Skin Wound Sites Research Articles

Effect of electroacupuncture Bangci on chloride flux at mouse skin incisional wounds

The effect of electroacupuncture Bangci on chloride flux (Cl- flux) at a skin wound site was investigated in mice. Ninety male C57BL/6 mice were randomly divided into three groups: electroacupuncture Bangci group, Bangci group, and normal unwound group, with each group containing 30 mice. Next, the mice in each group were divided into 5 subgroups according to different time points. In the electroacupuncture Bangci and Bangci groups, a 5-mm lancet wound was made by cutting into full-thickness skin. Then, Cl- flux was measured using ion-selective probes at the skin wound. The results showed that Cl- flux was significantly higher in the electroacupuncture Bangci group than that in the Bangci group at days 1 and 3 after wound induction (P < 0.05). The results of this study suggest that electroacupuncture Bangci positively affects the wound healing of mouse skin wounds.

MiR-142 Is Required for Staphylococcus aureus Clearance at Skin Wound Sites via Small GTPase-Mediated Regulation of the Neutrophil Actin Cytoskeleton

MicroRNAs (miRNAs) are small noncoding RNAs that regulate protein translation by binding to complementary target mRNAs. We previously identified two mature members of the miR-142 family, miR-142-5p and miR-142-3p, as inflammation-related miRNAs with potential roles in wound healing. Here, we demonstrated that these two miRNAs are prominently expressed in wound-infiltrated neutrophils and macrophages and play central roles in wound healing. We generated miR-142-/- mice using the exchangeable gene-trap method and showed that healing of Staphylococcus aureus-infected skin wounds was significantly delayed in miR-142-/- mice compared with that in wild-type mice. MiR-142-/- mice exhibited abnormal abscess formation at S.aureus-infected skin wound sites and were also more susceptible to horizontal transmission of wound infections. MiR-142-/- neutrophils showed altered phagocytosis as a consequence of chemotactic behavior, including enhanced F-actin assembly, disturbed cell polarity, and increased cell motility. We showed that these changes were linked to cytoskeletal regulation, and that expression of the small GTPases was markedly increased in miR-142-/- neutrophils. Collectively, our data demonstrate that the miR-142 family is indispensable for protection against S.aureus infection and its clearance at wound sites. MiR-142-3p and miR-142-5p play nonredundant roles in actin cytoskeleton regulation by controlling small GTPase translation in neutrophils at wound sites.

Zinc Oxide Nanorod‐Based Piezoelectric Dermal Patch for Wound Healing

Current treatments for wound healing engage in passive healing processes and rarely participate in stimulating skin cell behaviors for active wound healing. Electric potential difference‐derived electrical fields (EFs) are known to modulate skin cell behaviors. Here, a piezoelectric dermal patch is developed that can be applied on skin wound site and EF is generated to promote wound healing. The one‐directionally aligned zinc oxide nanorod‐based piezoelectric patch generates piezoelectric potential upon mechanical deformations induced by animal motion, and induces EF at the wound bed. In vitro and in vivo data demonstrate that the piezoelectric patch promotes the wound healing process through enhanced cellular metabolism, migration, and protein synthesis. This modality may lead to a clinically relevant piezoelectric dermal patch therapy for active wound healing.

Accelerating skin wound healing by M-CSF through generating SSEA-1 and -3 stem cells in the injured sites

Wound healing is a complicated process requiring the collaborative efforts of different cell lineages. Our recent studies have found that one subset of hematopoietic cells can be induced to dedifferentiate into multipotent stem cells by means of a proliferating fibroblast releasable factor, M-CSF. Understanding the importance of stem cells on skin wound healing, here we evaluate the biological significance of M-CSF on skin wound healing. In an in vivo mouse skin excisional wound model, we found that SSEA-positive stem cells were present in wounded but not normal skin. After isolating skin cells from either normal or wounded skin by collagenase digestion, and analyzing the SSEA-1 positive cells by flow cytometry, we found a significant increase in the number of SSEA-1 positive cells in wounded skin. Topical application of M-CSF in skin wounds accelerated healing remarkably, while application of M-CSF-neutralizing antibody slowed wound healing. Furthermore, injection of EGFP-labeled hematopoietic cell-derived stem cells generated from M-CSF treated splenocytes resulted in EGFP-labeled cells being enriched in the skin wound site and further differentiated into functional organ-specific cells. Together, these data demonstrated that M-CSF makes a significant contribution to the healing process by inducing hematopoietic cell dedifferentiation into stem cells.

