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: 8760703
• 负责人: NEIL J. FREEDMAN
• 金额: $ 46.72万
• 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/8760703
• 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; 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; public health relevance; response; rho; scaffold; tandem mass spectrometry

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Kalrn基因中的多态性与冠状动脉疾病和缺血性中风有关。 〜320 kDa蛋白kalirin包含两个鸟嘌呤核苷酸交换因子（GEF）结构域-RHOGEF1激活RAC，Rhogef2激活了RhoA，以及许多蛋白质 - 蛋白质相互作用域。我们发现卡利林在血管平滑肌细胞（SMC），巨噬细胞和内皮细胞中表达，卡利林促进了SMC Rac1信号传导，迁移和增殖。我们还发现，在电线介导的颈动脉内皮剥离后，Kalrn（ - /+）小鼠会出现较少的新内膜增生，但是Kalrn（ - /+）/apoE（ - / - ）小鼠的动脉粥样硬化少于先天性APOE（ - / - ）小鼠。因此，该项目将检验以下假设：Kalirin通过其Rhogef或其他蛋白质 - 蛋白质相互作用域，特别是在内皮细胞或巨噬细胞中预防动脉粥样硬化的假设。为此，AIM 1将研究对抑制Kalirin的Rhogef1结构域或iNOS的主动脉动脉粥样硬化的影响，这是一种在KALN LOCUS的APOE（ - / - ）小鼠中抑制Kalirin的活性的酶。 AIM 2将通过比较apoE（ - / - ）/kalrn（Flox/+）小鼠与他莫昔芬可诱导的，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抗动脉生育机制。在内皮细胞中定义Kalirin的RAC和Rho-GEF活性，可以在体外识别卡利林的潜在内皮细胞特异性抗动脉粥样硬化机制，从而比较kalrn（ - /+）（ - /+）和WT内皮细胞之间的kalrn（ - +）和WT内皮细胞之间的抗激素细胞之间的抗炎活性，并通过启动型细胞蛋白质与局部质量相关。内皮细胞和质谱法。 AIM 3将通过比较lysm-CRE+的apoE（ - / - ）/kalrn（flox/+）小鼠来确定巨噬细胞卡利林是否会影响动脉粥样硬化。潜在的巨噬细胞特异性卡利林机制将通过比较（a）细胞因子诱导的RhoA和RAC激活以及（b）抗炎性细胞因子肠毒intleukin-100的分泌，通过比较Kalrn（ - /+）和WT巨噬细胞来测试。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，并可能揭示出抗动脉粥样硬化治疗的新靶标。