Analysis of Age-Related Changes in Lower Facial Fat Compartments and of the Course of Blood Vessels Using Computed Tomography.

BACKGROUND: According to the volume restoration theory, lower facial fat compartments tend to selectively atrophy or hypertrophy with age. The aim of this study was to demonstrate age-related changes in lower facial fat compartments using computed tomography, with strict control of the body mass index and underlying diseases. METHODS: This study included 60 adult women in three age-based categories. The thicknesses of the jowl, labiomandibular, and chin fat compartments were measured using computed tomographic images. The distribution and arrangement of facial blood vessels were further analyzed to provide evidence of the safety of rejuvenation strategies based on the facial volumetric theory. RESULTS: The inferior part of the superficial jowl fat compartment and deep jowl fat compartment thickened with age. The deep layer of the labiomandibular fat compartment thinned with age, and the superficial layer thickened with age. The deep and superficial layers of the chin compartments thickened with age. The facial vein passes through the lower mandibular border at the anterior edge of the masseter muscle and moves upward, perpendicular to the lower mandibular border. The high-risk area of the facial artery had an angle of approximately 45 degrees to the lower mandibular border. CONCLUSIONS: This study suggests that with age, selective thickening or thinning occurs in different lower facial fat compartments. The mandible and masseter muscle were used as reference markers to analyze the courses of the facial artery and facial vein, which can help clinicians to reduce vascular injury.

CD14 Involvement in Third-degree Skin Burn-induced Myocardial Injury via the MAPK Signaling Pathway.

This study investigated the potential genes and related pathways in burn-induced myocardial injury. Rat myocardial injury induced by third-degree burn and the histopathological structures, apoptosis, and cardiac injury markers were then identified using hematoxylin & eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining, and enzyme-linked immunosorbent assay. Next, differentially expressed mRNAs were screened through next-generation sequencing (NGS), followed by functional annotation and key gene validation through quantitative reverse transcription-polymerase chain reaction. Subsequently, CD14 was screened out, and small interfering RNAs against CD14 were transfected to H9C2 cells to further verify the role of CD14 in burn-induced injury. The results showed that third-degree burn could markedly damage the structure of myocardial tissue, induce the apoptosis of myocardial cells, and increase the levels of myocardial injury-related markers, suggesting that burns could induce myocardial injury in rats. Besides, NGS data discovered that third-degree burn could result in 416 differentially upregulated mRNAs and 285 differentially downregulated mRNAs in myocardial tissue. It was also disclosed that differentially expressed mRNAs were mainly enriched in the phosphatidylinositol 3-kinase/Akt, mitogen-activated protein kinase (MAPK), and tumor necrosis factor signaling pathways. Furthermore, cell viability was significantly decreased in H9C2 cells treated with 10% rat burn serum. CD14 was significantly differentially expressed and screened out for further studies. Treatment with burn serum can significantly upregulate the phosphorylation level of extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase and the expression of cleaved caspase-3 and downregulate the expression of Bcl2 when compared with those in negative control of small interfering RNA transfected H9C2 cells, whereas interfering with CD14 expression reversed the effects of burn serum. The study demonstrated that burn serum treatment could activate the MAPK signaling pathway to promote cell apoptosis, and it can be reversed by interfering with the expression of CD14.

Blockade of LINC01605-enriched exosome generation in M2 macrophages impairs M2 macrophage-induced proliferation, migration, and invasion of human dermal fibroblasts.

