Extracellular matrix-derived tripeptide proline-glycine-proline inhibits keratinocyte proliferation and migration

Keratinocytes are the predominant cell type in epidermis, and are primarily responsible for the epithelialization phase of wound healing. Previous studies by our group showed a positive correlation between IL‐8 concentration and delayed healing of porcine cutaneous partial‐thickness wounds. Interleukin‐8 and collagen‐breakdown product N‐acetyl‐Pro‐Gly‐Pro (PGP) are known as chemoattractant molecules for neutrophils during inflammation. The activity of both molecules is dependent on chemokine receptors CXCR1 and CXCR2. In addition to neutrophils, keratinocytes also express CXCR1 and CXCR2. Here we investigated the effects of IL‐8 and PGP on keratinocyte proliferation and migration. Our results showed that IL‐8 up to 100 ng/mL does not have any significant impact on keratinocyte proliferation or migration. ECM‐derived tripeptide PGP chemotactically attracts neutrophils but not keratinocytes. PGP strongly inhibits keratinocyte proliferation and migration in a cell‐type specific manner. Thus, collagen breakdown product PGP plays a key role in modulating both the inflammatory and epithelialization phases of wound healing.     

Potential molecular mechanisms of negative pressure in promoting wound healing

Negative pressure wound therapy (NPWT) has been widely used in various lesions. This study aimed to explore the biological effects of negative pressure on the polymorphonuclear neutrophils (PMNs), macrophages, and epidermal keratinocyte cells involved in wound healing. PMNs differentiated from HL‐60, macrophages were derived from THP‐1 monocytes, and keratinocytes were cultured in vitro, and they were treated with 0, ?0.03 mp, and ?0.05 mp, respectively. Cell ultrastructure; viability; apoptosis; and protein factors such as tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), interleukin‐17 (IL‐17), and cell division cycle 42 (Cdc42) were determined by transmission electron microscopy (TEM), CCK8, flow cytometry (FCM), ELISA, and simple Western assays, respectively. After negative pressure stimulation, the cell ultrastructure of PMNs and macrophages cells was presented with a marked increase of lysosomes and a relative decrease of mitochondria. In addition, the cell viability was enhanced in PMNs and macrophages in a pressure‐dependent manner and apoptosis ratios were significantly reduced in PMNs and macrophages. In addition, under ?0.05 negative pressure, IFN‐γ and IL‐17 were significantly increased in PMNs or macrophages. Moreover, increased EGF and EGFR and Cdc42 levels in keratinocytes induced by the ?0.05 mpa were detected, indicating that the migration chemotaxis of keratinocyte cells was enhanced. Negative pressure might promote cell proliferation, accelerate inflammatory responses, and promote epithelialisation during wound healing by increasing IFN‐γ, IL‐17, Cdc42, EGF, and EGFR in PMNs, macrophages, or keratinocytes under different negative pressures.     

Cell surface engineering using glycosylphosphatidylinositol anchored tissue inhibitor of matrix metalloproteinase‐1 stimulates cutaneous wound healing

The balance between matrix metalloproteinases and their endogenous tissue inhibitors (TIMPs) is an important component in effective wound healing. The biologic action of these proteins is linked in part to the stoichiometry of TIMP/matrix metalloproteinases/surface protein interactions. We recently described the effect of a glycosylphosphatidylinositol (GPI) anchored version of TIMP‐1 on dermal fibroblast biology. Here, cell proliferation assays, in vitro wound healing, electrical wound, and impedance measurements were used to characterize effects of TIMP‐1‐GPI treatment on primary human epidermal keratinocytes. TIMP‐1‐GPI stimulated keratinocyte proliferation, as well as mobilization and migration. In parallel, it suppressed the migration and matrix secretion of dermal myofibroblasts, and reduced their secretion of active TGF‐β1. Topical application of TIMP‐1‐GPI in an in vivo excisional wound model increased the rate of wound healing. The agent positively influenced different aspects of wound healing depending on the cell type studied. TIMP‐1‐GPI counters potential negative effects of overactive myofibroblasts and enhances the mobilization and proliferation of keratinocytes essential for effective wound healing. The application of TIMP‐1‐GPI represents a novel and practical clinical solution for facilitating healing of difficult wounds.     

