Progressive degenerative role of Nox and thrombospondin-1 in the aging vasculature

项目摘要 衰老是周围血管疾病（PVD）的公认危险因素。人类衰老的一个标志是 血管功能部分地通过抑制内皮细胞（EC）的自我更新来反映。未满足的需求是 出现了阻止或逆转内皮衰老（细胞分裂能力降低， 自我更新）和血管功能不全。衰老的第二个特征是反应性增加 氧物种（ROS）。病理性活性氧损伤血管血流，但这些机制 内皮中与年龄相关的适应不良变化仍然未知。血小板反应蛋白-1（TSP 1）升高 在外周血管疾病（PVD）患者的血浆、动脉壁和间质液中， 疾病的严重程度和血管的损失。我们已经发现，随着年龄的增长，野生型（WT）小鼠显示出诱导 TSP 1及其同源细胞膜受体CD 47，这与血管扩张因子- 在TSP 1-/-和CD 47-/-小鼠中未观察到这种效应。我们还报道说， TSP 1通过CD 47有效刺激NADPH氧化酶1（Nox 1）衍生的ROS并阻碍后肢血流。 我们小组的初步数据显示，TSP 1在老年人中上调，它抑制内皮细胞自我调节， 复制并促进外周血管中的内皮衰老。这些发现促使我们提出 总体假设，TSP 1，通过CD 47的参与和Nox 1的激活，损害自我更新， 加剧ROS的产生，最终导致EC衰老和血管病变。三个具体目标将是 （1）体外试验表明，随着年龄的增长，TSP 1诱导Nox 1介导的关键自我抑制， 更新基因，促进内皮细胞（EC）衰老;（2）在体内探索抑制 TSP 1-CD 47轴以及减弱其下游Nox 1效应子恢复自我更新， 研究TSP 1-CD 47-Nox 1在老年动物血管生成和灌注中的作用;（3）研究TSP 1-CD 47-Nox 1 轴在老年人组织改善年龄相关的内皮和血管功能障碍。该提案 将采用多种分子和遗传工具来询问TSP 1-CD 47介导的 Nox 1在衰老的人和小鼠血管功能、自我更新和血管生成中的活化，以及 产生两个EC特异性敲除小鼠来测试我们的假设。这些研究也将提供强大的预- 合理开发新的有效治疗EC衰老的临床数据， 血管病变

PROJECT SUMMARY Aging is a recognized risk factor in peripheral vascular disease (PVD). A hallmark of aging in humans is loss of vascular function reflected, in part, by inhibited self-renewal of endothelial cells (EC). An unmet need is the emergence of therapies that stop or reverse endothelial senescence (decreased capacity for cellular division, self-renewal) and vascular insufficiency. A second pathognomonic feature of aging is increased reactive oxygen species (ROS). Pathologic ROS impair vascular flow, but the mechanisms responsible for these maladaptive age-related changes in the endothelium remain unknown. Thrombospondin-1 (TSP1) is increased in the plasma, arterial walls and interstitial fluid of peripheral vascular disease (PVD) patients and is linked to disease severity and loss of vascularity. We have found that with aging, wild type (WT) mice show induction of TSP1 and its cognate cell membrane receptor CD47 and this was associated with decreased vasodilator- mediated changes in blood flow, an effect not observed in TSP1-/- and CD47-/- mice. We also reported that TSP1, via CD47, potently stimulates NADPH oxidase 1 (Nox1)-derived ROS and impedes hind-limb blood flow. Preliminary data by our group show that TSP1 is upregulated in older adults, and it inhibits endothelial self- replication and promotes endothelial senescence in peripheral vessels. These findings led us to propose the overarching hypothesis that TSP1, via CD47 engagement and Nox1 activation, impairs self-renewal through exacerbated ROS production culminating in EC senescence and vasculopathy. Three specific aims will be pursued to: (1) test in vitro that with aging TSP1 induces Nox1-mediated suppression of key self- renewal genes and promotes endothelial cell (EC) senescence; (2) explore in vivo that inhibition of the TSP1-CD47 axis as well as attenuating its downstream Nox1 effector restores self-renewal, angiogenesis and perfusion in aged animals; and (3) investigate that perturbing the TSP1-CD47-Nox1 axis in aged human tissue ameliorates age-related endothelial and vascular dysfunction. The proposal will employ multiple molecular and genetic tools to interrogate mechanisms into TSP1-CD47-mediated activation of Nox1 in aging human and mouse vessel function, self-renewal and angiogenesis, as well as generate two EC-specific knockout mice to test our hypotheses. These studies will also provide robust pre- clinical data towards rational development of new and effective treatments for EC senescence and vasculopathy.