Keloid Fibroblasts Research Articles

Isorhamnetin attenuates the proliferation, invasion, migration and fibrosis of keloid fibroblasts by targeting S1PR1.

Isorhamnetin (IH) is a type of flavonoid with multiple biological activities, including cardioprotective, antitumor, anti-inflammatory and antioxidant activities. However, the role and potential mechanism of IH in keloids are still not completely understood. The aim of the present study was to explore how IH affects keloid progression. In the present study, cell proliferation was evaluated using the Cell Counting Kit-8 assay and immunofluorescence. Wound healing and Transwell assays were performed to assess cell migration and invasion, respectively. The expression levels of fibrosis-related proteins were measured using western blot analysis and immunofluorescence. In addition, the binding between IH and sphingosine-1-phosphate receptor-1 (S1PR1) was analyzed using the TargetNet database, and molecular docking was performed using Zinc, PubChem, AutoDockTools 1.5.6 and Discovery Studio 4.5 software. The expression levels of proteins in the PI3K/AKT pathway were detected by western blot analysis. The results showed that IH inhibited the proliferation, invasion, migration and fibrosis of keloid fibroblasts. The binding of IH and S1PR1 was verified and molecular docking was performed. Notably, IH significantly suppressed the expression levels of S1PR1, phosphorylated (p)-PI3K and p-AKT. Furthermore, the silencing of S1PR1 suppressed the cell proliferation, migration, invasion and fibrosis of keloid fibroblasts, as well as the expression of the PI3K/AKT pathway proteins. Conversely, S1PR1 upregulation reversed the inhibitory effects of IH on keloid fibroblast proliferation, migration, invasion and fibrosis. In conclusion, the results revealed that IH suppressed the proliferation, migration, invasion and fibrosis of keloid fibroblasts by targeting the S1PR1/PI3K/AKT pathway, suggesting that IH may be a promising drug for the treatment of keloids.

Evidence of inter- and intra-keloid heterogeneity through analysis of dermal fibroblasts: A new insight in deciphering keloid physiopathology.

Keloid scars are hypertrophic and proliferating pathological scars extending beyond the initial lesion and without tendency to regression. Usually, keloids are considered and treated as a single entity but clinical observations suggest heterogeneity in keloid morphologies with distinction of superficial/extensive and nodular entities. Within a keloid, heterogeneity could also be detected between superficial and deep dermis or centre and periphery. Focusing on fibroblasts as main actors of keloid formation, we aimed at evaluating intra- and inter-keloid fibroblast heterogeneity by analysing their gene expression and functional capacities (proliferation, migration, traction forces), in order to improve our understanding of keloid pathogenesis. Fibroblasts were obtained from centre, periphery, papillary and reticular dermis from extensive or nodular keloids and were compared to control fibroblasts from healthy skin. Transcriptional profiling of fibroblasts identified a total of 834 differentially expressed genes between nodular and extensive keloids. Quantification of ECM-associated gene expression by RT-qPCR brought evidence that central reticular fibroblasts of nodular keloids are the population which synthesize higher levels of mature collagens, TGFβ, HIF1α and αSMA as compared to control skin, suggesting that this central deep region is the nucleus of ECM production with a centrifuge extension in keloids. Although no significant variations were found for basal proliferation, migration of peripheral fibroblasts from extensive keloids was higher than that of central ones and from nodular cells. Moreover, these peripheral fibroblasts from extensive keloids exhibited higher traction forces than central cells, control fibroblasts and nodular ones. Altogether, studying fibroblast features demonstrate keloid heterogeneity, leading to a better understanding of keloid pathophysiology and treatment adaptation.

CircSLC8A1 targets miR-181a-5p/HIF1AN pathway to inhibit the growth, migration and extracellular matrix deposition of human keloid fibroblasts.

