The wound healing and haemocyte response in the skin of the lesser octopus Eledone cirrhosa (Mollusca: Cephalopoda) in the presence of Vibrio tubiashii

The sequential response of wound closure in the skin of the lesser octopus Eledone cirrhosa is described following experimentally induced infections by the Gram-negative bacterium, Vibrio tubiashii. Results show that the post-infection healing response varied considerably from the response observed in non-infected wounds reported previously. Prominent among the findings was the much more extensive haemocyte response noted throughout healing when compared with non-infected wounds. In addition, there was a generalized inhibition of epidermal migration so that wound closure was never completed during the experiment. The presence of a 'double tier' amorphous zone was evident at certain stages of the healing response and the implications of this finding in relation to post-infection wound closure is discussed.     

Healing of skin wounds in the African catfish Clarias gariepinus

The African catfish Clarias gariepinus was used as a model for wound healing and tissue regeneration in a scale-less fish. A temporal framework of histological and cell proliferation markers was established after wound induction in the dorsolateral cranial region, by removing the epidermal and dermal layers, including stratum adiposum (SA). Wound closure and epidermis formation was initiated within 3 h post-procedure (hpp) with migration and concomitant proliferation of epidermal cells from the wound borders. The wound was covered by this primary epidermal front 12 hpp and fusion of the opposing epidermal fronts occurred within 24 hpp. Attachment of the newly formed epidermal layer to the underlying dermis was observed 48 hpp concomitant with a second wave of cell proliferation at the wound edge. Normal epidermal thickness within the wound was achieved 72 hpp. Formation of a basement membrane occurred by 120 hpp with concomitant emergence of the SA from the wound borders. Wound healing in C. gariepinus skin involved closure of the wound and re-epithelization through cell migration with a single wave of early cell proliferation not documented in other species. Furthermore, covering of the wound by epithelium as well as the reappearance of the basement membrane and SA occurred sooner than in other fish species.     

Wound healing in tadpole tailfin pieces in vitro.

Explants of tail fins from R. catesbeiana tadpoles undergo reepithelialization of their cut surfaces (healing) when cultured in vitro in Hanks' balanced salt solution at 22 degrees C. Healing is initiated early and closure of the wound is complete by 12 to 24 hours. Morphogenesis continues for several days as further reorganization and migration of epidermal cells from the regions adjacent to the wound margins take place. The addition of serum to the culture media improves the general appearance of these tissues and promotes healing. The rate of healing is affected by temperature. Tail fins maintained at 10 degrees C do not heal while fins maintained at 30 degrees and 37 degrees, although healing more rapidly than at 22 degrees, undergo progressive degeneration in culture. Epidermal cell movements were also studied in explants consisting of a combination of intact tail fin plus tail fin deprived of its epithelium. Rapid and extensive migration of epidermal cells from the intact tail fin across the collagen lamella of the stripped fin is observed.     

A high-throughput migration assay reveals HER2-mediated cell migration arising from increased directional persistence

Human epidermal growth factor receptor 2 (HER2) overexpression has been associated with increased invasiveness in mammalian breast cancer cell lines, but the effects of overexpression on key underlying cell migration properties such as translational speed and directional persistence are not understood. Moreover, the differential effect of HER2 activation through heterodimerization with epidermal growth factor receptor versus human epidermal growth factor receptor 3 (HER3) on cell speed and persistence has not been studied. To investigate these issues, we developed a high-throughput wound closure assay in which individual cell locomotion and wound closure kinetics were quantified in human mammary epithelial cells with varying levels of HER2 under epidermal growth factor or heregulin (a HER3 ligand) stimulation. Increasing levels of HER2 elevated wound closure with closure kinetics dependent on ligand treatment. Cell speed increased with HER2 levels under epidermal growth factor treatment, but decreased under heregulin treatment. In contrast, directional persistence increased with HER2 levels under both ligand treatments. Increasing persistence quantitatively accounted for observed elevated wound closure, as measured by the effective diffusion of the cells. Taken together, the data show that the HER2 overexpression mediates cell migration through differential control of translational speed and directional persistence dependent on epidermal growth factor receptor-HER2 versus HER2-HER3 heterodimerization. Observed consistent increases in persistence associated with HER2 overexpression indicate a prospective mechanism for invasiveness previously documented in HER2-overexpressing human breast tumors.     

