Sustained Physiological Stretch Induces Abdominal Skin Growth in Pregnancy.

Supraphysiological stretches are exploited in skin expanders to induce tissue growth for autologous implants. As pregnancy is associated with large levels of sustained stretch, we investigated whether skin growth occurs in pregnancy. Therefore, we combined a mechanical model of skin and the observations from suction experiments on several body locations of five pregnant women at different gestational ages. The measurements show a continuous increase in stiffness, with the largest change observed during the last trimester. A comparison with numerical simulations indicates that the measured increase in skin stiffness is far below the level expected for the corresponding deformation of abdominal skin. A new set of simulations accounting for growth could rationalize all observations. The predicted amount of tissue growth corresponds to approximately 40% area increase before delivery. The results of the simulations also offered the opportunity to investigate the biophysical cues present in abdominal skin along gestation and to compare them with those arising in skin expanders. Alterations of the skin mechanome were quantified, including tissue stiffness, hydrostatic and osmotic pressure of the interstitial fluid, its flow velocity and electrical potential. The comparison between pregnancy and skin expansion highlights similarities as well as differences possibly influencing growth and remodeling.

Mechanical properties pattern the skin.

Morphogens induce variations in tissue mechanics to promote feather budding.

An active traveling wave of Eda/NF-κB signaling controls the timing and hexagonal pattern of skin appendages in zebrafish.

Periodic patterns drive the formation of a variety of tissues, including skin appendages such as feathers and scales. Skin appendages serve important and diverse functions across vertebrates, yet the mechanisms that regulate their patterning are not fully understood. Here, we have used live imaging to investigate dynamic signals regulating the ontogeny of zebrafish scales. Scales are bony skin appendages that develop sequentially along the anterior-posterior and dorsal-ventral axes to cover the fish in a hexagonal array. We have found that scale development requires cell-cell communication and is coordinated through an active wave mechanism. Using a live transcriptional reporter, we show that a wave of Eda/NF-κB activity precedes scale initiation and is required for scale formation. Experiments decoupling the propagation of the wave from dermal placode formation and osteoblast differentiation demonstrate that the Eda/NF-κB activity wavefront controls the timing of the sequential patterning of scales. Moreover, this decoupling resulted in defects in scale size and significant deviations in the hexagonal patterning of scales. Thus, our results demonstrate that a biochemical traveling wave coordinates scale initiation and proper hexagonal patterning across the fish body.

Mechanical tension mobilizes Lgr6+ epidermal stem cells to drive skin growth.

Uniquely among mammalian organs, skin is capable of marked size change in adults, yet the mechanisms underlying this notable capacity are unclear. Here, we use a system of controlled tissue expansion in mice to uncover cellular and molecular determinants of skin growth. Through machine learning-guided three-dimensional tissue reconstruction, we capture morphometric changes in growing skin. We find that most growth is driven by the proliferation of the epidermis in response to mechanical tension, with more limited changes in dermal and subdermal compartments. Epidermal growth is achieved through preferential activation and differentiation of Lgr6+ stem cells of the epidermis, driven in part by the Hippo pathway. By single-cell RNA sequencing, we uncover further changes in mechanosensitive and metabolic pathways underlying growth control in the skin. These studies point to therapeutic strategies to enhance skin growth and establish a platform for understanding organ size dynamics in adult mammals.

Understanding and Harnessing Epithelial‒Mesenchymal Interactions in the Development of Palmoplantar Identity.

Palmoplantar skin has several unique characteristics such as increased thickness, high resilience, hypopigmentation, and lack of hair follicles. The establishment of palmoplantar identity occurs through keratinocyte‒fibroblast interactions, with keratin 9 expression and Wnt signaling playing key roles. Understanding how palmoplantar features develop may help efforts to reproduce them at both palmoplantar and nonpalmoplantar body sites.