Circular RNAs (circRNAs) are identified as important regulators in human diseases, including keloid. The purpose of this study is to reveal the role and molecular mechanism of circSLC8A1 in keloid formation. Expression of circSLC8A1, microRNA (miR)-181a-5p, and hypoxia inducible factor 1 alpha inhibitor (HIF1AN) were detected by quantitative real-time PCR. Protein expression of extracellular matrix (ECM) deposition markers and HIF1AN was detected by western blot analysis. Furthermore, the interaction between miR-181a-5p and circSLC8A1 or HIF1AN was confirmed by dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Expression of circSLC8A1 was downregulated in keloid tissues and HKFs. Overexpression of circSLC8A1 suppressed HKFs proliferation, migration, ECM deposition, and promoted apoptosis. MiR-181a-5p is targeted by circSLC8A1, and its mimic reversed the effect of circSLC8A1 on the biological function of HKFs. HIF1AN was a target of miR-181a-5p, and it was positively regulated by circSLC8A1. Knockdown of HIF1AN also reversed the negatively regulation of circSLC8A1 on the biological functions of HKFs. Our data showed that circSLC8A1 regulates the miR-181a-5p/HIF1AN axis to restrain HKFs biological functions, confirming that circSLC8A1 might serve as a novel therapeutic target for keloids.

Keloid Core Factor CTRP3 Overexpression Significantly Controlled TGF-β1-Induced Propagation and Migration in Keloid Fibroblasts.

Keloid is a type of benign fibrous proliferative tumor characterized by excessive scarring. C1q/TNF-related protein 3 (CTRP3) has been proven to possess antifibrotic effect. Here, we explored the role of CTRP3 in keloid. In the current research, we examined the influence of CTRP3 on keloid fibroblasts (KFs) and investigated the potential molecular mechanism. KF tissue specimens and adjacent normal fibroblast (NF) tissues were collected cultured from 10 keloid participants. For the TGF-β1 stimulation group, KFs were processed with human recombinant TGF-β1. Cell transfection of pcDNA3.1-CTRP3 or pcDNA3.1 was performed. The siRNA of CTRP3 (si-CTRP3) or negative control siRNA (si-scramble) was transfected into KFs. CTRP3 was downregulated in keloid tissues and KFs. CTRP3 overexpression significantly controlled TGF-β1-induced propagation and migration in KFs. Col I, α-SMA, and fibronectin mRNA and protein levels were enhanced by TGF-β1 stimulation, whereas they were inhibited by CTRP3 overexpression. In contrast, CTRP3 knockdown exhibited the opposite effect. In addition, CTRP3 attenuated TGF-β receptors TRI and TRII in TGF-β1-induced KFs. Furthermore, CTRP3 prevented TGF-β1-stimulated nuclear translocation of smad2 and smad3 and suppressed the expression levels of p-smad2 and p-smad3 in KFs. CTRP3 exerted an antifibrotic role through inhibiting proliferation, migration, and ECM accumulation of KFs via regulating TGF-β1/Smad signal path.

Adipose-derived mesenchymal stem cells-sourced exosomal microRNA-7846-3p suppresses proliferation and pro-angiogenic role of keloid fibroblasts by suppressing neuropilin 2.

Exosomes (Exos) and their contained microRNAs (miRNAs) have been emergingly recognized as key regulators in spanning biological processes, including proliferation and angiogenesis. This work investigates the function of Exos derived from adipose-derived mesenchymal stem cells (adMSCs) in viability of keloid fibroblasts (KFs). Abnormally expressed miRNAs in keloid tissues were screened using the GEO dataset GSE113620. Meanwhile, miRNAs enriched in adMSC-Exos were predicted by bioinformatics system. Exos were extracted from acquired adMSCs and identified, which were co-incubated with KFs. Uptake of Exos by KFs was examined by fluorescence staining. Viability, proliferation, and apoptosis of KFs were analyzed by CCK-8, EdU labeling, and TUNEL assays. Conditioned medium of KFs was collected to stimulate angiogenesis of human umbilical vein endothelial cells (HUVECs). Binding between miR-7846-3p and neuropilin 2 (NRP2) was validated by luciferase assay. Protein levels of NRP2 and the Hedgehog pathway molecules were analyzed by western blot analysis. miR-7846-3p was predicted as an exosomal miRNA aberrantly expressed in keloids. AdMSC-Exos reduced viability, proliferation, and apoptosis resistance of KFs, and they blocked the angiogenesis of HUVECs. miR-7846-3p targeted NRP2 mRNA. miR-7846-3p upregulation in KFs suppressed NRP2 expression and reduced the expression of Hedgehog pathway molecules SHH, SMO, and GLI1. Either miR-7846-3p inhibition in Exos or NRP2 overexpression in KFs blocked the effects of Exos and restored the viability, proliferation, and pro-angiogenic role of KFs. This work unravels that adMSC-Exos-derived miR-7846-3p suppresses NRP2 and inactivates the Hedgehog signaling to reduce proliferation and pro-angiogenic role of KFs.