Activated M2 macrophages are involved in hypertrophic scar (HS) formation via manipulating the differentiation of fibroblasts to myofibroblasts having the proliferative capacity and biological function. However, the function of exosomes derived from M2 macrophages in HS formation is unclear. Thus, this study aims to investigate the role of exosomes derived by M2 in the formation of HS. To understand the effect of exosomes derived from M2 macrophages on formation of HS, M2 macrophages were co-cultured with human dermal fibroblast (HDF) cells. Cell Counting Kit-8 assay was performed to evaluate HDF proliferation. To evaluate the migration and invasion of HDFs, wound-healing and transwell invasion assays were performed, respectively. To investigate the interaction between LINC01605 and miR-493-3p, a dual-luciferase reporter gene assay was adopted; consequently, an interaction between miR-493-3p and AKT1 was detected. Our results demonstrated that exosomes derived from M2 macrophages promoted the proliferation, migration, and invasion of HDFs. Additionally, we found that long noncoding RNA LINC01605, enriched in exosomes derived from M2 macrophages, promoted fibrosis of HDFs and that GW4869, an inhibitor of exosomes, could revert this effect. Mechanistically, LINC01605 promoted fibrosis of HDFs by directly inhibiting the secretion of miR-493-3p, and miR-493-3p down-regulated the expression of AKT1. Exosomes derived from M2 macrophages promote the proliferation and migration of HDFs by transmitting LINC01605, which may activate the AKT signaling pathway by sponging miR-493-3p. Our results provide a novel approach and basis for further investigation of the function of M2 macrophages in HS formation.

Analysis of the differential expression profile of miRNAs in myocardial tissues of rats with burn injury.

Fifteen percent third-degree burn rat model was used to identify miRNAs that are markers of burn injury-induced myocardial damage. Cardiac tissues were evaluated to determine miRNA profile sequencing. Pearson's correlation analysis was used between miRNAs and injury markers. ROC curve analysis was used to estimate miRNA's sensitivity and specificity for the diagnosis of myocardial damage caused by burn injury. The sequencing analysis revealed 23 differentially expressed miRNAs. Pearson's correlation analysis revealed that rno-miR-190b-3p and C5b9, rno-miR-341, rno-miR-344b-3p and TnI, rno-miR-344b-3p and CK-MB were significantly positively correlated, respectively. ROC curve analysis demonstrated that rno-miR-341, rno-miR-344b-3p, and rno-miR-190b-3p exhibited high sensitivity and specificity for the diagnosis of myocardial damage caused by burn injury. In conclusion, our results suggest that rno-miR-341, rno-miR-344b-3p, and rno-miR-190b-3p have the potential to be used as sensitive and specific biomarkers to diagnose myocardial damage caused by burn injury.

Smad interacting protein 1 influences transforming growth factor-ß1/Smad signaling in extracellular matrix protein production and hypertrophic scar formation.

The transforming growth factor (TGF)-ß/Smad signal transduction pathway is closely associated with hypertrophic scar (HS) formation. Smad interacting protein 1 (SIP1) is a cytoplasmic protein that efficiently regulates Smad2-/3-dependent signaling within the TGF-ß1 pathway. SIP1 influences collagen synthesis in the HS through a heretofore unknown mechanism. This study investigated the role of the SIP1-mediated TGF-ß1/Smad signaling pathway in extracellular matrix (ECM) protein production and hypertrophic scarring. SIP1 expression was markedly lower in HS vs. normal skin (NS) tissue, and α-smooth muscle actin (α-SMA) content and collagen I/III (Col I/III) synthesis were inversely correlated with SIP1 expression. Furthermore, SIP1 inhibited Smad2/3 phosphorylation in vitro, and improved the collagen-based architecture of the scar while reducing collagen expression and overall scar formation in a rabbit ear model of HS. Based on these findings, we propose that SIP1 acts as a molecular modulator capable of altering Smad2-/3-facilitated signaling through the control of Smad phosphorylation, thus inhibiting α-SMA and collagen upregulation in fibroblasts and, ultimately, HS formation. The low SIP1 content in scar tissue also suggests that SIP1 (and positive regulation thereof) is a prospective target for selective HS drug therapy.

Notch1 Pathway Protects against Burn-Induced Myocardial Injury by Repressing Reactive Oxygen Species Production through JAK2/STAT3 Signaling.

