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特征。本项目的目的是验证糖尿病患者血管基底膜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所起的作用。这一假设是基于最近的发现，即人类视网膜血管表现出可能由增厚的脑转移瘤引起的改变，进而可能损害血管内壁的完整性。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）、胶原IV （coil 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地下调高糖培养基中生长的微血管内皮细胞中三种BM成分的过表达。此外，用FN反义寡核苷酸抑制FN过表达部分阻止了半乳糖喂养大鼠（糖尿病视网膜病变动物模型）视网膜毛细血管中增厚的血管BM的发展，对组织学病变有有益的影响。为了完全预防或逆转血管BM增厚，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中FN、coil IV或LM表达的反义寡核苷酸后，我们计划在拟开展的研究中：(1)确定联合反义寡核苷酸途径是否能阻止大鼠视网膜血管BM增厚，并影响基质中FN、coil IV、LM的转换。(2)确定特异性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。Western blot和免疫组化检测FN、线圈IV和LM蛋白水平；RT-PCR检测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计量学分析来测量。血管病变将通过视网膜胰蛋白酶消化和图像分析进行评估。本项目的研究结果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之间的病理联系。

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.