Accelerated transplant arteriosclerosis (TA), a manifestation of chronic rejection, is the leading cause of graft failure. The development of anti-HLA antibodies to donor HLA antigens is a major risk factor associated with TA. The overall goal of this proposal is to elucidate the signal transduction pathways that mediate HLA class I induced cell proliferation and the development of TA.
Under specific aim #1, we will establish whether anti- HLA antibodies induce tyrosine phoshphorylation of focal adhesion proteins and assembly of signaling complexes via an actin cytoskeleton dependent pathway in endothelium and smooth muscle. For this, we will establish whether exposure of cultured endothelial cells (EC) and smooth muscle cells (SMC) to monoclonal anti-HLA antibodies and anti-HLA antibodies from transplant patients with chronic rejection is accompanied by FAK phosphorylation and the generation of FAK/Src/Paxillin signaling complexes. We will determine if the phosphorylation of focal adhesion proteins is accompanied by alterations in the organization of the actin cytoskeleton and in the assembly of focal adhesions. We will explore the role of ROK, MLC phosphatase and ERK as upstream components of the class I signaling pathway. We will also determine if class I signaling stimulates anti-apoptotic signals by inducing tyrosine phosphorylation of PI3-kinase, Akt and Bad.
Under specific aim #2, we will identify the signaling pathways leading to MHC class I induced FGF receptor translocation in EC and SMC. For this, we will determine the role of the actin cytoskeleton, phosphorylation of FAK, Src, paxillin and assembly of focal adhesions in class I mediated FGFR translocation to distinct subcellular locations using flow cytometry and confocal microscopy. We will also assess the importance of ROK, ERK, MLC phosphatase in class I mediated FGF receptor translocation. The contribution of the FGF receptor tyrosine kinase activity and ERK phosphorylation to class I induced cell proliferation will be established.
Under aim #3, we will assess the expression of class I induced tyrosine phosphorylation, FGF receptors and anti-apoptotic proteins in clinical biopsy specimens from cardiac allografts with and without evidence of transplant arteriosclerosis. We will determine the correlation between protein phosphorylation events and protein expression with the incidence and time of onset of transplant arteriosclerosis and development of anti-HLA antibodies.
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