Knockdown of sodium channel Nax reduces dermatitis symptoms in rabbit skin.

The skin plays a critical role in maintenance of water homeostasis. Dysfunction of the skin barrier causes not only delayed wound healing and hypertrophic scarring, but it also contributes to the development of various skin diseases. Dermatitis is a chronic inflammatory skin disorder that has several different subtypes. Skin of contact dermatitis and atopic dermatitis (AD) show epidermal barrier dysfunction. Nax is a sodium channel that regulates inflammatory gene expression in response to perturbation of barrier function of the skin. We found that in vivo knockdown of Nax using RNAi reduced hyperkeratosis and keratinocyte hyperproliferation in rabbit ear dermatitic skin. Increased infiltration of inflammatory cells (mast cells, eosinophils, T cells, and macrophages), a characteristic of dermatitis, was reduced by Nax knockdown. Upregulation of PAR-2 and thymic stromal lymphopoietin (TSLP), which induce Th2-mediated allergic responses, was inhibited by Nax knockdown. In addition, expression of COX-2, IL-1ß, IL-8, and S100A9, which are downstream genes of Nax and are involved in dermatitis pathogenesis, were also decreased by Nax knockdown. Our data show that knockdown of Nax relieved dermatitis symptoms in vivo and indicate that Nax is a novel therapeutic target for dermatitis, which currently has limited therapeutic options.

Liposome-encapsulated statins reduce hypertrophic scarring through topical application.

Hypertrophic scar is an important clinical problem with limited therapeutic options. Aside from their roles as 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, statins have also been demonstrated to decrease scarring by reducing connective tissue growth factor (CTGF) expression. However, poor penetrative ability limits their utility as topical treatments for hypertrophic scar. Here, we aim to develop novel statin formulations using liposomes to enhance dermal penetrative ability and to evaluate their efficacy against formation of hypertrophic scar utilizing our validated rabbit ear hypertrophic scar model. Liposomal simvastatin or pravastatin were compounded using a novel, flexible liposomal formulation and applied topically to rabbit ear hypertrophic scars daily from postoperation day (POD) 14 until POD 25. Scar color, including erythema and melanin, was measured using reflectance spectrophotometry on POD 28, and scar tissue was harvested for evaluation of scar elevation index as well as gene and protein expression. Human foreskin fibroblasts were also treated with statin formulations and CCN2 expression was determined by quantitative PCR. Both simvastatin and pravastatin were efficiently encapsulated in liposomes, forming nanometer-scale particles possessing highly negative charges. Topical treatment with liposomal simvastatin and pravastatin at 6.5% concentration significantly reduced scar elevation index and decreased type I/III collagen content and myofibroblast persistence in the wound. The erythema/vascularity of scars was reduced by liposomal statin treatment, with concomitant decrease of CD31 expression as measured histologically. Expression levels of transcripts encoding CTGF, collagen I, and collagen III collagen in scar tissue were also decreased by liposomal pravastatin treatment, as were myofibroblast persistence and the type I/III collagen ratio as assessed by immunofluorescence and picrosirus red staining, respectively. Treatment of human foreskin fibroblasts with simvastatin or with liposome-encapsulated pravastatin resulted in decreased expression of transcript encoding CTGF. Overall, our novel statin formulations encapsulated in liposomes were successfully delivered through topical application, significantly reducing hypertrophic scarring in a rabbit ear model.

S100A8 and S100A9 Are Induced by Decreased Hydration in the Epidermis and Promote Fibroblast Activation and Fibrosis in the Dermis.

The most critical function of the epidermis is to prevent water loss and maintain skin homeostasis. Disruption of the functional skin barrier causes delayed wound healing, hypertrophic scarring, and many skin diseases. Herein, we show that reduced hydration increases the expression of S100 protein family members, S100A8/S100A9, in stratified keratinocyte culture and human ex vivo skin culture. Immunohistological analyses show that S100A8/A9 are highly expressed in the epidermis of human hypertrophic scar and keloid tissues. Reduced hydration demonstrates activation of fibroblasts in the keratinocyte-fibroblast co-culture. In contrast, knockdown of S100A8 or S100A9 by RNA interference in keratinocytes failed to activate fibroblasts. Pretreatment with pharmacological blockers of S100A8/A9 receptors, Toll-like receptor 4 and receptor for advanced glycation end products, inhibits fibroblast activation induced by recombinant S100A8/A9 proteins. Moreover, we observe that local delivery of S100A8 protein results in a marked increase in hypertrophic scarring in the in vivo rabbit ear scar model. Our results indicate that hydration status promotes fibroblast activation and fibrosis by directly affecting the expression of inflammatory signaling in keratinocytes, thereby strongly suggesting S100A8/A9 to be novel targets in preventing scarring.

