Hydrocellular Foam Dressing Promotes Wound Healing along with Increases in Hyaluronan Synthase 3 and PPARα Gene Expression in Epidermis

Background

Hydrocellular foam dressing, modern wound dressing, induces moist wound environment and promotes wound healing: however, the regulatory mechanisms responsible for these effects are poorly understood. This study was aimed to reveal the effect of hydrocellular foam dressing on hyaluronan, which has been shown to have positive effects on wound healing, and examined its regulatory mechanisms in rat skin.

Methodology/Principal Findings

We created two full-thickness wounds on the dorsolateral skin of rats. Each wound was covered with either a hydrocellular foam dressing or a film dressing and hyaluronan levels in the periwound skin was measured. We also investigated the mechanism by which the hydrocellular foam dressing regulates hyaluronan production by measuring the gene expression of hyaluronan synthase 3 (Has3), peroxisome proliferator-activated receptor α (PPARα), and CD44. Hydrocellular foam dressing promoted wound healing and upregulated hyaluronan synthesis, along with an increase in the mRNA levels of Has3, which plays a primary role in hyaluronan synthesis in epidermis. In addition, hydrocellular foam dressing enhanced the mRNA levels of PPARα, which upregulates Has3 gene expression, and the major hyaluronan receptor CD44.

Conclusions/Significance

These findings suggests that hydrocellular foam dressing may be beneficial for wound healing along with increases in hyaluronan synthase 3 and PPARα gene expression in epidermis. We believe that the present study would contribute to the elucidation of the mechanisms underlying the effects of hydrocellular foam dressing-induced moist environment on wound healing and practice evidence-based wound care.     

Epistasis with HLA DR3 implicates the P2X7 receptor in the pathogenesis of primary Sj?gren's syndrome

Introduction

The aim of this study was to examine the association between functional polymorphisms in the pro-inflammatory P2X7 receptor and the Ro/La autoantibody response in primary Sjögren''s syndrome (pSS).

Methods

Twelve functional P2RX7 polymorphisms were genotyped in 114 pSS patients fulfilling the Revised American-European Consensus Criteria for pSS, and 136 controls. Genotyping of the A1405G (rs2230912) polymorphism was performed on a replication cohort consisting of 281 pSS patients and 534 controls. P2X7 receptor function in lymphocytes and monocytes was assessed by measurement of ATP-induced ethidium+ uptake. Serum IL-18 levels were determined by ELISA.

Results

The minor allele of P2RX7 A1405G is a tag for a common haplotype associated with gain in receptor function, as assessed by ATP-induced ethidium+ uptake. A positive association between 1405G and anti-Ro±La seropositive pSS patients was observed in Cohort 1. Although not replicated in Cohort 2, there was a consistent, significant, negative epistatic interaction effect with HLA-DR3 in seropositive pSS patients from both cohorts, thereby implicating this gain of function variant in the pathogenesis of pSS. Serum IL-18 was elevated in seropositive pSS patients, but was not influenced by P2RX7 A1405G.

Conclusions

The P2RX7 1405G gain-of-function haplotype may be a risk factor for seropositive pSS in a subset of subjects who do not carry HLA risk alleles, but has no effect in subjects who do (epistasis). Potential mechanisms relate to autoantigen exposure and inflammatory cytokine expression. The observed elevation of IL-18 levels is consistent with P2X7 receptor activation in seropositive pSS patients. Collectively these findings implicate P2X7 receptor function in the pathogenesis of pSS.     

Involvement of the P2X7 Purinergic Receptor in Colonic Motor Dysfunction Associated with Bowel Inflammation in Rats

Background and Purpose

Recent evidence indicates an involvement of P2X7 purinergic receptor (P2X7R) in the fine tuning of immune functions, as well as in driving enteric neuron apoptosis under intestinal inflammation. However, the participation of this receptor in the regulation of enteric neuromuscular functions remains undetermined. This study was aimed at investigating the role of P2X7Rs in the control of colonic motility in experimental colitis.

Experimental Approach

Colitis was induced in rats by 2,4-dinitrobenzenesulfonic acid. P2X7R distribution was examined by immunofluorescence analysis. The effects of A804598 (selective P2X7R antagonist) and BzATP (P2X7R agonist) were tested on contractions of longitudinal smooth muscle evoked by electrical stimulation or by carbachol in the presence of tetrodotoxin.

