The Role of The Thrombospondins In Intimal Hyperplasia

血小板反应蛋白在内膜增生中的作用

• 批准号: 9260484
• 负责人: Vivian Gahtan
• 金额: $ 31.5万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-12-09 至 2020-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9260484
• 关键词: ADAMTS1 gene; Address; Affect; Amino Acid Motifs; Anastomosis - action; Angioplasty; Animal Model; Animals; Arterial Fatty Streak; Arteries; Balloon Angioplasty; Binding; Biological Assay; Biological Availability; Biology; Blood Vessels; CD36 gene; CD47 gene; Cell Surface Receptors; Cell physiology; Cell-Matrix Junction; Cessation of life; Chemotaxis; Clinical; Common carotid artery; Data; Development; Digestion; Disease; Disintegrins; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Extracellular Matrix Proteins; Foundations; Gene Expression; Gene Targeting; Genes; Goals; Hyperplasia; In Vitro; Injury; Intervention; Knockout Mice; Knowledge; Laboratories; Left; Ligation; Limb structure; Literature; Longevity; Mediating; Messenger RNA; Metalloproteases; Methodology; Methods; MicroRNAs; Mission; Molecular; Molecular and Cellular Biology; Mus; Muscle Cells; Operative Surgical Procedures; Outcome; Pathology; Patient Care; Patients; Peptide Hydrolases; Peripheral; Peripheral arterial disease; Physiological; Polymerase Chain Reaction; Population; Positioning Attribute; Prevention; Process; Proteins; Public Health; Publications; Quality of life; RNA; RNA Binding; Rattus; Regulation; Research; Role; Secondary to; Signal Pathway; Signal Transduction; Small Interfering RNA; Specimen; Staining method; Stains; Structure; System; Techniques; Technology; Testing; Therapeutic; Thrombospondin 1; Thrombospondins; Time; Tissues; Translations; Treatment Efficacy; United States National Institutes of Health; Untranslated RNA; Vascular Diseases; Vascular Smooth Muscle; Western Blotting; Work; artery occlusion; base; cell motility; cytokine; disability; improved; in vivo; innovation; knock-down; migration; novel; prevent; protein expression; restenosis; therapeutic target; thrombospondin 2; translational impact; vascular smooth muscle cell proliferation

Restenosis secondary to intimal hyperplasia (IH) after balloon angioplasty to treat arterial blockages in peripheral arteries is a significant cause of disability and death. The thrombospondins (TSPs) are multifunctional matricellular proteins central to the development of intimal hyperplasia. They are not part of the arterial wall structure, but exert their physiologic effects on arterial structure by binding cytokines, cell-surface receptors, proteases and other proteins. This proposal focuses on three TSPs integral to the development of intimal hyperplasia – TSP-1, TSP-2 and TSP-5. We have studied the effects of TSP-1 on vascular smooth muscle cell (VSMC) proliferation and migration and its importance in the development of intimal hyperplasia; however, increasing evidence exists that TSP-2 and TSP-5 have separate and contributory roles in this pathology. All three TSPs are substrates of ADAMTS (A Disintegrin And Metalloproteinase with Thrombospondin Motifs) proteins. ADAMTSs digest TSPs, enhancing or inhibiting TSP function, since the fragments left after digestion have distinct effects themselves on intimal hyperplasia. Therefore, ADAMTS-1,-4, and-7 will also be studied as they are involved in PAD and were also identified to be regulated by TSPs in our prior VSMC gene study. Our long-term goal is to understand how TSPs can be manipulated therapeutically to prevent intimal hyperplasia in vivo. The objective of this proposal is to determine how TSP-1, TSP-2 and TSP-5 specifically contribute to the development of intimal hyperplasia. Our central hypothesis is that the expression, bioavailability, signaling pathways and changes in gene expression induced by TSP-1, TSP-2 and TSP-5 and their interactions with ADAMTSs have distinct effects on regulating the development of IH. This hypothesis was formulated on the basis of our strong preliminary data, our publications and the literature. The rationale for the proposed project is that understanding the roles of TSPs on intimal hyperplasia will result in identification of therapeutic targets to inhibit intimal hyperplasia and restenosis after balloon angioplasty. Our hypothesis will be tested by pursuing the following Specific Aims: 1) determine the role that TSP-1, TSP-2 and TSP-5 each have on intimal hyperplasia; 2) determine the differential effects of TSP-1, TSP-2 and TSP-5 on protein and microRNA expression (i.e., miR-17~92 cluster), and their downstream effects on VSMC function; and 3) establish the role of ADAMTSs in TSP-1, TSP-2 and TSP-5 activity and in the development of intimal hyperplasia. The methodologies utilized to investigate these Specific Aims include: 1) modified Boyden chamber to assess chemotaxis and colorimetric assay to assess for proliferation in VSMCs; 2) western blot, ELISA and immunoPCR for cell signaling and protein expression; 3) quantitative real time polymerase chain reaction for gene expression; 4) two animal models of intimal hyperplasia – common carotid artery balloon injury in rats and ligation in mice; 5) use of knockout mice and siRNA for knockdown of TSP and ADAMTS genes in vitro and in vivo to see effects on VSMCs and intimal hyperplasia, respectively; and 6) morphometric analysis, western blot and immunohistochemical staining for analysis of arterial specimens. The siRNA work will also involve testing our novel siRNAs directed at TSP/ADAMTS combinations that may prove to be a highly effective method of blocking intimal hyperplasia at the time of angioplasty. The significance of the proposed research is that the findings will provide a major advance toward identifying new strategies for preventing restenosis due to intimal hyperplasia. The proposed research in this application is innovative, in our opinion, because the findings will define the interactions of multiple TSPs and peptidases with TSP motifs (ADAMTSs) in vascular disease and mechanisms through which these systems can be manipulated with novel siRNAs to improve clinical outcomes. The findings from this application will advance efforts to improve quality of life and longevity by providing safer and more effective therapeutic options for patients with peripheral arterial disease.

