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Abstract
PURPOSE: To examine the effects of high glucose concentrations on retinal endothelial permeability in an in vitro model of the retinal microvasculature. METHODS: The permeability of the endothelial barrier to small solutes was measured in a chromatographic cell column consisting of bovine retinal endothelial cells cultured on porous fibronectin-coated microcarriers. In each cell column, permeability changes were evaluated by comparing the treatment permeability response over time with the initial baseline permeability. Short-term (2-hour) barrier effects of glucose were examined by measuring permeability at 15-minute intervals after an increase in perfusate concentration from baseline (5.5 mM) to high (25 mM) glucose. Long-term (to 57 days) effects were tested by addition of 25 mM glucose to microcarrier cultures. The effect of glucose on beta-receptor signaling was tested by measuring its effect on the permeability decrease produced by 1 microM isoproterenol. RESULTS: An increase from 5.5 mM to 25 mM glucose concentration did not change retinal endothelial cell monolayer permeability (n=6) during 2 hours. However, an increase in monolayer permeability was observed after 19 days (n=8) in the 25-mM glucose culture. Paralleling this time course, short-term exposure to 25 mM glucose did not prevent a decrease in permeability triggered by the beta-receptor agonist isoproterenol. However, the permeability effect of the agonist was blocked by long-term culture in 25 mM glucose. Permeability of retinal endothelial monolayers cultured in 5.5 mM glucose and treated with 1 microM isoproterenol decreased significantly to 0.71+/-0.06 of baseline (n=4; mean+/-SEM). However, permeability did not change in parallel cell columns made from microcarriers cultured in 25 mM glucose (0.97+/-0.2 of baseline permeability; n=4; mean+/-SEM). CONCLUSIONS: High-glucose culture decreases the retinal endothelial barrier and blocks the response to beta-adrenergic receptors. This model may prove valuable in exploring other hypotheses of increased permeability associated with diabetic retinopathy or other retinal diseases that break down the retinal vascular barrier.