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.

球囊血管成形术治疗动脉阻塞后继发于内膜增生（IH）的复发 外周动脉是残疾和死亡的重要原因。血小板反应蛋白（TSP）是 多功能的基质细胞蛋白质的发展内膜增生的核心。他们不属于 动脉壁结构，但通过结合细胞因子、细胞表面 受体、蛋白酶和其他蛋白质。本提案侧重于发展中国家不可或缺的三个贸易战略计划， 内膜增生- TSP-1、TSP-2和TSP-5。我们研究了TSP-1对血管平滑肌的影响， 肌细胞（VSMC）增殖和迁移及其在内膜增生发展中的重要性; 然而，越来越多的证据表明，TSP-2和TSP-5在这一过程中起着独立的作用 病理所有三种TSP都是ADAMTS（一种解整合素和金属蛋白酶， 血小板反应蛋白基序）蛋白。ADAMTS消化TSP，增强或抑制TSP功能，因为 消化后留下的碎片本身对内膜增生有明显的影响。因此，ADAMTS-1，-4， 和-7也将被研究，因为它们参与PAD，并且在我们的研究中也被确定为受TSP调节。 VSMC基因研究。我们的长期目标是了解如何在治疗上操纵TSP， 防止体内内膜增生。本提案的目的是确定TSP-1、TSP-2和TSP-5 特别是有助于内膜增生的发展。我们的中心假设是， TSP-1、TSP-2和TSP-5诱导的生物利用度、信号通路和基因表达变化， 它们与ADAMTS的相互作用在调节IH的发展中具有明显的作用。这一假设 是根据我们强有力的初步数据、出版物和文献制定的。的理由 该项目的建议是，了解TSP对内膜增生的作用将导致识别 治疗目标是抑制球囊血管成形术后内膜增生和再狭窄。我们的假设是 通过追求以下具体目标进行测试：1）确定TSP-1、TSP-2和TSP-5各自具有的作用 2）确定TSP-1、TSP-2和TSP-5对蛋白质和内皮细胞增殖的不同影响， 微小RNA表达（即，miR-17~92簇），以及它们对VSMC功能的下游影响;以及3） 确定ADAMTS在TSP-1、TSP-2和TSP-5活性以及内膜发育中的作用 增生用于研究这些特定目的的方法包括：1）修改的Boyden 室以评估趋化性和比色测定以评估VSMC中的增殖; 2）蛋白质印迹， ELISA和免疫PCR用于细胞信号传导和蛋白质表达; 3）定量真实的时间聚合酶链 基因表达的反应; 4）两种内膜增生的动物模型-颈总动脉球囊 大鼠损伤和小鼠结扎; 5）使用敲除小鼠和siRNA敲除TSP和ADAMTS 分别在体外和体内观察基因对VSMC和内膜增生的影响;以及6）形态测量 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组合的新型siRNA，这可能被证明是一种高度有效的方法。 在血管成形术时阻断内膜增生的有效方法。建议的意义 这项研究的结果将为确定预防艾滋病的新策略提供重大进展。 内膜增生导致的再狭窄。在我们看来，这项应用中提出的研究是创新的， 因为这些发现将定义多种TSP和肽酶与TSP基序（ADAMTS）的相互作用， 在血管疾病和机制，通过这些系统可以操纵新的siRNA， 改善临床结果。这项应用的发现将推动改善生活质量的努力， 通过为外周动脉疾病患者提供更安全、更有效的治疗选择，