Cicatrization of the skin of the 7-day-old chick embryo cultured in vitro

A morphological study of in vitro wound healing has been performed by light, transmission and scanning electron microscopy in dorsal thoraco-lumbar skin of 7-day chick embryos. A circular wound, 750 microns in diameter, was punched out of dorsal skin, removing epidermis and the underlying dense dermis. Wound closure was completed within 96 to 120 hours. Feather bud development was not observed at the wound site. The epidermis began to migrate some 24 h after the wounding; the migration of peridermal cells preceded that of basal epidermal cells by some 12 hours. Mechanisms of the epidermal migration were similar to those observed in situ during wound healing of the integument in 5-day chick embryos (THEVENET, 1981), Superficial epithelization of bare dermis occurred as soon as 12 h after the injury. Cytoplasm of dermal cells exhibited many microtubules and a dilated rough endoplasmic reticulum. During the first 48 h, the epidermal cells established direct contacts and zones of close parallel apposition with epithelized dermal cell processes. The basement membrane lamina densa was maintained at the edges of the wound without retraction or ruffling. It was reconstituted concomitantly with the epidermal migration within 72 h. Cytoplasm of migratory epidermal and epithelized dermal cells exhibited many cytoskeleton structures.     

Epidermal growth factor-stimulated intestinal epithelial cell migration requires Src family kinase-dependent p38 MAPK signaling

Members of the epidermal growth factor (EGF) family of ligands and their receptors regulate migration and growth of intestinal epithelial cells. However, our understanding of the signal transduction pathways determining these responses is incomplete. In this study we tested the hypothesis that p38 is required for EGF-stimulated intestinal epithelial monolayer restitution. EGF-stimulated migration in a wound closure model required continuous presence of ligand for several hours for maximal response, suggesting a requirement for sustained signal transduction pathway activation. In this regard, prolonged exposure of cells to EGF activated p38 for up to 5 h. Furthermore genetic or pharmacological blockade of p38 signaling inhibited the ability of EGF to accelerate wound closure. Interestingly p38 inhibition was associated with increased EGF-stimulated ERK1/ERK2 phosphorylation and cell proliferation, suggesting that p38 regulates the balance of proliferation/migration signaling in response to EGF receptor activity. Activation of p38 in intestinal epithelial cells through EGF receptor was abolished by blockade of Src family tyrosine kinase signaling but not inhibition of phosphatidylinositol 3-kinase or protein kinase C. Taken together, these data suggest that Src family kinase-dependent p38 activation is a key component of a signaling switch routing EGF-stimulated responses to epithelial cell migration/restitution rather than proliferation during wound closure.     

Epidermal cell migration during wound healing in Dugesia lugubris

The epidermal cells that migrate over the surface during the wound closure stage of head regeneration in Dugesia lugubris s.l. were examined by scanning electron microscopy. The effect of cytochalasin B on epidermal cell migration was also examined. During the first few hours after decapitation epidermal cells at the edges of the wound showed significant changes of shape related to the process of migration that was accomplished approximately 10 h after wounding. Flattening of the marginal cells was associated with active epidermal spreading throughout the healing period. Suitable support for migrating cells appeared to be a rhabditic network attached to the wound tissue. Epidermal cell migration was inhibited by cytochalasin B. These results demonstrate that the basis for cell movement in planarians is similar to that of many other systems.     

Nonmuscle myosin II localization is regulated by JNK during Drosophila larval wound healing

We investigated cell shape changes during wound closure in the Drosophila larval epidermis. During reepithelialization, epidermal cells permanently change shape from pentagonal or hexagonal to irregular forms. This process requires zipper, a gene encoding the Drosophila nonmuscle myosin II heavy chain. Following wounding, myosin II is localized at the wound margin and at the rear end of individual cells located within several rows from the wound hole. The c-Jun N-terminal kinase (JNK) pathway is essential for this myosin II localization. These results suggest that not only the wound leading edge but also the cells lying distal to the leading edge cells actively participate in epithelial cell sheet migration during wound hole closure.     

