Human skin long noncoding RNA WAKMAR1 regulates wound healing by enhancing keratinocyte migration.

An increasing number of studies reveal the importance of long noncoding RNAs (lncRNAs) in gene expression control underlying many physiological and pathological processes. However, their role in skin wound healing remains poorly understood. Our study focused on a skin-specific lncRNA, LOC105372576, whose expression was increased during physiological wound healing. In human nonhealing wounds, however, its level was significantly lower compared with normal wounds under reepithelialization. We characterized LOC105372576 as a nuclear-localized, RNAPII-transcribed, and polyadenylated lncRNA. In keratinocytes, its expression was induced by TGF-ß signaling. Knockdown of LOC105372576 and activation of its endogenous transcription, respectively, reduced and increased the motility of keratinocytes and reepithelialization of human ex vivo skin wounds. Therefore, LOC105372576 was termed "wound and keratinocyte migration-associated lncRNA 1" (WAKMAR1). Further study revealed that WAKMAR1 regulated a network of protein-coding genes important for cell migration, most of which were under the control of transcription factor E2F1. Mechanistically, WAKMAR1 enhanced E2F1 expression by interfering with E2F1 promoter methylation through the sequestration of DNA methyltransferases. Collectively, we have identified a lncRNA important for keratinocyte migration, whose deficiency may be involved in the pathogenesis of chronic wounds.

The Immune Functions of Keratinocytes in Skin Wound Healing.

As the most dominant cell type in the skin, keratinocytes play critical roles in wound repair not only as structural cells but also exerting important immune functions. This review focuses on the communications between keratinocytes and immune cells in wound healing, which are mediated by various cytokines, chemokines, and extracellular vesicles. Keratinocytes can also directly interact with T cells via antigen presentation. Moreover, keratinocytes produce antimicrobial peptides that can directly kill the invading pathogens and contribute to wound repair in many aspects. We also reviewed the epigenetic mechanisms known to regulate keratinocyte immune functions, including histone modifications, non-protein-coding RNAs (e.g., microRNAs, and long noncoding RNAs), and chromatin dynamics. Lastly, we summarized the current evidence on the dysregulated immune functions of keratinocytes in chronic nonhealing wounds. Based on their crucial immune functions in skin wound healing, we propose that keratinocytes significantly contribute to the pathogenesis of chronic wound inflammation. We hope this review will trigger an interest in investigating the immune roles of keratinocytes in chronic wound pathology, which may open up new avenues for developing innovative wound treatments.

Transition from inflammation to proliferation: a critical step during wound healing.

The ability to rapidly restore the integrity of a broken skin barrier is critical and is the ultimate goal of therapies for hard-to-heal-ulcers. Unfortunately effective treatments to enhance healing and reduce scarring are still lacking. A deeper understanding of the physiology of normal repair and of the pathology of delayed healing is a prerequisite for the development of more effective therapeutic interventions. Transition from the inflammatory to the proliferative phase is a key step during healing and accumulating evidence associates a compromised transition with wound healing disorders. Thus, targeting factors that impact this phase transition may offer a rationale for therapeutic development. This review summarizes mechanisms regulating the inflammation-proliferation transition at cellular and molecular levels. We propose that identification of such mechanisms will reveal promising targets for development of more effective therapies.

Single-cell profiling and functional screening reveal crucial roles for lncRNAs in the epidermal re-epithelialization of human acute wounds.

Objectives: Re-epithelialization is an important physiological process for repairing skin barrier function during wound healing. It is primarily mediated by coordinated migration, proliferation, and differentiation of keratinocytes. Long noncoding RNAs (lncRNAs) are essential components of the noncoding genome and participate in various biological processes; however, their expression profiles and function in re-epithelialization during wound healing have not been established. Methods: We investigated the distribution of lncRNAs during wound re-epithelialization by comparing the genomic profiles of uninjured skin and acute wound (AW) from healthy donors. We performed functional screening of differentially expressed lncRNAs to identify the important lncRNAs for re-epithelialization. Results: The expression of multiple lncRNAs is changed during human wound re-epithelialization process. We identified VIM-AS1, SMAD5-AS1, and LINC02581 as critical regulators involved in keratinocyte migration, proliferation, and differentiation, respectively. Conclusion: LncRNAs play crucial regulatory roles in wound re-epithelialization. We established lncRNA expression profile in human acute wounds compared with intact skin, offering valuable insights into the physiological mechanisms underlying wound healing and potential therapeutic targets.

