Abstract
Elevated intraocular pressure (IOP) narrows Schlemm’s canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress. In parallel, human anterior segments were perfused under controlled flow. After delivering adenoviruses to the SC lumen by retroperfusion, the flow rate in one anterior segment of pair was increased to double pressure. In response to high shear stress, HUVECs and SC cells expressed more SEAP and GFP than control. Similarly, human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. This is the first in situ demonstration of shear-mediated SC mechanobiology as a key IOP-sensing mechanism necessary for IOP homeostasis.
Highlights
Elevated intraocular pressure (IOP) narrows Schlemm’s canal (SC), theoretically increasing luminal shear stress
To determine if shear stress in SC could detect changes in IOP, we first designed and engineered two adenovirus reporters and validated them in cell culture models using known levels of applied shear stress. We introduced these adenoviruses into a physiological model of outflow facility, the anterior segment perfusion model, to determine if we could utilise these reporters, and shear stress, to detect changes in IOP induced by an increased flow rate
human umbilical vein endothelial cells (HUVECs) were transduced with endothelial nitric oxide synthase (eNOS)-secreted alkaline phosphatase (SEAP) or eNOS-green fluorescent protein (GFP) adenovirus and subjected to shear stress
Summary
Elevated intraocular pressure (IOP) narrows Schlemm’s canal (SC), theoretically increasing luminal shear stress. Human anterior segments perfused at higher flow rates released significantly more nitrites and SEAP into perfusion effluent, and SC cells expressed increased GFP near collector channel ostia compared to control. These data establish that engineered adenoviruses have the capacity to quantify and localize shear stress experienced by endothelial cells. The primary risk factor for glaucoma is elevated intraocular pressure (IOP)[2,3], which is caused by increased outflow resistance to aqueous humor through the conventional outflow pathway Resistance in this pathway is generated principally in the juxtacanalicular region (JCT), where the trabecular meshwork (TM) and Schlemm’s canal (SC) cells interact[4,5].
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