Inhibiting Neovascularization and Subretinal Fibrosis in Neovascular Age-Related Macular Degeneration

项目总结 老年性黄斑变性(AMD)是一种眼神经退行性疾病，其原因是 发达国家约有一半的失明和视力残疾病例。据估计，大约 到2040年，全球将有2.88亿人被诊断出患有AMD。AMD的新血管形式--会计 对于大约80%的严重视力丧失的AMD患者来说，其特点是新生血管在亚 视网膜间隙。视网膜下纤维化是终末期新生AMD的分界，甚至会导致不可挽回的视力损失 接受抗血管内皮生长因子治疗。因此，了解视网膜下的分子机制势在必行。 纤维化的形成和基于药物的治疗不仅是针对黄斑新生血管(MNV)，也是为了 预防或治疗视网膜下纤维化。越来越多的证据表明，上皮向间充质转化 视网膜上皮细胞(RPE)和内皮-间充质转化(EndoMT) 在视网膜下纤维性病变中，脉络膜或视网膜内皮细胞均构成间充质细胞群。 此外，EndoMT导致内皮屏障通透性增加，并可能提供一个解释 随着时间的推移，抗血管内皮生长因子治疗的有效性降低。因此，旨在抑制或逆转 EMT和EndoMT可稳定内皮屏障功能，预防或减轻视网膜下纤维化。五花八门 细胞外细胞因子和生长因子参与激活MNV、EMT和EndoMT。我们已经确定了 小的GTP酶ARF6作为这些细胞因子激活的信号通路的汇聚点 增长因素。因此，我们假设ARF6激活诱导MNV、EMT和EndoMT，从而 导致视网膜下纤维化的发生，ARF6的基因缺失或药物抑制将 减少新生血管性AMD的MNV和视网膜下纤维化。为了验证这一假设，我们将追求三个目标。 在目标1中，我们将确定脉络膜和视网膜内膜和视网膜下是否需要ARF6激活 激光诱导的CNV和JR5558小鼠模型中的纤维化。我们还将使用激光诱导的CNV模型， 内皮细胞谱系追踪和单细胞RNAseq以确定ARF6在EndoMT中的作用。在目标2中，我们 将确定激光诱导的CNV的RPE EMT和视网膜下纤维化是否需要ARF6的激活 和JR5558小鼠模型，并用细胞谱系示踪和单细胞RNAseq来确定其功能 ARF6在EMT中的应用。在目标3中，我们将调查EMT和EndoMT是相加还是协同作用来促进 新生血管性AMD及ARF6的药理抑制能否减少新生血管性AMD 模特们。这项研究可能通过识别ARF6对未来新生血管性AMD的治疗产生重大影响 作为药物开发的潜在目标。此外，这项研究也可能对 治疗其他眼血管疾病，如增殖性玻璃体视网膜病变，视网膜前膜，以及 纤维血管疾病中的视网膜纤维化，如增殖性糖尿病视网膜病变和早产儿视网膜病变。

PROJECT SUMMARY Age-related macular degeneration (AMD) is an ocular neurodegenerative disorder that accounts for approximately half of all cases of blindness and visual disability in developed nations. It is estimated that around 288 million people will be diagnosed with AMD worldwide by 2040. The neovascular form of AMD, accounting for about 80% of the severe vision loss in patients with AMD, is characterized by neovascularization in the sub- retinal space. Subretinal fibrosis demarcates end-stage neovascular AMD, causing irreparable vision loss, even with anti-VEGF therapy. Thus, it is imperative to unravel the molecular mechanisms underpinning subretinal fibrosis formation and develop drug-based therapies not only for macular neovascularization (MNV) but also to prevent or treat subretinal fibrosis. Accumulating evidence suggests that epithelial-to-mesenchymal transition (EMT) of the retinal epithelial cell (RPE) and endothelial-to-mesenchymal transition (EndoMT) of either the choroidal or retinal endothelial cells all contribute to the mesenchymal cell population in subretinal fibrotic lesions. Additionally, EndoMT leads to an increase in endothelial barrier permeability and could provide an explanation for the reduced efficacy of anti-VEGF treatment over time. Therefore, therapies designed to inhibit or reverse EMT and EndoMT could stabilize endothelial barrier function and prevent or reduce subretinal fibrosis. Various extracellular cytokines and growth factors are involved in activating MNV, EMT, and EndoMT. We have identified the small GTPase ARF6 as a convergence point in signaling pathways activated by many of these cytokines and growth factors. Thus, we hypothesize that ARF6 activation induces MNV, EMT, and EndoMT, thereby contributing to the onset of subretinal fibrosis, and that genetic loss or pharmacological inhibition of ARF6 will reduce both MNV and subretinal fibrosis in neovascular AMD. To test this hypothesis, we will pursue three aims. In Aim 1, we will determine whether ARF6 activation is required for choroidal and retinal EndoMT and subretinal fibrosis in laser-induced CNV and JR5558 mouse models. We will also use the laser-induced CNV model, endothelial cell lineage tracing, and single cell RNAseq to determine the role of ARF6 in EndoMT. In Aim 2, we will determine whether ARF6 activation is required for RPE EMT and subretinal fibrosis in laser-induced CNV and JR5558 mouse models and use cell lineage tracing and single cell RNAseq to determine the function of ARF6 in EMT. In Aim 3, we will investigate whether EMT and EndoMT act additively or synergistically to promote neovascular AMD and whether pharmacologic inhibition of ARF6 can reduce neovascular AMD in mouse models. This study could have a major impact on the future treatment of neovascular AMD by identifying ARF6 as a potential target for drug development. Moreover, this study could also have important implications for the treatment of other ocular vascular diseases, e.g., proliferative vitreoretinopathy, epiretinal membranes, and retinal fibrosis in fibrovascular diseases, such as proliferative diabetic retinopathy and retinopathy of prematurity.