Melatonin enhances the human mesenchymal stem cells motility via melatonin receptor 2 coupling with Gαq in skin wound healing.

Melatonin, a circadian rhythm-promoting molecule, has a variety of biological functions, but the functional role of melatonin in the motility of mesenchymal stem cells (MSCs) has yet to be studied. In a mouse skin excisional wound model, we found that transplantation of umbilical cord blood (UCB)-MSCs pretreated with melatonin enhanced wound closure, granulation, and re-epithelialization at mouse skin wound sites, where relatively more UCB-MSCs which were engrafted onto the wound site were detected. Thus, we identified the signaling pathway of melatonin, which affects the motility of UCB-MSCs. Melatonin (1 μm) significantly increased the motility of UCB-MSCs, which had been inhibited by the knockdown of melatonin receptor 2 (MT2). We found that Gαq coupled with MT2 and that the binding of Gαq to MT2 uniquely stimulated an atypical PKC isoform, PKCζ. Melatonin induced the phosphorylation of FAK and paxillin, which were concurrently downregulated by blocking of the PKC activity. Melatonin increased the levels of active Cdc42 and Arp2/3, and it has the ability to stimulate cytoskeletal reorganization-related proteins such as profilin-1, cofilin-1, and F-actin in UCB-MSCs. Finally, a lack of MT2 expression in UCB-MSCs during a mouse skin transplantation experiment resulted in impaired wound healing and less engraftment of stem cells at the wound site. These results demonstrate that melatonin signaling via MT2 triggers FAK/paxillin phosphorylation to stimulate reorganization of the actin cytoskeleton, which is responsible for Cdc42/Arp2/3 activation to promote UCB-MSCs motility.

Skin Electroporation of a Plasmid Encoding hCAP-18/LL-37 Host Defense Peptide Promotes Wound Healing

Host defense peptides, in particular LL-37, are emerging as potential therapeutics for promoting wound healing and inhibiting bacterial growth. However, effective delivery of the LL-37 peptide remains limiting. We hypothesized that skin-targeted electroporation of a plasmid encoding hCAP-18/LL-37 would promote the healing of wounds. The plasmid was efficiently delivered to full-thickness skin wounds by electroporation and it induced expression of LL-37 in the epithelium. It significantly accelerated reepithelialization of nondiabetic and diabetic wounds and caused a significant VEGFa and interleukin (IL)-6 induction. IL-6 was involved in LL-37-mediated keratinocyte migration in vitro and IL-6 neutralizing antibodies delivered to mice were able to suppress the wound healing activity of the hCAP-18/LL-37 plasmid. In a hindlimb ischemia model, electroporation of the hCAP-18/LL-37 plasmid increased blood perfusion, reduced muscular atrophy, and upregulated the angiogenic chemokines VEGFa and SDF-1a, and their receptors VEGF-R and CXCR-4. These findings demonstrate that a localized gene therapy with LL-37 is a promising approach for the treatment of wounds.

Wound repair and anti-inflammatory potential of Lonicera japonica in excision wound-induced rats

BackgroundLonicera japonica Thunb. (Caprifoliaceae), a widely used traditional Chinese medicinal plant, is used to treat some infectious diseases and it may have uses as a healthy food and applications in cosmetics and as an ornamental groundcover. The ethanol extract of the flowering aerial parts of L. japonica (LJEE) was investigated for its healing efficiency in a rat excision wound model.MethodsExcision wounds were inflicted upon three groups of eight rats each. Healing was assessed by the rate of wound contraction in skin wound sites in rats treated with simple ointment base, 10% (w/w) LJEE ointment, or the reference standard drug, 0.2% (w/w) nitrofurazone ointment. The effects of LJEE on the contents of hydroxyproline and hexosamine during healing were estimated. The antimicrobial activity of LJEE against microorganisms was also assessed. The in vivo anti-inflammatory activity of LJEE was investigated to understand the mechanism of wound healing.ResultsLJEE exhibited significant antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Candida albicans, and Candida tropicalis. The ointment formulation prepared with 10% (w/w) LJEE exhibited potent wound healing capacity as evidenced by the wound contraction in the excision wound model. The contents of hydroxyproline and hexosamine also correlated with the observed healing pattern. These findings were supported by the histopathological characteristics of healed wound sections, as greater tissue regeneration, more fibroblasts, and angiogenesis were observed in the 10% (w/w) LJEE ointment-treated group. The results also indicated that LJEE possesses potent anti-inflammatory activity, as it enhanced the production of anti-inflammatory cytokines that suppress proinflammatory cytokine production.ConclusionsThe results suggest that the antimicrobial and anti-inflammatory activities of LJEE act synergistically to accelerate wound repair.

