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David Krizaj, Trine Lisberg Toft-Bertelsen, Aruna gorusupudi, Nanna Macaulay, Paul S Bernstein, Monika Lakk; Cholesterol regulates TRPV4-dependent signaling in the trabecular meshwork. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3424.
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Cholesterol is a primary determinant of membrane fluidity, compressibility and elastic modulus whereas cholesterol -rich domains serve as focal points for the coupling between the membrane and the cytoskeleton. Given that high serum cholesterol increases the risk for open angle glaucoma whereas statin drugs were suggested to inhibit actin polymerization in the trabecular meshwork (TM) and lower IOP, we tested the hypothesis that cholesterol controls the tensile homeostasis within the TM by modulating activation of stretch-activated TRPV4 channels and focal adhesions.
Primary human TM cells were stimulated with biaxial strains (6-10%) and/or exposed to TRPV4 agonists & antagonists, Rho modulators or simvastatin. Lipid rafts were tracked with filipin-1, actin with fluorescent phalloidin and cytoskeletal/focal adhesion transcripts & proteins with qRT-PCR, immunoblots and immunohistochemistry. [Ca2+]TM measured in in cells exposed to m-b-cyclodextrin (MbCD) and cholesterol/ MbCD supplementation (1:10). Membrane cholesterol was measured by DSQ-II GC-MS and c modulation of TRPV4-mediated currents was assessed in heterologously expressing X. laevis oocytes.
Mechanical stretch induced time- and TRPV4-dependent changes in membrane cholesterol and phosphatidylcholine levels. Cholesterol depletion dose-dependently reduced the lipid raft content and facilitated GSK1016790A-, stretch- and swelling-induced [Ca2+]i increases. The effects of MbCD on stress fiber formation were modulated by tensile stretch and TRPV4 blockers whereas extraneous cholesterol/MbCD reversed the effects of MbCD on TRPV4- and force-induced calcium and cytoskeletal signals. Cholesterol depletion interfered with aquaporin-dependent facilitation of TRPV4 currents in the presence of osmotic stress whereas statins reduced filipin fluorescence, phalloidin staining and TRPV4 -mediated calcium signals.
These results suggest that cholesterol regulates TM tensile homeostasis via mechanosensitive channels, focal adhesions and cytoskeletal proteins. Effects on actin polymerization and crosslinking were amplified by osmotic gradients and tensile forces, in part by through altered gating of the TRPV4 channel and interactions with actin, auxiliary proteins (aquaporins and focal anchoring/adapter proteins). Thus, the elasticity of the plasma membrane could play a role in the tensile homeostasis of the trabecular meshwork.
This is a 2020 ARVO Annual Meeting abstract.
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