Gender Differences in Post-Operative Human Skin.

Although the impact of age, gender, and obesity on the skin wound healing process has been extensively studied, the data related to gender differences in aspects of skin scarring are limited. The present study performed on abdominal human intact and scar skin focused on determining gender differences in extracellular matrix (ECM) composition, dermal white adipose tissue (dWAT) accumulation, and Foxn1 expression as a part of the skin response to injury. Scar skin of men showed highly increased levels of COLLAGEN 1A1, COLLAGEN 6A3, and ELASTIN mRNA expression, the accumulation of thick collagen I-positive fibers, and the accumulation of α-SMA-positive cells in comparison to the scar skin of women. However, post-injured skin of women displayed an increase (in comparison to post-injured men's skin) in collagen III accumulation in the scar area. On the contrary, women's skin samples showed a tendency towards higher levels of adipogenic-related genes (PPARγ, FABP4, LEPTIN) than men, regardless of intact or scar skin. Intact skin of women showed six times higher levels of LEPTIN mRNA expression in comparison to men intact (p < 0.05), men post-injured (p < 0.05), or women post-injured scar (p < 0.05) skin. Higher levels of FOXN1 mRNA and protein were also detected in women than in men's skin. In conclusion, the present data confirm and extend (dWAT layer) the data related to the presence of differences between men and women in the skin, particularly in scar tissues, which may contribute to the more effective and gender-tailored improvement of skin care interventions.

Dermal white adipose tissue development and metabolism: The role of transcription factor Foxn1.

Intradermal adipocytes form dermal white adipose tissue (dWAT), a unique fat depot localized in the lower layer of the dermis. However, recognition of molecular factors regulating dWAT development, homeostasis, and bioactivity is limited. Using Foxn1-/- and Foxn1+/+ mice, we demonstrated that epidermally expressed Foxn1 regulates dWAT development and defines the adipogenic capacity of dermal fibroblasts. In intact and post-wounded skin, Foxn1 contributes to the initial stimulation of dWAT adipogenesis and participates in the modulation of lipid metabolism processes. Furthermore, Foxn1 activity strengthens adipogenic processes through Bmp2 and Igf2 signaling and regulates lipid metabolism in differentiated dermal fibroblasts. The results reveal the contribution of Foxn1 to dWAT metabolism, thus identifying possible targets for modulation and regulation of dWAT in physiological and pathological processes in the skin.

Hypoxia reveals a new function of Foxn1 in the keratinocyte antioxidant defense system.

Skin exposed to environmental threats, including injuries and oxidative stress, develops an efficient but not fully recognized system of repair and antioxidant protection. Here, using mass spectrometry analysis (LC-MS/MS), followed by in vitro and in vivo experiments, we provided evidence that Foxn1 in keratinocytes regulates elements of the electron transport chain and participates in the thioredoxin system (Txn2, Txnrd3, and Srxn1) induction, particularly in a hypoxic environment. We first showed that Foxn1 in keratinocytes upregulates glutathione thioredoxin reductase 3 (Txnrd3) protein expression, and high levels of Txnrd3 mRNA were detected in injured skin of Foxn1+/+ mice. We also showed that Foxn1 strongly downregulated the Ccn2 protein expression, participating in epidermal reconstruction after injury. An in vitro assay revealed that Foxn1 controls keratinocyte migration, stimulating it under normoxia and suppressing it under hypoxia. Keratinocytes overexpressing Foxn1 and exposed to hypoxia displayed a reduced ability to promote angiogenesis by downregulating Vegfa expression. In conclusion, this study showed a new mechanism in which Foxn1, along with hypoxia, participates in the activation of antioxidant defense and controls the functional properties of keratinocytes.

Comparative studies on the effect of pig adipose-derived stem cells (pASCs) preconditioned with hypoxia or normoxia on skin wound healing in mice.

