Abstract
Purpose: :
Nitric Oxide (NO) is a free radical that is produced by the enzyme, endothelial NO synthase (eNOS). eNOS activity and abundance is regulated by shear stress in vascular endothelia, and the resulting increase in NO production has a variety of physiological consequences including smooth muscle relaxation and vasodilation. In humans, shear stress levels in Schlemm’s Canal (SC) are calculated to be comparable to shear levels attained in large arteries, particularly at elevated intraocular pressure (IOP). We investigated the relationship between NO production and shear stress in cultured human SC cells.
Methods: :
Human Schlemm’s Canal endothelial cells were seeded into Ibidi flow chambers at confluence, allowed to mature for one week and then assayed for effects of continuous shear (0.1, 1, 5, 10 and 15 dynes/cm^2) on cell alignment, NO production and eNOS expression. Cell alignment was assessed using phase-contrast microscopy, NO production was measured directly using an NO electrode (Innovative Instruments) paired with DAF-FM Fluorescence (Invitrogen), and eNOS expression was evaluated using SDS-PAGE and Western blot analysis. HUVECs were used as a positive control.
Results: :
Like HUVECs, SC cells aligned in parallel with the direction of flow, a behavior that was time and shear-dependent. For example, SC alignment at 10.0 dynes/cm^2 began within three days, with the majority of cells aligned within a week. By comparison, HUVECS fully aligned within 24 hours. The synthesis of NO (0.05-3.2 nmol/10^5 cells) in HUVECS and SC cells was directly proportional to the shear-stress magnitude. Likewise, the expression of eNOS was found to increase in a shear-stress -dependent manner in both HUVECS and SC cells.
Conclusions: :
Human SC cells respond to shear stress similar to other vascular endothelia; aligning with flow, up-regulating eNOS and increasing NO production. Due to known effects of NO on vascular permeability, these results suggest that shear stress and NO production in Schlemm’s Canal may participate in the regulation of aqueous outflow across the inner wall.
Keywords: stress response • optic flow • gene/expression