Biomechanics of Schlemm's canal endothelium and intraocular pressure reduction

Abstract

Ocular hypertension in glaucoma develops due to age-related cellular dysfunction in the conventional outflow tract, resulting in increased resistance to aqueous humor outflow. Two cell types, trabecular meshwork (TM) and Schlemm's canal (SC) endothelia, interact in the juxtacanalicular tissue (JCT) region of the conventional outflow tract to regulate outflow resistance. Unlike endothelial cells lining the systemic vasculature, endothelial cells lining the inner wall of SC support a transcellular pressure gradient in the basal to apical direction, thus acting to push the cells off their basal lamina. The resulting biomechanical strain in SC cells is quite large and is likely to be an important determinant of endothelial barrier function, outflow resistance and intraocular pressure. This review summarizes recent work demonstrating how biomechanical properties of SC cells impact glaucoma. SC cells are highly contractile, and such contraction greatly increases cell stiffness. Elevated cell stiffness in glaucoma may reduce the strain experienced by SC cells, decrease the propensity of SC cells to form pores, and thus impair the egress of aqueous humor from the eye. Furthermore, SC cells are sensitive to the stiffness of their local mechanical microenvironment, altering their own cell stiffness and modulating gene expression in response. Significantly, glaucomatous SC cells appear to be hyper-responsive to substrate stiffness. Thus, evidence suggests that targeting the material properties of SC cells will have therapeutic benefits for lowering intraocular pressure in glaucoma.

Keywords: Aqueous humor; Conventional outflow; Glaucoma; Ocular hypertension; Schlemm's canal; Trabecular meshwork.

Fig. 1

Aqueous humor flow pathway. Left panel: schematic of anterior segment of eye showing the direction of aqueous humor flow in red. Center panel: an enlargement of the angle region of the eye (boxed region in left panel) showing the conventional outflow pathway; SC: Schlemm's canal. Right panel: Transmission electron micrograph of the inner wall of Schlemm's canal showing a giant vacuole through which the aqueous humor passes; V: giant vacuoles. Panel C is reproduced with permission (Overby et al., 2014) [Permission needed].

Fig. 2

Structured Illumination Microscopy images of normal and glaucomatous SC cells labeled with actin filament marker, rAV-LifeAct-TagGFP2 (IBIDI, Verona, WI) (Riedl et al., 2008) before and after application of the actin-depolymerizing agent Latrunculin-A (1 μM) for 30 min(Overby et al., 2014) Large arrows point to cortical cytoskeleton, while small arrows indicated subcortical cytoskeletal structures. [Permission needed].

Fig. 3

Schematic of the “funneling” of aqueous humor through the JCT caused by inner wall pores. If the inner wall were not present, the flow would pass uniformly through the upstream matrix of the JCT and generate a lower flow resistance (Overby et al., 2002; Overby et al., 2009) [Permission needed].

Fig. 4

The density of pores in the inner wall endothelium of Schlemm's canal in post-mortem normal (blue) and glaucomatous (red) human eyes. Fixation was done under conditions of constant flow (2 ml/min) (Johnson et al., 2002).

Fig. 5

(A) Representative image of transcellular and paracellular pores in normal and glaucomatous cultured SC cells; (B) Pore density increases in 3 non-glaucomatous SC cell strains as a function of pressure drop in the basal-to-apical direction measured following monolayer perfusion at 0, 2 or 6 mmHg; perfused in the apical-to-basal direction (“6 mmHg AB”), pore densities are similar to the unperfused control at 0 mmHg; (C) Pore density is lower in glaucomatous compared to normal SC cells following perfusion at 6 mmHg in the basal-to-apical direction (Overby et al., 2014) [Permission needed].

Fig. 6

Conceptual model of the sequential process (A to D) of formation of a giant vacuole and pore in an SC cell in the face of a pressure gradient in the basal (intraocular pressure) to apical (episcleral venous pressure) direction.

Fig. 7

Methods utilized to examine mechanical properties of SC cells: (A) Magnetic pulling cytometry: a magnetic bead is bound to the surface of a cell while a magnetic force (arrow) is applied to the bead using an electromagnetic microneedle or a microneedle attached to a permanent magnet. The resulting bead displacement is optically recorded using a microscope and used to determine cell modulus (Overby et al., 2005)[Permission needed; image modified]. (B) Magnetic twisting cytometry: A bead is bound to the surface of a cell and initially magnetized in the horizontal direction. It is then subjected to a vertical oscillatory magnetic field and the resulting torque, T, causes the bead to pivot back and forth. This motion is resisted by the cell, and the resultant bead motion is recorded using a microscope and used to determine cell modulus (Smith et al., 2003) [Permission needed; image modified]. (C) AFM: a rounded (not shown) or sharp tip is used to indent the surface of a cell. The relationship between indenting force and magnitude of indentation is used to deduce cell stiffness [Permission needed; image modified].

Fig. 8

SC cell exerts traction when plated on a deformable substrate. Phase-contrast image is shown in (A) and traction stress intensity map in (B) (Zhou et al., 2012) [Permission needed].

Fig. 9

Y-27632 induces changes in actin stress fibers in cultured TM (A) and SC (B) cells. Cells were treated with 10 μM Y27632 for 1 h at 37 °C under serum-free conditions and subsequently stained for actin (with rhodamine-conjugated phalloidin). In both TM and SC cells, Y-27632 caused significant decreases in staining for actin stress fibers. Magnification, ×400 (Rao et al., 2001) [Permission needed].

Fig. 10

Comparison of cell stiffness of different SC cell strains isolated from normal and glaucomatous donor eyes: (A) Mean and standard errors of Young's modulus (E, a measure of stiffness) of 6 normal and 5 glaucomatous non-confluent SC cell strains as measured with three different AFM tips. Modulus is determined from force–deformation curves using a modified Hertzian analysis; note the log scale on the vertical axis (Vargas-Pinto et al., 2013) (B) Results show a significant correlation between pore density and Young's modulus of the subcortical cytoskeleton, as measured by atomic force microcopy using a 10 μm spherical tip. Bars represent 95% confidence intervals on pore density and Young's modulus. Light curves in panel B represent 95% confidence intervals on the slope of the general linear model regression (dark curve) (Overby et al., 2014) [Permission needed].

Fig. 11

Elastic modulus of the TM as a function of donor age and disease status (glaucoma versus normal) as measured using a 2 μm spherical AFM tip on (A) normal (inset: data plotted on a smaller y scale) and (B) glaucomatous samples (Last et al., 2011) [Permission needed].

Fig. 12

Increases in substrate stiffness differentially modulated SC cell gene expression differentially between normal SC cells strains (blue) and glaucomatous SC cell strains (red). All expression levels were normalized to that on the softest gel. Mean ± SEM (Overby et al., 2014) [Permission needed].