Dasatinib Attenuates Fibrosis in Keloids by Decreasing Senescent Cell Burden

Keloids are skin tumours caused by aberrant growth of dermal fibroblasts. Cellular senescence contributes to aging and various pathological conditions, including cancer, atherosclerosis, and fibrotic diseases. However, the effects of cellular senescence and senolytic drugs on keloids remain largely unknown. This study investigated senescent fibroblasts in keloids and assessed the effects of dasatinib on these cells. Tissues acquired from keloid removal surgery were analysed for senescence-associated β-galactosidase-positive cells, p16 expression, and the effects of dasatinib treatment on keloids. Keloid tissue was xenotransplanted into mice, and the effect of intralesional dasatinib injection on keloid growth was observed. The results showed that the numbers of β-galactosidase-positive and p16-expressing cells were higher in the keloids compared with in the controls. Dasatinib induced selective clearance of senescent cells and decreased procollagen expression in cultured keloid fibroblasts. In this xenotransplant keloid mouse model, intralesional injection of dasatinib reduced gross keloid tissue weight and the expression of both procollagen and p16. In addition, dasatinib-treated keloid fibroblasts conditioned medium reduced procollagen and p16 expression in cultured keloid fibroblasts. In conclusion, these results suggest that an increased number of senescent fibroblasts may play an important role in the pathogenesis of keloids. Therefore, dasatinib could be an alternative treatment for patients with keloids.

Osteomodulin contributes to keloid development by regulating p38 MAPK signaling.

A keloid is a classic fibrotic skin disease characterized by excessive deposition of extracellular matrix (ECM). Osteomodulin (OMD) is a heterologous protein that is a part of osteoadherin and plays a role in modulating ECM deposition. In this study, we investigated the effects of OMD on ECM synthesis and the tumor-like phenotype of keloid fibroblasts. We enrolled 10 patients with keloids and 10 age- and sex-matched healthy individuals, whose keloid or normal skin tissues were collected during surgery. Real-time quantitative polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemical staining were performed to analyze OMD expression in skin tissues. Cell transfection, CCK-8 assay, EdU staining, Transwell assay, qRT-PCR, western blotting, and immunofluorescence were performed to study the effects of OMD on primary keloid-derived fibroblasts (KFs). OMD exhibited greater expression in human keloid specimens than in normal skin tissues. Consistently, higher expression of OMD was observed in KFs, compared to that in normal fibroblasts. Silencing OMD expression in transforming growth factor (TGF)-β1-treated KFs inhibited cell proliferation and migration, as well as collagen and fibronectin expression; however, overexpression of OMD had the opposite effect. p38 mitogen-activated protein kinase (MAPK) was activated in keloid tissues but not in normal skin. OMD was positively correlated with p38 MAPK activation. Adding SB203580, p38 MAPK inhibitor, significantly reversed the effects of OMD on the regulation of KF phenotype. The high expression of OMD may contribute to hyperproliferation of KFs, their migration, and excess ECM synthesis in KFs via regulation of the p38 MAPK signaling pathway.

Tonic repression of collagen I by the bradykinin receptor 2 in skin fibroblasts.

Imbalance of collagen I expression results in severe pathologies. Apart from activation by the TGFβ-receptor/Smad pathway, control of collagen I expression remains poorly understood. Here, we used human dermal fibroblasts expressing a mCherry fluorescent protein driven by endogenous COL1A1 promoter to functionally screen the kinome and phosphatome. We identify 8 negative regulators, revealing that collagen is under tonic repression. The cell surface receptor BDKRB2 represses collagen I and other pro-fibrotic genes. Interestingly, it also promotes other basal membrane ECM genes. This function is independent of the natural ligand, bradykinin, and of SMAD2/3 factors, instead requiring constant ERK1/2 repression. TGFβ stimulation induces rapid BDKRB2 transcriptional downregulation. Human fibrotic fibroblasts have reduced BDKRB2 levels and enhancing its expression in keloid fibroblasts represses COL1A1. We propose that tonic signalling by BDKRB2 prevents collagen overproduction in skin fibroblasts.

IL13RA2 downregulation in fibroblast promotes keloid fibrosis via JAK/STAT6 activation.

