Twist2 contributes to skin regeneration and hair follicle formation in mouse fetuses.

Unlike adult mammalian wounds, early embryonic mouse skin wounds completely regenerate and heal without scars. Analysis of the underlying molecular mechanism will provide insights into scarless wound healing. Twist2 is an important regulator of hair follicle formation and biological patterning; however, it is unclear whether it plays a role in skin or skin appendage regeneration. Here, we aimed to elucidate Twist2 expression and its role in fetal wound healing. ICR mouse fetuses were surgically wounded on embryonic day 13 (E13), E15, and E17, and Twist2 expression in tissue samples from these fetuses was evaluated via in situ hybridization, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction. Twist2 expression was upregulated in the dermis of E13 wound margins but downregulated in E15 and E17 wounds. Twist2 knockdown on E13 left visible marks at the wound site, inhibited regeneration, and resulted in defective follicle formation. Twist2-knockdown dermal fibroblasts lacked the ability to undifferentiate. Furthermore, Twist2 hetero knockout mice (Twist + /-) formed visible scars, even on E13, when all skin structures should regenerate. Thus, Twist2 expression correlated with skin texture formation and hair follicle defects in late mouse embryos. These findings may help develop a therapeutic strategy to reduce scarring and promote hair follicle regeneration.

The Prospective Natural History Study of Patients with Intractable Venous Malformation and Klippel-Trenaunay Syndrome to Guide Designing a Proof-of-Concept Clinical Trial for Novel Therapeutic Intervention.

Background: The natural history of venous malformation (VM) and Klippel-Trenaunay Syndrome (KTS) has not been quantitatively studied. To obtain benchmarks to guide designing clinical trials to assess safety and efficacy of novel drug candidates, the clinical course of the patients was followed for 6 months. Methods and Results: This is a multicenter prospective observational study evaluating the change rate in lesion volume from baseline with magnetic resonance images, as the primary endpoint. In addition, disease severities, performance status (PS), pain visual analog scale (VAS) score, quality of life (QoL), infections, and coagulation markers were also evaluated. Thirty-four patients (VM = 17, KTS = 17, 1-53 of age; median 15.9 years) with measurable lesion volume were analyzed. There was no statistically significant difference in the lesion volume between baseline and day 180, and the mean change rate (standard deviation) was 1.06 (0.28). There were no baseline characteristics that affected the change in lesion volume over 6 months. However, there were patients who showed more than 20% volume change and it was suggested that the lesion volume was largely impacted by local infection. There were no statistically significant changes in pain VAS score, severity, PS, QoL score, D-dimer, and platelet count over 6 months within all patients analyzed. Conclusion: The results showed the representative natural course of VM and KTS for a 6-month period with objective change of lesion volume and other factors, suggesting that it is scientifically reasonable to conduct a Phase 2 proof-of-concept study without a placebo arm, using the results of this study as the control. Clinical Trial Registration: NCT04285723, NCT04589650.

Regeneration of Panniculus Carnosus Muscle in Fetal Mice Is Characterized by the Presence of Actin Cables.

Mammalian skin, including human and mouse skin, does not regenerate completely after injury; it is repaired, leaving a scar. However, it is known that skin wounds up to a certain stage of embryonic development can regenerate. The mechanism behind the transition from regeneration to scar formation is not fully understood. Panniculus carnosus muscle (PCM) is present beneath the dermal fat layer and is a very important tissue for wound contraction. In rodents, PCM is present throughout the body. In humans, on the other hand, it disappears and becomes a shallow fascia on the trunk. Fetal cutaneous wounds, including PCM made until embryonic day 13 (E13), regenerate completely, but not beyond E14. We visualized the previously uncharacterized development of PCM in the fetus and investigated the temporal and spatial changes in PCM at different developmental stages, ranging from full regeneration to non-regeneration. Furthermore, we report that E13 epidermal closure occurs through actin cables, which are bundles of actomyosin formed at wound margins. The wound healing process of PCM suggests that actin cables may also be associated with PCM. Our findings reveal that PCM regenerates through a similar mechanism.

Additional Wide Resection of Infantile Dermatofibrosarcoma Protuberans after Unplanned Excision: A Case Report.

