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Maria Gomez-Caraballo, Kristin Marie Perkumas, Joan Kalnitsky, Michael H Elliott, William D Stamer; Caveolin-mediated mechanosensation in Schlemm’s canal endothelia. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3183.
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© ARVO (1962-2015); The Authors (2016-present)
Caveolae are specialized membrane structures that respond to mechanical perturbations by protein component disassembly, releasing molecules such as the transcription factor, PTRF and the nitric oxide (NO) producing enzyme, endothelial nitric oxide synthase (eNOS). Our laboratory has previously shown that mechanical stretch of primary human trabecular meshwork cells induces phosphorylation of CAV1 and shear stress of primary human Schlemm’s canal (SC) cells stimulates NO production; however, this mechanism of activation is unknown. We hypothesize that caveolae are the mechanosensors that link biomechanical stimulation of SC to eNOS activation upon intraocular pressure elevation, and that caveola-dependent eNOS activation enhances conventional outflow.
To study the effect of Cav-1 silencing in SC cells, we tested a novel Cav-1 shRNA adenovirus with a GFP tag (Vector Biolabs) and a scrambled shRNA with GFP tag (control virus) in primary human SC cells, and in human umbilical vein endothelial cells (HUVECs, Clonetics). GFP expression by transduced and control cells was detected indirectly via Western blotting using anti-GFP IgGs or directly using flow cytometry to determine transduction efficiency. Total Cav-1 protein expression was monitored by Western blotting using antibodies that specifically recognize Cav-1. Western blot densitometry analyses were performed using Image Lab software.
We optimized timing and dosing with the Cav-1 shRNA silencing adenovirus for each cell type, finding that transduction efficiency of HUVECs was 99% at a multiplicity of infection (MOI) of 10, while that of SC cells was 78% at an MOI of 50. Both cell types were incubated with control and Cav-1 shRNA silencing adenovirus for 8hr. SC cells were tested 72hr post-transduction, while HUVECs were viewed at 60hr. Under these conditions, the Cav-1 shRNA silencing adenovirus decreased Cav-1 levels by 50% in SC cells compared to control cells, while knockdown of Cav-1 in HUVECs decreased by 70%.
We have optimized conditions to efficiently knockdown the long-lived membrane scaffolding protein, Cav-1 in SC cells using Cav-1shRNA GFP tagged adenovirus. Now the specific contribution of Cav-1 in SC cells to eNOS activation/NO production during times of elevated shear stress can be studied.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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