Release of TNF‐α from macrophages is mediated by small GTPase Rab37

Activated macrophages at wound sites release many cytokines which positively affect skin wound healing. However, the molecular mechanisms controlling cytokine secretion from macrophages have not been elucidated. In the present study, we performed an RT-PCR analysis and found that 19 small GTPase Rab isoforms were expressed at skin wound sites, with six of them (i.e. Rab3B, Rab27B, Rab30, Rab33A, Rab37, and Rab40C) being upregulated during the inflammation and proliferation/migration phase of skin repair. We also found that gene expression of Rab37 in murine primary and RAW264.7 macrophages was significantly induced after stimulation with LPS. Overexpression of wild type and constitutively active Rab37 in RAW264.7 cells significantly increased TNF-α secretion, whereas knockdown of Rab37 by siRNA significantly decreased it. We also identified 29 putative Rab37-interacting proteins, including the membrane fusion regulating Munc13-1, using liquid chromatography/linear ion trap mass spectrometry (LC-MS/MS). Immunocytochemical analysis further revealed that TNF-α-containing vesicles were colocalized with both Rab37 and Munc13-1 in activated macrophages. Knockdown of Munc13-1 by siRNA significantly decreased TNF-α secretion. Taken together, these findings demonstrate that Rab37 interacts with Munc13-1 to control TNF-α secretion from activated macrophages.

The use of morphometric and fractal parameters to assess the effects of 5-fluorouracil, interferon and dexamethasone treatment on colonic anastomosis healing: an experimental study in rats

Adjuvant chemotherapy and steroid therapy have been demonstrated to interfere with the wound healing process. The aim of this study was to evaluate the effects of 5-fluorouracil, interferon, and dexamethasone, on the healing of colon anastomosis by assessing morphometric and fractal parameters of the colonic wall. An experimental anastomosis of the ascending colon was performed in 60 male Wistar rats, which were then randomly assigned to four groups. On the second to sixth post-operative days, the rats were administered 5-fluorouracil, interferon-α, dexamethasone, or 0.9% NaCl solution as a control. Macroscopic, histomorphometric and microbiological evaluation was performed in order to assess healing of the anastomosis. In three animals from the dexamethasone group, there was leakage of anastomosis; adhesion formation was highest in the interferon group, and significantly higher than in the control and 5-fluorouracil groups. Histomorphometric parameter alterations were most pronounced on the seventh and fourteenth post-operative days in all treatment groups, with submucosal thickness the most affected parameter. Connective tissue fractal dimension was significantly decreased in those animals treated with interferon and dexamethasone. All three pharmaceutical agents impaired healing of anastomosis, and promoted infection in the anastomosis and skin wound sites. As dexamethasone induced both morphometric and macroscopic alterations, it was considered the most detrimental in this study.

Effects of Red Deer Antlers on Cutaneous Wound Healing in Full-thickness Rat Models

