Hypertrophic Scar-derived Fibroblasts Research Articles

Transcription factor c-Maf drives macrophages to promote hypertrophic scar formation.

Hypertrophic scar (HS) is caused by the abnormal proliferation of fibroblasts and excessive deposition of extracellular matrix (ECM). Emerging evidence demonstrates that c-Maf positive M2 macrophages were mainly located in the hypertrophic scar tissues of proliferative phase. But whether c-Maf positive M2 macrophages can promote hypertrophic scar formation through modulating hypertrophic scar fibroblasts remains elusive. The aim of this study is to investigate the effects of c-Maf positive M2 macrophages on the biological behaviors and functions of hypertrophic scar fibroblasts and the potential mechanism. HE and Masson trichrome staining were used to examine the histological features of human hypertrophic scar. Immunofluorescence staining was employed to label and quantify the c-Maf+ /CD68+ M2 macrophages. CCK8, wound healing, and transwell assays were utilized to test the effects of c-Maf overexpressed M2 macrophages or the cell culture supernatants on the proliferation and migration of hypertrophic scar derived fibroblasts (HFBs) and normal skin derived fibroblasts (NFBs). Western blot and qPCR were harnessed to test the expressions of COL1, COL3, and α-SMA in the co-cultivated fibroblasts and TGF-β1 in the c-Maf overexpressed M2 macrophages. Increased number of c-Maf+ /CD68+ M2 macrophages were found in HS in contrast to the normal skin (NS). Elevated proliferation and migration were observed in the HFBs or NFBs co-cultured with c-Maf overexpressed macrophages or the cell culture supernatants. A higher mRNA and protein expressions of COL1, COL3, and α-SMA were recorded in the HFBs co-cultured with c-Maf overexpressed macrophages or treated with its culture supernatants. In addition, augmented mRNA and protein expressions of TGF-β1 were also investigated in the c-Maf overexpressed macrophages. c-Maf positive macrophages promote hypertrophic scar formation through regulating HFBs proliferation, migration, and ECM deposition via the secreted TGF-β1.

Heterogeneous response to TGF-β1/3 isoforms in fibroblasts of different origins: implications for wound healing and tumorigenesis

Identification of therapeutic targets for treating fibrotic diseases and cancer remains challenging. Our study aimed to investigate the effects of TGF-β1 and TGF-β3 on myofibroblast differentiation and extracellular matrix deposition in different types of fibroblasts, including normal/dermal, cancer-associated, and scar-derived fibroblasts. When comparing the phenotype and signaling pathways activation we observed extreme heterogeneity of studied markers across different fibroblast populations, even within those isolated from the same tissue. Specifically, the presence of myofibroblast and deposition of extracellular matrix were dependent on the origin of the fibroblasts and the type of treatment they received (TGF-β1 vs. TGF-β3). In parallel, we detected activation of canonical signaling (pSMAD2/3) across all studied fibroblasts, albeit to various extents. Treatment with TGF-β1 and TGF-β3 resulted in the activation of canonical and several non-canonical pathways, including AKT, ERK, and ROCK. Among studied cells, cancer-associated fibroblasts displayed the most heterogenic response to TGF-β1/3 treatments. In general, TGF-β1 demonstrated a more potent activation of signaling pathways compared to TGF-β3, whereas TGF-β3 exhibited rather an inhibitory effect in keloid- and hypertrophic scar-derived fibroblasts suggesting its clinical potential for scar treatment. In summary, our study has implications for comprehending the role of TGF-β signaling in fibroblast biology, fibrotic diseases, and cancer. Future research should focus on unraveling the mechanisms beyond differential fibroblast responses to TGF-β isomers considering inherent fibroblast heterogeneity.

Exosome from adipose-derived mesenchymal stem cells attenuates scar formation through microRNA-181a/SIRT1 axis

Pathological scarring is the greatest challenge after injury. Exosome from adipose-derived mesenchymal stem cells has been reported effective to improve hypertrophic scar. This study focused on the possible mechanisms during this process. Exosomes from adipose-derived mesenchymal stem cells were extracted first. Hypertrophic scar tissue and paired normal skin tissue were collected from patients. Mice skin incision model and fibroblasts model were established. TGF-β1 was used to stimulate fibroblasts to myofibroblasts transdifferentiation. It was found that exosomes injection could decrease collagen sediment after wound healing. During which, the expression of microRNA-181a decreased. Further, we found that expression of microRNA-181a in scar tissue was higher than in normal skin. Then hypertrophic scar-derived fibroblasts were used for in vitro study. It was found that similar to the use of exosomes, microRNA-181a inhibitor decreased the expression of collagen and α-SMA. While microRNA-181a mimics suppressed the effects of exosomes. During fibroblast to myofibroblast trans-differentiation, level of microRNA-181a well as levels of scar-related molecules also decreased with the use of exosomes and vice versa. SIRT1 was confirmed one of the downstream targets of microRNA-181a. Suppression of SIRT1 led to diminished effects of exosomes in hypertrophic scar derived fibroblasts. In mice skin incision model, injection of SIRT1 inhibitor led to increased collagen synthesis. In conclusion, exosomes from Adipose-derived mesenchymal stem cells are promising to antagonize scarring through the regulation of microRNA-181a/SIRT1 axis.