Adipose-derived stem cells (ASCs) from human and animal fat have emerged as therapeutic alternatives for damaged tissues. Pre-conditioning of ASCs with hypoxia results in their functional enhancement, which might facilitate the process of healing. However, there is still a critical need for large-scale preclinical studies to reinforce the translation of these findings into clinical practice for humans and in veterinary medicine. Here, we adapted a full-thickness excisional skin wound mouse model to evaluate and compare the effect of pig adipose-derived stem cells (pASCs) cultured under normoxia (pASCs-Nor) or hypoxia (pASCs-Hyp) on the healing process. We show that pASCs-Hyp accelerated re-epithelialization, increased hyaluronic acid (HA) content, and decreased scar elevation index (SEI) during the late stage of healing (day 21). Transplantation of pASCs-Hyp also promoted expression of angiogenic marker VegfA and decreased levels of pro-scarring Tgfß1. Mice tolerated xenotransplantation of the pASCs with no impact on macrophage (CD68 -positive cell) content. However, wounds treated with pASCs-Hyp exhibited decreased elasticity at the early stage of healing and increased expression of Wnt signaling members including Wnt10a, Wnt11, and ß-catenin, which are associated with scar-forming wound repair. In conclusion, pASCs treatment may provide a critical step toward the evaluation of pASCs as therapeutically relevant cells in the context of wound healing.

Effect of Pig-Adipose-Derived Stem Cells' Conditioned Media on Skin Wound-Healing Characteristics In Vitro.

The primary mechanism by which adipose-derived stem cells (ASCs) exert their reparative or regenerative potential relies predominantly on paracrine action. Secretory abilities of ASCs have been found to be amplified by hypoxia pre-conditioning. This study investigates the impact of hypoxia (1% O2) on the secretome composition of pig ASCs (pASCs) and explores the effect of pASCs' conditioned media (CM) on skin cell functions in vitro and the expression of markers attributed to wound healing. Exposure of pASCs to hypoxia increased levels of vascular endothelial growth factor (VEGF) in CM-Hyp compared to CM collected from the cells cultured in normoxia (CM-Nor). CM-Hyp promoted the migratory ability of pig keratinocytes (pKERs) and delayed migration of pig dermal fibroblasts (pDFs). Exposure of pKERs to either CM-Nor or CM-Hyp decreased the levels of pro-fibrotic indicators WNT10A and WNT11. Furthermore, CM-Hyp enhanced the expression of KRT14, the marker of the basal epidermis layer. In contrast, CM-Nor showed a stronger effect on pDFs manifested by increases in TGFB1, COL1A1, COL3A1, and FN1 mRNA expression. The formation of three-dimensional endothelial cell networks was improved in the presence of CM-Hyp. Overall, our results demonstrate that the paracrine activity of pASCs affects skin cells, and this property might be used to modulate wound healing.

Histology Scoring System for Murine Cutaneous Wounds.

Monitoring wound progression over time is a critical aspect for studies focused on in-depth molecular analysis or on evaluating the efficacy of potential novel therapies. Histopathological analysis of wound biopsies can provide significant insight into healing dynamics, yet there is no standardized and reproducible scoring system currently available. The purpose of this study was to develop and statistically validate a scoring system based on parameters in each phase of healing that can be easily and accurately assessed using either Hematoxylin & Eosin (H&E) or Masson's Trichrome (MT) staining. These parameters included re-epithelization, epithelial thickness index, keratinization, granulation tissue thickness, remodeling, and the scar elevation index. The initial phase of the study was to (1) optimize and clarify healing parameters to limit investigator bias and variability; (2) compare the consistency of parameters assessed using H&E versus MT staining. During the validation phase of this study, the accuracy and reproducibility of this scoring system was independently iterated upon and validated in four different types of murine skin wound models (Excisional; punch biopsy; pressure ulcers; burn wounds). A total of n = 54 histology sections were randomized, blinded, and assigned to two groups of independent investigators (n = 5 per group) for analysis. The sensitivity of each parameter (ranging between 80% and 95%) is reported with illustrations on the appropriate assessment method using ImageJ software. In the validated scoring system, the lowest score (score:0) is associated with an open/unhealed wound as is evident immediately and within the first day postinjury, whereas the highest score (score:12) is associated with a completely closed and healed wound without excessive scarring. This study defines and describes the minimum recommended criteria for assessing wound healing dynamics using the SPOT skin wound score. The acronym SPOT refers to the academic and scientific institutions that were involved in the development of the scoring system, namely, Stellenbosch University, Polish Academy of Sciences, Obatala Sciences, and the University of Texas Southwestern.

Impairment of the Hif-1α regulatory pathway in Foxn1-deficient (Foxn1-/- ) mice affects the skin wound healing process.