A pre‐clinical functional assessment of an acellular scaffold intended for the treatment of hard‐to‐heal wounds

The majority of the population experience successful wound‐healing outcomes; however, 1–3% of those aged over 65 years experience delayed wound healing and wound perpetuation. These hard‐to‐heal wounds contain degraded and dysfunctional extracellular matrix (ECM); yet, the integrity of this structure is critical in the processes of normal wound healing. Here, we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that could replace dysfunctional ECM. In this regard, the synthetic protein was subjected to adsorption and diffusion assays using collagen and human dermal tissues; evaluated for its ability to influence keratinocyte and fibroblast attachment, migration and proliferation and assessed for its ability to influence in vivo wound healing in a porcine model. Critically, these experiments demonstrate that the matrix protein adsorbed to collagen and human dermal tissue but did not diffuse through human dermal tissue within a 24‐hour observation period, and facilitated cell attachment, migration and proliferation. In a porcine wound‐healing model, significantly smaller wound areas were observed in the test group compared with the control group following the third treatment. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound‐healing purposes.     

Exogenous CLASP2 protein treatment enhances wound healing in vitro and in vivo

Proliferative and migratory abilities of fibroblasts are essential for wound healing at the skin surface. Cytoplasmic linker‐associated protein‐2 (CLASP2) was originally found to interact with cytoplasmic linker protein (CLIP)‐170. CLASP2 plays an important role in microtubule stabilization and the microtubule‐stabilizing activity of CLASP2 depends on its interactions with end binding (EB)‐1 and CLIP‐170. Although the microtubule‐stabilizing role of CLASP2 is well established, the effects of CLASP2 on the migration and proliferation of fibroblasts remain unclear in the context of wound healing. Therefore, we tested the utilization of CLASP2 as a directly applied protein drug to improve wound healing by promoting the migration of effector cells, including skin fibroblasts, to the site of repair or injury using an in vivo excisional wound mouse model and in vitro Hs27 skin fibroblast model. Epidermal growth factor, which is a recognized contributor to cell proliferation and migration, was used as positive control. In vitro and in vivo, CLASP2 treatment significantly enhanced cell migration and accelerated wound closure. Furthermore, in vivo, the CLASP2‐treated animal group displayed enhanced epidermal repair and collagen deposition. Next, we studied the mechanism of CLASP2 for wound healing. Increasing the abundance of intracellular free CLASP2 in skin fibroblasts by supplying exogenous CLASP2 seemed to stabilize microtubules through an interaction between CLASP2 and CLIP‐170, as well as EB1. Exogenous CLASP2 also showed direct binding with IQGAP1, increasing both cyclic adenosine monophosphate activity and phosphorylation of glycogen synthase kinase 3β, which in turn reinstated the binding between free CLASP2 and IQGAP1. In summary, exogenous CLASP2 increased Hs27 skin fibroblast migration by interacting with IQGAP1 and other cytoskeletal linker proteins, such as CLIP‐170 and EB1. Our results strongly suggest that CLASP2 can be developed in wound healing drugs for skin repair and/or regenerating cosmetic products.     

Acute and chronic wound fluids influence keratinocyte function differently

Wound healing requires a proper functioning of keratinocytes that migrate, proliferate and lead to a competent wound closure. Impaired wound healing might be due to a disturbed keratinocyte function caused by the wound environment. Basically, chronic wound fluid (CWF) differs from acute wound fluid (AWF). The aim of this study was to analyse the effects of AWF and CWF on keratinocyte function. We therefore investigated keratinocyte migration and proliferation under the influence of AWF and CWF using MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] test and scratch assay. We further measured the gene expression by qRT‐PCR regarding growth factors and matrixmetalloproteinases (MMPs) involved in regeneration processes. AWF had a positive impact on keratinocyte proliferation over time, whereas CWF had an anti‐proliferative effect. Keratinocyte migration was significantly impaired by CWF in contrast to an undisturbed wound closure under the influence of AWF. MMP‐9 expression was strongly upregulated by CWF compared with AWF. Keratinocyte function was significantly impaired by CWF. An excessive induction of MMP‐9 by CWF might lead to a permanent degradation of extracellular matrix and thereby prevent wounds from healing.     

