MAPK inhibitors as well as anti-IL-4 antibody inhibited APN-induced T cell proliferation. P38 MAPK inhibitors reduced IL-4 production in mature DCs but enhanced IL-4 expression in immature DCs. In EL-4 T cells, APN increased nuclear expressions of GATA-3 and p-STAT6 and augmented IL-4 expression, and the phenomenon was suppressed by target specific knockdown of AdipoR I and II. In summary, current study provides new mechanistic insights of PPAR-γ activation and APN signaling in the modulation of adaptive and transplantation immunity, establishing a link between metabolism and immune regulation. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

Background: Currently, the optimal long-term therapy for end stage heart failure is heart transplantation. Cardiac allograft vasculopathy contributes to a significant number of deaths following transplantation. This vasculopathy is related to early endothelial injury sustained at the time of organ transplantation and to persistent endothelial injury as a result of cytotoxic immunosuppression, as well as chronic rejection. Epidermal growth factor-like domain 7 (Egfl7), is expressed in endothelial cells upon arterial injury and may have a role in maintaining vascular endothelial integrity and regeneration following injury. Similarly, novel pharmacologic agents such as Bosentan, an endothelin-1 antagonist, and Cilostazol, a phosphodiesterase 3 inhibitor, have been demonstrated to attenuate calcineurin inhibition induced endothelial dysfunction and neointimal hyperplasia, respectively. We hypothesized that, 1) Egfl7 will attenuate endothelial activation, cell adhesion molecule expression and neutrophil adhesion following simulated ischemia-reperfusion injury or exposure to calcineurin inhibition and that, 2) Bosentan and Cilostazol will inhibit neointimal hyperplasia following endothelial injury in a mouse model of vascular injury. Methods: Human coronary artery endothelial cells were subjected to hypoxia-reoxygenation injury or the calcineurin inhibitors Cyclosporine A and Tacrolimus to examine the effects of Egfl7 on these injury mechanisms. Cell adhesion molecule expression, neutrophil adhesion to endothelial cells, and NF-κB activation were measured. Cell adhesion molecule and Egfl7 expression were also examined in a mouse model of neointimal. This model was used to examine the effects of Bosentan and Cilostazol on neointimal hyperplasia. Results: Egfl7 had potent anti-inflammatory properties including inhibition of NF-κB pathway activation, ICAM-1 expression and neutrophil adhesion to injured endothelium. Within vessels exhibiting neointimal hyperplasia, Egfl7 was expressed in regions lacking ICAM-1 expression. Both cilostazol and bosentan attenuated neointimal hyperplasia in isolation as well as during co-treatment with CNI therapies. Conclusions: Egfl7 is an endothelial protective signaling protein with anti-inflammatory properties effective against simulated ischemia-reperfusion injury and calcineurin inhibition mediated injury. Cilostazol and Bosentan are pharmacologic strategies demonstrating efficacy against the development of neointimal hyperplasia. These observations provide a novel therapeutic target and strategies that may be relevant to endothelial protection and prevention of cardiac allograft vasculopathy following heart transplantation.

Background: Currently, the optimal long-term therapy for end stage heart failure is heart transplantation. Cardiac allograft vasculopathy contributes to a significant number of deaths following transplantation. This vasculopathy is related to early endothelial injury sustained at the time of organ transplantation and to persistent endothelial injury as a result of cytotoxic immunosuppression, as well as chronic rejection. Epidermal growth factor-like domain 7 (Egfl7), is expressed in endothelial cells upon arterial injury and may have a role in maintaining vascular endothelial integrity and regeneration following injury. Similarly, novel pharmacologic agents such as Bosentan, an endothelin-1 antagonist, and Cilostazol, a phosphodiesterase 3 inhibitor, have been demonstrated to attenuate calcineurin inhibition induced endothelial dysfunction and neointimal hyperplasia, respectively. We hypothesized that, 1) Egfl7 will attenuate endothelial activation, cell adhesion molecule expression and neutrophil adhesion following simulated ischemia-reperfusion injury or exposure to calcineurin inhibition and that, 2) Bosentan and Cilostazol will inhibit neointimal hyperplasia following endothelial injury in a mouse model of vascular injury. Methods: Human coronary artery endothelial cells were subjected to hypoxia-reoxygenation injury or the calcineurin inhibitors Cyclosporine A and Tacrolimus to examine the effects of Egfl7 on these injury mechanisms. Cell adhesion molecule expression, neutrophil adhesion to endothelial cells, and NF-κB activation were measured. Cell adhesion molecule and Egfl7 expression were also examined in a mouse model of neointimal. This model was used to examine the effects of Bosentan and Cilostazol on neointimal hyperplasia. Results: Egfl7 had potent anti-inflammatory properties including inhibition of NF-κB pathway activation, ICAM-1 expression and neutrophil adhesion to injured endothelium. Within vessels exhibiting neointimal hyperplasia, Egfl7 was expressed in regions lacking ICAM-1 expression. Both cilostazol and bosentan attenuated neointimal hyperplasia in isolation as well as during co-treatment with CNI therapies. Conclusions: Egfl7 is an endothelial protective signaling protein with anti-inflammatory properties effective against simulated ischemia-reperfusion injury and calcineurin inhibition mediated injury. Cilostazol and Bosentan are pharmacologic strategies demonstrating efficacy against the development of neointimal hyperplasia. These observations provide a novel therapeutic target and strategies that may be relevant to endothelial protection and prevention of cardiac allograft vasculopathy following heart transplantation.

