? Anastomotic intimal hyperplasia (AIH) remains the most common cause of delayed prosthetic arterial graft failure, a consequence of focal, unregulated gene expression. As graft healing occurs, genes are either up or down-regulated compared to a quiescent arterial wall. Our hypothesis is that this altered gene expression results in cellular proliferation, migration, and extracellular matrix production by smooth muscle cells, leading to AIH. The significance and unique aspect of this work is that our group was the first to identify specific genes that are altered following prosthetic arterial grafting in vivo. This is continuing study of AIH following prosthetic arterial grafting, uses microarray analysis and quantitative real-time RT-PCR (qPCR) to define temporal gene expression at the anastomotic region compared with control artery. This proposal builds on a foundation of molecular data originally derived by our group using differential display and Northern blot analysis to understand the molecular changes that occur at the anastomoses of prosthetic arterial grafts compared with normal arterial wall. Our group was the first to identify alterations in gene expression in the proteosome-ubiquitin pathway following prosthetic arterial grafting. Cell cycle regulators including retinoblastoma susceptibility protein were found to have altered expression following prosthetic arterial grafting. These two pathways subsequently have recently been identified by other investigators as codependent in terms of cell homeostatic functions. Our continued investigation will assess gene expression at additional time points using microarray analysis and computational software techniques. Laser Capture Microdissection will be utilized as an adjunct to localize altered gene expression within the medial wall and within the neointima of the prosthetic graft. In addition, our previous time points of gene expression using qPCR will be validated. Further, downstream products will be assessed using in situ hybridization, and protein expression will be examined using immunohistochemistry. Identification of alterations in gene expression, their time course, cellular localization and computational analysis provides a valuable guide to a comprehensive understanding of anastomotic intimal hyperplasia. ? ?
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