We have previously shown that topical opioids including morphine and its congeners promote healing of full thickness ischemic wounds in rats. We examined the contribution of mu opioid receptor (MOPr)–mediated healing of full thickness ischemic wounds using MOPr and delta or kappa opioid receptor knockout (KO) mice. Wound closure in the early (day 5) as well as later phases was delayed in topical morphine or PBS-treated MOPr-KO mice compared with reciprocal treatments of wounds in wild-type (WT) mice. MOPr expression was significantly upregulated at 30 min in the wound margins and colocalized with wound margins and vasculature in the epidermal and dermal layers of the skin. We next examined whether neuropeptide expression was involved in the mechanism of MOPr-mediated wound closure. Substance P (SP) and calcitonin gene-related peptide immunoreactivity (ir) was significantly increased in the skin of MOPr-KO mice as compared with WT mice. Neuropeptide-ir was increased significantly in PBS-treated wounds of MOPr and WT mice, but morphine treatment reduced neuropeptide immunoreactivity in both as compared with PBS. Wounding of keratinocytes led to the release of opioid peptide beta-endorphin (β-END) in conditioned medium, which stimulated the proliferation of endothelial cells. MOPr-selective (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, CTOP) and nonselective OPr antagonist naloxone-inhibited endothelial proliferation induced by wounded keratinocyte-conditioned medium. In addition, accelerated wound area closure in vitro by morphine was suppressed by methylnaltrexone, a nonselective OPr antagonist with high affinity for MOPr. Morphine and its congeners stimulated the proliferation of endothelial cells from WT mice but not those from MOPr-KO mice. Furthermore, morphine-induced mitogen-activated protein kinase/extracellular signal-regulated kinase phosphorylation in endothelial cells was significantly decreased in MOPr-KO mice as compared with WT mice. Collectively, these data suggest that MOPr plays a critical role in the proliferation phase with the formation of granulation tissue during wound healing.
Bibliographical noteFunding Information:
K. Gupta is a consultant for Fera Pharmaceuticals LLC and the remaining authors declare no conflicts of interest. This study was supported by NIH RO1 HL68802 and UO1 HL117664 and Institute for Engineering in Medicine grants to KG. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Author contributions are as follow: Y. Wang, M. Gupta, and S. Rao wrote the manuscript, Y. Wang, T. Poonawala, M. Farooqui, and Y. Li performed research, F. Peng analyzed data and prepared illustrations, M. Ansonoff and J. E. Pintar provided opioid receptor knockout mice, Y. Wang, M. Gupta, and K. Gupta interpreted the data, and conceived, supervised, and edited the manuscript. The authors thank Dariel J. Irizarry for help with scratch assays, Michael Franklin and Barb Benson for editorial assistance, and Georgi Walberg and Josette Fontana for help with communicating the manuscript.