Hypoxia and hypoxia-regulated factors (eg, hypoxia-inducible factor-1α [Hif-1α], factor inhibiting Hif-1α [Fih-1], thioredoxin-1 [Trx-1], aryl hydrocarbon receptor nuclear translocator 2 [Arnt-2]) have essential roles in skin wound healing. Using Foxn1-/- mice that can heal skin injuries in a unique scarless manner, we investigated the interaction between Foxn1 and hypoxia-regulated factors. The Foxn1-/- mice displayed impairments in the regulation of Hif-1α, Trx-1, and Fih-1 but not Arnt-2 during the healing process. An analysis of wounded skin showed that the skin of the Foxn1-/- mice healed in a scarless manner, displaying rapid re-epithelialization and an increase in transforming growth factor ß (Tgfß-3) and collagen III expression. An in vitro analysis revealed that Foxn1 overexpression in keratinocytes isolated from the skin of the Foxn1-/- mice led to reduced Hif-1α expression in normoxic but not hypoxic cultures and inhibited Fih-1 expression exclusively under hypoxic conditions. These data indicate that in the skin, Foxn1 affects hypoxia-regulated factors that control the wound healing process and suggest that under normoxic conditions, Foxn1 is a limiting factor for Hif-1α.

Dermal White Adipose Tissue (dWAT) Is Regulated by Foxn1 and Hif-1α during the Early Phase of Skin Wound Healing.

Dermal white adipose tissue (dWAT) is involved in the maintenance of skin homeostasis. However, the studies concerning its molecular regulation are limited. In the present paper, we ask whether the introduction of two transcription factors, Foxn1 and Hif-1α, into the post-wounded skin of Foxn1-/- mice regulates dWAT during wound healing (days 3 and 6). We have chosen lentivirus vectors (LVs) as a tool to deliver Foxn1 and Hif-1α into the post-wounded skin. We documented that combinations of both transgenes reduces the number, size and diameter of dermal adipocytes at the wound bed area. The qRT-PCR analysis of pro-adipogenic genes, revealed that LV-Hif-1α alone, or combined with LV-Foxn1, increases the mRNA expression of Pparγ, Glut 4 and Fasn at post-wounding day 6. However, the most spectacular stimulatory effect of Foxn1 and/or Hif-1α was observed for Igf2, the growth factor participating in adipogenic signal transduction. Our data also shows that Foxn1/Hif-1α, at post-wounding day 3, reduces levels of CD68 and MIP-1γ mRNA expression and the percentage of CD68 positive cells in the wound site. In conclusion, the present data are the first to document that Foxn1 and Hif-1α cooperatively (1) regulate dWAT during the proliferative phase of skin wound healing through the Igf2 signaling pathway, and (2) reduce the macrophages content in the wound site.

Wnt signaling and the transcription factor Foxn1 contribute to cutaneous wound repair in mice.

Aim: The transcription factor Foxn1 is a regulator of scar-ended cutaneous wound healing in mice. However, the link between Foxn1 and Wnt signaling has not been explored in the context of cutaneous repair. Here, we investigate the effects of ß-catenin-dependent and -independent Wnt signaling represented by Wnt10a and Wnt11, respectively, in healing of full-thickness cutaneous wounds in C57BL/6 mice. Material and Methods: Quantitative polymerase chain reaction, western blot, and immunostaining were performed to assess the spatial and temporal distribution of Wnt10a, Wnt11, and ß-catenin in skin during wound healing. A co-culture system consisting of keratinocytes transfected with an adenoviral vector carrying Foxn1-GFP and dermal fibroblasts (DFs) was employed to determine the influence of epidermal signals on the capacity of DFs to produce extracellular matrix (ECM) proteins in vitro. The levels of types I and III collagen in conditioned media from DFs cultures were examined via enzyme-linked immunosorbent assay. Results: The expression of Wnt10a, Wnt11, and ß-catenin increased at post-wounding days 14 and 21 when tissue remodeling occurred. Foxn1::Egfp transgenic mice experiments demonstrated that Wnts were abundant in the epidermis adjacent to the wound margin and to a lesser extent in the dermis. The Wnt10a signal colocalized with Foxn1-eGFP in the epithelial tongue and neo-epidermis during the initial stage of wound healing. Foxn1 overexpression in keratinocytes affected DFs function related to collagen synthesis. Conclusions: Wnt ligands contribute to cutaneous wound repair, predominantly by engagement in ECM maturation. The data indicates a possible relationship between Foxn1 and Wnts in post-traumatic skin tissue.

