Abstract
Abstract: :
Purpose: Elevated levels of vascular endothelial growth factor (VEGF) contribute to vascular permeability in diabetic retinopathy, but the molecular mechanisms by which VEGF affects endothelial tight junctions are currently unknown. The aim of this study was to test the hypothesis that VEGF and diabetes cause movement of the tight junction protein occludin from the cell border to the cytoplasm as a membrane microdomain, where it enters a classic endosomal trafficking pathway. Methods: Confluent cultures of primary bovine retinal endothelial cells (BREC) were treated with 1.2 nM VEGF for 15 or 60 minutes. BREC grown on coverslips were fixed in paraformaldehyde and immunostained for occludin and ZO-1. To fractionate cells, BREC or 2 month streptozotocin diabetic rat retinas were homogenized in MES-buffered saline containing 1% Triton X-100, diluted to 40% sucrose, and layered under 5-35% discontinuous sucrose gradients. Following centrifugation, fractions were TCA-precipitated and proteins were analyzed by western blot. Co-immunoprecipitation experiments allowed a determination of specific proteins associated with occludin. Results: Control BREC cultures had continuous ZO-1 and occludin immunoreactivity at cell borders. After VEGF treatment for 15 minutes, discontinuous occludin and ZO-1 immunoreactivity at the cell borders and punctate immunoreactivity adjacent to the cell borders was increased compared with untreated cells. After VEGF treatment for 60 minutes, occludin immunoreactivity had a perinuclear distribution. Cell fractionation studies revealed a shift in occludin from predominately 40% sucrose fractions in control conditions to 25% and 35% sucrose fractions following VEGF treatment. In addition, 2 months of diabetes induced the same shift in occludin migration on sucrose step gradients. This shift to buoyant fractions suggests the formation of a membrane microdomain. Additionally, occludin associates with dynamin, a protein required for fission of several types of vesicles from the plasma membrane, in total cell homogenates of control and VEGF-treated BREC. Conclusion: VEGF and diabetes cause internalization of occludin as a membrane microdomain and relocation from the cell border to the cytoplasm. The trafficking pathway employed by this microdomain is currently under investigation. These studies begin to elucidate the mechanism by which VEGF increases vascular permeability in diabetic retinopathy.
Keywords: 339 cell adhesions/cell junctions • 388 diabetic retinopathy • 423 growth factors/growth factor receptors