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Nicole Ashpole, William Stamer; . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2460.
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© ARVO (1962-2015); The Authors (2016-present)
Nitric oxide (NO) is a free radical that increases conventional outflow facility and lowers intraocular pressure. It appears that a major contributor to the IOP-lowering effects of NO is the shear stress-regulated enzyme, endothelial nitric oxide synthase (eNOS) in Schlemm's canal (SC). To specifically examine the eNOS promoter activity in SC, we constructed two reporter constructs, eNOS-GFP (green fluorescent protein) and eNOS-SEAP (secretory alkaline phosphatase) adenoviruses and tested them in human SC monolayers exposed to shear stress.
Human SC cells were seeded into Ibidi flow chambers at confluence and allowed to mature before adenovirus transduction with - eNOS-GFP, eNOS-SEAP or control (ubiquitin-GFP). After two days cells were exposed to 0.1 or 10 dynes/cm2 of continuous shear for 6, 12 or 24 hours. GFP expression was assessed by confocal microscopy and Western Blot. SEAP was quantified from conditioned media with a SEAP Reporter assay kit. HUVECs (human umbilical vein endothelial cells) were tested in parallel experiments for comparisons.
Both endothelial cell types (HUVECs and SC cells) transduced with eNOS-GFP adenovirus displayed amplified GFP fluorescence when exposed to 10 dynes/cm2 compared to 0.1 dynes/cm2. This behavior was time-dependent, with the largest increase in GFP expression occurring at 24 hours. GFP protein analyzed in cell lysates displayed a similar pattern (~2-fold increase). Surprisingly, shear stress also induced GFP expression in cells transduced with ubiquitin-GFP, which contains a single consensus shear-stress response element. The greatest shear-driven increase in expression (4000-fold) was observed in experiments using the eNOS-SEAP adenovirus.
Results suggest that the eNOS promoter in SC cells is shear responsive, and reporter constructs can be used as a sensitive tool to estimate and localize shear forces in SC in situ and in vivo.
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