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Nicole Ashpole, Darryl Overby, C Ethier, W Daniel Stamer; Shear Stress Stimulation of NO release from Schlemm’s Canal Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1996.
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Nitric Oxide (NO) has important physiological effects, including increasing endothelial permeability and smooth muscle relaxation. In vascular endothelia, NO is produced by endothelial NO synthase (eNOS), whose activity and abundance are regulated by shear stress. In Schlemm’s Canal (SC) shear stress is calculated to be comparable to those in large arteries (2-20 dynes/cm^2). Here we investigate the relationship between NO production and shear stress in cultured human SC cells.
Two strains of human SC endothelial cells isolated from non-glaucomatous donor eyes were seeded into Ibidi flow chambers at confluence, allowed to acclimate for at least 7 days and subjected to continuous shear stress (0.1, 10 and 15 dynes/cm^2) for 7 days. Cell alignment was assessed using phase-contrast microscopy coupled with Cell Profiler analysis (Broad Institute). NO production was measured by two methods: (i) DAF-FM Fluorescence (Invitrogen) monitored using ImageJ analysis, giving a qualitative measure of NO concentration; and (ii) Griess Reagent reaction (Invitrogen), which measures nitrite concentration, a product of the spontaneous oxidation of NO. HUVECs were used as a positive control.
SC cells, like HUVECs, aligned with the direction of flow, a behavior that was both time and shear-dependent. While HUVECs aligned within hours, SC cells took days. NO production by SC cells increased with shear stress as assayed by both DAF-FM Fluorescence and Griess Reagent. A similar effect was seen in HUVECs (Table).
Human SC cells respond to shear stress similar to other vascular endothelia, aligning with flow and increasing NO production in a shear-dependent manner. When IOP increases (and SC collapses, thus increasing shear stress in SC), our data suggest that NO production by SC cells will increase. This could relax trabecular cells and increase permeability of the inner wall to increase outflow facility. It will be important to see if the shear-eNOS response is compromised in glaucomatous SC cells.
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