Isoprocurcumenol Supports Keratinocyte Growth and Survival through Epidermal Growth Factor Receptor Activation.

Although proliferation of keratinocytes, a major type of skin cells, is a key factor in maintaining the function of skin, their ability to proliferate tends to diminish with age. To solve such a problem, researchers in medical and skin cosmetic fields have tried to utilize epidermal growth factor (EGF), but achieved limited success. Therefore, a small natural compound that can mimic the activity of EGF is highly desired in both medical and cosmetic fields. Here, using the modified biosensor system, we observed that natural small-compound isoprocurcumenol, which is a terpenoid molecule derived from turmeric, can activate EGFR signaling. It increased the phosphorylation of ERK and AKT, and upregulated the expression of genes related to cell growth and proliferation, such as c-myc, c-jun, c-fos, and egr-1. In addition, isoprocurcumenol induced the proliferation of keratinocytes in both physical and UVB-induced cellular damage, indicative of its function in skin regeneration. These findings reveal that EGF-like isoprocurcumenol promotes the proliferation of keratinocytes and further suggest its potential as an ingredient for medical and cosmetics use.

Developmental genetics of color pattern establishment in cats.

Intricate color patterns are a defining aspect of morphological diversity in the Felidae. We applied morphological and single-cell gene expression analysis to fetal skin of domestic cats to identify when, where, and how, during fetal development, felid color patterns are established. Early in development, we identify stripe-like alterations in epidermal thickness preceded by a gene expression pre-pattern. The secreted Wnt inhibitor encoded by Dickkopf 4 plays a central role in this process, and is mutated in cats with the Ticked pattern type. Our results bring molecular understanding to how the leopard got its spots, suggest that similar mechanisms underlie periodic color pattern and periodic hair follicle spacing, and identify targets for diverse pattern variation in other mammals.

Regional Specific Differentiation of Integumentary Organs: Regulation of Gene Clusters within the Avian Epidermal Differentiation Complex and Impacts of SATB2 Overexpression.

The epidermal differentiation complex (EDC) encodes a group of unique proteins expressed in late epidermal differentiation. The EDC gave integuments new physicochemical properties and is critical in evolution. Recently, we showed ß-keratins, members of the EDC, undergo gene cluster switching with overexpression of SATB2 (Special AT-rich binding protein-2), considered a chromatin regulator. We wondered whether this unique regulatory mechanism is specific to ß-keratins or may be derived from and common to EDC members. Here we explore (1) the systematic expression patterns of non-ß-keratin EDC genes and their preferential expression in different skin appendages during development, (2) whether the expression of non-ß-keratin EDC sub-clusters are also regulated in clusters by SATB2. We analyzed bulk RNA-seq and ChIP-seq data and also evaluated the disrupted expression patterns caused by overexpressing SATB2. The results show that the expression of whole EDDA and EDQM sub-clusters are possibly mediated by enhancers in E14-feathers. Overexpressing SATB2 down-regulates the enriched EDCRP sub-cluster in feathers and the EDCH sub-cluster in beaks. These results reveal the potential of complex epigenetic regulation activities within the avian EDC, implying transcriptional regulation of EDC members acting at the gene and/or gene cluster level in a temporal and skin regional-specific fashion, which may contribute to the evolution of diverse avian integuments.

Hyaluronan Hydrogels for Injection in Superficial Dermal Layers: An In Vitro Characterization to Compare Performance and Unravel the Scientific Basis of Their Indication.