Acceleration of cutaneous wound healing by brassinosteroids

Brassinosteroids are plant growth hormones involved in cell growth, division, and differentiation. Their effects in animals are largely unknown, although recent studies showed that the anabolic properties of brassinosteroids are possibly mediated through the phosphoinositide 3‐kinase/protein kinase B signaling pathway. Here, we examined biological activity of homobrassinolide (HB) and its synthetic analogues in in vitro proliferation and migration assays in murine fibroblast and primary keratinocyte cell culture. HB stimulated fibroblast proliferation and migration and weakly induced keratinocyte proliferation in vitro. The effects of topical HB administration on progression of wound closure were further tested in the mouse model of cutaneous wound healing. C57BL/6J mice were given a full‐thickness dermal wound, and the rate of wound closure was assessed daily for 10 days, with adenosine receptor agonist CGS‐21680 as a positive control. Topical application of brassinosteroid significantly reduced wound size and accelerated wound healing in treated animals. mRNA levels of transforming growth factor beta and intercellular adhesion molecule 1 were significantly lower, while tumor necrosis factor alpha was nearly suppressed in the wounds from treated mice. Our data suggest that topical application of brassinosteroids accelerates wound healing by positively modulating inflammatory and reepithelialization phases of the wound repair process, in part by enhancing Akt signaling in the skin at the edges of the wound and enhancing migration of fibroblasts in the wounded area. Targeting this signaling pathway with brassinosteroids may represent a promising approach to the therapy of delayed wound healing.     

Purinergic substances promote murine keratinocyte proliferation and enhance impaired wound healing in mice

As membrane-bound receptors for adenosine, purines, and pyrimidines, purinoceptors are expressed in nearly all cell types throughout the mammalian organism. Previous studies showed that purinoceptors are involved in the regulation of proliferation and differentiation of most target cells. The present study was performed to elucidate their role in keratinocyte proliferation and wound healing. The expression of the mRNA of several adenosine and P2Y receptors was shown in the immortalized murine keratinocyte cell line MSC-P5 and primary cultured keratinocytes of four different mouse strains. The nonselective adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine enhanced the growth of MSC-P5 cells in vitro via the A2B receptor. The proliferative stimulus of adenosine triphosphate and uridine triphosphate on this cell line was mediated by the P2Y2 receptor. The mitogenic effect of the purinergic substances was inhibited by simultaneous treatment with respective antagonists. Studies in a mouse model of dexamethasone-induced impaired wound healing showed the in vivo efficacy of the purinoceptor agonists. These studies confirm that pharmacological actions via purinoceptors offer an intriguing possibility in the treatment of impaired wound healing. Nevertheless, further investigations are needed to elucidate fully the role of purinergic mechanisms involved in wound healing.     

Chronic wounds – is cellular ‘reception’ at fault? Examining integrins and intracellular signalling

As with all physiologic processes, chronic wounds are associated with unique intracellular and cellular/extracellular matrix (ECM) receptor types and signalling messages. These cellular receptors mediate responses of the epidermis to provisional wound matrix and change in form and number in cases of impaired wound healing. Integrins are the major cell‐surface receptors for cell adhesion and migration and epidermal keratinocytes express several integrins that bind ECM ligands in provisional wound ECM. Integrin receptors and more particularly integrin clusters and focal adhesion points appear to influence epidermal and dermal cell matrix interactions, cell motility, cell phenotype and ultimate healing trajectory. In chronic wounds, a variety of changes in receptors have been identified: decreased integrin α5β1 receptors affect the integration of fibronectin and subsequent keratinocyte migration; integrin αvβ6 stimulate transforming growth factor (TGF)‐β and may increase the susceptibility to ulceration and fibrosis; however, TGF‐β signal receptors have been found to be dysfunctional in many chronic wounds; additionally receptor interactions result in increased senescent cells including fibroblasts, myofibroblasts and even keratinocytes – this produces a degradative ECM and wound bed and corrosive chronic wound fluid. The activation or inhibition of integrin receptors by various agents may provide an excellent means of influencing wound healing. This process offers an earlier intervention into the wound healing cascade promoting intrinsic healing and elaboration of growth factors and ECM proteins, which may be more cost effective than the traditional attempts at extrinsic addition of these agents.     

