Rixing Zhan

Controlled water vapor transmission rate promotes wound-healing via wound re-epithelialization and contraction enhancement

A desirable microenvironment is essential for wound healing, in which an ideal moisture content is one of the most important factors. The fundamental function and requirement for wound dressings is to keep the wound at an optimal moisture. Here, we prepared serial polyurethane (PU) membrane dressings with graded water vapor transmission rates (WVTRs), and the optimal WVTR of the dressing for wound healing was identified by both in vitro and in vivo studies. It was found that the dressing with a WVTR of 2028.3 ± 237.8 g/m2·24 h was able to maintain an optimal moisture content for the proliferation and regular function of epidermal cells and fibroblasts in a three-dimensional culture model. Moreover, the dressing with this optimal WTVR was found to be able to promote wound healing in a mouse skin wound model. Our finds may be helpful in the design of wound dressing for wound regeneration in the future.

Nitric Oxide Enhances Keratinocyte Cell Migration by Regulating Rho GTPase via cGMP-PKG Signalling

Objective Nitric oxide (NO) has been shown to improve wound healing, but the mechanism underlying this function is not well defined. Here, we explored the effect of NO on the migration of a human keratinocyte cell line (HaCaT) and its possible mechanism. Methods The effects of NO on HaCaT cells in the presence of different concentrations of the NO donor sodium nitroprusside (SNP) were evaluated in a cell migration assay. Subsequently, the cytoskeleton reorganization of cultured HaCaT cells stained with rhodamine-phalloidin was observed with a confocal laser scanning microscope. The mRNA expression and active proteins of CDC42, Rac1 and RhoA in the cultured cells were determined via RT-PCR and pull-down assays, respectively. Furthermore, the roles of various inhibitors or agonists specific to cGMP, PKG and CDC42, Rac1, RhoA in the effects of NO on HaCaT cell migration, F-actin stress fibre formation, and Rho GTPase expression were observed. Results It was also found HaCaT cell migration was increased by SNP in a dose-dependent manner, and the other two NO donors either spermine NONOate or SNAP had almost the same effects on HaCat cell migrations. The formation of F-actin stress fibres in SNP-treated HaCaT cells was increased. The mRNA expression and the active proteins of CDC42, Rac1 and RhoA were found to be upregulated after SNP treatment. Similar effects were observed after the cells were treated with a cGMP or PKG agonist. Additionally, the SNP-mediated upregulation of the mRNA expression and the active proteins of CDC42, Rac1 and RhoA were inhibited by the addition of an inhibitor of cGMP or PKG. Moreover, the SNP-mediated promoting effects of migration and cytoskeleton reorganization were inhibited by treatment with inhibitors of cGMP, PKG, CDC42, Rac1 and RhoA respectively. Conclusion Our data indicated that the stimulatory effects of NO on cell migration of HaCaT cells are mediated by the cGMP signalling pathway via the upregulation of Rho-GTPase expression, which might promote cytoskeleton reorganization.

P311 promotes renal fibrosis via TGFβ1/Smad signaling

P311, a gene that was identified in 1993, has been found to have diverse biological functions in processes such as cell proliferation, migration and differentiation. However, its role in fibrosis is unknown. We previously observed that P311 is highly expressed in skin hypertrophic scars. In this study, P311 over-expression was detected in a subset of tubular epithelial cells in clinical biopsy specimens of renal fibrosis; this over-expression, was found concurrent with α-smooth muscle actin (α-SMA) and transforming growth factor beta1 (TGFβ1) expression. Subsequently, these results were verified in a mouse experimental renal fibrosis model induced by unilateral ureteral obstruction. The interstitial deposition of collagen, α-SMA and TGF-β1 expression, and macrophage infiltration were dramatically decreased when P311 was knocked out. Moreover, TGFβ/Smad signaling had a critical effect on the promotion of renal fibrosis by P311. In conclusion, this study demonstrate that P311 plays a key role in renal fibrosis via TGFβ1/Smad signaling, which could be a novel target for the management of renal fibrosis.

