Statins and VEGF-induced cytoskeletal remodeling

他汀类药物和 VEGF 诱导的细胞骨架重塑

• 批准号: 7007327
• 负责人: FARHAD R DANESH
• 金额: $ 33.25万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7007327
• 关键词: G protein; HMG coA reductases; actins; biological signal transduction; biosensor device; cytoskeleton; diabetes mellitus; diabetic nephropathy; disease /disorder model; enzyme activity; fluorescence resonance energy transfer; growth factor receptors; guanosinetriphosphatases; laboratory mouse; myosins; oxidoreductase inhibitor; phosphorylation; renal glomerulus; simvastatin; vascular endothelial growth factors

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, also known as statins, are potent inhibitors of cholesterol biosynthesis. Recent experimental and clinical data, however, indicate that the overall benefits of statin therapy may exceed its cholesterol-lowering properties. In this proposal, we hypothesize that statins may attenuate the detrimental effects of vascular glomerular endothelial growth factor (VEGF) in diabetic nephropathy. This hypothesis is based on our recent observations indicating that 1) VEGF increases glomerular endothelial cell (GEC) hyperpermeability, 2) Rho GTPase proteins play a pivotal role in VEGF signaling pathway, and 3) Statins have multiple modulatory effects on Rho-mediated cell signaling pathways (Danesh et al. Proc Natl Acad Sci U S A. 2002, 99:8301-5, and Appendix #4). Based upon those initial observations, the investigators now propose an integrated in vitro and in vivo study to characterize the molecular mechanism of VEGF-induced GEC hyperpermeability, and to examine the modulatory effects of statins on VEGF-induced signaling pathway. The three specific aims of this proposal are: Aim 1: To delineate VEGF-induced GEC hyperpermeability: We hypothesize that VEGF stimulation elicits an increase in Rho GTPase activity and myosin regulatory light chain phosphorylation. This sequence of events causes significant actin-myosin cytoskeletal remodeling leading to increased GEC hyperpermeability. Using rat GECs as the experimental model, we will establish the role of Rho family of small GTPases in the VEGF-induced endothelial cell hyperpermeability. The effect of VEGF on cytoskeletal remodeling and myosin II contraction will be assessed using high resolution fluorescent live cell imaging. Aim 2: To determine the modulatory effects ofstatins on VEGF-induced signaling pathway: We hypothesize that statins, by preventing geranylgeranylation of small Rho GTPases, will attenuate VEGF-induced hyperpermeability in GECs. The modulatory effects of statins on Rho GTPase activation will be investigated. The effects of statins on myosin regulatory light chain phosphorylation and actin cytoskeletal remodeling will be assessed dynamically by measuring myosin light chain kinase activity in situ using a newly developed fluorescent resonant energy transfer-based biosensor. Aim 3: To assess renoprotective effects of statins in animal models of diabetes: Functional and biochemical analyses will determine whether renoprotective effects of statins in animal models of diabetic nephropathy correlate with their modulatory effects on VEGF-induced Rho-regulated signaling cascade. The broad and long term goal of this project is to investigate the role of small G proteins in the pathogenesis of diabetic nephropathy. The proposed study will impact the current management of this disease by identifying VEGF and small Rho GTPases as potential therapeutic targets, and by providing a new rationale for the use of statins in the early stages of diabetic nephropathy.

3-羟基-3-甲基戊二酰辅酶 A (HMG-CoA) 还原酶抑制剂，也称为他汀类药物，是胆固醇生物合成的有效抑制剂。然而，最近的实验和临床数据表明，他汀类药物治疗的总体益处可能超过其降低胆固醇的特性。在本提案中，我们假设他汀类药物可能会减轻有害的 血管性肾小球内皮生长因子（VEGF）在糖尿病肾病中的作用。这一假设基于我们最近的观察结果，表明 1) VEGF 增加肾小球内皮细胞 (GEC) 通透性过高，2) Rho GTPase 蛋白在 VEGF 信号通路中发挥关键作用，3) 他汀类药物对 Rho 介导的多种调节作用 细胞信号传导途径（Danesh 等人，Proc Natl Acad Sci U S A. 2002, 99:8301-5 和附录#4）。基于这些初步观察，研究人员现在提出一项综合体外和体内研究，以表征 VEGF 诱导的 GEC 通透性过高的分子机制，并检查他汀类药物对 VEGF 诱导的 GEC 通透性过高的调节作用。 信号通路。该提案的三个具体目标是： 目标 1：描述 VEGF 诱导的 GEC 通透性过高： 我们假设 VEGF 刺激会引起 Rho GTP 酶活性和肌球蛋白调节轻链磷酸化的增加。这一系列事件导致显着的肌动蛋白-肌球蛋白细胞骨架重塑，从而导致 GEC 通透性过高。使用大鼠GEC作为实验模型，我们将建立小GTP酶Rho家族在VEGF诱导的内皮细胞通透性过高中的作用。将使用高分辨率荧光活细胞成像来评估 VEGF 对细胞骨架重塑和肌球蛋白 II 收缩的影响。目标 2：确定他汀类药物对 VEGF 诱导的信号通路的调节作用：我们假设他汀类药物通过阻止小 Rho GTP 酶的香叶基香叶基化，将减弱 VEGF 诱导的 GEC 通透性过高。他汀类药物对 Rho GTPase 的调节作用 将调查激活情况。他汀类药物对肌球蛋白调节轻链磷酸化和肌动蛋白细胞骨架重塑的影响将通过使用新开发的基于荧光共振能量转移的生物传感器原位测量肌球蛋白轻链激酶活性来动态评估。目标 3：评估他汀类药物在动物模型中的肾脏保护作用 糖尿病：功能和生化分析将确定他汀类药物在糖尿病肾病动物模型中的肾脏保护作用是否与其对 VEGF 诱导的 Rho 调节信号级联的调节作用相关。该项目的广泛和长期目标是研究小G蛋白在糖尿病肾病发病机制中的作用。 拟议的研究将通过确定 VEGF 和小 Rho GTPases 作为潜在的治疗靶点，并为在糖尿病肾病早期阶段使用他汀类药物提供新的原理，从而影响该疾病的当前治疗。