TSG-6 inhibits hypertrophic scar fibroblast proliferation by regulating IRE1α/TRAF2/NF-κB signalling.

TNF-stimulated gene (TSG-6) was reported to suppress hypertrophic scar (HS) formation in a rabbit ear model, and the overexpression of TSG-6 in human HS fibroblasts (HSFs) was found to induce their apoptotic death. The molecular basis for these findings, however, remains to be clarified. HSFs were subjected to TSG-6 treatment. Treatment with TSG-6 significantly suppressed HSF proliferation and induced them to undergo apoptosis. Moreover, TSG-6 exposure led to reductions in collagen I, collagen III, and α-SMA mRNA and protein levels, with a corresponding drop in proliferating cell nuclear antigen (PCNA) expression indicative of impaired proliferative activity. Endoplasmic reticulum (ER) stress was also suppressed in these HSFs as demonstrated by decreases in Bip and p-IRE1α expression, downstream inositol requiring enzyme 1 alpha (IRE1α) -Tumor necrosis factor receptor associated factor 2 (TRAF2) pathway signalling was inhibited and treated cells failed to induce NF-κB, TNF-α, IL-1ß, and IL-6 expression. Overall, ER stress was found to trigger inflammatory activity in HSFs via the IRE1α-TRAF2 axis, as confirmed with the specific inhibitor of IRE1α STF083010. Additionally, the effects of TSG-6 on apoptosis, collagen I, collagen III, α-SMA, and PCNA of HSFs were reversed by the IRE1α activator thapsigargin (TG). These data suggest that TSG-6 administration can effectively suppress the proliferation of HSFs in part via the inhibition of IRE1α-mediated ER stress-induced inflammation (IRE1α/TRAF2/NF-κB signalling).

p75NTR silencing inhibits proliferation, migration, and extracellular matrix deposition of hypertrophic scar fibroblasts by activating autophagy through inhibiting the PI3K/Akt/mTOR pathway.

Hypertrophic scar (HS) results from abnormal wound healing, accompanied by excessive hypercellularity, migration, and extracellular matrix (ECM) deposition. Autophagy dysregulation plays crucial roles during HS formation. The overexpressed p75 neurotrophin receptor (p75NTR) in injured skin tissue after wound healing becomes a factor aggravating scar. This study was designed to investigate the role of p75NTR and p75NTR-mediated autophagy in the process of HS. The results revealed that p75NTR expression was significantly upregulated while that of autophagy proteins was downregulated in cicatrix at 3 and 6 months after a burn, which was recovered at 12 months. p75NTR silencing inhibited proliferation, migration, and ECM deposition of hypertrophic scar fibroblasts (HSF), whereas p75NTR overexpression presented the opposite results. Silencing of p75NTR reduced the expression of PI3K/Akt/mTOR signaling molecules while enhancing that of autophagy proteins. Importantly, PI3K agonist (IGF-1) intervention notably decreased the levels of LC3B II/I and Beclin-1 and restored the inhibitory effects of p75NTR silencing on proliferation, migration, and ECM deposition of HSF. Concurrently, autophagy inhibitor 3-methyladenine (3-MA) treatment exhibited the same variation trends with IGF-1. Taken together, these findings demonstrated that p75NTR silencing inhibits proliferation, migration, and ECM deposition of HSF by activating autophagy by inhibiting the PI3K/Akt/mTOR pathway.

Long non-coding RNA H19 acts as a microRNA-194 sponge to inhibit the apoptosis and promote the proliferation of hypertrophic scar fibroblasts.

The effects of long non-coding RNAs (lncRNAs) on the proliferation of hypertrophic scars have been described, however, the underlying mechanisms are not well characterized. The present study aimed to investigate the mechanisms of lncRNA H19 in hypertrophic scars. The effects of the lncRNA H19 on the proliferation and apoptosis of hypertrophic scar fibroblasts (HSFs) were analyzed using 5'-ethynyl-2'-deoxyuridine staining, flow cytometry, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT). The results revealed H19 promoted the proliferation and inhibited the apoptosis in HSF. In addition, the binding associations between H19 and microRNA-194 (miR-194), and miR-194 and insulin-like growth factor I receptor (IGF1R) were identified using bioinformatics screening and verified using dual-luciferase assays. Furthermore, the effects of the IGF1R knockdown on H19-induced HSF phenotypes and regulation over the p38 MAPK pathway were determined. Mechanistically, miR-194 was identified as the downstream effector of the H19-mediated phenotypes of HSFs through its ability to directly target IGF1R, thus modulating the p38 MAPK signaling pathway. In conclusion, the findings suggested that H19 may inhibit the apoptosis and promote the proliferation of HSFs through the miR-194/IGF1R/p38 MAPK signaling axis, thereby contributing to the progression of hypertrophic scars. These findings may provide novel targets for the treatment of hypertrophic scars.

