Migration Of Keloid Fibroblasts Research Articles

SDC1 Influences Keloid Fibroblasts Migration and Invasion via Targeting Wnt/β-Catenin Signaling Pathway Mediated EMT.

Keloid is the result of abnormal wound healing, puzzled by the invasive growth and high recurrence rate attributed to its complex pathogenic mechanism. Syndecan1 (SDC1) contributes to regulating cell migration and invasion by activating epithelial-mesenchymal transition (EMT) in tumor and fibrotic disease. Herein, using western blot analysis, the authors assessed the role of SDC1 on EMT in keloid and its underlying mechanism. The authors found keloid fibroblasts exhibited a higher proportion of mesenchymal phenotypes, and SDC1 was significantly upregulated in keloid fibroblasts. Then, the authors transfected small interfering RNA targeting SDC1 in keloid fibroblasts and tested the abilities of cell migration and invasion, as well as the expression of EMT-related markers, including N-cadherin, vimentin, and E-cadherin. The results showed that the knockdown of SDC1 markedly suppressed the migration and invasion abilities of keloid fibroblasts and reduced the phenotypes of EMT by inhibiting Wnt/β-catenin signaling pathway. The authors' findings suggest that SDC1 may influences keloid fibroblasts migration and invasion through targeting Wnt/β-catenin signaling pathway mediated EMT, which supports its potential value as a therapeutic target for the treatment of keloid.

NEDD4L Inhibits the Proliferation and Migration of Keloid Fibroblasts by Regulating YY1 Ubiquitination-Mediated Glycolytic Metabolic Reprogramming.

Keloid scarring is a complex fibroproliferative disorder characterised by excessive fibroblast proliferation. Inhibition of cellular glycolysis effectively suppresses the proliferation of keloid fibroblasts (KFs). Neural precursor cell-expressed developmentally downregulated gene 4-like (NEDD4L), a ubiquitin ligase, regulates cell proliferation in different diseases. This study investigated the effects of NEDD4L on glucose metabolism, proliferation and migration in KFs. Primary KFs were isolated from keloid skin tissues obtained from patients with active-stage keloids. Cell transfection was used to upregulate or downregulate NEDD4L and Yin Yang 1 (YY1) in KFs. Protein expression was assessed by immunohistochemistry and western blotting. The viability, proliferative capacity and migration ability of KFs were evaluated using the MTT method and the EdU and wound healing assays, respectively. The regulatory effect of NEDD4L on YY1 ubiquitination was examined by coimmunoprecipitation. The interaction between YY1 and hexokinase 2 (HK2) was confirmed by a dual-luciferase reporter assay. NEDD4L was downregulated, whereas YY1 and HK2 were highly expressed in keloid tissues compared with normal skin. Overexpression of NEDD4L inhibited the proliferation and migration of KFs. NEDD4L promoted YY1 degradation in KFs by inducing its ubiquitination. Upregulation of YY1 induced glucose consumption and lactate production in KFs via the transcriptional regulation of HK2. Increased expression of YY1 reversed the reduced viability, proliferation, and migration of KFs overexpressing NEDD4L. YY1 also reversed the NEDD4L-induced inhibition of glucose consumption and lactate production in KFs. Additionally, an invivo study confirmed the inhibitory roles of NEDD4L overexpression and YY1 knockdown in keloid formation. NEDD4L suppressed the viability, proliferation and migration of KFs by regulating YY1 ubiquitination-mediated glycolysis through HK2. These findings suggest a novel regulatory axis, NEDD4L/YY1/HK2, that mediates glucose metabolism in keloid formation.

AURKA Activates FOXO3a to Form a Positive Feedback Loop in the Proliferation and Migration of Keloid Fibroblasts.