Keloid is considered as a fibro-proliferative disease characterized by chronic inflammation that is induced following skin injury. Deciphering the underlying mechanism of keloid formation is essential for improving treatment outcomes. Here, we found that more macrophages were activated towards M2 subtype in keloid dermis when compared to normal dermis. Western Blot revealed that the level of phosphorylated STAT6, a known inducer of M2 polarization, was higher in keloid fibroblasts as opposed to fibroblasts from normal dermis. Moreover, keloid fibrosis was shown to be positively correlated with the level of phosphorylated STAT6. Further, we identified downregulation of IL13RA2, a 'decoy' receptor of IL13, in keloid fibroblasts compared to fibroblasts from normal dermis. Ectopic expression of IL13RA2 in keloid fibroblasts resulted in inhibition of STAT6 phosphorylation, cell proliferation, migration, invasion, extracellular matrix secretion and myofibroblast marker expression, as well as an increase in apoptosis. Consistently, knockdown of IL13RA2 in normal fibroblasts induced a 'keloidal' status. Furthermore, both in vitro application and intra-tumoral injection of pSTAT6 inhibitor AS1517499 in a PDX keloid-implantation mouse model, resulted in proliferation inhibition, tissue necrosis, apoptosis and myofibroblast marker reduction. Collectively, this study elucidates the key role of IL13RA2 in keloid pathology and inspire further translational research of keloid treatment concerning JAK/STAT6 inhibition.

An updated review of the immunological mechanisms of keloid scars.

Keloid is a type of disfiguring pathological scarring unique to human skin. The disorder is characterized by excessive collagen deposition. Immune cell infiltration is a hallmark of both normal and pathological tissue repair. However, the immunopathological mechanisms of keloid remain unclear. Recent studies have uncovered the pivotal role of both innate and adaptive immunity in modulating the aberrant behavior of keloid fibroblasts. Several novel therapeutics attempting to restore regulation of the immune microenvironment have shown variable efficacy. We review the current understanding of keloid immunopathogenesis and highlight the potential roles of immune pathway-specific therapeutics.

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.

The mechanism of oxidative stress in keloid fibroblasts and the experimental study of early application of angiotensin-converting enzyme inhibitor.

Objective To investigate the protective effects of an angiotensin-converting enzyme inhibitor after inducing oxidative stress on keloid fibroblasts. Methods Primary keloid fibroblasts were isolated and cultured by enzyme digestion combined with the tissue adhesion method in vitro, and the third to fifth generations of cells were selected for the experiment. For 24 hours, keloid fibroblasts were treated with different concentrations of hydrogen peroxide. Different concentrations of angiotensin-converting enzyme inhibitor were added to the keloid fibroblast culture medium, and then the cells were treated with hydrogen peroxide for 24 hours. Results With the increase of hydrogen peroxide concentration, the growth of keloid fibroblasts was inhibited and the levels of malondialdehyde, superoxide dismutase, and reactive oxygen species increased gradually, accompanied by an increase in the expression of nicotinamide adenine dinucleotide phosphate oxidase and collagen I mRNA. The expression of nicotinamide adenine dinucleotide phosphate oxidase-mRNA in keloid fibroblasts and the formation of reactive oxygen species in keloid fibroblasts were induced by different concentrations of angiotensin II, and the most significant effect was at 10-5 mmol/mL. The effects of diphenyleneiodonium chloride (NOX inhibitor), N-acetylcysteine (reactive oxygen species inhibitor) and nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) RNA treatment on angiotensin II-induced nicotinamide adenine dinucleotide phosphate oxidase and collagen I increased significantly. Hydrogen peroxide and angiotensin II alone or combined can induce NADPH oxidase and reactive oxygen species expression in keloid fibroblasts. When the angiotensin-converting enzyme inhibitor was added, the expression of NADPH oxidase and reactive oxygen species in keloid induced by hydrogen peroxide and angiotensin II could be inhibited. Conclusion Oxidative stress can lead to increased expression of reactive oxygen species, NADPH oxidase and collagen I in keloid fibroblasts, suggesting oxidative stress mediates the migration of human keloid fibroblasts and extracellular matrix synthesis.

Oleanolic acid regulates the proliferation and extracellular matrix of keloid fibroblasts by mediating the TGF-β1/SMAD signaling pathway.