Inhibition of bone morphogenetic protein signalling promotes wound healing in a human ex vivo model

Background

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) play roles in embryonic development and postnatal remodelling of the skin. Many indications suggest that BMP signalling regulates keratinocyte proliferation and differentiation. Chronic wounds have been shown to exhibit high levels of BMP ligands; however, the effect of BMP pathway modulation on human skin healing remains undefined.

Methods

A human ex vivo skin wound healing model was used to analyse the expression of BMP signalling pathway components during healing and to investigate the effects of BMPs and the BMP antagonist Noggin on skin repair. Additionally, the effects of BMP signalling on keratinocyte proliferation, apoptosis and migration were tested using in vitro flow cytometry and ‘scratch’ migration assays, respectively.

Results

BMP receptor-1B (BMPR-1B) and downstream signalling protein phosphorylated-Smad-1/5/8 were highly expressed in healing epidermis. Treatment of human skin with exogenous BMPs impaired wound closure by reducing keratinocyte proliferation and increasing apoptosis. The BMP antagonist Noggin negated the inhibitory effects of BMP ligands, and when used alone, Noggin reduced keratinocyte apoptosis in the wound bed. In vitro, BMP ligands suppressed keratinocyte proliferation whilst Noggin stimulated proliferation. Keratinocyte migration was slowed following BMP treatment; in contrast, migration was significantly accelerated due to inhibition of BMP activity by either Noggin or BMPR-1B silencing.

Conclusions

BMP signalling is inherently involved in wound healing. BMPs slow skin repair by suppressing keratinocyte proliferation and migration. Thus, modulation of BMP signalling using BMP inhibitors such as Noggin may serve as a new approach to promote cutaneous wound repair.Level of evidence: Not ratable.

     

Time course pathogenesis of sulphur mustard‐induced skin lesions in mouse model

Sulphur mustard (SM) is a bifunctional alkylating agent that causes cutaneous blistering in humans and animals. In this study, we have presented closer views on pathogenesis of SM‐induced skin injury in a mouse model. SM diluted in acetone was applied once dermally at a dose of 5 or 10 mg/kg to Swiss albino mice. Skin was dissected out at 0, 1, 3, 6, 12, 24, 48, 72 and 168 hours, post‐SM exposure for studying histopathological changes and immunohistochemistry of inflammatory‐reparative biomarkers, namely, transforming growth factor alpha (TGF‐α), fibroblast growth factor (FGF), endothelial nitric oxide synthase (eNOS) and interlukin 6 (IL‐6). Histopathological changes were similar to other mammalian species and basal cell damage resembled the histopathological signs observed with vesication in human skin. Inflammatory cell recruitment at the site of injury was supported by differential expressions of IL‐6 at various stages. Time‐dependent expressions of eNOS played pivotal roles in all the events of wound healing of SM‐induced skin lesions. TGF‐α and FGF were strongly associated with keratinocyte migration, re‐epithelialisation, angiogenesis, fibroblast proliferation and cell differentiation. Furthermore, quantification of the tissue leukocytosis and DNA damage along with semiquantitative estimation of re‐epithelialisation, fibroplasia and neovascularisation on histomorphologic scale could be efficiently used for screening the efficacy of orphan drugs against SM‐induced skin injury.     