Objective: Keloids are benign fibroproliferative disorders with invasive growth exceeding the wound boundary. Aurora kinase A (AURKA) is a serine/threonine kinase highly expressed in various tumors, facilitating tumor growth and invasion. Currently, the role of AURKA in keloid remains unclear. Approach: Fibroblasts were isolated from keloid and normal skin samples. AURKA was evaluated by qPCR, Western blot, and immunohistochemistry. Transcriptome sequencing and dual-luciferase reporter assays were applied to figure out targets of AURKA. Following expression alteration and MLN8237 (an AURKA kinase inhibitor, AKI) treatment, phenotypical experiments were conducted to clarify biological functions of AURKA along with its target, and to probe into the clinical potential of AURKA inhibition. Results: AURKA was upregulated in keloid tissues and fibroblasts. Forkhead box O 3a (FOXO3a) was verified as a downstream of AURKA. Further experiments demonstrated that AURKA transactivated FOXO3a by binding to FOXO3a, while FOXO3a directly transactivated AURKA. Functionally, AURKA and FOXO3a cooperated in enhancing the proliferation and migration of keloid fibroblasts via protein kinase B (AKT) phosphorylation. Although MLN8237 weakened the proliferation and migration in keloid fibroblasts, the transactivation of AURKA on FOXO3a was independent of kinase activity. Innovation: This study reveals that AURKA and FOXO3a compose a transactivation loop in enhancing the proliferative and migrative properties of keloid fibroblasts, and proposes AURKA as a promising target. Conclusion: AURKA/FOXO3a loop promotes the proliferation and migration of keloid fibroblasts via AKT signaling. Despite the anti-keloid effects of AKIs, AURKA acts as a transcription factor independently of kinase activity, deepening our understanding on AKI insensitivity.

Targeting Nuclear Mechanics Mitigates the Fibroblast Invasiveness in Pathological Dermal Scars Induced by Matrix Stiffening.

Pathological dermal scars such as keloids present significant clinical challenges lacking effective treatment options. Given the distinctive feature of highly stiffened scar tissues, deciphering how matrix mechanics regulate pathological progression can inform new therapeutic strategies. Here, it is shown that pathological dermal scar keloid fibroblasts display unique metamorphoses to stiffened matrix. Compared to normal fibroblasts, keloid fibroblasts show high sensitivity to stiffness rather than biochemical stimulation, activating cytoskeletal-to-nuclear mechanosensing molecules. Notably, keloid fibroblasts on stiff matrices exhibit nuclear softening, concomitant with reduced lamin A/C expression, and disrupted anchoring of lamina-associated chromatin. This nuclear softening, combined with weak adhesion and high contractility, facilitates the invasive migration of keloid fibroblasts through confining matrices. Inhibiting lamin A/C-driven nuclear softening, via lamin A/Coverexpression or actin disruption, mitigates such invasiveness of keloid fibroblasts. These findings highlight the significance of the nuclear mechanics of keloid fibroblasts in scar pathogenesis and propose lamin A/C as a potential therapeutic target for managing pathological scars.

Indocyanine green based photodynamic therapy for keloids: Fundamental investigation and clinical improvement

BackgroundKeloid, a prevalent pathological skin lesion, presents significant challenges in terms of treatment efficacy. Photodynamic therapy (PDT), an increasingly popular adjuvant treatment, has shown significant potential in the management of various disorders, including cancer. However, the therapeutic potential of indocyanine green-mediated photodynamic therapy (ICG-PDT) for keloids has not yet been demonstrated. MethodsIn this study, we divided the experimental groups into control group, Photothermal Therapy group, Photodynamic Therapy group, and Combined Therapy group. The in vitro investigation aimed to optimize the clinical application of PDT for keloid treatment by elucidating its underlying mechanism. Subsequently, on this basis, we endeavored to manage a clinical case of keloid by employing surgical intervention in conjunction with modified ICG-PDT. ResultsOur investigation revealed an unexpected outcome that ICG-PDT maximally inhibited the cellular activity and migration of keloid fibroblasts only when photodynamic mechanism took effect. Additionally, the induction of autophagy and apoptosis, as well as the inhibition of collagen synthesis, were particularly evident in this experimental group. Furthermore, the above therapeutic effect could be achieved at remarkably low drug concentrations. Building upon the aforementioned experimental findings, we successfully optimized the treatment modality for the latest case and obtained a more favorable treatment outcome. ConclusionsThis study investigated the mechanism of ICG-PDT treatment and optimized the in vivo treatment regimen, demonstrating the significant therapeutic potential of ICG-PDT treatment in clinical keloid treatment.