BACKGROUND: Skinboosters represent the latest category of hyaluronan (HA) hydrogels released for aesthetic purposes. Different from originally developed gels, they are intended for more superficial injections, claiming a skin rejuvenation effect through hydration and possibly prompting biochemical effects in place of the conventional volumetric action. Here, three commercial skinboosters were characterized to unravel the scientific basis for such indication and to compare their performances. METHODS: Gels were evaluated for water-soluble/insoluble-HA composition, rheology, hydration, cohesivity, stability and effect, in vitro, on human dermal fibroblasts towards the production of extracellular matrix components. RESULTS: Marked differences in the insoluble-hydrogel amount and in the hydrodynamic parameters for water-soluble-HA chains were evidenced among the gels. Hydration, rigidity and cohesivity also varied over a wide range. Sensitivity to hyaluronidases and Reactive Oxygen Species was demonstrated allowing a stability ranking. Slight differences were found in gels' ability to prompt elastin expression and in ColIV/ColI ratio. CONCLUSIONS: A wide panel of biophysical and biochemical parameters for skinboosters was provided, supporting clinicians in the conscious tuning of their use. Data revealed great variability in gels' behavior notwithstanding the same clinical indication and unexpected similarities to the volumetric formulations. Data may be useful to improve customization of gel design toward specific uses.

Analysis of Angiogenesis in Mouse Embryonic Dorsal Skin by Whole-Mount Fluorescent Staining.

The blood vascular system is a tree-like hierarchical branching structure and needs to function even before fully established. Abnormal formation of blood vessels results in embryonic lethality and also contributes to the pathogenesis of a number of human diseases, including cancer metastasis. To understand the molecular events associated with blood vessel formation, we established a fluorescence staining-based protocol on mouse embryonic skin. We harvested mouse embryonic skin and performed whole-mount staining. The reconstructed three-dimensional vascular structure provided detailed information on angiogenesis.

Asiaticoside-laden silk nanofiber hydrogels to regulate inflammation and angiogenesis for scarless skin regeneration.

Scarless skin regeneration remains a challenge due to the complicated microenvironment involved in wound healing. Here, the hydrophobic drug, asiaticoside (AC), was loaded inside silk nanofiber hydrogels to achieve bioactive and injectable matrices for skin regeneration. AC was dispersed in aqueous silk nanofiber hydrogels with retention of biological functions that regulated inflammatory reactions and vascularization in vitro. After implantation in full-thickness wound defects, these AC-laden hydrogel matrices achieved scarless wound repair. Inflammatory reactions and angiogenesis were regulated during inflammation and remodeling, which was responsible for wound regeneration similar to normal skin. Both in vitro and in vivo studies demonstrated promising applications of these AC-laden silk hydrogels towards scarless tissue regeneration.

Multitargeted Approach for the Optimization of Morphogenesis and Barrier Formation in Human Skin Equivalents.

In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen-chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.

Effects of mesenchymal stem cell-derived exosomes on oxidative stress responses in skin cells.

The mechanism by which reactive oxygen species (ROS) produced by oxidative stress promote cellular senescence has been studied in detail. This study aimed to verify the preventive or therapeutic effects of mesenchymal stem cell-derived exosomes (MSC-Ex) on the production of ROS induced by oxidative stress in human skin fibroblasts and clarify the mechanisms that promote cellular senescence. In a system where H2O2 was applied to skin fibroblasts, we assessed the effects of the application of MSC-Ex before and after oxidative stress and measured the fluctuations in several signaling molecules involved in subsequent intracellular stress responses. Exosomes were isolated from MSCs (MSC-Ex) and normal human dermal fibroblasts (NHDFs, NHDF-Ex) before and after exposure to H2O2. NHDFs were treated with exosomes before and after exposure to H2O2. mRNA expression (aquaporin-1 and aquaporin-3) and hyaluronan secretion associated with skin moisturization were reduced by H2O2 treatment, whereas MSC-Ex reversed these effects. The cellular senescence induced by H2O2 was also reproduced in fibroblasts. Specifically, the downregulation of SIRT1 led to increased acetylated p53 expression over time, which induced the expression of p21, a downstream molecule of p53, and arrested the cell cycle, leading to cell senescence. MSC-Ex enhanced these signal transduction systems. MSC-Ex was also effective at blocking the increase of ß-galactosidase activity and accumulation of ROS in cells. This effect was stronger than that of NHDF-Ex. MSC-Ex were found to act defensively against epidermal and cellular senescence induced by oxidative stress.

