TSP-1 and ROS: CD47 and SIRP-alpha as Mediators of Vascular Dysfunction

TSP-1 和 ROS：CD47 和 SIRP-α 作为血管功能障碍的介质

• 批准号: 8588484
• 负责人: Jeffrey S Isenberg
• 金额: $ 36.3万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-13 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8588484
• 关键词: Agonist; Antibodies; Aorta; Arteries; Binding; Blood; Blood Vessels; Blood flow; CD47 gene; Cardiovascular Diseases; Cells; Co-Immunoprecipitations; Data; Disease; Electron Spin Resonance Spectroscopy; Foundations; Functional disorder; GTP-Binding Proteins; Gene Silencing; Generations; Human; Hydrogen Peroxide; Immunofluorescence Immunologic; Impairment; Inflammatory; Injury; Ischemia; Knockout Mice; Knowledge; Laboratories; Lasers; Lead; Limb structure; Link; MAP Kinase Gene; MAPK14 gene; Measures; Mediating; Mediator of activation protein; Membrane; Mitogens; Modeling; Molecular Genetics; Monitor; Mus; Myocardial Infarction; Myosin Light Chain Kinase; Myosin Light Chains; NADPH Oxidase; Neurons; Nitric Oxide; Null Lymphocytes; Organ Transplantation; Oxidants; Oxidases; Oxidative Stress; PTPN11 gene; PTPN6 gene; PTPNS1 gene; Pathogenesis; Pathway interactions; Patients; Phenylephrine; Phosphorylation; Phosphotransferases; Play; Preparation; Process; Production; Proteins; Reactive Oxygen Species; Receptor Signaling; Relaxation; Reperfusion Injury; Reperfusion Therapy; Reporting; Research; Resistance; Role; SHPS-1 protein; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Source; Stroke; Superoxides; Testing; Thrombospondin 1; Time; Vasodilation; Vasodilator Agents; Western Blotting; base; constriction; cytochrome c; human PTPNS1 protein; in vivo; innovation; monocyte; new therapeutic target; novel; overexpression; protein activation; protein aminoacid sequence; public health relevance; receptor; restoration; therapeutic target; tissue trauma; tool; vasoconstriction

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) are broadly implicated in the pathogenesis of cardiovascular disease (CVD). ROS-mediated vascular dysfunction occurs, in part, via inactivation of the vasodilator nitric oxide (NO) by ROS superoxide anion and/or direct downstream signaling pathways promoting vasoconstriction. A major source of vascular ROS is the NADPH oxidases or Nox proteins. The matricellular protein thrombospondin-1 (TSP1) is significantly elevated in the vasculature in CVD and is associated with vascular dysfunction. We reported that TSP1, via its cognate receptor CD47, inhibits vasodilatation, however the exact mechanism remain unclear. In addition to CD47, vascular smooth muscle cells (VSMCs) also express signal regulatory protein alpha (SIRP- ¿), a membrane receptor protein that has been linked to ROS production in inflammatory cells, but SIRP-¿'s role in VSMC ROS is entirely unknown. Ischemia reperfusion (I/R) is a disease in which increased ROS leads to impairment in vascular flow. The mouse hind-limb preparation is a widely-accepted I/R model and previous data from our laboratory show that CD47 blockade protects vessels from I/R-associated flow impairment. SIRP-¿'s role in I/R is not known. Preliminary data show that TSP1 potently stimulates (1) Nox-derived superoxide anion production in VSMCs via CD47; (2) VSMC hydrogen peroxide via SIRP-¿-dependent signaling; and (3) ROS-mediated vascular tone dysfunction. These findings inform our overarching hypothesis that TSP1 promotes ROS production in VSMCs via CD47- and SIRP-¿-dependent signaling, leading to marked impairment in vascular relaxation and/or enhanced constriction in I/R. This wholly innovative proposal investigates via multi-faceted novel actions of TSP1 on distinct synergizing receptor/signaling pathways, leading to pathological ROS formation and I/R-induced vascular dysfunction. This will be tested via the following aims: (1) examining for the first time whether TSP1 binding to VSMC CD47 increases superoxide anion levels via G-protein activation and mitogen-activated kinase pathways and, in turn, Nox activation; (2) interrogating for the first time whether SIRP-¿, and SHP-1/2 signaling, plays a role in TSP1-induced hydrogen peroxide production in VSMCs and via Nox; (3) exploring in vivo whether CD47 and SIRP-¿ activation lead to decreased blood flow in the mouse hind limb I/R model. Based on a strong foundation of preliminary findings, the current proposal employs multiple molecular and genetic tools to explore (a) a novel role for matricellular protein TSP1, in vascular ROS production via CD47 and SIRP-¿; (b) novel downstream mediators and oxidase sources involved; and, in turn, identify novel therapeutic targets in I/R-induced vascular injury. This research plan is novel at al levels and has potential implications for the role of myriad other matricellular proteins in CVD.

描述（由申请人提供）：活性氧（ROS）广泛参与心血管疾病（CVD）的发病机制。ROS介导的血管功能障碍部分地通过ROS超氧阴离子和/或促进血管收缩的直接下游信号传导途径使血管扩张剂一氧化氮（NO）失活而发生。血管ROS的主要来源是NADPH氧化酶或Nox蛋白。基质细胞蛋白血小板反应蛋白-1（TSP 1）在CVD的血管系统中显著升高，并与血管功能障碍相关。我们报道了TSP 1通过其同源受体CD 47抑制血管舒张，但确切机制尚不清楚。除了CD 47，血管平滑肌细胞（VSMC）还表达信号调节蛋白α（SIRP-<$），这是一种与炎症细胞中ROS产生有关的膜受体蛋白，但SIRP-<$在VSMC ROS中的作用完全未知。缺血再灌注（I/R）是其中增加的ROS导致血管流动受损的疾病。小鼠后肢制备物是广泛接受的I/R模型，并且来自我们实验室的先前数据显示，CD 47阻断保护血管免受I/R相关的血流损伤。SIRP在I/R中的作用尚不清楚。初步数据显示，TSP 1有效刺激（1）通过CD 47在VSMC中产生Nox衍生的超氧阴离子;（2）通过SIRP-1依赖性信号传导刺激VSMC过氧化氢;和（3）ROS介导的血管张力功能障碍。这些发现为我们的总体假设提供了信息，即TSP 1通过CD 47和SIRP-1依赖性信号促进VSMC中ROS的产生，导致血管舒张的显著损害和/或I/R中收缩的增强。这一完全创新的提议通过TSP 1对不同的协同受体/信号通路的多方面新作用进行研究，导致病理性ROS形成和I/R诱导的血管功能障碍。这将通过以下目的进行测试：（1）首次检查TSP 1与VSMC CD 47结合是否通过G蛋白活化和丝裂原活化激酶途径增加超氧阴离子水平，进而增加Nox活化;（2）首次询问SIRP-1和SHP-1/2信号传导是否在TSP 1诱导的VSMC中过氧化氢产生中起作用并通过Nox;（3）在小鼠后肢I/R模型中，体内探讨CD 47和SIRP-<$活化是否导致血流量减少。基于初步研究结果的坚实基础，目前的建议采用多种分子和遗传工具来探索（a）基质细胞蛋白TSP 1在通过CD 47和SIRP-1产生血管ROS中的新作用;（B）涉及的新下游介质和氧化酶来源;并进而确定I/R诱导的血管损伤的新治疗靶点。这项研究计划在al水平上是新颖的，并对无数其他基质细胞蛋白在CVD中的作用具有潜在的影响。