Thermal injury model in the rabbit ear with quantifiable burn progression and hypertrophic scar.

Hypertrophic scar is a major clinical outcome of deep-partial thickness to full thickness thermal burn injury. Appropriate animal models are a limitation to burn research due to the lack of, or access to, animal models which address the endpoint of hypertrophic scar. Lower species, such as rodents, heal mainly by contracture, which limits the duration of study. Higher species, such as pigs, heal more similarly to humans, but are associated with high cost, long duration for scar development, challenges in quantifying scar hypertrophy, and poor manageability. Here, we present a quantifiable deep-partial thickness burn model in the rabbit ear. Burns were created using a dry-heated brass rod for 10 and 20 seconds at 90 °C. At the time of eschar excision on day 3, excisional wounds were made on the contralateral ear for comparison. Burn wound progression, in which the wound size expands over time is a major distinction between excisional and thermal injuries, was quantified at 1 hour and 3 days after the injuries using calibrated photographs and histology and the size of the wounds was found to be unchanged from the initial wound size at 1 hour, but 10% in the 20 seconds burn wounds at 3 days. A quantifiable hypertrophic scar, measured by histology as the scar elevation index, was present in both 20 seconds burn wounds and excisional wounds at day 35. ImageJ measurements revealed that the 20 seconds burn wound scars were 22% larger than the excisional wound scars and the 20 seconds burn scar area measurements from histology were 26% greater than in the excisional wound scar. The ability to measure both burn progression and scar hypertrophy over a 35-day time frame suits this model to screening early intervention burn wound therapeutics or scar treatments in a burn-specific scar model.

Systemic administration of hemoglobin improves ischemic wound healing.

BACKGROUND: Oxygen plays multifaceted roles in wound healing, including effects on cell proliferation, collagen synthesis, angiogenesis, and bacterial killing. Oxygen deficit is a major factor in the pathogenesis of chronic wounds. MATERIALS AND METHODS: We present a novel mechanism for oxygen delivery to ischemic wounds by systemic administration of an oxygen carrier substitute derived from bovine hemoglobin (IKOR 2084) in our ischemic rabbit ear wound model. The wound healing indexes, including epithelial gap and neo-granulation tissue area, were histologically analyzed. In situ expression of endothelial cells (CD31+) and proliferative cells (Ki-67+) were examined by immunohistochemistry analysis. The messenger RNA expression of collagen I, III, and vascular endothelial growth factor was measured by quantitative RT-PCR. Sirius Red staining was implemented for detection of collagen deposition, and terminal deoxynucleotidyl transferase dUTP nick end labeling analysis was performed to examine dermal cellular apoptosis. RESULTS: Systemic administration of IKOR 2084 significantly improved oxygen tension of ischemic tissue. When compared with saline controls, IKOR 2084 treatment enhanced wound repair as demonstrated by a reduced epithelial gap and increased granulation tissue area. The expression of Ki-67+, CD31+, vascular endothelial growth factor and collagen was also enhanced by IKOR 2084 administration. Moreover, apoptosis analysis in the wounds showed that cell survival in the dermis was increased by systemic IKOR 2084 administration. CONCLUSIONS: Our study suggests that systemic delivery of IKOR 2084 ameliorates hypoxic state, subsequently promotes angiogenesis, cellular proliferation, and collagen synthesis, attenuates hypoxia-induced apoptosis, and improved ischemic wound healing.

Response of human mature adipocytes to hypoxia-reoxygenation.

