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Kristin Marie Perkumas, Nicole E Ashpole, Joan Kalnitsky, W Daniel Stamer; Performance of shear stress-responsive reporter viruses in human Schlemm's canal monolayers. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1062.
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© ARVO (1962-2015); The Authors (2016-present)
Laminar shear stress across Schlemm's Canal (SC) endothelial cells initiates the release of nitric oxide (NO). Such effects are mediated in the long term by shear stress-responsive elements in the promoter region of endothelial nitric oxide synthase (eNOS). As a tool to study SC responses to shear stress in situ, we designed two adenovirus constructs, one which contains 1057 kb of the eNOS promoter driving the expression of either Green Florescent Protein (eNOS-GFP) or Secretory Alkaline Phosphatase (eNOS-SEAP) reporter proteins. The two adenoviruses were tested in experiments to visualize and quantify the effects of shear stress on SC cell monolayers in vitro.
Human SC and umbilical vein endothelial cells (HUVECs, positive control) were transduced with adenovirus (MOI=50 for 8 hr) after being seeded into Ibidi µ-slide chambers. The cells were exposed to one of two levels of shear stress (0.1 or 10 dynes/cm2) for 24 hours using the Ibidi pump system. GFP expression was monitored with confocal fluorescence microscopy, flow cytometry and western blot analyses. SEAP was measured using a SEAP assay kit. An adenovirus encoding a cytomegalovirus promoter driving GFP was used as a shear-insensitive negative control.
In response to a shear stress increase from 0.1 to 10 dynes/cm2, SEAP secretion increased by 36% from HUVECs, compared to a 102% increase from SC cells; despite dramatically different transduction efficiencies between cell types (~80% in HUVECs vs ~2% in SC cells). Exposure to increased shear stress also resulted in an increase in GFP expression in both endothelial cell types as measured using three different methods: In HUVECs, GFP fluorescence increased via confocal microscopy by 62%, and via flow cytometry by 70%; whereas GFP protein expression increased by 75%. In SC cells, GFP fluorescence increased via confocal microscopy by 68%, and via flow cytometry, by 211%; while protein expression increased by 133%.
The results suggest that both the eNOS-GFP and the eNOS-SEAP adenoviruses are effective tools to study SC responses to shear stress, having the capacity to localize and quantify shear stress exposure of SC cells in situ or in vivo at different intraocular pressures.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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