Thrombospondin-1, VSMC Migration and Intimal Hyperplasia - a role for statins

描述(由申请人提供)： 在退伍军人中，外周动脉疾病和球囊血管成形术治疗动脉阻塞后的再狭窄是导致残疾和死亡的重要原因。血管内介入治疗是外周动脉疾病发展最快的治疗方法之一；然而，继发于内膜增生(IH)的再狭窄仍然是这些治疗失败的主要原因。凝血酶敏感蛋白-1(TSP-1)通过调节动脉对损伤的反应而明显参与IH的发生。然而，血脂异常增强TSP-1‘S作用的机制，TSP-1如何诱导IH，以及预防周围动脉疾病患者血管成形术后IH的最佳治疗方法，代表了我们知识中的重要空白，并通过这一应用得到解决。我们的长期目标是了解TSP-1信号通路如何在体内通过治疗来预防IH。本研究的目的是探讨TSP-1、血脂异常和他汀类药物释放增加调节IH发生的机制。我们的中心假设是血脂异常增强，他汀类药物治疗将有效抑制TSP-1的前狭窄效应。这一假设是在我们强大的初步数据的基础上提出的。该项目的理论基础是，了解TSP-1和血脂异常对IH的潜在机制，并建立抑制IH的最佳他汀类药物释放，将导致预防血管成形术后再狭窄的新颖和创新的方法。我们的假设将通过追求以下特定目标来验证：1)研究血脂异常如何改变TSP-1诱导的趋化作用和信号转导；2)阐明急性他汀类药物抑制TSP-1诱导的信号转导的机制；3)确定TSP-1‘S在他汀类药物疗效中的作用幅度；以及4)确定他汀类药物的最佳给药方式以减少IH。用来研究这些特定目的的方法包括：1)改良的博伊登小室评估血管平滑肌细胞的趋化作用；2)蛋白质印迹和ELISA法检测细胞信号；3)实时定量聚合酶链式反应检测基因表达；4)2 TSP-1基因敲除小鼠的IH-颈动脉结扎和正常及血脂异常大鼠颈总动脉球囊损伤的动物模型；5)体内不同的他汀类药物给药方法-口服、腔内(使用靶向胶束)和局部给药(使用Pluronic凝胶)；6)形态计量分析、免疫组织化学染色和原位杂交分析动脉标本。这项拟议研究的意义在于，这些发现将提供一项重大进展，有望导致开发新的治疗策略，以防止外周动脉疾病病变的血管成形术后再狭窄。我们认为，在这一应用中拟议的研究是创新的，因为拟议的使用他汀类药物治疗这些皮损的靶向给药系统的开发和测试将是一种新的治疗模式。这一应用的结果将通过为患有外周动脉疾病的退伍军人提供更安全和更有效的治疗选择来推动提高生活质量和寿命的努力。

DESCRIPTION (provided by applicant): Peripheral arterial disease and restenosis after balloon angioplasty to treat arterial blockages is a significant cause of disability and death in the veteran population. Endovascular interventions are among the fastest growing treatments for peripheral arterial disease; however, restenosis secondary to intimal hyperplasia (IH) remains a major cause of failure from these procedures. Thrombospondin-1 (TSP-1) clearly contributes to IH by regulating the arterial response to injury. However, the mechanisms by which dyslipidemia enhances TSP- 1's actions, how TSP-1 induces IH, and optimal therapies to prevent IH after angioplasty in patients with peripheral arterial disease represent important gaps in our knowledge and is addressed by this application. Our long-term goal is to understand how TSP-1 signaling pathways can be manipulated therapeutically to prevent IH in vivo. The objective of this proposal is to investigate the mechanisms by which TSP-1, dyslipidemia and enhanced statin delivery regulate IH development. Our central hypothesis is that dyslipidemia enhances and that statin therapy will effectively inhibit the pro-stenotic effects of TSP-1. This hypothesis has been formulated on the basis of our strong preliminary data. The rationale for the proposed project is that understanding the underlying mechanisms of TSP-1 and dyslipidemia on IH and establishing optimal statin delivery to inhibit IH, will result in novel and innovative approaches to prevent restenosis after angioplasty. Our hypothesis will be tested by pursuing the following Specific Aims: 1) investigate how dyslipidemia modifies TSP-1-induced chemotaxis and signaling; 2) elucidate the mechanisms of acute statin inhibition on TSP-1-induced signaling; 3) determine the magnitude of TSP-1's role in statin efficacy; and 4) ascertain the optimal modality of statin delivery to reduce IH. The methodologies utilized to investigate these Specific Aims include: 1) modified Boyden chamber to assess vascular smooth muscle cell chemotaxis; 2) western blot and ELISA for cell signaling; 3) quantitative real time polymerase chain reaction for gene expression; 4) two animal models of IH - carotid artery ligation in the TSP-1 knockout mouse and balloon injury of the common carotid artery in normal and dyslipidemic rats; 5) different statin delivery methods in vivo - oral, intraluminal (using targeted micelles) and topical (using pluronic gel); and 6) morphometric analysis, immunohistochemical staining and in situ hybridization for analysis of the arterial specimens. The significance of the proposed research is that the findings will provide a major advance which is expected to result in the development of new treatment strategies to prevent restenosis after angioplasty for peripheral arterial disease lesions. The proposed research in this application is innovative, in our opinion, because the proposed development and testing of a targeted drug delivery system using statins for the treatment of these lesions would be a new treatment modality. The findings from this application will advance efforts to improve the quality of life and longevity by providing safer and more effective therapeutic options for veterans with peripheral arterial disease.