LncRNA PICSAR binds to miR-485-5p and activates TGF-ß1/Smad to promote abnormal proliferation of hypertrophic scar fibroblasts (HSFs) and excessive deposition of extracellular matrix (ECM).

The purpose of this study is to explore whether LncRNA PICSAR binds to miR-485-5p and thereby activates TGF-ß1/Smad signaling pathway, influencing the abnormal proliferation of fibroblasts and excessive deposition of ECM in hypertrophic scar formation. PICSAR and miR-485-5p expressions were detected by qPCR. Cell proliferation was examined by CCK-8. Protein expressions were determined by western blot. Immunofluorescence detected the Ki-67 expression. Dual-luciferase followed by immunoprecipitation was performed to verify the interaction between PICSAR and miR-485-5p. Interference with PICSAR inhibited the abnormal proliferation of hypertrophic scar fibroblasts (HSFs) and the excessive deposition of ECM. It was also confirmed in our study that MiR-485-5p is a direct target of PICSAR in HSFs. Additionally, inhibition of miR-485-5p reversed the effect of PICSAR knockdown in HSFs. LncRNA PICSAR binds to miR-485-5p and thereby activates TGF-ß1/Smad signaling pathway, promoting the abnormal proliferation of fibroblasts and excessive deposition of ECM in hypertrophic scar formation.

Inactivation of Beclin-1-dependent autophagy promotes ursolic acid-induced apoptosis in hypertrophic scar fibroblasts.

A hypertrophic scar (HS) is caused by abnormal proliferation of dermal fibroblasts. Thus, promoting hypertrophic scar fibroblast (HSFB) apoptosis is an effective strategy for HS therapy. Ursolic acid (UA) has been widely used as an inducer of apoptosis in diverse cancers. However, whether UA plays an inhibitory role in HS formation is still unknown. In our study, UA was used to treat HSFBs and the cell viability, apoptosis, and collagen synthesis were determined by a Cell Counting Kit 8 assay, flow cytometry, and an H3 -proline incorporation assay, respectively. Autophagy activity was detected by LC3 immunoblotting and electron microscopy, and siRNAs targeting Beclin-1 were used to inhibit autophagy. Western blotting was performed to investigate the molecular changes in HSFBs after various treatments. We found that UA inhibited collagen synthesis and induced cell apoptosis in HSFBs, evidenced by the deregulated expression of Bim, Bcl-2 and Cyto C. Furthermore, we demonstrated that UA induced autophagy and inactivation of autophagy promoted UA-induced apoptosis and collagen synthesis inhibition in HSFBs. Molecular investigation indicated that UA-induced autophagy through upregulation of Beclin-1 and knockdown of Beclin-1 prevent UA-induced autophagy. Overexpression of Bcl-2 prevents UA-induced autophagy, Beclin-1 upregulation, apoptosis and collagen synthesis inhibition in HSFBs. Collectively, our study demonstrated that UA is a novel agent for inhibiting HS formation by promoting apoptosis, especially in combination with an autophagy inhibitor. Our results provide strong evidence of the application of UA in clinical HS treatment.

Functional characterization of TRAP1-like protein involved in modulating fibrotic processes mediated by TGF-ß/Smad signaling in hypertrophic scar fibroblasts.

