Bmx tyrosine kinase transgene induces skin hyperplasia, inflammatory angiogenesis, and accelerated wound healing

The Bmx gene, a member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in arterial endothelium and in certain hematopoietic and epithelial cells. Previous in vitro studies have implicated Bmx signaling in cell migration and survival and suggested that it contributes to the progression of prostate carcinomas. However, the function of Bmx in normal tissues in vivo is unknown. We show here that Bmx expression is induced in skin keratinocytes during wound healing. To analyze the role of Bmx in epidermal keratinocytes in vivo, we generated transgenic mice overexpressing Bmx in the skin. We show that Bmx overexpression accelerates keratinocyte proliferation and wound reepithelialization. Bmx expression also induces chronic inflammation and angiogenesis in the skin, and gene expression profiling suggests that this occurs via cytokine-mediated recruitment of inflammatory cells. Our studies provide the first data on Bmx function in vivo and form the basis of evaluation of its role in epithelial neoplasia.     

Mesoglycan induces keratinocyte activation by triggering syndecan-4 pathway and the formation of the annexin A1/S100A11 complex

Wound healing is a dynamic process comprising multiple events, such as inflammation, re-epithelialization, and tissue remodeling. Re-epithelialization phase is characterized by the engagement of several cell populations, mainly of keratinocytes that sequentially go through cycles of migration, proliferation, and differentiation to restore skin functions. Troubles can arise during the re-epithelialization phase of skin wound healing particularly in keratinocyte migration, resulting in chronic non-healing lesions, which represent a serious clinical problem. Over the last decades, the efforts aimed to find new pharmacological approaches for wound care were made, yet almost all current therapeutic strategies used remain inadequate or even ineffective. As such, it is crucial to identify new drugs that can enable a proper regeneration of the epithelium in wounded skin. Here, we have investigated the effects of the fibrinolytic drug mesoglycan, a glycosaminoglycans mixture derived from porcine intestinal mucosa on HaCaT human keratinocytes that were used as in vitro experimental model of skin re-epithelialization. We found that mesoglycan induces keratinocyte migration and early differentiation by triggering the syndecan-4/PKCα pathway and that these effects were at least in part, because of the formation of the annexin A1/S100A11 complex. Our data suggest that mesoglycan may be useful as a new pro-healing drug for skin wound care.     

A novel function of angiotensin II in skin wound healing. Induction of fibroblast and keratinocyte migration by angiotensin II via heparin-binding epidermal growth factor (EGF)-like growth factor-mediated EGF receptor transactivation

The role of angiotensin II (Ang II) in the control of systemic blood pressure and volume homeostasis is well known and has been extensively studied. Recently, Ang II was suggested to also have a function in skin wound healing. In the present study, the in vivo function of Ang II in skin wound healing was investigated using Ang II type 1 receptor (AT1R) knock-out mice. Wound healing in these mice was found to be markedly delayed. Keratinocytes and fibroblasts play important roles in wound healing, and thus the effect of Ang II on the migration of these cells was examined. Ang II stimulated keratinocyte and fibroblast migration in a dose-dependent manner. It has been reported that G protein-coupled receptor (GPCR) activation induces epidermal growth factor (EGF) receptor (EGFR) transactivation through the shedding of heparin-binding EGF-like growth factor (HB-EGF). As AT1R is a GPCR, it was hypothesized that Ang II-induced keratinocyte and fibroblast migration is mediated by EGFR transactivation. Ang II induced EGFR phosphorylation, which was inhibited by an AT1R antagonist, HB-EGF neutralizing antibody, and an HB-EGF antagonist in both keratinocytes and in fibroblasts. Moreover, Ang II-induced migration of keratinocytes and fibroblasts was also prevented by these inhibitors. Taken together, these findings clearly demonstrate, for the first time, that Ang II plays an important role in skin wound healing and that it functions by accelerating keratinocyte and fibroblast migration in a process mediated by HB-EGF shedding.     

