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N. Tane, A. Ohira, S. Roy; Fibronectin Overexpression and Its Effect on Schlemm’s Canal Cell Monolayer Permeability . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1342.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study whether fibronectin (FN) overexpression plays a role in Schlemm's canal (SC) cell monolayer permeability. Methods: Human SC cells were grown in normal (N) medium (5 mM glucose) or high glucose (HG) medium (30 mM glucose) for 10 days. To inhibit high glucose–induced FN overexpression, cells grown in HG medium were transfected with FN antisense phosphorothioate oligonucleotides (AS–FN oligos) in the presence of lipofectin, and analyzed 3 days after transfection. As control for antisense oligo specificity random oligonucleotides (Ran–oligos) were similarly transfected in these cells and analyzed 3 days posttransfection. FN protein expression was determined using Western Blot (WB) analysis. Parallel cultures of SC cells grown on membrane inserts of transwell plates or coverslips in N or HG medium and transfected accordingly with AS–FN oligos were analyzed for in vitro permeability (IVP) assay, transelectrical resistance (TER), and FN immunostaining. Results: WB analysis indicated that cells grown in HG medium exhibited increased FN protein expression (141±17 % of control, p=0.003) compared to cells grown in N medium. When cells grown in HG medium were transfected with AS–FN oligos, FN protein level was significantly reduced (113±6 % of control, p=0.02) compared to cells grown in HG medium. Similarly, immunofluorescence microscopy data showed increased FN immunostaning in cells grown in HG medium (125±20 % of control, p=0.01) compared to cells grown in N medium. In cells grown in HG medium and transfected with AS–FN oligos FN immunostaning was significantly reduced (105±13 % of control, p=0.02) compared to cells grown in HG medium. IVP assay indicated reduced monolayer permeability in cells grown in HG medium compared to cells grown in N medium (81±3 % of control, p=0.001). When cells grown in HG medium were transfected with AS–FN oligos IVP was increased (94±4 % of control vs 81±3 % of control, p=0.02) compared to cells grown in HG medium. In cells grown in HG medium, TER was significantly increased compared to TER of cells grown in N medium (129±9 % of control, p=0.004). When cells grown in HG medium were transfected with AS–FN oligos, TER was significantly reduced (107±5 % of control, p=0.02) compared to cells grown in HG medium. Cells grown in HG medium and transfected with Ran–oligos showed no change in FN protein expression, IVP, and TER. Conclusions: Increased FN expression by SC cells is a biosynthetic change that may contribute to blockage in the aqueous humor outflow pathway.
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