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Olya Pokrovskaya, Deborah M Wallace, Colm J O'Brien; Modulation of the fibrotic response of the human lamina cribrosa cell to cyclical mechanical stretch using the Rho kinase inhibitor Y-27632. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5032.
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The purpose of our study was to investigate the role of the Rho kinase specific inhibitor Y-27632 in modulating the fibrotic response of human lamina cribrosa cells following cyclical mechanical stretch (CMS). CMS has previously been used to model the in vivo biomechanical environment of the LC cell subjected to raised intraocular pressure in primary open angle glaucoma (POAG). Pathological hallmarks of glaucomatous optic neuropathy include distorted lamina cribrosa architecture, associated with an exaggerated fibrotic response of LC cells and excessive extracellular matrix (ECM) deposition. This can lead to stiffening of the LC, and compromise the retinal ganglion cell axons as they traverse this structure. Genes linked to ECM deposition and assembly have been shown to be up regulated in LC cells subjected to CMS in vitro. These include TGFβ1 and collagen type 1A1. By influencing the actin cytoskeleton, Rho kinase inhibitors may also be implicated in regulating how a cell interacts with its environment - for example, cell-ECM interaction and ECM assembly and rigidity. In this study, we want to investigate the ability of Rho kinase inhibitors to modulate the fibrotic response of lamina cribrosa cells.
Glial fibrillary acid protein (GFAP) negative primary LC cells were generated from the optic nerve head tissue of three normal human donors. Confluent cells from passages 4-8 were exposed to 15% stretch at 1Hz or static conditions for 24 hours using the Flexercell tension plus FX-4000T system. Cells were stretched in the presence or absence of Y-27632 and q-PCR was used to assess the expression of genes, including collagen type 1A1 and TGFβ1. Inhibition of the Rho pathway was confirmed in treated cells versus controls using Western Blot by assessing myosin light chain phosphorylation.
Compared with static controls, the expression of pro-fibrotic genes was upregulated in LC cells exposed to CMS. Pre-treatment with Y-27632 resulted in significantly reduced expression of the genes of interest, including collagen type 1A1 and TGFβ1 (p < 0.05).
These findings suggest that Y-27632 modulates the fibrotic response of LC cells to mechanical stretch. Rho kinase inhibitors may hence act in a protective way on the lamina cribrosa in the setting of mechanical strain induced by raised intraocular pressure.
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