Adipose-Derived Stromal/Stem Cells from Large Animal Models: from Basic to Applied Science.

Adipose-derived stem cells (ASCs) isolated from domestic animals fulfill the qualitative criteria of mesenchymal stem cells, including the capacity to differentiate along multiple lineage pathways and to self-renew, as well as immunomodulatory capacities. Recent findings on human diseases derived from studying large animal models, have provided evidence that administration of autologous or allogenic ASCs can improve the process of healing. In a narrow group of large animals used in bioresearch studies, pigs and horses have been shown to be the best suited models for study of the wound healing process, cardiovascular and musculoskeletal disorders. To this end, current literature demonstrates that ASC-based therapies bring considerable benefits to animal health in both spontaneously occurring and experimentally induced clinical cases. The purpose of this review is to provide an overview of the diversity, isolation, and characterization of ASCs from livestock. Particular attention has been paid to the functional characteristics of the cells that facilitate their therapeutic application in large animal models of human disease. In this regard, we describe outcomes of ASCs utilization in translational research with pig and horse models of disease. Furthermore, we evaluate the current status of ASC-based therapy in veterinary practice, particularly in the rapidly developing field of equine regenerative medicine. In conclusion, this review presents arguments that support the relevance of animal ASCs in the field of regenerative medicine and it provides insights into the future perspectives of ASC utilization in animal husbandry.

Age, Diet and Epidermal Signaling Modulate Dermal Fibroblasts' Adipogenic Potential.

The recognition of a distinct fat depot, the dermal white adipose tissue (dWAT), points out the complexity of the interaction among skin resident cells: keratinocytes, dermal fibroblasts (DFs) and adipocytes in response to physiological (diet, age) and pathological (injury) stimulations. dWAT has been recognized as a significant contributor to thermoregulation, hair cycle, immune response, wound healing and scarring. In this study, we examined age- and diet-related changes in dWAT modulation and DFs' adipogenic potential. The data showed that diet modulates dWAT expansion predominantly by hypertrophy, whereas age affects the pool of adipocyte progenitor cells in the skin indicating its role in dWAT hyperplasia. Analysis of DFs' migratory abilities in the model of skin explants isolated from the skin of young, old, low (LFD)- or high (HFD)-fat diet C56BL/6 mice revealed that HFD, regardless of animal age has the most profound stimulatory impact of DF migration. We determined that the adipogenic potential of DFs is comparable to stromal vascular fraction (SVF) of inguinal fat depot and ear mesenchymal stem cells (EMSC). We also showed the stimulatory role of epidermally expressed transcription factor Foxn1 on adipogenic signaling: bone morphogenetic protein 2 (Bmp2) and insulin-like growth factor 2 (Igf2) in keratinocytes.

The effect of hypoxia on the proteomic signature of pig adipose-derived stromal/stem cells (pASCs).

Human adipose-derived stem cells (ASCs) have potential to improve wound healing; however, their equivalents from domestic animals have received less attention as an alternative cell-based therapy for animals or even humans. Hypoxia is essential for maintaining stem cell functionality in tissue-specific niches. However, a cellular response to low oxygen levels has not been demonstrated in pig ASCs. Hence, the goal of our study was to characterize ASCs isolated from the subcutaneous fat of domestic pigs (pASCs) and examine the effect of hypoxia on their proteome and functional characteristics that might reproduce pASCs wound healing ability. Analysis of immunophenotypic and functional markers demonstrated that pASCs exhibited characteristics of mesenchymal stem cells. Proteomic analysis revealed 70 differentially abundant proteins between pASCs cultured under hypoxia (1% O2) or normoxia (21% O2). Among them, 42 proteins were enriched in the cells exposed to low oxygen, whereas 28 proteins showed decrease expression following hypoxia. Differentially expressed proteins were predominantly involved in cell metabolism, regulation of focal and intracellular communication, and attributed to wound healing. Functional examination of hypoxic pASCs demonstrated acquisition of contractile abilities in vitro. Overall, our results demonstrate that hypoxia pre-conditioning impacts the pASC proteome signature and contractile function in vitro and hence, they might be considered for further cell-based therapy study on wound healing.

