Altered mechanobiology of Schlemm’s canal endothelial cells in glaucoma

Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm’s canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell—likely accounting for increased outflow resistance—that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness—and its biomechanical effects on pore formation—as a therapeutic target in glaucoma

Fluidization and Resolidification of the Human Bladder Smooth Muscle Cell in Response to Transient Stretch

Background: Cells resident in certain hollow organs are subjected routinely to large transient stretches, including every adherent cell resident in lungs, heart, great vessels, gut, and bladder. We have shown recently that in response to a transient stretch the adherent eukaryotic cell promptly fluidizes and then gradually resolidifies, but mechanism is not yet understood. Principal Findings: In the isolated human bladder smooth muscle cell, here we applied a 10% transient stretch while measuring cell traction forces, elastic modulus, F-actin imaging and the F-actin/G-actin ratio. Immediately after a transient stretch, F-actin levels and cell stiffness were lower by about 50%, and traction forces were lower by about 70%, both indicative of prompt fluidization. Within 5min, F-actin levels recovered completely, cell stiffness recovered by about 90%, and traction forces recovered by about 60%, all indicative of resolidification. The extent of the fluidization response was uninfluenced by a variety of signaling inhibitors, and, surprisingly, was localized to the unstretch phase of the stretch-unstretch maneuver in a manner suggestive of cytoskeletal catch bonds. When we applied an “unstretch-restretch” (transient compression), rather than a “stretch-unstretch” (transient stretch), the cell did not fluidize and the actin network did not depolymerize. Conclusions: Taken together, these results implicate extremely rapid actin disassembly in the fluidization response, and slow actin reassembly in the resolidification response. In the bladder smooth muscle cell, the fluidization response to transient stretch occurs not through signaling pathways, but rather through release of increased tensile forces that drive acute disassociation of actin

Hypercompliant Apical Membranes of Bladder Umbrella Cells

Urinary bladder undergoes dramatic volume changes during filling and voiding cycles. In the bladder the luminal surface of terminally differentiated urothelial umbrella cells is almost completely covered by plaques. These plaques (500 to 1000 nm) are made of a family of proteins called uroplakins that are known to form a tight barrier to prevent leakage of water and solutes. Electron micrographs from previous studies show these plaques to be interconnected by hinge regions to form structures that appear rigid, but these same structures must accommodate large changes in cell shape during voiding and filling cycles. To resolve this paradox, we measured the stiffness of the intact, living urothelial apical membrane and found it to be highly deformable, even more so than the red blood cell membrane. The intermediate cells underlying the umbrella cells do not have uroplakins but their membranes are an order of magnitude stiffer. Using uroplakin knockout mouse models we show that cell compliance is conferred by uroplakins. This hypercompliance may be essential for the maintenance of barrier function under dramatic cell deformation during filling and voiding of the bladder

Altered mechanobiology of Schlemm’s canal endothelial cells in glaucoma

Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that altered biomechanical behavior of Schlemm’s canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell—likely accounting for increased outflow resistance—that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness—and its biomechanical effects on pore formation—as a therapeutic target in glaucoma

Vascular smooth muscle cells exhibit a progressive loss of rigidity with serial culture passaging

One drawback of in vitro cell culturing is the dedifferentiation process that cells experience. Smooth muscle cells (SMC) also change molecularly and morphologically with long term culture. The main objective of this study was to evaluate if culture passages interfere in vascular SMC mechanical behavior. SMC were obtained from five different porcine arterial beds. Optical magnetic twisting cytometry (OMTC) was used to characterize mechanically vascular SMC from different cultures in distinct passages and confocal microscopy/western blotting, to evaluate cytoskeleton and extracellular matrix proteins. We found that vascular SMC rigidity or viscoelastic complex modulus (G) decreases with progression of passages. A statistically significant negative correlation between G and passage was found in four of our five cultures studied. Phalloidin-stained SMC from higher passages exhibited lower mean signal intensity per cell (confocal microscopy) and quantitative western blotting analysis showed a decrease in collagen I content throughout passages. We concluded that vascular SMC progressively lose their stiffness with serial culture passaging. Thus, limiting the number of passages is essential for any experiment measuring viscoelastic properties of SMC in culture

Cell volume change through water efflux impacts cell stiffness and stem cell fate

Cells alter their mechanical properties in response to their local microenvironment; this plays a role in determining cell function and can even influence stem cell fate. Here, we identify a robust and unified relationship between cell stiffness and cell volume. As a cell spreads on a substrate, its volume decreases, while its stiffness concomitantly increases. We find that both cortical and cytoplasmic cell stiffness scale with volume for numerous perturbations, including varying substrate stiffness, cell spread area, and external osmotic pressure. The reduction of cell volume is a result of water efflux, which leads to a corresponding increase in intracellular molecular crowding. Furthermore, we find that changes in cell volume, and hence stiffness, alter stem-cell differentiation, regardless of the method by which these are induced. These observations reveal a surprising, previously unidentified relationship between cell stiffness and cell volume that strongly influences cell biology