Recombinant nAG (a salamander-derived protein) decreases the formation of hypertrophic scarring in the rabbit ear model

Abstract

nAG (newt-Anterrior Gradient) protein is the key mediator of regrowth of amputated limbs in salamanders. In a previous work in our lab, a new nAG gene (suitable for humans) was designed and cloned. The cloned vector was transfected into primary human fibroblasts. The expression of nAG in human primary fibroblasts was found to suppress collagen expression. The current study shows that local injection of recombinant nAG reduces scar hypertrophy in the rabbit ear model. This is associated with lower scar elevation index (SEI), lower levels of collagen I & III, higher levels of MMP1, and a higher degree of scar maturation in experimental wounds compared to controls.

Figure 1

The degree of scar hypertrophy is reflected by SEI. After treatment of wounds with recombinant nAG for 13 days after complete epithelialization, SEI was measured for treated and untreated wounds. There was significant decrease in SEI in treated wounds: 1.168 ± 0.063 compared to the untreated wounds 1.35 ± 0.07 (P = 0.003).

Figure 2

Masson trichrome staining. (a) Collagen fibers in control wounds are thick and disorganized (magnification 40x). (b) Collagen fibers in experimental wounds are thin and organized parallel to each other (magnification 40x).

Figure 3

Quantitative real-time PCR measuring relative mRNA expression level of collagen I, collagen III, and MMP-1 in nAG treated wounds and untreated wounds. Total RNA was reverse-transcribed and target genes expression was measured in multiplex, one-step RT-PCR by using TaqMan probes with FAM and HEX reporter dyes and BHQ1 quencher. Relative mRNA expression was related to the reference gene, GAPDH. The relative expression of collagen I was decreased by 95% (P < 0.05), collagen III expression was decreased by 48% (P < 0.05), and MMP-1 expression was increased by 27% (P > 0.05) in nAG treated wounds compared to untreated wounds.