Adipose-derived stem cells enriched with therapeutic mRNA TGF-β3 and IL-10 synergistically promote scar-less wound healing in preclinical models.

Skin wound healing often leads to scar formation, presenting physical and psychological challenges for patients. Advancements in messenger RNA (mRNA) modifications offer a potential solution for pulsatile cytokine delivery to create a favorable wound-healing microenvironment, thereby preventing cutaneous fibrosis. This study aimed to investigate the effectiveness of human adipose-derived stem cells (hADSCs) enriched with N 1-methylpseudouridine (m1ψ) modified transforming growth factor-β3 (TGF-β3) and interleukin-10 (IL-10) mRNA in promoting scar-free healing in preclinical models. The results demonstrated that the modified mRNA (modRNA)-loaded hADSCs efficiently and temporarily secreted TGF-β3 and IL-10 proteins. In a dorsal injury model, hADSCs loaded with modRNA TGF-β3 and IL-10 exhibited multidimensional therapeutic effects, including improved collagen deposition, extracellular matrix organization, and neovascularization. In vitro experiments confirmed the ability of these cells to markedly inhibit the proliferation and migration of keloid fibroblasts, and reverse the myofibroblast phenotype. Finally, collagen degradation mediated by matrix metalloproteinase upregulation was observed in an ex vivo keloid explant culture model. In conclusion, the synergistic effects of the modRNA TGF-β3, IL-10, and hADSCs hold promise for establishing a scar-free wound-healing microenvironment, representing a robust foundation for the management of wounds in populations susceptible to scar formation.

Hsa_circ_0000129 knockdown attenuates proliferation and migration in keloid fibroblasts by targeting miR-485–3p/SGMS2 pathway

BackgroundAberrant biofunction of circular RNAs (circRNAs) is potently implicated in keloid formation. However, their roles have been underinvestigated. Recent evidence has demonstrated the pro-tumor role of circ_0000129 in cancers, and yet its role in keloid remains elusive. MethodsRT-qPCR analysis and or western blotting of miR-485–3p, circ_0000129, and SGMS2 in keloid tissues and keloid fibroblasts was implemented. CCK8, EdU, scratch wound healing, and Transwell migration assays were perfomed to determine the keloid fibroblast proliferation and migration. Luciferase reporter and RIP assays were adopted to analyze the interaction among circ_0000129, miR-485–3p and SGMS2. ResultsIn keloid tissues and keloid fibroblasts, circ_0000129 and SGMS2 were amplified, although miR-485–3p expression was downregulated. Furthermore, siRNAs-targeting endogenous circ_0000129 resulted in proliferation and migration defect of keloid fibroblasts. MiR-485–3p was simultaneously recognized by circ_0000129 and SGMS2 3′UTR. Rescued functional assays also illustrated that miR-485–3p loss was beneficial to the proliferation and migration of keloid fibroblasts, and these promoting changes were nullified by accompanied silence circ_0000129 or SGMS2. ConclusionCirc_0000129 sponges miR-485–3p and releases expression of SGMS2 from the miR-485–3p suppression, promoting migration and proliferation of keloid fibroblasts, suggesting targeting circ_0000129/miR-485–3p/SGMS2 might be a promising strategy against keloid fibroblasts. Availability of data and materialAll data generated or analyzed during this study are included in this article.

PTB Regulates the Metabolic Pathways and Cell Function of Keloid Fibroblasts through Alternative Splicing of PKM

