Graft arteriosclerosis (GA), defined as progressive loss of lumen in allograft conduit arteries, is the major cause of chronic cardiac allograft failure. Several recent lines of evidence have implicated the cytokine IFN-y as a pro-arteriosclerotic factor and that a key functional effect of IFN-Y on endothelial cells (EC) is an early impairment of EC-dependent relaxation which occurs prior to and is causally linked to smooth muscle cell (SMC) accumulation. Specifically, my colleagues have reported IFN-y-dependent reduction in the function/expression of endothelial nitric oxide synthase (eNOS) in graft EC. Interestingly, IFN-y by itself does not affect eNOS. It acts in concert with INF, another proinflammatoy cytokine, to reduce eNOS expression and NO release by EC. However, the mechanism by which IFN-Y and TNF synergistically reduce NO production by EC is not known, and is the subject of this project. Our data suggest that SOCS1, a member of suppressor of cytokine signaling proteins, is a critical mediator in IFN-y and TNF-induced EC dysfunction. We propose the following hypotheses: 1). In resting (and IFN-y-exposed) EC, SOCS1 binds to inactive (tyrosine phosphorylated) ASK1 leading to mutual degradation of both SOCS1 and ASK1. 2). In response to TNF, ASK1 is dissociated from SOCS1 leading to activation of ASK1-JNK signaling which in turn phosphorylates and activates SOCS1. 3). Activated SOCS1 and ASK1-JNK independently and synergistically inhibit growth factors (e.g. VEGF and IGF-1 )-mediated NO release leading to EC dysfunction and GA progression. We propose the following specific aims to test our hypothesis: 1) Determine the mechanism(s) by which SOCS1 induces ASK1 degradation in EC and how IFN-y and TNF modify this response. 2) Determine the mechanism(s) by which SOCS1 impairs NO function. 3) Determine the role of SOCS1 in EC function in allograft and xenograft models. These studies should facilitate the development of new therapeutic approaches to control GA and graft failure as well as other vascular diseases such as atherosclerosis.
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