Hypoxia impairs wound healing by inducing changes in calcium mobilization and adhesion proteins

Abstract

To achieve successful restoration of injured tissue, wound healing processes must be tightly regulated. Previously, we demonstrated that when injury to corneal epithelium occurs, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca2+ wave from the origin of the wound to neighboring cells. Ca2+ coordinates early wound repair mechanisms important for cell migration and re-adhesion to the basement membrane. The communication between innervating neurons and epithelial cells appears to be mediated by Ca2+ mobilization post-injury through signaling between purinergic and N-methyl-D-aspartate (NMDA) receptors. Using the cornea as a wound model, we examined how a pathological condition such as hypoxia impedes reepithelialization after injury. We hypothesized that hypoxia causes delayed wound closure by inducing changes in early cellular responses after injury such as Ca2+ mobilization, eventually leading to changes in the regeneration of injured tissue. We used both in vitro and ex vivo models including primary neuronal cultures, epithelial cultures and organ cultures. A signal-sorting algorithm was developed to determine dynamics of Ca2+ signaling between neuronal and epithelial cells post-injury. The location and crosstalk between activated cells in response to neuronal wound media under normoxic and hypoxic conditions were determined and injury-induced Ca2+ dynamic patterns changed in response to decreased oxygen levels. Alterations in Ca2+ dynamics were associated with an overall decrease in ATP, changes in purinergic receptor-mediated Ca2+ mobilization and localization of NMDA receptors. There was a change in the activation of paxillin and deposition of fibronectin along the basal lamina, both factors involved in cell migration. Furthermore, we observed changes in the extracellular matrix proteins in the stroma including collagens and proteoglycans. Our results indicate that hypoxia induces changes in nucleotide/glutamate-induced Ca2+ mobilization that ultimately attenuates cell-cell communication and wound closure.