One of the early features of diabetic retinopathy is the alteration of the blood-retinal barrier (BRB), which may involve the breakdown of endothelial cell tight junctions. The aim of this study was to examine the expression of extracellular proteinases in an animal model of early diabetic retinopathy and to determine their role in the alteration of the BRB. Matrix metalloproteinase (MMP) expression was studied in the retinas of rats with 12 weeks of diabetes. The role of MMPs in regulating tight junction function was investigated in retinal endothelial and pigment epithelial cells by measuring transepithelial electrical resistance (TER). The retinas of diabetic animals demonstrated elevated levels of MMP-2, MMP-9 and MMP-14 messenger RNA. A significant increase in the production of MMP-9 was seen when cells were exposed to high glucose conditions. Both cell types treated with purified MMP-2 or MMP-9 were found to have alterations of tight junction function as shown by decreased TER. Western blot analysis of cell extracts treated with MMP-2 or MMP-9, revealed specific degradation of the tight junction protein, occludin. Results suggest that elevated expression of MMPs in the retina may facilitate an increase in vascular permeability by a mechanism involving proteolytic degradation of the tight junction protein occludin followed by disruption of the overall tight junction complex.

OBJECTIVE-Increased vascular permeability due to alteration of the blood-retinal barrier (BRB) is one of the major complications in early diabetes. The aim of the present study was to determine whether diabetes alters the cellular expression and distribution of the adherens junction protein vascular endothelial (VE)-cadherin in retinal endothelial cells and if this alteration is mediated by proteinase activity.RESEARCH DESIGN AND METHODS-Diabetes was induced in Brown Norway rats using streptozotocin, and retinal vascular permeability was measured by the Evans blue technique. The expression of matrix metalloproteinases (MMPs) and VE-cadherin was examined in isolated retinal vessels or cultured endothelial cells in response to diabetes and advanced glycation end products (AGEs). The cleavage of VE-cadherin from the endothelial cell surface was monitored by Western blotting following MMP or AGE treatment.RESULTS-Retinal vascular permeability was significantly increased in rats following 2 weeks of diabetes coincident with a decrease of VE-cadherin expression. This increased vascular permeability could be inhibited with an MMP inhibitor. Treatment of endothelial cells with AGE-BSA led to a reduction of VE-cadherin staining on the cell surface and increased permeability, which was MMP mediated. Treatment of cells with specific MMPs or AGEs resulted in cleavage of VE-cadherin from the cell surface. T he normal permeability of the retinal microvasculature is tightly controlled by a series of complex interactions between adjacent endothelial cells and by additional interactions between the endothelial cells and other cell types in the retina, including vascular pericytes, astrocytes, and Mü ller glial cells (1). These interactions form the structural basis of the inner blood-retinal barrier (BRB), which facilitates normal functioning of the neural retina. Disruption of these interactions is anticipated to lead to a compromise of the barrier function, resulting in leakage of plasma constituents into the surrounding retinal tissues and retinal edema. The alteration of the BRB followed by retinal edema are key early events in the pathogenesis of diabetic retinopathy, and macular edema is the leading cause of vision loss in diabetic patients. CONCLUSIONS-TheseEndothelial cells of the retinal microvasculature contribute to the function of the BRB through the formation of specialized intercellular junctions including adherens and tight junctions. The cadherin family of proteins, of which there are more than 80 members, is a primary component of the adherens junctions (2). The vascular endothelial (VE)-cadherin mediates calcium-dependent homophilic adhesion between endothelial cells. In addition, VE-cadherin functions as a plasma membrane attachment site for the cytoskeleton through its interactions with the cytoplasmic proteins ␤-and ␥-catenin (3-5). In addition to contributing to the mechanical attachment of cells, VEcadherin also plays a role in the regulation of a variety of endothelial cell behaviors including motility, m...

One of the major complications of diabetes is the alteration of the blood-retinal barrier, leading to retinal edema and consequent vision loss. The aim of this study was to evaluate the role of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system in the regulation of retinal vascular permeability. Biochemical, molecular, and histological techniques were used to examine the role of uPA and uPAR in the regulation of retinal vascular permeability in diabetic rats and cultured retinal endothelial cells. The increased retinal vascular permeability in diabetic rats was associated with a decrease in vascular endothelial (VE) -cadherin expression in retinal vessels. Treatment with the uPA/uPAR-inhibiting peptide (A6) was shown to reduce diabetes-induced permeability and the loss of VE-cadherin. The increased permeability of cultured cells in response to advanced glycation end products (AGEs) was significantly inhibited with A6. Treatment of endothelial cells with specific matrix metalloproteinases or AGEs resulted in loss of VE-cadherin from the cell surface, which could be inhibited by A6. uPA/uPAR physically interacts with AGEs/receptor for advanced glycation end products on the cell surface and regulates its activity. uPA and its receptor uPAR play important roles in the alteration of the blood-retinal barrier through proteolytic degradation of VE-cadherin. The ability of A6 to block retinal vascular permeability in diabetes suggests a potential therapeutic approach for the treatment of diabetic macular edema.

These studies suggest that HGF may play an important role in the initial stages of retinal angiogenesis by stimulating a migratory phenotype in endothelial cells mediated by increased urokinase activity.

Purpose Angiogenesis, or the formation of new retinal blood vessels is a key feature of many proliferative retinal diseases including diabetic retinopathy, retinal vein occlusions, and retinopathy of prematurity. The aim of the present study was to investigate the role of the serine proteinase inhibitor PAI-1 in facilitating retinal angiogenesis. Methods The temporal expression of PAI-1 was examined by real time PCR, western blotting and immunohistochemistry in retinal tissues from mice with oxygen-induced retinopathy. The requirement for PAI-1 in facilitating the retinal angiogenic response in this model was examined by quantitating the angiogenic response using both wildtype and PAI-1 null mice. The mechanism by which PAI-1 mediates angiogenesis was further investigated using isolated human retinal vascular endothelial cells. Results PAI-1 expression is up regulated in the retina of mice with oxygen-induced retinopathy. This coincides with a significant increase in the expression of vitronectin in the retina of the experimental mice. There was significant reduction in the angiogenic response of PAI-1−/− mice as compared to wild type mice. PAI-1 promotes endothelial cell migration in vitro and facilitates migration of cells on a vitronectin substrate by regulating αv integrin cell surface expression. Conclusions These observations suggest a role for PAI-1 during retinal angiogenesis and point to a potential new therapeutic target in the prevention or treatment of retinal neovascularization seen in many ocular diseases.