CD86 Is an Activation Receptor for NK Cell Cytotoxicity against Tumor Cells

CTLA4Ig has been successfully used in the clinic for suppression of T cell activation. However, patients treated with CTLA4Ig experienced reduced incidence of tumors than predicted, but the underlying mechanism remains unknown. In this paper, we showed that brief administration of CTLA4Ig significantly reduced tumor metastasis and prolonged the survival of host mice bearing B16 melanoma. Depletion of NK cells prior to CTLA4Ig administration eliminated the CTLA4Ig-mediated anti-tumor activity. CTLA4Ig enhanced NK cell cytotoxicity to tumor cells via up-regulation of NK cell effecter molecules CD107a and perforin in vivo. In addition, we demonstrated that, upon activation, NK cells could significantly increase the expression of CD86 both in vitro and in vivo, and ligation of CD86 with CTLA4Ig significantly increased the ability of NK cells to kill tumor cells. Furthermore, a human NK cell line that expressed high level of CD86 was directly activated by CTLA4Ig so that killing of tumor targets was enhanced; this enhanced killing could be inhibited by blocking CD86. Our findings uncover a novel function of CTLA4Ig in tumor immunity and suggest that CD86 on NK cells is an activating receptor and closely involved in the CTLA4Ig-mediated anti-tumor response.

Three-Dimensional Histological Structures of the Human Dermis.

Spatial information has been shown to be critical for cell differentiation and function. Therefore, a better understanding of skin microstructures is very important for biomimetic and bioengineered scaffolds of engineering skin. The purpose of the study was to generate collagen/elastin-based three-dimensional (3D) images of human dermis to further understand the microstructures of the skin, which is believed to be helpful in the fabrication of bionic engineered skin. Skin samples were fixed, embedded, serially sectioned, stained with aldehyde-fuchsin, and photographed as serial panoramas. Dermal subregions were divided according to dermal depth and distance to hair follicle. The porosity, pore diameters, and wall thickness of human acellular dermal matrix (ADM) were measured by microcomputed tomography (micro-CT). Three-dimensional reconstructed images of collagen and elastic fibers were generated. Our results showed that there were fewer elastic fibers in the subregions close to hair follicles than in the subregions far away from hair follicles (p<0.001), but the collagen fibers were evenly distributed. Both collagen and elastic fibers were found in fewer numbers in the layers either close to the epidermis or close to the hypodermis. The mean proportions of collagen fibers and elastic fibers in the whole dermis were 28.96%±14.63% and 8.06%±3.75%, respectively. The porosity of ADM calculated by micro-CT was 68.3%±5.8%. The mean pore diameter of ADM was 131.2±96.8 μm, and the wall thickness of pores was 207.2±251.7 μm. This study represents for the first time that 3D histological cutaneous structures have been presented, which may be helpful for the next generation of skin engineering.

Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signalling.

The migration and reepithelization of epidermal stem cells (ESCs) are the most critical processes in wound healing. The gaseous messenger nitric oxide (NO) has multiple biological effects, but its actions on ESCs are poorly understood. In this study, an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), was found to facilitate the in vitro migration of human ESCs (huESCs) in both live-imaging and scratch models. In addition, pull-down assays demonstrated that SNAP could activate the small GTPases RhoA and Rac1 of the Rho family, but not Cdc42. Moreover, the effects of SNAP on the migration and F-actin polymerization of ESCs could be blocked by inhibitors of cGMP, PKG, RhoA or Rac1, and by a specific siRNA of RhoA or Rac1, but not by a Cdc42 inhibitor or siRNA. Furthermore, the roles of NO in ESC migration via cGMP-Rho GTPase signalling in vivo were confirmed by tracing 5-bromo-2-deoxyuridine (BrdU)-labelled cells in a superficial, partial-thickness scald mouse model. Thus, the present study demonstrated that the NO donor SNAP could promote huESC migration in vitro. Furthermore, NO was found to induce ESC migration via cGMP-Rho GTPase RhoA and Rac1 signalling, but not Cdc42 signalling, both in vivo and in vitro.

