Keratinocyte Integrin α3ß1 Promotes Efficient Healing of Wound Epidermis.

To date, studies of the role for epidermal integrin α3ß1 in cutaneous wound re-epithelialization have produced conflicting results: wound studies in skin from global α3-null neonatal mice have implicated the integrin in promoting timely wound re-epithelialization, whereas studies in adult mice with constitutive, epidermal-specific α3ß1 deletion have not. The objective of this study was to utilize a model of inducible α3ß1 deletion in the epidermis to clarify the role of α3ß1 in the healing of adult wounds. We utilized the recently developed transgenic K14Cre-ERT::α3flx/flx mice (ie, inducible α3 epidermal knockout), permitting us to delete floxed Itga3 alleles (α3flx/flx) from epidermis just prior to wounding with topical treatment of 4-hydroxytamoxifen. This allows for the elucidation of α3ß1-dependent wound healing in adult skin, free from compensatory mechanisms that may occur after embryonic deletion of epidermal α3ß1 in the widely used constitutive α3ß1-knockout mouse. We found that re-epithelializing wound gaps are larger in inducible α3 epidermal knockout mice than in control mice, indicating delayed healing, and that epidermal integrin α3ß1 promotes healing of wounds, at least in part by enhancing keratinocyte proliferation. This work provides essential rationale for future studies to investigate integrin α3ß1 as a therapeutic target to facilitate wound healing.

Genome-wide RNA-seq, DNA methylation and small RNA-seq analysis unraveled complex gene regulatory networks in psoriasis pathogenesis.

Psoriasis is a complex inflammatory skin disease characterized by reversible albeit relapsing red scaly plaques in the skin of a patient. In addition to the genetic predisposition, involvement of epigenetic and non-coding RNAs have also been liked with the disease. Nevertheless, any comprehensive study involving transcriptomic, small-RNA and DNA methylation at the genomic level from same patients is lacking. To investigate the complex regulation of molecular pathways in psoriasis, we carried out multi-omics integrative analysis of RNA-sequencing, small RNA-sequencing and DNA methylation profiling from the psoriatic and adjacent normal skin tissues. Our multi-omics analysis identified the genes and biological processes regulated either independently or in combination by DNA methylation and microRNAs. We identified miRNAs that specifically regulated keratinocyte hyper-proliferation, and cell cycle progression and checkpoint signaling in psoriasis. On contrary, DNA methylation was found to be more predominant in regulating immune and inflammatory responses, another causative factor in psoriasis pathogenesis. Many characteristic pathways in psoriasis e.g., Th17 cell differentiation and JAK-STAT signaling, were found to be regulated by both miRNAs and DNA methylation. We carried out functional characterization of a downregulated miRNA hsa-let-7c-5p, predicted to target upregulated genes in psoriasis involved in cell cycle processes, Th17 cell differentiation and JAK-STAT signaling pathways. Overexpression of hsa-let-7c-5p in keratinocytes caused the downregulation of its target genes, resulting in reduced cell proliferation and migration rates, demonstrating potential of miRNAs in regulating psoriasis pathogenesis. In conclusion, our findings identified distinct and shared gene-networks regulated by DNA methylation and miRNAs of a complex disease with reversible phenotype.

Epoxytiglianes induce keratinocyte wound healing responses via classical protein kinase C activation to promote skin re-epithelialization.