A Roadmap for a Consensus Human Skin Cell Atlas and Single-Cell Data Standardization.

Single-cell technologies have become essential to driving discovery in both basic and translational investigative dermatology. Despite the multitude of available datasets, a central reference atlas of normal human skin, which can serve as a reference resource for skin cell types, cell states, and their molecular signatures, is still lacking. For any such atlas to receive broad acceptance, participation by many investigators during atlas construction is an essential prerequisite. As part of the Human Cell Atlas project, we have assembled a Skin Biological Network to build a consensus Human Skin Cell Atlas and outline a roadmap toward that goal. We define the drivers of skin diversity to be considered when selecting sequencing datasets for the atlas and list practical hurdles during skin sampling that can result in data gaps and impede comprehensive representation and technical considerations for tissue processing and computational analysis, the accounting for which should minimize biases in cell type enrichments and exclusions and decrease batch effects. By outlining our goals for Atlas 1.0, we discuss how it will uncover new aspects of skin biology.

Beyond the Code: Noncoding RNAs in Skin Wound Healing.

An increasing number of noncoding RNAs (ncRNAs) have been found to regulate gene expression and protein functions, playing important roles in diverse biological processes and diseases. Their crucial functions have been reported in almost every cell type and all stages of skin wound healing. Evidence of their pathogenetic roles in common wound complications, such as chronic nonhealing wounds and excessive scarring, is also accumulating. Given their unique expression and functional properties, ncRNAs are promising therapeutic and diagnostic entities. In this review, we discuss current knowledge about the functional roles of noncoding elements, such as microRNAs, long ncRNAs, and circular RNAs, in skin wound healing, focusing on in vivo evidence from studies of human wound samples and animal wound models. Finally, we provide a perspective on the outlook of ncRNA-based therapeutics in wound care.

Circular RNA Signatures of Human Healing and Nonhealing Wounds.

Venous ulcers (VUs) have complex and obscure pathogenicity, and effective VU therapies are still lacking. Circular RNAs (circRNAs) have emerged as powerful gene regulators with important roles in health and disease. In this study, we used paired total RNA and small RNA sequencing to profile circRNAs, protein-coding mRNAs, and microRNAs expression in a unique collection of clinical samples: healthy skin and acute wounds at inflammatory and proliferative phases and wound-edge VU biopsies. We unravel a dynamically changed expression pattern of circRNAs during human skin repair and their abnormal expression signature in VU, which are presented as a searchable web resource (www.xulandenlab.com/humanwounds-circrna). We analyzed the coexpression relationship between the circRNAs and mRNAs with weighted correlation network analysis and constructed circRNA‒mRNA‒microRNA networks. This allowed us to expose the regulatory networks specific to the inflammatory and proliferative phases of wound repair and VU, the biological processes the circRNAs may regulate, and the circRNAs that could sponge microRNAs in human wounds. Importantly, we found that hsa-CHST15_0003 and hsa-TNFRSF21_0001, two circRNAs upregulated in VU, hampered epidermal keratinocyte migration while promoting proliferation by modulating gene networks underpinning these cellular processes. This study paves the way to decipher the functional significance of circRNAs in tissue repair.

Interleukin-17 governs hypoxic adaptation of injured epithelium.

Mammalian cells autonomously activate hypoxia-inducible transcription factors (HIFs) to ensure survival in low-oxygen environments. We report here that injury-induced hypoxia is insufficient to trigger HIF1α in damaged epithelium. Instead, multimodal single-cell and spatial transcriptomics analyses and functional studies reveal that retinoic acid-related orphan receptor γt+ (RORγt+) γδ T cell-derived interleukin-17A (IL-17A) is necessary and sufficient to activate HIF1α. Protein kinase B (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling proximal of IL-17 receptor C (IL-17RC) activates mammalian target of rapamycin (mTOR) and consequently HIF1α. The IL-17A-HIF1α axis drives glycolysis in wound front epithelia. Epithelial-specific loss of IL-17RC, HIF1α, or blockade of glycolysis derails repair. Our findings underscore the coupling of inflammatory, metabolic, and migratory programs to expedite epithelial healing and illuminate the immune cell-derived inputs in cellular adaptation to hypoxic stress during repair.

Investigation of Skin Wound Healing Using a Mouse Model.