BACKGROUND AIMS: Adipocytes are metabolically active cells and have endocrine functions, such as cytokine secretion. Notably, adipocytes are found underneath skin and are thought to be involved in the body's response to ischemia-reperfusion (I-R). I-R injury is an important factor in the pathogenesis of chronic skin wounds. In this study, we investigated the response of human adipocytes to hypoxia-reoxygenation (H-R), the in vitro equivalent of I-R. METHODS: We cultured human mature adipocytes by enclosing them in hydrogel composed of hyaluronan and collagen and analyzed their proliferation and response to H-R. RESULTS: The average diameter of mature adipocytes isolated from abdominal subcutaneous fat tissue was between 60 and 109 µm, and a positive correlation was found between adipocyte size and body mass index. Hydrogel-enclosed human adipocytes displayed viability in in vitro culture and were capable of expressing foreign genes for at least 1 month. Proliferation analysis revealed 5-bromo-2'-deoxy-uridine labeling and positive Ki67 signaling. vascular endothelial growth factor expression was differentially altered in adipocytes in response to hypoxia and H-R. Adipocyte messenger RNA expression of pro-inflammatory cytokines, such as interleukin-1, interleukin-8 and tumor necrosis factor-α, was upregulated in response to H-R. In addition, the expression of heat shock protein 70, a cytoprotective gene, and inducible nitric oxide synthase, a proapoptotic gene, were both increased in H-R. Survival of hydrogel-enclosed adipocytes was found at 2 months after delivery into athymic mice. CONCLUSIONS: These and previous results from our group show that mature adipocytes can be cultured in vitro within a matrix and that they are functionally active cells that respond to environmental changes.

Intravenous curcumin efficacy on healing and scar formation in rabbit ear wounds under nonischemic, ischemic, and ischemia-reperfusion conditions.

Curcumin, a spice found in turmeric, is widely used in alternative medicine for its purported anti-inflammatory and antioxidant activities. The goal of this study was to test the curcumin efficacy on rabbit ear wounds under nonischemic, ischemic, and ischemia-reperfusion conditions. Previously described models were utilized in 58 New Zealand White rabbits. Immediately before wounding, rabbits were given intravenous crude or pure curcumin (6 µg/kg, 30 µg/kg, or 60 µg/kg) dissolved in 1% ethanol. Specimens were collected at 7-8 days to evaluate the effects on wound healing and at 28 days to evaluate the effects on hypertrophic scarring. Student's t test was applied to screen difference between any treatment and control group, whereas analysis of variance was applied to further analyze for all treatment groups in aggregate in some specific experiments. Treatment with crude curcumin suggested accelerated wound healing that reached significance for reepithelialization in lower and medium doses and granulation tissue formation in lower dose. Purified curcumin became available and was used for all later experiments. Treatment with pure curcumin suggested accelerated wound healing that reached significance for reepithelialization in lower and medium doses and granulation tissue formation in lower dose. Treatment with pure curcumin significantly promoted nonischemic wound healing in a dose-response fashion compared with controls as judged by increased reepithelialization and granulation tissue formation. Improved wound healing was associated with significant decreases in pro-inflammatory cytokines interleukin (IL)-1 and IL-6 as well as the chemokine IL-8. Curcumin also significantly reduced hypertrophic scarring. The effects of curcumin were examined under conditions of impaired healing including ischemic and ischemia-reperfusion wound healing, and beneficial effects were also seen, although the dose response was less clear. Systemically administrated pure curcumin significantly promotes nonischemic wound healing and reduces hypertrophic scarring. Improvements in wound healing were associated with decreased inflammatory markers in wounds. Further study is needed to optimize dosing in ischemic and ischemia-reperfusion wound healing. In aggregate, the studies strongly support the systemic administration of curcumin to improve wound healing.

Evaluating the effects of subclinical, cyclic ischemia-reperfusion injury on wound healing using a novel device in the rabbit ear.

OBJECTIVE: This study aimed to evaluate the effect of cyclic ischemia-reperfusion (IR) injury on wound healing using a novel rabbit ear model. MATERIALS AND METHODS: A lightweight clamp apparatus was developed for reversible occlusion of the central ear artery. Ventral ear wounds were analyzed postoperatively for epithelialization and granulation as well as gene expression after 3 consecutive days of IR cycling. RESULTS: By postoperative day #7, ears showed no gross tissue necrosis, but histologic analysis of wounds confirmed a significant impairment in epithelial and granulation tissue gaps as well as total epithelial and granulation tissue areas (P < 0.001). Quantitative polymerase chain reaction analysis of IR wounds indicated significant up-regulation of heat shock protein-70 and down-regulation of superoxide dismutase 1 relative to sham controls (P < 0.05). CONCLUSIONS: A novel rabbit ear model for the induction of subclinical, cyclic IR injury in cutaneous tissue has been developed that will serve as a valuable tool for the testing of new therapeutics.

The Human Soluble NKG2D Ligand Differentially Impacts Tumorigenicity and Progression in Temporal and Model-Dependent Modes.

