title: Biomechanical strain as a trigger for pore formation in Schlemm's canal endothelial cells.
abstract: The bulk of aqueous humor passing through the conventional outflow pathway must cross the inner wall endothelium of Schlemm's canal (SC), likely through micron-sized transendothelial pores. SC pore density is reduced in glaucoma, possibly contributing to obstructed aqueous humor outflow and elevated intraocular pressure (IOP). Little is known about the mechanisms of pore formation; however, pores are often observed near dome-like cellular outpouchings known as giant vacuoles (GVs) where significant biomechanical strain acts on SC cells. We hypothesize that biomechanical strain triggers pore formation in SC cells. To test this hypothesis, primary human SC cells were isolated from three non-glaucomatous donors (aged 34, 44 and 68), and seeded on collagen-coated elastic membranes held within a membrane stretching device. Membranes were then exposed to 0%, 10% or 20% equibiaxial strain, and the cells were aldehyde-fixed 5 min after the onset of strain. Each membrane contained 3-4 separate monolayers of SC cells as replicates (N = 34 total monolayers), and pores were assessed by scanning electron microscopy in 12 randomly selected regions (∼65,000 μm(2) per monolayer). Pores were identified and counted by four independent masked observers. Pore density increased with strain in all three cell lines (p < 0.010), increasing from 87 ± 36 pores/mm(2) at 0% strain to 342 ± 71 at 10% strain; two of the three cell lines showed no additional increase in pore density beyond 10% strain. Transcellular "I-pores" and paracellular "B-pores" both increased with strain (p < 0.038), however B-pores represented the majority (76%) of pores. Pore diameter, in contrast, appeared unaffected by strain (p = 0.25), having a mean diameter of 0.40 μm for I-pores (N = 79 pores) and 0.67 μm for B-pores (N = 350 pores). Pore formation appears to be a mechanosensitive process that is triggered by biomechanical strain, suggesting that SC cells have the ability to modulate local pore density and filtration characteristics of the inner wall endothelium based on local biomechanical cues. The molecular mechanisms of pore formation and how they become altered in glaucoma may be studied in vitro using stretched SC cells.
authors.affiliation: Department of Bioengineering, Imperial College London, London, United Kingdom.
authors.forename: Sietse T
authors.initials: ST
authors.lastname: Braakman
authors.affiliation: Department of Bioengineering, Imperial College London, London, United Kingdom.
authors.forename: Ryan M
authors.initials: RM
authors.lastname: Pedrigi
authors.affiliation: Department of Ophthalmology and Vision Sciences, University of Toronto, Canada.
authors.forename: A Thomas
authors.initials: AT
authors.lastname: Read
authors.affiliation: Department of Bioengineering, Imperial College London, London, United Kingdom.
authors.forename: James A E
authors.initials: JA
authors.lastname: Smith
authors.affiliation: Department of Ophthalmology, Duke University Medical School, USA.
authors.forename: W Daniel
authors.initials: WD
authors.lastname: Stamer
authors.affiliation: Department of Bioengineering, Imperial College London, London, United Kingdom; Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, USA.
authors.forename: C Ross
authors.initials: CR
authors.lastname: Ethier
authors.affiliation: Department of Bioengineering, Imperial College London, London, United Kingdom. Electronic address: d.overby@imperial.ac.uk.
authors.forename: Darryl R
authors.initials: DR
authors.lastname: Overby
mesh_terms: D000328:Adult; D000368:Aged; D001082:Aqueous Humor / Q000378:metabolism*; D002450:Cell Communication; D002452:Cell Count; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000502:physiology* / Q000648:ultrastructure; D005110:Extracellular Space; D006801:Humans; D042541:Intracellular Space; D016850:Limbus Corneae / Q000166:cytology*; D008855:Microscopy, Electron, Scanning; D016062:Porosity; D012590:Sclera / Q000502:physiology* / Q000648:ultrastructure; D013314:Stress, Mechanical; D014019:Tissue Donors; D014617:Vacuoles
keywords: Schlemm's canal endothelium; biomechanics; endothelium; pore; strain; stretch