Key Results

P2X7Rs were predominantly located in myenteric neurons, but, in the presence of colitis, their expression increased in the neuromuscular layer. In normal preparations, A804598 elicited a negligible increase in electrically induced contractions, while a significant enhancement was recorded in inflamed tissues. In the presence of N^ω-propyl-L-arginine (NPA, neuronal nitric oxide synthase inhibitor) the A804598 effects were lost. P2X7R stimulation with BzATP did not significantly affect electrical-induced contractions in normal colon, while a marked reduction was recorded under inflammation. The inhibitory effect of BzATP was antagonized by A804598, and it was also markedly blunted by NPA. Both P2X7R ligands did not affect carbachol-induced contractions.

Conclusions and Implications

The purinergic system contributes to functional neuromuscular changes associated with bowel inflammation via P2X7Rs, which modulate the activity of excitatory cholinergic nerves through a facilitatory control on inhibitory nitrergic pathways.     

Modulation of Mouse Embryonic Stem Cell Proliferation and Neural Differentiation by the P2X7 Receptor

Background

Novel developmental functions have been attributed to the P2X7 receptor (P2X7R) including proliferation stimulation and neural differentiation. Mouse embryonic stem cells (ESC), induced with retinoic acid to neural differentiation, closely assemble processes occurring during neuroectodermal development of the early embryo.

Principal Findings

P2X7R expression together with the pluripotency marker Oct-4 was highest in undifferentiated ESC. In undifferentiated cells, the P2X7R agonist Bz-ATP accelerated cell cycle entry, which was blocked by the specific P2X7R inhibitor KN-62. ESC induced to neural differentiation with retinoic acid, reduced Oct-4 and P2X7R expression. P2X7R receptor-promoted intracellular calcium fluxes were obtained at lower Bz-ATP ligand concentrations in undifferentiated and in neural-differentiated cells compared to other studies. The presence of KN-62 led to increased number of cells expressing SSEA-1, Dcx and β3-tubulin, as well as the number of SSEA-1 and β3-tubulin-double-positive cells confirming that onset of neuroectodermal differentiation and neuronal fate determination depends on suppression of P2X7R activity. Moreover, an increase in the number of Ki-67 positive cells in conditions of P2X7R inhibition indicates rescue of progenitors into the cell cycle, augmenting the number of neuroblasts and consequently neurogenesis.

Conclusions

In embryonic cells, P2X7R expression and activity is upregulated, maintaining proliferation, while upon induction to neural differentiation P2X7 receptor expression and activity needs to be suppressed.     

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.     

Crosstalk between chemokine receptor CXCR4 and cannabinoid receptor CB2 in modulating breast cancer growth and invasion

Background

Cannabinoids bind to cannabinoid receptors CB₁ and CB₂ and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB₂ may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis.

Methodology/Principal Findings

We observed high expression of both CB₂ and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB₂-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems.

Conclusions/Significance

This study provides novel insights into the crosstalk between CB₂ and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB₂ receptors could be used for developing innovative therapeutic strategies against breast cancer.     

Phenotypic overlap between MMP-13 and the plasminogen activation system during wound healing in mice

Background

Proteolytic degradation of extracellular matrix is a crucial step in the healing of incisional skin wounds. Thus, healing of skin wounds is delayed by either plasminogen-deficiency or by treatment with the broad-spectrum metalloproteinase (MP) inhibitor Galardin alone, while the two perturbations combined completely prevent wound healing. Both urokinase-type plasminogen activator and several matrix metallo proteinases (MMPs), such as MMP-3, -9 and -13, are expressed in the leading-edge keratinocytes of skin wounds, which may account for this phenotypic overlap between these classes of proteases.

Methodology

To further test that hypothesis we generated Mmp13;Plau and Mmp13;Plg double-deficient mice in a cross between Mmp13- and Plau-deficient mice as well as Mmp13- and Plg-deficient mice. These mice were examined for normal physiology in a large cohort study and in a well-characterized skin wound healing model, in which we made incisional 20 mm-long full-thickness skin wounds.

Principal Findings

While mice that are deficient in Mmp13 have a mean healing time indistinguishable to wild-type mice, wound healing in both Plau- and Plg-deficient mice is significantly delayed. Histological analysis of healed wounds revealed a significant increase in keratin 10/14 immunoreactive layers of kerationcytes in the skin surface in Mmp13;Plau double-deficient mice. Furthermore, we observe, by immunohistological analysis, an aberrant angiogenic pattern during wound healing induced by Plau-deficiency, which has not previously been described.