The transforming growth factor-ß1 (TGF-ß)-mediated signaling pathway is believed to be closely associated with wound healing and scar formation, in which TRAP1-like protein (TLP) plays a role in regulating the balance of Smad2 vs. Smad3 signaling. Our previous study revealed the relation between TLP and collagen synthesis in normal human skin fibroblasts. Here, we present a detailed analysis of the effects of TLP on the process of hypertrophic scar formation and contraction. To explore and verify a contribution of TLP to the pathological mechanism of hypertrophic scar fibroblasts (HSFb), we constructed lentiviral vectors that either overexpressed TLP or encoded small hairpin RNAs (shRNAs) targeting TLP, then we transfected them into HSFb. TLP knockdown in HSFb resulted in reduced levels of cell contraction, type I and type III collagen mRNA transcripts and protein expression, and higher levels of fibronectin (FN) compared to control groups. In addition, knockdown of TLP promoted the phosphorylation of Smad3 but repressed Smad2 and Erk-1/2 phosphorylation in human hypertrophic scar fibroblasts compared to control groups. The reduction of TLP did not interfere with HSF proliferative ability, but exogenous TLP cooperated with TGF-ß1 to increase cell viability. Together, our findings demonstrate evidence for a contribution of TLP expression in hypertrophic scar formation and contraction.

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.

A New Strategy to Inhibit Scar Formation by Accelerating Normal Healing Using Silicate Bioactive Materials.

Inspired by the scar-free wound healing in infants, an anti-scar strategy is proposed by accelerating wound healing using silicate bioactive materials. Bioglass/alginate composite hydrogels are applied, which significantly inhibit scar formation in rabbit ear scar models. The underlining mechanisms include stimulation of Integrin Subunit Alpha 2 expression in dermal fibroblasts to accelerate wound healing, and induction of apoptosis of hypertrophic scar fibroblasts by directly stimulating the N-Acylsphingosine Amidohydrolase 2 expression in hypertrophic scar fibroblasts, and indirectly upregulating the secretion of Cathepsin K in dermal fibroblasts. Considering specific functions of the bioactive silicate materials, two scar treatment regimes are tested. For severe scars, a regenerative intervention is applied by surgical removal of the scar followed by the treatment with bioactive hydrogels to reduce the formation of scars by activating dermal fibroblasts. For mild scars, the bioactive dressing is applied on the formed scar and reduces scar by inducing scar fibroblasts apoptosis.

Hepatocyte growth factor-modified adipose-derived mesenchymal stem cells inhibit human hypertrophic scar fibroblast activation.

PURPOSE: Nucleoside-modified messenger RNA (modRNA) holds the potential for facilitating genetic enhancement of stem cells. In this study, modRNA encoding hepatocyte growth factor (modHGF) was used to chemically modify adipose-derived mesenchymal stem cells (ADSCs) and the effect of modified ADSCs on the activation of hypertrophic scar fibroblasts (HSFs) was evaluated. METHODS: CCK-8, wound healing, and transwell assays were utilized to evaluate the viability and migratory potential of modHGF-engineered ADSCs and their effect on HSF activation. Reverse transcription-polymerase chain reaction, western blot, and immunofluorescence staining were performed to detect the expression of collagen-I (Col I), collagen-III (Col III), alpha-smooth muscle actin (α-SMA), matrix metallopeptidase 1 (MMP-1), and MMP-3. RESULTS: Transfection of ADSCs with modHGF (HGF-ADSC) resulted in enhanced production of HGF. Meanwhile, modHGF modification enhanced the viability and migration of ADSCs. Notably, culture media from HGF-ADSCs exhibited a more potent inhibitory effect on the proliferation and migration of HSFs. In addition, culture media from HGF-ADSCs inhibited extracellular matrix synthesis of HSFs, as evidenced by reduced expression levels of Col I, Col III, and α-SMA, while increasing expression of MMP-1 and MMP-3. Conversely, neutralization experiments confirmed that these effects could be effectively alleviated by blocking HGF activity. CONCLUSION: modHGF modification optimizes the inhibitory effect of ADSCs on HSF activation, which provides a promising alternative for preventing and treating hyperplastic scars.

Desferrioxamine Enhances 5-Aminolaevulinic Acid- Induced Protoporphyrin IX Accumulation and Therapeutic Efficacy for Hypertrophic Scar.