NKG2D is an activating receptor expressed by all human NK cells and CD8 T cells. Harnessing the NKG2D/NKG2D ligand axis has emerged as a viable avenue for cancer immunotherapy. However, there is a long-standing controversy over whether soluble NKG2D ligands are immunosuppressive or immunostimulatory, originating from conflicting data generated from different scopes of pre-clinical investigations. Using multiple pre-clinical tumor models, we demonstrated that the impact of the most characterized human solid tumor-associated soluble NKG2D ligand, the soluble MHC I chain-related molecule (sMIC), on tumorigenesis depended on the tumor model being studied and whether the tumor cells possessed stemness-like properties. We demonstrated that the potential of tumor formation or establishment depended upon tumor cell stem-like properties irrespective of tumor cells secreting the soluble NKG2D ligand sMIC. Specifically, tumor formation was delayed or failed if sMIC-expressing tumor cells expressed low stem-cell markers; tumor formation was rapid if sMIC-expressing tumor cells expressed high stem-like cell markers. However, once tumors were formed, overexpression of sMIC unequivocally suppressed tumoral NK and CD8 T cell immunity and facilitated tumor growth. Our study distinguished the differential impacts of soluble NKG2D ligands in tumor formation and tumor progression, cleared the outstanding controversy over soluble NKG2D ligands in modulating tumor immunity, and re-enforced the viability of targeting soluble NKG2D ligands for cancer immunotherapy for established tumors.

Application of a partial-thickness human ex vivo skin culture model in cutaneous wound healing study.

A number of in vivo and ex vivo skin models have been applied to human wound healing studies. A reliable skin model, which recapitulates the features of human wound repair, is essential for the clinical and mechanical investigation of human cutaneous wound healing. Full-skin ex vivo culture systems have been used in wound healing studies. However, important structures of the skin, such as the differentiation of keratinocytes and epidermis-dermis junction, are poorly characterized in this model. This study aims to develop an optimized partial-thickness human ex vivo skin culture (HESC) model to maintain human skin characteristics in vitro. During our culture, the basal layer, suprabasal layer, and stratum granulosum layer of epidermis were preserved until day 8. Analyses of hemidesmosome proteins, bullous pemphigoid antigen 1 (BP180) and 2 (BP230), showed that the integrity of the basement membrane of the epidermis was well preserved in the HESC model. In contrast, an organotypic culture with human keratinocytes and fibroblasts failed to show an integrated basement membrane. Maintenance of skin structure by histological analysis and proliferation of epidermal keratinocytes by Ki67 staining were observed in our model for 12 days. Complete re-epithelialization of the wounding area was observed at day 6 post wounding when a superficial incisional wound was created. The expression of Ki-67 and keratin 6, indicators of activated keratinocytes in epidermis, was significantly upregulated and new collagen synthesis was found in the dermis during the wound healing process. As control, we also used organotypic culture in studying the differentiation of the keratinocyte layers and incisional wound repair. It turned out that our model has advantage in these study fields. The results suggest that our HESC model retains important elements of in vivo skin and has significant advantages for the wound healing studies in vitro.

The effects of collagenase ointment on the prevention of hypertrophic scarring in a rabbit ear scarring model: a pilot study.

UNLABELLED:  Collagen synthesis and degradation are important in scar maturation. Collagenase has been used for debridement of wounds and treatment of Dupuytren's disease but its effects on scar prevention are unknown. OBJECTIVE: The objective of this study was to determine the effect of collagenase ointment on hypertrophic scar prevention by using a rabbit ear-scarring model. METHODS: Four 7-mm, full-thickness dermal punches were made on the inner surface of each ear of 8 young adult New Zealand white rabbits. The wounds from one ear were treated with collagenase in petrolatum or petrolatum alone, while the wounds from the other ear of the same rabbits were not treated, serving as control. Tissues were collected at 28 days after surgery and histological analysis was performed. Total area of new scar and scar elevation index were determined. A paired two-tailed Student's t test was performed to compare treated scars with their own controls and a non-paired Student's t test was performed to compare collagenase-treated with petrolatum-treated scars. RESULTS: Collagenase-treated wounds developed scars with less hypertrophy than untreated scars. The total area of new scar was smaller in collagenase-treated wounds than in untreated wounds (531 ± 32 vs. 617 ± 51; P = 0.03). The scar elevation index was lower in collagenase-treated wounds than in untreated wounds (2.08 ± 0.15 vs. 2.45 ± 0.20; P = 0.015). The adjusted scar elevation index was lower in collagenase-treated wounds than in petrolatum-treated wounds, and the difference showed a trend toward statistical significance (0.88 ± 0.04 vs. 1.03 ± 0.07; P = 0.10). CONCLUSION: Treatment with collagenase ointment reduces hypertrophic scar formation in a rabbit ear model. .

