Anti-atherogenic Mechanisms of the Dual Rho-GEF Kalirin

描述（由申请人提供）：人类KALRN基因的多态性与冠状动脉疾病和缺血性中风有关。~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——以及许多蛋白质相互作用结构域。我们发现Kalirin在血管平滑肌细胞（SMCs）、巨噬细胞和内皮细胞中表达，Kalirin促进SMC Rac1信号转导、迁移和增殖。我们还发现，在金属丝介导的颈动脉内皮剥脱后，Kalrn(-/+)/Apoe（-/-）小鼠发生的新内膜增生较少，但与同源Apoe（-/-）小鼠相比，Kalrn(-/+)/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（特别是内皮细胞或巨噬细胞）防止动脉粥样硬化的假设。为此，Aim 1将在Kaln位点为（+/+）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。Aim 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。通过确定Kalirin在内皮细胞中的Rac-和Rho-GEF活性，比较Kalrn（-/+）和WT内皮细胞之间的血流促进抗炎活性，以及使用代谢标记内皮细胞和质谱法确定与Kalirin相关的内皮细胞蛋白，可以在体外识别Kalirin潜在的内皮细胞特异性抗动脉粥样硬化机制。Aim 3将通过比较Apoe(-/-)/Kalrn（flox/+）小鼠LysM- Cre+和非LysM- Cre+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(a)细胞因子诱导的RhoA和Rac激活，以及(b)抗炎细胞因子白细胞介素-10的分泌，将测试潜在的巨噬细胞特异性Kalirin机制。总之，这些研究将建立加里林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.