Anti-atherogenic Mechanisms of the Dual Rho-GEF Kalirin

描述(由申请人提供)：人类KALRN基因的多态与冠状动脉疾病和缺血性中风有关。约320 kDa的Kalirin蛋白含有两个鸟嘌呤核苷酸交换因子结构域--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表达于血管平滑肌细胞(SMC)、巨噬细胞和内皮细胞，并且Kalirin促进SMC rac1信号转导、迁移和增殖。我们还发现Kalrn(-/+)小鼠在电线介导的颈动脉内皮剥脱后新生内膜增生较少，但Kalrn(-/+)/APOE(-/-)小鼠较同基因APOE(-/-)小鼠动脉粥样硬化较少。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将研究抑制Kalirin的RhoGEF1结构域或iNOS对位于Kaln基因(+/+)或(-/+)的APOE(-/-)小鼠的动脉粥样硬化的影响。iNOS是一种被Kalirin抑制活性的酶。目的2将通过比较APOE(-/-)/Kalrn(FLOX/+)小鼠和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抗动脉粥样硬化的机制。通过确定Kalirin在内皮细胞中的RAC-和Rho-Egf活性，比较Kalrn(-/+)和WT内皮细胞之间促进流动的抗炎活性，以及通过使用代谢标记内皮细胞和质谱学来定义与Kalirin相关的内皮细胞蛋白，将在体外识别Kalirin潜在的内皮细胞特有的抗动脉粥样硬化机制。目的3将通过比较APOE(-/-)/Kalrn(FLOX/+)小鼠和非LysM-Cre+小鼠来确定巨噬细胞Kalirin是否影响动脉粥样硬化。将通过比较Kalrn(-/+)和WT巨噬细胞在(A)细胞因子诱导的RhoA和Rac激活，以及(B)抗炎细胞因子IL-10的分泌方面来测试潜在的巨噬细胞特异性Kalirin机制。总之，这些研究将建立卡利林减轻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。

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.