Thickening of basement membrane in diabetic retinopathy

描述(申请人提供)：血管基底膜增厚是糖尿病视网膜病变的显著和特征性病变。本项目的目的是验证糖尿病患者血管基底膜(BM)增厚在糖尿病视网膜病变严重结构和功能异常的发生和发展中起作用的假说。这一假说是基于最近的发现，即人类视网膜血管表现出可由增厚的BMS引起的变化，进而可能损害血管内层的完整性。在糖尿病患者的视网膜毛细血管细胞中，BM蛋白、纤维连接蛋白(FN)、IV型胶原(COIL IV)和层粘连蛋白(LM)的产生增加。本实验室研制的反义寡核苷酸可特异性下调高糖培养的微血管内皮细胞中三种BM组分的过度表达。此外，用FN反义寡核苷酸抑制FN的过度表达部分阻止了半乳糖喂养的大鼠视网膜毛细血管BM增厚的发展，这是一种糖尿病视网膜病变的动物模型，对组织学损害有利。为了完全防止或逆转血管BM增厚，并调节功能异常，开发更有效的反义策略是必要的。在确定了独立下调视网膜血管细胞FN、COIL IV或Lm表达的反义寡核苷酸后，在拟议的研究中，我们计划：(1)确定联合反义寡核苷酸方法是否能防止大鼠视网膜血管BM增厚，并影响基质中FN、COIL IV、Lm的周转。(2)确定特定BM基因的下调是否逆转糖尿病大鼠视网膜的BM增厚，减少血管病变，并影响细胞过程。(3)确定高糖诱导或糖尿病诱导的BM成分表达改变是否在血管通透性中起作用。FN、COIL IV和LM的蛋白水平将通过Western印迹分析和免疫组织化学进行监测，RNA水平将通过RT-PCR进行分析。视网膜毛细血管BM宽度将通过电子显微镜的形态计量学分析来测量。血管损伤将通过视网膜胰酶消化和图像分析进行评估。该项目的发现将建立或排除离散生物合成异常与糖尿病视网膜病变的结构和功能损害的发展之间的致病联系。

DESCRIPTION (provided by applicant): Vascular basement membrane thickening is a prominent and characteristic lesion of diabetic retinopathy. The goal of this project is to test the hypothesis that the thickened vascular basement membrane (BM) occurring in diabetes plays a role in the development and progression of serious structural and functional abnormalities of diabetic retinopathy. The hypothesis is based on recent findings that human retinal vessels manifest alterations that can be induced by the thickened BMs and, in turn, can compromise the integrity of the vessels' inner lining. In retinal capillary cells of diabetic individuals there is increased production of the BM proteins, fibronectin (FN), collagen IV (coil IV), and laminin (LM). Antisense oligonucleotides developed in our laboratory specifically down-regulate overexpression of the three BM components in microvascular endothelial cells grown in high glucose medium. Furthermore, inhibition of FN overexpression with the FN antisense oligonucleotide partially prevented the development of thickened vascular BM in retinal capillaries of galactose-fed rats, an animal model of diabetic retinopathy, with beneficial consequences to histological lesions. To completely prevent or reverse vascular BM thickening, and regulate functional abnormalities, the development of a more effective antisense strategy is necessary. Having identified antisense oligos that independently down-regulate FN, coil IV or LM expression in retinal vascular cells, in the proposed studies we plan to: (1) Establish whether combined antisense oligo approach prevents vascular BM thickening in rat retinas, and affects FN, coil IV, LM turnover in matrix. (2) Determine if downregulation of the specific BM genes reverses BM thickening, reduces vascular lesions, and affects cellular processes in retinas of diabetic rats. (3) Determine whether high glucose-induced or diabetes-induced altered expression of BM components plays a role in vascular permeability. FN, coil IV, and LM protein level will be monitored by Western blot analysis and immunohistochemistry; RNA level will be analyzed by RT-PCR. Retinal capillary BM width will be measured by morphometric analysis of electron micrographs. Vascular lesions will be assessed from retinal trypsin digests and image analysis. Findings from this project will establish or exclude a pathogenetic link between a discrete biosynthetic abnormality and the development of structural and functional lesions of diabetic retinopathy.