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D. D. Simon, D. R. Overby; In Vitro Formation of "Giant Vacuole-like" Structures in Schlemm's Canal vs. HUVEC Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4854.
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© ARVO (1962-2015); The Authors (2016-present)
Giant vacuoles and pores in Schlemm’s canal (SC) endothelium likely contribute to outflow resistance by influencing flow pathways across the inner wall, but the biomechanics governing the formation of these structures are unknown. In this project, we examined the time and pressure dependence of "giant vacuole-like" structures that form in an in vitro perfusion model of SC endothelium, and we compared GVL dynamics between SC cells and HUVECs (human umbilical vein endothelial cells).
Our existing in vitro perfusion system (ARVO 2008 #1605) was modified for pressure and temperature control. Human SC cells or HUVECs were seeded at confluence (105 cells/cm2) on track-etched membranes and cultured for up to 3 days. Cells were stained with cytoplasmic and nuclear fluorescent dyes, and placed within the perfusion apparatus. 6 HUVEC and 6 SC monolayers were perfused at high (6mmHg) or low (2mmHg) pressure in the basal-to-apical direction with DMEM+20mM HEPES at 37°C while imaging cytoplasmic and nuclear deformation (2 frames/min, 20min). Viability and membrane rupture was assayed by dye exclusion.
During perfusion, circular void regions appeared in the cytoplasm that we interpret as focal sites of apical cell displacement into thin domes or "GVLs". For HUVECs at 6mmHg, GVL diameter increased monotonically to 19.6±4.4µm (mean±SD). At 2mmHg, GVL size was bimodal with a more prevalent smaller population (6.9±3.0µm) and a larger population (19.4±3.7µm) that appeared similar to GVLs at 6mmHg. For SC cells at 2mmHg, no GVLs were observed. At 6mmHg, SC cells exhibited fewer and smaller (14.7±5.3µm; p=0.002) GVLs than HUVECs, and SC GVLs were more dynamic, often contracting and expanding repeatedly during perfusion.
GVL dimensions appear bimodal, shifting from smaller to larger populations with increasing pressure. Compared to HUVECs at the same pressure, SC GVLs were fewer, smaller and more contractile. These data suggest that SC cells may be better adapted to withstand pressure gradients, possibly due to greater contractility, stiffness or adhesion.Acknowledgements: We thank Dr. Dan Stamer (U. of Arizona) for providing SC cells.
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