Genetic Engineering of Vein Bypass Grafts in Vascular and Cardiovascular Surgery

描述（由申请人提供）：动脉化静脉旁路移植物中的内膜增生（IH）是静脉移植物（VG）狭窄和延迟性移植物失效的重要原因。植入时的损伤或移植到高压动脉系统的结果导致了这些延迟事件。在犬模型中，我们已经确定了植入/动脉化损伤后转录组的改变，并将内皮细胞中的遗传事件与中膜平滑肌细胞（SMC）中的遗传事件分开。使用系统生物学，我们已经确定了上调的基因是最重要的损伤反应。通过反向传播，从在最新时间点即30天（D）差异表达的基因开始，然后添加来自每个先前时间点的上游相互作用基因，构建了整合网络。这确定了30 D时的胶原蛋白1A 1（Col 1A 1），作为反向传播的中心基石和IH病变的主要贡献者，以及白细胞介素（IL）-6，IL-8和PKC作为在所有时间点（从术后2小时（H）-12小时开始）差异上调的焦点枢纽基因。这些结果建立了因果关系，澄清VG植入损伤的发病机制，并确定新的目标，其预防。我们的假设是局灶性枢纽和最终病变的沉默 基因将减少与VG植入损伤相关的过程，从而减少IH。为了实现这一目标，我们设计并改进了在手术室限制下沉默一个或多个基因的方法。在本研究中，我们将应用siRNA技术（i）系统地评估沉默靶基因的有效性和持久性，（IL-6、IL-8和PKC）和Col 1A 1），并在人隐静脉内皮细胞（EC）和SMC培养物中实现蛋白质敲低，并在人隐静脉的完整壁中离体证实了这种有效性，（ii）使用小鼠静脉旁路移植物模型确定可以预防IH的最成功的siRNA混合物，和（iii）在犬静脉旁路移植物转化模型中测试最有效的siRNA混合物并确定遗传后遗症。将采用最先进的微阵列，激光捕获显微切割（LCM），以及使用系统生物学的复杂和创新的全球转录组分析。此外，将使用标准化的免疫组织化学、细胞、生物化学和分子技术。我们的调查小组已经证明了多学科合作对成功实施这一建议至关重要。我们坚信，这项工作将大大加强基因沉默在VG患者中的应用，预测其扩展到血管外科的其他临床问题。此外，这项工作无疑将拓宽我们对血管壁生物学的理解。

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.