Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.