Oxidative stress plays an important role in burn-induced myocardial injury, but the cellular mechanisms that control reactive oxygen species (ROS) production and scavenging are not fully understood. This study demonstrated that blockade of Notch signaling via knockout of the transcription factor RBP-J or a pharmacological inhibitor aggravated postburn myocardial injury, which manifested as deteriorated serum CK, CK-MB, and LDH levels and increased apoptosis in vitro and in vivo. Interruption of Notch signaling increased intracellular ROS production, and a ROS scavenger reversed the exacerbated myocardial injury after Notch signaling blockade. These results suggest that Notch signaling deficiency aggravated postburn myocardial injury through increased ROS levels. Notch signaling blockade also decreased MnSOD expression in vitro and in vivo. Notably, Notch signaling blockade downregulated p-JAK2 and p-STAT3 expression. Inhibition of JAK2/STAT3 signaling with AG490 markedly decreased MnSOD expression, increased ROS production, and aggravated myocardial injury. AG490 plus GSI exerted no additional effects. These results demonstrate that Notch signaling protects against burn-induced myocardial injury through JAK2/STAT3 signaling, which activates the expression of MnSOD and leads to decreased ROS levels.

The amelioration of composite tissue allograft rejection by TIM-3-modified dendritic cell: Regulation of the balance of regulatory and effector T cells.

T cell-dependent immune responses play a central role in allograft rejection. Exploring ways to disarm alloreactive T cells represents a potential strategy to promote long-term allograft acceptance and survival. T cell Ig domain and mucin domain 3 (TIM-3) has previously been demonstrated as a central regulator of T helper 1 (Th1) responses and immune tolerance. Hence, TIM-3 may be an important molecule for decreasing immunological rejection during composite tissue allotransplantation (CTA). In this study, BALB/c and C57BL/6 mice were chosen as the experimental animals. The effects of TIM-3 on allograft rejection were explored using TIM-3-modified mature dendritic cells (TIM-3 mDCs). A laser speckle blood flow (LSBF) imager was used to evaluate blood distribution of the BALB/c mice. ELISA, MTT, ELISPOT assays and flow cytometry analysis were carried out for further researches. We found that TIM-3 could obviously prolong the survival time of the transplanted limbs. And TIM-3 could mitigate the immune response and thus enhance immune tolerance after CTA. Also, TIM-3 can induce lymphocyte hyporesponsiveness, including facilitating lymphocyte apoptosis, decreasing lymphocyte proliferation, and influencing the secretion of inflammatory cytokines by CD4(+) T cells. Furthermore, TIM-3 overexpression could induce CD4(+) T cells to differentiate into regulatory T cells (Tregs), which recalibrate the effector and regulatory arms of the alloimmune response. In summary, we concluded that TIM-3 can mitigate allograft rejection and thus enhance immune tolerance by inducing lymphocyte hyporesponsiveness and increasing the number of Tregs of the alloimmune response. TIM-3 may be a potential therapeutic molecule for allograft rejection in CTA.

Protective effect of crocetin against burn-induced intestinal injury.

BACKGROUND: Oxidative stress and inflammation exert central roles in burn-induced intestinal injury. Crocetin, a natural carotenoid compound from gardenia fruits and saffron, has been shown to inhibit oxidative stress and inflammatory response. However, the possibility of crocetin to be used in the treatment of intestinal injury after burn injury has not been investigated. The purpose of the present study was to investigate the effects and potential mechanisms of crocetin in burn-induced intestinal injury. MATERIALS AND METHODS: Several free radical-generating and lipid peroxidation models were used to systematically assess the antioxidant activities of crocetin in vitro. A common burn model was used to induce the intestinal injury in rats. Changes in the levels of malondialdehyde, superoxidase dismutase, catalase, glutathione peroxidase, tumor necrosis factor α, interleukin 6, polymorphonuclear neutrophil accumulation, intestinal permeability, and intestinal histology were examined. RESULTS: In several models of antioxidant activity, crocetin exhibited marked inhibitory action against free radicals and lipid peroxidation. Crocetin increased levels of antioxidant enzymes and reduced intestinal oxidative injury in burn models. In addition, crocetin inhibited polymorphonuclear neutrophil accumulation, ameliorated tumor necrosis factor α and interleukin 6 levels, intestinal permeability, and histological changes. CONCLUSIONS: Crocetin treatment may protect against burn-induced small intestinal injury, possibly by inhibiting burn-induced oxidative stress and inflammatory response.

