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: 10044162
• 负责人: Vivian Gahtan
• 金额: $ 38.23万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10044162
• 关键词: ADAMTS; 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; 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; Smooth Muscle Myocytes; Specimen; 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; novel; optimal treatments; 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的再狭窄 外周动脉是致残和死亡的重要原因。凝血酶原蛋白(TSP)是 多功能基质细胞蛋白在内膜增生症的发生发展中起中心作用。它们不是 动脉壁结构，但通过结合细胞因子、细胞表面而对动脉结构产生生理作用 受体、蛋白水解酶和其他蛋白质。本提案侧重于三个TSP，这三个TSP是 内膜增生-TSP-1、TSP-2、TSP-5。我们研究了TSP-1对血管平滑肌的影响。 肌细胞(VSMC)的增殖和迁移及其在内膜增生症发生发展中的重要性； 然而，越来越多的证据表明，TSP-2和TSP-5在这一过程中具有单独的和有贡献的作用 病理学。这三个TSP都是ADAMTS(一种具有 凝血酶原蛋白基序)蛋白质。ADAMTS消化TSP，增强或抑制TSP功能，因为 消化后的残留物对内膜增生有明显的影响。因此，ADAMTS-1，-4， 和-7也将被研究，因为它们参与了PAD，并被确定为受我们的 先前的VSMC基因研究。我们的长期目标是了解如何通过治疗来操纵TSP 预防体内血管内膜增生。本提案目标是确定TSP-1、TSP-2和TSP-5如何 特别是对内膜增生的发展有促进作用。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的变化 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 是在我们强大的初步数据、我们的出版物和文献的基础上制定的。其基本原理是 建议的项目是，了解TSP在内膜增生中的作用将导致识别 抑制球囊血管成形术后内膜增生和再狭窄的治疗目标。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各自所起的作用 2)比较TSP-1、TSP-2和TSP-5对血管内膜细胞蛋白质和蛋白表达的影响。 MicroRNA表达(即miR-17~92簇)及其下游对VSMC功能的影响； 确定ADAMTS在TSP-1、TSP-2和TSP-5活性以及在血管内膜发育中的作用 增生症。用于调查这些特定目标的方法包括：1)修改后的博伊登 评估趋化性的小室和评估VSMCs增殖的比色法；2)蛋白质印迹， 细胞信号和蛋白表达的ELISA法和免疫PCR法；3)实时定量聚合酶链式反应 基因表达反应；4)两种动脉内膜增生动物模型--颈总动脉球囊 大鼠损伤和小鼠结扎；5)利用基因敲除小鼠和siRNA敲除TSP和ADAMTS 分别观察体外和体内基因对血管平滑肌细胞和内膜增生的影响；以及6)形态计量学 动脉标本的分析、免疫印迹和免疫组织化学染色。SiRNA起作用 还将涉及测试我们针对TSP/ADAMTS组合的新型siRNA，这些组合可能被证明是高度 血管成形术时阻断内膜增生的有效方法。建议的重要意义 研究表明，这些发现将在确定新的预防策略方面取得重大进展 血管内膜增生性再狭窄。在本申请中提出的研究是创新的，在我们看来， 因为这些发现将定义多个TSP和肽酶与TSP基序(ADAMTS)的相互作用 在血管疾病中以及通过新的siRNA操纵这些系统的机制 改善临床结果。这项申请的结果将推动改善生活质量和 为外周动脉疾病患者提供更安全、更有效的治疗选择，从而延长寿命。