Lipopolysaccharide induces skin scarring through the TLR4/Myd88 inflammatory signaling pathway in dermal fibroblasts

Skin scarring is a frequent complication of the wound healing process. Bacterial contamination and prolonged inflammation in wounds are thought to play significant roles during scar formation, but little is known about their specific mechanisms of action. In this study, hypertrophic scar derived fibroblasts (HSFs) and paired normal skin derived fibroblasts (NSFs) were used to evaluate the effects of lipopolysaccharide (LPS) on inflammation-induced skin scarring and explore the inflammation-mediated mechanism of activity of LPS on dermal fibroblasts. LPS was found to significantly upregulate the expression of the proinflammatory molecules TLR4, Myd88, TRAF6, and p65, and the fibrosis-related proteins Col I, Col III, and α-SMA, in NSFs. Blocking Myd88 expression with T6167923 downregulated the expression of Col I, Col III, and α-SMA, whereas activating Myd88 expression with CL075 significantly upregulated their expression in LPS-treated NSFs. LPS was found to delay wound healing and increase skin scarring in cell and mouse models. These results showed that LPS could induce scar formation through the TLR4/Myd88 signaling pathway in dermal fibroblasts, suggesting that the downregulation of excessive inflammation in wound tissues inhibits skin scarring and improves scar appearance.

Salvianolic Acid B Attenuates Hypertrophic Scar Formation In Vivo and In Vitro.

Hypertrophic scars (HTSs) are a fibroproliferative disorder that occur following skin injuries. Salvianolic acid B (Sal-B) is an extractant from Salvia miltiorrhiza that has been reported to ameliorate fibrosis in multiple organs. However, the antifibrotic effect on HTSs remains unclear. This study aimed to determine the antifibrotic effect of Sal-B in vitro and in vivo. In vitro, hypertrophic scar-derived fibroblasts (HSFs) were isolated from human HTSs and cultured. HSFs were treated with (0, 10, 50, 100 μmol/L) Sal-B. Cell proliferation and migration were evaluated by EdU, wound healing, and transwell assays. The protein and mRNA levels of TGFβI, Smad2, Smad3, α-SMA, COL1, and COL3 were detected by Western blots and real-time PCR. In vivo, tension stretching devices were fixed on incisions for HTS formation. The induced scars were treated with 100 μL of Sal-B/PBS per day according to the concentration of the group and followed up for 7 or 14 days. The scar condition, collagen deposition, and α-SMA expression were analyzed by gross visual examination, H&E, Masson, picrosirius red staining, and immunofluorescence. In vitro, Sal-B inhibited HSF proliferation, migration, and downregulated the expression of TGFβI, Smad2, Smad3, α-SMA, COL1, and COL3 in HSFs. In vivo, 50 and 100 μmol/L Sal-B significantly reduced scar size in gross and cross-sectional observations, with decreased α-SMA expression and collagen deposition in the tension-induced HTS model. Our study demonstrated that Sal-B inhibits HSFs proliferation, migration, fibrotic marker expression and attenuates HTS formation in a tension-induced HTS model in vivo. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Transdermal delivery of Protocatechuic aldehyde using hyaluronic acid/gelatin-based microneedles for the prevention and treatment of hypertrophic scars

The formation of hypertrophic scar (HS) involves many pathological processes, such as reduced apoptosis in fibroblasts, excessive collagen deposition by fibroblasts, over-abundant angiogenesis, etc. The therapeutic effects of current treatments targeting one single pathological process are limited. Due to their diverse biological activities, natural products offer a potential solution to this issue. In this study reported herein, we investigated the effects of Protocatechuic aldehyde (PA) on both hypertrophic scar-derived fibroblasts (HSF) and vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs). Microneedles (MN) containing PA and hyaluronic acid (HA) or containing PA, HA, and gelatin were prepared by mixing PA stock solution with HA or HA/gelatin at a ratio of 1:10. The HS prevention and treatment outcomes of these HA-PA-MN and HA/gelatin-PA-MN were tested using a rabbit ear HS model. Our data indicate that PA induces apoptosis and reduces collagen deposition in HSF. In addition, PA attenuates VEGF-stimulated angiogenesis of HUVECs. Furthermore, HA-PA-MN or HA/gelatin-PA-MN are able to effectively penetrate the epidermis of the HS tissues and then quickly dissolve, enabling the fast release of PA directly into the dermis of the HS tissues. HA-PA-MN or HA/Gelatin-PA-MN have also been found to effectively prevent or alleviate HS in a rabbit ear HS model. In conclusion, this study demonstrates that PA can be used to prevent and treat HS by simultaneously regulating HSF and HUVECs, which offers a potential novel reagent for HS management.