Streptolysin O enhances keratinocyte migration and proliferation and promotes skin organ culture wound healing in vitro

ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is currently being investigated as a treatment for collagen-related disorders such as scleroderma and fibrosis. Furthermore, ML-05 may be effective in promoting wound healing and alleviating the formation of hypertrophic scars and keloids. To investigate the effects of ML-05 on wound-healing processes, in vitro wound-healing scratch assays (using human primary epidermal keratinocytes and dermal fibroblasts) and a human skin organ culture wound model were utilized. ML-05 markedly enhanced keratinocyte migration and proliferation in wound scratch assays. ML-05 did not affect either proliferation or migration of dermal fibroblasts, indicating that ML-05's effects on cell migration/proliferation may be keratinocyte-specific. ML-05 was tested in a dose-dependent manner in a skin organ culture wound model using two different application methods: Through the culture media (dermal exposure) or direct topical treatment of the wound surface. ML-05 was found to accelerate wound healing as measured by reepithelialization, particularly after topical application. Therefore, ML-05 may have potential as a wound-healing agent that promotes reepithelialization through stimulation of keratinocyte migration and proliferation.     

071TGF‐ß‐Mediated Paracrine Regulation of Keratinocyte Wound Closure

The importance of stromal‐epithelial interactions in wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the TGF‐ß family. TGF‐ß1, ß2 and ß3 isoforms signal by sequentially binding to the TGF‐ß type II and type I receptors, respectively. We address the role of TGF‐ß signaling in dermal fibroblasts using a conditional fibroblastic TGF‐ß type II receptor knockout mouse model (termed FßKO). We found that the loss of TGF‐ß signaling in the dermal fibroblasts results in accelerated excision‐wound closure compared with similar wounds in wild type mice. The mechanism of the altered rate of re‐epitheliaization in the FßKO mice was examined with regard to keratiocyte motility and proliferation. The migration of keratinocytes through collagen I coated 8 μm pore filters in the presence or absence of fibroblast‐conditioned media was tested. These experiments showed increased keratinocyte migration when incubated with FßKO dermal fibroblast conditioned media compared to media conditioned in wild type fibroblasts. Immuno‐histochemical staining of paraffin embedded intact skin indicated both wild type and FßKO mice had similar low levels of keratinocyte proliferation, based on Ki67 staining. In healing wounds, only the distal wound edges of wild type mice were proliferative. In contrast, the FßKO mice exhibited elevated proliferation across the length of the wound, including the leading edge of epithelial closure. Together our results suggest TGF‐ß signaling by the dermal fibroblasts suppresses re‐epithelialization of excision wounds by regulating keratinocyte motility and proliferation through paracrine mechanisms.
Funding: DOD BC99184 and NIH CA85492.     

Effects of an aqueous extract from the leaves of Chromolaena odorata (Eupolin) on the proliferation of human keratinocytes and on their migration in an in vitro model of reepithelialization

Eupolin ointment, prepared from the leaves of Chromolaena odorata, has been shown to promote the healing of soft tissue wounds and burns in Vietnam. However, the mechanism by which this agent affects cells involved in the wound healing process is unknown. Cultured human keratinocytes were used in this study to investigate the effects of the Eupolin extract in vitro on processes involved in wound reepithelialization. Keratinocyte proliferation was monitored by a colorimetric assay and migration by the closure of a denuded area scratched in a confluent monolayer. Human keratinocyte proliferation was stimulated by low concentrations of the extract (from 0.1 to 5 microg/ml), cell differentiation by higher concentrations (50 to 300 microg/ml), and migration by intermediate concentrations (5 to 60 microg/ml). The increased proliferation and migration of human keratinocytes observed in vitro might explain, in part, the beneficial effects that have been observed in the clinic.     

CCN2 is transiently expressed by keratinocytes during re-epithelialization and regulates keratinocyte migration in vitro by the ras-MEK-ERK signaling pathway

Background

CCN2 (previously known as connective tissue growth factor) is a multifunctional matricellular protein that has numerous effects on cell life and cell interactions with the connective tissue. Although the importance of CCN2 for the fibrotic process in wound healing has been well studied, the involvement of CCN2 in keratinocyte function has not yet been explored. Therefore, the aim of the present study was to investigate the role of CCN2 in the epidermis during wound healing.

