The EGF Motif With CXDXXXXYXCXC Sequence Suppresses Fibrosis in a Mouse Skin Wound Model

Fibrosis is an essential process for wound healing, but excessive fibrosis, such as keloids and hypertrophic scars, can cause cosmetic and functional problems. These lesions are caused by abnormal deposition and shrinkage of collagen fibers. The light chain of FIX, a plasma protein essential for hemostasis, has the amino acid sequence CXDXXXXYXCXC in the EGF domain. Peptides containing this sequence inhibited stromal growth in a mouse transplant tumor model. In this study, the effect of the FIX light chain on wound healing was studied. A full-layer wound was made on the back of each mouse, and cDNA encoding the light chain of mouse FIX (F9-LC) in an expression vector was injected locally once each week using a non-viral vector. Histochemical analysis of the wound was then performed to assess the effects on wound healing. Moreover, the effect of F9-LC on fibroblasts was studied in vitro. Macroscopic observation showed that wounds with forced expression of F9-LC appeared flatter and had fewer wrinkles than control wounds. Tissue collagen staining and immunostaining revealed that administration of F9-LC suppressed collagen 1 and 3 deposition and decreased α-smooth muscle actin expression. Electron microscopy revealed sparse and disorganized collagen fibers in the F9-LC-treated mice. In experiments using fibroblasts, addition of a recombinant protein of the FIX light chain disrupted the typical spindle shape and alignment of fibroblasts. F9-LC is a new candidate for use in treatments to regulate excessive fibrosis and contraction in wound healing.