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AT Tsai, SW Hassan, R Haimovici, T Sato, A-F Li, S Roy; Antisense Fibronectin Oligonucelotides Reduce High Glucose-Induced Endothelial Cell Monolayer Permeability . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1319.
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Purpose: To establish whether high glucose-induced overexpression of fibronectin, an extracellular matrix component, plays a role in the breakdown of endothelial barrier permeability. Methods: Rat microvascular endothelial cells (RMEC) were grown in normal (5mM) or high (30mM) glucose medium on semipermeable inserts of transwell plates. Cells grown in high glucose medium for 8 days were transfected with 0.4uM phosphorothioate (PS) antisense fibronectin oligonucleotides (AS-FN oligos) or PS sense oligonucleotides or PS random oligonucleotides in the presence of 8 mM lipofectin. After 3 days post-transfection, fibronectin protein level was determined by Western blot analysis, and in vitro permeability assay was performed in which FITC-dextran clearance from upper chamber into lower chamber was measured at specific time points 30, 60, 90, and 150 min. Aliquots in duplicate were measured from the lower chamber at 492nm absorbance. Results: At all time points, cells grown in high glucose medium exhibited a small but consistent increase in permeability compared to cells grown in normal medium, with maximum permeability observed at 60 min (111±2% of control, p=0.02). When cells grown in high glucose medium were transfected with AS-FN oligos, a significant reduction in permeability was observed (100±4% of control, P=0.001); no effect on permeability level was observed in cells transfected with sense or random oligos. Cells grown in high glucose medium showed increased fibronectin protein level compared to cells grown in normal medium (159±15% of control, p=0.002); cells grown in high glucose medium transfected with AS-FN oligos showed reduced fibronectin protein level (120±19% of control), whereas, cells transfected with sense or random oligos showed no change in fibronectin protein level. Conclusions: High glucose-induced fibronectin overexpression may contribute to increased endothelial cell permeability associated with blood retinal barrier breakdown in diabetic retinopathy.
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