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.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p.44-49

Thesis (Ph.D.)--Ohio University, November, 2007. / Abstract only has been uploaded to OhioLINK. Includes bibliographical references (leaves 199-226)

Thesis (Ph.D.)--Ohio University, November, 2007. / Title from PDF t.p. Abstract only has been uploaded to OhioLINK. Includes bibliographical references (leaves 199-226)

Gender differences in myocardial apoptosis of the patients after heart transplantation Background: Many functions of the cardiovascular apparatus are influenced by gender. The aim of our study was to find out the sensitivity to perioperative ischemia of the donor female and male myocardium; and determine how the organism affects the donor myocardium of the other sex after heart transplantation (detection of apoptosis), and whether the investigated biomarkers can predict primary graft dysfunction (PGD). Methods: The research was divided into three prospective studies. The Study 1 included 81 patients undergoing heart transplantation from September 2010 to January 2013. Patients were divided into two groups according to male allograft and female allograft. In order to prove myocardial necrosis the high-sensitive cardiac troponin T (hs-cTnT) method was used. Apoptosis was determined by immunohistochemical detection of caspase-3, Bcl-2, and by the TUNEL method. The Study 2 includeded 58 patients divided into four groups according to gender; both of the recipient and the donor. Apoptosis (caspase-3, Bcl-2, TUNEL) was analysed in these groups during the two-year follow-up. Into Study 3 64 patients were involved. We investigated the relationship in between these biomarkers and the development of PGD after...

INTRODUÇÃO: O significado clínico das vasculites, lesões isquêmicas, efeito Quilty e da presença de eosinófilos em biópsias endomiocardicas de receptores de transplante cardíaco com rejeições leves não foi ainda estabelecido. OBJETIVOS: Verificar se estes achados histológicos encontrados nas biópsias endomiocardicas (eosinófilos, vasculites, efeito Quilty e lesões isquêmicas) são capazes de predizer rejeição aguda do enxerto acompanhada ou não de grave comprometimento hemodinâmico e morte por rejeição aguda. MÉTODOS: Foram reavaliadas 939 biópsias endomiocardicas consecutivas classificadas como OR ou 1R pela de 2005 da Nomenclatura da Sociedade Internacional de Transplante de Coração e Pulmão e dividimos estas em dois grupos (1) Biópsias preditoras: aquelas que precederam rejeição aguda, rejeição aguda associada à grave comprometimento hemodinâmico ou morte e (2) Biópsias não preditoras aquelas que não precederam eventos clínicos. Comparamos a ocorrência dos seguintes achados histológicos: vasculites, lesões isquêmicas, efeito Quilty e eosinófilos por análise uni e multivariada entre os grupos. RESULTADOS: Após análise estatística verificou-se que a presença de vasculite intensa e de eosinófilos como maiores preditores tanto para rejeição aguda futura, apresentando respectivamente as seguintes razões de chance: 10,60 (IC95%: 3,62 31,06. p<0,001) e 6,26 (IC95%:3,16 12,43. p< 0,001) , quanto para rejeição aguda associada á grave comprometimento hemodinâmico, que para este desfecho clínico apresentaram respectivamente as seguintes razões de chance 7,52 (IC95%: 1,45-38,93. p=0,016) e 6,61 (IC95%: 2,38 18,31. p< 0,001), e também para morte em decorrência a rejeição aguda com as respectivas razões de chance: 11,20 (IC95%: 3,53 36,17. p < 0, 001) e 14,50 (IC95%: 2,19 36,17. p = 0,006). CONCLUSÕES: Vasculites intensas e eosinófilos em biópsias do miocárdio são os principais fatores preditores de rejeição aguda, rejeição aguda associada à grave comprometimento hemodinâmico e morte pós - transplante cardíaco / INTRODUCTION: The clinical meaning of vasculitis, ischemic lesions, Quilty effect and the presence of eosinophils in endomyocardial biopsies of transplant recipients with mild rejections have not been established yet. OBJECTIVES: Verify if these histological findings (eosinophils, vasculitis, Quilty effect and ischemic lesions), whose clinical meaning remains unknown so far, are able to predict acute rejection of the transplanted organ, accompanied or not by severe hemodynamic compromise and death due to acute rejection. METHODS: We reevaluated 939 consecutive endomyocardial biopsies classified as 0R or 1R, according to the nomenclature that the International Society for Heart and Lung Transplantation established in 2005. We divided these biopsies in 2 groups, as they follow: (1) Predictor biopsies, which are preceded by acute rejection, acute rejection associated to severe hemodynamic compromise or death and (2) Non-predictor biopsies that did not precede any clinical events. We compared the occurrence of the histological findings studied (eosinophils, vasculitis, Quilty effect and ischemic lesions) through univariate and multivariate analysis among the groups. RESULTS: After an appropriate statistical analysis, the result obtained was the presence of intense vasculitis and eosinophils as the greatest predictors of future acute rejection, presenting the respective odds ratio: 10,60 (IC95%: 3,62 31,06. p<0,001) and 6,26 (IC95%:3,16 12,43. p< 0,001), as well as acute rejection associated to severe hemodynamic compromise, which presented the respective odds ratio for this clinical outcome: 7,52 (IC95%: 1,45-38,93. p=0,016) and 6,61 (IC95%: 2,38 18,31. p< 0,001) and death due to acute rejection, presenting the respective odds ratio: 11,20 (IC95%: 3,53 36,17. p < 0, 001) and 14,50 (IC95%: 2,19 36,17. p = 0,006). CONCLUSIONS: Intense vasculitis and eosinophils in myocardial biopsies post-cardiac transplantation are the chief factors that can predict acute rejection, acute rejection associated to severe hemodynamic compromise or death