Keloids, benign fibroproliferative cutaneous lesions, are characterized by abnormal growth and reprogramming of the metabolism of keloid fibroblasts (KFb). However, the underlying mechanisms of this kind of metabolic abnormality have not been identified. Our study aimed to investigate the molecules involved in aerobic glycolysis and its exact regulatory mechanisms in KFb. We discovered that polypyrimidine tract binding (PTB) was significantly upregulated in keloid tissues. siRNA silencing of PTB decreased the mRNA levels and protein expression levels of key glycolytic enzymes and corrected the dysregulation of glucose uptake and lactate production. In addition, mechanistic studies demonstrated that PTB promoted a change from pyruvate kinase muscle 1 (PKM1) to PKM2, and silencing PKM2 substantially reduced the PTB-induced increase in the flow of glycolysis. Moreover, PTB and PKM2 could also regulate the key enzymes in the tricarboxylic acid (TCA) cycle. Assays of cell function demonstrated that PTB promoted the proliferation and migration of KFb in vitro, and this phenomenon could be interrupted by PKM2 silencing. In conclusion, our findings indicate that PTB regulates aerobic glycolysis and the cell functions of KFb via alternative splicing of PKM.

Bioinformatics analysis and identification of dysregulated POSTN in the pathogenesis of keloid.

Keloid is a benign fibro-proliferative dermal tumour formed by an abnormal scarring response to injury and characterised by excessive collagen accumulation and invasive growth. The pathophysiology of keloids is complex, and the treatment for keloids is still an unmet medical need. Here, we investigated the transcriptional gene that influences keloid development by comparing keloid, non-lesioned keloid skin and normal skin as well as keloid fibroblast and normal fibroblast (GSE83286, GSE92566, GSE44270). Based on the analysis, 146 up-regulated genes and 48 down-regulated genes were found in keloid tissue compared with normal skin and keloid no-lesioned skin. Eleven genes were further identified by overlapping the DEGs from keloid tissue described previously with DEGs in keloid fibroblast. The overlapped genes included PRR16, SFRP2, EDIL3, GERM1, POSTN, PDE3A, GALNT5, F2RL2, EYA4, ZFHX4, and AIM2. POSTN is the most crucial node in PPI network, which mainly correlate to collagen-related genes. Moreover, siRNA knockdown identified POSTN is a crucial regulatory gene that regulates keloid fibroblast migration and collagen I, collagen III expression level. In conclusion, our study identified 11 hub genes that play crucial role in keloid formation and provided insights for POSTN to be the therapeutic target for keloid through bioinformatic analysis of three datasets. Additionally, our results would support the development of future therapeutic strategies.

Long non-coding RNA HOXA11-AS contributes to the formation of keloid by relieving the inhibition of miR-182-5p on ZNF217

BackgroundLong non-coding RNA (lncRNA) dysregulation is demonstrated to be associated with disease progression. Mounting studies show that lncRNA promotes or inhibits the development of keloid. We aimed to disclose the role of homebox A11 antisense RNA (HOXA11-AS) in the formation of keloid. MethodsQuantitative real-time PCR (qPCR) was adopted for expression analysis of HOXA11-AS, miR-182-5p and zinc finger protein 217 (ZNF217) mRNA, and the expression of ZNF protein and marker proteins was detected by western blot. Cell proliferation, cell migration and cell apoptosis were investigated using CCK-8 assay, wound healing assay and flow cytometry assay, respectively. The potential interplay between miR-182-5p and HOXA11-AS or ZNF217 was verified by dual-luciferase reporter assay, RIP assay and pull-down assay. The role of HOXA11 in vivo was studied by establishing animal models. ResultsHOXA11-AS was highly expressed in tissues and fibroblasts of keloid. Deficiency of HOXA11-AS blocked the proliferation and migration of keloid fibroblasts and induced fibroblast apoptosis. HOXA11-AS directly combined to miR-182-5p whose downregulation reversed the effects of HOXA11-AS knockdown. ZNF217 was a target of miR-182-5p, and HOXA11-AS indirectly promoted ZNF217 expression by binding to miR-182-5p. MiR-182-5p enrichment also blocked keloid fibroblast proliferation, survival and migration, while further ZNF217 overexpression abolished these effects. HOXA11-AS knockdown also hindered the growth of keloid in mouse models. ConclusionHigh expression of HOXA11-AS promoted the formation and growth of keloid through the upregulation of ZNF217 by targeting miR-182-5p, and the inhibition of HOXA11-AS might be a novel strategy to prevent keloid development.

