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.

Keywords: Schlemm's canal endothelium; biomechanics; endothelium; pore; strain; stretch.

Figure 1

The membrane stretching device used to apply equibiaxial strain to adherent Schlemm’s canal endothelial cells. A) A schematic vertical cross-section through the device originally described by Lee et al. (1996). A silicone elastic membrane is clamped into the device and cells are seeded on the upward-facing surface of the membrane. The membrane is stretched by turning the threaded membrane holder (grey) along the outer cylinder (white), thereby pulling the membrane over the indenter (black) to impose equibiaxial strain. B) A representative calibration curve obtained from one of the three cell stretching devices used in this study. The measured components of the Green-Lagrange strain tensor (E_cc, E_rr, E_cr) are plotted against the number of turns of the membrane stretching device. The normal components of the strain tensor in the circumferential (E_cc) and radial (E_rr) directions increase with each turn and remain statistically identical, whereas the shear component (E_cr) remains close to zero, indicating that equibiaxial strain is applied to the membrane. The calibration closely follows the analytical solution (Appendix A). Error bars show the standard deviation of each strain component measured over 5 different regions on the membrane.

Figure 2

Method to covalently coat the elastic PDMS membrane with collagen. A) Representative brightfield images (scale bar 50 μm) showing the attachment and spreading of Schlemm’s canal (SC) cells on bare and collagen-coated PDMS relative to tissue culture plastic. SC cells do not adhere to bare PDMS but attach and spread on collagen-coated PDMS similar to as when on tissue culture plastic. B) The diagram summarizes the method to covalently coat the PDMS membrane with collagen, after Wipff et al., 2009. PDMS is exposed to oxygen plasma to create hydroxide groups on the surface. The hydroxide groups are then reacted with APTES to present an amino group that can be cross-linked to the amino groups of collagen using glutaraldehyde. PDMS, poly(dimethylsiloxane); APTES: (3-aminopropyl)triethoxysilane.

Figure 3

Scanning electron micrographs of pores in Schlemm’s canal (SC) endothelial cells. The top two rows are representative images of pores observed in cultured SC cells following stretch, while the bottom row is representative of pores observed in the inner wall in situ following perfusion at 8 mmHg (ostensibly normal human eye, unpublished data). Transcellular “I” pores, paracellular “B” pores and unknown “U” pores are indicated by yellow text. White arrowheads mark the border between adjacent cells. Note that the pores and cell surfaces observed in culture are more irregular than those observed in situ. Unmarked openings were considered to be artifacts. The cell line and strain-level are specified in the upper left of each micrograph. Scale bars: 1 μm.

Figure 4

Pore density increases from 0% to 10% Green-Lagrange strain in cultured Schlemm’s canal (SC) cells for total pores (p ≤ 0.0003, E-test; panel A), paracellular “B” pores (p ≤ 0.0070; panel B), and transcellular “I” pores (p ≤ 0.038; panel C). Each symbol (circle, plus or cross) denotes a pore density measurement from a single specimen (based on 12 regions-of-interest per specimen), while the lines connect the mean pore density at each strain level for each cell line as given in Table 1. The difference in total pore, I-pore and B-pore density between 10% and 20% strain is not statistically significant for SC58 or SC65 (p ≥ 0.10), but is statistically significant for SC67 (p ≤ 0.013). Note that all vertical axes are presented on the same scale and that symbols are shifted slightly on the horizontal axes so as to avoid overlap (bottom braces). Pore density data are normalized by the original (undeformed) membrane area, as described in Methods. Statistically significant changes in pore density between 0% and 10% or 20% strain are indicated by (*) for p < 0.05, (†) for p < 0.01 and (‡) for p < 0.001, where the reported p-values are the maximum p-values across all three cell lines.

