A Molecular Approach to Promote Wound Healing in the Sulfur Mustard–exposed Human Keratinocyte Model

Endogenous nitric oxide (NO) produced by three major NO synthases (NOSs) is a pleiotrophic signaling molecule in various tissue under normal and pathological conditions. In the skin, increased NO synthesized by inducible NOS (iNOS/NOS2), a NOS isozyme, has been considered to have pivotal roles in the inflammatory response to heat, infection and wound healing. It has also been shown that iNOS regulates the expression of several biomarkers, including Regulated upon Activation Normal T-cell Expressed and Secreted (RANTES) and vascular endothelial growth factor (VEGF), to exert its effect in accelerating normal skin wound healing. However, there is conflicting evidence as to the roles of iNOS in the wound healing. The effects of sulfur mustard (SM), a chemical warfare agent, on the skin include formation of blisters and slow-healing skin injuries due to delayed reepithelialization. These are the major therapeutic targets of SM-caused skin damage. Re-epithelialization of the skin following SM injury is an important but challenging goal in treatment in order to protect the slow healing wound from infection. The effects of SM on the expression of NOSs, VEGF and RANTES for wound healing were studied in an in vitro human skin model using normal human epidermal keratinocytes (NHEK). The cells were exposed to 1–20 μM SM immediately after scraping the monolayer of cells to wound them. SM delayed the healing of the wounded area, which, in an untreated (no SM exposure) cell layer, was closed (healed) through migration and proliferation of the cells by 48 h after scraping. The expression of iNOS, RANTES, and VEGF was monitored by Western blotting and real-time RT-PCR. The levels of both protein and mRNA expression of iNOS and RANTES peaked at 7 h after wounding. Exposure to 20 μM SM abolished the enhanced expression at both the protein and mRNA levels. There were no significant changes in VEGF protein or mRNA expression levels. Fluorescence microscopy also showed strong iNOS expression 7 h after wounding in the NHEK, and SM substantially inhibited iNOS expression. Taken together, these results suggest that iNOS has an important role in skin wound healing, and the prevention of SM-induced alterations in iNOS may be a potential treatment for SM skin injuries.