Circular RNA hsa_circ_0057452 facilitates keloid progression by targeting the microRNA-1225-3p/AF4/FMR2 family member 4 axis

ABSTRACT The circular RNA, hsa_circ_0057452, is highly expressed in keloids, but its specific mechanism of action remains unknown. The levels of hsa_circ_0057452, microRNA (miR)-1225-3p, and AF4/FMR2 family member 4 (AFF4) in keloid tissues and keloid fibroblasts (KFs) were determined using quantitative reverse transcription-polymerase chain reaction. Changes in KFs viability, proliferation, apoptosis, and migration were investigated using the cell counting kit-8, bromodeoxyuridine, flow cytometry, and Transwell assays. Luciferase, RNA immunoprecipitation, and RNA pull-down assays were performed to identify the binding relationship among hsa_circ_0057452, miR-1225-3p, and AFF4. We found that hsa_circ_0057452 and AFF4 expression levels were upregulated, whereas miR-1225-3p expression levels were downregulated in keloids. Knockdown of hsa_circ_0057452 or AFF4 suppressed the viability, proliferation, and migration of KFs and induced apoptosis, whereas hsa_circ_0057452 overexpression and miR-1225-3p knockdown showed the opposite trend. Furthermore, hsa_circ_0057452 affected the biological behavior of KFs by releasing AFF4 via sponging of miR-1225-3p. Therefore, our results show that hsa_circ_0057452 promotes keloid progression by targeting miR-1225-3p and regulating AFF4 levels.

HOXA11-AS facilitates the proliferation, cell cycle process and migration of keloid fibroblasts through sponging miR-188–5p to regulate VEGFA

BackgroundAbnormal expression of long non-coding RNA (lncRNA) has been proved to be related to the formation of keloid. Homeobox A11 antisense (HOXA11-AS) has been found to be a significantly upregulated lncRNA in keloid tissues. ObjectiveTo explore the mechanism of HOXA11-AS regulates keloid formation. MethodsPrimary fibroblasts were isolated from keloid tissues and normal skin tissues. The expression of HOXA11-AS, microRNA (miR)−188–5p and vascular endothelial growth factor A (VEGFA) was determined using quantitative real-time PCR (qRT-PCR). Cell counting kit 8 (CCK8) assay, EdU staining, flow cytometry and wound healing assay were performed to assess the proliferation, cell cycle process, apoptosis and migration of keloid fibroblasts. Importantly, some marker protein levels and VEGFA protein level were examined by western blot (WB) analysis. The interaction between miR-188–5p and HOXA11-AS or VEGFA was confirmed using dual-luciferase reporter assay, RNA pull-down assay and RIP assay. Animal experiments were performed to further confirm the role of HOXA11-AS in keloid growth. ResultsHOXA11-AS was markedly upregulated in keloid tissues and fibroblasts. Knockdown of HOXA11-AS repressed the proliferation, cell cycle process, migration and promoted the apoptosis of keloid fibroblasts. Further analysis suggested that HOXA11-AS could sponge miR-188–5p to positively regulate VEGFA. The inhibition of HOXA11-AS silencing on the biological functions of keloid fibroblasts could be reversed by miR-188–5p inhibitor. In addition, VEGFA overexpression also abolished the suppressive effect of miR-188–5p on the biological functions of keloid fibroblasts. Interferences of HOXA11-AS suppressed keloid growth in vivo. ConclusionHOXA11-AS might regulate the miR-188–5p/VEGFA axis to promote keloid formation.

Multitranscriptome analyses of keloid fibroblasts reveal the role of the HIF-1α/HOXC6/ERK axis in keloid development.