Hypertrophic scar is a common problem after skin burns or trauma which brings physical, psychological, and cosmetic problems to patients. Photodynamic therapy with 5-aminolevulinic acid (5-ALA) is a promising therapy for hypertrophic scar. However, clinical applications of 5-ALA are limited because of the low permeability of 5-ALA in the skin stratum corneum and the rapid binding of protoporphyrin IX (PpIX) with iron ions, which lead to insufficient PpIX production in target tissues. Herein, a mixture of 5-ALA and DFO (deferoxamine, a special iron chelator) was applied for the treatment of hypertrophic scar. 5-ALA/DFO could efficiently block the biotransformation of PpIX to heme, thus realizing a significant accumulation of photosensitizer. In addition, injection locally into the lesion was applied, which combined with enhanced photodynamic therapy to destroy hypertrophic scar fibroblasts. In vitro experiments showed that 5-ALA/DFO could increase more ROS generation by increasing the accumulation of PpIX, resulting in the apoptosis of hypertrophic scar fibroblasts. Furthermore, 5-ALA/DFO inhibited the proliferation and migration of hypertrophic scar fibroblasts. In vivo study showed that 5-ALA/DFO could effectively inhibit the formation of proliferative scar. Therefore, 5-ALA/DFO has the potential to enhance the photodynamic therapy of 5-ALA and provides a new treatment strategy for hypertrophic scar.

Ellagic acid inhibits the formation of hypertrophic scars by suppressing TGF-ß/Smad signaling pathway activity.

Hypertrophic scar (HS) is a benign fibroproliferative skin disease, which lacks the ideal treatment and drugs. Ellagic acid (EA) is a natural polyphenol that prevents fibroblasts from proliferating and migrating. This study aimed to determine the role of EA in HS formation and its possible mechanism by in vitro experiments. HS fibroblasts (HSFs) and normal fibroblasts (NFs) were separated from HS tissue and normal skin tissue, respectively. HSFs were treated with 10 and 50 µM EA to assess their effect on HS formation. In particular, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and scratch assay were used to detect the viability and migration ability of HSFs. Quantitative reverse transcriptase real-time polymerase chain reaction was used to measure the mRNA expression level of basic fibroblast growth factor (bFGF), extracellular matrix (ECM)-related gene collagen-I (COL-I), and fibronectin 1 (FN1) in HSFs. Finally, Western blot was utilized to measure the expression level of TGF-ß/Smad signaling pathway-related proteins in HSFs. The viability of HSFs was significantly increased compared with NFs. 10 and 50 µM EA treatment markedly inhibition the cell viability and migration of HSFs. EA treatment upregulated the bFGF expression level and downregulated the COL-I and FN1 expression level in HSFs. In addition, p-Smad2, p-Smad3, and transforming growth factor (TGF)-ß1 expression levels as well as p-Smad2/Smad2 and p-Smad3/Smad3 ratios remarkably decreased in HSFs after EA treatment. EA inhibited the formation of HSs by suppressing the viability and migration of HSFs and ECM deposition as well as by preventing the activation of TGF-ß/Smad signaling.

Interferon-α2b-Induced RARRES3 Upregulation Inhibits Hypertrophic Scar Fibroblasts' Proliferation and Migration Through Wnt/ß-Catenin Pathway Suppression.

Hypertrophic scar (HS) is a severe skin fibrotic disorder with unclear pathogenesis. Interferon-α2b (IFN-α2b) exerts inhibitory effects on HS in vivo and in vitro; however, the exact mechanism remains unclear. In this study, we aimed to evaluate the inhibitory effects of IFN-α2b on hypertrophic scar fibroblasts' (HSFs) proliferation and migration, and to further investigate the associated molecular mechanism. Cell Counting Kit-8 and CyQUANT assays were used to assess HSFs' proliferation; wound healing and Transwell assays were used to assess HSFs' migration; real-time quantitative polymerase chain reaction and Western blotting were used to detect messenger RNA and protein levels, respectively, of related genes; bioinformatics analysis was performed to predict the downstream target of IFN-α2b. Our findings are as follows: (1) IFN-α2b inhibited HSFs' proliferation and migration in a dose-dependent manner. (2) IFN-α2b inhibited HSFs' proliferation and migration by suppressing the Wnt/ß-catenin pathway. (3) Retinoic-acid receptor responder 3 (RARRES3) was predicted as a functional downstream molecule of IFN-α2b, which was low in HSFs. (4) IFN-α2b inhibited HSF phenotypes and the Wnt/ß-catenin pathway by upregulating RARRES3 expression. (5) RARRES3 restrained HSFs' proliferation and migration by repressing the Wnt/ß-catenin pathway. In conclusion, IFN-α2b-induced RARRES3 upregulation inhibited HSFs' proliferation and migration through Wnt/ß-catenin pathway suppression.