Activities of the matrix metalloproteinase stromelysin-2 (MMP-10) in matrix degradation and keratinocyte organization in wounded skin

The matrix metalloproteinase stromelysin-2 is expressed in keratinocytes of the epithelial tongue of skin wounds, suggesting a role in keratinocyte migration. Here, we show that stromelysin-2 enhances migration of cultured keratinocytes. To gain insight into the in vivo activities of stromelysin-2 in epithelial repair, we generated transgenic mice expressing a constitutively active stromelysin-2 mutant in keratinocytes. These animals had no alterations in skin architecture, and the healing rate of skin wounds was normal. Histologically, however, we found abnormalities in the organization of the wound epithelium. Keratinocytes at the migrating epidermal tip were scattered in most sections of mice with high expression level, and there was a reduced deposition of new matrix. In particular, the staining pattern of laminin-5 at the wound site was altered. This may be due to proteolytic processing of laminin-5 by stromelysin-2, because degradation of laminin-5 by this enzyme was observed in vitro. The inappropriate matrix contact of keratinocytes was accompanied by aberrant localization of beta1-integrins and phosphorylated focal adhesion kinase, as well as by increased apoptosis of wound keratinocytes. These results suggest that a tightly regulated expression level of stromelysin-2 is required for limited matrix degradation at the wound site, thereby controlling keratinocyte migration.     

Exploring Hypotheses of the Actions of TGF-β1 in Epidermal Wound Healing Using a 3D Computational Multiscale Model of the Human Epidermis

In vivo and in vitro studies give a paradoxical picture of the actions of the key regulatory factor TGF-β1 in epidermal wound healing with it stimulating migration of keratinocytes but also inhibiting their proliferation. To try to reconcile these into an easily visualized 3D model of wound healing amenable for experimentation by cell biologists, a multiscale model of the formation of a 3D skin epithelium was established with TGF-β1 literature–derived rule sets and equations embedded within it. At the cellular level, an agent-based bottom-up model that focuses on individual interacting units (keratinocytes) was used. This was based on literature-derived rules governing keratinocyte behavior and keratinocyte/ECM interactions. The selection of these rule sets is described in detail in this paper. The agent-based model was then linked with a subcellular model of TGF-β1 production and its action on keratinocytes simulated with a complex pathway simulator. This multiscale model can be run at a cellular level only or at a combined cellular/subcellular level. It was then initially challenged (by wounding) to investigate the behavior of keratinocytes in wound healing at the cellular level. To investigate the possible actions of TGF-β1, several hypotheses were then explored by deliberately manipulating some of these rule sets at subcellular levels. This exercise readily eliminated some hypotheses and identified a sequence of spatial-temporal actions of TGF-β1 for normal successful wound healing in an easy-to-follow 3D model. We suggest this multiscale model offers a valuable, easy-to-visualize aid to our understanding of the actions of this key regulator in wound healing, and provides a model that can now be used to explore pathologies of wound healing.     

Cellular and molecular facets of keratinocyte reepithelization during wound healing

Cutaneous wound healing is a highly coordinated physiological process that rapidly and efficiently restores skin integrity. Reepithelization is a crucial step during wound healing, which involves migration and proliferation of keratinocytes to cover the denuded dermal surface. Recent advances in wound biology clarified the molecular pathways governing keratinocyte reepithelization at wound sites. These new findings point towards novel therapeutic targets and provide suitable methods to promote faster tissue regeneration in vivo.     

Bombesin: a possible role in wound repair

During tissue regeneration and wound healing of the skin, migration, proliferation and differentiation of keratinocytes are important processes. Here we assessed the effect of a neuropeptide, bombesin, on keratinocytes during regeneration from scratch wounding. Bombesin purified from amphibian skin, is homologous of mammalian gastrin-releasing peptide and is active in mammals. Its pharmacological effects mediate various physiological activities: hypertensive action, stimulating action on gastric secretion, hyperglycemic effect or increased insulin secretion. In vitro it shows a hyperproliferative effect on different experimental models and is involved in skin repair. The aim of this study was to elucidate the effect of Bombesin in an in vitro experimental model on a mechanically injured human keratinocyte monolayer. We evaluated different mediators involved in wound repair such as IL-8, TGFbeta, IL-1, COX-2, VEGF and Toll-like receptors 2 and 4 (TLR2 and TLR4). We also studied the effects of bombesin on cell proliferation and motility and its direct effect on wound repair by observing the wound closure after mechanical injury. The involvement of the bombesin receptors neuromedin receptor (NMBR) and gastrin-releasing peptide receptor (GRP-R) was also evaluated. Our data suggest that bombesin may have an important role in skin repair by regulating the expression of healing markers. It enhanced the expression of IL-8, TGFbeta, COX-2 and VEGF. It also enhanced the expression of TLR2, while TLR4 was not expressed. Bombesin also increased cell growth and migration. In addition, we showed that NMBR was more involved in our experimental model compared to GRP-R.     