P311 induces the transdifferentiation of epidermal stem cells to myofibroblast-like cells by stimulating transforming growth factor β1 expression

BackgroundEpithelial to mesenchymal transition, especially to myofibroblasts, plays an important role in wound healing, fibrosis, and carcinogenesis. Epidermal stem cells (EpSCs) are responsible for epidermal renewal and wound re-epithelialization. However, it remains unclear whether and how EpSCs transdifferentiate into myofibroblasts or myofibroblast-like cells (MFLCs). Here, we provide the first evidence showing that P311 induces EpSC to MFLC transdifferentiation (EpMyT) via TGFβ1/Smad signaling.MethodsWound healing and mesenchymal features were observed in the P311 KO and P311 WT mouse model of superficial second-degree burns. After the primary human or mouse EpSCs were forced to highly express P311 using an adenoviral vector, EpMyT was observed by immunofluorescence, real-time PCR, and western blot. The activity of TGFβ1 and Smad2/3 in EpSCs with different P311 levels was observed by western blot. The TβRI/II inhibitor LY2109761 and Smad3 siRNA were applied to block the EpMyT in P311-overexpressing EpSCs and exogenous TGFβ1 was to restore the EpMyT in P311 KO EpSCs. Furthermore, the mechanism of P311 regulating TGFβ1 was investigated by bisulfite sequencing PCR, luciferase activity assay, and real-time PCR.ResultsP311 KO mouse wounds showed delayed re-epithelialization and reduced mesenchymal features. The human or mouse EpSCs with overexpressed P311 exhibited fusiform morphological changes, upregulated expression of myofibroblast markers (α-SMA and vimentin), and downregulated expression of EpSC markers (β1-integrin and E-cadherin). P311-expressing EpSCs showed decreased TGFβ1 mRNA and increased TGFβ1 protein, TβRI/II mRNA, and activated Smad2/3. Moreover, LY2109761 and Smad3 siRNA reversed P311-induced EpMyT. Under the stimulation of exogenous TGFβ1, the phosphorylation of Smad2 and Smad3 in P311 KO EpSCs was significantly lower than that in P311 WT EpSCs and the EpMyT in P311 KO EpSCs was restored. Furthermore, P311 enhanced the methylation of TGFβ1 promoter and increased activities of TGFβ1 5′/3′ untranslated regions (UTRs) to stimulate TGFβ1 expression. P311+α-SMA+ cells and P311+vimentin+ cells were observed in the epidermis of human burn wounds. Also, P311 was upregulated by IL-1β, IL-6, TNFα, and hypoxia.ConclusionsP311 is a novel TGFβ1/Smad signaling-mediated regulator of transdifferentiation in EpSCs during cutaneous wound healing. Furthermore, P311 might stimulate TGFβ1 expression by promoting TGFβ1 promoter methylation and by activating the TGFβ1 5′/3′ UTR.

Biomimetic thermoplastic polyurethane porous membrane with hierarchical structure accelerates wound healing by enhancing granulation tissue formation and angiogenesis

Thermoplastic polyurethane (TPU) is an appropriate material for wound dressings, and has been processed into a variety of forms to match the structural and morphological features required by current clinical demands. The aim of the present work was to manufacture biomimetic porous membranes composed of TPU and investigate the effects on wound healing. The hierarchical thermoplastic polyurethane porous membrane (HTPM) were prepared using a novel, simple and tunable method that combines immersion precipitation with particle leaching. Physical testing indicated that the HTPM possess is more favorable mechanical properties than conventional products, and the inner membrane structure is more similar to that of natural skin. Meanwhile, the HTPM exerted no adverse effects on fibroblast viability and proliferation by a Cell Counting Kit-8 assay. Furthermore, the histological and Western blot results indicated that wound re-epithelialization, granulation tissue formation and angiogenesis were enhanced when skin defects were covered with the HTPM, which significantly accelerated wound healing. These results demonstrated that the bilayer HTPM have therapeutic promise as wound dressings.

