Genetic Engineering of Vein Bypass Grafts in Vascular and Cardiovascular Surgery

描述(申请人提供)：动脉化静脉搭桥术中的内膜增生(IH)是移植静脉(VG)狭窄和延迟性移植失败的重要原因。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变化，并将内皮细胞的遗传事件与中膜平滑肌细胞(SMC)的遗传事件分开。利用系统生物学，我们已经确定了对伤害反应最重要的上调基因。通过反向传播，从最新时间点，即30天(D)差异表达的基因开始，然后添加每个先前时间点的上游交互基因，建立一个整合的网络。这发现30D的胶原1A1(Col1A1)是背向繁殖的中心基石和IH病变的主要贡献者，以及IL-6、IL-8和PKC？作为焦点中心基因，从术后2小时(H)-12小时开始在所有时间点差异上调。这些结果建立了因果关系，阐明了VG植入损伤的发病机制，并确定了其预防的新靶点。我们的假设是焦点中枢和最终病变的沉默 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。在拟议的研究中，我们将应用siRNA技术来(I)系统地评估沉默靶基因(IL-6、IL-8、PKC和Col1A1)的有效性和持久性，并在人大隐静脉内皮细胞(EC)和SMC培养中实现蛋白质下调，并在体外证实这一有效性，(Ii)使用小鼠静脉旁路移植模型确定可以预防IH的最成功的siRNA鸡尾酒，(Iii)测试最有效的siRNA鸡尾酒并确定犬静脉搭桥移植翻译模型中的遗传后遗症。将使用最先进的微阵列、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，还将使用标准化的免疫组织化学、细胞、生化和分子技术。我们的调查团队已经证明，多学科合作对于成功实施这项提案至关重要。我们坚信，这项工作将极大地加强基因沉默在患者VG中的应用，并有望将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.