Role of Hsp-70 responses in cold acclimation of HUVEC-12 cells.

BACKGROUND: Endothelial recovery is a central feature of tissues after frostbite injuries. Thermo tolerance plays an important role in protecting cells against injury after frozen and thawing. The present study aimed to quantitatively assess the injury of human umbilical vein endothelial cells HUVEC-12 after repeated low temperature. MATERIAL AND METHOD: Pretreatments (HUVEC-12) cells were repeatedly exposed to cold (1°C/min decrement to -20°C). Their proliferation, death, apoptosis, and protein and mRNA expressions of HSP70 were determined. RESULTS: Endothelial cells after repeated cold exposures were more resistant to apoptosis and necrosis than normal cells. The expressions of HSP70 in cells after repeated cold exposures were significantly higher than in normal HUVEC-12 cells (P < 0.05). CONCLUSION: Cold acclimation may induce the expression of HSP-70 which plays a protective role in the temperature tolerance.

Rho kinase inhibitor Y-27632 promotes the differentiation of human bone marrow mesenchymal stem cells into keratinocyte-like cells in xeno-free conditioned medium.

INTRODUCTION: Bone marrow mesenchymal stem cells (BMSCs), which have the ability to self-renew and to differentiate into multiple cell types, have recently become a novel strategy for cell-based therapies. The differentiation of BMSCs into keratinocytes may be beneficial for patients with burns, disease, or trauma. However, the currently available cells are exposed to animal materials during their cultivation and induction. These xeno-contaminations severely limit their clinical outcomes. Previous studies have shown that the Rho kinase (ROCK) inhibitor Y-27632 can promote induction efficiency and regulate the self-renewal and differentiation of stem cells. In the present study, we attempted to establish a xeno-free system for the differentiation of BMSCs into keratinocytes and to investigate whether Y-27632 can facilitate this differentiation. METHODS: BMSCs isolated from patients were cultured by using a xeno-free system and characterised by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Human primary keratinocytes were also isolated from patients. Then, the morphology, population doubling time, and ß-galactosidase staining level of these cells were evaluated in the presence or absence of Y-27632 to determine the effects of Y-27632 on the state of the keratinocytes. Keratinocyte-like cells (KLCs) were detected at different time points by immunocytofluorescence analysis. Moreover, the efficiency of BMSC differentiation under different conditions was measured by quantitative real-time-polymerase chain reaction (RT-PCR) and Western blot analyses. RESULTS: The ROCK inhibitor Y-27632 promoted the proliferation and lifespan of human primary keratinocytes. In addition, we showed that keratinocyte-specific markers could be detected in BMSCs cultured in a xeno-free system using keratinocyte-conditioned medium (KCM) independent of the presence of Y-27632. However, the efficiency of the differentiation of BMSCs into KLCs was significantly higher in the presence of Y-27632 using immunofluorescence, quantitative RT-PCR, and Western blot analyses. CONCLUSIONS: This study demonstrated that Y-27632 could promote the proliferation and survival of human primary keratinocytes in a xeno-free culture system. In addition, we found that BMSCs have the ability to differentiate into KLCs in KCM and that Y-27632 can facilitate this differentiation. Our results suggest that BMSCs are capable of differentiating into KLCs in vitro and that the ROCK pathway may play a critical role in this process.

SIRT1 is a regulator in high glucose-induced inflammatory response in RAW264.7 cells.