A novel mathematical model to predict prognosis of burnt patients based on logistic regression and support vector machine.

OBJECTIVE To develop a mathematical model of predicting mortality based on the admission characteristics of 6220 burn cases. METHODS Data on all the burn patients presenting to Institute of Burn Research, Southwest Hospital, Third Military Medical University from January of 1999 to December of 2008 were extracted from the departmental registry. The distributions of burn cases were scattered by principal component analysis. Univariate associations with mortality were identified and independent associations were derived from multivariate logistic regression analysis. Using variables independently and significantly associated with mortality, a mathematical model to predict mortality was developed using the support vector machine (SVM) model. The predicting ability of this model was evaluated and verified. RESULTS The overall mortality in this study was 1.8%. Univariate associations with mortality were identified and independent associations were derived from multivariate logistic regression analysis. Variables at admission independently associated with mortality were gender, age, total burn area, full thickness burn area, inhalation injury, shock, period before admission and others. The sensitivity and specificity of logistic model were 99.75% and 85.84% respectively, with an area under the receiver operating curve of 0.989 (95% CI: 0.979-1.000; p<0.01). The model correctly classified 99.50% of cases. The subsequently developed support vector machine (SVM) model correctly classified nearly 100% of test cases, which could not only predict adult group but also pediatric group, with pretty high robustness (92%-100%). CONCLUSION A mathematical model based on logistic regression and SVM could be used to predict the survival prognosis according to the admission characteristics.

Effective symptomatic treatment for severe and intractable pruritus associated with severe burn-induced hypertrophic scars : a prospective, multicenter, controlled trial

BACKGROUND Burn-induced hypertrophic scars are disfiguring and can be associated with severe and intractable pruritus. No effective treatment modalities are currently available for symptomatic control of pruritus for most patients. We assessed the effect of the Antipruritic Hydrogel (CQ-01) in the symptomatic treatment of severe and intractable pruritus associated with burn-induced hypertrophic scars in a prospective, multicenter, controlled trial. METHODS A pilot study was conducted in healthy adult volunteers to identify the most appropriate hydrogel formulation. A selected preparation called Chongqing No. 1 (CQ-01; a guar gum-based hydrogel impregnated with peppermint oil, menthol, and methyl salicylate by a nanoemulsion), showed an excellent symptomatic relief in an exploratory study in 2 patients with intractable pruritus. A statistically powered, prospective, multicenter, controlled study was then conducted in 74 patients to evaluate the efficacy and safety of a 24-h application of CQ-01 compared to a gel control and a negative control on three separate areas in each patient. Symptom assessment was based on our visual analog JW scale (ranging from 0 to 100) at baseline and various time points up to 7 days after application. Follow-up studies were conducted to determine the reproducibility of CQ-01 in repeated applications. RESULTS Of the 74 enrolled subjects, the only observed adverse event was skin irritation reported in 6 patients (8%) and resolved shortly after gel removal. Compared to the baseline, the gauze negative control had a mean JW score reduction of 7; while the gel control and CQ-01 had a drop of 18 (p<0.001) and 36 (p<0.001), respectively. The CQ-01 clinical effect was significant for up to 3 days and waned slowly from 3 to 7 days. There was no statistical correlation between the treatment response and any of the demographic, patient or burn-related factors. Further studies showed a trend that repeated applications might be more effective, suggesting the absence of tachyphylaxis. CONCLUSIONS This prospective, multicenter, controlled study showed that this novel hydrogel CQ-01 is safe and provides significant symptomatic relief for severe and intractable pruritus associated with hypertrophic scars, an unmet medical need for these patients. This effect is independent of the etiology of the burn trauma, extent of the scarring, and duration of the scar formation.