Cutaneous wound healing in aged, high fat diet-induced obese female or male C57BL/6 mice.

Since there are limited studies analyzing the impact of age, sex and obesity on cutaneous repair, the current study evaluated excisional skin wound healing as a function of age, sex and diet in C57BL/6 mice subjected to either low (LFD) or high (HFD) fat diet. Older mice accumulated increased body fat relative to younger mice under HFD. Skin wound healing at particular stages was affected by age in the aspect of Tgfß-1, MCP-1, Mmp-9 and Mmp-13 expression. The most profound, cumulative effect was observed for the combination of two parameters: age and sex. While skin of younger males displayed extremely high collagen 1 and collagen 3 expression, younger females showed exceptionally high Mmp-13 expression at day 3 and 7 after injury. Diet as a single variable modified the thickness of dermis due to increased dermal White Adipose Tissue (dWAT) accumulation in mice fed HFD. The combination of age and diet affected the re-epithelialization and inflammatory response of injured skin. Overall, our data indicate that age has the most fundamental impact although all components (age, sex and diet) contribute to skin repair.

The Transcription Factor FOXN1 Regulates Skin Adipogenesis and Affects Susceptibility to Diet-Induced Obesity.

FOXN1, a transcription factor expressed in the epidermis, regulates keratinocyte differentiation and participates in skin wound healing. In this study, we explored the impact of FOXN1 insufficiency on diet-stimulated weight gain and dermal white adipose tissue regulation in the intact and wounded skin of FOXN1eGFP/+ (heterozygotes, FOXN1-insufficient) mice in the context of age and diet. The results showed that on a high-fat diet, FOXN1eGFP/+ mice gained significantly less body weight than their FOXN1+/+ counterparts (FOXN1-sufficient mice). The intact and wounded skin of FOXN1eGFP/+ mice displayed abrogated expression of the master regulators of adipogenesis, PPARγ, FABP4, and leptin, which decreased with age in FOXN1+/+ mice. FOXN1 insufficiency also resulted in a decreased percentage of adipocyte-committed precursor cells (CD24+) in the skin. The proadipogenic pathway genes Bmp2, Igf2, and Mest showed a gradual decrease in expression that accompanied the gradual inactivation of FOXN1 in the skin of FOXN1+/+, FOXN1eGFP/+, and FOXN1eGFP/eGFP (lack of FOXN1) mice. Bone morphogenetic protein 2 and insulin-like growth factor 2 signals colocalized with FOXN1-eGFP in the epidermis and in hair follicles. These data demonstrated that FOXN1 initiates the cascade of adipogenic signaling that regulates skin homeostasis and wound healing and affects susceptibility to diet-induced obesity.

Foxn1 in Skin Development, Homeostasis and Wound Healing.

Intensive research effort has focused on cellular and molecular mechanisms that regulate skin biology, including the phenomenon of scar-free skin healing during foetal life. Transcription factors are the key molecules that tune gene expression and either promote or suppress gene transcription. The epidermis is the source of transcription factors that regulate many functions of epidermal cells such as proliferation, differentiation, apoptosis, and migration. Furthermore, the activation of epidermal transcription factors also causes changes in the dermal compartment of the skin. This review focuses on the transcription factor Foxn1 and its role in skin biology. The regulatory function of Foxn1 in the skin relates to physiological (development and homeostasis) and pathological (skin wound healing) conditions. In particular, the pivotal role of Foxn1 in skin development and the acquisition of the adult skin phenotype, which coincides with losing the ability of scar-free healing, is discussed. Thus, genetic manipulations with Foxn1 expression, specifically those introducing conditional Foxn1 silencing in a Foxn1+/+ organism or its knock-in in a Foxn1−/− model, may provide future perspectives for regenerative medicine.

Effect of TGFß1, TGFß3 and keratinocyte conditioned media on functional characteristics of dermal fibroblasts derived from reparative (Balb/c) and regenerative (Foxn1 deficient; nude) mouse models.

