The Role of Sustained Calcium Oscillations in the Wound Healing Response

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PROJECT ABSTRACT The cornea is the most anterior structure of the eye, which makes it susceptible to injury from scratching, infections, and dystrophy. In fact, corneal blindness from injury and disease is the fourth highest cause of preventable blindness globally. The only curative treatment for this condition is corneal transplantation or grafting, but viable eye tissues are not readily available worldwide. Therefore, studying the mechanism of normal wound healing in the cornea could give us information on development of potential novel therapeutic targets. Upon injury, corneal epithelial cells release nucleotides (e.g. ATP) that activate purinergic receptors P2Y2 and P2X7; P2Y2 is a G-protein coupled receptor, while P2X7 is a ligand-gated ion channel. Both of these receptors induce a transient calcium wave that has been established to be a critical step in the wound healing process. However, little is known about the long-term role of calcium signaling in wound repair. Given that the expression and localization of P2X7 and P2Y2 change after the initial calcium wave, we speculated that these receptors may also be involved in the later stages of the wound healing process. We have now identified a long-term role of calcium propagation that when inhibited attenuates the healing process. This secondary response, which occurs 10-30 minutes after the primary calcium wave is induced, consists of a series of calcium oscillations that propagate within multiple clusters of cells, and it was seen to last for several hours. Deactivating extracellular nucleotides with ectonucleotidases inhibited the secondary response, indicating that P2Y2 and P2X7 are required in this process. Unlike the primary response, the secondary calcium response depended on cell-cell contact and thus was inhibited by sub-confluent conditions and gap junction inhibitors. These results demonstrate that the calcium response after wounding is more complex than that of either receptor alone. The goal of my project is to investigate how the purinergic receptors P2X7 and P2Y2 coordinate and produce the secondary calcium response and how that response contributes to the wound healing process. Therefore, I hypothesize that purinergic receptors P2X7 and P2Y2 differentially mediate the secondary calcium response and contribute to multiple processes in wound repair.