Prevention of Hypertrophic Scarring by Compounds Modulating Circadian Gene Npas2
Yvonne Roca1, Yoichiro Shibuya2, Adam Clements2, Ichiro Nishimura3, Akishige Hokugo2, Reza Jarrahy2 1 David Geffen School of Medicine at UCLA 2 Regenerative Bioengineering and Repair Laboratory, Division of Plastic and Reconstructive Surgery, Department of Surgery, David Geffen School of Medicine at UCLA 3 Weintraub Center for Reconstructive Biotechnology, UCLA School of Dentistry
Background: Abnormal collagen biosynthesis and deposition can result in hypertrophic scar (HTS) formation. HTS demonstrate increased collagen biosynthesis compared to normal scar (Craig et al., 1975) and over-expression of collagen I and III (Wong et al., 2014). The circadian rhythm helps to maintain physiological homeostasis and disruption of the circadian rhythm can lead to impaired wound healing (Sapahi et al., 2014). Neuronal PAS domain 2 (Npas2), a core circadian clock gene, is expressed in dermal fibroblasts (Landgraf et al., 2016) and has been shown to play a critical role in regulating collagen synthesis. (Sasaki et al., 2019). Previous studies showed Npas2 knockout mice demonstrated faster dermal wound closure suggesting that Npas2 can be a novel therapeutic target for pathological scarring and wound healing. (Sasaki et al., 2019). We have performed high throughput drug screening using mouse dermal fibroblasts with Npas2-lacZ reporter system and identified 5 FDA-approved hit compounds (Dwn1, Dwn2, Dwn3, Dwn4, and Dwn5) that suppress Npas2 expression in fibroblasts. We hypothesized that the therapeutic suppression of Npas2 by hit compounds will result in collagen synthesis downregulation and reduced collagen-related gene expression.
Methods: Collagen synthesis and collagen-related gene expression in clonal and primary human fibroblasts was compared between 5 groups treated with 5 compounds at 0, 0.1, 1, and 10 ÁM. Fibroblast behaviors were characterized by quantitative RT-PCR (qPCR) for collagen-related genes and in vitro collagen accumulation was evaluated by Picrosirius red staining. The statistical analysis was performed by one-way ANOVA and Dunnett's test.
Results: In primary human adult dermal fibroblast, Dwn1 was found to simultaneously down-regulated collagen synthesis, Npas2 gene expression and collagen type I and III gene expression at 10ÁM. Dwn2 was discovered to suppress collagen synthesis in all concentrations and showed collagen type1A2 and type III suppression at 1ÁM. Interestingly, Dwn2 did not significantly decreased Npas2 gene expression. Dwn3 exhibited increased collagen synthesis at 0.1ÁM but no significant differences were observed in collagen gene expression or Npas2 gene expression. Dwn4 and Dwn5 did not show down-regulation of collagen synthesis and collagen-related gene expression. Dwn4 and Dwn5 effects on Npas2 gene expression are still pending.
Conclusion: Our study demonstrated that Npas2 suppression in human dermal fibroblasts by some compounds (Dwn1 and Dwn2) modified cell behaviors resulting in changes in collagen deposition and collagen-related gene expression. This study suggests that Dwn1, and Dwn2 may be novel therapeutic agents for promoting collagen homeostasis and accelerated wound healing with minimal hypertrophic scarring.
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