Shikonin promotes hypertrophic scar repair by autophagy of hypertrophic scar-derived fibroblasts.

To investigate the Shikonin (SHI) induce autophagy of hypertrophic scar-derived fibroblasts (HSFs) and the mechanism of which in repairing hypertrophic scar. This study showed that SHI induced autophagy from HSFs and repaired skin scars through the AMPK/mTOR pathway. Alamar Blue and Sirius red were used to identify cell activity and collagen. Electron microscopy, label-free quantitative proteomic analysis, fluorescence and other methods were used to identify autophagy. The differences in the expression of autophagy and AMPK/mTOR pathway-related proteins after SHI treatment were quantitatively analyzed by Western blots. A quantitative real-time polymerase chain reaction assay was used to detect the expression of LC3, AMPK and ULK after adding chloroquine (CQ) autophagy inhibitor. After treatment with SHI for 24 hours, it was found that the viability of HSFs was significantly reduced, the protein expression of LC3-II/LC3-I and Beclin1 increased, while the protein expression of P62 decreased. The expression of phosphorylated AMPK increased and expression of phosphorylated mTOR decreased. After the use of CQ, the cell autophagy caused by SHI was blocked. The key genes LC3 and P62 were then reexamined by immunohistochemistry using a porcine full-thickness burn hypertrophic scar model, and the results verified that SHI could induce autophagy in vivo. These findings suggested that SHI promoted autophagy of HSFs cells, and the potential mechanism may be related to the AMPK/mTOR signal pathway, which provided new insights for the treatment of hypertrophic scars.

Platycodin D inhibits the proliferation and migration of hypertrophic scar-derived fibroblasts and promotes apoptosis through a caspase-dependent pathway

Abnormal fibroblast proliferation and excessive extracellular matrix (ECM) deposition lead to the formation of hypertrophic scars (HSs). However, there is no satisfactory method to inhibit the occurrence and development of HSs. In our study, platycodin D (PD), a natural compound extracted from Platycodon grandiflorus, inhibited HSs formation both in vitro and in vivo. First, qRT-PCR and Western blot were used to confirm PD dose-dependently downregulated the expression of Col I, Col III and α-SMA in human hypertrophic scar-derived fibroblasts (HSFs) (p < 0.05). Second, cck-8, transwell and wound healing assays verified PD suppressed the proliferation (p < 0.05) and migration of HSFs (p < 0.05), and inhibited the differentiation of HSFs into myofibroblasts. Moreover, PD-induced HSFs apoptosis were analyzed by flow cytometry and the apoptosis was activated through a caspase-dependent pathway. The rabbit ear scar model was used to further confirm the inhibitory effect of PD on collagen and α-SMA deposition. Finally, Western blot analysis showed that PD reduced TGF-β RI expression (p < 0.05) and affected matrix metalloproteinase 2 (MMP2) protein levels (p < 0.05). In conclusion, our study showed that PD inhibited the proliferation and migration of HSFs by inhibiting fibrosis-related molecules and promoting apoptosis via a caspase-dependent pathway. The TGF-β/Smad pathway also mediated the inhibition of HSFs proliferation and HSFs differentiation into myofibroblasts. Therefore, PD is a potential therapeutic agent for HSs and other fibrotic diseases.

