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Darryl R Overby, Amir Vahabikashi, Rocio Vargas-Pinto, Sietse T Braakman, Ryan Pedrigi, Ritika Gupta, Kristin Marie Perkumas, W Daniel Stamer, C Ross Ethier, Mark Johnson; The Effect of Cell Stiffness on I- and B-Pore Formation in Schlemm’s Canal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5982.
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© ARVO (1962-2015); The Authors (2016-present)
Aqueous humor enters Schlemm’s canal (SC) by passing through micron-sized pores in SC inner wall. Pores may be transcellular “I-pores” through individual cells or paracellular “B-pores” between neighboring cells. Pore density is reduced in glaucoma, possibly contributing to elevated outflow resistance. We reported previously that glaucomatous SC cells in vitro have increased stiffness and reduced pore-forming capability, and that reduced total pore density correlates with increased SC cell stiffness (ARVO, 2013). Here, we examine these relationships for I- and B-pores.
Six SC cell lines were isolated from 3 normal and 3 glaucomatous human donors. SC cells were seeded at confluence on track-etch filters, cultured for 2 days, and perfused in the basal-to-apical direction at 6 mmHg for 30 minutes prior to fixation. Pore density (pores/mm2) and area (% of total cell area) were measured by masked observers from scanning electron micrographs. Cell stiffness was measured in the same cell lines by atomic force microscopy using spherical 10μm tips on subconfluent SC cells, and Young’s modulus (E) was calculated using a modified Hertz model. One normal cell line was treated with latrunculin-A (0.1µM), an actin depolymerizing agent, to directly examine the effect of decreasing cell stiffness on pores. Statistical analysis used the linear mixed model on log-transformed pore data and the geometric mean for E.
Compared to normal SC cells, glaucomatous SC cells had significantly lower density of I-pores (96±63 vs 9±4 pores/mm2; p=0.02) and B-pores (234±114 vs 59±11 pores/mm2; p=0.03). Similar results held for pore area (I-pores: 0.021±0.014% vs 0.001±0.001%, p=0.13; B-pores: 0.056±0.024% vs. 0.007±0.003%, p=0.02). Increasing cell stiffness correlated with decreasing I-pore density (p=0.06) and area (p=0.01), with the former being only marginally significant. For B-pores, there was a marginally significant decrease in pore area with increasing cell stiffness (p=0.06) but no observed relationship with pore density (p=0.26). Treatment with latrunculin decreased cell stiffness while increasing I-pore density and I- and B-pore area.
Cell stiffness appears to influence the formation of both I- and B-pores, and impaired pore formation in glaucomatous SC cells may arise from increased cell stiffness. Drugs that selectively lower SC cell stiffness may therefore be useful glaucoma therapies.
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