BackgroundA keloid is a disease of excessive fibrosis that is characterized by the aberrant proliferation of fibroblasts. However, the molecular mechanisms of fibroblasts during the development of keloids remain unclear. This study aims to identify new molecular targets that promote the proliferation and migration of keloid fibroblasts, providing new ideas for the prevention and treatment of keloids.MethodsWe utilized bioinformatics tools to analyze data from keloid fibroblasts (KFs) available in the Gene Expression Omnibus (GEO) database to identify the key genes involved in keloid development. Homeobox C6 (HOXC6) emerged as a hub gene in KFs from the GEO database was verified in keloid tissue samples and KFs using reverse transcription-quantitative polymerase chain reaction, western blot (WB) and immunohistochemistry. Subsequently, the effects of downregulated HOXC6 expression on the cellular behaviors of KFs were examined by performing Cell Counting Kit-8, flow cytometry, transwell migration and WB assays. Meanwhile, we performed transcriptome sequencing and gene set enrichment analysis (GSEA) to further explore HOXC6-related mechanisms and validated the signaling pathways by performing a series of experiments.Results HOXC6 was the top-ranking hub gene of KFs in microarray datasets from GEO and was upregulated in keloid tissue samples and KFs. Downregulation of HOXC6 inhibited proliferation, migration and extracellular matrix (ECM) accumulation and promoted KF apoptosis. GSEA predicted that the hypoxia signaling pathway was associated with HOXC6 in KFs. Transcriptome sequencing suggested that the extracellular regulated protein kinase (ERK) pathway was one of the downstream pathways of HOXC6 in KFs. Our experiments confirmed that hypoxia-inducible factor-1α (HIF-1α) upregulates HOXC6, contributing to KFs proliferation, migration, apoptosis inhibition and collagen accumulation through the ERK signaling pathway.ConclusionsOur findings first revealed that HOXC6 acts as an oncogenic driver in the molecular mechanisms of fibroblasts in keloids. The HIF-1α/HOXC6/ERK axis promotes proliferation, migration and ECM production by KFs, contributing to the progression of keloids. Taken together, HOXC6 may serve as a promising novel therapeutic target and new focus for research designed to understand the pathogenesis of keloids.

Warburg effect in keloids: A unique feature different from other types of scars

Keloid fibroblasts (KFs) undergo reprogramming of the metabolic phenotype from oxidative phosphorylation to the Warburg effect. However, more studies are needed to demonstrate whether there is a Warburg effect in KFs and to determine whether there is a similar phenomenon in other types of scars or in the proliferative stage of scars. In our study, the mRNA and protein expression of key glycolytic enzymes, glucose consumption and lactate production in KFs, normal skin fibroblasts (NFs), atrophic scar fibroblasts (ASFs), proliferative stage scar fibroblasts (PSSFs), and hypertrophic scar fibroblasts (HSFs) were detected. In addition, the effects of 2-deoxy-d-glucose (2-DG, a glycolysis inhibitor) on cell proliferation in KFs and NFs were studied. We found that the mRNA and protein expression of key glycolytic enzymes in KFs were significantly upregulated compared with those in NFs. Glucose consumption and lactate production in KFs were also higher than that in NFs. However, we found no similar phenomenon in ASFs, PSSFs, or HSFs. When treated with 2mmol/l 2-DG, the cell viability of KFs decreased more than that of NFs. What's more, treatment with increasing concentrations of 2-DG could inhibit cell viability and migration of KFs in a dose-dependent manner. In conclusion, the Warburg effect in KFs is a feature different from ASFs, PSSFs, or HSFs. Keloids are essentially different from other types of scars in terms of energy metabolism. This characteristic of KFs could provide new hope for the early diagnosis and treatment of keloids.

A selective small-molecule inhibitor of c-Met suppresses keloid fibroblast growth in vitro and in a mouse model

Keloids, tumor-like lesions that result from excessive scar formation, have no definitive treatment modality. Activation of c-mesenchymal-epithelial transition factor (c-Met) promotes cell proliferation and survival. Selective c-Met inhibitors, such as PHA-665752, may attenuate the activity of keloid fibroblasts and reduce keloid formation. Here, we aimed to evaluate the effect of PHA-665752, a second-generation selective small-molecule inhibitor of c-Met, on human keloid fibroblasts in vitro and in a mouse model. We performed in vitro cytotoxicity assays, scratch tests, western blotting, and immunofluorescence on human keloid fibroblasts. We also injected human fibroblasts into severe combined immunodeficient mice and measured the degree of nodule formation and skin histologic characteristics. We found that keloid fibroblast migration was inhibited by PHA-665752. Inhibitor treatment was also associated with lower expression of members of the hepatocyte growth factor/c-Met pathway, and lower fibroblast activity and collagen synthesis. In the in vivo experiments, PHA-665752—treated mice had lower nodule volumes and weights, accompanied by less inflammatory cell infiltration and collagen deposition, than those in control mice. These findings showed that although an in vivo model may not accurately represent the pathophysiology of human keloid development, PHA-665752 suppressed keloid fibroblast activity by inhibiting the c-Met—related tyrosine kinase pathway.