Dermatofibrosarcoma protuberans (DFSP) is a locally aggressive intermediate soft tissue neoplasm that occurs in the dermis. DFSP generally occurs in young to middle-aged adults and rarely in infancy. Because of its extreme rarity, DFSP is difficult to diagnose and treat, especially when it occurs in infancy. In this paper, we reported a case of infantile DFSP in which we performed additional wide resection with a 3-cm horizontal margin for a mass that had previously undergone unplanned excision. No tumor recurrence has been seen for 3 years postoperatively. We suggest that the possibility of DFSP should always be considered when an enlarging superficial mass is identified on the trunk, even in an infant. Additionally, radical local treatment is as important for DFSP in infancy as it is for DFSP in adults, even after unplanned excision.

Compound 13 Promotes Epidermal Healing in Mouse Fetuses via Activation of AMPK.

Unlike adults, early developing fetuses can completely regenerate tissue, and replicating this could lead to the development of treatments to reduce scarring. Mice epidermal structures, including wound healing patterns, are regenerated until embryonic day (E) 13, leaving visible scars thereafter. These patterns require actin cable formation at the epithelial wound margin through AMP-activated protein kinase (AMPK) activation. We aimed to investigate whether the administration of compound 13 (C13), a recently discovered AMPK activator, to the wound could reproduce this actin remodeling and skin regeneration pattern through its AMPK activating effect. The C13 administration resulted in partial formations of actin cables, which would normally result in scarring, and scar reduction during the healing of full-layer skin defects that occurred in E14 and E15 fetuses. Furthermore, C13 was found to cause AMPK activation in these embryonic mouse epidermal cells. Along with AMPK activation, Rac1 signaling, which is involved in leaflet pseudopodia formation and cell migration, was suppressed in C13-treated wounds, indicating that C13 inhibits epidermal cell migration. This suggests that actin may be mobilized by C13 for cable formation. Administration of C13 to wounds may achieve wound healing similar to regenerative wound healing patterns and may be a potential candidate for new treatments to heal scars.

Corrigendum: Single-cell RNA-seq analysis reveals cellular functional heterogeneity in dermis between fibrotic and regenerative wound healing fates.

[This corrects the article DOI: 10.3389/fimmu.2022.875407.].

Fetal Fibroblast Transplantation via Ablative Fractional Laser Irradiation Reduces Scarring.

Scar treatments include fractional laser treatment, cell transplantation, surgery, skin needling, and dermal fillers. Fractional laser treatments are used to reduce scarring and blurring. Cell transplantation is promising, with mature fibroblasts and adipose-derived stem cells being used clinically, while embryonic fibroblasts are used experimentally. Herein, we developed a combination of ablative CO2 (carbon dioxide) fractional laser and cell transplantation for the treatment of scars. Eight-week-old male C57Bl/6 mice were used to create a full-layer skin defect in the back skin and create scars. The scar was then irradiated using a CO2 fractional laser. The cells were then transplanted onto the scar surface and sealed with a film agent. The transplanted cells were GFP-positive murine fetal fibroblasts (FB), fetal fibroblasts with a long-term sphere-forming culture (LS), and fetal skin with a short-term sphere-forming culture (SS). After transplantation, green fluorescent protein (GFP)-positive cells were scattered in the dermal papillary layer and subcutis in all the groups. LS significantly reduced the degree of scarring, which was closest to normal skin. In conclusion, the combination of ablative fractional laser irradiation and fetal fibroblast transplantation allowed us to develop new methods for scar treatment.

Effect of Sonic Hedgehog on the Regeneration of Epidermal Texture Patterns.