Wound repair is a fundamental physiological process to keep the integrity of the skin, and its dysregulation results in diseases, such as chronic nonhealing wounds or excessive scarring. To study the underlying cellular and molecular mechanisms and identify new therapeutic targets, animal models are often used in the wound healing research. In this chapter, we describe an easy step-by-step protocol to generate skin wounds in a mouse model. Briefly, two full-thickness wounds extending through the panniculus carnosus are made on the dorsum on each side of the midline of a mouse, which is followed by monitoring and quantifying the wound closure. Moreover, the biopsy tissues of skin and wound-edges are collected at different time points for subsequent histology and gene expression analysis.

MicroRNA-34 Family Enhances Wound Inflammation by Targeting LGR4.

Venous ulcers are the most common type of human chronic nonhealing wounds and are stalled in a constant and excessive inflammatory state. The molecular mechanisms underlying the chronic wound inflammation remain elusive. Moreover, little is known about the role of regulatory RNAs, such as microRNAs, in the pathogenesis of venous ulcers. We found that both microRNA (miR)-34a and miR-34c were upregulated in the wound-edge epidermal keratinocytes of venous ulcers compared with normal wounds or the skin. In keratinocytes, miR-34a and miR-34c promoted inflammatory chemokine and cytokine production. In wounds of wild-type mice, miR-34a-mimic treatment enhanced inflammation and delayed healing. To further explore how miR-34 functions, LGR4 was identified as a direct target mediating the proinflammatory function of miR-34a and miR-34c. Interestingly, impaired wound closure with enhanced inflammation was also observed in Lgr4 knockout mice. Mechanistically, the miR-34-LGR4 axis regulated GSK-3ß-induced p65 serine 468 phosphorylation, changing the activity of the NF-κB signaling pathway. Collectively, the miR-34-LGR4 axis was shown to regulate keratinocyte inflammatory response, the deregulation of which may play a pathological role in venous ulcers.

MicroRNAs in skin wound healing.

Wound healing is a fundamental physiological progress to keep the integrity of the skin. The transition from inflammation to proliferation is a critical step during skin wound repair process. Impairment of this transition has been known as a common dominator in the pathophysiology of chronic non-healing wounds. MicroRNAs (miRNAs) are short non-coding RNAs regulating gene expression. Emerging evidence has revealed that miRNAs play important roles in both normal skin wound healing and in the pathogenesis of chronic wounds. We focus on the miRNAs regulating the inflammation-proliferation transition during wound healing and propose that these miRNAs may be promising targets for development of more effective wound therapy.

MicroRNA-132 promotes fibroblast migration via regulating RAS p21 protein activator 1 in skin wound healing.

MicroRNA (miR)-132 has been identified as a top up-regulated miRNA during skin wound healing and its inhibition impairs wound repair. In a human in vivo surgical wound model, we showed that miR-132 was induced in epidermal as well as in dermal wound-edge compartments during healing. Moreover, in a panel of cells isolated from human skin wounds, miR-132 was found highly expressed in human dermal fibroblasts (HDFs). In HDFs, miR-132 expression was upregulated by TGF-ß1. By overexpression or inhibition of miR-132, we showed that miR-132 promoted HDF migration. Mechanistically, global transcriptome analysis revealed that RAS signaling pathway was regulated by miR-132 in HDFs. We found that RAS p21 protein activator 1 (RASA1), a known target of miR-132, was downregulated in HDFs upon miR-132 overexpression. Silencing of RASA1 phenocopied the pro-migratory effect of miR-132. Collectively, our study reveals an important role for miR-132 in HDFs during wound healing and indicates a therapeutic potential of miR-132 in hard-to-heal skin wounds.

Intratracheal Instillation of Perfluorohexane Modulates the Pulmonary Immune Microenvironment by Attenuating Early Inflammatory Factors in Patients With Smoke Inhalation Injury: A Randomized Controlled Clinical Trial.

Smoke inhalation injury (SII) is associated with significant morbidity and mortality in burn patients, and effective treatments are lacking. Perfluorocarbons (PFCs) have a protective effect against acute lung injury. We aimed to assess the therapeutic effects of perfluorohexane on burn patients with SII. Patients with burns complicated by moderately severe SII were randomly divided into control (n = 11) and PFC groups (n = 12). The control group received conventional treatment (anti-infection, nutritional support, antishock measures, and supportive treatment). The PFC group received endotracheal perfluorohexane instillation in addition to conventional treatment. On admission and 3 days later, therapeutic effects were evaluated and inflammatory mediators in bronchoalveolar lavage fluid and plasma were analyzed. There was no significant difference between the control and PFC group on admission. After 3 days, perfluorohexane treatment significantly (P < .05) increased lung dynamic compliance, and reduced alveolar-arterial oxygen gradient, Acute Physiology and Chronic Health Evaluation II score, percentage of neutrophils, and levels of interleukin-6, interleukin-8, and tumor necrosis factor alpha in bronchoalveolar lavage fluid; there was no significant change in the control group before and after treatment. Intratracheal instillation of perfluorohexane modulates the pulmonary immune microenvironment and supplements current conventional treatments for burn patients with SII.

