Identifying a gene orchestrating skin regeneration via tissue rebuilding
~ Inspiration from aesthetic treatments ~
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Presented by: Satomi Kiuchi
Introduction
Many cosmetic users desire high efficacy in skin improvement. In aesthetic treatments by physicians, the mechanism that leads to remarkable skin improvement often involves damage to the skin. Following the damage, the treated skin is remodeled, and the fibrous structures show a histologically rejuvenated phenotype. In contrast to that, wounding damage to the skin often results in fibrotic scar tissue formation. Although both phenomena are caused by tissue damage, studies explaining the underlying mechanisms of the former are still scarce. In this study, we elucidated a mechanism of aesthetic treatment and identified a steering gene that triggers a series of processes that lead to tissue remodeling. By applying these findings to cosmetics via the modulation of these genes, we anticipate that cosmetics will have novel functions to induce tissue remodeling - we call this concept Rebuilding Induction.
Methods
In this study, we focused on the aesthetic treatment provided by subcutaneous injection of adipose-derived stem cells (ASCs). To develop an evaluation system that mimics this treatment, fresh subcutaneous adipose tissues from 5 human donors were cultured with and without ASCs for 7 days. Each donor provided informed consent. To examine the details of the fibrous structures from these specimens, we performed a sequential confocal immunofluorescence assay on the subcutaneous adipose tissue. To identify the genes involved in tissue rebuilding, we used RNA sequencing to detect the differences in the gene expression response of subcutaneous adipose tissue in the presence and absence of ASCs. To analyze the function of the differentially expressed genes, we used Gene Ontology and pathway databases. Key regulatory genes were determined through biological network analysis algorithms. For subsequent investigation of a function of candidate gene, quantitative real-time polymerase chain reaction, immunohistochemistry, and scanning electron microscopy were used.
Results
From the sequential confocal microscopy, we observed type I collagen degradation at 3 days and its subsequent synthesis at 7 days after the start of the ASC co-culture, indicating that the rebuilding phenomenon was initiated by ASCs. On the other hand, in the absence of ASC, only synthesis was observed, and the level of synthesis was higher than in the ASC co-culture group. Next, to identify the steering gene involved in tissue rebuilding, we investigated the changes in the ASC co-culture at the gene expression level. We found that several wound healing-like reaction marker genes were up-regulated, while inflammation-related genes and profibrotic genes were down-regulated. In addition, protein-protein network analysis revealed that TSG-6 was a humoral factor differentially expressed in the ASC co-culture, and is central to several biological reactions. Notably, the addition of TSG-6 neutralizing antibody into the developed evaluation system inhibited the effect of ASC co-culture, indicating that TSG-6 controls collagen renewal which leads to tissue rebuilding. Finally, to elucidate the underlying mechanism leading to tissue rebuilding, based on gene expression analysis, we focused on neutrophil extracellular traps (NETs). Results indicate that TSG-6 inhibits the formation of NETs, and that NETs cause the deterioration of the subcutaneous fibrous structure, suggesting that TSG-6 regulates biological reactions in neutrophils, implying TSG-6 mediated regulation on several cell species.
Discussion/Conclusion
The present study demonstrates that TSG-6 controls a variety of biological reactions leading to rebuilding, such as inhibiting the formation of NETs released by neutrophils and promoting transient degradation of collagen as well as collagen synthesis not directed toward fibrosis. Previously, it was reported that TSG-6 detects tissue inflammation and turns it down, suggesting that the key mechanism is that TSG-6 acts as a steering gene switching several types of immune cells to a rebuilding state. Our finding is useful for promoting the development of cosmetics with superior effects for various skin issues caused by changes of fibrous structures such as wrinkles and sagging, furthermore, it has the potential of expanding applications of cosmetics on fibrotic skin problems such as an acne scars that are believed to be challenging. In summary, this study pioneers a new approach method of elucidation of the biological mechanisms underlying skin improvement, whereas the dominant research targets in the conventional research are the causes of skin problems. In all, this study paves the way for developing innovative cosmetics that go far beyond conventional cosmetics.
