Abstract
Purpose :
Micron-sized pores are conduits for aqueous humor flow across Schlemm’s Canal (SC) endothelium. SC pore reduction in glaucoma contributes to increased outflow resistance and elevated IOP. SC pore formation is triggered by mechanical stretch (Braakman, EER 2014) and stretch increases intracellular [Ca2+]i by opening stretch-activated Ca2+ channels. Ca2+ regulates vesicle exocytosis and membrane fusion in neurons and other cell types. As membrane fusion is necessary for transcellular “I” pore formation, we hypothesize that stretch-induced Ca2+ influx promotes I-pore formation in SC cells.
Methods :
In this study, we compare I-pore formation between SC cells depleted of [Ca2+]i using BAPTA vs. vehicle treated SC cells exposed to the same level of mechanical stretch. Human SC cells were isolated from 2 non-glaucomatous donors (SC69 and SC75) and characterized following established protocols (Perkumas & Stamer, EER 2012). SC cells were seeded (8x103 cells/cm2) onto 6mm ø islands of biotinylated gelatin on elastic PDMS membranes and cultured for 24hrs. Test samples were incubated with 5 µM BAPTA-AM (30 min) and 1 mM EGTA (5min) in Ca2+ free PBS at 37°C. Samples were exposed to 0% or 20% equibiaxial stretch for 2 mins followed by incubation with streptavidin (SA) for 2 mins and then fixed. I-pores were identified based on transcellular movement of SA across the cell body where it binds to the underlying biotinylated substrate. SA labelling patterns were imaged at 20x by epifluorescence microscopy. One masked observer (JB) identified I-pores and measured the area of SA labelling within the perimeter (“pore area”) normalized by total cell area (N = 229 and 206 cells in SC69 and SC75). Statistical significance was determined by 3-way ANOVA.
Results :
Without BAPTA, increasing stretch from 0% to 20% led to doubling of pore area (0.0025±0.007 vs. 0.0041±0.008; p = 0.004). BAPTA decreased pore area at both 0% and 20% stretch (0.0021±0.001 and 0.0035±0.002; p < 0.001) relative to vehicle. BAPTA eliminated the increase in pore area in response to stretch (p > 0.7).
Conclusions :
Transcellular pore formation is mechanosensitive in SC cells and is triggered by stretch. Ca2+ is a necessary component of pore formation, such that depletion of [Ca2+]i inhibits formation of transcellular pores in SC cells exposed to stretch. This suggests that alterations in [Ca2+]i may contribute to impaired pore formation in glaucomatous SC cells.
This is a 2021 ARVO Annual Meeting abstract.