Psoriasis Skin Inflammation-Induced microRNA-26b Targets NCEH1 in Underlying Subcutaneous Adipose Tissue.

Psoriasis is an immune-mediated inflammatory disease, which is associated with a high risk of developing systemic comorbidities, such as obesity, cardiovascular disease, and diabetes mellitus. However, the mechanistic links between psoriatic skin inflammation and systemic comorbidities remain largely unknown. MicroRNAs (miRNAs) are recently discovered gene regulators that play important roles in psoriasis skin inflammation. In this study we aimed to explore whether the skin inflammation in psoriasis affects miRNA expression of the underlying subcutaneous adipose tissue and whether this may be a link between psoriasis and comorbidities. To this end, we compared the miRNA expression profile of subcutaneous adipose tissue underneath lesional and nonlesional psoriatic skin. We further validated the differential expression of several miRNAs and characterized their expression patterns in different cell types present in subcutaneous adipose tissue. We focused on miR-26b-5p, which was highly up-regulated in subcutaneous adipose tissue underneath lesional psoriasis skin. We showed that it targets and down-regulates neutral cholesterol ester hydrolase 1, an enzyme essential for cholesterol efflux, in monocytes/macrophages, adipocytes, vascular endothelial cells, and fibroblasts. We conclude that this miRNA may serve as a mechanistic link between psoriatic skin inflammation and its systemic comorbidities.

MicroRNA-203 Inversely Correlates with Differentiation Grade, Targets c-MYC, and Functions as a Tumor Suppressor in cSCC.

Cutaneous squamous cell carcinoma (cSCC) is the second most common cancer and a leading cause of cancer mortality among solid organ transplant recipients. MicroRNAs (miR) are short RNAs that regulate gene expression and cellular functions. Here, we show a negative correlation between miR-203 expression and the differentiation grade of cSCC. Functionally, miR-203 suppressed cell proliferation, cell motility, and the angiogenesis-inducing capacity of cSCC cells in vitro and reduced xenograft tumor volume and angiogenesis in vivo. Transcriptomic analysis of cSCC cells with ectopic overexpression of miR-203 showed dramatic changes in gene networks related to cell cycle and proliferation. Transcription factor enrichment analysis identified c-MYC as a hub of miR-203-induced transcriptomic changes in squamous cell carcinoma. We identified c-MYC as a direct target of miR-203. Overexpression of c-MYC in rescue experiments reversed miR-203-induced growth arrest in cSCC, which highlights the importance of c-MYC within the miR-203-regulated gene network. Together, miR-203 acts as a tumor suppressor in cSCC, and its low expression can be a marker for poorly differentiated tumors. Restoration of miR-203 expression may provide a therapeutic benefit, particularly in poorly differentiated cSCC.

MicroRNA-31 Promotes Skin Wound Healing by Enhancing Keratinocyte Proliferation and Migration.

Wound healing is a basic biological process restoring the integrity of the skin. The role of microRNAs during this process remains largely unexplored. By using an in vivo human skin wound healing model, we show here that the expression of miR-31 is gradually upregulated in wound edge keratinocytes in the inflammatory (1 day after injury) through the proliferative phase (7 days after injury) in comparison with intact skin. In human primary keratinocytes, overexpression of miR-31 promoted cell proliferation and migration, whereas inhibition of miR-31 had the opposite effects. Moreover, we identified epithelial membrane protein 1 (EMP-1) as a direct target of miR-31 in keratinocytes. The expression of EMP-1 in the skin was negatively correlated with the level of miR-31 during wound healing. Silencing of EMP-1 mimicked the effects of overexpression of miR-31 on keratinocyte proliferation and migration, indicating that EMP-1 is a critical target mediating the functions of miR-31 in keratinocytes. Finally, we demonstrated that transforming growth factor-ß2, which is highly expressed in skin wounds, upregulated miR-31 expression in keratinocytes. Collectively, we identify miR-31 as a key regulator for promoting keratinocyte proliferation and migration during wound healing.