Conclusions

We demonstrate a phenotypic overlap, defined as an additional delay in wound healing in the double-deficient mice compared to the individual single-deficient mice, between MMP-13 and the plasminogen activation system in the process of wound healing, but not during gestation and in postnatal development. Thus, a dual targeting of uPA and MMP-13 might be a possible future strategy in designing therapies aimed at tissue repair or other pathological processes, such as cancer invasion, where proteolytic degradation is a hallmark.     

Valproic Acid Induces Cutaneous Wound Healing In Vivo and Enhances Keratinocyte Motility

Background

Cutaneous wound healing is a complex process involving several signaling pathways such as the Wnt and extracellular signal-regulated kinase (ERK) signaling pathways. Valproic acid (VPA) is a commonly used antiepileptic drug that acts on these signaling pathways; however, the effect of VPA on cutaneous wound healing is unknown.

Methods and Findings

We created full-thickness wounds on the backs of C3H mice and then applied VPA. After 7 d, we observed marked healing and reduced wound size in VPA-treated mice. In the neo-epidermis of the wounds, β-catenin and markers for keratinocyte terminal differentiation were increased after VPA treatment. In addition, α-smooth muscle actin (α-SMA), collagen I and collagen III in the wounds were significantly increased. VPA induced proliferation and suppressed apoptosis of cells in the wounds, as determined by Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining analyses, respectively. In vitro, VPA enhanced the motility of HaCaT keratinocytes by activating Wnt/β-catenin, ERK and phosphatidylinositol 3-kinase (PI3-kinase)/Akt signaling pathways.

Conclusions

VPA enhances cutaneous wound healing in a murine model and induces migration of HaCaT keratinocytes.     

Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier Breakdown

Background

Intestinal barrier failure may lead to systemic inflammation and distant organ injury in patients following severe injury. Enteric glia cells (EGCs) have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO). We have recently shown than Vagal Nerve Stimulation (VNS) increases EGC activation, which was associated with improved gut barrier integrity. Thus, we sought to further study the mechanism by which EGCs prevent intestinal barrier breakdown utilizing an in vitro model. We postulated that EGCs, through the secretion of GSNO, would improve intestinal barrier function through improved expression and localization of intestinal tight junction proteins.

Methods

Epithelial cells were co-cultured with EGCs or incubated with GSNO and exposed to Cytomix (TNF-α, INF-γ, IL-1β) for 24 hours. Barrier function was assessed by permeability to 4kDa FITC-Dextran. Changes in tight junction proteins ZO-1, occludin, and phospho-MLC (P-MLC) were assessed by immunohistochemistry and immunoblot.

Key Results

Co-culture of Cytomix-stimulated epithelial monolayers with EGCs prevented increases in permeability and improved expression and localization of occludin, ZO-1, and P-MLC. Further, treatment of epithelial monolayers with GSNO also prevented Cytomix-induced increases in permeability and exhibited a similar improvement in expression and localization of occludin, ZO-1, and P-MLC.

Conclusions & Inferences

The addition of EGCs, or their secreted mediator GSNO, prevents epithelial barrier failure after injury and improved expression of tight junction proteins. Thus, therapies that increase EGC activation, such as VNS, may be a novel strategy to limit barrier failure in patients following severe injury.     

P2X7 Integrates PI3K/AKT and AMPK-PRAS40-mTOR Signaling Pathways to Mediate Tumor Cell Death

Background

Extracellular adenosine triphosphate (ATP) functions as a novel danger signal that boosts antitumor immunity and can also directly kill tumor cells. We have previously reported that chronic exposure of tumor cells to ATP provokes P2X7-mediated tumor cell death, by as yet incompletely defined molecular mechanisms.

Methodology/Principal Findings

Here, we show that acute exposure of tumor cells to ATP results in rapid cytotoxic effects impacting several aspects of cell growth/survival, leading to inhibition of tumor growth in vitro and in vivo. Using agonist and antagonist studies together with generation of P2X7 deficient tumor cell lines by lentiviral shRNA delivery system, we confirm P2X7 to be the central control node transmitting extracellular ATP signals. We identify that downstream intracellular signaling regulatory networks implicate two signaling pathways: the known P2X7-PI3K/AKT axis and remarkably a novel P2X7-AMPK-PRAS40-mTOR axis. When exposed to high levels of extracellular ATP, these two signaling axes perturb the balance between growth and autophagy, thereby promoting tumor cell death.