Fibrin sealant combined with fibroblasts and platelet-derived growth factor enhance wound healing in excisional wounds.

We test the hypothesis that the fibrinogen-thrombin formulation of fibrin sealant combined with fibroblasts and PDGF-BB enhance cutaneous wound healing. Four formulations varying in fibrinogen and thrombin concentration were applied to full-thickness biopsy wounds in the rabbit ear cutaneous wound healing model with or without cultured rabbit dermal fibroblasts (RDFs; 3 x 10(5) cells/wound) embedded in the fibrinogen component. At post-wounding day 7, there was no difference in the diluted vs. non-diluted formulations for either the promotion of granulation tissue coverage of the open wounds or total granulation tissue area when tested without embedded cells. Including the RDFs, the highest degree of wound coverage by granulation tissue was observed in the combined dilution formulation (17.3 mg/mL fibrinogen, 167 U/mL thrombin; n=10 wounds) that was 167% (p<0.05) of the nondiluted FS containing cells (50 mg/mL fibrinogen, 250 U/mL thrombin; n=10 wounds). Inclusion of fibroblasts increased granulation tissue area within the wounds vs. FS alone (p<0.05) for each diluted formulation although no differences in this parameter were observed within each group (FS alone or with embedded cells). However, addition of the vulnerary growth factor PDGF-BB (3 mg; n=4) with the embedded RDFs in the combined dilution formulation increased granulation tissue area over two-fold (p<0.01) over FS alone. Additionally, the presence of the RDFs promoted incorporation of the granulation tissue with and epithelial migration over the FS suggesting an active interaction between cells delivered to the wound by FS and the host repair cells. The findings suggest the progress of cutaneous defect repair can be enhanced by ex vivo cell delivery in fibrin sealant.

Activation of JAK/STAT Signaling in Megakaryocytes Sustains Myeloproliferation In Vivo.

PURPOSE: The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. EXPERIMENTAL DESIGN: We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. RESULTS: JAK2V617F -mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. CONCLUSIONS: Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Local Application of Statins Significantly Reduced Hypertrophic Scarring in a Rabbit Ear Model.

BACKGROUND: We previously showed that intradermal injection of statins is a successful treatment for hypertrophic scarring. Topical application has many advantages over intradermal injection. In this study, we demonstrate the efficacy of topical statin treatment in reducing scar in our validated rabbit ear scar model. METHODS: Twenty New Zealand White rabbits were divided into 2 study groups, with 6 rabbits receiving 10 µm pravastatin intradermally at postoperative days 15, 18, and 21, and 14 rabbits receiving 0.4%, 2%, and 10% simvastatin topical application at postoperative days 14-25. Four or 6 full-thickness circular dermal punches 7 mm in diameter were made on the ventral surface of the ear down to but not including the perichondrium. Specimens were collected at 28 days to evaluate the effects of statins on hypertrophic scarring. RESULTS: Treatment with pravastatin intradermal administration significantly reduced scarring in terms of scar elevation index. Topical treatment with both medium- and high-dose simvastatin also significantly reduced scarring. High-dose simvastatin topical treatment showed a major effect in scar reduction but induced side effects of scaling, erythema, and epidermal hyperplasia, which were improved with coapplication of cholesterol. There is a dose response in scar reduction with low-, medium- and high-dose simvastatin topical treatment. High-dose simvastatin treatment significantly reduced the messenger ribonucleic acid (mRNA) expression of connective tissue growth factor, consistent with our previously published work on intradermally injected statins. More directly, high-dose simvastatin treatment also significantly reduced the mRNA expression of collagen 1A1. CONCLUSIONS: Topical simvastatin significantly reduces scar formation. The mechanism of efficacy for statin treatment through interference with connective tissue growth factor mRNA expression was confirmed.

Topical application of Dermatophagoides farinae or oxazolone induces symptoms of atopic dermatitis in the rabbit ear.

