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

蛋白质-S-谷胱甘肽化在内皮功能障碍和动脉粥样硬化中的作用

基本信息

批准号：
9883986
负责人：
JINGYAN HAN
金额：
$ 41.25万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-04 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9883986
关键词：
Acetylcholine Adult Affect American Apolipoprotein E Atherosclerosis Attenuated Biogenesis Biological Availability Blood Vessels CRISPR/Cas technology Cardiovascular Diseases Cardiovascular system Cause of Death Cell physiology Cysteine Data Development Diet Dietary Intervention Down-Regulation Endothelial Cells Endothelium Enzymes Equilibrium Event Functional disorder Gatekeeping Generations Grx1 protein Hemostatic function Homeostasis Human Impairment Intervention Knock-out Knockout Mice Lead Measures Mediating Molecular Monitor Mus Myocardial Infarction NADPH Oxidase Nitric Oxide Oxidases Oxidation-Reduction Oxidative Stress Pathogenicity Pathway interactions Pharmacology Physiological Population Post-Translational Protein Processing Production Proteins Publishing Reactive Oxygen Species Recombinant Proteins Reporting Resistance Risk Factors Role Signal Transduction Stimulus Stroke System Testing Time Transgenes Transgenic Mice Transgenic Organisms Treatment Efficacy Ubiquitin United States Vascular Diseases Vascular Endothelium Vasodilation adenoviral-mediated antioxidant enzyme atherogenesis atherosclerosis risk clinically relevant combat driving force endothelial dysfunction femoral artery human subject imaging approach improved in vivo mouse model multicatalytic endopeptidase complex mutant novel novel strategies novel therapeutic intervention overexpression preservation rac1 GTP-Binding Protein response success therapeutic target transgene expression vascular endothelial dysfunction vascular injury western diet

项目摘要

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.

项目摘要动脉粥样硬化的路径（AS）始于血管内皮功能障碍（ED），可以逆转；但是，仍缺乏针对ED的有效治疗策略。小rhogtpase rac1，已建立的内皮功能的主要调节剂，是血管稳态不可或缺的，管理血管完整性和一氧化氮（NO）生物发生系统，但异常RAC1信号传导，特别是Rac1触发的NADPH-氧化酶（NOX）/活性氧（ROS）产生，与血管氧化应激和因此，可能是CVD处理的有希望的目标。但是，要成功确定新的将Rac1信号归一化的特定方法，该信号传导可以打击血管氧化应激以逆转ED和因为，我们必须对Rac1的监管机制有深刻的了解，以便能够平衡Rac1的关键其在内皮细胞（EC）中具有促进质基的后果。在此提案中，我们将测试是否：（1）在半胱氨酸上稳定但可逆的翻译后修饰（PRS-SG），一种稳定但可逆的氧化后氧化后修饰 RAC1的残留物是一个有前途的氧化还原靶标，（2）谷胱甘肽-1（GRX1），一种突出的脱甲基二硫代二硫代化靶标酶，可以保留Rac1的氧化还原状态和正常信号传导。我们和其他人报告了这项活动和 Rac1的稳定性对氧化还原敏感，并且容易受到PRS-SG的影响。我们的新初步数据表明，这两个 RAC1的小鼠模型中的人主动脉EC（HAEC）和主动脉进行了PRS-SG和降解与GRX1下调并行； HAEC中增加的GRX1保留了Rac1氧化还原状态/表达。在小鼠中增加内皮GRX1会减弱饮食引起的ED和AS。因此，我们假设在AS期间 Rac1上的PRS-SG诱导促进了ED和AS，可以通过在内皮中补充GRX1来逆转。我们将测试三个目标：（1）表征内皮Rac1/grx1轴在ED和AS中的体内作用；（2）阐明 Rac1上PRS-SG如何驱动ED的分子机制；（3）将Rac1/Grx1轴的电位作为A测试逆转ED和AS的治疗目标。定义氧化还原状态/表达/活动之间的关系 Rac1/grx1和ED的发作/进展和AS（AIM 1），我们将使用EC特异性GRX1转基因和敲除小鼠以及Rac1的药理抑制作用。测试Rac1上的PRS-SG在ED中是否具有因果作用（AIM 2），我们将确定Rac1-SSG-MIMIC-MIMIC和耐药突变体在产生的Rac1 - / - HAEC中的影响在NOX/ROS信号传导上，eNOS功能障碍和超透明度。测试是否补充内皮GRX1 可以逆转血管功能障碍，而随着进展的速度缓慢（AIM 3），我们将使用诱导的EC-GRX1转基因小鼠和EC从具有既定为AS的人类受试者中新鲜分离出来。预计该提议的成功是建立一种新型的氧化还原机制，该机制可以专门介导Rac1 proathathothenit Signal传导，并提供概念证明GRX1可以用作逆转心血管功能障碍的新治疗策略。