Altered Mechanics of Schlemm’s Canal Endothelial Cells and Trabecular Meshwork in GlaucomaPublic Deposited
Glaucoma is a neurodegenerative blinding disease associated with increased intraocular pressure, which is caused by an increased resistance to the outflow of aqueous humor. Although the cause for increased resistance remains unknown, it has been associated with a decreased density of pores in the cells of the inner wall endothelium of Schlemm's canal (SC). These pores are thought to form when SC cells experience large deformations in response to a transcellular pressure gradient.00- Previous studies established a correlation between the elevated cell stiffness in glaucomatous SC cells and their reduced pore formation capability in-vitro. Here, we extend these studies by (i) mechanically characterizing the SC cell and it substrate properties in-situ and (ii) further characterizing the cyto-mechanical behavior of these cells in vitro, particularly as to how they are affect by changes to their cytoskeleton. In the first of these studies, we combined atomic force microscopy (AFM) and optical microscopy to establish a new technique to measure the stiffness of SC cells and their underlying substrate, the trabecular meshwork (TM), in-situ. To interpret AFM measurements, finite element modeling (FEM) is used to simulate AFM indentation on a cell with cortex that is sitting on the TM. Our results show that both SC cell stiffness and that of their substrate can be measured simultaneously in-situ. Our in-situ measurements and computational simulations confirm previous in-vitro findings suggesting that the stiffness is elevated in TM in-situ but further show that SC cells stiffness in-situ is increased in glaucoma, as was already reported in-vitro. To better understand the cytomechanics of the SC cells, we used agents that altered their cytoskeleton. Dexamethasone is an anti-inflammatory glucocorticoid that can cause steroid-induced glaucoma in some individuals and has been reported to alter the cytoskeleton. We focused on investigating the effect of dexamethasone on SC cell cytoskeleton and mechanics using imaging, AFM, optical magnetic twisting cytometry (OMTC), and traction force microscopy (TFM) studies. Using FEM we have previously shown that AFM sharp probes measure the mechanics of the cell cortex whereas rounded probes mainly characterize the subcortical stiffness of the cell; here we show that OMTC measurements behave similar to AFM rounded probes and thus, probe the mechanics of the subcortical region as opposed to some studies that suggest OMTC characterizes cortex mechanics. Our studies show that dexamethasone treatment alters cytoskeletal distribution and significantly increases cortical stiffness in SC cells, which can potentially impede pore formation. Finally, to further understand the role of F-actin and vimentin in cell mechanics, the same experimental approaches were used to show that promoting RhoA and α-actinin increases cortex stiffness and traction forces in SC cells and that vimentin plays an integral role in regulating cortex mechanics and traction forces in mouse embryonic fibroblasts. These findings improve our understanding from the pathogenesis of glaucoma and open a new venue for developing new therapeutics for the disease by targeting SC cell mechanics.
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