The process of wound repair involves an ordered sequence of events such as overlapping biochemical and cellular events that, in the best of circumstances, result in the restoration of both the structural and functional integrity of the damaged tissue. An important event during wound healing is the contraction of newly formed connective tissues by fibroblasts. The polypeptide growth factors, like transforming growth factor-β (TGF-β), insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF), play very important mediator roles in the process of wound contraction. Deer antlers, as models of mammalian regeneration, are cranial appendages that develop after birth as extensions of a permanent protuberance (pedicle) on the frontal bone. Antlers contain various growth factors which stimulate dermal fibroblast growth. They are involved in digestion and respiration and are necessary for normal wound healing and skin health. In order to investigate and evaluate the effects of red deer antlers on skin wound site, the speed of full-thickness skin wound healing and the expression of IGF-I, TGF-β, and EGF in skin wounds, three groups of skin full-thickness rat models with a high concentration of antler ointment, a low concentration of antler ointment and without antler ointment were compared. At post-injury days 0, 2, 4, 8, 16, 20, 32, 40 and 60, the skin wound area was measured, the expressions of IGF-I, TGF-β, and EGF mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR) and collagen formation by sirius red dye and the localization of IGF-I, TGF-β, and EGF peptides were inspected by histological immunohistochemical techniques. Wound healing was significantly more rapid in antler treated skins. In addition, the wound treated with a high concentration antler ointment, a low concentration antler ointment, and the control closed completely at post-injury day 40, day 44 and day 60, respectively. Via RT-PCR, the expressions of IGF-I (day 8 and day 16), TGF-β (day 8, day 16 and day 20) and EGF (day 4, day 8, day 16, and day 32) were obviously up-regulated in high concentration antler-treated skins compared to control skins. Similar results could be seen in the histological detection of collagen dye and immunohistochemical methods using the corresponding polyclone antibodies of IGF-I, TGF-β, and EGF. These results illustrate that antlers stimulate and accelerate the repair of cutaneous wounds.

Antimicrobial Peptides Human β-Defensins Stimulate Epidermal Keratinocyte Migration, Proliferation and Production of Proinflammatory Cytokines and Chemokines

Besides their microbicidal functions, human beta-defensins (hBD) and LL-37 activate different immune and inflammatory cells, and their expression is enhanced in inflamed skin and cutaneous wound sites. To protect against pathogens, the skin produces antimicrobial peptides including hBDs and LL-37. Therefore, the aim of our study was to investigate whether hBDs participate in cutaneous inflammation and wound healing by inducing keratinocyte migration, proliferation, and production of proinflammatory cytokines/chemokines. We found that hBD-2, -3, and -4 but not hBD-1 stimulated human keratinocytes to increase their gene expression and protein production of IL-6, IL-10, IP-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-3alpha, and RANTES. This stimulatory effect was markedly suppressed by pertussis toxin and U-73122, inhibitors for G protein and phospholipase C, respectively. We also demonstrated that hBDs elicited intracellular Ca2+ mobilization, and increased keratinocyte migration, and proliferation. In addition, these peptides induced phosphorylation of EGFR, signal transducer and activator of transcription (STAT)1, and STAT3, which are intracellular signaling molecules involved in keratinocyte migration and proliferation. In our study, inhibition of these molecules significantly reduced hBD-mediated keratinocyte migration and proliferation. In conclusion, this study provides evidence that human antimicrobial peptides may be involved in skin immunity through stimulating cytokine/chemokine production, and participate in wound healing by promoting keratinocyte migration and proliferation.

Acute downregulation of connexin43 at wound sites leads to a reduced inflammatory response, enhanced keratinocyte proliferation and wound fibroblast migration

Experimental downregulation of connexin43 (Cx43) expression at skin wound sites appears to markedly improve the rate and quality of healing, but the underlying mechanisms are currently unknown. Here, we have compared physiological and cell biological aspects of the repair process with and without Cx43 antisense oligodeoxynucleotide treatment. Treated wounds exhibited accelerated skin healing with significantly increased keratinocyte and fibroblast proliferation and migration. In vitro knockdown of Cx43 in a fibroblast wound-healing model also resulted in significantly faster healing, associated with increased mRNA for TGF-beta1, and collagen alpha1 and general collagen content at the wound site. Treated wounds showed enhanced formation of granulation tissue and maturation with more rapid angiogenesis, myofibroblast differentiation and wound contraction appeared to be advanced by 2-3 days. Recruitment of both neutrophils and macrophages was markedly reduced within treated wounds, concomitant with reduced leukocyte infiltration. In turn, mRNA levels of CC chemokine ligand 2 and TNF-alpha were reduced in the treated wound. These data suggest that, by reducing Cx43 protein with Cx43-specific antisense oligodeoxynucleotides at wound sites early in the skin healing process repair is enhanced, at least in part, by accelerating cell migration and proliferation, and by attenuating inflammation and the additional damage it can cause.

