Investigating the role of mitochondrial dysfunction in the pathogenesis of retinal vascular diseases

项目摘要/摘要 血管生成是血管形成的过程，在这个过程中增殖的内皮细胞(ECs)从 预先存在的血管来延伸血管网络。血管生成受损或过度与 人类视网膜疾病的发病机制，如视网膜低血管家族性渗出性玻璃体视网膜病变和 高血管糖尿病视网膜病变。EC主要使用糖酵解途径，在 血管生成。最近的研究表明，线粒体活性对血管生成至关重要，但其机制 目前还不完全清楚。我们研究了三种不同的线粒体蛋白的血管生成功能。 活性-线粒体转录因子(TFAM)、呼吸复合体IV组分(COX10)和a 线粒体氧化还原蛋白硫氧还蛋白2(TRX2)。我们的数据显示：1)Tfam、Cox10或Trx2在 在体外，3D发芽试验减弱EC发芽，这与EC增殖减少有关，但与 ROS生成或EC凋亡；2)Tfam、Cox10或Trx2可诱导缺失的小鼠表现出发育迟缓 早期(P5-P12)视网膜血管生长不能穿透深层神经丛，这会削弱视网膜。 发芽与mtROS的产生无关；3)三个突变的小鼠发生了动静脉病变 微血管动脉化增强和微动脉瘤形成的畸形(AVM) 高龄(P12-P30)。血管减少和微动脉瘤的表型与老年人相似。 视网膜和人类视网膜血管异常，如Coats病、Leber‘s军事动脉瘤和家族性 渗出性玻璃体视网膜病变(FEVR)；4)此外，视网膜内皮细胞的单细胞RNA-SEQ分析表明 这三个突变小鼠都有共同的EC簇，在血管生成和代谢方面基因表达降低 但增加转化生长因子受体信号转导途径。基于这些数据，我们假设线粒体的活动 调节血管生长和代谢中常见的血管生成和代谢途径(而不是ROS/凋亡) 成熟。我们提出了以下具体目标：1)确定关键的血管生成和代谢途径 线粒体功能障碍所致的视网膜新生血管、动静脉畸形和微动脉瘤 2)确定线粒体功能障碍如何调节转化生长因子R-Smad2/3 3)确定转化生长因子R-Smad2/3信号在线粒体功能障碍中的作用 药物阻断和遗传缺陷导致的血管发育迟缓和畸形。我们建议的研究 将定义线粒体活动调节正常视网膜血管生长和 这将为人类视网膜血管疾病提供一种新的模型和治疗手段。 与许多病理并发症有关，如糖尿病、高血压和衰老，这些并发症可能导致 视力丧失。 1

PROJECT SUMMARY/ABSTRACT Angiogenesis is the process of blood vessel formation in which proliferating endothelial cells (ECs) sprout from preexisting vessels to extend a vascular network. Either impaired or excessive angiogenesis is associated with the pathogenesis of human retina diseases such as retinal hypovascular familial exudative vitreoretinopathy and hypervascular diabetic retinopathy. EC primarily uses the glycolysis pathway which is further enhanced during angiogenesis. Recent studies suggest that mitochondrial activity is critical for angiogenesis, but its mechanism is not entirely clear. We have investigated the angiogenic functions of three mitochondrial proteins with distinct activities - mitochondrial transcriptional factor (TFAM), respiratory complex IV component (COX10) and a mitochondrial redox protein thioredoxin 2 (TRX2). Our data show that: 1) silencing of Tfam, Cox10, or Trx2 in an in vitro 3D sprouting assay attenuates EC sprouting, which correlated with reduced EC proliferation, but not with ROS generation or EC apoptosis; 2) mice with an inducible deletion of Tfam, Cox10, or Trx2 exhibit retarded retinal vessel growth without penetration into the deep plexi at early ages (P5–P12), and this attenuated retinal sprouting was not correlated with mtROS production; 3) the three mutant mice develop arteriovenous malformations (AVM) with enhanced arterialization and microaneurysm formation in the microvessels at advanced ages (P12–P30). The hypovasculature and microaneurysm phenotypes resemble that of aged human retinas and human retinal vascular abnormalities such as Coats' disease, Leber's military aneurysms and familial exudative vitreoretinopathy (FEVR); 4) Furthermore, single-cell RNA-seq analyses of retinal ECs suggest that the three mutant mice have common EC clusters with reduced gene expression in angiogenic and metabolic pathways but increased TGFR signaling. Based on these data, we hypothesize that mitochondrial activity regulates common angiogenic and metabolic pathways (rather than ROS/apoptosis) in vascular growth and maturation. We propose the following specific aims: 1) To identify critical angiogenic and metabolic pathways in mitochondrial dysfunction-induced defects in retinal sprouting angiogenesis, AVM and microaneurysm formation as well as in retinal function; 2) To define how mitochondrial dysfunction regulates TGFR-Smad2/3 signaling; 3) To determine the role of the TGFR-Smad2/3 signaling in mitochondrial dysfunction-induced vascular retardation and malformation by pharmacological blockade and genetic deficiency. Our proposed study will define the mechanism by which mitochondrial activities regulate normal retinal vascular growth and maturation, and will provide a novel model and therapeutic intervention for human retinal vascular diseases associated with many pathological complications such as diabetes, hypertension and aging that can result in vision loss. 1