The Galvanotactic Migration Of Keratinocytes Is Enhanced By Hypoxic Preconditioning

Background:To migrate effectively to heal a wound, keratinocytes must sense not only when to migrate but also the required direction. Endogenous electric fields(EFs) have been proposed as a directional cue guiding the migration of keratinocytes in wound healing. Endogenous EFs are generated instantaneously after an injury due to the collapse of the trans-epithelial potentials, with the wound center being more negative than the surrounding tissue and thus acting as the cathode of the endogenous EF until wound re-epithelialization is complete. Endogenous EFs measure between 42 and 100 mV/mm and are regulated spatially and temporally. In vitro, many cell types respond to applied EFs at the strength equivalent to those measured in vivo by directional migration, a phenomenon termed galvanotaxis or electrotaxis. Keratinocytes migrate toward the cathode in an applied EF in vitro, which is the same direction as migration occurs under an endogenous EF. Significantly, recent studies found that EFs could override other cues in guiding cell migration during epithelial wound healing, indicating that the role for EFs in wound healing is far more important than previously thought.The wound microenvironment is, however, complex. Keratinocyte migration into wounds may therefore involve the integration of several relevant factors that co-exist in the wound. Although a number of membrane-bound and cytosolic proteins have been identified as being involved in the intracellular signaling that underpins keratinocyte galvanotaxis, whether or how the mechanisms of galvanotaxis could be modulated by other factors in the wound microenvironment is mainly unknown. Hypoxia is a microenvironmental stress that occurs immediately after injury, likely due to the vascular disruption and increased oxygen consumption by cells at the wound’s edge. The oxygen tension is approximately 0-10 mmHg(0-1.3% O2) in the wound center and shows an increased gradient toward the wound periphery, where it is approximately 60 mmHg(7.9% O2). As keratinocytes do not migrate immediately but a few hours post-wounding, hypoxia may act as an initial stimulant for the migration of keratinocytes in a wound. In fact, hypoxia has been shown to increase the motility of both keratinocytes and fibroblasts in vitro and may play a beneficial role in wound re-epithelialization in vivo. Wound re-epithelialization depends on the movement and, more importantly, the migratory direction of keratinocytes. However, the question of whether hypoxia is functional in terms of keratinocyte directionality when migrating over the wound site has not been addressed. Numerous studies have described the EF-guided directional migration of keratinocytes in vitro. Those studies, however, were all performed under normoxia, ignoring the wound-induced hypoxia that keratinocytes encounter prior to migration in vivo.Objective:We wonder whether hypoxic preconditioning is functional in terms of keratinocyte galvanotactic migration and the mechnasim involved in.Methods and Results:First, we established the experiment model for the study of keratinocytes galvanotactic migration, and then explored the characteristics of keratinocyte galvanotaxis under normoxic conditions. We found that the cathodal migration of keratinocytes was induced by EFs in a voltage- and time- dependent manner and the threshold voltage for keratinocyte galvanotaxis was between 25 and 50 mV/mm. Second, we examined the effects of various oxygen tensions and durations of hypoxic preconditioning on the EF-guided migration of keratinocytes and the threshold voltage of galvanotaxis. We showed that the galvanotactic migration of keratinocytes was promroted by hypoxia preconditioning, and the promoting effects were both oxygen tension- and preconditioning time-dependent, with the maximum effects achieved using 2% O2 preconditioning for 6 hours, while 12 hours of 2% O2 treatment inhibited the galvanotactic migration of keratinocyte, which may be the result of decreased activity of keratinocyte following 12 hours of 2% O2 treatment. We also showed that hypoxic preconditioning(2% O2, 6 hours) decreased the threshold voltage of keratinocytes galvanotaxis to < 25 mV/mm, whereas this value was between 25 and 50 mV/mm in the normal culture control. Third, we monitored the monolayer wound healing and migration of keratinocytes in a scratch-wound monolayer assay to test the physiological significance of hypoxia in keratinocyte galvanotaxis during wound healing. We showed that hypoxic preconditioning accelerated EF-guided wound healing by 1.38-fold and promoted the EF-guided migration of keratinocytes. Forth, we examined whether ROS were involved in hypoxic preconditioning-promoted keratinocyte galvanotaxis. We showed that ROS were essential for keratinocyte migration directionality rather than motility when guided by EF, and hypoxic preconditioning promoted keratinocyte galvanotaxis through the induction of ROS.Conclusion:Our results demonstrate that hypoxic preconditioning promotes keratinocyte galvanotactic migration through the induction of ROS, and the promoting effects are both oxygen tensionand preconditioning time-dependent, with the maximum effects achieved using 2% O2 preconditioning for 6 hours. Furthermore, hypoxic preconditioning accelerated the healing process in a scratch-wound monolayer assay. These results provide novel insights into our understanding of the importance of hypoxia in wound re-epithelialization. Clinically, enhancing the galvanotactic response of cells might be an attractive approach for improved wound healing.