Atopic dermatitis (AD) is a chronically relapsing inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of the epidermis, and dermal infiltration of inflammatory cells. Appropriate animal models that recapitulate human AD and allow the analysis of disease processes in a reliable manner are essential to the study of AD. In this study, we established two AD models in rabbits by applying an allergen, Dermatophagoides farinae (Der f), or a hapten, oxazolone (OXZ). Application of the allergen or hapten induced a rapid onset and a chronically sustained AD-like skin lesion. The clinical symptoms, which include skin erythema, scaling, papula and edema, of AD-like rabbit skin were similar to those in human AD. Histological analysis showed that allergen- or hapten-treated rabbit skin showed increased epidermal thickening and inflammatory cell infiltration. Furthermore, PCNA and keratin 10 (K10) staining revealed excessive proliferation and insufficient differentiation of the epidermis in the rabbit AD-like skin. Western blot analysis showed decreased expression of thymic stromal lymphopoietin (TSLP), an AD cytokine, in the rabbit AD-like skin. Our results suggest that the allergen- or hapten-induced rabbit AD models have pathological features of human AD-like symptoms and will be useful for evaluating both pathogenic mechanisms and potential therapeutic agents for human AD.

S100A12 Induced in the Epidermis by Reduced Hydration Activates Dermal Fibroblasts and Causes Dermal Fibrosis.

Disruption of the barrier function of skin increases transepidermal water loss and up-regulates inflammatory pathways in the epidermis. Consequently, sustained expression of proinflammatory cytokines from the epidermis is associated with dermal scarring. We found increased expression of S100A12 in the epidermis of human hypertrophic and keloid scar. Exposing a stratified keratinocyte culture to a reduced-hydration environment increased the expression and secretion of S100A12 by nearly 70%, which in turn activated dermal fibroblasts in vitro. Direct treatment of fibroblasts with conditioned medium collected from stratified keratinocyte culture under reduced-hydration conditions activated fibroblasts, shown by up-regulation of α-smooth muscle actin, pro-collagen 1, and F-actin expression. However, this fibroblast activation was not found when S100A12 was knocked down by RNA interference in keratinocytes. Pharmacological blockade of S100A12 receptors, RAGE, or TLR4 inhibited S100A12-induced fibroblast activation. Local delivery of S100A12 resulted in a marked hypertrophic scar formation in a validated rabbit hypertrophic scar model compared with saline control. Our findings indicate that S100A12 functions as a proinflammatory cytokine and suggest that S100A12 is a potential therapeutic target for dermal scarring.

Adipose Tissue Drives Response to Ischemia-Reperfusion Injury in a Murine Pressure Sore Model.

BACKGROUND: Ischemia-reperfusion injury contributes significantly to the pathogenesis of chronic wounds such as pressure sores and diabetic foot ulcers. The authors' laboratory has previously developed a cyclical murine ischemia-reperfusion injury model. The authors here use this model to determine factors underlying tissue response to ischemia-reperfusion injury. METHODS: C57BL/6 mice were subjected to cycles of ischemia-reperfusion that varied in number (one to four cycles) and duration of ischemia (1 to 2 hours). For each ischemia-reperfusion condition, the following variables were analyzed: (1) digital photographs for area of necrosis; (2) hematoxylin and eosin staining and immunohistochemistry for inflammatory infiltrate; and (3) expression of inflammatory markers by quantitative polymerase chain reaction. In addition, human adipocytes and fibroblasts were cultured in vitro under conditions of hypoxia and reoxygenation, and expression of inflammatory markers was analyzed by quantitative polymerase chain reaction. RESULTS: Increases in both ischemia-reperfusion cycle number and ischemia duration correlated with increased areas of epithelial necrosis both grossly and histologically, and with an increase in cellularity and neutrophil density. This increased inflammatory infiltrate and a significant increase in the expression of proinflammatory markers (Hmox1, interleukin-6, interleukin-1, and monocyte chemoattractant protein-1) was observed in adipose tissue subjected to ischemia-reperfusion injury, but not in dermis. These results were mirrored in human adipose tissue. CONCLUSIONS: The authors further characterize a novel, reproducible murine model of ischemia-reperfusion injury. The results of their study indicate that adipose tissue is less tolerant of ischemia-reperfusion than dermal tissue. Rather than being an "innocent bystander," adipose tissue plays an active role in driving the inflammatory response to ischemia-reperfusion injury.

