Mechanisms of Shear-Regulated Vein Graft Remodeling

描述(由申请人提供)： 这项拟议的为期5年的培训计划旨在扩大申请者在高级分子遗传学方面的知识，主要有两个目标：1)将他的基础研究推进到对血管重构的假设驱动的、机械性的理解；2)为他提供所需的技能，以开发将基因疗法应用于临床环境的转化性研究计划。为了实现这些目标，已整合了关键的培训部分，包括高级遗传学的教学指导、临床研究过程中的基础课程以及专注于体内基因传递和调节技术的研究经验。该培训经过深思熟虑，旨在帮助申请者实现其长期职业目标--独立的基础研究计划，该计划旨在开发潜在的策略，防止静脉移植物狭窄和闭塞。拟议中的研究项目包含了佛罗里达大学的几个独特的组成部分： 1)创伤研究所，在细胞外基质重组的生长因子信号方面具有专业知识；2)国家载体实验室，熟练掌握病毒基因传递技术；以及3)在应用核酶进行体内基因抑制方面的开创性研究。 这项拟议的研究集中在静脉搭桥失败的临床相关问题上，并检查了加速内膜增生发展的机制。生物力学已被认为是内膜增厚的有力调节因素，但对其潜在的信号机制的了解仍然有限。这项建议建立在我们建立的血流调节静脉移植物重塑的兔模型基础上，初步数据表明结缔组织生长因子(CTGR)和明胶酶(MMP-2和-9)是重塑过程的关键调节因子。我们推测，转化生长因子(转化生长因子-β1)通过CTGF发挥作用，是重建静脉移植物壁内基质金属蛋白酶活性的主要调节因子。具体地说，我们假设静脉移植物内的壁切应力升高会导致转化生长因子-β1的产生增加。通过上调CTGF起作用，导致血管壁中的基质金属蛋白酶-2和-9活性降低，抑制了平滑肌细胞的迁移和增殖，从而减少了内膜增生。利用反义寡核苷酸和核酶技术在体内抑制转化生长因子-β1和结缔组织生长因子，将描述内皮细胞剪切力改变引起中膜改变和新血管内膜形成的介体。对这些信号通路的深入了解，结合这些强大的基因抑制技术的开发，为直接应用于临床环境提供了潜力。

DESCRIPTION (provided by applicant): This proposed 5-year training program seeks to expand the applicant's knowledge in advanced molecular genetics with two primary goals: 1) advance his basic research into a hypothesis-driven, mechanistic understanding of vascular remodeling; and 2) provide him the skills needed to develop a translational research program for the application of gene therapy to the clinical environment. Key training components have been integrated to accomplish these goals and include didactic instruction in advanced genetics, fundamental coursework in the conduct of clinical investigation, and a research experience focusing on the techniques of in vivo gene delivery and modulation. The training has been thoughtfully constructed to help the applicant reach his long-term career goal of an independent basic research program that develops potential strategies to prevent vein graft narrowing and occlusion. The proposed research program incorporates several unique components at the University of Florida: 1) the Institute for Wound Research with expertise in the growth factor signaling of extracellular matrix reorganization; 2) the national Vector Laboratory with proficiency in viral gene delivery techniques; and 3) pioneering research in the application of ribozymes for in vivo gene inhibition. The proposed research focuses on the clinically relevant problem of vein bypass graft failure and examines the mechanisms underlying accelerated intimal hyperplasia development. Biomechanical forces have been identified as potent regulators of intimal thickening, yet an understanding of the underlying signaling mechanism remains limited. This proposal builds on our established rabbit model of flow-regulated vein graft remodeling and preliminary data suggesting connective tissue growth factor (CTGR) and gelatinases (MMP-2 and -9) to be key regulators of the remodeling process. We hypothesize that transforming growth factor (TGF-beta1), acting through CTGF, is a primary regulator of MMP activity within the remodeling vein graft wall. Specifically, we hypothesize that elevated wall shear stress within vein grafts induces an increase in TGF-beta1 production. Acting through an up-regulation of CTGF, this results in a decrease in MMP-2 and -9 activities within the vessel wall, inhibiting smooth muscle cell migration and proliferation and leading to a reduction in intimal hyperplasia. Using antisense oligonucleotide and ribozyme techniques to inhibit TGF-beta1 and CTGF In vivo, the mediators through which alterations in shear at the endothelium induce changes in the media and developing neointima will be characterized. Insight into these signaling pathways, in combination with development of these powerful gene inhibition techniques, offers the potential for direct application to the clinical setting.