Review on various combination therapies for the treatment of Hypertrophic scars and keloids

Keloids and hypertrophic scars are mild proliferative growth of dermal collagen that results in various physical, physiological and cosmetic concerns. Till the date the basis behind the formation of these scars has not been fully defined, it is assumed that keloids- and hypertrophic scar-derived fibroblasts produce increased amount of collagen per cell compared with normal fibroblast. Thus suppression of the fibroblast becomes the basic approach for therapeutic treatment of the wound responses. There is no permanent treatment for ablation of keloids and hypertrophic scars. A large number of trials have been taken on different drugs and different drug delivery systems and all of them are having variable results. Trials such as laser treatment, surgical procedures, intralesional injections of steroids have been taken for ablation of these scars. But most of these treatments are not permanent (intralesional injection), time taking (surgical procedures) and have a large probability of recurrence. This review article summarizes the information on various therapies for the treatment of keloids and hypertrophic scars as well as their pros and cons Keywords: Keloids, Hypertrophic Scars, Intralesional Corticosteroid Injection, Laser therapy, Surgical excision, Radio therapy, Cryotherapy

The role of altered fatty acid in pathological scars and their dermal fibroblasts

PurposeThe proposed pathological mechanism for scar formation is controversial, and increased attention has been paid to the fatty acids (FAs) in the formation of pathological scars. Notably, FAs are known to be important in inflammation and mechanotransduction, which is closely related to scar formation. Therefore, it is necessary to clarify the roles of FA in scar formation. MethodsHypertrophic scar and keloid formed for more than a year and without other treatment, as well as normal skin samples were obtained from patients who underwent plastic surgery. Finally, keloids (n = 10), hypertrophic scars (n = 10), and normal skin samples (n = 10) were collected under informed consent. Primary dermal fibroblasts were isolated and cultured. The amount and variety of FAs were detected by lipid chromatography-mass spectrometry. Immunohistochemistry, real-time PCR, and western blotting were used to verify the expression of sterol regulatory element-binding protein-1 (SREBP1) and fatty acid synthase (FASN) in the samples and their fibroblasts. Student's t-test, ANOVA, and orthogonal partial least square discriminant analysis were performed for statistical analysis (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). ResultsCompared with full-thickness normal skin, there were 27 differential FAs in keloids and 15 differential FAs in hypertrophic scars (∗p < 0.05 and variable influence on projection >1.0). The expression of SREBP1 and FASN was lower in pathological scars both at mRNA and protein levels (all ∗p < 0.05). However, the mRNA levels of SREBP1 (∗∗∗p = 0.0002) and FASN (∗∗∗p = 0.0021) in keloid-derived fibroblasts were higher than that in normal skin fibroblasts (NFBs), while the expression in hypertrophic scar-derived fibroblasts was lower than that in NFBs (both ∗p < 0.05). Whereas there was no significant difference in FASN protein expression between keloid-derived fibroblasts and NFBs (p > 0.05). ConclusionFAs involved in pathological scars are abnormally changed in scar formation. Thus, fatty acid-derived inflammation and de novo synthesis pathway of FA may play a key role in the formation of pathological scars.

Dramatic Effect of Botulinum Toxin Type A on Hypertrophic Scar: A Promising Therapeutic Drug and Its Mechanism Through the SP-NK1R Pathway in Cutaneous Neurogenic Inflammation.

BackgroundHypertrophic scar formation may be related to cutaneous neurogenic inflammation (CNI) through the substance P-neurokinin 1 receptor (SP-NK1R) signaling pathway. As a widely used drug in aesthetic clinical work, botulinum toxin type A (BTX-A) has a therapeutic effect on scars, but the actual mechanism remains unclear. This study aimed to clarify the potential mechanism by which BTX-A inhibits CNI in hypertrophic scars both in vitro and in vivo.MethodsTissue samples were obtained from surgical excisions. Immunohistological analysis was used to locate SP in human hypertrophic scars and normal skin. RT-PCR and western blot analysis were used to evaluate the expression of collagens after SP/BTX-A treatment. A rabbit ear scar model was used to explore the in vivo effect of BTX-A on scar treatment.ResultsSP and NK-1R were overexpressed in hypertrophic scars compared to normal skin tissues. Collagen secretion of hypertrophic scar-derived fibroblasts increased with increasing doses of SP. However, BTX-A may downregulate collagen expression through SP-NK1R pathway with or without the presence of SP inducing agent capsaicin. Meanwhile, SP inhibited the expression of NK-1R, and this inhibition was blocked by pretreatment with BTX-A. In vivo, intralesional BTX-A injection can also reduce the volume of scars and inhibit collagen secretion. Capsaicin may cause more severe scar manifestations, while the therapeutic effect of BTX-A remains.ConclusionOur research confirms that CNI stimulates fibroblasts during scar formation, while BTX-A can reduce collagen secretion by inhibiting the SP-NK1R signaling pathway, thus identifying a novel therapeutic target for this benign solid skin tumor.

Epidermal growth factor effect on lipopolysaccharide-induced inflammation in fibroblasts derived from diabetic foot ulcer.