Conclusions

Our study defines novel molecular mechanisms underpinning the antitumor actions of P2X7 and provides a further rationale for purine-based drugs in targeted cancer therapy.     

Excisional Wound Healing Is Delayed in a Murine Model of Chronic Kidney Disease

Background

Approximately 15% of the United States population suffers from chronic kidney disease (CKD), often demonstrating an associated impairment in wound healing. This study outlines the development of a surgical murine model of CKD in order to investigate the mechanisms underlying this impairment.

Methods

CKD was induced in mice by partial cauterization of one kidney cortex and contralateral nephrectomy, modifying a previously published technique. After a minimum of 6-weeks, splinted, dorsal excisional wounds were created to permit assessment of wound healing parameters. Wounds were harvested on postoperative days (POD) 0, 3, 7, and 14 for histological, immunofluorescent, and quantitative PCR (qPCR).

Results

CKD mice exhibited deranged blood chemistry and hematology profiles, including profound uremia and anemia. Significant decreases in re-epithelialization and granulation tissue deposition rates were found in uremic mice wounds relative to controls. On immunofluorescent analysis, uremic mice demonstrated significant reductions in cellular proliferation (BrdU) and angiogenesis (CD31), with a concurrent increase in inflammation (CD45) as compared to controls. CKD mice also displayed differential expression of wound healing-related genes (VEGF, IL-1β, eNOS, iNOS) on qPCR.

Conclusions

These findings represent the first reported investigation of cutaneous healing in a CKD animal model. Ongoing studies of this significantly delayed wound healing phenotype include the establishment of renal failure model in diabetic strains to study the combined effects of CKD and diabetes.     

From a Traditional Medicinal Plant to a Rational Drug: Understanding the Clinically Proven Wound Healing Efficacy of Birch Bark Extract

Background

Birch bark has a long lasting history as a traditional medicinal remedy to accelerate wound healing. Recently, the efficacy of birch bark preparations has also been proven clinically. As active principle pentacyclic triterpenes are generally accepted. Here, we report a comprehensive study on the underlying molecular mechanisms of the wound healing properties of a well-defined birch bark preparation named as TE (triterpene extract) as well as the isolated single triterpenes in human primary keratinocytes and porcine ex-vivo wound healing models.

Methodology/Principal Findings

We show positive wound healing effects of TE and betulin in scratch assay experiments with primary human keratinocytes and in a porcine ex-vivo wound healing model (WHM). Mechanistical studies elucidate that TE and betulin transiently upregulate pro-inflammatory cytokines, chemokines and cyclooxygenase-2 on gene and protein level. For COX-2 and IL-6 this increase of mRNA is due to an mRNA stabilizing effect of TE and betulin, a process in which p38 MAPK and HuR are involved. TE promotes keratinocyte migration, putatively by increasing the formation of actin filopodia, lamellipodia and stress fibers. Detailed analyses show that the TE components betulin, lupeol and erythrodiol exert this effect even in nanomolar concentrations. Targeting the actin cytoskeleton is dependent on the activation of Rho GTPases.

Conclusion/Significance

Our results provide insights to understand the molecular mechanism of the clinically proven wound healing effect of birch bark. TE and betulin address the inflammatory phase of wound healing by transient up-regulation of several pro-inflammatory mediators. Further, they enhance migration of keratinocytes, which is essential in the second phase of wound healing. Our results, together with the clinically proven efficacy, identify birch bark as the first medical plant with a high potential to improve wound healing, a field which urgently needs effective remedies.     

Modification of collagen by 3-deoxyglucosone alters wound healing through differential regulation of p38 MAP kinase

Background

Wound healing is a highly dynamic process that requires signaling from the extracellular matrix to the fibroblasts for migration and proliferation, and closure of the wound. This rate of wound closure is impaired in diabetes, which may be due to the increased levels of the precursor for advanced glycation end products, 3-deoxyglucosone (3DG). Previous studies suggest a differential role for p38 mitogen-activated kinase (MAPK) during wound healing; whereby, p38 MAPK acts as a growth kinase during normal wound healing, but acts as a stress kinase during diabetic wound repair. Therefore, we investigated the signaling cross-talk by which p38 MAPK mediates wound healing in fibroblasts cultured on native collagen and 3DG-collagen.

