GENE AND STEM CELL THERAPY IN CORONARY ARTERY BYPASS GRAFT

 描述（由申请人提供）：大隐静脉（SV）移植失败是接受冠状动脉旁路移植术（CABG）患者的常见临床问题。潜在的机制尚不清楚。包括靶向基因治疗在内的各种疗法的失败可能是由于基因的错误选择和/或使用与人类疾病不同的动物模型。此外，没有考虑促进内皮细胞增殖。我们观察到在人SV中随着SMC增殖的增加而使PTEN失活，并且PTEN过表达限制了细胞增殖。由于PTEN调节细胞信号传导和细胞生长，因此在SV移植物SMC中的PTEN转基因表达将防止内膜增生的发展。此外，用间充质干细胞（MSC）修复内皮细胞（EC）可以抑制血栓形成。假设在CABG之前，通过PTEN转基因抑制内膜增生，同时通过在自体静脉移植物中递送MSC来保护内皮，这将是维持静脉移植物通畅性的最佳策略。我们将在我们实验室中建立的和常规使用的猪动脉粥样硬化模型中进行这些研究，并使用“无接触技术”分离的腹壁浅静脉（SEV）进行CABG。目标1：我们的假设是，在动脉粥样硬化猪冠状动脉移植静脉平滑肌细胞中过表达PTEN转基因可以防止旁路移植静脉新生内膜增生的发展。目标二：我们的假设是，在动脉粥样硬化猪冠状动脉移植静脉的SMC中，MSC与PTEN转基因一起修复内皮细胞可以防止旁路移植静脉中的血栓形成和新生内膜增生，这将优于单独的PTEN转基因的效果上级。目标3：我们的假设预测，PTEN转基因的过表达和MSC诱导的内皮细胞再生将减少炎症和抑制收缩性重塑，同时抑制冠状动脉中静脉移植物的新生内膜增生。将自体SEV暴露于PTEN载体和MSC，然后在动脉粥样硬化猪中进行冠状动脉移植。心电图和超声心动图监测心脏功能，冠状动脉造影和最佳相干断层扫描量化段内最小管腔直径，直径狭窄，晚期损失和内膜增生将检查这种疗法的治疗效果。组织学参数将包括内膜厚度、管腔面积、内膜-中膜比、斑块的发展和溃疡、血栓形成、细胞外基质和再闭塞。将在切除的静脉移植物中检查促炎和抗炎以及促增生途径的表达，并将其与新生内膜增生和收缩性重塑的程度相关联。 这些研究的结果将为我们的假设提供概念上的支持，并使我们能够将我们的研究转化为临床1期研究，用于使用PTEN工程化的自体MSC治疗和可能治愈CABG后的静脉移植物疾病。

 DESCRIPTION (provided by applicant): Saphenous vein (SV) graft failure is a common clinical problem in patients undergoing coronary artery bypass graft (CABG) surgery. Underlying mechanisms are still unclear. Failure of various therapies, including targeted gene therapy, could be due to the bad choice of the gene and/or the use of animal model dissimilar to human disease. Also, there was no consideration to promote endothelial cell proliferation. We observed inactivation of PTEN with increased proliferation of SMCs in human SV and PTEN overexpression limits cell proliferation. Since PTEN modulates cell signaling and cell growth, PTEN transgene expression in SV graft SMCs would prevent the development of intimal hyperplasia. Also, the repair of endothelial cells (ECs) with mesenchymal stem cells (MSCs) would inhibit thrombosis. The hypothesis is that the inhibition of intimal hyperplasia by PTEN transgene while preserving endothelium with the delivery of MSCs in autologous vein grafts prior to CABG would be the best strategy to maintain vein graft patency. We will do these studies in a well-established and routinely used in our laboratory the swine model of atherosclerosis and perform CABG using superficial epigastric vein (SEV) isolated with "no touch technique". Aim 1: Our hypothesis predicts that the overexpression of PTEN transgene in the SMCs of vein graft in coronary arteries in atherosclerotic swine will prevent the development of neointimal hyperplasia in bypass vein graft. Aim 2: Our hypothesis predicts that the repair of endothelial cells with MSCs together with PTEN transgene in the SMCs of vein graft in coronary arteries of atherosclerotic swine will prevent thrombosis and neointimal hyperplasia in bypass vein graft, and this would be superior to the effect of PTEN transgene alone. Aim 3: Our hypothesis predicts that overexpression of PTEN transgene and MSCs-induced endothelial cell regeneration would reduce inflammation and inhibit constrictive remodeling together with inhibition of neointimal hyperplasia in the vein graft in coronary arteries. The autologous SEV will be exposed to PTEN vector and MSCs followed by aorto-coronary grafting in atherosclerotic swine. EKG and echocardiography to monitor heart function, and coronary angiography and optimal coherence tomography to quantify in-segment minimal luminal diameter, diameter stenosis, late loss and intimal hyperplasia will examine the therapeutic efficacy of this therapy. Histological parameters will include the intimal thickness, lumen area, intima-media ratio, development and ulceration of plaque, thrombosis, extracellular matrix, and re-occlusion. Expression of pro- and anti-inflammatory and pro-hyperplasia pathways will be examined in excised vein graft and correlated with the degree of neointimal hyperplasia and constrictive remodeling. Findings from these studies will provide the conceptual support for our hypothesis, and position us to translate our investigation into a clinical phase 1 study for the use of PTEN-engineered autologous MSCs for the treatment and possibly cure of the vein graft disease following CABG.