Anti-atherogenic Mechanisms of the Dual Rho-GEF Kalirin

描述（申请人提供）：人KALRN基因多态性与冠状动脉疾病和缺血性卒中相关。约320 kDa的Kalirin蛋白含有两个鸟嘌呤核苷酸交换因子（GEF）结构域-RhoGEF 1激活Rac和RhoGEF 2激活RhoA-以及许多蛋白质-蛋白质相互作用结构域。我们已经发现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 Rac 1信号传导、迁移和增殖。我们还发现，Kalrn（-/+）小鼠在导丝介导的颈动脉内皮剥脱后发生的新生内膜增生较少，但Kalrn（-/+）/Apoe（-/-）小鼠比同类Apoe（-/-）小鼠发生的动脉粥样硬化较少。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，目的1将研究抑制Kalirin的RhoGEF 1结构域或iNOS（一种其活性被Kalirin抑制的酶）在Kaln基因座为（+/+）或（-/+）的Apoe（-/-）小鼠中对主动脉粥样硬化的影响。目的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抗动脉粥样硬化的机制。通过定义Kalirin在内皮细胞中的Rac-和Rho-GEF活性，比较Kalrn（-/+）和WT内皮细胞之间的流动促进的抗炎活性，以及通过使用代谢标记的内皮细胞和质谱法定义与Kalirin相关的内皮细胞蛋白，将在体外识别Kalirin的潜在内皮细胞特异性抗动脉粥样硬化机制。目的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。将通过比较Kalrn（-/+）和WT巨噬细胞的（a）苦参碱诱导的RhoA和Rac活化和（B）抗炎细胞因子白细胞介素-10的分泌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。

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.