在气球血管成形术后发生内膜增生（IH）的再狭窄，以治疗动脉堵塞 周围动脉是残疾和死亡的重要原因。血小板传播（TSP）是 多功能基质蛋白是内膜增生发展的中心。他们不是 动脉壁结构，但通过结合细胞因子，细胞表面对动脉结构产生生理影响 受体，蛋白质和其他蛋白质。该提案侧重于三个TSP的发展 内膜增生 - TSP-1，TSP-2和TSP-5。我们研究了TSP-1对血管平滑的影响 肌肉细胞（VSMC）增殖和迁移及其在内膜增生发展中的重要性； 但是，越来越多的证据表明TSP-2和TSP-5在这方面具有独立和贡献的作用 病理。这三个TSP都是ADAMTS的底物（一种拆解蛋白和金属蛋白酶， 血小板传播基序）蛋白质。 Adamtss Digest TSP，增强或抑制TSP功能，因为 消化后留下的碎片对内膜增生具有明显的影响。因此，Adamts-1，-4， 和-7也将研究，因为它们参与了PAD，并且还被确定为在我们的 先前的VSMC基因研究。我们的长期目标是了解如何热能操纵TSP 预防体内内膜增生。该提案的目的是确定TSP-1，TSP-2和TSP-5如何 特别有助于内膜增生的发展。我们的中心假设是表达， TSP-1，TSP-2和TSP-5诱导的生物利用度，信号通路以及基因表达的变化和 他们与Adamts的相互作用对重新计算IH的发展有明显的影响。这个假设 基本原理是根据我们强大的初步数据，出版物和文献提出的。 拟议的项目是了解TSP在内膜增生中的作用将导致鉴定 在球囊血管成形术后抑制内膜增生和再狭窄的治疗靶标。我们的假设将是 通过追求以下特定目的测试：1）确定TSP-1，TSP-2和TSP-5具有的作用 关于内膜增生； 2）确定TSP-1，TSP-2和TSP-5对蛋白质和 microRNA表达（即miR-17〜92簇）及其对VSMC功能的下游影响； 3） 确定ADAMTS在TSP-1，TSP-2和TSP-5活性以及内膜发展中的作用 增生。用于研究这些特定目的的方法包括：1）修改 评估趋化性和比色评估的腔室，以评估VSMC中的增殖； 2）Western印迹， ELISA和免疫可用于细胞信号传导和蛋白质表达； 3）定量实时聚合酶链 基因表达的反应； 4）两种内膜增生的动物模型 - 常见的颈动脉气球 大鼠损伤和小鼠的结扎； 5）使用敲除小鼠和siRNA进行TSP和ADAMTS的敲除 在体外和体内基因分别对VSMC和内膜增生的影响； 6）形态计量学 分析，蛋白质印迹和免疫组织化学染色，用于分析动脉标本。 sirna工作 还将涉及测试我们针对TSP/ADAMTS组合的小说SIRNA，这可能被证明是一种高度 在血管成形术时阻断内膜增生的有效方法。提议的意义 研究是，这些发现将为确定防止新的策略提供重大进步 由于内膜增生而引起的再狭窄。在我们看来，本应用程序中提出的研究是创新的 因为发现将定义多个TSP和PETIDass与TSP基序（ADAMTSS）的相互作用 在血管疾病和机制中，这些系统可以用新型siRNA操纵到 改善临床结果。该应用程序的发现将促进改善生活质量和 通过为周围动脉疾病的患者提供更安全，更有效的治疗选择，寿命。