Wounds on embryonic mouse fetuses regenerate up to embryonic day (E) 13, but after E14, the pattern is lost and a visible scar remains. We hypothesized that the sonic hedgehog (Shh), which is involved in patterning during development, is involved in the regeneration of texture. Embryos of ICR mice were surgically injured at E13, E14, and E15 and analyzed for the expression of Shh. For external Shh administration, recombinant Shh-containing slow-release beads were implanted in the wounds of mice. In contrast, cyclopamine was administered to wounds of adult mice to inhibit Shh. The expression of Shh was unaltered at E13, whereas it was upregulated in the epidermis of the wound from E14 onward. Implantation of recombinant Shh-containing beads into E13 wounds inhibited skin texture regeneration. Cyclopamine treatment inhibited epithelialization and thickening of the epidermis in the wounds of adult mice. In vitro, Shh promoted proliferation and inhibited the migration of epidermal keratinocytes through the activation of cyclin D proteins. Thus, our results suggested that the expression of Shh is involved in the regeneration of texture during wound healing, especially in epidermal keratinocyte migration and division, and could inhibit skin texture regeneration after E14.

Effect of All-trans Retinoic Acid on Panniculus Carnosus Muscle Regeneration in Fetal Mouse Wound Healing.

The dermal panniculus carnosus (PC) muscle is critical for wound contraction in lower mammals and is a useful model of muscle regeneration owing to its high cellular metabolic turnover. During wound healing in mice, skin structures, including PC, are completely regenerated up to embryonic day (E) 13, but PC is only partially regenerated in fetuses or adult animals after E14. Nevertheless, the mechanisms underlying wound repair for complete regeneration in PC have not been fully elucidated. We hypothesized that retinoic acid (RA) signaling, which is involved in muscle differentiation, regulates PC regeneration. Methods: Surgical injury was induced in ICR mice on E13 and E14. RA receptor alpha (RARα) expression in tissue samples from embryos was evaluated using immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. To evaluate the effects of RA on PC regeneration, beads soaked in all-trans RA (ATRA) were implanted in E13 wounds, and tissues were observed. The effects of RA on myoblast migration were evaluated using a cell migration assay. Results: During wound healing, RARα expression was enhanced at the cut surface in PCs of E13 wounds but was attenuated at the cut edge of E14 PCs. Implantation of ATRA-containing beads inhibited PC regeneration on E13 in a concentration-dependent manner. Treatment of myoblasts with ATRA inhibited cell migration. Conclusions: ATRA inhibits PC regeneration, and decreased RARα expression in wounds after E14 inhibits myoblast migration. Our findings may contribute to the development of therapies to promote complete wound regeneration, even in the muscle.

Comprehensive targeted next-generation sequencing in patients with slow-flow vascular malformations.

Recent studies have shown that the PI3K signaling pathway plays an important role in the pathogenesis of slow-flow vascular malformations (SFVMs). Analysis of genetic mutations has advanced our understanding of the mechanisms involved in SFVM pathogenesis and may identify new therapeutic targets. We screened for somatic variants in a cohort of patients with SFVMs using targeted next-generation sequencing. Targeted next-generation sequencing of 29 candidate genes associated with vascular anomalies or with the PI3K signaling pathway was performed on affected tissues from patients with SFVMs. Fifty-nine patients with SFVMs (venous malformations n = 21, lymphatic malformations n = 27, lymphatic venous malformations n = 1, and Klippel-Trenaunay syndrome n = 10) were included in the study. TEK and PIK3CA were the most commonly mutated genes in the study. We detected eight TEK pathogenic variants in 10 samples (16.9%) and three PIK3CA pathogenic variants in 28 samples (47.5%). In total, 37 of 59 patients (62.7%) with SFVMs harbored pathogenic variants in these three genes involved in the PI3K signaling pathway. Inhibitors of this pathway may prove useful as molecular targeted therapies for SFVMs.

Actin cable formation and epidermis-dermis positional relationship during complete skin regeneration.

Up to a certain developmental stage, a fetus can completely regenerate wounds in the skin. To clarify the mechanism of fetal skin regeneration, identifying when the skin switches from fetal-type wound regeneration to adult-type wound repair is necessary. We hypothesized that this switch occurs at several time points and that complete skin regeneration requires epidermal-dermal interactions and the formation of actin cables. We compared normal skin and wound morphology at each developmental stage. We examined two parameters: epidermal texture and dermal structure. We found that the three-dimensional structure of the skin was completely regenerated in full-thickness skin incisions made before embryonic day (E) 13. However, the skin texture did not regenerate in wounds made after E14. We also found that the dermal structure regenerates up to E16, but wounds created after E17 heal as scars with dermal fibrosis. By controlling the activity of AMP-activated protein kinase and altering actin cable formation, we could regulate scar formation in utero. These findings may contribute to therapies that allow complete skin regeneration without scarring.

