An-Fei Li1, Nobuhiro Tane, Sayon Roy. 1. Department of Ophthalmology, Boston University School of Medicine, Boston, MA 02118, USA.
Abstract
PURPOSE: To study whether excess synthesis of an extracellular matrix (ECM) component, fibronectin (FN), underlying the monolayer of human trabecular meshwork (HTM) cells, influences permeability. METHODS: To upregulate FN expression, HTM cells were grown in high glucose (30 mM) medium for 10 days. In parallel, cells were grown in normal (5 mM) medium as control, and two separate groups of HTM cells were grown in high glucose medium for transfection with FN antisense phosphorothioate oligonucleotides (AS-FN oligos) to modulate high glucose induced FN overexpression, or random phosphorothioate oligonucleotides (Ran oligos) as control. FN protein expression and distribution was assessed by western blot analysis and immunofluorescence microscopy. In parallel, HTM cells were grown in transwell plates in normal or high glucose medium to perform in vitro permeability (IVP) assays and to assess transelectrical resistance (TER). RESULTS: Western blot analysis showed FN expression was upregulated by 27% (p=0.018) in HTM cells grown in high glucose medium compared to cells grown in normal medium. Immunofluorescence microscopy showed intense FN immunostaining, and IVP results showed a consistent reduction in monolayer permeability (13% reduction, p=0.004) in cells grown in high glucose medium compared to cells grown in normal medium. When cells grown in high glucose medium were transfected with AS-FN oligos FN expression was reduced by 33% (p=0.009) and resulted in increased permeability to near normal levels (98+/-7% of control, p=0.01), whereas random oligos had no effect on either FN expression or IVP. TER was significantly increased across TM cell monolayers grown in high glucose compared to those grown in normal medium (143+/-11% of control, p=0.001), which was reduced when cells were transfected with AS-FN oligos (109+/-7% of control, p=0.02) whereas cells transfected with random oligos showed no change. CONCLUSIONS: Excess FN synthesis by trabecular meshwork cells may contribute to blockage in aqueous outflow associated with the development of primary angle open glaucoma (POAG).
PURPOSE: To study whether excess synthesis of an extracellular matrix (ECM) component, fibronectin (FN), underlying the monolayer of human trabecular meshwork (HTM) cells, influences permeability. METHODS: To upregulate FN expression, HTM cells were grown in high glucose (30 mM) medium for 10 days. In parallel, cells were grown in normal (5 mM) medium as control, and two separate groups of HTM cells were grown in high glucose medium for transfection with FN antisense phosphorothioate oligonucleotides (AS-FNoligos) to modulate high glucose induced FN overexpression, or random phosphorothioate oligonucleotides (Ran oligos) as control. FN protein expression and distribution was assessed by western blot analysis and immunofluorescence microscopy. In parallel, HTM cells were grown in transwell plates in normal or high glucose medium to perform in vitro permeability (IVP) assays and to assess transelectrical resistance (TER). RESULTS: Western blot analysis showed FN expression was upregulated by 27% (p=0.018) in HTM cells grown in high glucose medium compared to cells grown in normal medium. Immunofluorescence microscopy showed intense FN immunostaining, and IVP results showed a consistent reduction in monolayer permeability (13% reduction, p=0.004) in cells grown in high glucose medium compared to cells grown in normal medium. When cells grown in high glucose medium were transfected with AS-FNoligosFN expression was reduced by 33% (p=0.009) and resulted in increased permeability to near normal levels (98+/-7% of control, p=0.01), whereas random oligos had no effect on either FN expression or IVP. TER was significantly increased across TM cell monolayers grown in high glucose compared to those grown in normal medium (143+/-11% of control, p=0.001), which was reduced when cells were transfected with AS-FNoligos (109+/-7% of control, p=0.02) whereas cells transfected with random oligos showed no change. CONCLUSIONS: Excess FN synthesis by trabecular meshwork cells may contribute to blockage in aqueous outflow associated with the development of primary angle open glaucoma (POAG).