Abstract
Purpose :
Epithelial wound healing is essential to repair the barrier function of the cornea after injury. We found that Pannexin1 (Panx1), a channel that moves ATP into the extracellular space, is localized along cell borders in unwounded tissue and becomes concentrated near the wound margin. We propose that the region of intense localization of Panx1 is where Ca2+ mobilization occurs and hypothesize that the interaction of Panx1 and P2X7 is important in corneal wound healing. Our goal is to determine if the interaction is altered in moderately obese NONcNZO10/LtJ mice that model type 2 diabetes in humans.
Methods :
Epithelial debridement wounds were performed on corneas from C57BL/6J and from NONcNZO10/LtJ mice. Immunohistochemical assays were performed before and after injury and imaged using confocal microscopy. Corneas were incubated in the presence or absence of inhibitors against Panx1 and P2X7 to examine Ca2+ mobilization and communication between cells. Proximity ligation assays (PLA) were performed to determine the interaction of the proteins before and after injury. Analyses were performed using ImageJ and CellProfiler.
Results :
Panx1 was present along cell borders in unwounded tissue in both models with more diffuse staining in NONcNZO10/LtJ mice. An epithelial abrasion induced a striking difference in the localization of Panx1 and there was intense staining near the leading edge where Ca2+ mobilizations were detected. However, Panx1 was not intense in the epithelium of NONcNZO10/LtJ female and male mice near the wound. Inhibition caused a change in cell shape distal to the wound. It also attenuated calcium mobilizations near the leading edge that led to a fold reduction in cell-cell communication. PLA revealed a reduction in the interaction between P2X7 and Panx1 in epithelium of NONcNZ010/Ltj mice.
Conclusions :
Injury induces a change in localization of Panx1 that is not detected in the epithelium from the moderately obese type 2 diabetic mice suggesting that the protein is dysregulated. The decreased interaction of the two channel proteins suggests that both are critical for the migration of epithelium and suggest that the decrease in cell-cell communication depends on this interaction. The results demonstrate that Panx1 is a critical component to the healing response, and suppression is associated with cell-cell communication and migration.
This is a 2021 ARVO Annual Meeting abstract.