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Casey Kopczynski, Feryan Ahmed, Dhruba Bharali, Karen Torrejon, Cheng-Wen Lin; Anti-fibrotic Effects of AR-13324 in a 3D Bioengineered Human Trabecular Meshwork Model of Steroid-induced Glaucoma. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5639.
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© ARVO (1962-2015); The Authors (2016-present)
Fibrosis at the human trabecular meshwork (HTM) has been associated with elevated intraocular pressure (IOP) in glaucoma. AR-13324, a Rho kinase/Norepinephrine transporter inhibitor, is in Phase 3 trials for the treatment of glaucoma. This study explores the potential anti-fibrotic effects of AR-13324 in a 3D Bioengineered HTM model of steroid-induced glaucoma.
3D HTM was treated with the steroid prednisolone acetate (PA) and simultaneously treated with 0, 100, 250 or 500nM of AR-13324 for 72 hours. Separately, 3D HTM was treated with PA for 144 hours, with 100nM AR-13324 added after the first 72 hours of PA treatment. HTM samples were analyzed by immunocytochemistry and confocal microscopy for expression of paxillin (focal adhesions), F-actin stress fibers, and the extracellular matrix (ECM) proteins fibronectin and collagen IV. Transcriptional and protein expression levels of fibronectin and collagen IV were also evaluated. Scanning electron microscopy (SEM) was used to investigate the morphology and topography of the 3D HTM. Perfusion studies were performed to measure the outflow facility of 3D HTM.
The PA-treated 3D HTM responded to AR-13324 treatment in a dose-dependent manner. Compared to PA-only treated samples, the presence of AR-13324 significantly decreased fibronectin and collagen IV transcript levels (p<0.01) and protein accumulation and secretion (p<0.001). Confocal microscopy of treated HTM showed AR-13324 prevented the increased accumulation of focal adhesions and actin stress fibers induced by PA. Addition of AR-13324 to HTM that had been pre-treated with PA for 72 hours also significantly inhibited PA-induced increases in fibronectin and collagen IV transcript levels (p<0.005) and protein secretion (p<0.001). SEM revealed that addition of AR-13324 to PA-treated HTM produced openings between HTM cells and cell-layers. Perfusion studies showed that 100nM of AR-13324 increased the outflow facility of PA-treated 3D HTM tissue.
AR-13324 prevented overproduction of ECM, focal adhesions and actin stress fibers in steroid-treated 3D bioengineered HTM. The biochemical and morphological changes induced by AR-13324 correlated with an increase in outflow facility in steroid-treated HTM. AR-13324 countered the effects of steroid treatment on the HTM whether added prior to or after the onset of the steroid-induced changes.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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