Polyphyllin VII induces fibroblasts apoptosis via the ERK/JNK pathway.

Hypertrophic scar (HS) is a pathological scar that often occurs in burn patients. Its histology is characterized by the excessive proliferation of fibroblasts (FB) and excessive accumulation of extracellular matrix (ECM). Inhibition of proliferation and activation of FB is essential for the treatment of HS. The crude extracts of traditional Chinese medicines have beneficial therapeutic effects on HS besides possessing fewer side effects and being easily available. Polyphyllin VII (PP7) is an isoprene saponin isolated from Rhizoma paridis. It has a pro-apoptotic effect on cancer cells. In the present study, we demonstrate that PP7 exerts a significant inhibitory effect on hypertrophic scar fibroblasts (HSFs) in vitro. We also demonstrate that PP7 considerably induces the apoptosis of HSFs and inhibits their activity. Our data show that the PP7-induced HSFs cell apoptosis was mainly due to the enhanced expression of apoptotic genes (Bax, Caspase-3, Caspase-9) and decreased expression of Bcl-2. Moreover, PP7 treatment also enhances the expression of JNK, but that of extracellular protein kinases (ERK) was reduced, and induces apoptosis through ERK/JNK pathways. Thus, PP7 can be used as a drug to prevent the formation of HS.

MicroRNA-18a-5p represses scar fibroblast proliferation and extracellular matrix deposition through regulating Smad2 expression.

The aim of the present study was to investigate the expression and role of microRNA-18a-5p (miR-18a-5p) during the formation of hypertrophic scar (HS), and to further explore the molecular mechanisms involved. Downregulation of miR-18a-5p in HS tissues and human HS fibroblasts (hHSFs) was detected by reverse transcription-quantitative polymerase chain reaction. The binding sites between miR-18a-5p and the 3'-untranslated region of SMAD family member 2 (Smad2) were predicted by TargetScan and confirmed by dual-luciferase reporter assay. To investigate the role of miR-18a-5p in HS formation, the effects of miR-18a-5p downregulation or upregulation on hHSFs were subsequently determined. Cell proliferation was detected by an MTT assay, while cell apoptosis was measured by flow cytometry. In addition, the protein expression levels of Smad2, Collagen I (Col I) and Col III were examined by western blot assay. The findings indicated that miR-18a-5p downregulation in hHSFs significantly promoted the cell proliferation, decreased cell apoptosis and enhanced the expression levels of Smad2, Col I and Col III protein and mRNA, whereas miR-18a-5p upregulation in hHSFs exerted opposite effects. Notably, the effects of miR-18a-5p upregulation on hHSFs were eliminated by Smad2 upregulation. In conclusion, the data indicated that miR-18a-5p was downregulated during HS formation, and its upregulation repressed scar fibroblast proliferation and extracellular matrix deposition by targeting Smad2. Therefore, miR-18a-5p may serve as a novel therapeutic target for the treatment of HS.

Umbilical cord-derived mesenchymal stem cells exert anti-fibrotic action on hypertrophic scar-derived fibroblasts in co-culture by inhibiting the activation of the TGF ß1/Smad3 pathway.