Topical Administration of Oxygenated Hemoglobin Improved Wound Healing in an Ischemic Rabbit Ear Model.

BACKGROUND: Localized oxygen deficiency plays a central role in the pathogenesis of chronic wounds; thus, rectifying localized ischemia with oxygen therapy has been postulated to be an integral aspect of the management of chronic wounds. The efficacy of a novel approach for oxygen therapy on chronic wound healing was evaluated. METHODS: Oxygen was delivered to ischemic wounds by means of the topical application of oxygenated, chemically modified bovine hemoglobin (IKOR 2084) in a validated rabbit ear ischemic wound model. The wound healing was evaluated histologically by measuring epithelial gap and neo-granulation tissue area. In situ expression of endothelial cells (CD31) and proliferative cells (Ki-67) was examined by immunohistochemistry analysis. The mRNA of vascular endothelial growth factor, endothelial nitric oxide synthase, and matrix metalloproteinase-9 was quantified by real-time reverse-transcriptase polymerase chain reaction. The collagen was detected by Sirius red staining. RESULTS: In comparison with topical application of saline, the administration of oxygenated IKOR 2084 increases wound reepithelialization and formation of neo-granulation tissue in a dose-dependent manner, and cellular proliferation (Ki-67). Conversely, the administration of deoxygenated IKOR 2084 aggravated the ischemic wound healing process. Moreover, the topical administration of oxygenated IKOR 2084 induces angiogenesis as evidenced by concomitant increases in CD31 protein and vascular endothelial growth factor and endothelial nitric oxide synthase mRNA expression in treated wounds. Oxygenated IKOR 2084 administration also increased collagen deposition in wounds, with decreases in the expression of matrix metalloproteinase-9 mRNA. CONCLUSION: This study suggests that the topical application of oxygenated IKOR 2084 ameliorates the reparative progress of ischemic wounds through enhanced angiogenesis, cellular proliferation, and collagen deposition.

Hydration status regulates sodium flux and inflammatory pathways through epithelial sodium channel (ENaC) in the skin.

Although it is known that the inflammatory response that results from disruption of epithelial barrier function after injury results in excessive scarring, the upstream signals remain unknown. It has also been observed that epithelial disruption results in reduced hydration status and that the use of occlusive dressings that prevent water loss from wounds decreases scar formation. We hypothesized that hydration status changes sodium homeostasis and induces sodium flux in keratinocytes, which result in activation of pathways responsible for keratinocyte-fibroblast signaling and ultimately lead to activation of fibroblasts. Here, we demonstrate that perturbations in epithelial barrier function lead to increased sodium flux in keratinocytes. We identified that sodium flux in keratinocytes is mediated by epithelial sodium channels (ENaCs) and causes increased secretion of proinflammatory cytokines, which activate fibroblast via the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2) pathway. Similar changes in signal transduction and sodium flux occur by increased sodium concentration, which simulates reduced hydration, in the media in epithelial cultures or human ex vivo skin cultures. Blockade of ENaC, prostaglandin synthesis, or PGE2 receptors all reduce markers of fibroblast activation and collagen synthesis. In addition, employing a validated in vivo excessive scar model in the rabbit ear, we demonstrate that utilization of either an ENaC blocker or a COX-2 inhibitor results in a marked reduction in scarring. Other experiments demonstrate that the activation of COX-2 in response to increased sodium flux is mediated through the PIK3/Akt pathway. Our results indicate that ENaC responds to small changes in sodium concentration with inflammatory mediators and suggest that the ENaC pathway is a potential target for a strategy to prevent fibrosis.

Sodium channel Nax is a regulator in epithelial sodium homeostasis.

The mechanisms by which the epidermis responds to disturbances in barrier function and restores homeostasis are unknown. With a perturbation of the epidermal barrier, water is lost, resulting in an increase in extracellular sodium concentration. We demonstrate that the sodium channel Nax functions as a sodium sensor. With increased extracellular sodium, Nax up-regulates prostasin, which results in activation of the sodium channel ENaC, resulting in increased sodium flux and increased downstream mRNA synthesis of inflammatory mediators. Nax is present in multiple epithelial tissues, and up-regulation of its downstream genes is found in hypertrophic scars. In animal models, blocking Nax expression results in improvement in scarring and atopic dermatitis-like symptoms, both of which are pathological conditions characterized by perturbations in barrier function. These findings support an important role for Nax in maintaining epithelial homeostasis.