The hallmark of graft arteriosclerosis (GA) is vascular remodeling, which includes intimal and medial expansion by matrix deposition and medial smooth muscle cell (VSMC) proliferation with migration into the neointima. Interferon-gamma (IFN-y) plays a causative and key role in immune-mediated remodeling responses typical of those observed in GA. Our cDNA array and immunohistochemical analyses of arteriopathic, explanted human allograft coronary vessels identified vascular endothelial growth factor receptor-2 (VEGFR2) and Neuropilin-2, both VEGF-responsive receptors, and endothelial/smooth muscle cell-derived neuropilin-like molecule (ESDN), as potential specific GA markers. Because of the known VEGFR2-alpha-v--beta3 interactions and the role of alpha-v-beta3 in VSMC migration/proliferation, we utilized the chimeric human arterial transplant/SCID mouse model of GA to address whether alpha-v-beta3 is modulated during GA. We demonstrated that alpha-v-beta3, ESDN and VEGFR2 are all highly upregulated and/or activated as early events in GA, consistent with cell proliferation kinetics. VEGF can induce Th1 cell polarization and activate alpha-v-beta3 integrin. alpha-v-beta3 engagement can, in turn, enhance VEGFR2-mediated signaling. We hypothesize that VEGF, induced by tissue hypoxia and/or alloreactivity, contributes to T cell differentiation, IFN-y elaboration and consequent DTH-like responses. In parallel, VEGF, through its classical receptors and neuropilin-like molecules, modulates VSMC integrin activation and alpha-v-beta3-mediated cell proliferation/migration, a hallmark feature of GA. Specific proposals now include to: (1) determine whether VEGF contributes to the development of IFN-y- producing T cells and GA utilizing VEGF/VEGFR antagonists in the chimeric SCID mouse/human arterial graft model, and in allogeneic T cell-endothelial cell cultures;(2) assess the causative role of alpha-v-beta3 integrin activation/function in allogeneic peripheral blood mononuclear cell (PBMC)- and IFN-y-induced GA and its relationship to IFN-yR and VEGFR signaling, using the SCID/human arterial transplant model and alpha-v-beta3 antibody-mediated blockade, as well as in vivo GA imaging with an activated alpha-v-beta3-targeted agent and microSPECT technology;(3) determine whether ESDN induction in GA is IFN-y-mediated or VEGF-dependent, utilizing the allogeneic human PBMC or IFN-y stimuli in the chimeric mouse model, and whether ESDN expression enhances or terminates maximal GA responses;and (4) address the IFN-y- VEGFR/ESDN-alpha- v-beta3 axis in cardiac transplant patients, correlating intravascular ultrasound- documented GA with microvessel-localized IFN-y, IFN-y-regulated chemokines, VEGF/VEGFRs, alpha-v-beta3 and ESDN expression in endomyocardial biopsy specimens and, for secreted molecules, serum samples. Molecular mechanisms of the pathways identified in vivo in Aims 1-3 will be studied in vitro, and relevance to human GA will be addressed in Aim 4. The proposed studies will establish the validity of an IFN-y - alpha-v-beta3 - VEGFR/ESDN axis in the development of GA, thus defining sensitive and practical markers of early GA.
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