Fibronectin overexpression inhibits trabecular meshwork cellmonolayer permeability

[摘要] Purpose: To study whether excess synthesis of an extracellularmatrix (ECM) component, fibronectin (FN), underlying the monolayer ofhuman trabecular meshwork (HTM) cells, influences permeability.Methods: To upregulate FN expression, HTM cells were grown in highglucose (30 mM) medium for 10 days. In parallel, cells were grown innormal (5 mM) medium as control, and two separate groups of HTM cellswere grown in high glucose medium for transfection with FN antisensephosphorothioate oligonucleotides (AS-FN oligos) to modulate highglucose induced FN overexpression, or random phosphorothioateoligonucleotides (Ran oligos) as control. FN protein expression anddistribution was assessed by western blot analysis andimmunofluorescence microscopy. In parallel, HTM cells were grown intranswell plates in normal or high glucose medium to perform in vitropermeability (IVP) assays and to assess transelectrical resistance(TER).Results: Western blot analysis showed FN expression was upregulatedby 27% (p=0.018) in HTM cells grown in high glucose medium compared tocells grown in normal medium. Immunofluorescence microscopy showedintense FN immunostaining, and IVP results showed a consistent reductionin monolayer permeability (13% reduction, p=0.004) in cells grown inhigh glucose medium compared to cells grown in normal medium. When cellsgrown in high glucose medium were transfected with AS-FN oligos FNexpression was reduced by 33% (p=0.009) and resulted in increasedpermeability to near normal levels (98±7% of control, p=0.01),whereas random oligos had no effect on either FN expression or IVP. TERwas significantly increased across TM cell monolayers grown in highglucose compared to those grown in normal medium (143±11% ofcontrol, p=0.001), which was reduced when cells were transfected withAS-FN oligos (109±7% of control, p=0.02) whereas cells transfectedwith random oligos showed no change.Conclusions: Excess FN synthesis by trabecular meshwork cells maycontribute to blockage in aqueous outflow associated with thedevelopment of primary angle open glaucoma (POAG).

[学科分类] 生物化学/生物物理