Methodology/Principal Findings

Using human dermal fibroblasts cultured on 3DG-collagen as a model of diabetic wounds, we demonstrated that p38 MAPK can promote either cell growth or cell death, and this was dependent on the activation of AKT and ERK1/2. Wound closure on native collagen was dependent on p38 MAPK phosphorylation of AKT and ERK1/2. Furthermore, proliferation and collagen production in fibroblasts cultured on native collagen was dependent on p38 MAPK regulation of AKT and ERK1/2. In contrast, 3DG-collagen decreased fibroblast migration, proliferation, and collagen expression through ERK1/2 and AKT downregulation via p38 MAPK.

Conclusions/Significance

Taken together, the present study shows that p38 MAPK is a key signaling molecule that plays a significantly opposite role during times of cellular growth and cellular stress, which may account for the differing rates of wound closure seen in diabetic populations.     

Expression of TGF-β3 in isolated fibroblasts from foreskin

Background:

The multifunctional transforming growth factor beta (TGF-β) is a glycoprotein that exists in three isoforms. TGF-β3 expression increases in fetal wound healing and reduces fibronectin and collagen I and III deposition, and also improves the architecture of the neodermis which is a combination of blood vessels and connective tissue during wound healing. Fibroblasts are key cells in the wound healing process. TGF-β3 plays a critical role in scar-free wound healing and fibroblast actions in the wound healing process. The aim of this study was to express the TGF-β3 gene (tgf-b3) in human foreskin fibroblasts (HFF’s).

Methods:

We obtained HFF’s from a newborn and a primary fibroblast culture was prepared. The cells were transfected with TGF-β3-pCMV6-XL5 plasmid DNA by both lipofection and electroporation. Expression of TGF-β3 was measured by enzyme-linked immunosorbent assay (ELISA).

Results:

The highest TGF-β3 expression (8.3-fold greater than control) was obtained by lipofection after 72 hours using 3 µl of transfection reagent. Expression was 1.4-fold greater than control by electroporation.

Conclusions:

In this study, we successfully increased TGF-β3 expression in primary fibroblast cells. In the future, grafting these transfected fibroblasts onto wounds can help the healing process without scarring.Key Words: Fibroblasts, Gene expression, TGF-β3     

Acute Stress Reduces Wound-Induced Activation of Microbicidal Potential of Ex Vivo Isolated Human Monocyte-Derived Macrophages

Background

Psychological stress delays wound healing but the precise underlying mechanisms are unclear. Macrophages play an important role in wound healing, in particular by killing microbes. We hypothesized that (a) acute psychological stress reduces wound-induced activation of microbicidal potential of human monocyte-derived macrophages (HMDM), and (b) that these reductions are modulated by stress hormone release.

Methods

Fourty-one healthy men (mean age 35±13 years) were randomly assigned to either a stress or stress-control group. While the stress group underwent a standardized short-term psychological stress task after catheter-induced wound infliction, stress-controls did not. Catheter insertion was controlled. Assessing the microbicidal potential, we investigated PMA-activated superoxide anion production by HMDM immediately before and 1, 10 and 60 min after stress/rest. Moreover, plasma norepinephrine and epinephrine and salivary cortisol were repeatedly measured. In subsequent in vitro studies, whole blood was incubated with norepinephrine in the presence or absence of phentolamine (norepinephrine blocker) before assessing HMDM microbicidal potential.

Results

Compared with stress-controls, HMDM of the stressed subjects displayed decreased superoxide anion-responses after stress (p’s <.05). Higher plasma norepinephrine levels statistically mediated lower amounts of superoxide anion-responses (indirect effect 95% CI: 4.14–44.72). Norepinephrine-treated HMDM showed reduced superoxide anion-production (p<.001). This effect was blocked by prior incubation with phentolamine.

Conclusions

Our results suggest that acute psychological stress reduces wound-induced activation of microbicidal potential of HMDM and that this reduction is mediated by norepinephrine. This might have implications for stress-induced impairment in wound healing.     

Immunohistochemical Monitoring of Wound Healing in Antibiotic Treated Buruli Ulcer Patients

Background

While traditionally surgery has dominated the clinical management of Buruli ulcer (BU), the introduction of the combination chemotherapy with oral rifampicin and intramuscular streptomycin greatly improved treatment and reduced recurrence rates. However management of the often extensive lesions after successful specific therapy has remained a challenge, in particular in rural areas of the African countries which carry the highest burden of disease. For reasons not fully understood, wound healing is delayed in a proportion of antibiotic treated BU patients. Therefore, we have performed immunohistochemical investigations to identify markers which may be suitable to monitor wound healing progression.