Downregulation of Lhx2 Markedly Impairs Wound Healing in Mouse Fetus.

Multiple transitions occur in the healing ability of the skin during embryonic development in mice. Embryos up to embryonic day 13 (E13) regenerate completely without a scar after full-thickness wounding. Then, up to E16, dermal structures can be formed, including skin appendages such as hair follicles. However, after E17, wound healing becomes incomplete, and scar formation is triggered. Lhx2 regulates the switch between maintenance and activation of hair follicle stem cells, which are involved in wound healing. Therefore, we investigated the role of Lhx2 in fetal wound healing. Embryos of ICR mice were surgically wounded at E13, E15, and E17, and the expression of Lhx2 along with mitotic (Ki67 and p63) and epidermal differentiation (keratin-10 and loricrin) markers was analyzed. The effect of Lhx2 knockdown on wound healing was observed. Lhx2 expression was not noticed in E13 due to the absence of folliculogenesis but was evident in the epidermal basal layer of E15 and E17 and at the base of E17 wounds, along with Ki67 and p63 expression. Furthermore, Lhx2 knockdown in E15 markedly prolonged wound healing and promoted clear scar formation. Therefore, Lhx2 expression is involved in cell division associated with wound healing and may contribute to scar formation in late embryos.

Role of Wnt Signaling in Mouse Fetal Skin Wound Healing.

Wnt proteins secrete glycoproteins that are involved in various cellular processes to maintain homeostasis during development and adulthood. However, the expression and role of Wnt in wound healing have not been fully documented. Our previous studies have shown that, in an early-stage mouse fetus, no scarring occurred after cutaneous wounding, and complete regeneration was achieved. In this study, the expression and localization of Wnt proteins in a mouse fetal-wound-healing model and their associations with scar formation were analyzed. Wnt-related molecules were detected by in-situ hybridization, immunostaining, and real-time polymerase chain reaction. The results showed altered expression of Wnt-related molecules during the wound-healing process. Moreover, scar formation was suppressed by Wnt inhibitors, suggesting that Wnt signaling may be involved in wound healing and scar formation. Thus, regulation of Wnt signaling may be a possible mechanism to control scar formation.

Fibroblast Growth Factor 7 Suppresses Fibrosis and Promotes Epithelialization during Wound Healing in Mouse Fetuses.

Adult mammalian wounds leave visible scars, whereas skin wounds in developing mouse fetuses are scarless until a certain point in development when complete regeneration occurs, including the structure of the dermis and skin appendages. Analysis of the molecular mechanisms at this transition will provide clues for achieving scarless wound healing. The fibroblast growth factor (FGF) family is a key regulator of inflammation and fibrosis during wound healing. We aimed to determine the expression and role of FGF family members in fetal wound healing. ICR mouse fetuses were surgically wounded at embryonic day 13 (E13), E15, and E17. Expression of FGF family members and FGF receptor (FGFR) in tissue samples from these fetuses was evaluated using in situ hybridization and reverse transcription-quantitative polymerase chain reaction. Fgfr1 was downregulated in E15 and E17 wounds, and its ligand Fgf7 was upregulated in E13 and downregulated in E15 and E17. Recombinant FGF7 administration in E15 wounds suppressed fibrosis and promoted epithelialization at the wound site. Therefore, the expression level of Fgf7 may correlate with scar formation in late mouse embryos, and external administration of FGF7 may represent a therapeutic option to suppress fibrosis and reduce scarring.

Single-Cell RNA-seq Analysis Reveals Cellular Functional Heterogeneity in Dermis Between Fibrotic and Regenerative Wound Healing Fates.

