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Jennifer Faralli, Mark S Filla, Donna M Peters; Controlling Fibronectin Fibrillogenesis May Also Control IOP. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3491.
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© ARVO (1962-2015); The Authors (2016-present)
An increase in the deposition of extracellular matrix (ECM) proteins is thought to be involved in the reduction of outflow facility and elevation in intraocular pressure (IOP) observed in primary open angle glaucoma. Since fibronectin (FN) often serves as a template for ECM formation, we examined if disruption of FN fibril formation can effect IOP.
Normal confluent human trabecular meshwork (HTM) cells were treated with 2ng/ml TGFβ2 for 2 days to induce an increase in ECM formation. Some cells were also incubated with a peptide derived from the functional upstream domain (FUD) of Streptococcus pyogenes adhesin F1 protein, which is a known inhibitor of FN fibrillogenesis or a mutated, inactive FUD. Cells were then extracted with a 1% deoxycholate (DOC) lysis buffer and FN fibrils were detected by immunofluorescence microscopy and/or an On-cell western (OCW) assay. To determine if FUD could affect IOP, a mouse model of ocular hypertension and a porcine organ cultured anterior segment (POCAS) model were used. In the POCAS model, cultured anterior segments (AS) were treated with TGFβ2 +/- FUD and outflow facility was measured for 24 hr. Some AS were paraffin embedded and sections were labeled for FN. In a mouse model of ocular hypertension, Balb/c mice were injected intravitreally with the Ad5CMV-TGFβ2 (226/228) virus. Two weeks after injection, some mice were then injected intracamerally with FUD or a mutated FUD. IOP was measured weekly in mice anesthetized with a ketamine/xylazine mix using a rebound tonometer (TonoLab).
As expected, TGFβ2 increased the deposition of FN into the matrix of HTM cells and FUD prevented this increase. In POCAS treated with TGFβ2 + FUD, 7 out of 9 POCAS had an increase in outflow facility compared to baseline. Immunofluorescence microscopy indicated that TGFβ2 + FUD treated AS had less FN labeling than TGFβ2 only treated AS. Expression of constitutively active TGFβ2 in mice caused a significant increase in IOP 2 weeks post injection. Two days after injection of FUD, however, the TGFβ2 induced IOP increase was significantly decreased. The decrease, however, was temporary and IOP returned to pre-FUD injection levels four days after FUD injection, presumably because the FUD was washed out. In contrast, injection of the mutated FUD had no effect on IOP.
These studies suggest that targeting the assembly of fibronectin fibrillogenesis may represent a way to control IOP.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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