Anti-atherogenic Mechanisms of the Dual Rho-GEF Kalirin

双 Rho-GEF Kalirin 的抗动脉粥样硬化机制

• 批准号: 8894586
• 负责人: NEIL J. FREEDMAN
• 金额: $ 46.6万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-18 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8894586
• 关键词: Address; Affect; Anti-Inflammatory Agents; Anti-inflammatory; Antiatherogenic; Apolipoprotein E; Atherosclerosis; Attenuated; Benzimidazoles; Blood Vessels; Candidate Disease Gene; Carotid Arteries; Cell Adhesion Molecules; Cells; Chronic Disease; Coronary Arteriosclerosis; Data; Diet; EP300 gene; Endothelial Cells; Enzymes; Genes; Genetic; Genetic Polymorphism; Genomics; Goals; Growth Factor; Guanine Nucleotide Exchange Factors; Health; Human; Hyperlipidemia; Hyperplasia; In Vitro; Individual; Inflammatory; Interleukin-10; Ischemic Stroke; Label; Liquid substance; Mass Spectrum Analysis; Mediating; Metabolic; Molecular; Monomeric GTP-Binding Proteins; Mus; Neurons; Protein Binding Domain; Protein Isoforms; Proteins; Rattus; Regulation; Role; Signal Transduction; Smooth Muscle Myocytes; TNF gene; Tamoxifen; Testing; Variant; atherogenesis; benzimidazole; carbene; congenic; cytokine; epidemiology study; feeding; genetic epidemiology; human NOS2A protein; human NOS3 protein; inhibitor/antagonist; macrophage; migration; new therapeutic target; novel; osmotic minipump; p65; promoter; protein protein interaction; response; rho; scaffold; tandem mass spectrometry; western diet

DESCRIPTION (provided by applicant): Polymorphisms in the human KALRN gene have been associated with both coronary artery disease and ischemic stroke. The ~320 kDa protein Kalirin contains two guanine nucleotide exchange factor (GEF) domains--RhoGEF1 activates Rac and RhoGEF2 activates RhoA--as well as numerous protein-protein interaction domains. We have found that Kalirin is expressed in vascular smooth muscle cells (SMCs), macrophages and endothelial cells, and that Kalirin promotes SMC Rac1 signaling, migration and proliferation. We also found that Kalrn(-/+) mice develop less neointimal hyperplasia after wire-mediated carotid artery endothelial denudation, but that Kalrn(-/+)/Apoe(-/-) mice develop less atherosclerosis than congenic Apoe(-/-) mice. This project will therefore test the hypothesis that Kalirin protects against atherogenesis, either through its RhoGEF or other protein-protein interaction domains, specifically in endothelial cells or macrophages. To that end, Aim 1 will study the effects on aortic atherosclerosis of inhibiting Kalirin's RhoGEF1 domain or iNOS, an enzyme whose activity is inhibited by Kalirin, in Apoe(-/-) mice that are (+/+) or (-/+) at the Kaln locus. Aim 2 will determine endothelial cell-specific Kalirin anti-atherogenic mechanisms by comparing Apoe(-/- )/Kalrn(flox/+) mice with tamoxifen-inducible, endothelial cell-specific Cre expression (VECad-Cre-ER[T2]). Potential endothelial cell-specific anti-atherogenic mechanisms of Kalirin will be discerned in vitro by defining Kalirin's Rac- and Rho-GEF activity in endothelial cells, comparing flow-promoted anti-inflammatory activity between Kalrn(-/+) and WT endothelial cells, and by defining the endothelial cell proteins that associate with Kalirin using metabolically labeled endothelial cells and mass spectrometry. Aim 3 will determine whether macrophage Kalirin affects atherosclerosis, by comparing Apoe(-/-)/Kalrn(flox/+) mice that are either LysM- Cre+ or not. Potential macrophage-specific Kalirin mechanisms will be tested by comparing Kalrn(-/+) and WT macrophages with regard to (a) cytokine-induced RhoA and Rac activation, and (b) secretion of the anti- inflammatory cytokine interleukin-10. Together, these studies will establish cellular and molecular mechanisms by which Kalirin reduces atherosclerosis, and may reveal novel targets for anti-atherosclerosis therapy.

描述（由申请人提供）：人类 KALRN 基因的多态性与冠状动脉疾病和缺血性中风相关。约 320 kDa 的 Kalirin 蛋白包含两个鸟嘌呤核苷酸交换因子 (GEF) 结构域（RhoGEF1 激活 Rac，RhoGEF2 激活 RhoA）以及许多蛋白质-蛋白质相互作用结构域。我们发现 Kalirin 在血管平滑肌细胞 (SMC)、巨噬细胞和内皮细胞中表达，并且 Kalirin 促进 SMC Rac1 信号传导、迁移和增殖。我们还发现，Kalrn(-/+)小鼠在导线介导的颈动脉内皮剥脱后发生较少的内膜增生，但Kalrn(-/+)/Apoe(-/-)小鼠比同系Apoe(-/-)小鼠发生更少的动脉粥样硬化。因此，该项目将测试 Kalirin 通过其 RhoGEF 或其他蛋白质-蛋白质相互作用结构域（特别是在内皮细胞或巨噬细胞中）防止动脉粥样硬化形成的假设。为此，目标 1 将在 Kaln 基因座为 (+/+) 或 (-/+) 的 Apoe(-/-) 小鼠中研究抑制 Kalirin 的 RhoGEF1 结构域或 iNOS（一种活性被 Kalirin 抑制的酶）对主动脉粥样硬化的影响。目标 2 将通过比较 Apoe(-/- )/Kalrn(flox/+) 小鼠与他莫昔芬诱导的内皮细胞特异性 Cre 表达 (VECAD-Cre-ER[T2]) 来确定内皮细胞特异性 Kalirin 抗动脉粥样硬化机制。通过定义内皮细胞中 Kalirin 的 Rac- 和 Rho-GEF 活性，比较 Kalrn(-/+) 和 WT 内皮细胞之间的血流促进抗炎活性，并通过使用代谢标记的与 Kalirin 相关的内皮细胞蛋白来确定 Kalirin 潜在的内皮细胞特异性抗动脉粥样硬化机制。 内皮细胞和质谱。目标 3 将通过比较 LysM-Cre+ 或非 LysM-Cre+ 的 Apoe(-/-)/Kalrn(flox/+) 小鼠来确定巨噬细胞 Kalirin 是否影响动脉粥样硬化。通过比较 Kalrn(-/+) 和 WT 巨噬细胞的 (a) 细胞因子诱导的 RhoA 和 Rac 激活，以及 (b) 抗炎细胞因子 IL-10 的分泌，可以测试潜在的巨噬细胞特异性 Kalirin 机制。这些研究将共同​​建立 Kalirin 减少动脉粥样硬化的细胞和分子机制，并可能揭示抗动脉粥样硬化治疗的新靶点。