Endothelial Transmigration in Neovascular Age-related Macular Degeneration

抗VEGF治疗，新生血管性年龄相关性黄斑变性（nvAMD）的标准治疗， 不到50%的患者的结果，并且不能预防由于纤维化或黄斑变性导致的视力丧失进展。 萎缩我们使用人类生理学相关模型来了解 参与脉络膜内皮细胞（CEC）迁移激活的信号通路和串扰 并在nvAMD中形成黄斑新生血管。支架蛋白，IQGAP 1，维持激活的细胞， GTTRac 1，CEC迁移所必需的。Rac1被AMD相关应激激活， 炎症、氧化和血管生成因子，以及氧化固醇、7-酮胆固醇（7KC）， 随着年龄的增长和在AMD中在血液和布鲁赫膜中积累。7KC导致CEC改变 细胞标志物从内皮细胞到间充质细胞的表达，表明内皮-间充质细胞 过渡（EndMT）。IQGAP 1似乎参与其中。7KC还在激光诱导的损伤模型中引起纤维化。我们 数据支持将在下一个资助期进行测试的假设框架：（1）IQGAP 1是 氧化固醇7KC对EndMT的诱导至关重要;（2）7KC触发转录事件， CEC不能维持内皮标志物的表达，而是发展成一种新的迁移表型， 间充质细胞发展成纤维化。我们还将测试两种潜在的疗法来减少纤维化： 与抗VEGF组合抑制TGF β信号传导和（B）在新的免疫抑制剂中靶向IQGAP 1的磷酸化， 我们通过CRISPR-Cas9诱导的基因突变创建的突变IQGAP 1小鼠。具体目标1是测试 预测IQGAP 1介导暴露于7KC的CEC中EndMT诱导的迁移。具体目标2是测试 预测7KC通过IQGAP 1介导，减少标记的内皮细胞阳性的比例， 间充质阳性细胞激光后内皮特异性黄色荧光蛋白报告小鼠。具体 目的3是测试预测，增加年龄，TGF β信号传导，或IQGAP 1丝氨酸磷酸化将 在7KC治疗的眼睛中激光后α SMA标记的病变增加，并测试未来可能的策略 治疗。我们还将评估Müller细胞、眼周和RPE的参与。工具包括隔离 人CEC;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;黄色荧光蛋白内皮细胞 报告小鼠;条件诱导内皮细胞Iqgap1基因敲除小鼠;突变IQGAP 1小鼠，通过 CRISPR-Cas9技术;药物的玻璃体内注射;微米IV激光诱导的损伤测试 7KC诱导的EndMT和损伤形成。这些研究将测试IQGAP 1在7KC诱导的EndMT中的作用。 作为纤维化的潜在原因，其在nvAMD中对抗VEGF反应不良，并将测试两种新的 治疗以减少nvAMD和EndMT。

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.