MESENCHYMAL STEM CELLS IN THE PREVENTION OF THROMBOSIS AND NEOINTIMAL HYPERPLASIA

描述(申请人提供)：药物洗脱支架导致较少的内膜增生和较少的晚期管腔丢失，但抑制支架节段的再内皮化，使其更容易发生血栓。因此，在再狭窄和晚期支架血栓形成之间存在权衡，需要更长时间的抗血小板治疗。显然，一种更好的治疗方法可以预防内膜增生并诱导再内皮化，这将有助于提供长期无症状的冠状动脉疾病控制，而不需要长期的抗血小板治疗。由于PTEN调控细胞信号转导和细胞生长，因此PTEN转基因在动脉平滑肌细胞中的过表达将阻止新生内膜的发展。此外，血栓形成可以通过用间充质干细胞(MSCs)修复内皮细胞来预防，MSCs已经准备好分化为内皮细胞。我们假设，将MSCs和PTEN转基因局部转移到血管成形术和支架损伤的部位可以防止血栓形成和新生内膜增生的发展。骨髓间充质干细胞中Wnt/2连环蛋白信号的下调促进内皮细胞分化和再内皮化。这一假设得到了我们的数据的支持，该数据表明血管内皮细胞生长因子(Vgef)诱导的MSCs向内皮细胞分化，PTEN的过表达抑制了动脉平滑肌细胞的增殖。我们将在实验室常规使用的猪高脂血症和动脉粥样硬化血管成形术和支架内再狭窄模型中进行这些研究。目的：验证PTEN转基因与MSCs在冠状动脉球囊成形术局部过表达可预防血栓形成和新生内膜增生的假说。目的：验证PTEN转基因与骨髓间充质干细胞在动脉粥样硬化冠状动脉支架裸露部位过表达可预防血栓形成和支架内再狭窄的假说，其效果优于单纯药物洗脱支架。目的：验证Wnt/2-catenin信号下调促进MSC向冠状动脉损伤部位内皮细胞分化的假说。我们将研究Wnt/β-catenin信号分子，特别是β-catenin、LRP5和Kremen1，作为MSCs在体外和体内猪冠状动脉内皮细胞分化和再内皮化的潜在机制。这些研究将使我们能够将我们的研究转化为临床第一阶段研究，使用PTEN工程的自体MSCs治疗冠状动脉介入手术引起的并发症，PTCA和PTEN工程的自体MSCs可能会消除冠状动脉疾病对支架的需求。 公共卫生相关性：球囊血管成形术或支架置入后心脏冠状动脉再次狭窄是一个严重的问题。这主要是由于球囊血管成形术或冠状动脉支架置入术导致损伤部位的平滑肌细胞不受控制地生长所致。另一个主要问题是形成血栓的血小板的沉积。在这个项目中，我们建议在动脉粥样硬化猪的心脏损伤部位植入自体间充质干细胞和一个新的基因，以防止冠状动脉再狭窄并使其保持更长时间的通畅。目的是验证这样一种假设，即使用干细胞进行基因治疗比使用药物洗脱支架更好。拟议的研究将为我们的假设提供概念性支持，并使我们能够将我们的研究转化为临床第一阶段研究，用于在冠状动脉疾病患者中输送带有新基因的干细胞。

DESCRIPTION (provided by applicant): Drug-eluting stents cause less intimal hyperplasia and less late luminal loss, but inhibit re- endothelialization of the stented segment making it more susceptible to thrombosis. Thus, there is trade-off between restenosis and late stent thrombosis requiring longer periods of anti-platelet therapy. Clearly, a better therapeutic approach that can prevent intimal hyperplasia and induce re-endothelialization would be useful to provide long-term symptom-free control of coronary artery disease without the need for prolonged anti- platelet therapy. Since PTEN modulates cell signaling and cell growth, PTEN transgene overexpression in arterial smooth muscle cells would prevent the development of neointimal hyperplasia. In addition, thrombosis could be prevented by the repair of endothelial cells with mesenchymal stem cells (MSCs) which have been primed to differentiate into endothelial cells. We hypothesize that the delivery of MSCs and PTEN transgene locally to the site of angioplasty and stent injury prevents thrombosis and the development of neointimal hyperplasia. Down-regulation of Wnt/2 catenin signaling in MSCs promotes endothelial cell differentiation and re-endothelialization. This hypothesis has been supported by our data demonstrating that vascular endothelial growth factor (VGEF)-primed MSCs differentiate into endothelial cells and PTEN overexpression inhibits proliferation of arterial smooth muscle cells. We will do these studies in a well-established and routinely used in our laboratory the swine model of hyperlipidemia and atherosclerosis with angioplasty and in-stent restenosis. Aim 1: Test the hypothesis that overexpression of PTEN transgene with MSCs at the site of balloon angioplasty in atherosclerotic coronary artery prevents thrombosis and the development of neointimal hyperplasia. Aim 2: Test the hypothesis that overexpression of PTEN transgene with MSCs at the site of bare metal stenting in atherosclerotic coronary artery prevents thrombosis and in-stent restenosis and this would be superior to the effect of drug-eluting stent alone. Aim 3: Test the hypothesis that down-regulation of Wnt/2-catenin signaling promotes MSC differentiation to endothelial cells at the injury site in coronary arteries. We will examine the role of Wnt/¿-catenin signaling molecules, particularly ¿-catenin, LRP5 and Kremen1, as the underlying mechanism of MSCs differentiation into ECs and re-endothelialization both in vitro and in vivo in swine coronary arteries. These studies would position us to translate our investigation into a clinical phase 1 study for the use of PTEN-engineered autologous MSCs for the treatment of the complications due to coronary interventional procedures, and PTCA with PTEN-engineered autologous MSCs might eliminate the need of stents in coronary artery disease. PUBLIC HEALTH RELEVANCE: Re-narrowing of coronary arteries in the heart after balloon angioplasty or placement of stents is serious problem. This is primarily due to uncontrolled growth of smooth muscle cells at the site of injury due to balloon angioplasty or the placement of stent in coronary artery. Another major problem is the deposition of platelets that develop thrombosis. In this project we proposed to deliver autologous mesenchymal stem cells together with a novel gene at the site of injury in the heart of atherosclerotic pig during these procedures to prevent the re-narrowing of the coronary arteries and keep them patent for longer period. The goal is to test the hypothesis that the administration of stem cells with gene therapy is superior to drug-eluting stents. The proposed studies will provide conceptual support of our hypothesis and position us to translate our investigation into a clinical phase 1 study for the delivery of stem cells with the novel gene in patients with coronary artery disease.