BackgroundDiabetic foot ulcers (DFU) are characterised by high levels of inflammatory mediators, resulting from sustained hyperglycaemic insult and the local microbial biofilm. The intralesional administration of epidermal growth factor (EGF) has emerged as an effective treatment that stimulates granulation and closure of DFU, reducing the risk of amputation. Within the wound, fibroblasts play key roles during the healing process, promoting granulation and contraction. The aim of the present study was to examine the anti-inflammatory effect of EGF in DFU-derived fibroblasts, challenged with lipopolysaccharide (LPS), under hyperglycaemic conditions, recreating in vitro what happens in a clinical scenario.MethodsHealthy skin (HS) and DFU granulation tissue biopsies were used to isolate primary fibroblasts. The effect of LPS on cell proliferation was analysed. Transcriptional expression of toll-like receptor (TLR) pathway mediators (TLR4, TLR2, CD14, MYD88 and NFKB) and pro-inflammatory cytokines (TNF, IL-6 and IL-1B) were measured by semi-quantitative polymerase chain reaction (qPCR), in cells treated with appropriate concentrations of LPS, EGF and their combination. IL-6 protein concentration was quantified by ELISA.ResultsLPS stimulated proliferation of HS-derived fibroblasts, while inhibiting the proliferation of cells derived from DFU at the highest assayed concentration of 1 µg/mL. Regarding the TLR signalling pathway, LPS increased messenger RNA levels of mediators and pro-inflammatory genes, while EGF, alone or in the presence of LPS, downregulated them, except for IL-1B.ConclusionThe results suggest that EGF might elicit an anti-inflammatory response in LPS-challenged fibroblasts, even in a hyperglycaemic milieu. Collectively, our findings contribute to explain newly observed effects of EGF in the clinical arena.Lay SummaryIn this research article, we analyse the putative anti-inflammatory effect of epidermal growth factor (EGF) on fibroblast isolated from diabetic foot ulcer (DFU) granulation tissue. To induce the inflammatory response, the cells were treated with lipopolysaccharide (LPS), simulating the gram-negative bacterial infection that takes place in the wounds of diabetic patients. We studied the expression of genes involved in bacterial recognition receptors signalling pathway and those that code for different pro-inflammatory cytokines.We obtained primary fibroblasts from biopsies of a neuropathic diabetic ulcer and from healthy skin, the former was used as the control. Cells were isolated and grown in high glucose Dulbecco’s Modified Eagle Medium (DMEM) culture medium, to simulate the hyperglycaemic insult. The effect of increasing concentrations of LPS on cell proliferation was analysed. Relative transcriptional expression of genes in the study was quantified by quantitative polymerase chain reaction (qPCR) in cells treated with LPS, EGF or a combination. Untreated cells served to normalise the expression.In the present study, we demonstrated that EGF modulated the primary immune response by reducing the activation of pathogen-recognition receptors and common genes involved in these signalling pathways, even in hyperglycaemic conditions. This effect translated in a decreased expression of pro-inflammatory cytokines. These results contribute to explain our previous observations about the reduction of circulating levels of inflammatory cytokines after local administration of human recombinant EGF in DFU. Further molecular studies should be carried out to fully understand the biological mechanisms elicited by EGF in this clinical scenario.

USP15 Enhances the Proliferation, Migration, and Collagen Deposition of Hypertrophic Scar-Derived Fibroblasts by Deubiquitinating TGF-βR1 In Vitro.

Background:Hypertrophic scar is a fibroproliferative disorder caused by skin injury. The incidence of hypertrophic scar following trauma or burns is 40 to 70 percent or 70 percent, respectively. It has been shown that transforming growth factor (TGF) β1/Smad signaling plays a crucial role in hypertrophic scar, and that USP15 can regulate the activity of TGFβ1/Smad signaling to affect the progression of the disease. However, the underlying mechanism of USP15 in hypertrophic scar remains unclear. The authors hypothesized that USP15 was up-regulated and enhanced the proliferation, migration, invasion, and collagen deposition of hypertrophic scar–derived fibroblasts by deubiquitinating TGF-β receptor I (TβRI) in vitro.Methods:Fibroblasts were isolated from human hypertrophic scars in vitro. The knockdown and overexpression of USP15 in hypertrophic scar–derived fibroblasts were performed using lentivirus infection. The effect of USP15 on hypertrophic scar–derived fibroblast proliferation, migration, and invasion, and the expression of TβRI, Smad2, Smad3, α-SMA, COL1, and COL3, were detected by Cell Counting Kit-8, scratch, invasion, quantitative real-time polymerase chain reaction, and Western blot assays. The interaction between USP15 and TβRI was detected by co-immunoprecipitation and ubiquitination assays.Results:The authors demonstrated that USP15 knockdown significantly inhibited the proliferation, migration, and invasion of hypertrophic scar–derived fibroblasts in vitro and down-regulated the expression of TβRI, Smad2, Smad3, α-SMA, COL1, and COL3; in addition, USP15 overexpression showed the opposite trends (p < 0.05). Co-immunoprecipitation and ubiquitination assays revealed that USP15 interacted with TβRI and deubiquitinated TβRI.Conclusion:USP15 enhances the proliferation, migration, invasion, and collagen deposition of hypertrophic scar–derived fibroblasts by deubiquitinating TβRI in vitro.