Skin injuries in mammals are healed through repair or regeneration. Our previous studies demonstrated that deficient expression of the transcription factor Foxn1 in epidermis of nude mice accounts for their skin's pronounced regenerative properties. Since homeostasis within the skin depends on complex interactions between the epidermal and underlying dermal layers, the present study characterizes and compares isolated dermal fibroblasts (DFs) between regenerative nude (Foxn1 deficient) mice and their wild-type Balb/c counterparts. Nude DFs exhibited a higher cumulative number of population doublings (cumulative PD) at low seeding density and increased adipogenic differentiation capacity relative to their Balb/c DF counterparts. Nude DFs displayed reduced migration and gel contraction, functional features associated with wound healing. The comparison of transforming growth factor ß family (TGFß) expression showed significantly higher levels of Tgfß3 transcript between nude and Balb/c mice but no differences were detected for Tgfß1. Nude DFs were specifically sensitive to the presence of the pro-regenerative TGFß3 isoform, showing increased collagen I deposition and alpha smooth muscle actin expression. Viability of Balb/c DFs was stimulated by keratinocyte conditioned media (KCM) from Balb/c (Foxn1 active) but inhibited by nude (Foxn1 deficient) KCM. In contrast, nude DFs did not respond to either KCMs with respect to their metabolic activity. Collectively, the enhanced plasticity and greater sensitivity of nude DFs to TGFß3 stimulation are indicative of and consistent with their pro-regenerative characteristics. These data support the hypothesis that epidermal Foxn1 plays a critical role in determining the DFs regenerative phenotype.

Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing.

Recent studies have shown that the transcription factor Foxn1, which is expressed in keratinocytes, is involved in the skin wound healing process, yet how Foxn1 functions remains largely unknown. Our latest data indicate that Foxn1 drives skin healing via engagement in re-epithelization and the epithelial-mesenchymal transition (EMT) process. In the present study, 2D-DIGE proteomic profiling analysis of in vitro cultured keratinocytes transfected with adenoviral vector carrying Foxn1-GFP or GFP alone (control) revealed forty proteins with differential abundance between the compared groups. Among the proteins with Foxn1-dependent expression, several enable adaptation to hypoxia. Subsequent experiments revealed that hypoxic conditions (1% O2) stimulate endogenous and exogenous (transfected Ad-Foxn1) Foxn1 expression in cultured keratinocytes. A proteomics analysis also identified proteins that can act as a factors controlling the balance between cell proliferation, differentiation and apoptosis in response to Foxn1. We also showed that in C57BL/6 keratinocytes, the stimulation of Foxn1 by hypoxia is accompanied by increases in Mmp-9 expression. These data corroborate the detected co-localization of Foxn1 and Mmp-9 expression in vivo in post-wounding skin samples of Foxn1::Egfp transgenic mice. Together, our data indicate that Foxn1 orchestrates cellular changes in keratinocytes in both physiological (self-renewal) and pathological (skin wound healing) contexts.

Foxn1 and Mmp-9 expression in intact skin and during excisional wound repair in young, adult, and old C57Bl/6 mice.

The transcription factor Foxn1 is essential for skin development. Our previous studies performed on young C57BL/6J mice model showed that Foxn1 acts as regulator of the skin wound healing process. The present study extended our initial research regarding the expression and potential role of Foxn1 in the intact and wounded skin as a function of animal age and stage of the wound healing process. We analyzed Foxn1 and Mmp-9 expression in the intact and postinjured skin of young, adult, and old C57BL/6J and transgenic Foxn1::Egfp mice. The similar levels of epidermal Foxn1 mRNA expression were detected in young and adult C57BL/6J mice and higher levels in old animals. Postinjured skin tissues displayed a gradual decrease of Foxn1 mRNA expression at Days 1, 5, and 7 after injury. Foxn1-eGFP positive cells were abundant at wound margin and in re-epithelialized epidermis at postwounded Days 1, 5, and 7 and colocalized with E-cadherin and Mmp-9. Postwounded skin at Days 14-36 displayed Foxn1-eGFP cells in the epidermis and in the dermal part of the skin (papillary dermis). A subset of Foxn1-eGFP positive cells in the papillary dermis expressed the myofibroblast marker αSMA. Flow cytometric analysis of cells isolated from postwounded (Day 7) skin tissues showed a significant increase in the percentage of Foxn1-eGFP positive cells with phenotype of double positivity for E-cadherin/N-cadherin (epithelial/mesenchymal markers). Collectively, these data identify the transcription factor Foxn1 as a potential key epidermal regulator modifying both epidermal and dermal healing processes after cutaneous wounding.

