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

项目总结 衰老是公认的外周血管疾病(PVD)的危险因素。人类衰老的一个标志是失去 血管功能部分通过抑制内皮细胞(EC)的自我更新来反映。一个未得到满足的需求是 出现了阻止或逆转内皮衰老的治疗方法(细胞分裂能力下降， 自我更新)和血管功能不全。衰老的第二个病相学特征是反应性增加 氧物种(ROS)。病理性ROS损害血管流动，但导致这些的机制 血管内皮细胞的不适应年龄相关变化仍不清楚。凝血酶敏感蛋白-1(TSP1)升高 在外周血管疾病(PVD)患者的血浆、动脉壁和间质液体中，并与 疾病的严重性和血管的丧失。我们发现，随着年龄的增长，野生型(WT)小鼠表现出诱导 TSP1及其同源细胞膜受体CD47与血管扩张剂减少有关。 调节血流变化，这种影响在TSP1-/-和CD47-/-小鼠中没有观察到。我们还报道了， TSP1通过CD47有效地刺激NADPH氧化酶1(Nox1)来源的ROS，并阻碍后肢血流。 我们课题组的初步数据显示，TSP1在老年人中上调，它抑制内皮自身 复制并促进外周血管内皮细胞衰老。这些发现促使我们提出了 最重要的假设是，TSP1通过CD47参与和Nox1激活，通过 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 研究目的：(1)体外检测TSP1老化诱导NOX1介导的关键自我抑制。 更新基因，促进内皮细胞衰老；(2)在体内探讨抑制血管内皮细胞的作用 TSP1-CD47轴以及减弱其下游的Nox1效应器恢复自我更新， (3)研究TSP1-CD47-Nox1对TSP1-CD47-Nox1表达的影响。 老年人组织中的AXIS改善与年龄相关的内皮和血管功能障碍。这项建议 将使用多种分子和遗传工具来询问TSP1-CD47介导的机制 Nox1在衰老的人和小鼠血管功能、自我更新和血管生成中的激活 产生两只EC特异的基因敲除小鼠来测试我们的假设。这些研究还将提供强有力的预 临床数据为合理开发治疗EC衰老和营养不良的新的有效疗法提供依据 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.