Role of protein-S-glutathionylation in endothelial dysfunction and atherosclerosis

项目摘要 动脉粥样硬化(AS)的途径始于血管内皮功能障碍(ED)，这种功能障碍是可以逆转的； 然而，针对ED的有效治疗策略仍然缺乏。小RhoGTP酶rac1，一个已建立的 内皮功能的主要调节者，是血管内稳态不可或缺的一部分，管理血管的完整性和 一氧化氮(NO)生物发生系统，但异常的rac1信号，特别是rac1触发的NADPH-氧化酶 (NOx)/活性氧(ROS)的产生与血管氧化应激和 因此，AS可能是CVD治疗的一个有希望的靶点。然而，要想成功地确定新的 使rac1信号正常化以对抗血管氧化应激以逆转ED和 因此，我们必须对Rac1的调控机制有深刻的了解，才能平衡Rac1对S的批评 在内皮细胞(ECs)中发挥促动脉粥样硬化的作用。在这项建议中，我们将测试是否： (1)对半胱氨酸的一种稳定但可逆的氧化后修饰--S-谷胱甘肽修饰 Rac1的残基是一个很有希望的氧化还原靶标，以及(2)谷氧还蛋白-1(Grx1)，一个重要的谷胱甘肽脱氢酶 酶，可以维持rac1的氧化还原状态和正常的信号转导。我们和其他人报告了这一活动 Rac1的稳定性对氧化还原反应敏感，对PRS-SG敏感。我们新的初步数据显示，在这两个国家 人主动脉内皮细胞(HAECs)和AS、rac1小鼠模型的主动脉经PRS-SG和降解 在Grx1下调的同时，增加Grx1在HAECs中保留了rac1的氧化还原状态/表达。 增加小鼠内皮细胞Grx1水平可减轻饮食诱导的ED和AS。因此我们假设在AS期间， 在血管内皮细胞内补充Grx1可逆转这一作用。 我们将测试三个目标：(1)在体内表征内皮细胞rac1/Grx1轴在ED和AS中的作用；(2)阐明 Rac1上PsS-SG驱动ED的分子机制；(3)检测Rac1/Grx1轴作为 逆转ED和AS的治疗靶点。定义氧化还原状态/表达式/活动之间的关系 对于rac1/Grx1与ED和AS的发生/发展(目标1)，我们将使用EC特异性的Grx1转基因和敲除 小鼠，以及对rac1的药理抑制。为了测试rac1上的prs-sg是否在ED中起因果作用(目标2)， 我们将确定rac1-SSG模拟突变体和耐药突变体在产生的rac1-/-HAECs中的表达效果 关于NOx/ROS信号、eNOS功能障碍和高通透性。以测试补充内皮细胞Grx1是否 可以逆转血管功能障碍并减缓AS进展(目标3)，我们将使用可诱导的EC-Grx1转基因 小鼠和新鲜分离自已确诊AS的人的内皮细胞。这项提议有望获得成功 为了建立一种新的氧化还原机制，该机制可以特异性地介导rac1致动脉粥样硬化信号转导，并提供 Grx1可作为逆转心血管功能障碍的新治疗策略的概念验证。

Project Summary The path to atherosclerosis (AS) starts with vascular endothelial dysfunction (ED), which can be reversed; however, effective therapeutic strategies targeting ED are still lacking. Small RhoGTPase Rac1, an established master regulator of endothelial function, is integral to vascular homeostasis, governing vascular integrity and the nitric oxide (NO) biogenesis system, but aberrant Rac1 signaling, in particular, Rac1-triggered NADPH-oxidase (Nox)/reactive oxygen species (ROS) generation, has been strongly implicated in vascular oxidative stress and AS and may therefore be a promising target for CVD treatment. However, to be successful at determining new specific approaches to normalizing Rac1 signaling that can combat vascular oxidative stress to reverse ED and AS, we must have a deep understanding of regulatory mechanisms of Rac1 to be able to balance Rac1's critical functions with its proatherogenic consequences in endothelial cells (ECs). In this proposal, we will test whether: (1) S-glutathionylation (PrS-SG), a stable but reversible oxidative post-translational modification, on cysteine residues of Rac1 is a promising redox target, and (2) glutaredoxin-1 (Grx1), a prominent de-glutathionylation enzyme, can preserve redox status and normal signaling of Rac1. We and others reported that activity and stability of Rac1 are redox-sensitive and susceptible to PrS-SG. Our new preliminary data show that in both human aortic ECs (HAECs) and aortae from a mouse model of AS, Rac1 underwent PrS-SG and degradation in parallel with Grx1 downregulation; increasing Grx1 in HAECs preserved Rac1 redox status/expression. Increasing endothelial Grx1 in mice attenuated diet-induced ED and AS. We thus hypothesize that during AS, induction of PrS-SG on Rac1 promotes ED and AS, which can be reversed by replenishing Grx1 in endothelium. We will test three aims: (1) Characterize in vivo role of endothelial Rac1/Grx1 axis in ED and AS; (2) Elucidate the molecular mechanism of how PrS-SG on Rac1 drives ED; and (3) Test the potential of Rac1/Grx1 axis as a therapeutic target in reversing ED and AS. To define the relationship between redox status/expression/activity of Rac1/Grx1 and onset/progression of ED and AS (Aim 1), we will use EC-specific Grx1 transgenic and knockout mice, as well as pharmacological inhibition of Rac1. To test if PrS-SG on Rac1 has a causal role in ED (Aim 2), we will determine effects of expression of Rac1-SSG-mimic and -resistant mutants in generated Rac1-/- HAECs on Nox/ROS signaling, eNOS dysfunction, and hyperpermeability. To test whether replenishing endothelial Grx1 can reverse vascular dysfunction and slow AS progression (Aim 3), we will use inducible EC-Grx1 transgenic mice and ECs freshly isolated from human subjects with established AS. Success of this proposal is expected to establish a novel redox mechanism that can specifically mediate Rac1 proatherogenic signaling, and provide proof-of-concept that Grx1 can be used as a new therapeutic strategy for reversal of cardiovascular dysfunction.