| Diabetic retinopathy (DR) is the most common microvascular complication of diabetes mellitus. Increased permeability of the capillaries comprising the inner blood retinal barrier (iBRB) is one of the characteristics of the disease. Therefore, understanding of the transport properties of the iBRB is an essential step towards elucidating means to control vascular permeability in persons with DR.;We have developed an in-vitro model of the iBRB by culturing monolayers of bovine retinal endothelial cells (BREC) onto polyester porous inserts. We quantified the diffusive and the apparent permeability of (TAMRA, 70-kDa Dextran and LDL), as well as the hydraulic conductivity (Lp). Based on the results, we developed a 3-pore transport model to estimate the routes taken by the different molecules and water to cross the endothelium.;Culturing the cells in a high glucose (HG) environment for six days did not alter any of the measurements mentioned above, consistent with intact cell-cell junctions, as shown by immunostaining and protein and gene expression. However, incubation with vascular endothelial growth factor (VEGF) increased permeability and hydraulic conductivity under both normal and HG environments. The results support the existence of an indirect pathway by which iBRB permeability is increased through the established up-regulation of retinal VEGF in response to hyperglycemia.;In addition, an in-vitro model of aortic endothelium was developed by culturing bovine aortic endothelial cells (BAEC) onto polyester porous inserts. We studied how the shear induced Lp response, known to be mediated by increases in NO, is changed by enzymatically cleaving specific components of the glycocalyx (GCX). Both cleaving the heparan sulfate component and culturing the monolayers in HG, significantly attenuated the shear stress mediated Lp response while maintaining the baseline Lp unaltered. Consistently, HG incubation diminished the heparan sulfate component of the GCX as shown by immunostaining. Western blots showed that eNOS activation was significantly lower in monolayers exposed to physiological levels of shear stress that were incubated in HG media compared to monolayers incubated in normal glucose media. These results support the hypothesis that early GCX modifications in response to hyperglycemia can induce endothelial dysfunction that may accelerate atherosclerosis development. |
