Flagler, M. J. | bioRxiv Science

bioRxiv2021

Transcriptomic Analysis of Human Skin Wound Healing and Rejuvenation Following Ablative Fractional Laser Treatment

Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel(R)) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments resulted in continued changes in gene expression, with many genes either differentially regulated or continuously upregulated with increasing number of treatments, indicating that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of skin activates several biological processes involved in wound healing and tissue regeneration, all of which significantly contribute to the rejuvenating effect of fractional laser treatment on aged skin.

bioinformatics↗

bioRxiv2021

Combinations of Peptides Synergistically Activate the Regenerative Capacity of Skin Cells In Vitro

OBJECTIVETo explore synergistic effects related to skin regeneration, peptides with distinct biological mechanisms of action were evaluated in combination in different skin cell lines in the presence or absence of niacinamide (Nam). Furthermore, the synergistic responses of peptide combinations on global gene expression were compared to the changes that occur with fractional laser resurfacing treatment, a gold standard approach for skin rejuvenation, to further define optimal peptide combinations. METHODSMicroarray profiling was used to characterize the biological responses of peptide combinations (+/- Nam) relative to the individual components in epidermal keratinocyte and dermal fibroblast cell lines. Cellular functional assays were utilized to confirm the synergistic effects of peptide combinations. Bioinformatics approaches were used to link the synergistic effects of peptide combinations on gene expression to the transcriptomics of the skin rejuvenation response from fractional laser treatment. RESULTSMicroarray analysis of skin cells treated with peptide combinations revealed synergistic changes in gene expression compared to individual peptide controls. Bioinformatic analysis of synergy genes in keratinocytes revealed activation of NRF2-mediated oxidative stress responses by a combination of Ac-PPYL, Pal-KTTKS, and Nam. Additional analysis revealed direct downstream transcriptional targets of NRF2/ARE exhibiting synergistic regulation by this combination of materials, which was corroborated by a cellular reporter assay. NRF2-mediated oxidative stress response pathways were also found to be activated in the transcriptomics of the early skin rejuvenation response to fractional laser treatment, suggesting the importance of this biology in the early stages of tissue repair. Additionally, a second combination of peptides (pal-KT and Ac-PPYL) was found to synergistically restore cellular ATP levels that had been depleted due to the presence of ROS, indicating an additional mechanism whereby peptide synergies may accelerate skin repair. CONCLUSIONThrough combinatorial synergy studies, we have identified additional in vitro skin repair mechanisms beyond the previously described functions of individual peptides and correlated these to the transcriptomics of the skin rejuvenation response of fractional laser treatment. These findings suggest that specific peptides can act together, via complementary and synergistic mechanisms, to holistically enhance the regenerative capacity of in vitro skin cells.

molecular biology↗