Thyroid hormone regulates abrupt skin morphogenesis during zebrafish postembryonic development.

Thyroid hormone is a key regulator of post-embryonic vertebrate development. Skin is a biomedically important thyroid hormone target organ, but the cellular and molecular mechanisms underlying skin pathologies associated with thyroid dysfunction remain obscure. The transparent skin of zebrafish is an accessible model system for studying vertebrate skin development. During post-embryonic development of the zebrafish, scales emerge in the skin from a hexagonally patterned array of dermal papillae, like other vertebrate skin appendages such as feathers and hair follicles. We show here that thyroid hormone regulates the rate of post-embryonic dermal development through interaction with nuclear hormone receptors. This couples skin development with body growth to generate a well ordered array of correctly proportioned scales. This work extends our knowledge of thyroid hormone actions on skin by providing in-vivo evidence that thyroid hormone regulates multiple aspects of dermal development.

Exploration of novel candidate genes involved in epidermal keratinocyte differentiation and skin barrier repair in man.

A proper skin barrier function requires constant formation of stratum corneum, i.e. the outermost layer of epidermis composed of terminally differentiated keratinocytes. The complex process of converting proliferative basal keratinocytes into corneocytes relies on programmed changes in the activity of many well-established genes. Much remains however to be investigated about this process, e.g. in conjunction with epidermal barrier defects due to genetic errors as in ichthyosis. To this end, we re-analyzed two sets of microarray-data comparing altered gene expression in differentiated vs. proliferating keratinocytes and in the skin of patients with autosomal recessive congenital ichthyosis (ARCI) vs. healthy controls, respectively. We thus identified 24 genes to be upregulated in both sets of array and not previously associated with keratinocyte differentiation. For 10 of these genes (AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM45B, TMEM86A and VSNL1), qPCR analysis confirmed the array results and subsequent immunostainings of normal epidermis showed superficial expression of several of the proteins. Furthermore, induction of keratinocyte differentiation using phorbol esters (PMA) resulted in increased expression of eight of the genes, whereas siRNA silencing of PPARδ, a transcription factor supporting differentiation, had the opposite effect. In summary, our results identify ten new candidate genes seemingly involved in human epidermal keratinocyte differentiation and possibly important for epidermal repair in a genetic skin disease characterized by barrier failure.

CircAMD1 regulates proliferation and collagen synthesis via sponging miR-27a-3p in P63-mutant human dermal fibroblasts.

Transcription factor p63 has critical functions in epidermal, hindgut/anorectal, and limb development. Human mutations in P63 correlate with congenital syndromes affecting the skin, anorectal, and limbs. Nevertheless, less are detected regarding networks and functions controlled by P63 mutations in dermal fibroblasts, which are closely related to skin physiology. To screen for new targets, we employed microarray technology to investigate the R226Q P63 mutation with regards to the resulting circular RNA (circRNA) profiles from P63 point mutations in human dermal fibroblasts (HDFs). In this study, we show that P63-mutant HDFs display reduced proliferation, collagen synthesis, and myofibroblast differentiation; circAMD1 was also downregulated in P63-mutant HDFs compared with wild-type HDFs. Furthermore, overexpressing circAMD1 rescued the functional and phenotypic alterations of p63-mutant HDFs. We as well determined that miR-27a-3p was circAMD1 target involved in effects of circAMD1 in P63-mutant HDFs. Collectively, our data show that circAMD1 functions as a miR-27a-3p sponge that inhibits the functional and phenotypical alteration of P63-mutant HDFs and may be a critical marker in pathogenesis regarding P63-associated traits.

A Two-Step Protocol to Erase Human Skin Fibroblasts and Convert Them into Trophoblast-like Cells.

