TY - JOUR
T1 - A model of giant vacuole dynamics in human Schlemm's canal endothelial cells
AU - Pedrigi, Ryan M.
AU - Simon, David
AU - Reed, Ashley
AU - Stamer, W. Daniel
AU - Overby, Darryl R.
N1 - Funding Information:
We acknowledge funding support from the Whitaker International Scholars Program (RMP), National Glaucoma Research , a program of the American Health Assistance Foundation (DRO), US National Institutes of Health grants EY018373 (DRO), EY019696 (DRO, WDS), and EY17007 (WDS). We thank Profs. Mark Johnson and Ross Ethier for thoughtful comments and helpful discussions throughout this study and Dr. Thomas Read for providing the giant vacuole image shown in Fig. 1 . We thank Dr. Christina Abbott for providing porcine aortic endothelial cells, and the Facility for Imaging by Light Microscopy (FILM) at Imperial College for use of Volocity ® software. Finally, we thank Kristin Perkumas for her careful work isolating human Schlemm’s canal endothelial cells.
PY - 2011/1
Y1 - 2011/1
N2 - Aqueous humour transport across the inner wall endothelium of Schlemm's canal likely involves flow through giant vacuoles and pores, but the mechanics of how these structures form and how they influence the regulation of intraocular pressure (IOP) are not well understood. In this study, we developed an in vitro model of giant vacuole formation in human Schlemm's canal endothelial cells (HSCECs) perfused in the basal-to-apical direction (i.e., the direction that flow crosses the inner wall in vivo) under controlled pressure drops (2 or 6 mmHg). The system was mounted on a confocal microscope for time-lapse en face imaging, and cells were stained with calcein, a fluorescent vital dye. At the onset of perfusion, elliptical void regions appeared within an otherwise uniformly stained cytoplasm, and 3-dimensional reconstructions revealed that these voids were dome-like outpouchings of the cell to form giant vacuole-like structures or GVLs that reproduced the classic " signet ring" appearance of true giant vacuoles. Increasing pressure drop from 2 to 6 mmHg increased GVL height (14 ± 4 vs. 21 ± 7 μm, p < 0.0001) and endothelial hydraulic conductivity (1.15 ± 0.04 vs. 2.11 ± 0.49 μl min-1 mmHg-1 cm-2; p < 0.001), but there was significant variability in the GVL response to pressure between cell lines isolated from different donors. During perfusion, GVLs were observed " migrating" and agglomerating about the cell layer and often collapsed despite maintaining the same pressure drop. GVL formation was also observed in human umbilical vein and porcine aortic endothelial cells, suggesting that giant vacuole formation is not a unique property of Schlemm's canal cells. However, in these other cell types, GVLs were rarely observed " migrating" or contracting during perfusion, suggesting that Schlemm's canal endothelial cells may be better adapted to withstand basal-to-apical directed pressure gradients. In conclusion, we have established an in vitro model system to study giant vacuole dynamics, and we have demonstrated that this system reproduces key aspects of giant vacuole morphology and behaviour. This model offers promising opportunities to investigate the role of endothelial cell biomechanics in the regulation of intraocular pressure in normal and glaucomatous eyes.
AB - Aqueous humour transport across the inner wall endothelium of Schlemm's canal likely involves flow through giant vacuoles and pores, but the mechanics of how these structures form and how they influence the regulation of intraocular pressure (IOP) are not well understood. In this study, we developed an in vitro model of giant vacuole formation in human Schlemm's canal endothelial cells (HSCECs) perfused in the basal-to-apical direction (i.e., the direction that flow crosses the inner wall in vivo) under controlled pressure drops (2 or 6 mmHg). The system was mounted on a confocal microscope for time-lapse en face imaging, and cells were stained with calcein, a fluorescent vital dye. At the onset of perfusion, elliptical void regions appeared within an otherwise uniformly stained cytoplasm, and 3-dimensional reconstructions revealed that these voids were dome-like outpouchings of the cell to form giant vacuole-like structures or GVLs that reproduced the classic " signet ring" appearance of true giant vacuoles. Increasing pressure drop from 2 to 6 mmHg increased GVL height (14 ± 4 vs. 21 ± 7 μm, p < 0.0001) and endothelial hydraulic conductivity (1.15 ± 0.04 vs. 2.11 ± 0.49 μl min-1 mmHg-1 cm-2; p < 0.001), but there was significant variability in the GVL response to pressure between cell lines isolated from different donors. During perfusion, GVLs were observed " migrating" and agglomerating about the cell layer and often collapsed despite maintaining the same pressure drop. GVL formation was also observed in human umbilical vein and porcine aortic endothelial cells, suggesting that giant vacuole formation is not a unique property of Schlemm's canal cells. However, in these other cell types, GVLs were rarely observed " migrating" or contracting during perfusion, suggesting that Schlemm's canal endothelial cells may be better adapted to withstand basal-to-apical directed pressure gradients. In conclusion, we have established an in vitro model system to study giant vacuole dynamics, and we have demonstrated that this system reproduces key aspects of giant vacuole morphology and behaviour. This model offers promising opportunities to investigate the role of endothelial cell biomechanics in the regulation of intraocular pressure in normal and glaucomatous eyes.
KW - Aqueous humour outflow resistance
KW - Cellular biomechanics
KW - Endothelial transport
KW - Giant vacuole
KW - Intraocular pressure
KW - Schlemm's canal
UR - http://www.scopus.com/inward/record.url?scp=78650533822&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=78650533822&partnerID=8YFLogxK
U2 - 10.1016/j.exer.2010.11.003
DO - 10.1016/j.exer.2010.11.003
M3 - Article
C2 - 21075103
AN - SCOPUS:78650533822
SN - 0014-4835
VL - 92
SP - 57
EP - 66
JO - Experimental eye research
JF - Experimental eye research
IS - 1
ER -