Novel regulation and dynamics of myosin II activation during epidermal wound responses

Wound healing in the skin is an important and complex process that involves 3-dimensional tissue reorganization, including matrix and chemokine-triggered cell migration, paracrine signaling, and matrix remodeling. The molecular signals and underlying mechanisms that stimulate myosin II activity during skin wound healing have not been elucidated. To begin understanding the signaling pathways involved in the activation of myosin II in this process, we have evaluated myosin II activation in migrating primary human keratinocytes in response to scratch wounding in vitro. We report here that myosin II activation and recruitment to the cytoskeleton in wounded keratinocytes are biphasic. Post-wounding, a rapid phosphorylation of myosin II regulatory light chain (RLC) occurs with resultant translocation of myosin IIA to the cell cortex, far in advance of the later polarization and cell migration. During this acute-phase of myosin II activation, pharmacological approaches reveal p38-MAP kinase and cytosolic calcium as having critical roles in the phosphorylation driving cytoskeletal assembly. Although p38-MAPK has known roles in keratinocyte migration, and known roles in leading-edge focal complex dynamics, to our knowledge this is the first report of p38-MAPK acting as an upstream activator of myosin II phosphorylation and assembly during any type of wound response.     

Adipose tissue extract shows potential for wound healing: in vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma

Growth factors are the key elements in wound healing signaling for cell migration, differentiation and proliferation. Platelet-rich plasma (PRP), one of the most studied sources of growth factors, has demonstrated to promote wound healing in vitro and in vivo. Adipose tissue is an alternative source of growth factors. Through a simple lipoaspirate method, adipose derived growth factor-rich preparation (adipose tissue extract; ATE) can be obtained. The authors set out to compare the effects of these two growth factor sources in cell proliferation and migration (scratch) assays of keratinocyte, fibroblast, endothelial and adipose derived stem cells. Growth factors involved in wound healing were measured: keratinocyte growth factor, epidermal growth factor, insulin-like growth factor, interleukin 6, platelet-derived growth factor beta, tumor necrosis factor alfa, transforming growth factor beta and vascular endothelial growth factor. PRP showed higher growth factor concentrations, except for keratinocyte growth factor, that was present in adipose tissue in greater quantities. This was reflected in vitro, where ATE significantly induced proliferation of keratinocytes at day 6 (p < 0.001), compared to plasma and control. Similarly, ATE-treated fibroblast and adipose stem cell cultures showed accelerated migration in scratch assays. Moreover, both sources showed accelerated keratinocyte migration. Adipose tissue preparation has an inductive effect in wound healing by proliferation and migration of cells involved in wound closure. Adipose tissue preparation appears to offer the distinct advantage of containing the adequate quantities of growth factors that induce cell activation, proliferation and migration, particularly in the early phase of wound healing.     

miR-155 promotes cutaneous wound healing through enhanced keratinocytes migration by MMP-2

Inflammation, re-epithelization and tissue remodeling are three essential steps during wound healing. The re-epithelization process plays the most important role which mainly involves keratinocyte proliferation and migration. miR-155 has been reported to participate in cell migration and transformation, however, its function in skin wound healing is largely unknown. Here we hypothesize that overexpression of miR-155 at wound edges could accelerate wound healing mediated by enhanced keratinocyte migration. To test this hypothesis, direct local injection of miR-155 expression plasmid to wound edges was conducted to overexpress miR-155 in vivo. Results shown that miR-155 significantly promoted wound healing and re-epithelization compared to control, while did not affect wound contraction. Also, miR-155 overexpression accelerated primarily cultured keratinocyte migration in vitro, but had no effect on cell proliferation. Importantly, western blot analysis shown that MMP-2 was significantly upregulated whiles its inhibitor TIMP-1 downregulated after miR-155 treatment. Moreover, the use of ARP-101, an MMP-2 inhibitor, effectively attenuated the accelerative effects on cell migration induced by miR-155. Taken together, our results suggest that miR-155 has the promote effect on wound healing that is probably mediated by accelerating keratinocyte migration via upregulated MMP-2 level. This study provides a rationale for the therapeutic effect of miR-155 on wound healing.     

Comparison of proliferation and motile activity between human keratinocytes isolated from skin and oral mucosa

OBJECTIVE: Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. For healing of skin wounds the skin keratinocytes can be replaced by oral mucosa epithelial cells grown in vitro. The presented experiments were carried out in order to compare the proliferation, morphology, and migration between human keratinocytes isolated from human skin and oral mucosa. MATERIALS AND METHODS: Human epidermal and oral mucosa keratinocytes from primary culture were used in all experiments. Cell motility and shape were determined using computer-aided methods. RESULTS AND CONCLUSIONS: It was demonstrated that although both cell types exhibit the same typical epithelial morphology, oral mucosa keratinocytes locomote significantly faster than skin keratinocytes. They also differ in proliferation activity. Oral mucosa keratinocytes exhibited faster growth and different actin cytoskeleton organisation than skin keratinocytes under in vitro conditions. Autologous oral mucosa keratinocytes may be expanded in vitro and used for skin wound healing in vivo.     

