Disruption of fibronectin fibrillogenesis affects intraocular pressure (IOP) in BALB/cJ mice

Increased deposition of fibronectin fibrils containing EDA+fibronectin by TGFβ2 is thought to be involved in the reduction of aqueous humor outflow across the trabecular meshwork (TM) of the eye and the elevation in intraocular pressure (IOP) observed in primary open angle glaucoma (POAG). Using a fibronectin-binding peptide called FUD that can disrupt fibronectin fibrillogenesis, we examined if disrupting fibronectin fibrillogenesis would affect IOP in the TGFβ2 BALB/cJ mouse model of ocular hypertension. BALB/cJ mice that had been intravitreally injected with an adenovirus (Ad5) expressing a bioactive TGFβ2(226/228) showed a significant increase in IOP after 2 weeks. When 1μM FUD was injected intracamerally into mice 2 weeks post Ad5-TGFβ2 injection, FUD significantly reduced IOP after 2 days. Neither mutated FUD (mFUD) nor PBS had any effect on IOP. Four days after FUD was injected, IOP returned to pre-FUD injection levels. In the absence of TGFβ2, intracameral injection of FUD had no effect on IOP. Western blotting of mouse anterior segments expressing TGFβ2 showed that FUD decreased fibronectin levels 2 days after intracameral injection (p<0.05) but not 7 days compared to eyes injected with PBS. mFUD injection had no significant effect on fibronectin levels at any time point. Immunofluorescence microscopy studies in human TM (HTM) cells showed that treatment with 2ng/ml TGFβ2 increased the amount of EDA+ and EDB+ fibronectin incorporated into fibrils and 2μM FUD decreased both EDA+ and EDB+ fibronectin in fibrils. An on-cell western assay validated this and showed that FUD caused a 67% reduction in deoxycholate insoluble fibronectin fibrils in the presence of TGFβ2. FUD also caused a 43% reduction in fibronectin fibrillogenesis in the absence of TGFβ2 while mFUD had no effect. These studies suggest that targeting the assembly of fibronectin fibrillogenesis may represent a way to control IOP.