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是移植静脉狭窄和延迟性移植失败的重要原因。植入时的损伤或作为移植到高压动脉系统的结果可能导致这些延迟事件。基因表达的改变伴随着植入和动脉化损伤。这些改变导致内膜增生，包括内皮细胞向炎症状态的转变，以及启动平滑肌细胞从收缩状态向分泌状态的迁移和转变，从而造成内膜增生的病变。我们的假设是，静脉壁损伤上调的基因沉默将减少内膜增生。此外，基因沉默可以在手术室条件的限制下完成。我们的初步数据表明，我们有能力识别与内膜增生相关的候选基因，以及我们有能力用siRNA抑制基因的表达。使用激光捕获显微镜，我们已经将内皮细胞中的遗传事件与平滑肌中的遗传事件分开。在拟议的研究中，我们将应用这些技术来(1)系统地识别沉默靶基因，(2)在体外沉默靶基因，(3)在外科条件下沉默靶基因，(4)在体内证明抑制移植静脉内膜增生。将使用人体组织和犬类模型以及所有培养、微阵列、激光捕获、RT-PCR、Western Blot和相关技术。这项研究将大大加强基因沉默在血管外科临床问题中的应用，并拓宽我们对血管壁生物学的理解。手术创伤引起的瘢痕组织形成是心脏搭桥术和外周血管疾病搭桥术失败的主要原因。研究人员建议通过控制引起疤痕的基因的表达来防止这种疤痕组织的形成，方法是使用可以在手术室应用的技术。

DESCRIPTION (provided by applicant): Intimal hyperplasia in arterialized vein bypass grafts is a significant cause of vein graft stenosis and delayed graft failure. Injury at the time of implantation or as a consequence of transplantation into the high pressure arterial system may contribute to these delayed events. Alterations in gene expression accompany implantation and arterialization injury. These alterations lead to intimal hyperplasia, including transformation of endothelial cells to an inflammatory state and initiating migration and transformation of smooth muscle cells from the contractile to secretory states thus creating the lesion of intimal hyperplasia. It is our hypothesis that silencing of genes upregulated by injury to the vein wall will diminish intimal hyperplasia. Furthermore, gene silencing can be accomplished within the constraints of operating room conditions. Our preliminary data demonstrates our ability to identify candidate genes associated with intimal hyperplasia and our ability to knockdown gene expression with siRNA. Using laser capture microscopy we have separated genetic events in the endothelium from those in smooth muscle. In the proposed study we will apply these technologies to (1) systematically identify silencing targets, (2) to silence target genes in vitro, (3) to silence target genes under surgical conditions, (4) to demonstrate inhibition of intimal hyperplasia in vein grafts in vivo. Both human tissue and canine models will be used as well as all cultures, microarrays, laser capture, RT-PCR, Western Blot, and related technology. This study will greatly strengthen the application of gene silencing to clinical problems in vascular surgery, and broaden our understanding of vascular wall biology. Public Lay Summary: Scar tissue formation due to surgical injury is a major cause for failure of heart bypass grafts and bypass grafts for peripheral vascular disease. The investigators propose to prevent formation of this scar tissue by controlling expression of the genes that cause it, using techniques that can be applied in the operating room.