Endothelial Transmigration in Neovascular Age-related Macular Degeneration

抗血管内皮生长因子治疗是新生血管性老年性黄斑变性(NvAMD)的标准护理，改善了 不到50%的患者的预后，并且不能防止由于纤维化或黄斑而导致的视力丧失进展 萎缩。我们使用与人体生理相关的模型来了解 脉络膜内皮细胞(CECs)迁移激活的信号通路和串扰 在nvAMD形成黄斑新生血管。支架蛋白IQGAP1维持着对 GTPase rac1是CEC迁移所必需的。与AMD相关的压力激活了rac1，包括 炎症、氧化和血管生成因子，以及氧固醇，7-酮胆固醇(7KC)，这是 随着年龄的增长和AMD，在血液和Bruchs膜中积聚。7KC导致CEC发生变化 从内皮到间充质的细胞标志物的表达，提示内皮-间充质 过渡(EndMT)。IQGAP1似乎也参与其中。7KC还可导致激光损伤模型的纤维化。我们的 数据支持将在下一个资金期测试的假设框架：(1)IQGAP1是 对氧固醇7KC诱导的EndMT至关重要；以及(2)7KC触发转录事件，使 CECs不能维持内皮标记物的表达，而是发展为新的迁移表型， 间充质细胞发展成纤维化。我们还将测试两种可能的减少纤维化的疗法： 联合抗血管内皮生长因子抑制转化生长因子β信号转导和(B)靶向磷酸化IQGAP1 我们通过CRISPR-Cas9诱发基因突变而培育的突变型IQGAP1小鼠。具体目标1是测试 预测IQGAP1介导EndMT诱导的7KC细胞迁移。具体目标2是测试 通过IQGAP1介导的7KC降低标记内皮细胞阳性比例的预测 血管内皮细胞特异性黄色荧光蛋白报告小鼠激光照射后间充质阳性细胞。特定的 目标3是测试年龄增加、转化生长因子β信号转导或IQGAP1丝氨酸磷酸化将 7KC激光治疗后增加αSMA标记的损害并测试未来可能的策略 治疗。我们还将评估Müler细胞、视网膜周细胞和视网膜色素上皮的参与情况。工具包括隔离 人内皮细胞；高通量RNA测序；流式细胞术；光谱域光学相干 断层扫描(SdOCT)；7KC诱导的EndMT和纤维化模型；黄色荧光蛋白内皮 报道小鼠；条件诱导内皮细胞Iqgap1基因敲除小鼠；突变IQGAP1小鼠 CRISPR-CAS9-技术；玻璃体内注射药物；Micron IV激光损伤检测 7KC诱导的EndMT和病变形成。这些研究将测试IQGAP1在7KC诱导的EndMT中的作用 作为纤维化的潜在原因，它对抗血管内皮生长因子在nvAMD中的反应很差，并将测试两种新的 减少NvAMD和EndMT的治疗。

Anti-VEGF therapies, standard care in neovascular age-related macular degeneration (nvAMD), improve outcomes in less than 50% of patients and do not prevent vision-loss progression due to fibrosis or macular atrophy. We used human physiologically relevant models to gain understanding into the coordination of signaling pathways and cross-talk involved in the activation of choroidal endothelial cells (CECs) to migrate and form macular neovascularization in nvAMD. The scaffolding protein, IQGAP1, sustains activation of the GTPase Rac1, which is necessary for CEC migration. Rac1 is activated by AMD-related stresses involving inflammatory, oxidative and angiogenic factors, as well as by the oxysterol, 7-ketocholesterol (7KC), which accumulates in blood and Bruch’s membrane with increased age and in AMD. 7KC causes CECs to change expression of cell markers from endothelial to mesenchymal ones, suggesting endothelial-mesenchymal transition (EndMT). IQGAP1 appears involved. 7KC also causes fibrosis in models of laser induced injury. Our data support the hypothetical framework that will be tested in the next funding period: that (1) IQGAP1 is critical to EndMT induced by the oxysterol 7KC; and that (2) 7KC triggers transcriptional events that render CECs unable to maintain expression of endothelial markers but to develop into a new phenotype of migratory, mesenchymal cells that develop into fibrosis. We will also test two potential therapies to reduce fibrosis: (a) to inhibit TGFβ signaling in combination with anti-VEGF and (b) to target phosphorylation of IQGAP1 in a novel mutant IQGAP1 mouse that we created by CRISPR-Cas9-induced gene mutation. Specific Aim 1 is to test the prediction that IQGAP1 mediates EndMT-induced migration in CECs exposed to 7KC. Specific Aim 2 is to test the prediction that 7KC, mediated through IQGAP1, decreases the proportion of labeled endothelial positive to mesenchymal positive cells after laser in endothelial specific yellow-fluorescent protein reporter mice. Specific Aim 3 is to test predictions that increased age, TGFβ-signaling, or IQGAP1 serine phosphorylation will increase αSMA-labeled lesions after laser in 7KC-treated eyes and to test strategies as possible future treatments. We will also evaluate the involvement of Müller cells, pericyes, and RPE. Tools include isolated human CECs; high throughput RNA sequencing; flow cytometry; spectral domain optical coherence tomography (sdOCT); 7KC-induced models of EndMT and fibrosis; yellow-fluorescent protein endothelial reporter mice; conditional inducible endothelial Iqgap1 knockout mice; a mutant IQGAP1 mouse through CRISPR-Cas9-technology; intravitreal injections of pharmacologic agents; Micron IV laser induced injury to test 7KC-induced EndMT and lesion formation. These studies will test the role of IQGAP1 in 7KC-induced EndMT as a potential cause of fibrosis, which is poorly responsive to anti-VEGF in nvAMD, and will test two novel treatments to reduce nvAMD and EndMT.