Downregulation of CR6-interacting factor 1 suppresses keloid fibroblast growth via the TGF-\u03b2/Smad signaling pathway

Keloids are a type of aberrant skin scarring characterized by excessive accumulation of collagen and extracellular matrix (ECM), arising from uncontrolled wound healing responses. While typically non-pathogenic, keloids are occasionally regarded as a form of benign tumor. CR6-interacting factor 1 (CRIF1) is a well-known CR6/GADD45-interacting protein, that has both nuclear and mitochondrial functions, and also exerts regulatory effects on cell growth and apoptosis. In this study, cell proliferation, cell migration, collagen production and TGF-β signaling was compared between normal fibroblasts (NFs) and keloid fibroblasts (KFs). Subsequently, the effects of CRIF1 deficiency were investigated in both NFs and KFs. Cell proliferation, cell migration, collagen production and protein expressions of TGF-β, phosphorylation of Smad2 and Smad3 were all found to be higher in KFs compared to NFs. CRIF1 deficiency in NFs and KFs inhibited cell proliferation, migration, and collagen production. In addition, phosphorylation of Smad2 and Smad3, which are transcription factors of collagen, was decreased. In contrast, mRNA expression levels of Smad7 and SMURF2, two important inhibitory proteins of Smad2/3, were increased, suggesting that CRIF1 may regulate collagen production. CRIF1 deficiency decreases the proliferation and migration of KFs, thereby inhibiting their overgrowth via the transforming growth factor-β (TGF-β)/Smad pathway. CRIF1 may therefore represent a potential therapeutic target in keloid pathogenesis.

Knockdown of FOXM1 inhibits activation of keloid fibroblasts and extracellular matrix production via inhibition of TGF-β1/Smad pathway

Keloid is characterized by overactive fibroblasts. Forkhead box M1 (FOXM1) is transcription factor that plays important roles in the progression of fibrosis. However, the role of FOXM1 in keloid has not been elucidated. In the present study, we examined the expression levels of FOXM1 in clinical keloid tissue specimens and primary keloid fibroblasts (KFs). The results showed that FOXM1 levels were significantly increased in both keloid tissues and KFs. To further investigate the biological functions of FOXM1, FOXM1 was knocked down in KFs by transfection with small interfering RNA targeting FOXM1 (si-FOXM1). Knockdown of FOXM1 inhibited transforming growth factor-β1 (TGF-β1)-induced cell proliferation and migration of KFs. Besides, the increased expressions of collagen (coll I), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) in TGF-β1-induced KFs were suppressed by si-FOXM1 transfection. Furthermore, TGF-β1-induced increase in p-Smad2 and p-Smad3 expressions was attenuated by FOXM1 knockdown. These data indicated that knockdown of FOXM1 inhibited TGF-β1-induced KFs activation and extracellular matrix (ECM) accumulation, which was attributed to the inhibition of TGF-β1/Smad pathway.

PARP1 Inhibition as a Novel Therapeutic Target for Keloid Disease.

