Abstract
Purpose: :
To investigate whether high glucose-induced overexpression of vascular BM components alters the composition and function of the BM in a manner that may contribute to early changes in vascular permeability associated with DR.
Methods: :
To determine cell monolayer permeability, rat retinal endothelial cells (RRECs) were grown in normal (5 mM) or high (30 mM) glucose (HG) medium on transwell inserts for 4 days. When cells were confluent, monolayer permeability was determined by measuring diffusion of FITC-dextran from upper to lower chamber across the cell monolayer. Cells grown on inserts were fixed with 2% glutaraldehyde/2% paraformaldehyde, embedded in Poly/Bed 812 resin, stained with uranyl acetate and lead citrate, and viewed on a Hitachi H-7600 transmission electron microscope. BM thickness was measured at 59,800X. Fibronectin and collagen IV protein expression was determined in RRECs grown in parallel by Western Blot analysis.
Results: :
There was increased permeability of FITC-dextran through monolayers of RRECs grown in HG (127±9% of control, P=0.02) compared to cells grown in normal medium. Western blot analysis showed increased fibronectin and collagen IV expression (132±11% of control, P=0.01; 130±19% of control, P=0.03, respectively) in the RRECs grown in high glucose medium compared to cells grown in normal medium. However, EM analysis of these cells showed that the interendothelial cell tight junctions were intact, vesicles and coated pits were rare, and BM thickness was unchanged. Some gaps between the endothelial cells appeared more predominantly in cells grown in high glucose medium, possibly due to fluid accumulation between the cells or cell shrinkage. It is possible that the gaps may cause some tension on the tight junctions and allow minor leakage, but this cannot be determined, morphologically.
Conclusions: :
Our findings suggest that prior to breakdown of the tight junctions, upregulation of vesicular transport, or a change in ECM thickness, alteration in ECM composition from high glucose-induced increased synthesis of the BM components may contribute to early changes in permeability.
Keywords: diabetic retinopathy • retina • diabetes