Thickening of basement membrane in diabetic retinopathy

血管基底膜增厚是糖尿病视网膜病变的一个突出和特征性病变。 本项目的目的是检验增厚的血管基底膜（BM） 发生在糖尿病中起着重要的作用，在严重的结构和功能的发展和进展， 糖尿病性视网膜病变这一假设是基于最近的发现，人类视网膜血管 明显的变化，可以诱导增厚的BM，反过来，可以损害的完整性， 容器的内衬。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（螺旋IV）和层粘连蛋白（LM）。开发的反义寡核苷酸 在我们的实验室中，特异性下调微血管中三种BM组分的过表达， 内皮细胞在高糖培养基中生长。此外，用FN-100抑制FN过表达， 反义寡核苷酸部分阻止视网膜毛细血管中增厚的血管BM的发展 半乳糖喂养的大鼠，糖尿病视网膜病变的动物模型，具有有益的结果， 病变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。已经鉴定了反义寡核苷酸， 独立下调视网膜血管细胞中FN、coil IV或LM的表达， 我们计划：（1）确定联合反义寡核苷酸方法是否能防止大鼠血管BM增厚 视网膜，并影响FN，线圈IV，LM周转矩阵。(2)确定特定BM的下调是否 基因逆转BM增厚，减少血管病变，并影响糖尿病患者视网膜中的细胞过程 大鼠(3)确定是否高糖诱导或糖尿病诱导的BM组分表达改变 在血管渗透性中起作用。将通过Western印迹法监测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 可以通过电子显微照片的形态测定分析来测量。血管病变将从 视网膜胰蛋白酶电泳及图像分析。该项目的发现将确定或排除一种致病性 离散的生物合成异常与结构和功能病变的发展之间的联系， 糖尿病视网膜病变

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.