Lee A, Derricks K, Minns M, Ji S, Chi C, Nugent MA, Trinkaus-Randall V. Hypoxia-induced changes in Ca 2ϩ mobilization and protein phosphorylation implicated in impaired wound healing. Am J Physiol Cell Physiol 306: C972-C985, 2014. First published March 26, 2014; doi:10.1152/ajpcell.00110.2013.-The process of wound healing must be tightly regulated to achieve successful restoration of injured tissue. Previously, we demonstrated that when corneal epithelium is injured, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca 2ϩ wave from the origin of the wound to neighboring cells. In the present study we sought to determine how the communication between epithelial cells in the presence or absence of neuronal wound media is affected by hypoxia. A signal-sorting algorithm was developed to determine the dynamics of Ca 2ϩ signaling between neuronal and epithelial cells. The cross talk between activated corneal epithelial cells in response to neuronal wound media demonstrated that injury-induced Ca 2ϩ dynamic patterns were altered in response to decreased O 2 levels. These alterations were associated with an overall decrease in ATP and changes in purinergic receptor-mediated Ca 2ϩ mobilization and localization of N-methyl-D-aspartate receptors. In addition, we used the cornea in an organ culture wound model to examine how hypoxia impedes reepithelialization after injury. There was a change in the recruitment of paxillin to the cell membrane and deposition of fibronectin along the basal lamina, both factors in cell migration. Our results provide evidence that complex Ca 2ϩ -mediated signaling occurs between sensory neurons and epithelial cells after injury and is critical to wound healing. Information revealed by these studies will contribute to an enhanced understanding of wound repair under compromised conditions and provide insight into ways to effectively stimulate proper epithelial repair. hypoxia; wound healing; imaging; cell communication DAMAGE TO TISSUE activates intricate underlying mechanisms that mediate the healing process. Communication between cells is generated immediately after injury and continues during the migration phase and the later phases of proliferation and extracellular matrix reassembly (13,14,23). This reepithelialization requires the precise control of glutamatergic and purinergic signaling pathways. Injured cells release nucleotides that serve as ligands for cell-surface purinergic receptors, and injured neurons release components, including ATP and glutamate, that bind their respective receptors. The release of nucleotides can modulate Ca 2ϩ homeostasis (4,11,43,52,54).Neuronal transmitters such as glutamate bind to metabotropic G protein-coupled receptors or to ionotropic receptors, including 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptors. It has been reported that epithelial cells do not respond to AMPA or kainate, but they do respond to glutamate in neuronal wound media and to NM...

Vascular disease and its associated complications are the number one cause of death in the Western world. Both extracellular matrix stiffening and dysfunctional endothelial cells contribute to vascular disease. We examined endothelial cell calcium signaling in response to VEGF as a function of extracellular matrix stiffness. We developed a new analytical tool to analyze both population based and individual cell responses. Endothelial cells on soft substrates, 4 kPa, were the most responsive to VEGF, whereas cells on the 125 kPa substrates exhibited an attenuated response. Magnitude of activation, not the quantity of cells responding or the number of local maximums each cell experienced distinguished the responses. Individual cell analysis, across all treatments, identified two unique cell clusters. One cluster, containing most of the cells, exhibited minimal or slow calcium release. The remaining cell cluster had a rapid, high magnitude VEGF activation that ultimately defined the population based average calcium response. Interestingly, at low doses of VEGF, the high responding cell cluster contained smaller cells on average, suggesting that cell shape and size may be indicative of VEGF-sensitive endothelial cells. This study provides a new analytical tool to quantitatively analyze individual cell signaling response kinetics, that we have used to help uncover outcomes that are hidden within the average. The ability to selectively identify highly VEGF responsive cells within a population may lead to a better understanding of the specific phenotypic characteristics that define cell responsiveness, which could provide new insight for the development of targeted anti- and pro-angiogenic therapies.

Extracellular matrix remodeling is a continuous process that is critical to maintaining tissue homeostasis, and alterations in this process have been implicated in chronic diseases such as atherosclerosis, lung fibrosis, and emphysema. Collagen and elastin are subject to ascorbate-dependent hydroxylation. While this post-translational modification in collagen is critical for function, the role of hydroxylation of elastin is not well understood. A number of studies have indicated that ascorbate leads to reduced elastin synthesis. However, these studies were limited to analysis of cells grown under traditional 2D tissue culture conditions. To investigate this process we evaluated elastin and collagen synthesis in primary rat neonatal pulmonary fibroblasts in response to ascorbate treatment in traditional 2D culture and within 3D cross-linked gelatin matrices (Gelfoam). We observed little change in elastin or collagen biosynthesis in standard 2D cultures treated with ascorbate, yet observed a dramatic increase in elastin protein and mRNA levels in response to ascorbate in 3D cell-Gelfoam constructs. These data suggest that the cell-ECM architecture dictates pulmonary cell response to ascorbate, and that approaches aimed toward stimulating ECM repair or engineering functional cell-derived matrices should consider all aspects of the cellular environment.