Materials and methods

Immunohistochemistry was done on sections from full-thickness porcine wounds. The effect of CCN2 on the migration of cultured human keratinocytes exposed to scratch wounds, the effect on phosphorylation of extracellular signal-related kinases (ERK), and the effect of adding inhibitors to the ERK/mitogen-activated protein kinase pathway to human keratinocytes were studied.

Results

The CCN2 protein was transiently expressed in vivo at the leading keratinocyte edge during re-epithelialization of full-thickness porcine wounds. In vitro, exogenous addition of CCN2 to human keratinocyte cultures regulated keratinocyte migration and resulted in phosphorylation of ERK. The addition of inhibitors of ERK/mitogen-activated protein kinase counteracted the effect of CCN2 on migration.

Conclusions

CCN2 was transiently expressed at the leading keratinocyte edge in vivo. The biologic importance of this was supported in vitro, because CCN2 regulated human keratinocyte migration through activation of the Ras-mitogen-activated protein kinase kinase-ERK signal transduction pathway.     

Immunomodulatory potential of Nisin A with application in wound healing

Antimicrobial peptides can have a dual role with both antimicrobial activity against a broad range of bacteria and immunomodulatory effect, making them attractive as therapeutic treatment of difficult wounds. Nisin A is widely known for its antimicrobial activity, and a preliminary study demonstrated that it increased wound closure, but the mechanism behind its effect is unknown. The aim of this study is to elucidate the wound healing potential of Nisin A and the mechanism behind. First, an epithelial and endothelial cell line, human keratinocyte (HaCaT) and human umbilical vein endothelial cell, were used to demonstrate migration and proliferation effects in vitro. From HaCaT cells and peripheral blood mononuclear cell, changes in cytokine levels were shown by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. Second, the ex vivo porcine wound healing model was used to investigate the re‐epithelization potential of Nisin A. Finally, the model Galleria mellonella was used to confirm antimicrobial activity and to investigate potential immunomodulatory effects in vivo. Here, we demonstrated that Nisin A affected migration significantly of both human umbilical vein endothelial cell and HaCaT cells (p < 0.05) but not proliferation, potentially by decreasing the levels of proinflammatory cytokines tumor necrosis factor‐α, interleukin‐6, and interleukin‐8 (p < 0.001). Furthermore, Nisin A treatment diminished lipopolysaccharide‐induced tumor necrosis factor‐α levels from peripheral blood mononuclear cells and monocyte chemoattractant protein‐1 from HaCaT cells (p < 0.001). Furthermore, Nisin A did not affect proliferation ex vivo either but increased re‐epithelization of the porcine skin. Nisin A improved survival of G. mellonella significantly from Staphylococcus epidermidis (p < 0.001) but not from Escherichia coli, indicating that Nisin A did not help the larvae to survive the infection in a different than direct antimicrobial way. All together this makes Nisin A a potential treatment to use in wound healing, as it increases the mobility of skin cells, dampens the effect of lipopolysaccharide and proinflammatory cytokines, and decreases bacterial growth.     

Functional characterisation of bioactive peptide derived from terrestrial snail Cryptozona bistrialis and its wound‐healing property in normal and diabetic‐induced Wistar albino rats