Epoxytiglianes are a novel class of diterpene esters. The prototype epoxytigliane, EBC-46 (tigilanol tiglate), is a potent anti-cancer agent in clinical development for local treatment of a range of human and animal tumors. EBC-46 also consistently promotes wound re-epithelialization at the treatment sites, mediated via activation of classical protein kinase C (PKC) isoforms. We have previously shown that epoxytiglianes stimulate proliferative and wound repopulation responses in immortalized human skin keratinocytes (HaCaTs) in vitro, abrogated by pan-PKC inhibitor, bisindolylmaleimide-1. In this study, we further investigate the specific PKC isoforms responsible for inducing such wound healing responses, following HaCaT treatment with 1.51 nM-15.1 µM EBC-46 or analogue, EBC-211. Classical PKC inhibition by GÓ¦6976 (1 µM), significantly attenuated epoxytigliane induced, HaCaT proliferation and wound repopulation at all epoxytigliane concentrations. PKC-ßI/-ßII isoform inhibition by enzastaurin (1 µM), significantly inhibited HaCaT proliferation and wound repopulation responses induced by both epoxytiglianes, especially at 1.51-151 nM. PKC-α inhibitor, Ro 31-8220 mesylate (10 nM), exerted lesser inhibitory effects on HaCaT responses. Epoxytigliane changes in key keratin (KRT17) and cell cycle (cyclin B1, CDKN1A) protein levels were partly attenuated by GÓ¦6976 and enzastaurin. GÓ¦6976 also inhibited increases in matrix metalloproteinase (MMP-1, MMP-7, MMP-10) activities. Phospho-PKC (p-PKC) studies confirmed that epoxytiglianes transiently activated classical PKC isoforms (p-PKCα, p-PKC-ßI/-ßII, p-PKCγ) in a dose- and time-dependent manner. By identifying how epoxytiglianes stimulate classical PKCs to facilitate keratinocyte healing responses and re-epithelialization, these findings support further epoxytigliane development as topical therapeutics for clinical situations involving impaired re-epithelialization, such as non-healing wounds in skin.

Streptococcus canis transcriptomic modifications in host cell entry environments of human keratinocytes.

BACKGROUND: Streptococcus canis is a commensal bacterium in companion animals. This microorganism can infect humans who have been in deep contact with or bitten by pet dogs, suggesting that the skin/soft tissue is one of infection entry sites. To understand pathological process in human cells, we aimed to determine S. canis transcriptomic changes in invasive environments of human keratinocytes. METHODS: We selected one isolate from candidates with whole-genome sequences, based on re-obtained cell invasion ability (CIA) data into human keratinocytes along with bacterial cytotoxicity. RNA-sequencing was conducted for the samples at baselines and 2 h/5 hr post-inoculation using NovaSeq 6000. Global/differential gene expression analyses [principal component analysis (PCA)/k-means clustering analysis/differentially expressed gene (DEG) analyses] were performed. We classified DEGs into their functional categories. To validate transcriptomic results, we did quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. RESULTS: FU1 isolate was selected from seven candidates, based on re-obtained CIA data with less cytotoxicity. Total read bases of 6.17-9.02 Gbp were obtained by RNA-sequencing. PCA and k-means clustering analysis indicated clustering according to their inoculation times. Volcano plots and Venn diagrams revealed that S. canis invasion into keratinocytes produced altered distributions of many genes. Gene ontology enrichment analysis showed most of the gene expressions were downregulated. DEG functional analysis showed the downregulated DEGs belonging to energy production and conversion/carbohydrate transport and metabolism/amino acid transport and metabolism/nucleotide transport and metabolism, with the upregulated DEGs belonging to transcription. qRT-PCR assays for downregulated/upregulated expressions of four genes (pgk-slo/opuAA-kdpB) validated transcriptomic results. CONCLUSION: Our observations suggest that S. canis can downregulate its metabolism-associated gene expressions in human keratinocyte environments. The observed gene expression changes can imply the latent infection in human cells. Further investigation is needed to elucidate the underlying mechanisms for the latent infection.

Sustained Repigmentation in Vitiligo and Leukodermas Using Melanocyte-Keratinocyte Transplantation: 7 Years of Data.

Background: The autologous non-cultured melanocyte-keratinocyte transplantation procedure (MKTP) has emerged as an effective treatment for various types of vitiligo and leukodermas. However, there is limited data on the long-term outcomes of the MKTP, especially in Thai patients. Objective: To assess the long-term efficacy and safety of the MKTP in patients with vitiligo and other leukodermas. Methods: This retrospective observational study analyzed data from 23 patients who underwent the MKTP for vitiligo and other leukodermas at the Siriraj MKTP Clinic, Thailand, and had a follow-up period exceeding 12 months. Clinical characteristics and MKTP specifics were evaluated. Repigmentation outcomes were assessed using the Vitiligo Area Scoring Index (VASI). Results: Of the 23 patients (24 treated lesions), 78.3% had segmental vitiligo, while the others had nevus depigmentosus, nonsegmental vitiligo, or piebaldism. Most lesions (70.8%) were located on the face. At the 12-month follow-up, repigmentation showed an 80.8% ± 19.3% VASI improvement, which was sustained over 84 months with an 80%-90% VASI improvement. There was no statistically significant difference in repigmentation outcomes between facial and non-facial lesions. Conclusion: The MKTP demonstrated long-term efficacy and safety in treating vitiligo and other leukodermas, with sustained repigmentation over 84 months. These findings support the use of the MKTP as an effective treatment option for patients with refractory vitiligo and leukodermas, particularly within Thai populations.

