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Samuel Herberg, Haiyan Li, Tyler Bague, Alexander Kirschner, Daniel Stamer, Preethi S Ganapathy; Investigation of 3D Mechanotransduction using a Trabecular Meshwork Hydrogel. Invest. Ophthalmol. Vis. Sci. 2020;61(7):3454.
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© ARVO (1962-2015); The Authors (2016-present)
Abnormal trabecular meshwork (TM) cell function and extracellular matrix (ECM) remodeling contribute to glaucomatous TM stiffening. TM cell monolayers on stiffer polymer films (~100 µm thick) showed increased activation of the mechanotransducers YAP/TAZ in vitro. A gap in our understanding of TM stiffening is due to the reliance on 2D cell culture systems, although cells in 3D environments can show altered, even opposite, behavior vs. 2D. Therefore, we developed a TM cell-containing hydrogel using biomimetic ECM components to model TM cell interactions with their 3D environment. Here, we investigate the effects of 3D substratum stiffness on YAP/TAZ regulation using our TM hydrogel.
Human TM cells were derived from surgical discard corneal rims. Hydrogels were engineered by mixing functionalized collagen type I, elastin-like polypeptide, and hyaluronic acid in presence of photoinitiator followed by UV crosslinking (320-500 nm, 2.2 W/cm2) for 1 s or 60 s. Rheology analysis was performed using an oscillatory shear-stress sweep test (0.6–10.0 Pa, 1.0 Hz, 25°C).TM cells were seeded onto the preformed hydrogels (~500 µm thick) and cultured for 7 d. Cell spreading within hydrogels was assessed by longitudinal imaging. YAP, TAZ, CTGF (downstream target), and Piezo1 (controls YAP/TAZ activity) mRNA levels were investigated by qRT-PCR. YAP subcellular localization was determined by immunostaining.
Hydrogels showed higher storage vs. loss moduli in both groups (p<0.05), suggesting principally elastic properties of the polymer networks. A 16-fold difference in 3D substratum stiffness (0.1 kPa vs. 1.6 kPa; p<0.0001) between short (1 s) and long (60 s) UV crosslinking was noted, consistent with reported increased TM stiffness in glaucomatous vs. normal eyes. TM cells in softer hydrogels (1 s) exhibited more cell spreading vs. stiffer hydrogels (60 s). Next, TM cells in stiffer hydrogels showed lower YAP (0.3-fold, p<0.001) and TAZ (0.5-fold, p<0.05) mRNA expression vs. softer hydrogels. Similar trends were noted for CTGF and Piezo1 mRNA levels (0.5-fold, p<0.05). Lastly, TM cells in stiffer hydrogels showed less YAP nuclear localization/activity (p<0.001) vs. softer hydrogels.
Our data are contrary to previous 2D reports, and suggest that the interplay of culture dimensionality and substratum stiffness regulates TM cell YAP/TAZ activity in ways that conventional 2D systems cannot accurately model.
This is a 2020 ARVO Annual Meeting abstract.
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