A hypertrophic scar (HS) is a severe fibrotic skin disease that causes disfigurement and deformity. It occurs after deep cutaneous injury and presents a major clinical challenge. The present study aimed to evaluate the effects of umbilical cord-derived mesenchymal stem cells (UCMSCs) on hypertrophic scar fibroblasts (HSFs), one of the main effector cells for HS formation, in a co-culture system and to investigate the potential underlying molecular mechanism. Cultured HSFs were divided into control and co-culture groups. The proliferation ability of HSFs was evaluated using cell counting kit-8 and the percentage of Ki67-positive fibroblasts was assessed by immunofluorescence. The apoptosis of HSFs was determined using a TUNEL assay and by assessing the expression of capase-3 via western blotting. A scratch wound healing assay was employed to examine the migration of HSFs. The expression levels of HS-associated genes (collagen type Iα 2 chain, collagen type IIIα 1 chain and actin α 2 smooth muscle) and proteins (collagen I, collagen III and α-smooth muscle actin) were measured by reverse transcription-quantitative PCR (RT-qPCR) and western blotting, respectively, to assess the pro-fibrotic phenotype of HSFs. The modulation of the transforming growth factor ß1 (TGF ß1)/Smad3 pathway in HSFs was evaluated by measuring the protein levels of TGF ß1, Smad3 and phosphorylated Smad3 using western blotting, and the mRNA levels of TGFß1 and several other target genes (cellular communication network factor 2, metalloproteinase inhibitor 1 and periostin) were measured by RT-qPCR. The proliferative and migratory ability of co-cultured HSFs was suppressed compared with controls, and no significant difference in apoptosis was observed between the two groups. The pro-fibrotic phenotype of co-cultured HSFs was inhibited due to a decline in expression levels of HS-associated genes and proteins. Furthermore, co-culture with UCMSCs inhibited the activation of the TGF ß1/Smad3 pathway. In conclusion, the present study indicated that UCMSCs may exert an anti-fibrotic action on HSFs in co-culture through inhibition of the TGF ß1/Smad3 pathway, which suggests a potential use for UCMSCs in HS therapy.

Nitric oxide: Is it the culprit for the continued expansion of keloids?

Keloids are characterized by excessive proliferation of fibroblasts and invasion of surrounding healthy skin. High levels of Nitric Oxide (NO) are thought to be the crucial factor within the micro-environment in promoting keloid formation. However, the effects and mechanisms of NO on the proliferation of Keloid Fibroblasts (KDFs) remain unclear. In this study, we investigated the effect of NO on KDFs proliferation by Sodium Nitroprusside (SNP), an NO donor. Our results show that SNP significantly enhanced KDFs proliferation. Moreover, with prolonged treatment with SNP after cell confluence, the growth of KDFs escape contact inhibition and experience significant pile up growth. Furthermore, PTIO, an NO scavenger, attenuated SNP-enhanced cell proliferation effectively. The mechanism involved in SNP-induced KDFs proliferation was soluble Guanylyl Cyclase (sGC) and cGMP independent. ODQ, a specific sGC inhibitor, failed to suppress SNP-enhanced KDFs proliferation. 8-Bromo-c GMP, a cell-permeable cGMP analogue, could not stimulate KDFs proliferation. Erk and Akt provide important signaling for cell growth. U0126 and LY294002, inhibitors of Erk and Akt respectively, block SNP-enhanced KDFs proliferation effectively. As expected, a Western blot showed that SNP up-regulated the phosphorylation levels of Erk and Akt. Moreover, it decreased the expression of p27, a cell cycle inhibitor. Our results reveal that SNP induced KDFs proliferation and loss contact inhibition led to pile up growth via activation of the Erk and Akt pathways, as well as a decreased expression of p27. Thus, we speculate that the pathological feature of continuous expansion in keloids is caused by NO-induced KDFs sustained growth.

Inhibition of Pathological Phenotype of Hypertrophic Scar Fibroblasts Via Coculture with Adipose-Derived Stem Cells.

Hypertrophic scar (HS) is a dermal fibroproliferative disease characterized by fibroblast over-proliferation, overproduction, and deposition of the extracellular matrix. Growing evidence demonstrated that adipose-derived stem cells (ASCs) secrete a plethora of trophic and antifibrotic factors, which suppress inflammation and ameliorate fibrosis of different tissues. However, few studies investigate their effect on repressing HS activity. This study evaluated the suppressing effect of ASCs on HS fibroblast bioactivity and the possible mechanism via a coculture model. HS-derived fibroblasts (HSFs) and ASCs were isolated from individual patients. HSFs or HSFs treated with transforming growth factor-ß1 (TGF-ß1) were cocultured with ASCs and the change of HSF cellular behaviors, such as cell proliferation, migration, contractility, and gene/protein expression of scar-related molecules, were evaluated by cell counting assay, cell cycle analysis, scratch wound assay, fibroblast-populated collagen lattice (FPCL) contractility assay, real-time quantitative polymerase chain reaction, ELISA, and western blotting assay. After 5 days of ASC coculture treatment, the expression levels of collagen I (Col 1), collagen III (Col 3), fibronectin (FN), TGF-ß1, interleukin-6 (IL-6), interleukin-8 (IL-8), connective tissue growth factor (CTGF), and alpha-smooth muscle actin (α-SMA) in HSFs decreased significantly while the expression levels of decorin (DCN) and MMP-1/TIMP-1 (matrix metalloproteinase/tissue inhibitor of MMP) ratio increased significantly. Besides, after 5 days of exogenous TGF-ß1 stimulation, the expression levels of Col 1, FN, TGF-ß1, IL-6, CTGF, and α-SMA in HSFs increased significantly. Impressively, all these increased gene expression levels were reversed by 5 days of ASCs coculture treatment. Additionally, the proliferation, migration, and contractility of HSFs were all significantly reduced by ASC coculture treatment. Furthermore, the protein levels of TGF-ß1 and intracellular signal pathway-related molecules, such as p-smad2, p-smad3, p-Stat3, and p-ERK, were downregulated significantly in HSFs after 5 days of ASCs coculture treatment. This study demonstrated that coculture of HSFs with ASCs not only inhibited proliferation, migration, and contractility of HSFs but also decreased the expression levels of HSF-related or TGF-ß1-induced molecules. Additionally, the antifibrotic effect on HSFs was likely mediated by the inhibition of multiple intracellular signaling. The results of this study suggest the therapeutic potential of ASCs for HS treatment, which is worth of further investigation.

