TGF-beta in intimal hyperplasia after vascular bypass

描述（由申请人提供）：再狭窄，或动脉管腔再狭窄，在血管介入治疗动脉粥样硬化后，造成数千人的死亡率和发病率。转化生长因子- β (TGF-2)是再狭窄的重要介质，尽管其对动脉壁影响的机制尚不清楚。在我们完成的提案中，我们探索了这些机制，并假设抑制TGF-2的促纤维化作用和增强其对平滑肌细胞（SMC）迁移和增殖的抑制作用将为控制内膜增生提供一个场所。我们的研究为TGF-2的作用机制提供了新的见解。与我们最初的假设相反，TGF-2不是通过产生细胞外基质而产生内膜增生，而是通过信号分子Smad3刺激SMC增殖。我们的数据还表明，TGF-2通过Smad3信号传导，可能通过募集骨髓祖细胞（BMPC）进入内膜病变，从而促进初始增生。最后，我们发现TGF-2通过Smad3产生适应性重塑或动脉扩张，并通过结缔组织生长因子（CTGF）蛋白介导。这些新发现产生了三个假设。在Specific Aim I中，我们将验证Smad3水平在动脉损伤中升高的假设，以及持续高水平的Smad3通过将静止的smc转化为一种对TGF-2有增殖反应的表型而导致内膜增生。我们将首先在体内测试阻断内源性Smad3产生对新内膜形成的影响。为了更好地理解Smad3激活的SMCs从静止表型到增殖表型的转换，我们将研究Smad3与细胞周期蛋白依赖性激酶抑制剂p27之间的关系。最后，我们将探讨TGF-2和Smad3在人类再狭窄和动脉粥样硬化斑块中的功能是否相似。在Specific Aim II中，我们将测试TGF-2是否通过Smad3刺激动脉SMCs产生BMPCs的化学引诱剂，从而促进内膜增生。利用大鼠骨髓移植模型进行体内研究，旨在验证TGF-2和Smad3在祖细胞募集中的作用。此外，我们将验证单核细胞趋化蛋白-1 （MCP-1）是介导TGF-2和Smad3作用的趋化剂的假设。在Specific Aim III中，我们将测试SMCs中的TGF-2/Smad3通路是否刺激CTGF的产生，CTGF反过来通过差异调节I型和III型胶原的合成来刺激适应性重塑。我们将证明CTGF是适应性重塑的必要和充分条件，并探讨III型胶原在这一过程中的作用。通过这些研究，我们期望进一步了解TGF-2及其在细胞增殖、BMPC募集和动脉重塑中的作用。最终，我们的发现将导致能够抑制内膜增生和促进适应性重塑的治疗方法的发展，为再狭窄的破坏性问题提供解决方案。在美国，动脉粥样硬化是导致死亡的主要原因。动脉粥样硬化的治疗方法很多；然而，它们的长期成功受到动脉管腔再狭窄或再狭窄过程的阻碍，这种情况发生在30-50%的血管成形术或支架植入术后的患者中。我们建议的目标是建立在我们在过去几年中获得的关于TGF-2的分子机制及其在再狭窄过程中的作用的知识基础上。长期目标是开发特定的治疗方法来预防或阻止动脉再狭窄的进展，从而降低每年成千上万人的发病率和死亡率。

DESCRIPTION (provided by applicant): Restenosis, or re-narrowing of the arterial lumen following vascular interventions to treat atherosclerosis, produces significant mortality and morbidity for thousands of individuals. Transforming growth factor-beta (TGF-2) is an important mediator of restenosis, although the mechanisms that contribute to its effects on the arterial wall remain unclear. In our completed proposal, we explored these mechanisms and hypothesized that inhibition of TGF-2's profibrotic effects and enhancement of its inhibitory effects on smooth muscle cell (SMC) migration and proliferation would provide a venue for controlling intimal hyperplasia. Our studies have provided new insights into the mechanism of action of TGF-2. Contrary to our original hypothesis, TGF-2 produces intimal hyperplasia not through the production of extracellular matrix but rather by stimulating SMC proliferation through the signaling molecule Smad3. Our data also suggest that TGF-2, through Smad3 signaling, may contribute to initmal hyperplasia through the recruitment of bone marrow progenitor cells (BMPC) into the intimal lesion. Lastly we found that TGF-2, through Smad3, appears to produce adaptive remodeling or arterial enlargement, mediated through the protein connective tissue growth factor (CTGF). These novel findings have generated three hypotheses. In Specific Aim I, we will test the hypothesis that Smad3 levels are elevated in arterial injury and that sustained high levels of Smad3 lead to intimal hyperplasia by converting quiescent SMCs to a phenotype that responds to TGF-2 with proliferation. We will begin by testing the effect of blocking endogenous Smad3 production on neointimal formation in vivo. To better understand the switch of Smad3 activated SMCs from a quiescent to a proliferative phenotype, we will study the relationship between Smad3 and the cyclin-dependent-kinase inhibitor p27. Finally, we will explore whether TGF-2 and Smad3 function in a similar fashion in human restenotic and atherosclerotic plaque. In Specific Aim II, we will test whether TGF-2, through Smad3, enhances intimal hyperplasia by stimulating arterial SMCs to produce a chemoattractant of BMPCs. In vivo studies, using a rat bone marrow transplant model, have been designed to verify the role in TGF-2 and Smad3 in progenitor cell recruitment. Moreover, we will test the hypothesis that monocyte chemoattractant protein-1 (MCP-1) is the chemoattractant that mediates TGF-2 and Smad3's effect. In Specific Aim III, we will test whether the TGF-2/Smad3 pathway in SMCs stimulates the production of CTGF, which in turn stimulates adaptive remodeling by differentially regulating synthesis of collagen types I and III. We will show that CTGF is necessary and sufficient for adaptive remodeling and explore the role of collagen type III in this process. With these studies, we expect to gain further insight into TGF-2 and its role in cell proliferation, BMPC recruitment, and arterial remodeling. Ultimately, our findings will lead to the development of therapies that can inhibit intimal hyperplasia and promote adaptive remodeling, providing a solution to the devastating problem of restenosis. Atherosclerosis is the leading cause of death in the United States. Treatments for atherosclerosis are numerous; however, their long term success is impeded by the process of restenosis or re-narrowing of the arterial lumen, which occurs in 30-50% of patients following angioplasty or stenting. The goal of our proposal is to build upon knowledge that we have acquired over the past several years about the molecular mechanisms that underlie TGF-2 and its role in the process of restenosis. The long-term goal is to develop specific therapies that prevent or halt the progression of arterial restenosis, thus reducing morbidity and mortality for thousands of individuals each year.