An IFN-y-Integrin-Growth Factor Axis in GA Biomarker Development

Yale University, New Haven CT

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Abstract

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