Thickening of basement membrane in diabetic retinopathy

描述（由申请人提供）：血管基底膜增厚是糖尿病视网膜病变的一种突出和特征性病变。本项目的目的是验证以下假设：糖尿病中发生的增厚的血管基底膜（BM）在糖尿病视网膜病变的严重结构和功能异常的发展和进展中起作用。该假设基于最近的发现，即人类视网膜血管表现出可以由增厚的BM诱导的改变，并且反过来可以损害血管内层的完整性。在糖尿病个体的视网膜毛细血管细胞中，BM蛋白、纤连蛋白（FN）、胶原IV（卷曲IV）和层粘连蛋白（LM）的产生增加。我们实验室开发的反义寡核苷酸特异性下调高葡萄糖培养基中生长的微血管内皮细胞中三种BM组分的过表达。此外，用FN反义寡核苷酸抑制FN过表达部分地防止了半乳糖喂养的大鼠（糖尿病视网膜病变的动物模型）的视网膜毛细血管中增厚的血管BM的发展，对组织学病变具有有益的后果。为了完全防止或逆转血管BM增厚，并调节功能异常，需要开发更有效的反义策略。已经鉴定了独立地下调视网膜血管细胞中FN、coil IV或LM表达的反义寡核苷酸，在所提出的研究中，我们计划：（1）确定组合的反义寡核苷酸方法是否防止大鼠视网膜中的血管BM增厚，并影响基质中的FN、coil IV、LM周转。(2)确定特定BM基因的下调是否逆转BM增厚，减少血管病变，并影响糖尿病大鼠视网膜中的细胞过程。(3)确定高糖诱导或糖尿病诱导的BM组分表达改变是否在血管通透性中起作用。将通过Western印迹分析和免疫组织化学监测FN、coil IV和LM蛋白水平;将通过RT-PCR分析RNA水平。视网膜毛细血管BM宽度将通过电子显微照片的形态测量分析来测量。将通过视网膜胰蛋白酶检查和图像分析评估血管病变。本项目的发现将建立或排除离散的生物合成异常与糖尿病视网膜病变的结构和功能病变的发展之间的发病联系。

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.