Tether-guided lamellipodia enable rapid wound healing

Adhesion between animal cells and the underlying extracellular matrix is challenged during wounding, cell division, and a variety of pathological processes. How cells recover adhesion in the immediate aftermath of detachment from the extracellular matrix remains incompletely understood, due in part to technical limitations. Here, we used acute chemical and mechanical perturbations to examine how epithelial cells respond to partial delamination events. In both cases, we found that cells extended lamellipodia to establish readhesion within seconds of detachment. These lamellipodia were guided by sparse membrane tethers whose tips remained attached to their original points of adhesion, yielding lamellipodia that appear to be qualitatively distinct from those observed during cell migration. In vivo measurements in the context of a zebrafish wound assay showed a similar behavior, in which membrane tethers guided rapidly extending lamellipodia. In the case of mechanical wounding events, cells selectively extended tether-guided lamellipodia in the direction opposite of the pulling force, resulting in the rapid reestablishment of contact with the substrate. We suggest that membrane tether-guided lamellipodial respreading may represent a general mechanism to reestablish tissue integrity in the face of acute disruption.     

Activin B promotes epithelial wound healing in vivo through RhoA-JNK signaling pathway

Background

Activin B has been reported to promote the proliferation and migration of keratinocytes in vitro via the RhoA-JNK signaling pathway, whereas its in vivo role and mechanism in wound healing process has not yet been elucidated.

Principal Findings

In this study, we explored the potential mechanism by which activin B induces epithelial wound healing in mice. Recombinant lentiviral plasmids, with RhoA (N19) and RhoA (L63) were used to infect wounded KM mice. The wound healing process was monitored after different treatments. Activin B-induced cell proliferation on the wounded skin was visualized by electron microscopy and analyzed by 5′-bromodeoxyuridine (BrdU) incorporation assay. Protein expression of p-JNK or p-cJun was determined by immunohistochemical staining and immunoblotting analysis. Activin B efficiently stimulated the proliferation of keratinocytes and hair follicle cells at the wound area and promoted wound closure. RhoA positively regulated activin B-induced wound healing by up-regulating the expression of p-JNK and p-cJun. Moreover, suppression of RhoA activation delayed activin B-induced wound healing, while JNK inhibition recapitulated phenotypes of RhoA inhibition on wound healing.

Conclusion

These results demonstrate that activin B promotes epithelial wound closure in vivo through the RhoA-Rock-JNK-cJun signaling pathway, providing novel insight into the essential role of activin B in the therapy of wound repair.     

Changes in epithelial cells in Hirudo medicinalis during wound healing.

In the leech, Hirudo medicinalis, reepithelialization is an event which takes place early in the wound healing process, immediately after the formation of the pseudoblastema, 4-8 hr postinjury. Epithelial cells on the wound margins move into the wound, modifying their phenotypic characteristics. Cells lose their columnar shape and become flattened. Dermal junctions disrupt and tonofilaments regroup around the nucleus. Then, the epithelial cell sheet moves over the newly formed pseudoblastema by extending filopodia, formed by the cells on the edge, following the so-called "sliding model." When the wound is fully covered by the new epithelium, about 24 hr postinjury, a reorganization of the cytoskeleton occurs and the basal dermal junctions are reconstructed. Six days postinjury, the epidermal cells return to their original columnar shape.     

Adipose-derived stem cells for wound healing

Wound healing is a complex but a fine-tuned biological process in which human skin has the ability to regenerate itself following damage. However, in particular conditions such as deep burn or diabetes the process of wound healing is compromised. Despite investigations on the potency of a wide variety of stem cells for wound healing, adipose-derived stem cells (ASCs) seem to possess the least limitations for clinical applications, and literature showed that ASCs can improve the process of wound healing very likely by promoting angiogenesis and/or vascularisation, modulating immune response, and inducing epithelialization in the wound. In the present review, advantages and disadvantages of various stem cells which can be used for promoting wound healing are discussed. In addition, potential mechanisms of action by which ASCs may accelerate wound healing are summarised. Finally, clinical studies applying ASCs for wound healing and the associated limitations are reviewed.     

Rho kinases regulate corneal epithelial wound healing

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.     

Rhinovirus infection induces cytotoxicity and delays wound healing in bronchial epithelial cells

Background

Human rhinoviruses (RV), the most common triggers of acute asthma exacerbations, are considered not cytotoxic to the bronchial epithelium. Recent observations, however, have questioned this knowledge. The aim of this study was to evaluate the ability of RV to induce epithelial cytotoxicity and affect epithelial repair in-vitro.

Methods

Monolayers of BEAS-2B bronchial epithelial cells, seeded at different densities were exposed to RV serotypes 1b, 5, 7, 9, 14, 16. Cytotoxicity was assessed chromatometrically. Epithelial monolayers were mechanically wounded, exposed or not to RV and the repopulation of the damaged area was assessed by image analysis. Finally epithelial cell proliferation was assessed by quantitation of proliferating cell nuclear antigen (PCNA) by flow cytometry.