Myosin II activity is required for functional leading-edge cells and closure of epidermal sheets in fish skin ex vivo

Re-epithelialization in skin wound healing is a process in which epidermal sheets grow and close the wound. Although the actin–myosin system is thought to have a pivotal role in re-epithelialization, its role is not clear. In fish skin, re-epithelialization occurs around 500 μm/h and is 50 times faster than in mammalian skin. We had previously reported that leading-edge cells of the epidermal outgrowth have both polarized large lamellipodia and “purse string”-like actin filament cables in the scale-skin culture system of medaka fish, Oryzias latipes (Cell Tissue Res, 2007). The actin purse-string (APS) is a supracellular contractile machinery in which adherens junctions (AJs) link intracellular myosin II-including actin cables between neighboring cells. In this study, we developed a modified “face-to-face” scale-skin culture system as an ex vivo model to study epidermal wound healing, and examined the role of the actin–myosin system in the rapid re-epithelialization using a myosin II ATPase inhibitor, blebbistatin. A low level of blebbistatin suppressed the formation of APS and induced the dissociation of keratocytes from the leading edge without attenuating the growth of the epidermal sheet or the migration rate of solitary keratocytes. AJs in the superficial layer showed no obvious changes elicited by blebbistatin. However, two epidermal sheets without APSs did not make a closure with each other, which was confirmed by inhibiting the connecting AJs between the superficial layers. These results suggest that myosin II activity is required for functional leading-edge cells and for epidermal closure.     

Tumor necrosis factor regulates intestinal epithelial cell migration by receptor-dependent mechanisms

Altered mucosal integrity andincreased cytokine production, including tumor necrosis factor (TNF),are the hallmarks of inflammatory bowel disease (IBD). In this study,we addressed the role of TNF receptors (TNFR) on intestinal epithelialcell migration in an in vitro wound closure model. With mouse TNFR1 orTNFR2 knockout intestinal epithelial cells, gene transfection, andpharmacological inhibitors, we show a concentration-dependentreceptor-mediated regulation of intestinal cell migration by TNF. Aphysiological TNF level (1 ng/ml) enhances migration through TNFR2,whereas a pathological level (100 ng/ml) inhibits wound closure through TNFR1. Increased rate of wound closure by TNFR2 or inhibition by TNFR1cannot be explained by either increased proliferation orapoptosis, respectively. Furthermore, inhibiting Src tyrosine kinase decreases TNF-induced focal adhesion kinase (FAK) tyrosine phosphorylation and cellular migration. We therefore conclude thatTNFR2 activates a novel Src-regulated pathway involving FAK tyrosinephosphorylation that enhances migration of intestinal epithelial cells.

     

Topical application of plasma fibronectin in full-thickness skin wound healing in rats

Fibronectin (Fn) has been shown to play an important role in wound healing because it appears to be the stimulus for migration of fibroblasts and epidermal cells. The purpose of this study was to investigate whether topical application of plasma Fn (pFn) improves healing of full-thickness skin wounds in rats. A round section of full-thickness skin (diameter of approximately 15 mm) was resected in rats. Animals were then divided into two groups, and wounds were treated topically with a single application of human plasma albumin (control group) or human pFn (FN group). Wound closure rate, hydroxyproline concentration, and histologic features (immunohistochemical staining) were evaluated. The FN group had a significantly higher wound closure rate and hydroxyproline level in the skin than the control group. Histologic analysis of macrophage and fibroblast migration, collagen regeneration, and epithelialization were significantly increased in the FN group compared with the control group. A single topical application of pFn increased the migration of macrophages, myofibroblasts, and fibroblasts. Moreover, further release of transforming growth factor-beta1 from activated fibroblasts, keratinocytes, and epithelial cells may also contribute to the beneficial effect of pFn on wound healing.     