MicroRNA-132 enhances transition from inflammation to proliferation during wound healing.

Wound healing is a complex process that is characterized by an initial inflammatory phase followed by a proliferative phase. This transition is a critical regulatory point; however, the factors that mediate this process are not fully understood. Here, we evaluated microRNAs (miRs) in skin wound healing and characterized the dynamic change of the miRNome in human skin wounds. miR-132 was highly upregulated during the inflammatory phase of wound repair, predominantly expressed in epidermal keratinocytes, and peaked in the subsequent proliferative phase. TGF-ß1 and TGF-ß2 induced miR-132 expression in keratinocytes, and transcriptome analysis of these cells revealed that miR-132 regulates a large number of immune response- and cell cycle-related genes. In keratinocytes, miR-132 decreased the production of chemokines and the capability to attract leukocytes by suppressing the NF-κB pathway. Conversely, miR-132 increased activity of the STAT3 and ERK pathways, thereby promoting keratinocyte growth. Silencing of the miR-132 target heparin-binding EGF-like growth factor (HB-EGF) phenocopied miR-132 overexpression in keratinocytes. Using mouse and human ex vivo wound models, we found that miR-132 blockade delayed healing, which was accompanied by severe inflammation and deficient keratinocyte proliferation. Together, our results indicate that miR-132 is a critical regulator of skin wound healing that facilitates the transition from the inflammatory to the proliferative phase.

Therapeutic effect of intravenous infusion of perfluorocarbon emulsion on LPS-induced acute lung injury in rats.

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS) are the leading causes of death in critical care. Despite extensive efforts in research and clinical medicine, mortality remains high in these diseases. Perfluorocarbon (PFC), a chemical compound known as liquid ventilation medium, is capable of dissolving large amounts of physiologically important gases (mainly oxygen and carbon dioxide). In this study we aimed to investigate the effect of intravenous infusion of PFC emulsion on lipopolysaccharide (LPS) induced ALI in rats and elucidate its mechanism of action. Forty two Wistar rats were randomly divided into three groups: 6 rats were treated with saline solution by intratracheal instillation (control group), 18 rats were treated with LPS by intratracheal instillation (LPS group) and the other 18 rats received PFC through femoral vein prior to LPS instillation (LPS+PFC group). The rats in the control group were sacrificed 6 hours later after saline instillation. At 2, 4 and 6 hours of exposure to LPS, 6 rats in the LPS group and 6 rats in LPS+PFC group were sacrificed at each time point. By analyzing pulmonary pathology, partial pressure of oxygen in the blood (PaO2) and lung wet-dry weight ratio (W/D) of each rat, we found that intravenous infusion of PFC significantly alleviated acute lung injury induced by LPS. Moreover, we showed that the expression of pulmonary myeloperoxidase (MPO), intercellular adhesion molecule-1 (ICAM-1) of endothelial cells and CD11b of polymorphonuclear neutrophils (PMN) induced by LPS were significantly decreased by PFC treatment in vivo. Our results indicate that intravenous infusion of PFC inhibits the infiltration of PMNs into lung tissue, which has been shown as the core pathogenesis of ALI/ARDS. Thus, our study provides a theoretical foundation for using intravenous infusion of PFC to prevent and treat ALI/ARDS in clinical practice.

MicroRNA-31 is overexpressed in cutaneous squamous cell carcinoma and regulates cell motility and colony formation ability of tumor cells.

Cutaneous squamous cell carcinoma (cSCC) is a malignancy of epidermal keratinocytes that is responsible for approximately 20% of skin cancer-related death yearly. We have previously compared the microRNA (miRNA) expression profile of cSCC to healthy skin and found the dysregulation of miRNAs in human cSCC. In this study we show that miR-31 is overexpressed in cSCC (n = 68) compared to healthy skin (n = 34) and precancerous skin lesions (actinic keratosis, n = 12). LNA in situ hybridization revealed that miR-31 was specifically up-regulated in tumor cells. Mechanistic studies of inhibition of endogenous miR-31 in human metastatic cSCC cells revealed suppressed migration, invasion and colony forming ability, whereas overexpression of miR-31 induced these phenotypes. These results indicate that miR-31 regulates cancer-associated phenotypes of cSCC and identify miR-31 as a potential target for cSCC treatment.