Many cosmetic users desire high efficacy in skin improvement. In aesthetic treatments by physicians, the mechanism that leads to remarkable skin improvement often involves damage to the skin. Following the damage, the treated skin is remodeled, and the fibrous structures show a histologically rejuvenated phenotype. In contrast to that, wounding damage to the skin often results in fibrotic scar tissue formation. Although both phenomena are caused by tissue damage, studies explaining the underlying mechanisms of the former are still scarce. In this study, we elucidated a mechanism of aesthetic treatment and identified a steering gene that triggers a series of processes that lead to tissue remodeling. By applying these findings to cosmetics via the modulation of these genes, we anticipate that cosmetics will have novel functions to induce tissue remodeling - we call this concept Rebuilding Induction.
Methods
In this study, we focused on the aesthetic treatment provided by subcutaneous injection of adipose-derived stem cells (ASCs). To develop an evaluation system that mimics this treatment, fresh subcutaneous adipose tissues from 5 human donors were cultured with and without ASCs for 7 days. Each donor provided informed consent. To examine the details of the fibrous structures from these specimens, we performed a sequential confocal immunofluorescence assay on the subcutaneous adipose tissue. To identify the genes involved in tissue rebuilding, we used RNA sequencing to detect the differences in the gene expression response of subcutaneous adipose tissue in the presence and absence of ASCs. To analyze the function of the differentially expressed genes, we used Gene Ontology and pathway databases. Key regulatory genes were determined through biological network analysis algorithms. For subsequent investigation of a function of candidate gene, quantitative real-time polymerase chain reaction, immunohistochemistry, and scanning electron microscopy were used.
Results
From the sequential confocal microscopy, we observed type I collagen degradation at 3 days and its subsequent synthesis at 7 days after the start of the ASC co-culture, indicating that the rebuilding phenomenon was initiated by ASCs. On the other hand, in the absence of ASC, only synthesis was observed, and the level of synthesis was higher than in the ASC co-culture group. Next, to identify the steering gene involved in tissue rebuilding, we investigated the changes in the ASC co-culture at the gene expression level. We found that several wound healing-like reaction marker genes were up-regulated, while inflammation-related genes and profibrotic genes were down-regulated. In addition, protein-protein network analysis revealed that TSG-6 was a humoral factor differentially expressed in the ASC co-culture, and is central to several biological reactions. Notably, the addition of TSG-6 neutralizing antibody into the developed evaluation system inhibited the effect of ASC co-culture, indicating that TSG-6 controls collagen renewal which leads to tissue rebuilding. Finally, to elucidate the underlying mechanism leading to tissue rebuilding, based on gene expression analysis, we focused on neutrophil extracellular traps (NETs). Results indicate that TSG-6 inhibits the formation of NETs, and that NETs cause the deterioration of the subcutaneous fibrous structure, suggesting that TSG-6 regulates biological reactions in neutrophils, implying TSG-6 mediated regulation on several cell species.
Discussion/Conclusion
The present study demonstrates that TSG-6 controls a variety of biological reactions leading to rebuilding, such as inhibiting the formation of NETs released by neutrophils and promoting transient degradation of collagen as well as collagen synthesis not directed toward fibrosis. Previously, it was reported that TSG-6 detects tissue inflammation and turns it down, suggesting that the key mechanism is that TSG-6 acts as a steering gene switching several types of immune cells to a rebuilding state. Our finding is useful for promoting the development of cosmetics with superior effects for various skin issues caused by changes of fibrous structures such as wrinkles and sagging, furthermore, it has the potential of expanding applications of cosmetics on fibrotic skin problems such as an acne scars that are believed to be challenging. In summary, this study pioneers a new approach method of elucidation of the biological mechanisms underlying skin improvement, whereas the dominant research targets in the conventional research are the causes of skin problems. In all, this study paves the way for developing innovative cosmetics that go far beyond conventional cosmetics.