Knockdown of lysyl oxidase like 1 inhibits the proliferation and pro-fibrotic effects of transforming growth factor-β1-induced hypertrophic scar fibroblasts.

The excessive healing response during wound repair can result in hypertrophic scars (HS). Lysyl oxidase like 1 (LOXL1) has been reported to be associated with fibrosis via targeting transforming growth factor β1 (TGF-β1) signaling. This study aimed to investigate the effect of LOXL1 on HS formation. The expression of LOXL1 in HS tissues and TGF-β1-induced HS-derived fibroblasts (HSFs) was detected via reverse transcription quantitative PCR and Western blot. LOXL1 was silenced in HSFs using transfection with short hairpin RNA (shRNA), then wound healing process including cell proliferation, cell cycle distribution, migration, and extracellular matrix (ECM) deposition along with Smad expression were measured by cell counting kit-8, EdU staining, flow cytometry, transwell, immunofluorescence, and Western blot assays. LOXL1 was upregulated in HS tissues and TGF-β1-induced HSFs. Knockdown of LOXL1 inhibited proliferation and migration but promoted cell cycle G0/G1 phase arrest in TGF-β1-induced HSFs; it increased expression of cyclin D1, CDK4, MMP2, MMP9, COL1A1, COL1A2, fibronectin, COL3A1, α-SMA, but decreased expression of p27, and the phosphorylation of Smad2 and Smad3 caused by TGF-β1 were also blocked by LOXL1 silencing. Silence of LOXL1 could effectively inhibit TGF-β1-induced proliferation, migration, and ECM deposition in HSFs via inactivating Smad pathway. Targeting LOXL1 may have future therapeutic implications for HS treatment.

Corilagin alleviates hypertrophic scars via inhibiting the transforming growth factor (TGF)-β/Smad signal pathway

AimsExploring the effects of corilagin on hypertrophic scar (HS) and its underlying mechanisms. Main methodsHuman HS-derived fibroblasts (HSFs) were isolated and treated with corilagin. To investigate the effects of corilagin on HSFs, quantitative real time polymerase chain reaction (qRT-PCR), western blotting, wound healing, and immunofluorescence assays were performed. These effects were confirmed in a rabbit ear scar model by histological and immunohistochemical studies. Lastly, western blot assay was performed to detect the protein levels of several components of the transforming growth factor (TGF)-β/Smad signaling pathway, as well as the protein levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs). Key findingsCorilagin showed multiple effects on HSFs, including does-dependent inhibition of collagen production, cell proliferation, and migration, besides suppression of the activation of HSFs. Moreover, corilagin suppressed HS formation and collagen deposition in a rabbit ear scar model. Corilagin also inhibited fibroblast proliferation and α-smooth muscle actin (α-SMA) expression in vivo. Finally, western blot analysis revealed that corilagin downregulated the protein levels of TGF-β1 and TGF-β receptor type I (TGFβRI), thus lowering the level of p-smad2/3, also affected the protein levels of MMPs and TIMP1. SignificanceCorilagin could be a potential agent for HS treatment through the inhibition of extracellular matrix (ECM) deposition and multiple functions of fibroblasts.

Exosomes derived from human adipose mesenchymal stem cells attenuate hypertrophic scar fibrosis by miR-192-5p/IL-17RA/Smad axis