Warmer Temperatures Are More Predictive Than Solar Radiation for the Number of Skin Cancer Removal Procedures.

BACKGROUND: Solar radiation is the primary risk factor for skin cancer, with personal exposure influenced by environmental and behavioral factors. At higher temperatures, behavioral changes increase solar radiation exposure. OBJECTIVES: Examine the relationships between solar radiation, ambient temperature, age, and skin cancer. METHODS: For the contiguous United States, we obtained the state mean global horizontal irradiance (GHI), daily maximum temperature, and number of skin cancer removals in the Medicare population. For skin cancer removals, we defined more sun-exposed skin as the head, neck, hands, and feet, and less sun-exposed skin as the trunk, arms, and legs. RESULTS: By comparing the temperature thresholds 17°C, 20°C, 24°C, 27°C, 31°C, and 34°C, we found that the annual number of days above 24°C was the strongest temperature-related predictor of skin cancer removals. Multivariable linear regression showed that the number of days above 24°C predicted more skin cancer removals for all body locations and less sun-exposed skin (p = 0.008 and p = 0.003, respectively), while GHI did not (p = 0.1 and p = 0.8, respectively). GHI only predicted more skin cancer removals for more sun-exposed skin (p = 0.02). CONCLUSION: More days above 24°C was a better predictor of skin cancer removals than GHI for all skin locations and less sun-exposed skin, suggesting that the behavioral changes occurring at warmer temperatures are more predictive of skin cancer removals than solar irradiance. Due to rising global temperatures, skin cancer incidence may further increase. Temperature-related behavioral changes represent a potential target for skin cancer prevention efforts.

Radiofrequency Currents Modulate Inflammatory Processes in Keratinocytes.

Keratinocytes play an essential role in the inflammatory phase of wound regeneration. In addition to migrating and proliferating for tissue regeneration, they produce a large amount of cytokines that modulate the inflammatory process. Previous studies have shown that subthermal treatment with radiofrequency (RF) currents used in capacitive resistive electric transfer (CRET) therapy promotes the proliferation of HaCat keratinocytes and modulates their cytokine production. Although physical therapies have been shown to have anti-inflammatory effects in a variety of experimental models and in patients, knowledge of the biological basis of these effects is still limited. The aim of this study was to investigate the effect of CRET on keratinocyte proliferation, cytokine production (IL-8, MCP-1, RANTES, IL-6, IL-11), TNF-α secretion, and the expression of MMP9, MMP1, NF-κB, ERK1/2, and EGFR. Human keratinocytes (HaCat) were treated with an intermittent 448 kHz electric current (CRET signal) in subthermal conditions and for different periods of time. Cell proliferation was analyzed by XTT assay, cytokine and TNF-α production by ELISA, NF-κB expression and activation by immunofluorescence, and MMP9, MMP1, ERK1/2, and EGF receptor expression and activation by immunoblot. Compared to a control, CRET increases keratinocyte proliferation, increases the transient release of MCP-1, TNF-α, and IL-6 while decreasing IL-8. In addition, it modifies the expression of MMPs and activates EGFR, NF-κB, and ERK1/2 proteins. Our results indicate that CRET reasonably modifies cytokine production through the EGF receptor and the ERK1/2/NF-κB pathway, ultimately modulating the inflammatory response of human keratinocytes.

Linoleic Acid Induces Metabolic Reprogramming and Inhibits Oxidative and Inflammatory Effects in Keratinocytes Exposed to UVB Radiation.

