内皮间充质转化：血管内皮细胞参与脉络膜新生血管重塑的新机制

Choroidal neovascularization (CNV) is a major cause of irreversible blindness in various ocular diseases. Essentially, pathological stimuli initiate the growth of neo-vessels arising from choroid into subretinal space. The aberrant buddings fail in normal vascular remodeling and evolve into end-stage fibrous scar, which results in a permanent damage of retina. Currently, anti-vascular endothelial growth factor (VEGF) therapy is a preferred option for CNV treatment. However, as anti-VEGF might be way off target in consequent vascular remodeling/fibrosis progress, evidences reveal it is incapable to suppress subretinal scar formation and even aggravates the fibrosis. Hence, to reveal CNV remodeling/fibrosis and its underlying molecular mechanisms will benefit the new therapeutic strategies. Vascular endothelial cells (ECs) can lose their markers through the process of endothelial mesenchymal transition (EndoMT), and finally undergo the transition from mesenchymal cells into fibroblasts. Various conditions including inflammation, hypoxia and oxidative stress can induce EndoMT, in which transforming growth factor beta (TGF-beta) and its downstream transcription factor Snai1 are the major regulatory pathways. In this study, by using ECs specific genetic tracing mice combining with laser-induced CNV model, we firstly plan to confirm whether EndoMT contributes to CNV remodeling/fibrosis. Secondly, we adopt ECs specific Snai1 knockout mice to explore the mechanisms of EndoMT in CNV. Finally, by modifying the specific signal pathway, we tend to test whether targeting EndoMT could promote neovascular maturation or suppress subretinal fibrosis.

脉络膜新生血管(CNV)是多种致盲眼病共有的病理学特征，极易出血渗出并伴重塑异常，最终形成纤维化瘢痕，导致不可逆性视功能丧失。抗血管内皮生长因子药物是目前临床一线治疗方案，其机制是抑制新生血管形成，但不涉及血管重塑/纤维化等重要环节，这可能是其疗效有限、治疗后瘢痕形成严重等问题的症结所在。因此，研究CNV重塑/纤维化及其调控机制，或可为该类疾病的治疗提供新思路。血管内皮细胞(ECs)可通过内皮间充质转化(EndoMT)失去自身标志物，经间充质细胞最终转变为成纤维细胞等参与血管重塑/纤维化。炎症、缺氧和氧化应激是EndoMT的重要诱因，而转化生长因子β及转录因子Snai1是主要调控通路。本研究拟利用ECs特异性遗传示踪小鼠，证实EndoMT参与中晚期CNV重塑/纤维化；利用ECs特异性Snai1敲除小鼠，探讨EndoMT对CNV的作用及机制，并靶向EndoMT以促进血管成熟或减轻纤维化。

脉络膜新生血管（CNV）是多种致盲性眼病的共同病理特征，异常新生血管渗出、出血，导致不可逆性视功能丧失；其发病机制尚未完全阐明。内皮间充质转化（EndMT）指EC失去自身标志物而获得间充质细胞表达特征及部分功能；完全性EndMT参与胚胎发育、纤维化等生理病理过程，而部分性EndMT可能介导EC活化。本研究主要内容围绕EndMT关键转录因子SNAI1，检测CNV-EC中SNAI1的表达调控，探讨CNV内是否存在EndMT；在体内外干预SNAI1，阐明SNAI1调控CNV的功能机制。同时，拓展原计划研究内容，包括：①SNAI1调控视网膜新生血管（RNV）的功能机制；②骨髓来源细胞（BMC）募集并调控CNV的机制；③单细胞测序技术解析CNV中的细胞亚群、互作、命运转归与分子表达特征。本研究取得以下结果：①CNV-EC高表达SNAI1，但未发生完全性EndMT；SNAI1促进细胞骨架重组与ECM重构相关基因表达，增强EC迁移侵袭能力，促进CNV；②BMC募集分化调控CNV；CNV-EC/BMC上的整合素α5β1受SDF-1/CXCR4信号刺激升高；抑制CXCR4/α5β1减少BMC募集、降低EC迁移和管腔形成，减轻CNV；③CNV内BMC中miR-188-5p表达下调；miR-188-5p通过抑制靶基因MMP-2/13发挥抗血管生成作用；④单细胞转录组测序证实，CNV-EC存在SNAI1高表达，但无完全性EndMT证据；此外，CNV-EC呈线粒体基因上调的异常活化特征。综上所述，本研究以SNAI1入手，结合CNV模型、单细胞转录组测序分析及多种干预手段，阐明了SNAI1介导EC活化调控CNV的功能和机制，及病理性EC表达与转归特征，并进一步揭示了包括BMC在内的CNV复杂细胞分子调控网络，加深对其发病机制的理解，为新治疗靶点开发提供理论依据。