BackgroundHypertrophic scar (HS) is a fibro-proliferative disorder of dermis after burn or trauma and usually leads to esthetic disfiguration and functionary impairment for patients. Emerging evidences demonstrated ADSC-Exo could alleviate the visceral fibrosis, but little attention had been paid to its role in skin fibrosis. In the study, we would explore the effect of ADSC-Exo on HS and investigated the exact mechanism underlying the properties.MethodsADSC-Exo were isolated, identified, and internalized by HS-derived fibroblasts (HSFs). The effect of ADSC-Exo on the proliferation and migration of HSFs were detected by flow cytometry and Ki67 immunofluorescence staining, or scratch and trans-wells assays, respectively. RT-PCR, immunoblotting, immunofluorescence, and immunohistochemistry staining were used to evaluate the expression of IL-17RA, Col1, Col3, α-SMA, SIP1, and p-Smad2/p-Smad3 in HSFs stimulated with ADSC-Exo, miR-192-5p mimics, or inhibitors, IL-17RA siRNA and their negative controls. Digital morphology, H&E, Masson’s trichrome staining, and immunohistochemistry staining were performed to measure the effect of ADSC-Exo and Lv-IL-17RA shRNA on excisional wound of BALB/c mice.ResultsThe verified ADSC-Exo effectively inhibited the proliferation and migration of HSFs, decreased the expression of Col1, Col3, α-SMA, IL-17RA, and p-Smad2/p-Smad3 and increased the levels of SIP1 in HSFs. Besides, the mice in ADSC-Exo-treated group demonstrated faster wound healing and less collagen deposition. Furthermore, miR-192-5p was highly expressed in ADSC-Exo and ADSC-Exosomal miR-192-5p ameliorated hypertrophic scar fibrosis. Meanwhile, miR-192-5p targeted the expression of IL-17RA to decrease the pro-fibrotic proteins levels. Moreover, IL-17RA was overexpressed in HS and HSFs, and knockdown IL-17RA alleviated the expression of Col1, Col3, α-SMA, and p-Smad2/p-Smad3 and increased the expression of SIP1 in HSFs. Most importantly, IL-17RA silence also facilitated wound healing, attenuated collagen production, and modulated Smad pathway in HSFs.ConclusionsThis study illustrated ADSC-Exo attenuated the deposition of collagen, the trans-differentiation of fibroblasts-to-myofibroblasts, and the formation of hypertrophic scar by in vitro and in vivo experiments. ADSC-Exosomal miR-192-5p targeted IL-17RA to regulate Smad pathway in hypertrophic scar fibrosis. ADSC-Exo could be a promising therapeutic strategy for clinical treatment of hypertrophic scar and the anti-fibrotic properties could be achieved by miR-192-5p/IL-17RA/Smad axis.

IL-10 alleviates lipopolysaccharide-induced skin scarring via IL-10R/STAT3 axis regulating TLR4/NF-κB pathway in dermal fibroblasts.

Hypertrophic scar (HS) is a severe fibrotic skin disease. It has always been a major problem in clinical treatment, mainly because its pathogenesis has not been well understood. The roles of bacterial contamination and prolonged wound inflammation were considered significant. IL‐10 is a potent anti‐inflammatory cytokine and plays a pivotal role in wound healing and scar formation. Here, we investigate whether IL‐10 alleviates lipopolysaccharide (LPS)‐induced inflammatory response and skin scarring and explore the possible mechanism of scar formation. Our results showed that the expression of TLR4 and pp65 was higher in HS and HS‐derived fibroblasts (HSFs) than their counterpart normal skin (NS) and NS‐derived fibroblasts (NSFs). LPS could up‐regulate the expression of TLR4, pp65, Col I, Col III and α‐SMA in NSFs, but IL‐10 could down‐regulate their expression in both HSFs and LPS‐induced NSFs. Blocking IL‐10 receptor (IL‐10R) or the phosphorylation of STAT3, their expression was up‐regulated. In addition, in vitro and in vivo models results showed that IL‐10 could alleviate LPS‐induced fibroblast‐populated collagen lattice (FPCL) contraction and scar formation. Therefore, IL‐10 alleviates LPS‐induced skin scarring via IL‐10R/STAT3 axis regulating TLR4/NF‐κB pathway in dermal fibroblasts by reducing ECM proteins deposition and the conversion of fibroblasts to myofibroblasts. Our results indicate that IL‐10 can alleviate the LPS‐induced harmful effect on wound healing, reduce scar contracture, scar formation and skin fibrosis. Therefore, the down‐regulation of inflammation may lead to a suitable scar outcome and be a better option for improving scar quality.

Resveratrol inhibits hypertrophic scars formation by activating autophagy via the miR-4654/Rheb axis.