miR-21 promotes keratinocyte migration and re-epithelialization during wound healing

MicroRNAs involved in keratinocyte migration and wound healing are largely unknown. Here, we revealed the indispensable role of miR-21 in keratinocyte migration and in re-epithelialization during wound healing in mice. In HaCaT cell, miR-21 could be upregulated by TGF-β1. Similar to the effect of TGF-β1, miR-21 overexpression promoted keratinocyte migration. Conversely, miR-21 knockdown attenuated TGF-β1-induced keratinocyte migration, suggesting that miR-21 was essential for TGF-β-driven keratinocyte migration. Furthermore, we found that miR-21 was upregulated during wound healing, coincident with the temporal expression pattern of TGF-β1. Consistently, knockdown of endogenous miR-21 using a specific antagomir dramatically delayed re-epithelialization possibly due to the reduced keratinocyte migration. TIMP3 and TIAM1, direct targets of miR-21, were verified to be regulated by miR-21 in vitro and in vivo, indicating that these two molecules might contribute to miR-21-induced keratinocyte migration. Taken together, our results demonstrate that miR-21 promotes keratinocyte migration and boosts re-epithelialization during skin wound healing.     

The integrin αv-TGFβ signaling axis is necessary for epidermal proliferation during cutaneous wound healing

Proliferation and migration of epidermal keratinocytes are essential for proper cutaneous wound closure after injury. αv integrins and several of their ligands—vitronectin, TGFβ and thrombospondin—are up-regulated in healing wounds. However, the role of αv integrins in wound re-epithelialization is unknown. Here, we show that genetic depletion or antibody-mediated blockade of pan-integrin αv, or the specific heterodimer αvβ6, in keratinocytes limited epidermal proliferation at the wound edge and prevented re-epithelialization of wounded human organotypic skin both in vivo and in vitro. While we did not observe a migration defect upon αv blockade in vivo, αv was necessary for keratinocyte migration over longer distances in organotypic skin. Integrin αv is required for local activation of latent TGFβ, and the wound healing defect in the setting of integrin αv loss was rescued by exogenous, active TGFβ, indicating that the αv-TGFβ signaling axis is a critical component of the normal epidermal wound healing program. As chronic wounds are associated with decreased TGFβ signaling, restoration of TGFβ activity may have therapeutic utility in some clinical settings.     

Adiponectin Regulates Cutaneous Wound Healing by Promoting Keratinocyte Proliferation and Migration via the ERK Signaling Pathway

Diabetic patients are at high risk of developing delayed cutaneous wound healing. Adiponectin plays a pivotal role in the pathogenesis of diabetes and is considered to be involved in various pathological conditions associated with diabetes; however, its role in wound repair is unknown. In this study, we elucidated the involvement of adiponectin in cutaneous wound healing in vitro and in vivo. Normal human keratinocytes expressed adiponectin receptors, and adiponectin enhanced proliferation and migration of keratinocytes in vitro. This proliferative and migratory effect of adiponectin was mediated via AdipoR1/AdipoR2 and the ERK signaling pathway. Consistent with in vitro results, wound closure was significantly delayed in adiponectin-deficient mice compared with wild-type mice, and more importantly, keratinocyte proliferation and migration during wound repair were also impaired in adiponectin-deficient mice. Furthermore, both systemic and topical administration of adiponectin ameliorated impaired wound healing in adiponectin-deficient and diabetic db/db mice, respectively. Collectively, these results indicate that adiponectin is a potent mediator in the regulation of cutaneous wound healing. We propose that upregulation of systemic and/or local adiponectin levels is a potential and very promising therapeutic approach for dealing with diabetic wounds.     

Cathepsin B is essential for regeneration of scratch-wounded normal human epidermal keratinocytes