Sepsis is defined as a systemic inflammatory response syndrome that disorders the functions of host immune system, including the imbalance between pro- and anti-inflammatory responses mediated by immune macrophages. Sepsis could also induce acute hyperglycemia. Studies have shown that the silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent deacetylase, mediates NF-κb deacetylation and inhibits its function. Therefore, SIRT1 is likely to play an important role in high glucose-mediated inflammatory signalings. Here we demonstrate that high glucose significantly downregulates both the mRNA and protein levels of SIRT1 and upregulates the mRNA level and the release of two pro-inflammatory cytokines, IL-1ß and TNF-α, in RAW264.7 macrophages. Interestingly, the reduced level of SIRT1 by high glucose is remarkably upregulated by SIRT1 activator SRT1720, while the level and the release of IL-1ß and TNF-α significantly decrease with the use of SRT1720. However, when the function of SIRT1 is inhibited by EX527 or its expression is suppressed by RNAi, the upregulated level and release of IL-1ß and TNF-α by high glucose are further increased. Taken together, these findings collectively suggest that SIRT1 is an important regulator in many high glucose-related inflammatory diseases such as sepsis.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145.

The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at the gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs.

Effects of resveratrol on the treatment of inflammatory response induced by severe burn.

The aim of this study was to preliminarily investigate the effects of resveratrol on the treatment of systemic inflammatory response induced by severe burn wounding. Through the simulation experiment in vivo on burned mice and simulative experiment in vitro on mice macrophage respectively, differences of the related pro-inflammatory cytokines and SIRT1 expression levels between the resveratrol-treated group and the untreated control group were detected and analyzed. The results of the simulation experiment in vivo on burned mice manifested that the survival rate of the mice in the resveratrol-treated group was markedly higher than that of controls (p<0.05). Resveratrol could significantly reduce the levels of pro-inflammatory factors TNF-α, IL-1ß, and IL-6 in serum (p<0.01) and greatly elevate the expression level of SIRT1 (p<0.01). The results of the simulative experiment in vitro on mice macrophage showed no significant difference in TNF-α, IL-1ß, or IL-6 contents among three groups (C, mice macrophage control group; R, resveratrol-treated macrophage group; I, SIRT1-inhibitor-treated macrophage group). Whereas, after lipopolysaccharide (LPS) activation (L group), macrophage TNF-α, IL-1ß, and IL-6 levels were significantly increased in L group, dramatically higher than those in L+R group (LPS and resveratrol treatment group) (p<0.01). After adding SITR1 inhibitor, three pro-inflammatory cytokines in L+R+I group all showed significant increases compared with those in L+R group (p<0.01). LPS activated macrophages were able to promote the expression of pro-inflammatory cytokines. By upregulating the expression levels of SIRT1, resveratrol could effectively inhibit the inflammation cascade reaction and increase the survival rate of severe burn with bacterial infections in a large extent.

Expression, purification, and characterization of scar tissue neovasculature endothelial cell-targeted rhIL10 in Escherichia coli.

Interleukin 10 (IL10) plays a pivotal role in the anti-inflammatory response and immunosuppressive reactions. It has also been identified as a new promising therapy for scar formation. Treatment of scars with IL10 has significant effects, but there are some shortcomings, including poor tissue-binding specificity and low effectiveness. RGD peptide has been demonstrated to bind specifically to αvß3 integrin on neovasculature endothelial cells, and the excess production of neovasculature is crucial to scar formation. To increase efficacy against scar formation and to decrease the side effects on normal tissues, a novel hybrid protein combining human IL10 with RGD was designed. The DNA sequence encoding the recombinant fusion protein IL10-RGD (rhIL10-RGD) was subcloned into a pET22b (+) vector for protein expression in E. coli strain BL21 (DE3). SDS-PAGE analysis displayed an induced expression product band at a molecular weight of 19.3 kDa, which constituted 30 % of the total bacterial protein. We developed a procedure to purify rhIL10-RGD from inclusion bodies and then renatured the protein using dialysis against urea with a step-down concentration procedure. Hypertrophic scar fibroblasts (HSFs) were treated with rhIL10-RGD, and the fibrosis-related protein levels were assessed by Western blotting. The results indicated that rhIL10-RGD can downregulate the expression levels of Col1 and α-SMA in HSFs and suppress tube formation of HUVECs. These results indicate that rhIL10-RGD has anti-fibrosis effects and can potentially be used to treat the neovasculature in scar formation and improve the abnormal deposition of the extracellular matrix (ECM). Thus, rhIL10-RGD may be a more effective candidate for scar-improvement and anti-fibrosis therapy.