Linoleic acid (LA), the primary ω-6 polyunsaturated fatty acid (PUFA) found in the epidermis, plays a crucial role in preserving the integrity of the skin's water permeability barrier. Additionally, vegetable oils rich in LA have been shown to notably mitigate ultraviolet (UV) radiation-induced effects, including the production of reactive oxygen species (ROS), cellular damage, and skin photoaging. These beneficial effects are primarily ascribed to the LA in these oils. Nonetheless, the precise mechanisms through which LA confers protection against damage induced by exposure to UVB radiation remain unclear. This study aimed to examine whether LA can restore redox and metabolic equilibria and to assess its influence on the inflammatory response triggered by UVB radiation in keratinocytes. Flow cytometry analysis unveiled the capacity of LA to diminish UVB-induced ROS levels in HaCaT cells. GC/MS-based metabolomics highlighted significant metabolic changes, especially in carbohydrate, amino acid, and glutathione (GSH) metabolism, with LA restoring depleted GSH levels post-UVB exposure. LA also upregulated PI3K/Akt-dependent GCLC and GSS expression while downregulating COX-2 expression. These results suggest that LA induces metabolic reprogramming, protecting against UVB-induced oxidative damage by enhancing GSH biosynthesis via PI3K/Akt signaling. Moreover, it suppresses UVB-induced COX-2 expression in HaCaT cells, making LA treatment a promising strategy against UVB-induced oxidative and inflammatory damage.

Sphingosine 1-phosphate receptor 2 in keratinocytes plays a key role in reducing inflammation in psoriasis.

Background: Psoriasis is an inflammatory skin condition where immune cells play a significant role. The importance of the cross-talk between keratinocytes and immune cells in the pathogenesis of psoriasis has recently been reaffirmed. Recent studies have found that several S1PR functional antagonists, other than S1PR2, are effective in improving psoriasis. This study aims to investigate the role of S1PR2 in psoriasis, that has not been investigated before. Methods: Spatial transcriptomics, RT-qPCR, and flow cytometry were used to map the immune cell landscape and its association with metabolic pathways in an imiquimod (IMQ)-induced psoriasis-like inflammation in S1pr2fl/fl K14-Cre mice that could not sense sphingosine-1-phosphate (S1P) in the epidermis through the S1PR2 receptor. Results: Our analysis suggests that S1PR2 in keratinocytes plays a major role in psoriasis-like inflammation compared to other S1PRs. It acts as a down-regulator, inhibiting the recruitment of Th17 cells into the skin. In IMQ-induced psoriasis skin, both S1pr2-/- and S1pr2fl/fl K14-Cre mice showed higher expressions of proinflammatory cytokines such as TNF-α, IL-17A, and IL-1ß together with higher expressions of MyD88/NF-κB pathway compared to the wild-type mice. Remarkably, in IMQ-treated mice, the deletion of S1pr2 in keratinocytes only resulted in a larger population of Th17 cells in skin-draining lymph nodes. Other S1PR modulators did not improve the worsening of psoriasis-like inflammation caused by S1PR2 deficiency in keratinocytes. Conclusion: This study reaches two main conclusions: signals from keratinocytes play a central role in creating an immune environment that promotes the development of psoriasis, and stimulating S1PR2, instead of suppressing it, represents a potential therapeutic approach for psoriasis.

Trivalent arsenicals induce skin toxicity through thiol depletion.

Arsenic, a widespread environmental contaminant, is highly toxic to human health. Arsenic exposure is associated with the occurrence of skin lesions and diseases. This study investigated the dermal toxicity of trivalent arsenicals (AsIII and MMAIII) and its underlying mechanism using human keratinocyte cell line and ex vivo porcine skin. AsIII and MMAIII induced concentration-dependent cell apoptosis and necrosis in HaCaT cells, which was confirmed in ex vivo porcine skin. AsIII and MMAIII increased reactive oxygen species generation and GSH depletion. Interestingly, radical scavenger antioxidants such as Vitamin C failed to mitigate arsenic-induced cytotoxicity, while thiol-containing compounds effectively alleviated it, suggesting a key role of thiol depletion in the trivalent arsenical-induced dermal toxicity. DMSA showed the strongest protective effects against AsIII and MMAIII-induced cytotoxicity in HaCaT cells. Of note, DMSA restored arsenical-induced tissue damage, and reduced the apoptosis in ex vivo porcine skin, highlighting its potential use to alleviate arsenic-induced skin lesions and diseases.

Trehalose Prevents IL-4/IL-13-Induced Skin Barrier Impairment by Suppressing IL-33 Expression and Increasing NRF2 Activation in Human Keratinocytes In Vitro.