Methodology/Principal findings

Tissue specimens from eight BU patients with plaque lesions collected before, during and after chemotherapy were analyzed by immunohistochemistry for the presence of a set of markers associated with connective tissue neo-formation, tissue remodeling and epidermal activation. Several target proteins turned out to be suitable to monitor wound healing. While α-smooth muscle actin positive myofibroblasts were not found in untreated lesions, they emerged during the healing process. These cells produced abundant extracellular matrix proteins, such as pro-collagen 1 and tenascin and were found in fibronectin rich areas. After antibiotic treatment many cells, including myofibroblasts, revealed an activated phenotype as they showed ribosomal protein S6 phosphorylation, a marker for translation initiation. In addition, healing wounds revealed dermal tissue remodeling by apoptosis, and showed increased cytokeratin 16 expression in the epidermis.

Conclusion/Significance

We have identified a set of markers that allow monitoring wound healing in antibiotic treated BU lesions by immunohistochemistry. Studies with this marker panel may help to better understand disturbances responsible for wound healing delays observed in some BU patients.     

Botulinum Toxin to Improve Results in Cleft Lip Repair: A Double-Blinded,Randomized, Vehicle-Controlled Clinical Trial

Background

Most patients with facial scarring would value even a slight improvement in scar quality. Botulinum toxin A is widely used to alleviate facial dynamic rhytides but is also believed to improve scar quality by reducing wound tension during healing. The main objective was to assess the effect of Botulinum toxin on scars resultant from standardized upper lip wounds.

Methods

In this double-blinded, randomized, vehicle-controlled, prospective clinical trial, 60 consecutive consenting adults undergoing cleft lip scar revision (CLSR) surgery between July 2010 and March 2012 were randomized to receive botulinum toxin A (n = 30) or vehicle (normal saline; n = 30) injections into the subjacent orbicularis oris muscle immediately after wound closure. Scars were independently assessed at 6-months follow-up in blinded fashion using: Vancouver Scar Scale (VSS), Visual Analogue Scale (VAS) and photographic plus ultrasound measurements of scar widths.

Results

58 patients completed the trial. All scar assessment modalities revealed statistically significantly better scars in the experimental than the vehicle-control group.

Conclusion

Quality of surgical upper lip scars, which are oriented perpendicular to the direction of pull of the underlying orbicularis oris muscle, is significantly improved by its temporary paralysis during wound healing.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01429402","term_id":"NCT01429402"}}NCT01429402     

Macrophage Depletion Impairs Corneal Wound Healing after Autologous Transplantation in Mice

Purpose

Macrophages have been shown to play a critical role in the wound healing process. In the present study, the role of macrophages in wound healing after autologous corneal transplantation was investigated by depleting local infiltrated macrophages.

Methods

Autologous corneal transplantation model was used to induce wound repair in Balb/c mice. Macrophages were depleted by sub-conjunctival injections of clodronate-containing liposomes (Cl₂MDP-LIP). The presence of CD11b⁺ F4/80⁺ macrophages, α-smooth muscle actin⁺ (α-SMA⁺) myofibroblasts, CD31⁺ vascular endothelial cells and NG₂ ⁺ pericytes was examined by immunohistochemical and corneal whole-mount staining 14 days after penetrating keratoplasty. Peritoneal macrophages were isolated from Balb/c mice and transfused into conjunctiva to examine the recovery role of macrophages depletion on wound healing after autologous corneal transplantation.

Results

Sub-conjunctival Cl₂MDP-LIP injection significantly depleted the corneal resident phagocytes and infiltrated macrophages into corneal stroma. Compared with the mice injected with PBS-liposome, the Cl₂MDP-LIP-injected mice showed few inflammatory cells, irregularly distributed extracellular matrix, ingrowth of corneal epithelium into stroma, and even the detachment of donor cornea from recipient. Moreover, the number of macrophages, myofibroblasts, endothelial cells and pericytes was also decreased in the junction area between the donor and recipient cornea in macrophage-depleted mice. Peritoneal macrophages transfusion recovered the defect of corneal wound healing caused by macrophages depletion.

Conclusions

Macrophage depletion significantly impairs wound healing after autologous corneal transplantation through at least partially impacting on angiogenesis and wound closure.