[Effects of reactive by burn rat serum oxygen species on apoptosis of pulmonary microvascular endothelial cells induced].

OBJECTIVE: To observe the level of intracellular reactive oxygen species (ROS) in rats with severe burn and pulmonary microvascular endothelial cells (PMVECs) treated with serum of rat with burn injury, and to investigate the relationship between ROS and apoptosis of PMVECs. METHODS: (1) Twenty-four SD rats were divided into sham injury group ( n = 3) and burn group (n = 21) according to the random number table (the same grouping method below). Rats in burn group were inflicted with 30% TBSA full-thickness scald on the back, and rats in sham injury group were sham injured. Blood samples were collected from abdominal aorta at post injury hour 6, 12, 24, 36, 48, 60, 72 respectively from 3 rats of burn group. The serum content of ROS was assayed by ELISA. The same determination was performed in rats of sham injury group. (2) Five rats were subjected to scald injury as above, and burn serum was prepared 24 hours after injury. Another 5 rats without receiving any treatment were used to prepare normal serum. (3) Marginal pulmonary tissue was harvested from 20 SD young rats. Cells were cultured with tissue block method and indentified with immunohistochemical staining. The third passage of PMVECs in logarithmic phase were inoculated in 6-well plates and 12-well plates. PMVECs in both plates were divided into 4 groups: normal serum group, burn serum group, normal serum + MnTBAP group, and burn serum + MnTBAP group, with 3 wells in each group. Cells in the former 2 groups were respectively cultured with special nutrient solution of endothelial cells without serum added with 15% healthy rat serum or 15% burn rat serum. Cells in the latter 2 groups were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 µmol/L MnTBAP in the nutrient solution. After being cultured for 24 h, the content of ROS in PMVECs in 6-well plates was detected with flow cytometry. The apoptosis of PMVECs in 12-well plates was observed with acridine orange-ethidium bromide staining, and the apoptosis rate was calculated. Data were processed with one-way analysis of variance and LSD-t test. RESULTS: (1) The serum contents of ROS in rats of burn group were respectively (187 ± 21), (235 ± 22), (231 ± 25), (291 ± 20), (315 ±23) nmol/mL at post injury hour 24, 36, 48, 60, 72, which were significantly higher than that in sham injury group [(141 ± 19) nmol/mL, with t values respectively 7. 86, 9. 57, 13. 87, 14.98, 18.40, P values below 0.01]. (2) Primary cells grew slowly and showed a cobblestone appearance. After passages, cells grew with orderly distribution. The positive rate of coagulation factor VIII of cells was (96 ± 5)% , and thus they were identified as PMVECs. (3) In normal serum group, burn serum group, normal serum + MnTBAP group, and burn serum + MnTBAP group, the contents of ROS in PMVECs were respectively 798 ± 40, 1 294 ± 84, 763 ± 59, 926 ± 42 ( F =93.01, P <0.01), and the apoptosis rates of PMVECs were respectively (6.2 ± 1.3)%, (57.3 ± 6. 7)%, (3.7 ± 0. 8)%, (28.7 ± 5. 7)% (F = 224.50, P <0.01) after being cultured for 24 h. Compared with those of normal serum group, the content of ROS and apoptosis rate of PMVECs in burn serum group increased significantly (with t values respectively 10.40 and 49.06, P values below 0.01). The content of ROS and apoptosis rate of PMVECs in burn serum + MnTBAP group were significantly lower than those in burn serum group (with t values respectively 7.48 and 23.94, P values below 0.01). CONCLUSIONS: Serum content of ROS was increased in severely burned rats. Burn rat serum stimulation on PMVECs can lead to the increase of the intracellular ROS and induce apoptosis. However application of MnTBAP can scavenge ROS and reduce the apoptosis induced by burn rat serum.

