Graft arteriosclerosis (GA) is the major cause of late cardiac allograft failure. Although the precisepathogenesis of clinical GA is unknown, considerable evidence supports a role for IFN-gamma and fordysregulation of nitric oxide synthases (NOSs). In studies conducted during the past funding cycle using ourhumanized mouse model of GA, we have found that an unexpected component of NOS dysregulationinvolves IFN-gamma-dependent expression of inducible (i)-NOS by graft artery infiltrating T cells. The centralhypothesis of this project is that the status of the endothelial cells (ECs) of an allograft artery at the time of theirencounter with host effector or effector memory T cells determines whether those T cells that take upresidence within the vessel wall will secrete IFN-gamma and/or express i-NOS, two characteristic features of Tcells that mediate GA. In this continuation, our aims are: (1) to determine if two important innate immunesignals of tissue injury, namely C5a or HMGB1, act on ECs or T cells to favor the differentiation ofpathogenetic T cells that express IFN-gamma or i-NOS; (2) to identify specific EC molecules that contribute to therecruitment of pathogenetic T cells in vitro or in vivo; (3) to elucidate the control of i-NOS expression andactivity in human T cells and to identify EC signals that contribute to its regulation; and (4) to determine if andhow macrophages or dendritic cells (DCs) autologous to T cells influence their responses toallogeneic ECs in general and how they influence IFN-gamma or i-NOS expression in particular. Theseexperiments will utilize both in vitro assays (co-cultures and flow chambers) and in vivo assays, including ourestablished huPBL-SCID/bg mouse human allograft artery model of GA and two models under development,namely (i) combining adoptive transfer of human T memory cells with engraftment of human hematopoieticstem cells from the same volunteer donor in order to introduce macrophages and DCs, and (ii) transplantation oftissue-engineered synthetic human arteries containing genetically modified ECs into huPBL-SCID/bg mice inorder to assess the role of specific EC molecules. Successful completion of these studies may lead tofurther insights into pathogenesis and to new approaches for prevention or treatment of GA.
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