Figure 5

The cumulative distribution function (CDF) describing pore diameter (red) plotted with the best-fit predictions of the CDF from a logarithmic-normal (solid black line) and a Gaussian-normal (dashed black line) distribution. The logarithmic-normal distribution better represents the empirical CDF describing pore diameter than does the normal distribution. This is true for both transcellular “I” pores (p = 0.97 vs. p = 0.04, Shapiro-Wilk; left panel) and paracellular “B” pores (p = 0.44 vs. p < 10⁻⁵; right panel). Pore data taken from SC58 at 20% strain.

Figure 6

The pore diameter distributions for transcellular “I” pores and paracellular “B” pores across all three Schlemm’s canal (SC) cell lines. Because pore diameter was insensitive to strain, all measured pore diameters were aggregated to determine the empirical cumulative distribution function (CDF) for each cell line (circles, right axes). Each circle represents an individual pore diameter measurement. Each empirical CDF was then fitted to the theoretical CDF of the logarithmic-normal distribution (dashed curves, right axes), and that fit was used to estimate the best-fit logarithmic-normal probability distribution function (PDF) or “histogram” describing pore diameter for each cell line (solid curves, left axes). These data reveal differences in the pore diameter distributions between different SC cell lines, with SC65 tending to have smaller (p < 0.03, ANOVA) and more tightly distributed pore diameters relative to SC58 and SC67.

Figure 7

Empirical cumulative distribution functions (CDF) describing pore diameter for Schlemm’s canal (SC) cells in culture (thin lines) versus the inner wall in situ (thick lines). In situ data were taken from a prior study of 5 ostensibly normal human eyes perfusion fixed at 8 mmHg (Ethier et al., 2006), but only the maximum and minimum CDFs are shown to represent the physiologically observed range of pore diameters along the inner wall in situ. For both transcellular “I” pores (A) and paracellular “B” pores (B), the pore diameters observed in cultured SC cells are broadly consistent with the pore diameters measured along the inner wall in situ. The CDFs of pore diameter in culture are reproduced from Figure 6.

Figure 8

A comparison between the morphology of pores observed in cultured Schlemm’s canal (SC) cells and “pores” or transendothelial “tunnels” observed in vascular endothelial cells. A) In SC cells, protrusions (yellow dotted curves) and processes (yellow arrows) were occasionally observed extending from the perimeter of paracellular “B” pores. Images are scanning electron micrographs (SEMs) from SC58, with the cell borders indicated by white arrowheads. B) In vascular endothelial cells from a prior study (Martinelli et al., 2013), closure of transcellular “pores” and paracellular “gaps” often occurs by ‘ventral lamellipodia’ (white dotted curves) that extend across the cell surface (blue arrowheads) to cover and close the “pore” or “gap”. Note the similarity between the ventral lamellepodia in Panel B and the cellular protrusions and processes about B-pores in SC cells shown in Panel A. Panel B represents a time-lapse confocal fluorescence image sequence with the elapsed time in minutes displayed in the upper left of each frame. Reproduced with permission of the authors and the publisher. ©2013 Martinelli et al., Journal of Cell Biology. 201: 449–465. doi: 10.1083/jcb.201209077. C) In SC cells, protrusions (yellow dotted curves) were occasionally observed extending from the perimeter of transcellular “I” pores. Images are SEMs from SC67 (left and middle) and SC65 (right). D) In vascular endothelial cells from a prior study (Maddugoda et al, 2011), closure of transcellular “pores” or “tunnels” was often associated with actin-rich membrane waves that extended from the “pore” perimeter, as visualized in cells transfected with green fluorescent actin. Inset on the right shows time-lapse images from the start (top) to end (bottom) of closure. Note the similarity between the shape of the actin-rich protrusions in vascular endothelial cells and the protrusions surrounding I-pores in SC cells in Panel C. Reproduced from with permission of the authors and the publisher ©2011 Maddugoda et al., Cell Host & Microbe. 10: 464–474, doi: 10.1016/j.chom.2011.09.014. Scale bars: 1 μm (AμC), 5 μm (B) and 10 μm (D).