Skin barrier dysfunction initiates or deteriorates various cutaneous problems, such as atopic dermatitis. At high concentrations, the nonreducing disaccharide trehalose (α-d-glucopyranosyl α-d-glucopyranoside) induces a transient senescence-like state in fibroblasts and promotes wound repair. In this study, we investigated the effect of trehalose on normal human keratinocytes and demonstrated its specific role in the skin barrier. RNA-sequencing analysis revealed that trehalose regulates the expression of many skin barrier-associated genes. T helper 2 cytokines IL-4/IL-13 were observed to downregulate several differentiation markers (FLG, loricrin, keratin 1, and keratin 10) and epidermal antimicrobial proteins in monolayer-cultured keratinocytes and living skin equivalents and impaired skin barrier function in living skin equivalents, all of which were significantly upregulated or restored by trehalose. Trehalose inhibited IL-33 expression and reduced nuclear IL-33 levels by activating MAPK/extracellular signal-regulated kinase kinase 5-extracellular signal-regulated kinase 5 and suppressing extracellular signal-regulated kinase kinase 1/2-extracellular signal-regulated kinase pathway. It also increased NRF2 activation to trigger antioxidant enzyme production through JNK, thus neutralizing IL-4/IL-13-mediated oxidative stress. Trehalose prevented IL-4/IL-13-mediated signal transducer and activator of transcription 3/signal transducer and activator of transcription 6 activation and restored IL-4/IL-13-suppressed skin barrier molecules through IL-33 downregulation and NRF2 activation. This study demonstrated that trehalose may play a role in skin barrier repair in atopic dermatitis.

Vascular Endothelial Growth Factor a Promotes Chronic Itch via VEGFA-VEGFR2-PI3K-TRPV1 Axis in Allergic Contact Dermatitis.

Introduction: Allergic contact dermatitis (ACD), a prevalent skin disorder affecting up to 20% of the population, triggers significant discomfort and health implications. Our research investigates the pivotal role of Vascular Endothelial Growth Factor A (VEGFA) in chronic itching associated with ACD. Methods: Bioinformatics methods were utilized to identify differentially expressed genes (DEGs) between ACD models and patients. In vivo models of chronic pruritus in mice induced by 2,4-dinitrofluorobenzene (DNFB) were employed. Mice were administered subcutaneously with a VEGFA inhibitor, sFlt1, and compared to a control group. Real-time RT-PCR, Western blot, and immunohistochemical staining were performed to evaluate VEGFA expression and the impact of sFlt1 on itching behavior. Results: The analysis revealed that VEGFA is significantly upregulated in ACD skin, primarily expressed by keratinocytes. Administration of the VEGFA inhibitor sFlt1 in the ACD mouse model led to a substantial reduction in scratching behavior, indicating that VEGFA may mediate pruritus through the VEGFA-VEGFR2-PI3K-TRPV1 signaling pathway. Discussion: These findings suggest that VEGFA plays a crucial role in ACD-associated pruritus and may serve as a potential therapeutic target. However, further research is required to validate these findings and to explore additional molecular pathways involved in the pruritic response in ACD.

Transglutaminase 1: Emerging Functions beyond Skin.

Transglutaminase enzymes catalyze Ca2+- and thiol-dependent posttranslational modifications of glutamine-residues that include esterification, hydrolysis and transamidation, which results in covalent protein-protein crosslinking. Among the eight transglutaminase family members in mammals, transglutaminase 1 (TG1) plays a crucial role in skin barrier formation via crosslinking and insolubilizing proteins in keratinocytes. Despite this established function in skin, novel functions have begun merging in normal tissue homeostasis as well as in pathologies. This review summarizes our current understanding of the structure, activation, expression and activity patterns of TG1 and discusses its putative novel role in other tissues, such as in vascular integrity, and in diseases, such as cancer and fibrosis.

Isosilybin A exhibits anti-inflammatory properties in rosacea by inhibiting MAPK pathway and M1 macrophage polarization.