Results

RV1b, RV5, RV7, RV14 and RV16 were able to induce considerable epithelial cytotoxicity, more pronounced in less dense cultures, in a cell-density and dose-dependent manner. RV9 was not cytotoxic. Furthermore, RV infection diminished the self-repair capacity of bronchial epithelial cells and reduced cell proliferation.

Conclusion

RV-induced epithelial cytotoxicity may become considerable in already compromised epithelium, such as in the case of asthma. The RV-induced impairment on epithelial proliferation and self-repair capacity may contribute to the development of airway remodeling.     

A possible role for membrane depolarization in epithelial wound healing

Linear narrow wounds produced on cultured bovine corneal endothelial monolayers heal by actin cable formation at the wound border and lamellar crawling of cells into the injured area. We report the novel finding that membrane potential depolarization occurs at the leading edge of wounds and gradually extends inward toward the neighboring cells. We have determined that the replacement of extracellular Na⁺ by choline and the incorporation of phenamil, an inhibitor of the epithelial Na⁺ channel (ENaC), provoke a decrease in the actin cable and depolarization areas and in the lamellar activity of the wound edges. To the contrary, extracellular Li⁺ can successfully replace Na⁺ in the determination of the depolarization and cytoskeletal responses. This finding supports the idea that membrane depolarization, not the increase in intracellular Na⁺ concentration, is responsible for the formation of the actin cable, a result that is in agreement with previous evidence showing that nonspecific depolarization of the plasma membrane potential (PMP) of epithelial cells may promote characteristic cytoskeletal rearrangements per se (Chifflet S, Hernández JA, Grasso S, and Cirillo A. Exp Cell Res 282: 1–13, 2003). We suggest that spontaneous depolarization of the PMP of the cells at the wound borders determined by a rise in the ENaC activity of these cells constitutes an additional factor in the intermediate cellular processes leading to wound healing in some epithelia. actin; epithelial sodium channel     

Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation

Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.     

Cytoplasmic filaments and gap junctions in epithelial cells and myofibroblasts during wound healing

During the healing of an experimental skin wound, epidermal cells and granulation tissue fibroblasts (myofibroblasts) develop an extensive cytoplasmic contactile apparatus. Concurrently, the proportion of epidermal cell surface occupied by gap junctions increases when compared to normal skin, and newly formed gap junctions appear between myofibroblasts; this suggests that epidermal cell migration and granulation tissue contraction are synchronized phenomena.     

Vasoactive intestinal peptide enhances wound healing and proliferation of human bronchial epithelial cells

In the present study, we investigated the effects of vasoactive intestinal peptide (VIP) on wound healing of bronchial epithelium. Wound healing of the mechanical damaged human bronchial epithelial cells (HBEC) was observed in the absence or presence of VIP. Effects of VIP on chemotactic migration, cell proliferation of HBEC were also tested. HBEC chemotaxis was assessed by the blind well chamber technique, the cell cycle was determined by flow cytometry, and cell proliferation was determined by measuring the expression of proliferating cell nuclear antigen Ki67. Effects of VIP on epithelial E-cadherins protein and mRNA were also measured by immunohistochemistry and RT-PCR. The results showed that VIP accelerated the recovery of wound area of HBEC. VIP increased the migration and proliferation of HBEC, and these effects were blocked by a VPAC1 receptor antagonist. VIP also increased the expression of E-cadherin mRNA and protein in HBEC, suggesting that protective effects of VIP on wound healing may be related to its ability to increase the expression of E-cadherin. In conclusion, VIP has protective effects against human bronchial epithelial cell damage, and the beneficial effects of VIP might be mediated, at least in part, by VPAC1, and associated with increased expression of E-cadherin.     

Exosomes derived from human amniotic epithelial cells accelerate wound healing and inhibit scar formation

Wound healing is a highly orchestrated physiological process consisting of a complex events, and scarless wound healing is highly desired for the development and application in clinical medicine. Recently, we have demonstrated that human amniotic epithelial cells (hAECs) promoted wound healing and inhibited scar formation through a paracrine mechanism. However, exosomes (Exo) are one of the most important paracrine factors. Whether exosomes derived from human amniotic epithelial cells (hAECs-Exo) have positive effects on scarless wound healing have not been reported yet. In this study, we examined the role of hAECs-Exo on wound healing in a rat model. We found that hAECs, which exhibit characteristics of both embryonic and mesenchymal stem cells, have the potential to differentiate into all three germ layers. hAECs-Exo ranged from 50 to 150 nm in diameter, and positive for exosomal markers CD9, CD63, CD81, Alix, TSG101 and HLA-G. Internalization of hAECs-Exo promoted the migration and proliferation of fibroblasts. Moreover, the deposition of extracellular matrix (ECM) were partly abolished by the treatment of high concentration of hAECs-Exo (100 μg/mL), which may be through stimulating the expression of matrix metalloproteinase-1 (MMP-1). In vivo animal experiments showed that hAECs-Exo improved the skin wound healing with well-organized collagen fibers. Taken together, These findings represent that hAECs-Exo can be used as a novel hope in cell-free therapy for scarless wound healing.     