Background: Fibrotic scars are common in both human and mouse skin wounds. However, wound-induced hair neogenesis in the murine wounding models often results in regenerative repair response. Herein, we aimed to uncover cellular functional heterogeneity in dermis between fibrotic and regenerative wound healing fates. Methods: The expression matrix of single-cell RNA sequencing (scRNA-seq) data of fibrotic and regenerative wound dermal cells was filtered, normalized, and scaled; underwent principal components analysis; and further analyzed by Uniform Manifold Approximation and Projection (UMAP) for dimension reduction with the Seurat package. Cell types were annotated, and cell-cell communications were analyzed. The core cell population myofibroblast was identified and the biological functions of ligand and receptor genes between myofibroblast and macrophage were evaluated. Specific genes between fibrotic and regenerative myofibroblast and macrophage were identified. Temporal dynamics of myofibroblast and macrophage were reconstructed with the Monocle tool. Results: Across dermal cells, there were six cell types, namely, EN1-negative myofibroblasts, EN1-positive myofibroblasts, hematopoietic cells, macrophages, pericytes, and endothelial cells. Ligand and receptor genes between myofibroblasts and macrophages mainly modulated cell proliferation and migration, tube development, and the TGF-ß pathway. Specific genes that were differentially expressed in fibrotic compared to regenerative myofibroblasts or macrophages were separately identified. Specific genes between fibrotic and regenerative myofibroblasts were involved in the mRNA metabolic process and organelle organization. Specific genes between fibrotic and regenerative macrophages participated in regulating immunity and phagocytosis. We then observed the underlying evolution of myofibroblasts or macrophages. Conclusion: Collectively, our findings reveal that myofibroblasts and macrophages may alter the skin wound healing fate through modulating critical signaling pathways.

Decorin Inhibits Dermal Mesenchymal Cell Migration and Induces Scar Formation.

Background: Variations in skin healing capacities are observed during different murine embryonic developmental stages. Through embryonic day 16 (E16), embryos are able to regenerate dermal architecture following flank skin wounding; however, after E17, wounds heal incompletely, inducing scar formation. The regenerative ability of the E16 fetal dermis depends on the migration of dermal mesenchymal cells. Decorin is a small molecule known to affect tissue tensile strength, cell phenotype, and tissue repair, including skin wound healing. In the current study, we evaluated the expression and roles of decorin in wound healing. Methods: Surgical injury was induced at E16 and E17 in ICR mouse embryos. Decorin expression was evaluated in tissue samples from these embryos using immunohistochemistry and reverse transcription quantitative polymerase chain reaction. Cell migration assays were used to evaluate wound healing capability of separated dermal and fascial tissues. Results: Our results showed that decorin exhibited distinct expression patterns during wound healing at E16 versus E17. Additionally, decorin expression altered cell migration in vitro. Dermal and fascial mesenchymal cells were found to exhibit distinct migration patterns concomitant with altered decorin expression. Specifically, decorin inhibited migration and favored scar formation. Conclusion: Decorin expression may contribute to scar formation in the late stage of mouse embryos by inhibiting the migration of dermal mesenchymal cells.

Screening of Autophagy-Related Prognostic Genes in Metastatic Skin Melanoma.

Cutaneous melanoma refers to a common skin tumor that is dangerous to health with a great risk of metastasis. Previous researches reported that autophagy is associated with the progression of cutaneous melanoma. Nevertheless, the role played by genes with a relation to autophagy (ARG) in the prediction of the course of metastatic cutaneous melanoma is still largely unknown. We observed that thirteen ARGs showed relations to overall survival (OS) in the Cox regression investigation based on a single variate. We developed 2-gene signature, which stratified metastatic cutaneous melanoma cases to groups at great and small risks. Cases suffering from metastatic cutaneous melanoma in the group at great risks had power OS compared with cases at small risks. The risk score, T phase, N phase, and age were proved to be individual factors in terms of the prediction of OS. Besides, the risk scores identified by the two ARGs were significantly correlated with metastatic cutaneous melanoma. Receiver operating characteristic (ROC) curve analysis demonstrated accurate predicting performance exhibited by the 2-gene signature. We also found that the immunization and stromal scores achieved by the group based on large risks were higher compared with those achieved by the group based on small risks. The metastatic cutaneous melanoma cases achieving the score based on small risks acquired greater expression of immune checkpoint molecules as compared with the high-risk group. In conclusion, the 2-ARG gene signature indicated a novel prognostic indicator for prognosis prediction of metastatic cutaneous melanoma, which served as an important tool for guiding the clinical treatment of cutaneous melanoma.