A "traffic control" role for TGFbeta3: orchestrating dermal and epidermal cell motility during wound healing

Cell migration is a rate-limiting event in skin wound healing. In unwounded skin, cells are nourished by plasma. When skin is wounded, resident cells encounter serum for the first time. As the wound heals, the cells experience a transition of serum back to plasma. In this study, we report that human serum selectively promotes epidermal cell migration and halts dermal cell migration. In contrast, human plasma promotes dermal but not epidermal cell migration. The on-and-off switch is operated by transforming growth factor (TGF) beta3 levels, which are undetectable in plasma and high in serum, and by TGFbeta receptor (TbetaR) type II levels, which are low in epidermal cells and high in dermal cells. Depletion of TGFbeta3 from serum converts serum to a plasmalike reagent. The addition of TGFbeta3 to plasma converts it to a serumlike reagent. Down-regulation of TbetaRII in dermal cells or up-regulation of TbetaRII in epidermal cells reverses their migratory responses to serum and plasma, respectively. Therefore, the naturally occurring plasma-->serum-->plasma transition during wound healing orchestrates the orderly migration of dermal and epidermal cells.     

Loss of Epithelial Hypoxia-Inducible Factor Prolyl Hydroxylase 2 Accelerates Skin Wound Healing in Mice

Skin wound healing in mammals is a complex, multicellular process that depends on the precise supply of oxygen. Hypoxia-inducible factor (HIF) prolyl hydroxylase 2 (PHD2) serves as a crucial oxygen sensor and may therefore play an important role during reepithelialization. Hence, this study was aimed at understanding the role of PHD2 in cutaneous wound healing using different lines of conditionally deficient mice specifically lacking PHD2 in inflammatory, vascular, or epidermal cells. Interestingly, PHD2 deficiency only in keratinocytes and not in myeloid or endothelial cells was found to lead to faster wound closure, which involved enhanced migration of the hyperproliferating epithelium. We demonstrate that this effect relies on the unique expression of β₃-integrin in the keratinocytes around the tip of the migrating tongue in an HIF1α-dependent manner. Furthermore, we show enhanced proliferation of these cells in the stratum basale, which is directly related to their attenuated transforming growth factor β signaling. Thus, loss of the central oxygen sensor PHD2 in keratinocytes stimulates wound closure by prompting skin epithelial cells to migrate and proliferate. Inhibition of PHD2 could therefore offer novel therapeutic opportunities for the local treatment of cutaneous wounds.     

BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.     

Scanning electron microscopical observations on epidermal wound healing in the PlanarianDugesia tigrina

Summary Epidermal wound healing in regeneratingDugesia tigrina (Planaria) has been studied using scanning electron microscopy (SEM). The normal epidermal surface and its differentiations have been descrebed. Observations on living material reveal the highly dynamic state of the wound in invididual animals and its more or less continously changing size due to the state of activity of the animals. These observations show good agreement with the SEM studies, which allow a clear delineation of cellular details of the wound, the wound margins and the apposing epidermal regions. These details are described. The over-all picture of planarian wound healing that emerges is briefly as follows: Epithelization is characterized by absence of proliferation from the old intact epidermis. Variable contraction of smooth muscle cells reduces the wound size to a certain extent. Simultaneously with this and also during a longer period epidermal cells adjacent to the wound are extending and some become highly attenuated. These two processes together are only to a certain degree effective in wound closure because of a definite epidermal cell deficit which is reflected in the emergence of an epidermal wound edge reflecting the maximal contribution of these two processes to an attempt to close the wound. Complete epithelization is effected by the operation of a third mechanism: Recruitment of cell through flow of subjacent blastemal cells (including rhabdite-forming cells) along the wound border; these cells subsequently occupy a peripheral position in the wound. This process is supplemented by cell immigration and insertion into the adjacent old epidermis and in the wound cell sheet. Rhabdite-forming cells contribute predominantly to this process. Eventually integration between old epidermal cells and the newly recruited cells which differentiate into epidermal cells results in final epithelization. Complete wound healing is based on interactions between the epidermal cell system and the regenerating subepidermal membrane-connective tissue filament-muscle cell system.     