Absence of IL-1 Receptor Antagonist Impaired Wound Healing along with Aberrant NF-κB Activation and a Reciprocal Suppression of TGF-β Signal Pathway

Although enhanced expression of IL-1 family proteins, including IL-1alpha, IL-1beta, and IL-1 receptor antagonist (IL-1ra) during wound healing has been observed, the pathophysiological roles of these factors, particularly IL-1ra, still remain elusive. We explored skin wound-healing processes in IL-1ra-deficient mice. Compared to wild-type (WT) mice, IL-1ra-deficient mice exhibited impaired wound healing, as evidenced by attenuated collagen deposition and delayed neovascularization. In contrast, neutrophil recruitment was significantly exaggerated, with the augmented expression of IL-1s, TNF-alpha, and CXC chemokines, MIP-2 and KC, in IL-1ra-deficient mice compared with WT mice. Because the transcription of these proinflammatory cytokines and CXC chemokines requires the activation of NF-kappaB, a major target of IL-1- and TNF-alpha-mediated signal pathway, we examined the activation states of NF-kappaB. Nuclear translocation of NF-kappaB p65 was significantly enhanced and prolonged in IL-1ra-deficient mice, compared to that in WT mice. The cross-talk between NF-kappaB and TGF-beta-mediated signals has been proposed based on in vitro observations. Indeed, compared to WT mice, the amounts of total and phosphorylated Smad2 and Smad3 were decreased with a reciprocal increase in the amount of Smad7 in skin wound sites of IL-1ra-deficient mice. Moreover, the gene expression of vascular endothelial growth factor, a target gene of TGF-beta1, was decreased in IL-1ra-deficient mice. Thus, the absence of IL-1ra may suppress TGF-beta-mediated signaling pathway, which is crucial for collagen deposition and vascular endothelial growth factor-mediated neovascularization in wound healing.

Induction of Keratinocyte Migration via Transactivation of the Epidermal Growth Factor Receptor by the Antimicrobial Peptide LL-37

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 microg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.

Efficient Gene Expression in Skin Wound Sites Following Local Plasmid Injection

Transfection of the skin by local gene delivery, as well as widespread transfection of systemic tissues following intravenous injection of cationic liposome/DNA complexes have been reported. Here, we show that surgically wounded mouse skin can be transfected either by local injection of DNA alone or by intravenous injection of optimized cationic liposome/DNA complexes; however, direct cutaneous injection produces much higher levels of gene expression in the skin, which is targeted to dermal and subdermal layers. High levels of chloramphenicol acetyltransferase activity were present from 3 h to 2 wk following direct injection of a gene expression plasmid into wounded skin and were maintained at detectable levels up to 8 wk after injection. Expression of transferred chloramphenicol acetyltransferase as well as beta-GAL genes was localized to fibroblasts, macrophages, and adipocytes as determined by histochemistry and immunohistochemistry. Further- more, local injection of a human granulocyte- colony-stimulating factor gene expression plasmid produced high levels of the biologically relevant human granulocyte-colony-stimulating factor protein in wounded mouse skin. This efficient and simple method of site-specific gene transfer into wounds may lead to the development of cutaneous gene therapy directed against disorders of abnormal cutaneous wound healing.

76 Expression of presenilin 1 in rat cortical and hippocampal primary cultures: An immunocytochemical study

Besides their microbicidal functions, human β-defensins (hBD) and LL-37 activate different immune and inflammatory cells, and their expression is enhanced in inflamed skin and cutaneous wound sites. To protect against pathogens, the skin produces antimicrobial peptides including hBDs and LL-37. Therefore, the aim of our study was to investigate whether hBDs participate in cutaneous inflammation and wound healing by inducing keratinocyte migration, proliferation, and production of proinflammatory cytokines/chemokines. We found that hBD-2, -3, and -4 but not hBD-1 stimulated human keratinocytes to increase their gene expression and protein production of IL-6, IL-10, IP-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-3α, and RANTES. This stimulatory effect was markedly suppressed by pertussis toxin and U-73122, inhibitors for G protein and phospholipase C, respectively. We also demonstrated that hBDs elicited intracellular Ca2+ mobilization, and increased keratinocyte migration, and proliferation. In addition, these peptides induced phosphorylation of EGFR, signal transducer and activator of transcription (STAT)1, and STAT3, which are intracellular signaling molecules involved in keratinocyte migration and proliferation. In our study, inhibition of these molecules significantly reduced hBD-mediated keratinocyte migration and proliferation. In conclusion, this study provides evidence that human antimicrobial peptides may be involved in skin immunity through stimulating cytokine/chemokine production, and participate in wound healing by promoting keratinocyte migration and proliferation.