Clinical Analysis of Cultured Epidermal Autograft (JACE) Transplantation for Giant Congenital Melanocytic Nevus.

BACKGROUND: Cultured epidermal transplantation (JACE) is performed for giant congenital melanocytic nevus (GCMN), but there are few reports on its postoperative course and surgical content or indications. We aimed to investigate the postoperative course of GCMN patients undergoing cultured epidermal autograft transplantation and compare the outcomes between 2 nevus tissue resection methods. METHODS: Twelve GCMN patients aged 0 months to 8 years and 9 months were included in this single-center case series study. Cultured epidermal autograft transplantation was performed at 19 sites of the patients' extremities and trunks, after excision of the nevus either by using an electric dermatome, which we initially used in 2017, or by curettage with a sharp spoon and use of a hydrosurgery system (Versajet), which we started performing in 2018. Univariate and multivariate analyses were performed for factors associated with postoperative hypertrophic scar formation. RESULTS: In all cases, >90% of the grafts survived, and the dark brown color of the nevus was reduced. Average postoperative observation period was 16.5 months. Hypertrophic scar formation was observed postoperatively at 9 wound sites out of the 12 sites with GCMN removed with a dermatome and at only 1 site with GCMN removed by curettage with use of a hydrosurgery system. In the univariate and multivariate analyses, hypertrophic scar formation was associated with age at surgery. CONCLUSION: In cultured epidermal autograft transplantation for GCMN, nevus tissue removal at an early age by curettage with use of a hydrosurgery system can provide good results while reducing complications, including recurrence and hypertrophic scar formation.

Arteriovenous malformation on the sole of the foot treated successfully by embolization.

Arteriovenous malformations of the sole of the foot are rare and can cause disturbances in normal living activities. We report a case of a plantar arteriovenous malformation in a 24-year-old male with pain and difficulty in walking. The arteriovenous malformation was complex, with a large and poorly marginated nidus, so we considered that with surgical resection, walking disabilities would be inevitable. When surgical removal of vascular mass is difficult, embolization alone can be effective. Therefore, he was treated with 4 therapeutic embolization procedures. Transvenous approaches to the venous sac and direct punctures of the nidus was performed. The nidus was successfully eradicated by embolization using alcohol, resulting in the disappearance of associated symptoms. Appropriate imaging is essential for diagnosis and evaluation of treatment. We were successful in achieving improved quality of life and satisfaction for a rare and difficult case by percutaneous embolization and sclerotherapy.

Treatment of Deliberate Self-harm Scars with Rotated Thin-skin Graft and Minced-skin Graft.

Scars developing after wrist cutting (a deliberate action of self-harm) have various patterns and are difficult to treat. In addition, they can occur at anatomically prominent sites and are easily recognized as caused by self-harm; thus, scars can cause lifelong regrets. However, there are no standard treatment guidelines for wounds inflicted through self-harm. This study aimed to evaluate the effectiveness of our novel technique using 90-degree rotated skin grafts, which were thinly collected at a thickness of 250 µm from a wound site, together with minced-skin grafts. METHODS: Five regions on the forearm of 5 Japanese women (age, 19-29 years) were treated from July 2011 to April 2012. The skin at the scar site was cut with an electric dermatome at a thickness of 250 µm. The scar contained therein was excised, and the skin was rotated 90 degree and transplanted. The scar remaining in the dermis of the wound was resected and resurfaced. At the site where the skin graft was insufficient, the skin was processed into a minced shape and then transplanted (minced-skin graft). RESULTS: In all cases, skin grafting was performed. The scar was successfully camouflaged and transformed into a socially acceptable appearance. At the wound site, the skin texture was reproduced. Following skin grafting, nodules, pigmentation, and redness around the graft transiently occurred, which then disappeared over time. No scar contractures were observed. CONCLUSION: A combination of thin-skin graft rotated 90 degrees and minced-skin graft is useful in camouflaging a wide variety of deliberate self-harm scars.