Application of decellularized human reticular allograft dermal matrix promotes rapid re-epithelialization in a diabetic murine excisional wound model

Background aimsThe treatment and care of human wounds represent an enormous burden on the medical system and patients alike. Chronic or delayed healing wounds are characterized by the inability to form proper granulation tissue, followed by deficiencies in keratinocyte migration and wound re-epithelialization, leading to increased likelihood of infection and poor wound outcomes. Human reticular acellular dermal matrix (HR-ADM) is one type of tissue graft developed to enhance closure of delayed healing wounds that has demonstrated clinical utility through accelerating closure of lower extremity diabetic ulcers, but the mechanisms underlying this clinical success are not well understood.MethodsThe authors utilized a diabetic murine splinted excisional wound model to investigate the effects of HR-ADM application on wound closure.ResultsThe authors demonstrate that application of HR-ADM served as a dermal scaffold and promoted rapid re-epithelialization and keratinocyte proliferation, resulting in accelerated wound closure while minimizing granulation tissue formation. HR-ADM-applied wounds also demonstrated evidence of cellular infiltration, neovascularization and collagen remodeling by the host organism.ConclusionsThese data suggest that HR-ADM supports epidermal closure in delayed healing wounds and remodeling of the matrix into host tissue, lending further support to the clinical success of HR-ADM described in clinical reports.     

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.     

THE DYNAMICS OF WOUND CLOSURE AND ITS ROLE IN THE PROGRAMMING OF PLANARIAN REGENERATION. II — DISTALIZATION

When the dorsal and ventral epidermal layers join by first intention during the closure of the wound, the cells of their borders (M-cells) do not meet in the same manner in all sections. In anterior sections the dorsal M-cells attach themselves to the ventral basement membrane, so that only the dorsal epidermis is stretched. In posterior sections the dorsal and the ventral M-cells join by their apical edges without being closely apposed to the wound surface. Only the ventral cells are stretched because of their specific motility. In longitudinal sections the dorsal and the ventral M-cells also join by their apical edges, but since they are closely apposed to the wound surface both epidermal layers are stretched. The stretching is a process equivalent to distalization. The junction between the dorsal and the ventral epidermis is shifted ventrally in the anterior wounds (as in the intact heads) and dorsally in the posterior wounds (as in the intact tails). Some abnormalities of wound closure have been observed at levels where heteromorphic regeneration frequently occurs. These findings are consistent with the hypothesis previously advanced (3) that the modalities of wound closure establish the programme for regeneration.     

Efficacy of the designer antimicrobial peptide SHAP1 in wound healing and wound infection

Infected wounds cause delay in wound closure and impose significantly negative effects on patient care and recovery. Antimicrobial peptides (AMPs) with antimicrobial and wound closure activities, along with little opportunity for the development of resistance, represent one of the promising agents for new therapeutic approaches in the infected wound treatment. However, therapeutic applications of these AMPs are limited by their toxicity and low stability in vivo. Previously, we reported that the 19-amino-acid designer peptide SHAP1 possessed salt-resistant antimicrobial activities. Here, we analyzed the wound closure activities of SHAP1 both in vitro and in vivo. SHAP1 did not affect the viability of human erythrocytes and keratinocytes up to 200 μM, and was not digested by exposure to proteases in the wound fluid, such as human neutrophil elastase and Staphylococcus aureus V8 proteinase for up to 12 h. SHAP1 elicited stronger wound closure activity than human cathelicidin AMP LL-37 in vitro by inducing HaCaT cell migration, which was shown to progress via transactivation of the epidermal growth factor receptor. In vivo analysis revealed that SHAP1 treatment accelerated closure and healing of full-thickness excisional wounds in mice. Moreover, SHAP1 effectively countered S. aureus infection and enhanced wound healing in S. aureus-infected murine wounds. Overall, these results suggest that SHAP1 might be developed as a novel topical agent for the infected wound treatment.     

Annexins II and V Inhibit Cell Migration

Cell motility is a crucial component involved in wound healing, development, and tumor metastasis. This study investigated whether extracellular annexins, members of a calcium- and phospholipid-binding family of proteins, play a role in the migration of Lewis lung carcinoma cells. Using assays for wound closure and migration through 8-μm pores, it was found that annexins II and V significantly (>40%) inhibited migration of these highly metastatic cells. Additionally, anti-annexin II antibodies enhanced migration of these same cells in the wound closure assay, while an irrelevant antibody (anti-calmodulin) showed no effect. These effects may be due to annexin–membrane binding and inhibition of phospholipid movement that is necessary for the formation of membrane protrusions.