Abstract:
Age-related macular degeneration (AMD) is a progressive, degenerative disease of the macula. The formation of macular neovascularization (MNV) and subretinal fibrosis of AMD is the most classic cause of the loss of vision in older adults worldwide. While the underlying causes of MNV and subretinal fibrosis remain elusive, the common feature of many common retinal diseases is changes the proportions of protein deposition in extracellular matrix (ECM) when compared to normal tissue. In ECM, fibronectin (FN) is a crucial component and plays a pivotal part not only in fibrotic diseases but also in the process of angiogenesis. The study aims to understand the role of ligand FN and its common integrin receptor α5β1 on MNV, and to understand the molecular mechanism involved. To study this, the laser-induced MNV mouse model and the rhesus macaque choroid-retinal endothelial cell line (RF/6A) chemical hypoxia mode were established, and the FN-α5β1 expression levels were detected by immunohistochemistry (IHC) and quantitative real-time PCR analysis (qRT-PCR). Fibronectin expression was silenced using small interfering RNA (siRNA) targeting FN. The tube formation and vitro scratch assays were used to assess the ability to form blood vessels and cell migration. To measure the formation of MNV, immunofluorescence, and Western blot assays were used. These results revealed that the expressions of FN and integrin α5β1 were distinctly increased in the laser-induced MNV mouse model and in the RF/6A cytochemically induced hypoxia model, and the expression tendency was identical. After the use of FN siRNA, the tube formation and migration abilities of the RF/6A cells were lower, the ability of endothelial cells to proliferate was confined and the scope of damage caused by the laser in animal models was significantly cut down. In addition, FN gene knockdown dramatically inhibited the expression of Wnt/β-catenin signal. The interaction of FN with the integrin receptor α5β1 in the constructed model, which may act through the Wnt/β-catenin signaling pathway, was confirmed in this study. In conclusion, FN may be a potential new molecular target for the prevention and treatment of subretinal fibrosis and MNV.
摘要:
年龄相关性黄斑变性(AMD)是一种进行性、黄斑的退行性疾病。AMD的黄斑新生血管形成(MNV)和视网膜下纤维化是全球老年人视力丧失的最经典原因。虽然MNV和视网膜下纤维化的根本原因仍然难以捉摸,与正常组织相比,许多常见视网膜疾病的共同特征是改变了细胞外基质(ECM)中蛋白质沉积的比例。在ECM中,纤连蛋白(fibronectin,FN)是一个重要的组成部分,不仅在纤维化疾病中起着关键作用,而且在血管生成过程中起着重要作用。本研究旨在了解配体FN及其常见整合素受体α5β1在MNV,并了解所涉及的分子机制。为了研究这个,建立激光诱导MNV小鼠模型和恒河猴脉络膜-视网膜内皮细胞系(RF/6A)化学缺氧模式,免疫组织化学(IHC)和定量实时PCR(qRT-PCR)检测FN-α5β1的表达水平。使用靶向FN的小干扰RNA(siRNA)沉默纤连蛋白表达。管形成和体外划痕测定用于评估形成血管和细胞迁移的能力。为了测量MNV的形成,免疫荧光,并使用蛋白质印迹分析。这些结果表明,在激光诱导的MNV小鼠模型和RF/6A细胞化学诱导的缺氧模型中,FN和整合素α5β1的表达明显增加,表达倾向相同。使用FNsiRNA后,RF/6A细胞的管形成和迁移能力较低,在动物模型中,内皮细胞的增殖能力受到限制,激光引起的损伤范围显着降低。此外,FN基因敲除显著抑制了Wnt/β-catenin信号的表达。在构建的模型中,FN与整合素受体α5β1的相互作用,可能通过Wnt/β-catenin信号通路,在这项研究中得到了证实。总之,FN可能是预防和治疗视网膜下纤维化和MNV的潜在新分子靶标。