The first differentiation event in mammalian embryos is the formation of the trophectoderm, which is the progenitor of the outer epithelial component of the placenta and supports the fetus during intrauterine life. Our understanding of these events is limited, particularly in human, because of ethical and legal restrictions and availability of adequate in vitro models would be very advantageous. Here we describe a method that converts human fibroblasts into trophoblast-like cells, combining the use of 5-azacytidine-CR (5-aza-CR) to erase the original cell phenotype and a cocktail containing bone morphogenetic protein 4 (BMP4) with inhibitors of the Activin/Nodal/ERK signaling pathways, to drive erased fibroblasts into the trophoblastic differentiation. This innovative method uses very easily accessible cells to derive trophoblast-like cells and it can be useful to study embryo implantation disorders related to aging.

αßγδ T cells play a vital role in fetal human skin development and immunity.

T cells in human skin play an important role in the immune defense against pathogens and tumors. T cells are present already in fetal skin, where little is known about their cellular phenotype and biological function. Using single-cell analyses, we identified a naive T cell population expressing αß and γδ T cell receptors (TCRs) that was enriched in fetal skin and intestine but not detected in other fetal organs and peripheral blood. TCR sequencing data revealed that double-positive (DP) αßγδ T cells displayed little overlap of CDR3 sequences with single-positive αß T cells. Gene signatures, cytokine profiles and in silico receptor-ligand interaction studies indicate their contribution to early skin development. DP αßγδ T cells were phosphoantigen responsive, suggesting their participation in the protection of the fetus against pathogens in intrauterine infections. Together, our analyses unveil a unique cutaneous T cell type within the native skin microenvironment and point to fundamental differences in the immune surveillance between fetal and adult human skin.

Human Primary Dermal Fibroblasts Interacting with 3-Dimensional Matrices for Surgical Application Show Specific Growth and Gene Expression Programs.

Several types of 3-dimensional (3D) biological matrices are employed for clinical and surgical applications, but few indications are available to guide surgeons in the choice among these materials. Here we compare the in vitro growth of human primary fibroblasts on different biological matrices commonly used for clinical and surgical applications and the activation of specific molecular pathways over 30 days of growth. Morphological analyses by Scanning Electron Microscopy and proliferation curves showed that fibroblasts have different ability to attach and proliferate on the different biological matrices. They activated similar gene expression programs, reducing the expression of collagen genes and myofibroblast differentiation markers compared to fibroblasts grown in 2D. However, differences among 3D matrices were observed in the expression of specific metalloproteinases and interleukin-6. Indeed, cell proliferation and expression of matrix degrading enzymes occur in the initial steps of interaction between fibroblast and the investigated meshes, whereas collagen and interleukin-6 expression appear to start later. The data reported here highlight features of fibroblasts grown on different 3D biological matrices and warrant further studies to understand how these findings may be used to help the clinicians choose the correct material for specific applications.

Pannexin 3 regulates skin development via Epiprofin.

Pannexin 3 (Panx3), a member of the gap junction pannexin family is required for the development of hard tissues including bone, cartilage and teeth. However, the role of Panx3 in skin development remains unclear. Here, we demonstrate that Panx3 regulates skin development by modulating the transcription factor, Epiprofin (Epfn). Panx3-/- mice have impaired skin development and delayed hair follicle regeneration. Loss of Panx3 in knockout mice and suppression by shRNA both elicited a reduction of Epfn expression in the epidermis. In cell culture, Panx3 overexpression promoted HaCaT cell differentiation, cell cycle exit and enhanced Epfn expression. Epfn-/- mice and inhibition of Epfn by siRNA showed no obvious differences of Panx3 expression. Furthermore, Panx3 promotes Akt/NFAT signaling pathway in keratinocyte differentiation by both Panx3 ATP releasing channel and ER Ca2+ channel functions. Our results reveal that Panx3 has a key role factor for the skin development by regulating Epfn.