Neotenic phenomenon in gene expression in the skin of Foxn1- deficient (nude) mice - a projection for regenerative skin wound healing.

BACKGROUND: Mouse fetuses up to 16 day of embryonic development and nude (Foxn1- deficient) mice are examples of animals that undergo regenerative (scar-free) skin healing. The expression of transcription factor Foxn1 in the epidermis of mouse fetuses begins at embryonic day 16.5 which coincides with the transition point from scar-free to scar-forming skin wound healing. In the present study, we tested the hypothesis that Foxn1 expression in the skin is an essential condition to establish the adult skin phenotype and that Foxn1 inactivity in nude mice keeps skin in the immature stage resembling the phenomena of neoteny. RESULTS: Uninjured skin of adult C57BL/6J (B6) mice, mouse fetuses at days 14 (E14) and 18 (E18) of embryonic development and B6.Cg-Foxn1 nu (nude) mice were characterized for their gene expression profiles by RNA sequencing that was validated through qRT-PCR, Western Blot and immunohistochemistry. Differentially regulated genes indicated that nude mice were more similar to E14 (model of regenerative healing) and B6 were more similar to E18 (model of reparative healing). The up-regulated genes in nude and E14 mice were associated with tissue remodeling, cytoskeletal rearrangement, wound healing and immune response, whereas the down-regulated genes were associated with differentiation. E14 and nude mice exhibit prominent up-regulation of keratin (Krt23, -73, -82, -16, -17), involucrin (Ivl) and filaggrin (Flg2) genes. The transcription factors associated with the Hox genes known to specify cell fate during embryonic development and promote embryonic stem cells differentiation were down-regulated in both nude and E14. Among the genes enriched in the nude skin but not shared with E14 fetuses were members of the Wnt and matrix metalloproteinases (Mmps) families whereas Bmp and Notch related genes were down-regulated. CONCLUSIONS: In summary, Foxn1 appears to be a pivotal control element of the developmental program and skin maturation. Nude mice may be considered as a model of neoteny among mammals. The resemblance of gene expression profiles in the skin of both nude and E14 mice are direct or indirect consequences of the Foxn1 deficiency. Foxn1 appears to regulate the balance between cell proliferation and differentiation and its inactivity creates a pro-regenerative environment.

Foxn1 Transcription Factor Regulates Wound Healing of Skin through Promoting Epithelial-Mesenchymal Transition.

Transcription factors are key molecules that finely tune gene expression in response to injury. We focused on the role of a transcription factor, Foxn1, whose expression is limited to the skin and thymus epithelium. Our previous studies showed that Foxn1 inactivity in nude mice creates a pro-regenerative environment during skin wound healing. To explore the mechanistic role of Foxn1 in the skin wound healing process, we analyzed post-injured skin tissues from Foxn1::Egfp transgenic and C57BL/6 mice with Western Blotting, qRT-PCR, immunofluorescence and flow cytometric assays. Foxn1 expression in non-injured skin localized to the epidermis and hair follicles. Post-injured skin tissues showed an intense Foxn1-eGFP signal at the wound margin and in leading epithelial tongue, where it co-localized with keratin 16, a marker of activated keratinocytes. This data support the concept that suprabasal keratinocytes, expressing Foxn1, are key cells in the process of re-epithelialization. The occurrence of an epithelial-mesenchymal transition (EMT) was confirmed by high levels of Snail1 and Mmp-9 expression as well as through co-localization of vimentin/E-cadherin-positive cells in dermis tissue at four days post-wounding. Involvement of Foxn1 in the EMT process was verified by co-localization of Foxn1-eGFP cells with Snail1 in histological sections. Flow cytometric analysis showed the increase of double positive E-cadherin/N-cadherin cells within Foxn1-eGFP population of post-wounded skin cells isolates, which corroborated histological and gene expression analyses. Together, our findings indicate that Foxn1 acts as regulator of the skin wound healing process through engagement in re-epithelization and possible involvement in scar formation due to Foxn1 activity during the EMT process.