Gallic acid attenuates TGF-ß1-stimulated collagen gel contraction via suppression of RhoA/Rho-kinase pathway in hypertrophic scar fibroblasts.

AIMS: To examine the effect and molecular mechanism of gallic acid (GA) on transforming growth factor-ß1 (TGF-ß1)-stimulated hypertrophic scar fibroblast (HSF) contraction. MATERIALS AND METHODS: A fibroblast-populated collagen lattice (FPCL) was developed to examine the effect of GA on TGF-ß1-enhanced HSF contraction. The changes in crucial factors related to cell contraction including α-smooth muscle actin (α-SMA), F-actin, and the phosphorylation level of myosin light chain (MLC) were evaluated using western blot and immunostaining. The activation and expression of RhoA/ROCK after the TGF-ß1 challenge and GA insult were evaluated using RhoA-G-LISA and RhoA-ELISA kit while the phosphorylation level of MYPT1 and the expression of ROCK1 and ROCK2 were examined by western blot, respectively. KEY FINDINGS: GA significantly suppressed TGF-ß1-stimulated HSF contraction in a dose- and time-dependent manner. Moreover, the TGF-ß1-enhanced α-SMA expression, F-actin formation, and MLC phosphorylation were obviously attenuated by GA. TGF-ß1 significantly stimulated RhoA activation but did not alter the expression of RhoA in the HSFs. However, both the activation and expression of RhoA decreased obviously with GA pretreatment followed by TGF-ß1 stimulation. Furthermore, GA inhibited ROCK activity but did not affect its expression after TGF-ß1 stimulation. SIGNIFICANCE: These results suggest that GA exhibited the potential to prevent HSF contraction after TGF-ß1 stimulation by down regulating the RhoA/ROCK signal cascade, followed by the inhibition of the expression of α-SMA, F-actin formation, and phosphorylation of MLC.

Preliminary study of the effect of abnormal savda munziq on TGF-ß1 and Smad7 expression in hypertrophic scar fibroblasts.

BACKGROUND: To study the effect of abnormal savda munziq (ASMq) on TGF-ß1 and Smad7 expression in hypertrophic scar fibroblasts (HSFs) and to preliminarily assess the function of abnormal savda munziq in hypertrophic scar formation at the molecular biology level. METHODS: HSFs were cultured in vitro. RT-PCR and Western-blot were used to investigate the influence of 48-h treatment with ASMq at different concentrations (0 mg/mL, 0.1 mg/mL, 0.4 mg/mL, and 0.7 mg/mL) on TGF-ß1 and Smad7 mRNA and protein expression levels. RESULTS: After 48-h treatment with ASMq, the expression of TGF-ß1 mRNA and protein gradually decreased in HSFs as the concentration increased. In contrary, Smad7 mRNA and protein expression were positively correlated with ASMq concentration. CONCLUSIONS: ASMq reduces TGF-ß1, increases Smad7 mRNA and protein expression through regulating TGFß-1/Smad signaling pathway, inhibiting HSFs proliferation and reducing extracellular collagen deposition.