Effects of integrin ανß3 on differentiation and collagen synthesis induced by connective tissue growth factor in human hypertrophic scar fibroblasts.

CCN2 is a matricellular protein that appears to be important in scar formation. CCN2 mediates the pro-fibrotic effects in hypertrophic scars (HTSs) through an unknown mechanism. However, many activities of CCN2 protein are known to be mediated by direct binding to integrin receptors. In this study, we investigated the role of integrin α(ν)ß(3) in the differentiation of hypertrophic scar fibroblasts (HTSFs) induced by CCN2. The levels of integrin α(ν)ß(3) between normal skin and hypertrophic scar (HTS) tissues were compared, and integrin α(ν)ß(3) was found to be upregulated in HTS. CCN2 was shown to induce HTSF differentiation and collagen (COL) synthesis at the mRNA and protein levels. Based on these results, the expression of integrin α(ν)ß(3) was upregulated by CCN2 stimulation during HTSF differentiation. Blockade of integrin α(ν)ß(3) prevented CCN2-induced HTSF differentiation and COL synthesis. Furthermore, the CCN2-induced increase in contractility of the HTSF in COL lattices was inhibited by integrin α(ν)ß(3) blocking antibodies. HTSs were established in a rabbit ear model, and the inhibitor of integrin α(ν)ß(3) significantly improved the architecture of the rabbit ear scar. Results of the present study showed that integrin α(ν)ß(3) contributes to pro-fibrotic CCN2 signaling. Blocking this pathway may therefore be beneficial for the treatment of HTS.

Anti-fibrotic actions of interleukin-10 against hypertrophic scarring by activation of PI3K/AKT and STAT3 signaling pathways in scar-forming fibroblasts.

BACKGROUND: The hypertrophic scar (HS) is a serious fibrotic skin condition and a major clinical problem. Interleukin-10 (IL-10) has been identified as a prospective scar-improving compound based on preclinical trials. Our previous work showed that IL-10 has anti-fibrotic effects in transforming growth factor (TGF)-ß1-stimulated fibroblasts, as well as potential therapeutic benefits for the prevention and reduction of scar formation. However, relatively little is known about the mechanisms underlying IL-10-mediated anti-fibrotic and scar-improvement actions. OBJECTIVE: To explore the expression of the IL-10 receptor in human HS tissue and primary HS fibroblasts (HSFs), and the molecular mechanisms contributing to the anti-fibrotic and scar-improvement capabilities of IL-10. METHODS: Expression of the IL-10 receptor was assessed in HS tissue and HSFs by immunohistochemistry, immunofluorescence microscopy, and polymerase chain reaction analysis. Primary HSFs were treated with IL-10, a specific phosphatidylinositol 3 kinase (PI3K) inhibitor (LY294002) or a function-blocking antibody against the IL-10 receptor (IL-10RB). Next, Western blot analysis was used to evaluate changes in the phosphorylation status of AKT and signal transducers and activators of transcription (STAT) 3, as well as the expression levels of fibrosis-related proteins. RESULTS: HS tissue and primary HSFs were characterized by expression of the IL-10 receptor and by high expression of fibrotic markers relative to normal controls. Primary HSFs expressed the IL-10 receptor, while IL-10 induced AKT and STAT3 phosphorylation in these cells. In addition, LY294002 blocked AKT and STAT phosphorylation, and also up-regulated expression levels of type I and type III collagen (Col 1 and Col 3) and alpha-smooth muscle actin (α-SMA) in IL-10-treated cells. Similarly, IL-10RB reduced STAT3/AKT phosphorylation and blocked the IL-10-mediated mitigation of fibrosis in HSFs. CONCLUSION: IL-10 apparently inhibits fibrosis by activating AKT and STAT3 phosphorylation downstream of the IL-10 receptor, and by facilitating crosstalk between the PI3K/AKT and STAT3 signal transduction pathways.

