Papillary and reticular dermis show distinct extracellular matrix (ECM) and vascularization, and fibroblasts isolated from these compartments have different gene expression patterns and behaviour in vitro. However, due to lack of relevant models, the contribution of skin fibroblast sub-populations to vascularization remains unknown. We thus cultured human papillary and reticular fibroblasts as cell sheets. Differential transcriptomic analysis was performed by RNA sequencing to characterize their microenvironment. Bioinformatic analysis revealed that each fibroblast population expressed specific angiogenesis and matrisome gene expression signatures resulting in specific ECM that differed both in composition and structure. The impact of secreted and ECM-bound factors was then assessed using 3D angiogenesis assays. When co-cultivated with endothelial cells, the papillary and reticular microenvironments induced the formation of distinct capillary networks mimicking the characteristics of vasculature of native dermis subcompartments (vessel diameter and density, number of branch points). Whereas conditioned media of papillary fibroblasts displayed intrinsic high angiogenic potential, reticular ones only contributed to capillary formation induced by exogenous VEGF. These results show that skin fibroblast populations regulate angiogenesis via both secreted and ECM-bound factors. Our work emphasizes the importance of papillary and reticular fibroblasts, not only for modelling dermis microenvironment but also for its vascularization.

Gyeonggi-do, Korea (the Republic of) and 2 Dermatology, Hallym Institute for Translational Medicine, Anyang, Korea (the Republic of) Hypertrophic scar (HS) is a dermal fibroproliferative disease characterized by the overproduction and deposition of extracellular matrix, cell over-proliferation, enhanced angiogenesis, and enhanced differentiation of fibroblasts to myofibroblasts. Although there has been extensive research on botulinum toxin type A (BTX) treatment for the prevention of HS formation, its effectiveness in the attenuation of skin fibrosis and the related mechanism are unclear. In this study, we investigated the inhibitory effect of BTX on HS-derived fibroblasts (HSFs) in vitro and explored the possible associated molecular mechanism by examining cell proliferation, cell migration, protein expression of scar-related factors, and intracellular signaling. Human scar fibroblasts were cultured and stimulated with BTX. The MTS, scratch, and ELISA, and western blotting were performed to detect changes in fibroblast proliferation, migration, and protein expression of pro-fibrotic factors. Our study revealed that the proliferation, viability and migration of BTX-treated human scar fibroblasts were decreased compared with those of the untreated controls. And protein expression of pro-fibrotic factors including transforming growth factor b1, interleukin-6, and connective tissue growth factor was inhibited by BTX treatment, whereas JNK phosphorylation was activated. Blocking the JNK pathway rescued the inhibitory effects on human scar fibroblast proliferation and the production of pro-fibrotic factors. Therefore it is thought that the suppressive effects of BTX are closely associated with JNK pathway activation. This study showed that BTX has a suppressive effect on extracellular matrix production and scar-related factors in human scar fibroblasts in vitro. Moreover, regulation of JNK signaling played an important role in this process. Our results provide theoretical basis, at the cellular level, for HS treatment.

High mobility group box 1" (HMGB1) is a well-known nuclear protein that stabilizes DNA and facilitates gene transcription, but at outside the membrane, it functions as an alarmin, causing an inflammatory response in combination with other cytokines. Recently, we confirmed that HMGB1 shows the proinflammatory activity depending on the redox status, which is in the reduced, disulfide, or oxidized form. The reduced-HMGB1 exerts a chemoattractive effect, and the disulfide-HMGB1 has proinflammatory cytokine activity, but the oxidized form has no inflammatory activity. In our previous study, the proinflammatory effect of disulfide-HMGB1 was seen under the poly(I:C)-induced inflammation in keratinocyte, but reduced-HMGB1 showed the suppressive effect of poly(I:C)-induced inflammation. In addition, HMGB1 contains two homologous DNA-binding domains, the A and B-boxes. HMGB1 B-box induces strong proinflammatory activities, but HMGB1 A-box is a specific antagonist for HMGB1 and exerts an anti-inflammatory effect in macrophage. However, the direct effects of HMGB1 A-box and B-box for keratinocytes were not well elucidated. To investigate those effects, we established recombinant peptides for HMGB1 A-box and B-box. HMGB1 A and B-box alone did not induce inflammatory cytokines for keratinocyte on own. On the other hand, HMGB1 A-box had suppressive effect for IL-6 and IFN-beta expression on poly (I:C) induced inflammation in keratinocyte, but HMGB1 B-box had no inflammatory in keratinocytes. From these results, it is possible that HMGB1 A-box could become a new antiinflammatory material for some inflammatory skin diseases.

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Antiviral proteins (AVPs) including the oligoadenylate-synthase (OAS) and Interferon induced transmembrane protein (IFITM) families have protective roles within the innate immune system. However, little is known about their regulation in skin. BMAL1 and CLOCK, regulators of the circadian rhythm, have known importance in a number of immune functions. We hypothesized that the circadian clock may regulate cutaneous AVP expression. We demonstrate that murine skin displays homeostatic oscillations of AVP expression through the day, and that AVPs exhibit modest rhythmic expression in primary human keratinocytes post-circadian synchronization using serum starvation or dexamethasone shock with a periodicity of 20 to 24 hours. siRNA knockdown of CLOCK also decreased AVP expression in vitro. Further in silico analysis revealed that murine and non-human primate skin display circadian expression of AVPs. Notably, we have found that skin wounding at different times of day induces variable AVP expression. Woundinduced transcription of AVPs also was attenuated in ClockD19 circadian mutant mice. These findings support a paradigm where circadian rhythm may control time-of-day anticipatory AVP transcriptional production in order to prioritize cutaneous defenses when the host is more likely to be wounded and encounter pathogens. Further work is needed to establish the mechanistic links of wounding, circadian rhythm, and the AVP response to ultimately permit a better understanding of our skin's homeostatic and wound-induced viral defense mechanisms.

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