TRPV4 contributes to stiffness-induced upregulation of YAP in the Trabecular Meshwork

Abstract

Purpose : In primary open-angle glaucoma (POAG) the trabecular meshwork (TM) takes on a myofibroblast-like phenotype resulting in increased TM stiffness and increased expression of extracellular matrix proteins. The purpose of this study was to establish whether the calcium-permeable stretch-activated ion channel TRPV4 (transient receptor potential vanilloid isoform 4) contributes to sensing cytoskeletal strain and substrate stiffness in human TM cells and to establish a methodology to study TRPV4 and other mechanosensitive ion channels in primary cultured murine TM cells.

Methods : Human TM cells (hTM) were dissected from human post-mortem donors and cultured on polyacrylamide hydrogels set at stiffnesses of 4kPa and 100kPa in serum free media alone or in the presence of TRPV4 antagonist HC-067047 for 24 hours. The cells were fixed and stained using antibodies against fibroblast markers alpha smooth muscle actin (α-SMA) and fibronectin, and the Hippo pathway transcriptional effector Yes-associated protein (YAP). Primary mouse TM cells (mTM) from wild-type C57BL/6 mice were isolated using magnetic microbeads. Ca²⁺-imaging was used to measure the response of mTM cells to ion channel agonists and fluid shear flow stimuli.

Results : The expression of fibroblast markers α-SMA, Fibronectin, and YAP transcriptional activation was increased on hTM cells grown on 100kPa hydrogels compared to 4kPa gels. Inhibition of TRPV4 through co-culture with HC-06 did not affect α-SMA and Fibronectin expression on 100kPa gels, but partially rescued stiffness-induced upregulation of YAP activation. Primary cultured mTM cells exhibited Ca²⁺-influx in response to TRPV4 agonist GSK-1016790A and fluid shear-flow.

Conclusions : TRPV4 contributes to transduction of mechanical stress impelled by substrate stiffness. This involves, in part, the fibrogenic Hippo pathway that links membrane mechanoactivation with transcription. This finding supports the idea that mechanosensitive channels contribute POAG-related remodeling of the TM. mTM cells respond to mechanosensitive stimuli similarly to hTM cells and thus represent a viable in-vitro model to investigate biomechanical signaling in transgenic mouse models.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.