Abstract
Purpose :
After an injury, corneal epithelial cells engage in coordinated calcium signaling that precedes migration. These signals are attenuated with age and disease. Our goal is to explore the contributions of the mechanosensitive receptor TRPV4 to the wound response, with an emphasis on its interactions with P2X7. We hypothesize that inhibition of TRPV4 will significantly reduce signal initiation and propagation in the post-wound calcium signaling cascade.
Methods :
Experiments were performed using ex vivo globes from 10-week-old C57BL/6J mice and Human Corneal Limbal Epithelial (HCLE) cells. Samples were pre-incubated with Fluo-4 AM and the TRPV4 inhibitor RN-1734 or the P2X7 inhibitor A438019. Globes were pre-incubated with the membrane stain CellMask. A calcium signaling cascade was induced through scratch wounding or through application of the TRPV4 agonist GSK1016790A. Live cell imaging was performed at a rate of 1frame/3sec for 45 minutes using a Zeiss LSM 880 confocal microscope. Data was analyzed using Image J and MATLAB.
Results :
Preincubation with the TRPV4 inhibitor decreased cellular signaling events after injury in both cell culture and ex vivo models. In globes, there was decreased cell-cell signaling in both wing and basal layers. The P2X7 inhibitor decreased the clustering and coordination of cellular signaling in the epithelium of intact globes.
Conclusions :
The findings will be used to better understand the interaction between TRPV4 and P2X7 in the corneal wound healing response, and whether this interaction is affected in age and disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.