A peptide might be an exciting biomaterial or template for the development of novel wound‐healing agents. In this report, it was isolated from the terrestrial snail Cryptozona bistrialis by enzymatic digestion and was evaluated for its in vitro wound‐healing activity in NIH/3T3 mouse fibroblasts cell line and in vivo wound‐healing activity in normal and diabetic‐induced Wistar albino rats. The C. bistrialis protein was digested by the papain enzyme, and 21.79 kDa peptide (Cb‐peptide) was purified by reversed‐phase high‐performance liquid chromatography and identified by MALDI (matrix‐assisted laser desorption/ionization)‐TOF analysis. The isolated Cb‐peptide was characterised by various analytical methods. The peptide demonstrated a capacity to prevent the development of pathogenic bacterial and fungal cultures and proved that it promotes significant wound‐healing activity in the wound scratch assay method by rapid cell migration and closure of wound. Isolated Cb‐peptide was lyophilised and formulated to ointment and analysed for in vivo wound‐healing activity in normal and diabetic (alloxan monohydrate)‐induced Wistar albino rats. Cb‐peptide ointment‐treated groups showed a greater degree of wound healing and early and complete period of epithelialisation in normal and diabetic‐induced Wistar albino rats. Cb‐peptide ointment‐treated groups showed significant excision and incision wound‐healing activity. A conclusion was reached that the peptide isolated from C. bistrialis showed greater wound‐healing activity compared with vehicle control and standard control.     

Lesional skin in vitiligo exhibits delayed in vivo reepithelialization compared to the nonlesional skin

Vitiligo, a common skin disorder, is characterized by the loss of functional melanocytes resulting in the depigmentation of skin. Previous studies have demonstrated molecular and architectural alterations in the epidermal keratinocytes upon loss of melanocytes. The physiological implications of these “altered” keratinocytes are yet not known. We investigated the wound healing efficiency of lesional vs nonlesional skin in 12 subjects with stable nonsegmental vitiligo using histological and ultrastructural evaluation of partial‐thickness wounds. The wounds were examined 12 days postinjury, coinciding with the reepithelialization phase of healing marked primarily by keratinocyte migration and proliferation. This study demonstrated a significant difference in the reepithelialization potential between the lesional and nonlesional skin. While all 12 nonlesional wounds demonstrated considerable neoepidermis formation on the 12th day post wound, only four of the corresponding lesional samples showed comparable reepithelialization; the rest remaining in the inflammatory phase. Ultrastructural studies using transmission electron microscopy as well as immunohistochemical staining revealed a reduced number of desmosomes, shorter keratin tonofilaments and an increase in myofibroblast population in the dermis of lesional reepithelialized tissue compared to the nonlesional reepithelialized samples. This study implicates gross functional perturbations in the lesional skin during physiological wound healing in vitiligo, suggesting that the breakdown of keratinocyte‐melanocyte network results in delayed wound repair kinetics in the lesional skin when compared to patient‐matched nonlesional skin.     

KCNQ1OT1 mediates keratinocyte migration to promote skin wound healing through the miR-200b-3p/SERP1 axis

BackgroundThe basic functions of keratinocyte are crucial steps during skin wound healing. KCNQ1OT1 long noncoding RNA was found to accelerate the migration and proliferation of keratinocyte in psoriasis. Here, we elucidated the action and mechanism of KCNQ1OT1 in skin wound healing.MethodsExpression levels of genes and proteins were evaluated by quantitative real-time PCR (qRT-PCR) and western blotting. Cell migration was assessed by using scratch and transwell assays. The interaction between miR-200b-3p and KCNQ1OT1 or SERP1 (Stress Associated Endoplasmic Reticulum Protein 1) was confirmed by bioinformatics analysis, dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and pull-down assay.ResultsKCNQ1OT1 had increased significantly in wound edge 1 day and 7 day after injury. Functionally, overexpression of KCNQ1OT1 promoted keratinocyte migration. Mechanistically, KCNQ1OT1/miR-200b-3p/SERP1 constituted a competing endogenous RNA (ceRNA) network in keratinocytes. A series of rescue experiments showed that miR-200b-3p up-regulation in keratinocytes attenuated the pro-migration action of KCNQ1OT1 in cells. Moreover, knockdown of miR-200b-3p could promote keratinocyte migration, which was abolished by SERP1 silencing. KCNQ1OT1 competitively sponged for miR-200b-3p to elevate the expression of its target SERP1.ConclusionKCNQ1OT1 could promote keratinocyte migration by miR-200b-3p/SERP1 axis, suggesting that KCNQ1OT1 might play a crucial role in skin wound healing.