Keloid (KD) is a unique pathological fibroproliferative disease that seriously affects the appearance of patients. This study investigated the effect of oleanolic acid (OA) on the proliferation of keloid fibroblasts (KFs) and the expression of extracellular matrix (ECM)-related proteins. The proliferation of KFs was evaluated using an MTT assay. The effects of OA on intra- and extracellular levels of fibronectin (FN), procollagen I, matrix metalloproteinase-1 (MMP-1), and α-smooth muscle actin (α-SMA) were evaluated using Western blotting. To simulate the KD microenvironment, TGF-β1 was added to the serum-free culture medium, and KFs were incubated with TGF-β1 and OA for 24 h. The intra- and extracellular levels of the ECM-related proteins and the effect of OA on TGF-β1-induced phosphorylation of the SMAD2 and SMAD3 proteins were evaluated using Western blotting. OA inhibited the proliferation of KFs in a concentration- and time-dependent manner. Furthermore, OA treatment of KFs reduced the intra- and extracellular levels of FN, procollagen I, and α-SMA and increased those of MMP-1. OA also reduced TGF-β1-induced increases in the intra- and extracellular levels of FN, procollagen I, and α-SMA and increased the levels of the MMP-1 protein. Additionally, OA significantly reduced TGF-β1-induced phosphorylation of SMAD2 and SMAD3 in KFs. OA inhibited KF proliferation and reduced ECM deposition through the TGF-β1/SMAD pathway, which suggests that OA may be an effective drug for the prevention and treatment of KD.

The JAK-STAT pathway in keloid pathogenesis: a systematic review with qualitative synthesis.

Keloid tissues contain inflammatory cells and upregulated pro-inflammatory cytokines. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway mediate cellular responses to these cytokines. We performed a systematic review on the role of the JAK-STAT pathway in keloid pathogenesis and the evidence for JAK-STAT inhibitors in keloid treatment. The search combined the terms (1) keloid and (2) JAK or TYK or STAT and included MeSH terms and synonyms. Two reviewers screened the articles and assessed the full texts on eligibility. Data were collected on the tested drugs and molecules, the type of cells and tissues used in the experiments, and study findings on the association between the JAK-STAT pathway and keloid cells and tissues. A total of twenty preclinical studies were included. Eleven preclinical studies proved that STAT3 expression and phosphorylation are enhanced in keloid tissue and keloid fibroblasts. Thirteen different JAK and/or STAT inhibitors were investigated. Tested drugs inhibited keloid progression as demonstrated by different processes, including reduced collagen production, cell proliferation and migration, increased cell cycle arrest and apoptosis, enhanced antioxidant responses, decreased (paracrine) signalling, and decreased profibrotic gene expression. No clinical studies have been published to date. Preclinical studies indicate a role for the JAK-STAT pathway in keloid pathogenesis and a potential role for JAK-STAT inhibitors in keloid treatment. The effect of these drugs should be further investigated on relevant biomarkers in a human keloid skin model, preferably including immune cells besides keloid fibroblasts and keratinocytes and in clinical studies.

Relief of Extracellular Matrix Deposition Repression by Downregulation of IRF1-Mediated TWEAK/Fn14 Signaling in Keloids

Keloids represent a fibrotic disorder characterized by the excessive deposition of extracellular matrix (ECM). However, the mechanisms through which ECM deposition in keloids is regulated remain elusive. In this study, we found that the expression of both TWEAK and its cognate receptor Fn14 was significantly downregulated in keloids and that TWEAK/Fn14 signaling repressed the expression of ECM-related genes in keloid fibroblasts. The IRF1 gene was essential for this repression, and the TWEAK/Fn14 downstream transcription factor p65 directly bound to the promoter of the IRF1 gene and induced its expression. Furthermore, in patients with keloid, the expression of TWEAK and Fn14 was negatively correlated with that of ECM genes and positively correlated with that of IRF1. These observations indicate that relief of TWEAK/Fn14/IRF1-mediated ECM deposition repression contributes to keloid pathogenesis, and the identified mechanism and related molecules provide potential targets for keloid treatment in the future.

