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A. Li, A. Chen, S. Roy; High Glucose-Induced Fibronectin Overexpression Inhibits Trabecular Meshwork Cell Permeability . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1151.
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Purpose: To study whether high glucose-induced fibronectin (FN) overexpression plays a role in the cellular permeability of human trabecular meshwork (HTM) cells. Methods: HTM cells were grown in normal (5mM) or high glucose (30mM) medium for 10 days. In addition, two separate groups of HTM cells grown in high glucose medium for 7 days were transfected with FN antisense phosphorothioate oligonucleotides targeted against the translation initiation site of the FN transcript to modulate high glucose-induced fibronectin (FN) overexpression, or random phosphorothioate oligonucleotides. Cells treated with antisense or random oligos were analyzed 72 h after transfection. FN protein expression was assessed using Western blot analysis and immunofluorescense microscopy. In parallel, HTM cells were grown on polyester membrane inserts of 24 transwell plates in normal or high glucose medium. When cells reached confluency, in vitro permeability assay was performed by adding fluorescein (0.5 mg/ml) to the upper chamber and monitoring its passage into the lower chamber of the transwells. Presence of fluorescein in the lower chamber was determined by spectrophotometric readings performed at 60 minute time point. Results: Western blot analysis and immunofluorescence microscopy showed significant increase in FN expression in HTM cells grown in high glucose medium compared to cells grown in normal medium (127±14% of control, p=0.018; 118±11% of control, p=0.009, respectively). Permeability of fluorescein molecules in HTM cell monolayer decreased in cells grown in high glucose medium (87±9% of control, p=0.004). HTM cells transfected with antisense phosphorothioate oligonucleotide against FN showed significant reduction in FN expression to near normal level (94±15% of control, p=0.009) and an increase in permeability (98±7% of control, p=0.01); whereas, random oligonucleotide had no effect on FN expression or in vitro permeability. Conclusion: Excessive FN synthesis by trabecular meshwork cells may contribute to blockage in the outflow facility and the development of primary open angle glaucoma.
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