Inhibition of Notch signaling leads to increased intracellular ROS by up-regulating Nox4 expression in primary HUVECs.

The essential roles of Notch pathway in angiogenesis have been reported for years. However, how Notch pathway plays its role in regulating endothelial cells remains largely unknown. In this study we found that blockade of Notch signaling with a γ-secretase inhibitor increased reactive oxygen species (ROS) in primary human umbilical vein endothelial cells (HUVECs) under both normaxic and ischemia/reperfusion (I/R) conditions. Abruption of ROS generation with ROS scavengers or specific inhibitors of ROS production in HUVECs abolished Notch blockade-induced HUVEC proliferation, migration and adhesion, suggesting that the regulation of Notch pathway on endothelial cell behavior is at least partially dependent on its down-regulation of ROS level. We further showed that the enhanced generation of ROS after blocking Notch signal was accompanied by augmented expression of Nox4, which led to increased phosphorylation of VEGFR2 and ERK in HUVECs. In summary, our results have shown that Notch signaling regulates ROS generation by suppressing Nox4, and further modulates endothelial cell proliferation, migration and adhesion.

Inhibition of Na+/H+ exchanger 1 by cariporide alleviates burn-induced multiple organ injury.

BACKGROUND: Severe burns initiate an inflammatory response characterized by the upregulation of proinflammatory cytokine, which contributes to multiple organ injury. Na(+)/H(+) exchanger 1 (NHE1) plays a significant role in several inflammatory processes. This study was designed to investigate the role of NHE1 in burn-induced inflammation and multiple organ injury. MATERIALS AND METHODS: Rats were subjected to a 30% total body surface area full-thickness burn. Cariporide was used to assess the function of NHE1 in burn-induced multiple organ injury by biochemical parameters, histologic changes, and inflammatory cytokine production. RESULTS: We found that NHE1 expression was significantly increased after burn injury. Inhibition of NHE1 by cariporide attenuated burn-induced edema and tissue injury in heart, lung, kidney, and small intestine. Cariporide also inhibited plasma levels of tumor necrosis factor α, interleukin 6, and myeloperoxidase activity. CONCLUSIONS: These results indicate that NHE1 inhibition prevents burn-induced multiple organ injury. The salutary effects afforded by NHE1 inhibition, at least in part, are mediated by attenuating systemic inflammatory response.

[Systemic therapy for defects of skin and soft tissue on the knees after severe trauma or burn].

OBJECTIVE: To explore the methods of systemic treatment of defects of skin and soft tissue on the knees after severe trauma or burn. METHODS: Twenty patients with defects of skin and soft tissue on the knees after severe trauma or burn hospitalized in our center from January 2009 to December 2011. The injury areas on the knees ranged from 5 cm×4 cm to 30 cm×20 cm. The wounds were treated with radical debridement, vacuum sealing drainage, and douche through dripping to control infection in early stage. Then they were covered with transplantation of skin grafts plus flap or only with flap. Totally 8 local flaps (including 6 local rotation or transposition flaps and 2 saphenous artery flaps) and 12 free flaps (including 8 anterolateral thigh flaps and 4 latissimus dorsi musculocutaneous flaps) were used. The flap size ranged from 6 cm×5 cm to 32 cm×22 cm. The rehabilitation training of the knee joints was carried out in the early stage after wound healing. RESULTS: All free skin grafts and flaps used in 15 patients survived. Thirteen of them were primarily healed, while some small parts of skin grafts of the other two patients were in poor condition because of infection, and they healed after another session of skin transplantation. Infection occurred under the free flap in one of the 5 patients transplanted with flaps only, which was healed after continuous douche through dripping and another surgical debridement following wet dressing. The knee joints were in good function during the follow-up period of 1 - 3 years. CONCLUSIONS: The systemic therapy of radical debridement, vacuum sealing drainage technique, douche through dripping, transplantation of large autologous grafts and flaps, and the early rehabilitation training are effective and reliable in repairing defects of skin and soft tissue at the knee region after severe injuries.