Rosacea is a chronic inflammatory skin disease, which is prone to flares and requires continuous management and treatment. However, long-term use of drugs can lead to additional adverse drug reactions. Based on the comorbid relationship between rosacea and Parkinson's disease, bioinformatics and network pharmacology analysis were used to identify a safer drug for rosacea. It has been demonstrated that ISA has an ameliorative impact on the symptoms of Parkinson's disease. The results demonstrated that ISA exhibited anti-inflammatory properties, including reducing erythema areas and inflammatory cell infiltration in rosacea-like mice models, and inhibiting the expression of inflammatory factors in cellular inflammation models. Furthermore, the anti-inflammatory effect of ISA was associated with inhibition of the Erk, p38 and NF-κB signaling pathways and inhibition of macrophage polarization to M1 type. In addition, molecular docking and drug affinity responsive target stability experiment results indicated that VEGFA and RELA were the direct targets of ISA in the treatment for rosacea. In conclusion, these results suggested that ISA may be a potential therapeutic agent for rosacea.

High efficacy of red light photodynamic therapy with 10 % aminolevulinic acid gel irrespective of the extent of keratinocyte atypia in actinic keratosis - exploratory post-hoc analysis of three pivotal phase III trials.

BACKGROUND: Primary endpoints of clinical studies investigating treatments for actinic keratosis (AK) are mainly based on clinical evaluation, but a recent study showed that in AK, clinical classification according to Olsen and the extent of keratinocyte atypia do not necessarily correlate. The influence of the epidermal extent of atypia on treatment efficacy is usually not investigated and therefore remains largely unknown. OBJECTIVE: To evaluate whether the extent of keratinocyte atypia influences efficacy of photodynamic therapy (PDT) when treating AK. METHODS: We performed a post-hoc analysis of histological (keratinocyte intraepithelial neoplasia (KIN)), and clinical (Olsen) data of biopsied lesions of three pivotal studies evaluating PDT using 10 % aminolevulinic acid (ALA) gel or vehicle and narrow- or broad-spectrum red light lamps. RESULTS: Overall, 514 biopsied lesions were considered. Clearance rates after red light PDT with 10 % ALA gel were comparable for KIN I-III (88.2 %, 92.0 % and 87.9 %) and Olsen I-II lesions for any given lamp type. Generally, clearance rates were higher using narrow- compared to broad-spectrum lamps. For both lamp types, the variation in clearance rates from KIN I-III was low. Clearance was lower with vehicle. LIMITATIONS: Varying lesion numbers in the subgroups and a remaining risk of bias due to the biopsies are potential limitations. CONCLUSION: Our results suggest that red light PDT with 10 % ALA gel is an effective treatment option for AK regardless of the extent of keratinocyte atypia.

Epidermal RORα Maintains Barrier Integrity and Prevents Allergic Inflammation by Regulating Late Differentiation and Lipid Metabolism.

The skin epidermis provides a barrier that is imperative for preventing transepidermal water loss (TEWL) and protecting against environmental stimuli. The underlying molecular mechanisms for regulating barrier functions and sustaining its integrity remain unclear. RORα is a nuclear receptor highly expressed in the epidermis of normal skin. Clinical studies showed that the epidermal RORα expression is significantly reduced in the lesions of multiple inflammatory skin diseases. In this study, we investigate the central roles of RORα in stabilizing skin barrier function using mice with an epidermis-specific Rora gene deletion (RoraEKO). While lacking spontaneous skin lesions or dermatitis, RoraEKO mice exhibited an elevated TEWL rate and skin characteristics of barrier dysfunction. Immunostaining and Western blot analysis revealed low levels of cornified envelope proteins in the RoraEKO epidermis, suggesting disturbed late epidermal differentiation. In addition, an RNA-seq analysis showed the altered expression of genes related to "keratinization" and "lipid metabolism" in RORα deficient epidermis. A lipidomic analysis further uncovered an aberrant ceramide composition in the RoraEKO epidermis. Importantly, epidermal Rora ablation greatly exaggerated percutaneous allergic inflammatory responses to oxazolone in an allergic contact dermatitis (ACD) mouse model. Our results substantiate the essence of epidermal RORα in maintaining late keratinocyte differentiation and normal barrier function while suppressing cutaneous inflammation.

Non-electrophilic NRF2 activators promote wound healing in human keratinocytes and diabetic mice and demonstrate selective downstream gene targeting.

