Genetic Engineering of Vein Bypass Grafts in Vascular and Cardiovascular Surgery

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DESCRIPTION (provided by applicant): Intimal hyperplasia (IH) in arterialized vein bypass grafts is a significant cause of vein graft (VG) stenosis and delayed graft failure. Injury at the time of implantation or as a consequence of transplantation into the high- pressure arterial system contributes to these delayed events. In a canine model, we have identified alterations in the transcriptome following implantation/arterialization injury, and have separated genetic events in the endothelium from those in medial smooth muscle cells (SMC). Using Systems Biology, we have identified the upregulated genes that were most essential to the injury response. Through back propagation, an integrated network was built starting with genes differentially expressed at the latest time-points i.e. 30 days (D), followed by adding upstream interactive genes from each prior time-point. This identified collagen 1A1 (Col1A1) at 30D, as a central cornerstone of back propagation and dominant contributor to IH lesions, as well as Interleukin (IL)- 6, IL-8, and PKC¿ as focus hub genes that were differentially upregulated across all time-points, starting at 2 hours (H) -12H post-surgery. These results establish causality relationships clarifying the pathogenesis of VG implantation injury, and identifying novel targets for its prevention. It is our hypothesis that silencing of focal hub and final lesion genes will diminish processes associated with VG implantation injury and thereby IH. Toward this goal, we have devised and refined methodology for silencing one or more genes under operating room constrains. In the proposed study we will apply siRNA technology to (i) systematically evaluate effectiveness and durability of silencing target genes (IL-6, IL-8, and PKC¿, and Col1A1) and achieving protein knockdown in human saphenous vein endothelial cell (EC) and SMC cultures and confirming this effectiveness ex vivo in the intact wall of the human saphenous vein, (ii) determine the most successful siRNA cocktail that can prevent IH using a mouse vein bypass graft model and (iii) test the most effective siRNA cocktail and determine the genetic sequelae in a canine vein bypass graft translational model. State-of the art microarrays, Laser Capture Microdissection (LCM), as well as sophisticated and innovative global transcriptome analysis using Systems Biology will be employed. In addition, standardized immunohistochemistry, cellular, biochemical and molecular techniques will be used. Our investigative team has demonstrated the multidisciplinary collaboration essential to successful conduct of this proposal. We strongly believe this work will greatly strengthen the application of gene silencing to VG in patients, forecasting its expansion to other clinical problems in vascular surgery. In addition, this work will undoubtedly broaden our understanding of vascular wall biology.