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N. Tane, A.–F. Li, S. Dhar, A. Aohira, S. Roy; Laminin Overexpression by Trabecular Meshwork Cells: Possible Effect on Aqueous Outflow . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4381.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study whether overexpression of laminin (LM), an extracellular matrix (ECM) protein, plays a role in the development of outflow resistance through trabecular meshwork. Methods: Bovine trabecular meshwork (BTM) cells were grown in normal (N), (5 mM glucose) medium, high glucose (H), (30 mM glucose) medium, or 0.1 uM dexamethasone (D) medium for 7 days. To reduce LM overexpression, cells grown in H or D medium were transfected with LM antisense phosphorothioate oligonucleotides (AS–LM) in the presence of lipofectin, and analyzed 3 days after transfection. As control for antisense specificity random oligonucleotides were similarly transfected in these cells and analyzed 3 days later. LM protein expression was assessed using Western blot analysis. Parallel cultures of BTM cells grown on polyester membrane inserts of transwell plates in N, H, or D medium were analyzed for in vitro permeability (IVP) and transelectrical resistance (TER). Results: BTM cells grown in H or D medium showed significant increase in LM expression compared to cells grown in N medium (142±11 % of control, p=0.001, 127±14 % of control, p=0.002, respectively). Cells grown in H or D medium and transfected with AS–LM showed reduced LM protein level compared to cells grown in H or D medium (124±10 % of control, p=0.03, 106±6 % of control, p=0.035, respectively). IVP assay performed in cells grown in H or D medium indicated reduced monolayer permeability compared to cells grown in N medium (71±3 % of control, p=0.001, 77±10 % of control, p=0.002, respectively). When cells grown in H or D medium were transfected with AS–LM, IVP was significantly increased compared to cells grown in H or D medium (88±2 % of control, p=0.008, 91±5 % of control, p=0.04, respectively). In cells grown in H or D medium, TER was significantly increased compared to cells grown in N medium (156±7 % of control, p=0.028, 162±3 % of control, p=0.023, respectively). When cells grown in H or D medium were transfected with AS–LM, TER was significantly reduced compared to cells grown in H or D medium (138±14 % of control, p=0.04, 148±5 % of control, p=0.003, respectively). Cells transfected with random oligonucleotides showed no change in LM expression, IVP, or TER. Conclusions: Increased LM synthesis by trabecular meshwork cells may contribute to blockage in aqueous outflow and development of primary open angle glaucoma.
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