Wnt signaling induces epithelial differentiation during cutaneous wound healing

Background  

Cutaneous wound repair in adult mammals does not regenerate the original epithelial architecture and results in altered skin function. We propose that lack of regeneration may be due to the absence of appropriate molecular signals to promote regeneration. In this study, we investigated the regulation of Wnt signaling during cutaneous wound healing and the consequence of activating either the beta-catenin-dependent or beta-catenin-independent Wnt signaling on epidermal architecture during wound repair.     

Mechanical compression attenuates normal human bronchial epithelial wound healing

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.     

A model for the study of epithelial migration in wound healing.

A model is described which enables the detailed study of epithelial regeneration in experimentally produced lesions in the common bile duct of the rabbit. The circular on slightly oval defect of 1 mm diameter produced by a specially developed apparatus has a perfectly smooth base. Epithelial migration in this model has been investigated using light microscopy of transverse sections and scanning electron microscopy of whole preparations. Typical changes in the border cells, characterised by the formation of tapered protusions, can be observed as early as two hours after the lesion has been made. Later the cells in the flattened edge of the moving border also show various types of protrusion which rest on the substratum. Mitotic activity in the surface epithelium and crypts in the surrounding region only increases after closure of the lesion, which usually takes place within 16--24 h.     

Glucagon-like peptide 2 improves intestinal wound healing through induction of epithelial cell migration in vitro-evidence for a TGF--beta-mediated effect

BACKGROUND/AIMS: In vitro studies suggest that glucagon-like peptide 2 (GLP-2), secreted from enteroendocrine cells in the gastrointestinal tract after food intake, is able to ameliorate mucosal injury in settings of human disease characterized by injury and dysfunction of the intestinal mucosal epithelium. We evaluated this potential of GLP-2 after epithelial trauma by using two in vitro models measuring intestinal epithelial cell proliferation and cell migration. MATERIALS AND METHODS: Injuries were induced in confluent monolayers of the small intestinal cells lines IEC-6 and IEC-18, as well as in the colonic cell lines Caco-2 and Colo 320. GLP-2 (50-500 nM) or other peptides were added to the media. Wound healing was investigated after 24 h by quantification of the number of cells migrating across the wound edge. Proliferation of cells was assessed by using photometric mitochondrial incorporation measurement of MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide). Monoclonal TGF-beta antibodies were added to wounded monolayers to examine whether the GLP-2-induced wound healing was TGF-beta-mediated. RESULTS: Migration assessments revealed a significant stimulation of GLP-2-induced migration in IEC-6 and IEC-18 monolayers compared to the placebo group. No effect was observed in the colon cancer cell lines Caco-2 and Colo 320. Results of the proliferation assays show a significant inhibition of proliferation by GLP-2 in small intestinal cell lines whereas a dose-dependent stimulation of proliferation in colonic epithelial cells was observed. Addition of neutralizing TGF-beta1 antibodies to wounded IEC-6 and IEC-18 monolayers incubated with GLP-2 significantly reduced the number of migrating cells to the level of the placebo group. CONCLUSIONS: In our in vitro model, it was shown that the GLP-2-induced improvement of intestinal wound healing is TGF-beta-mediated. These effects were predominant in the epithelium of the small intestine compared to colonic epithelium. Our findings provide further insight into mechanisms leading to GLP-2-induced mucosal wound healing. These results suggest that GLP-2 or analogues of this peptide may potentially be useful for the treatment of intestinal disorders characterized by injury and ineffective repair of the intestinal mucosa.     

Intracellular signaling molecules involved in vasoactive intestinal peptide-mediated wound healing in human bronchial epithelial cells

Vasoactive intestinal peptide (VIP), a non-adrenergic, non-cholinergic neuromediator, plays an important role in maintaining the bronchial tone of the airway and has anti-inflammatory properties. Recently, we reported that VIP enhances wound repair in human bronchial epithelial cells (HBEC). In the present study, we have identified the intracellular signaling molecules that are involved in VIP-mediated wound healing in HBEC. The effects of VIP on wound repair of HBEC were partially blocked by H-7 (a protein kinase C (PKC) inhibitor), W-7 (a calmodulin inhibitor), H-89 (a protein kinase A (PKA) inhibitor), and PD98059 (a specific extracellular signal-regulated kinase (ERK) inhibitor). VIP-induced chemotactic migration was inhibited in the presence of W-7, H-89, PD98059 or H-7. H-7, W-7, and H-89 were also found to decrease VIP-induced expression of Ki67 as well as the proliferation index in HBEC. Furthermore, H-7, W-7, H-89, and PD98059 inhibited the expression of E-cd protein and mRNA induced by VIP. These results suggest that intracellular signaling molecules such as PKA, PKC, ERK, and calmodulin play important role in VIP-mediated wound healing of HBEC.