Research by scientists based at the University of California, Los Angeles, has found that compounds that target the circadian clock and affect the synthesis of collagen—a protein that is important for skin repair—could improve scar healing. The team carried out a screen to identify FDA-approved compounds that would suppress the expression of the gene neuronal PAS domain 2 (Npas2)—a core circadian clock gene that is expressed in dermal fibroblasts and has been shown to play a critical role in regulating collagen synthesis. Two of the compounds identified could, the researchers suggested, represent potential therapeutic candidates in future skin wound healing studies.

“Our aim was to find compounds that were able to increase the rate at which dermal wounds heal while mitigating the formation of hypertrophic scars,” said Akishige Hokugo, PhD, corresponding author of a study published in Frontiers in Medicine. “Scars can result in emotional distress following normal wound healing by serving as permanent reminders of the initial incident. Accounting for additional revision procedures, extended hospital stays, and increased incidences of infection following surgical operations, hypertrophic scars cause a quantifiable burden to healthcare institutions.” The researchers’ paper is titled, “In vitro assessment of neuronal PAS domain 2 mitigating compounds for scarless wound healing.” In their report they concluded, “This work has direct implications in the healing of surgical wounds, specifically following surgery, as the ability to prevent the formation of HTS will enhance both clinical success and patient experience.”

Skin wound healing following injury, surgical procedure, burn, or from systemic disease remains a common clinical problem that requires reliable wound management, the authors wrote. “The goal of wound management is to fully restore the protective function of the skin as quickly as possible and to optimize appearance.” Skin wound healing often leaves a scar. But the role of the scar itself in healing is often underestimated. A scar that doesn’t heal cleanly can be painful or upsetting or affect the range of movement of the affected body part. It may even require further surgical treatment. Normal wound healing involves three stages: the inflammatory phase, the proliferative phase, and the maturation phase. During the first two, different kinds of cells migrate toward the wound. First, cells that protect against infection, and then cells that lower inflammation and help the skin rebuild itself. During this second phase, cells migrate to refill the wound and collagen is generated to provide structure. But excess collagen deposition leads to scars that are thicker, raised, and less elastic than the surrounding skin.

Wounds that are received at different points in the circadian cycle heal at different rates, which is thought to be due to the links between circadian clock genes and the behavior of cells implicated in healing. “Recent studies have also established the relationship between circadian rhythm changes and the rate at which dermal wounds heal,” the investigators noted. “The role of specific cell types, including dermal fibroblasts, macrophages, keratinocytes, and subcutaneous adipocytes, all exhibit varying amounts in response to circadian gene activity levels.”  If the right cell types don’t reach a wound at the right time, healing is compromised. “Previous studies have shown that skin wounds in mice wounded during the circadian rest period healed less quickly than those wounded during the active period, and human burn injuries incurred during the night healed more slowly than wounds acquired during the day,” the authors wrote. “This phenomenon is believed to be due to the strong correlation between circadian cycles and activity of different immunological cell types.”

Npas2 is a core circadian gene, which means that it helps to regulate the natural rhythms of the body. And mice that are genetically modified so the Npas2 gene doesn’t express itself heal faster, with increased cell migration and less excessive collagen deposition. “In our previous study, we reported that mice lacking the Npas2 gene (Npas2 -/-) exhibited accelerated dermal wound healing mechanisms in comparison to those with fully functional circadian rhythm genes and showed increased cellular migration and contraction in vitro,” the investigators noted. “Previous experiments conducted in this area have shown that Npas2 knockout mice are able to produce significant decreases in total wound healing time, yet the lack of circadian control throughout all body systems in these subjects is not transferrable to clinical implementation,” said Hokugo.

For their newly reported work, the scientists aimed to change the expression of Npas2. “We have performed high throughput drug screening to identify genes responsible for downregulation of Npas2 while maintaining cell viability,” they explained. “From this, five FDA-approved hit compounds were shown to suppress Npas2 expression in fibroblasts.”

They named the five compounds Dwn1, Dwn2, Dwn3, Dwn4, and Dwn5, and each compound was screened using mouse fibroblasts to show that they did suppress Npas2 expression, before being applied to samples of scratched adult human dermal fibroblasts. Each different repeat of the experiment was conducted over 14 days, to monitor collagen development as it would happen for a real wound.

These results showed that while Dwn3, Dwn4, and Dwn5 had no apparent effect on collagen synthesis, Dwn1 and Dwn2 successfully modulated both cell migration and collagen synthesis without damaging the fibroblasts, improving the speed of cell migration and minimizing excess collagen synthesis. “Dwn1 and Dwn2 were found to significantly affect collagen synthesis and cell migration without any cytotoxicity,” the team stated.

The authors cautioned that more studies are needed that explain how the compounds work to speed up healing, test whether they work in patients, and determine the appropriate doses. But if the results can be translated into medicines, they offer promise for cleaner healing and less scarring.  “… although there is a few evidence in the current literature of Npas2-specific control of dermal wound healing, there is an extensive publication about the way elements of the circadian system interact with skin physiology to impact wound healing,” the scientists stated. “… this study puts forth a novel mechanism by which the circadian system is able to impact cutaneous wounds in order to accelerate dermal wound healing while focusing on the mitigation of excess collagen deposition … The results of this study suggest that Dwn1 and Dwn2 could be novel therapeutic agents capable of promoting collagen homeostasis and accelerating wound healing with minimal hypertrophic scarring.”

Hokugo added: “We hope that this work serves as a foundation for future investigations into how collagen expression may affect dermal wounds and impact the efficiency of native wound healing processes in postoperative patients.” The authors further suggested that further studies could be used to identify the specific pathways used by Dwn1 and Dwn2 to alter gene activity, “thereby providing insights into a possible cascade able to be utilized for more extensive wound healing studies.”

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