Anti-Atherogenic Mechanisms of Drebrin

Drebrin 的抗动脉粥样硬化机制

• 批准号: 9887940
• 负责人: NEIL J. FREEDMAN
• 金额: $ 52.33万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-16 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9887940
• 关键词: Actin-Binding Protein; Angiotensin II; Antiatherogenic; Apolipoprotein E; Arterial Fatty Streak; Arterial Injury; Arteries; Atherosclerosis; CX3CL1 gene; Carotid Arteries; Cell Lineage; Cell Proliferation; Cells; Cholesterol; Common carotid artery; Congestive Heart Failure; Development; Disease; Down-Regulation; Dynamin; Endocytosis; Enzymes; Exhibits; Foam Cells; Fractalkine; GKLF protein; GSTM1 gene; Generations; Genes; Glutathione S-Transferase; Goals; Human; In Vitro; Inflammatory; Knock-in; Knowledge; Lead; Mass Spectrum Analysis; Mediating; Medical; Memory; Metabolism; Microfilaments; Mission; Molecular; Morbidity - disease rate; Mus; Myocardial Infarction; NADPH Oxidase 1; Neurons; Nuclear; Nuclear Translocation; Phagocytes; Phenotype; Plant Roots; Prevalence; Process; Production; Proteins; Public Health; Reactive Oxygen Species; Research; Role; Signal Transduction; Smooth Muscle Myocytes; Stroke; Testing; United States National Institutes of Health; Vascular Smooth Muscle; ascending aorta; atherogenesis; burden of illness; cell motility; congenic; cytokine; disability; drebrins; in vivo; macrophage; mortality; novel therapeutic intervention; oxidized low density lipoprotein; response; transcription factor; transdifferentiation; vascular inflammation; vascular injury; western diet

We discovered that the actin-binding protein, Drebrin, is abundantly expressed in smooth muscle cells (SMCs) and is up-regulated in response to arterial injury in mice and to atherosclerosis in humans. Comparing WT with Dbn-/+ mice, we found that Drebrin inhibits SMC migration and proliferation, both in vitro and in vivo, through stabilization of actin filaments. In studies with SMC-specific Dbn-/- (SMC-Dbn-/-) mice, we found that SMC Drebrin limits angiotensin II-induced remodeling of the ascending aorta, in a manner that correlates with down-regulation of NADPH oxidase 1 (NOX1), decreased SMC reactive oxygen species (ROS) production and reduced vascular inflammation. Thus, Drebrin constrains not only the migratory/proliferative SMC phenotype but also the pro-inflammatory SMC phenotype evoked by vascular injury and angiotensin II. Congruently, we found that atherosclerosis is greater in SMC-Dbn-/-/Ldlr-/- than in congenic SMC-Dbn+/+/Ldlr-/- mice. Because SMC Drebrin negatively regulates atherosclerosis, our goals are to determine the mechanisms by which Drebrin regulates SMC pro-inflammatory signaling and whether enhanced SMC expression of Drebrin can inhibit atherogenesis. To this end, we performed SILAC/mass spectrometry studies on Dbn-/- and congenic WT SMCs. We found that whereas in Dbn-/- SMCs the ROS-defensive enzyme Glutathione-S-transferase µ1 (GSTM1) is down-regulated, the pro-inflammatory cytokine CX3CL1 (fractalkine) is up-regulated: thus we will investigate the role of these proteins in mediating the phenotype of Dbn-/- SMCs. Because endocytosis of NOX1 regulates ROS generation and activation of pro-inflammatory NFκB signaling and Drebrin has been shown to inhibit endocytosis, we will investigate whether Drebrin inhibits NOX1-mediated ROS generation by inhibiting endocytosis. A major focus of our studies will be pro-atherogenic SMC-derived foam cells, which SMC lineage tracing studies have shown, comprise ~40% of foam cells in atherosclerotic lesions. Our Preliminary Studies show that, compared with cognate Dbnflox/flox SMCs, Dbn-/- SMCs induced to transdifferentiate with cholesterol loading exhibited increased expression of the macrophage marker, CD68, and Kruppel-like factor 4 (KLF4), a transcription factor required for SMC-to-foam cell transdifferentiation. By grafting common carotid arteries from Dbnflox/flox and SMC-Dbn-/- mice into carotid arteries of congenic Apoe-/- mice, we also show that Drebrin deficiency augments transdifferentiation of SMCs to CD68+ cells in vivo. We will test the hypothesis that Drebrin inhibits atherogenesis by limiting SMC transdifferentiation into foam cells. To do so, we will establish whether Drebrin inhibits SMC-to-foam cell transdifferentiation and associated pro- inflammatory signaling, both in vitro and in vivo; determine if Drebrin inhibits SMC transdifferentiation through ROS-dependent mechanisms; and define the roles of GSTM1 and CX3CL1 in mediating Drebrin’s inhibitory effects on SMC transdifferentiation and pro-inflammatory signaling.

