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

描述(由申请人提供)：活性氧物种(ROS)广泛地与心血管疾病(CVD)的发病机制有关。ROS介导的血管功能障碍部分是通过ROS超氧阴离子失活血管扩张剂一氧化氮(NO)和/或促进血管收缩的直接下游信号通路发生的。血管ROS的一个主要来源是NADPH氧化酶或Nox蛋白。基质细胞蛋白血栓反应蛋白-1(TSP1)在CVD的血管系统中显著升高，并与血管功能障碍有关。我们报道了TSP1通过其同源受体CD47抑制血管扩张，但其确切机制尚不清楚。除CD47外，血管平滑肌细胞也表达信号调节蛋白α，这是一种膜受体蛋白，与炎症细胞中ROS的产生有关，但信号调节蛋白S在VSMC ROS中的作用尚不清楚。缺血再灌注(I/R)是一种ROS升高导致血管血流障碍的疾病。小鼠后肢准备是一种被广泛接受的I/R模型，我们实验室以前的数据表明，CD47拮抗剂可以保护血管免受I/R相关的血流损伤。SIRP-？S在I/R中的角色尚不清楚。初步数据显示，TSP1可通过CD47刺激VSMC产生超氧阴离子；(2)通过SIRP依赖的信号通路刺激VSMC产生过氧化氢；(3)通过ROS介导的血管张力障碍。这些发现提供了我们的总体假设，即TSP1通过CD47和SIRP依赖的信号促进VSMCs产生ROS，导致I/R血管松弛和/或收缩功能明显受损。这一全新的创新方案通过TSP1在不同的协同受体/信号通路上的多方面新作用来研究，导致病理性ROS形成和I/R诱导的血管功能障碍。将通过下列目的进行测试：(1)首次检测TSP1与VSMC CD47结合是否通过G蛋白激活和丝裂原激活激酶途径增加超氧阴离子水平，进而进而激活NOx；(2)首次询问SIRP-β和SHP-1/2信号通路是否在TSP1诱导的VSMC和NOx诱导的过氧化氢产生中扮演一个角色；(3)在体内探讨CD47和SIRP-β激活是否导致小鼠后肢I/R模型中的血流量减少。基于初步发现的坚实基础，目前的提案利用多种分子和遗传工具来探索(A)基质细胞蛋白TSP1在通过CD47和SIRP-1产生血管ROS中的新作用；(B)涉及的新的下游介质和氧化酶来源；以及反过来，在I/R诱导的血管损伤中确定新的治疗靶点。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意义。

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.