Anastomotic intimal hyperplasia remains as the most important cause of prosthetic arterial graft failure. This is a continuing investigation of the mechanism(s) contributing to intimal hyperplasia at the prosthetic graft/arterial anastomosis. The specific objective of the current study is to determine the sequence of gene-related events associated with the development of hyperplasia at the graft/arterial anastomosis using an in vivo, prosthetic arterial graft model. mRNA differential display will be utilized to identify alterations in gene expression between anastomotic sites and control arterial segments. These differences will then be confirmed and quantified using Northern Blot analysis. For genes that have altered expression, riboprobes will be produced for use in in situ hybridization and polyclonal antibodies formed for use in immunohistocytochemistry. Northern Blots generated from anastomotic sites will be screened using cDNA probes to platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF-beta), angiotensin Il (ATII) and vascular endothelial cell growth factor (VEGF). To evaluate actual levels of synthesized proteins, Western Blot analysis will be used. Simultaneous cell proliferation index will also be determined using two established antibodies: proliferative cell nuclear antigen (PCNA) and MIB- 1. This will allow direct correlation between proliferative events and altered expression of genes seen with differential display and confirmed on Northern Blots. Results from this study will greatly enhance our knowledge of the molecular mechanism(s) of prosthetic arterial graft failure and may provide the basis for future therapeutic strategies to improve graft patency. The fundamental cellular mechanisms identified will also have broad application to the field of vascular biology.
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