Human skin comprises of epidermis, dermis, and subcutaneous tissue and protects the body from extrinsic factors. A wound is a functional or structural damage to the skin and wound healing indicates the repair of the wound area. Wounds are generally classified as chronic wounds and acute wounds. Chronic wounds include vascular ulcers, diabetic ulcers, and pressure ulcers, while acute wounds involve abrasions, cut wounds, lacerations, and burns, etc. Severe wounds (e.g., burns) often result in irreversible scarring, leading to cosmetic and functional impairments as well as secondary complications such as reduced skin strength and itching. In the case of chronic wounds and diabetic ulcers, impaired healing capacity is the primary clinical concern, not scarring. These wounds frequently fail to heal, presenting a therapeutic challenge and life-threatening risks, including progression to infection and potential limb amputation. Despite ongoing research, the development of scarless, cost-effective, and clinically viable therapies for these complex wounds remains a significant challenge. Therefore, development of novel and effective methods for wound healing is necessary.
Wound healing is a sequential and highly complex cascade process, such as inflammation, proliferation, and remodeling phases. Also it is associated with various factors such as resident skin cells, extracellular matrix, chemokines, cytokines, and growth factors. Accordingly, several techniques including skin regenerative strategies have been explored to improve scarless wound healing outcomes.
Angiogenesis plays a crucial role in skin regeneration-based wound healing as oxygen, nutrients, and bioactive substances are supplied through angiogenesis. In this respects, research on angiogenesis and wound healing has received extensive attention. VEGF is a representative angiogenic factor and known to aid in skin regeneration by promoting keratinocyte proliferation. Visfatin, a kind of adipokine, induces activation of VEGF and possess angiogenic potential. It also promotes wound healing by enhancing proliferation and mobility of keratinocytes and human dermal fibroblasts.
Computer-aided drug design (CADD), a computer simulation-based material screening technology, is fast, convenient, and efficient in deriving candidate substances. It also aids in screening protein-derived peptides that are identical or superior to existing proteins using the affinity of ligands and receptors. In our previous study, we developed two peptides with superior angiogenic efficacy than the native visfatinbased on the active site of visfatin using computer simulation techniques. Therefore, this study is aimed to investigate the skin regeneration effects of these peptides through in vitro and in vivo experiments.
Two in vitro assays were conducted to evaluate the wound-healing effects of visfatin-derived peptides (Vis-1 and Vis-2) on HaCaT cells and HDFs. The first test assessed the proliferation effect using an MTT assay, and the other measured the degree of wound closure using a cell scratch assay in HaCaT cells and HDFs. In HaCaT cells, the groups treated with 0.1, 1.0, and 10 µM Vis-1 peptide showed 106.49%, 113.78%, and 109.25% cell proliferation, respectively, a significant increase compared to the 100% of the control group reference. In HDFs, 106.44% and 106.01% cell proliferation were noted in the groups treated with 0.1 and 10 µM Vis-1 peptide, respectively, which was higher than the control group, but the change was not significant (Supplementary Fig.1). These results suggest that Vis-1 peptide has a proliferation effect on these two cells. The degree of wound closure was measured by scratching the cultured cells and calculating the changes in the wound area before and after treatment with the peptides at various concentrations (0.1, 1, and 10 µM).
Wound closure after Vis-2 peptide treatment was not significantly different compared to that of the control group in both HaCaT and HDF, whereas wound closure after Vis-1 peptide treatment was significantly different at a rate of 64.8% at 0.1 µM and 63.7% at 1 µM compared to the control group (47.1%) and positive control group (hEGF, 49.1%) in HaCaT cells. Vis-1 peptide also significantly increased wound closure rates in HDFs by 71.7% at 0.1 µM and 67.0% at 1 µM compared to control (54.8%) and positive control (hFGF, 57.1%). These results indicate that Vis-1 peptide exerts a wound healing effect by promoting cell proliferation and migration of keratinocytes and skin fibroblasts.
Thereafter, western blot analysis was performed to further investigate whether Vis-1 peptide could affect the Wnt/β-Catenin and MAPK signaling pathway in HaCaT cells and HDFs in vitro. In HaCaT cells, the expressions of β-Catenin and p-p38 were increased at 0.1 and 1 µM, and the expressions of p-JNK was increased at 1 µM concentration, respectively. In HDFs, only β-Catenin expression was significantly increased at 1 µM concentration. These results indicate that treatment with Vis-1 peptide increases the expression of β-Catenin and p-p38 at low concentrations.
Since Vis-1 peptide showed potential in promoting cell proliferation on HDFs and HaCaT cells in vitro, the effect of Vis-1 peptide on the healing of full-thickness skin wounds in vivo was investigated by using an excision wound healing test in mice. In the preliminary experiments using 0.1, 1, 10, and 20 µM of Vis-1 peptide, we observed that 1 and 20 µM were more effective concentrations in promoting wound closure than other concentrations (Supplementary Fig.2). Therefore, the following experiments were conducted with concentrations of 1 and 20 µM of Vis-1. On post-injury day 8, wounds were almost healed at a low concentration (1µM) of Vis-1 peptide, but the wounds in the control group were not closed. On post-injury day 10, the wound closure rates were at 76.9% in the control group, 87.5% in the positive control group (20 µM hEGF), 90.8% using 1µM of Vis-1 peptide, and 86.9% using 20 µM of Vis-1 peptide. In wound tissues at post-injury, the wound area of mice treated with Vis-1 peptide and hEGF was smaller than that of the control group, suggesting that 1µM of Vis-1 peptide accelerated wound healing. Furthermore, there were no adverse effects on body weight, overall health status, or behavior of the mice during the treatment with Vis-1 peptide.
Considering that Vis-1 peptide accelerated wound healing in vivo, we hypothesized that this peptide may also contribute to the promotion of angiogenesis during wound healing. The analysis of subcutaneous wound tissues collected on days 10 post-injury revealed that the wounds in subcutaneous tissues showed a marked increase in new vessel formation, as evidenced by increased vessel size and number of vessels in the Vis-1 peptide-treated groups compared to the control group. These results suggest that the Vis-1 peptide appeared to effectively promote wound healing.