Hypertrophic scars (HSs) are a type of pathological scar which are induced by surgery, burn injuries or trauma during the healing process. Due to the high recurrence rates and strong invasive properties, HSs have become a major clinical issue. Resveratrol has been identified as a potential agent to suppress scar formation; however, the underlying mechanism of action remains unclear. Therefore, the present study aimed to investigate the effect of resveratrol on HS-derived fibroblasts (HSFBs) in vitro. MTT assay was performed to evaluate cell viability following the resveratrol treatment. Western blot and RT-qPCR analysis was used to identify the expression levels and the relationship among autophagic markers, miR-4654 and resveratrol treatment. Finally, GFP-LC3 stable HSFBs cells were generated to further assess the effect of resveratrol. The results revealed that resveratrol significantly induced cell death in a dose-dependent manner and induced autophagy by downregulating the expression levels of Rheb in HSFBs. Notably, microRNA-4654 (miR-4654) was significantly decreased in the HSFBs and re-upregulated by resveratrol treatment dose-dependently. Through the bioinformatic analysis and luciferase assay, miR-4654 was identified to directly target Rheb. Transfection studies showed that miR-4654 negative correlated with Rheb expression, suggesting that the autophagic process may be altered by the miR-4654/Rheb axis under the control of resveratrol. In conclusion, the results of the present study suggested that resveratrol may promote autophagy by upregulating miR-4654, which in turn may suppress Rheb expression via directly binding to the 3′-untranslated region of Rheb. These findings provided a novel insight into the development of potential therapeutic targets for HSs.

Hepatocyte Growth Factor Secreted from Human Adipose-Derived Stem Cells Inhibits Fibrosis in Hypertrophic Scar Fibroblasts.

To study the effect of Adipose-derived stem cells (ADSCs) on fibrosis of hypertrophic scar-derived fibroblasts (HSFs) and its concrete mechanism. ADSCs have been reported to reduce collagen production and fibroblast proliferation in co-culture experiments. Conditioned medium from adipose-derived stem cells (ADSCs-CM) has successfully inhibited fibrosis by decreasing the expression of collagen type І (Col1) and α-smooth muscle actin (α-SMA) in rabbit ear scar models. Hepatocyte growth factor (HGF), the primary growth factor in ADSCs-CM, has been shown to reverse fibrosis in various fibrotic diseases. To test the hypothesis that ADSCs inhibit fibrosis of HSFs through the secretion of HGF. HSFs were treated with DMEM containing 0%, 10%, 50% and 100% concentration of ADSCs-CM. The effect of ADSCs-CM on the viability was determined by cell viability assay, and the collagen production in HSFs was examined by Sirius red staining. Expression and secretion of fibrosis and degradation proteins were detected separately. After measuring the concentration of HGF in ADSCs-CM, the same number of HSFs were treated with 50% ADSCs-CM or HGF. HGF activity in ADSCs-CM was neutralized with a goat anti-human HGF antibody. The results demonstrated that ADSCs-CM dose-dependently decreased cell viability, expression of fibrosis molecules, and tissue inhibitor of metalloproteinases-1 (TIMP-1), and significantly increased matrix metalloproteinase-1 (MMP-1) expression in HSFs. Collagen production and the ratio of collagen type І and type III (Col1/Col3) were also suppressed by ADSCs-CM in a dose-dependent manner. When HSFs were cultured with either 50% ADSCs-CM or HGF (1 ng/ml), a similar trend was observed in gene expression and protein secretion. Adding an HGF antibody to both groups returned protein expression and secretion to basal levels but did not significantly affect the fibrosis factors in the control group. Our findings revealed that adipose-derived stem cell-secreted HGF effectively inhibits fibrosis-related factors and regulates extracellular matrix (ECM) remodeling in hypertrophic scar fibroblasts.

Tetramethylpyrazine Induces Apoptosis and Inhibits Proliferation of Hypertrophic Scar-Derived Fibroblasts via Inhibiting the Phosphorylation of AKT.

Hypertrophic scar (HS) is a serious fibrotic skin disease and often considered as a kind of benign skin tumor. Tetramethylpyrazine (TMP), the main chemical composition of the traditional Chinese medicine Chuanxiong Rhizoma, has shown significant clinical benefits in the treatment of fibrosis disease and tumor, while the role in HS and the concrete mechanisms remain elusive. Herein, the protective effects of TMP in the treatment of HS was investigated and the results showed that the protein expression levels of type I collagen (Col I), type III collagen (Col III), and α-smooth muscle actin (α-SMA) were all inhibited remarkably after addition of TMP in HS-derived fibroblasts (HFs). Moreover, TMP also suppressed fibroblast proliferative and induced cell apoptosis. The protein expression levels of Caspase-3 and Bcl-2 were all decreased comparing with the control group while proapoptotic proteins Bax and Cleaved Caspase-3 were increased. In addition, TMP treatment markedly reduced the phosphorylation levels of AKT. Taken together, our investigations demonstrated that TMP could down-regulate the expression of fibrosis-related molecules, inhibit scar fibroblast proliferation and activate cell apoptosis, during which AKT pathway was involved. Thus, this study shed more light on the pharmacological mechanisms of TMP, and provided a novel therapeutic alternative for prevention and treatment of HS.