Objective: Inactivation of poly(ADP-ribose) polymerase 1 (PARP1) has been found to have protective effect in several fibrotic diseases. But the effect is not studied yet in keloids. Herein, we evaluated the therapeutic effect of PARP1 inhibitor, rucaparib, for keloids.Approach: The protein expressions of PARP1 and smad3 were evaluated with western blotting in keloids and controls. The effect of rucaparib was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and migration assay. We further analyzed the effect of rucaparib on patient-derived keloid xenograft murine model.Results: The protein expressions of PARP1 and smad3 were significantly higher in keloid tissue. Rucaparib (20 μM) significantly suppressed the proliferation of keloid fibroblasts. Moreover, the combination of rucaparib (20 μM) and triamcinolone (50 μM) showed additive suppressive effect on keloid fibroblasts. Migration assay showed that rucaparib (10 μM) significantly suppressed the migration of keloid fibroblasts. Fibrosis markers in keloid fibroblasts significantly decreased after rucaparib treatment (20 μM). In patient-derived keloid xenograft model, rucaparib significantly reduced the size of keloid tissue.Innovation and Conclusion: The study data suggest PARP1 might be a novel therapeutic target for keloid disease. PARP1 inhibitor, rucaparib, might be a promising therapeutic drug for the treatment of keloid disease.

Increased Cthrc1 Activates Normal Fibroblasts and Suppresses Keloid Fibroblasts by Inhibiting TGF-β/Smad Signal Pathway and Modulating YAP Subcellular Location.

Keloid may induce severe impairment of life quality for the patients, although keloid is a cutaneous benign tumor. Collagen triple helix repeat containing protein 1 (Cthrc1) was identified as a novel gene that was originally found in adventitial fibroblasts after arterial injury. To address the role of Cthrc1 in keloid, the expression level of Cthrc1 was assessed in normal skin and keloid tissue, as well as in normal fibroblasts (NFs) and keloid fibroblasts (KFs) by using quantitative PCR, Western blotting and immunohistochemical analysis. The results showed that Cthrc1 was increased in keloid tissue and KFs as compared with normal skin and NFs. Meanwhile, CCK8 and Transwell assays found the cellular proliferation and migration of KFs were increased as compared with NFs. Further, to verify the function of Cthrc1 in NFs and KFs, we increased Cthrc1 expression by transfecting lentivirus (LV) vectors LV-Cthrc1. The cellular proliferation and migration, collagen synthesis and the influence on TGF-β and YAP signaling were tested. The cellular proliferation and migration were increased in NFs-Cthrc1 as compared with NFs-control. Meanwhile, YAP expression and nuclear-location was increased in NFs-Cthrc1. On the contrary, when Cthrc1 was overexpressed in KFs, the cellular migration was suppressed and YAP expression was reduced and transferred to cytoplasm in KFs-Cthrc1 as compared with KFs-control. But the expression level of collagen I was decreased and pSmad2/3 nucleus transfer was suppressed in both NFs-Cthrc1 and KFs-Cthrc1 by using Western blotting and immunofluorescence. Increased Cthrc1 activated NFs by promoting YAP nucleus translocation, whereas suppressed KFs by inhibiting YAP nucleus translocation. Enhanced Cthrc1 decreased collagen I in both NFs and KFs by inhibiting TGF-β/Smad pathway. In conclusion, Cthrc1 may play a role in the pathogenesis of keloid by inhibiting collagen synthesis and fibroblasts migration via suppressing TGF-β/Smad pathway and YAP nucleus translocation.

AMF siRNA treatment of keloid through inhibition signaling pathway of RhoA/ROCK1

A keloid (KD) is a benign dermal fibrotic tumor. Treatment of KDs is challenging and the recurrence rate is high; thus, there is an unmet need to explore new target sites and new treatment methods. As a tumor-associated cytokine, autocrine motility factor (AMF) can effectively stimulate the random and directional movement of cells. We first found that AMF was overexpressed in keloid fibroblasts (KFs) and the proliferation and migration of KFs were promoted by AMF stimulation. After treatment with Y-27632, RhoA kinase inhibitor, the proliferation and migration capacity of KFs declined significantly, and type I collagen protein, active RhoA and ROCK1 also were downregulated. In addition, a KD transplantation model was established under the skin of nude mice, with KD intramural injection AMF siRNA, we found that the weight of the KD was smaller than in the control group (P < 0.05), KD tissue sections stained by HE and Masson showed that fibers became loose and the blood vessels were visibly reduced. In conclusion, AMF siRNA is expected to be a novel strategy to treat KD by inhibiting signaling pathway of RhoA/ROCK1.