The transcription factor NRF2 plays an important role in many biological processes and is a promising therapeutic target for many disease states. NRF2 is highly expressed in the skin and is known to play a critical role in diabetic wound healing, a serious disease process for which treatment options are limited. However, many existing NRF2 activators display off-target effects due to their electrophilic mechanism, underscoring the need for alternative approaches. In this work, we investigated two recently described non-electrophilic NRF2 activators, ADJ-310 and PRL-295, and demonstrated their efficacy in vitro and in vivo in human keratinocytes and Leprdb/db diabetic mice. We also compared the downstream targets of PRL-295 to those of the widely used electrophilic NRF2 activator CDDO-Me by RNA sequencing. Both ADJ-310 and PRL-295 maintained human keratinocyte cell viability at increasing concentrations and maintained or improved cell proliferation over time. Both compounds also increased cell migration, improving in vitro wound closure. ADJ-310 and PRL-295 enhanced the oxidative stress response in vitro, and RNA-sequencing data showed that PRL-295 activated NRF2 with a narrower transcriptomic effect than CDDO-Me. In vivo, both ADJ-310 and PRL-295 improved wound healing in Leprdb/db diabetic mice and upregulated known downstream NRF2 target genes in treated tissue. These results highlight the non-electrophilic compounds ADJ-310 and PRL-295 as effective, innovative tools for investigating the function of NRF2. These compounds directly address the need for alternative NRF2 activators and offer a new approach to studying the role of NRF2 in human disease and its potential as a therapeutic across multiple disease states.

Mitochondrial Bioenergetics of Functional Wound Closure is Dependent on Macrophage-Keratinocyte Exosomal Crosstalk.

Tissue nanotransfection (TNT)-based fluorescent labeling of cell-specific exosomes has shown that exosomes play a central role in physiological keratinocyte-macrophage (mϕ) crosstalk at the wound-site. Here, we report that during the early phase of wound reepithelialization, macrophage-derived exosomes (Exomϕ), enriched with the outer mitochondrial membrane protein TOMM70, are localized in leading-edge keratinocytes. TOMM70 is a 70 kDa adaptor protein anchored in the mitochondrial outer membrane and plays a critical role in maintaining mitochondrial function and quality. TOMM70 selectively recognizes cytosolic chaperones by its tetratricopeptide repeat (TPR) domain and facilitates the import of preproteins lacking a positively charged mitochondrial targeted sequence. Exosomal packaging of TOMM70 in mϕ was independent of mitochondrial fission. TOMM70-enriched Exomϕ compensated for the hypoxia-induced depletion of epidermal TOMM70, thereby rescuing mitochondrial metabolism in leading-edge keratinocytes. Thus, macrophage-derived TOMM70 is responsible for the glycolytic ATP supply to power keratinocyte migration. Blockade of exosomal uptake from keratinocytes impaired wound closure with the persistence of proinflammatory mϕ in the wound microenvironment, pointing toward a bidirectional crosstalk between these two cell types. The significance of such bidirectional crosstalk was established by the observation that in patients with nonhealing diabetic foot ulcers, TOMM70 is deficient in keratinocytes of wound-edge tissues.

Revealing the Therapeutic Potential of Muscle-Derived Mesenchymal Stem/Stromal Cells: An In Vitro Model for Equine Laminitis Based on Activated Neutrophils, Anoxia-Reoxygenation, and Myeloperoxidase.

Laminitis in horses is a crippling condition marked by the deterioration of the dermal-epidermal interface, leading to intense lameness and discomfort, often necessitating euthanasia. This study aimed to establish an in vitro model of laminitis using a continuous keratinocyte cell line exposed to anoxia-reoxygenation and an activated neutrophil supernatant. A significant decrease in the keratinocytes' metabolism was noted during the reoxygenation period, indicative of cellular stress. Adding muscle-derived mesenchymal stem/stromal cells during the reoxygenation demonstrated a protective effect, restoring the keratinocytes' metabolic activity. Moreover, the incubation of the keratinocytes with either an activated neutrophil supernatant or myeloperoxidase alone induced increased keratinocyte myeloperoxidase activity, which was modulated by stem cells. These findings underscore the potential of muscle-derived mesenchymal stem/stromal cells in mitigating inflammation and restoring keratinocyte metabolism, offering insights for future cell therapy research in laminitis treatment.