我们发现肌动蛋白结合蛋白 Drebrin 在平滑肌细胞 (SMC) 中大量表达 并且在小鼠动脉损伤和人类动脉粥样硬化的反应中上调。比较WT 通过 Dbn-/+ 小鼠，我们发现 Drebrin 在体外和体内均可抑制 SMC 迁移和增殖， 通过稳定肌动蛋白丝。在对 SMC 特异性 Dbn-/- (SMC-Dbn-/-) 小鼠的研究中，我们发现 SMC Drebrin 限制血管紧张素 II 诱导的升主动脉重塑，其方式与 NADPH 氧化酶 1 (NOX1) 下调，SMC 活性氧 (ROS) 产生减少， 减少血管炎症。因此，Drebrin 不仅限制迁移/增殖 SMC 表型 还有血管损伤和血管紧张素 II 诱发的促炎 SMC 表型。相应地，我们 发现 SMC-Dbn-/-/Ldlr-/- 小鼠的动脉粥样硬化程度高于同系 SMC-Dbn+/+/Ldlr-/- 小鼠。因为 SMC Drebrin 对动脉粥样硬化有负调节作用，我们的目标是确定其机制 Drebrin 调节 SMC 促炎信号传导以及 Drebrin 增强 SMC 表达是否可以 抑制动脉粥样硬化形成。为此，我们对 Dbn-/- 和同类进行了 SILAC/质谱研究 WT SMC。我们发现，在 Dbn-/- SMC 中，ROS 防御酶谷胱甘肽-S-转移酶 µ1 (GSTM1) 下调，促炎细胞因子 CX3CL1 (fractalkine) 上调：因此我们将 研究这些蛋白质在介导 Dbn-/- SMC 表型中的作用。因为内吞作用 NOX1 调节 ROS 生成和促炎 NFκB 信号传导的激活，Drebrin 已被 被证明可以抑制内吞作用，我们将研究 Drebrin 是否通过以下方式抑制 NOX1 介导的 ROS 产生： 抑制内吞作用。我们研究的一个主要焦点是促动脉粥样硬化的 SMC 衍生的泡沫细胞， SMC 谱系追踪研究表明，动脉粥样硬化病变中约 40% 的泡沫细胞由 SMC 组成。我们的 初步研究表明，与同源 Dbnflox/flox SMC 相比，Dbn-/- SMC 诱导 胆固醇负荷转分化表现出巨噬细胞标记物 CD68 表达增加， 和 Kruppel 样因子 4 (KLF4)，一种 SMC 向泡沫细胞转分化所需的转录因子。经过 将 Dbnflox/flox 和 SMC-Dbn-/- 小鼠的颈总动脉移植到同源 Apoe-/- 的颈动脉中 在小鼠中，我们还发现 Drebrin 缺陷会增强体内 SMC 向 CD68+ 细胞的转分化。我们 将检验 Drebrin 通过限制 SMC 转分化为泡沫细胞来抑制动脉粥样硬化形成的假设。 为此，我们将确定 Drebrin 是否抑制 SMC 到泡沫细胞的转分化以及相关的促细胞分化。 体外和体内的炎症信号传导；确定 Drebrin 是否通过以下方式抑制 SMC 转分化 ROS依赖机制；并定义 GSTM1 和 CX3CL1 在介导 Drebrin 抑制中的作用 对 SMC 转分化和促炎症信号传导的影响。