Migration, proliferation and differentiation of keratinocytes are important processes during tissue regeneration and wound healing of the skin. Here, we focussed on proteases that contribute to extracellular matrix (ECM) remodeling as a prerequisite of keratinocyte migration. In particular, we assessed the significance of the mammalian cysteine peptidase cathepsin B for human keratinocytes during regeneration from scratch wounding. We describe the construction of a scratch apparatus that allows applying scratches of defined length, width and depth to cultured cells in a reproducible fashion. The rationale for our approach derived from our previous work where we have shown that HaCaT keratinocytes secrete cathepsin B into the extracellular space during spontaneous and induced migration. Here, we observed rapid removal of type IV collagen from underneath lamellipodial extensions of keratinocytes at the advancing fronts of regenerating monolayers, indicating that proteolytic ECM remodeling starts upon initiation of keratinocyte migration. Furthermore, we verified our previous results with HaCaT cells by using normal human epidermal keratinocytes (NHEK) and show that non-cell-permeant cathepsin B-specific inhibitors delayed full regeneration of the monolayers from scratch wounding in both cell systems, HaCaT and NHEK. Application of a single dose of cathepsin B inhibitor directly after scratch wounding of keratinocytes demonstrated that cathepsin B is essential during initial stages of wound healing, while its contribution to the subsequent processes of proliferation and differentiation of keratinocytes was of less significance. This notion was supported by our observation that the cathepsin B inhibitors used in this study did not affect proliferation rates of keratinocytes of regenerating cultures. Thus, we conclude that cathepsin B is indeed involved in ECM remodeling after its secretion from migrating keratinocytes. Cathepsin B might directly cleave ECM constituents or it may initiate proteolytic cascades that involve other proteases with the ability to degrade ECM components. Because cathepsin B is important for enabling migration of both, HaCaT cells and NHEK, our results support the notion that HaCaT keratinocytes represent an excellent cell culture model for analysis of human epidermal skin keratinocyte migration.     

Effects of low frequency pulsed electrical current on keratinocytes in vitro

The effects of low frequency pulsed electrical current on epidermal repair in vitro were examined. Charge-balanced current stimuli proposed for chronic wound treatment were tested on skin keratinocytes cultured at an air-liquid interface on dead human dermis. Results imply that the balance between proliferation and differentiation in electrically treated samples is significantly modified in favor of differentiation. More advanced differentiation, shown through epidermal histology, was obtained in cultures exposed to electrical current, whereas the culture growth, the result of keratinocyte migration and proliferation, was greater in control samples. Bioelectromagnetics 18:250–254, 1997. © 1997 Wiley-Liss, Inc.     

Growth-regulated peptide-alpha (GRO-alpha) production by oral keratinocytes: a comparison with skin keratinocytes

Growth regulated peptide (GRO-alpha) is chemotactic for neutrophils. It also stimulates keratinocyte proliferation and migration, and angiogenesis in cutaneous wound healing. We compared GRO-alpha production by normal human skin and oral keratinocytes, and the effects of cytokine stimulation. Resting keratinocytes produced little, if any, GRO-alpha. TNF-alpha induced a large increase in GRO-alpha mRNA and protein production in both cell types (P<0.001). However, the response of oral keratinocytes was significantly higher (P<0.01). Oral, but not skin, keratinocytes also produced significant amounts of GRO-alpha in response to IL-1 alpha (P<0.005) and IL-4 (P<0.01) stimulation. Indeed, there was an additive effect on GRO-alpha production when oral keratinocytes were stimulated with combinations of TNF-alpha and IL-1 alpha or TNF-alpha and IL-4. Neither cell type responded to interferon gamma. Keratinocyte GRO-alpha production may help selectively recruit neutrophils in mucocutaneous inflammatory diseases, and differences in production by skin and oral keratinocyte could explain the different presentation of these diseases at the two sites. The increased GRO-alpha responsiveness of oral keratinocytes may also help explain the excellent wound healing properties of oral mucosa.     

Characterization of the living skin equivalent as a model of cutaneous re-epithelialization

The living skin equivalent, a three-dimensional organotypic model, has been widely used to investigate many aspects of cutaneous biology. However, there are relatively few studies assessing how faithfully the skin equivalent reproduces normal skin biology. The skin equivalent was fabricated by seeding human epidermal keratinocytes onto the upper surface of a hydrated collagen lattice populated with human dermal fibroblasts and subsequently raised to the air-liquid interface where keratinocyte stratification and differentiation led to the formation of a tissue which showed many common morphological features to that of normal skin. Histology and immunohistochemical detection of keratinocyte integrins and matrix metalloproteinases (MMPs) were used as cytological markers to assess the accuracy of the model during cutaneous re-epithelialization. Analysis of expression of keratinocyte integrins revealed that whilst there were a number of similarities to normal skin, skin equivalent keratinocytes appeared to be 'activated' and hyper-proliferating. Wounding of the skin equivalent, by complete bisection, induced re-epithelialization from both wound edges within 8-12 h, which completely restored the epidermis within 4 days. This migration, like that in vivo, was associated with nascent expression of MMPs and upregulation of certain integrins. However, whilst integrin expression, was similar to in vivo re-epithelialization, there were subtle differences in the level of expression and distribution of certain integrins.