The Role of The Thrombospondins In Intimal Hyperplasia

血小板反应蛋白在内膜增生中的作用

基本信息

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
项目类别：
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

项目摘要

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) 的再狭窄外周动脉是导致残疾和死亡的重要原因。多功能基质细胞蛋白对于内膜增生的发展至关重要，它们不属于内膜增生的一部分。动脉壁结构，但通过结合细胞因子、细胞表面对动脉结构发挥生理作用该提案重点关注三种 TSP 的开发。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 在此过程中具有独立且共同的作用所有三种 TSP 都是 ADAMTS（一种解整合素和金属蛋白酶）的底物。 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。和-7 也将受到研究，因为它们涉及 PAD，并且在我们的研究中也被确定受 TSP 监管我们的长期目标是了解如何对 TSP 进行治疗性操作。本提案的目的是确定 TSP-1、TSP-2 和 TSP-5 如何预防内膜增生。我们的中心假设是，表达特别有助于内膜增生的发展。 TSP-1、TSP-2 和 TSP-5 诱导的生物利用度、信号传导途径和基因表达变化以及它们与 ADAMTS 的相互作用对调节 IH 的发展有明显的影响。是在我们强有力的初步数据、出版物和文献的基础上制定的。拟议的项目是了解 TSP 对内膜增生的作用将导致识别我们的假设是抑制球囊血管成形术后内膜增生和再狭窄的治疗目标。通过测试以下具体目标来追求： 1) 确定 TSP-1、TSP-2 和 TSP-5 各自的作用 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 活性以及内膜发育中的作用用于研究这些具体目标的方法包括：1) 改良博伊登。用于评估趋化性的腔室和用于评估 VSMC 增殖的比色测定；2) 蛋白质印迹，用于细胞信号转导和蛋白质表达的 ELISA 和免疫 PCR；3) 实时定量聚合酶链基因表达反应；4）两种内膜增生动物模型——颈总动脉球囊大鼠损伤和小鼠结扎；5) 使用敲除小鼠和 siRNA 敲低 TSP 和 ADAMTS 体外和体内基因分别观察对 VSMC 和内膜增生的影响；6) 形态测量；用于分析动脉样本的蛋白质印迹和免疫组织化学染色 siRNA 的作用。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血管成形术时有效阻断内膜增生的方法的提出的意义。研究表明，这些发现将为确定预防新策略提供重大进展我们认为，本申请中提出的研究具有创新性，因为这些发现将定义多种 TSP 和肽酶与 TSP 基序 (ADAMTS) 的相互作用血管疾病和机制，通过这些系统可以用新型 siRNA 来操纵改善临床结果该应用的结果将推动改善生活质量和通过为外周动脉疾病患者提供更安全、更有效的治疗选择来延长寿命。