TWEAK/Fn14 signaling may function as a reactive compensatory mechanism against extracellular matrix accumulation in keloid fibroblasts

Overabundance of the extracellular matrix resulting from hyperproliferation of keloid fibroblasts (KFs) and dysregulation of apoptosis represents the main pathophysiology underlying keloids. TWEAK is a weak apoptosis inducer, and it plays a critical role in pathological tissue remodeling via its receptor, Fn14. However, the role of TWEAK/Fn14 signaling in the pathogenesis of keloids has not been investigated. In this study, we confirmed the overexpression levels of TWEAK and Fn14 in clinical keloid tissue specimens and primary KFs. The extracellular matrix (ECM)-related genes were also evaluated between primary KFs and their normal counterparts to determine the factors leading to the formation or development of keloids. Unexpectedly, exogenous TWEAK significantly reduced the levels of collagen I and collagen III, as well as alpha-smooth muscle actin (α-SMA). Additionally, TWEAK promoted MMPs expression and apoptosis activity of KFs. Furthermore, we verified that the inhibitory effect of TWEAK on KFs is through down-regulation of Polo-like kinase 5, which modulates cell differentiation and apoptosis. The TWEAK-Fn14 axis seems to be a secondary, although less effective, compensatory mechanism to increase the catabolic functions of fibroblasts in an attempt to further decrease the accumulation of collagen. Data AvailabilityAll data generated or analyzed during this study are included in this published article (and its Supporting Information files).

PI3K/AKT pathway promotes keloid fibroblasts proliferation by enhancing glycolysis under hypoxia.

Our previous study demonstrated altered glucose metabolism and enhanced phosphorylation of the PI3K/AKT pathway in keloid fibroblasts (KFb) under hypoxic conditions. However, whether the PI3K/AKT pathway influences KFb cell function by regulating glucose metabolism under hypoxic conditions remains unclear. Here, we show that when PI3K/AKT pathway was inactivated with LY294002, the protein expression of glycolytic enzymes decreased, while the amount of mitochondria and mitochondrial membrane potential increased. The key parameters of extracellular acidification rate markedly diminished, and those of oxygen consumption rate significantly increased after inhibition of the PI3K/AKT pathway. When the PI3K/AKT pathway was suppressed, the levels of reactive oxygen species (ROS) and mitochondrial ROS (mitoROS) were significantly increased. Meanwhile, cell proliferation, migration and invasion were inhibited, and apoptosis was increased when the PI3K/AKT pathway was blocked. Additionally, cell proliferation was compromised when KFb were treated with both SC79 (an activator of the PI3K/AKT pathway) and 2-deoxy-d-glucose (an inhibitor of glycolysis), compared with the SC79 group. Moreover, a positive feedback mechanism was demonstrated between the PI3K/AKT pathway and hypoxia-inducible factor-1α (HIF-1α). Our data collectively demonstrated that the PI3K/AKT pathway promotes proliferation and inhibits apoptosis in KFb under hypoxia by regulating glycolysis, indicating that the PI3K/AKT signalling pathway could be a therapeutic target for keloids.

Targeting the Glycolytic Enzyme PGK1 to Inhibit the Warburg Effect: A New Strategy for Keloid Therapy.

Aerobic glycolysis (the Warburg effect) may play an important role in keloid pathogenesis, which may be aggravated by the hypoxic microenvironment in keloids. Phosphoglycerate kinase 1 (PGK1), a key glycolytic enzyme, is essential for cellular aerobic glycolysis, but its role in keloid formation remains unknown. This study aimed to detect PGK1 expression in keloid tissue and investigate the effects of inhibiting PGK1 expression on keloid fibroblasts (KFbs) under hypoxia and normoxia. Normal skin and keloid samples were separated into two parts, one was used for immunohistochemistry, and one for primary cell culture. PGK1 tissue expression was detected by immunohistochemistry. Reverse-transcriptase polymerase chain reaction and Western blotting were used to detect PGK1, GLUT1, LDHA, and COL1 expression, and glucose uptake and lactate production were detected with a microplate reader. Cell proliferation and apoptosis were investigated with IncuCyte and flow cytometry. Cell migration and invasion were detected with Transwell assays. Glycolytic function was explored with the Seahorse XF96 system. Immunohistochemistry showed PGK1 overexpression in keloid tissue compared with normal skin tissue ( P < 0.05). Consistently, PGK1 expression was significantly higher in KFbs than in normal skin fibroblasts (NFbs), and hypoxia stimulated PGK1 expression in KFbs and NFbs ( P < 0.05). PGK1 knockdown significantly inhibited KFb glycolysis, proliferation, migration, invasion, glucose consumption, and lactate production ( P < 0.05). Furthermore, GLUT1, LDHA, and COL1 expression was decreased in KFbs compared with NFbs ( P < 0.05). In addition, suppressing PGK1 may mediate the PI3K/AKT pathway to down-regulate GLUT1, LDHA, and COL1 expression ( P < 0.05). These findings provide new evidence that suppressing PGK1, inhibiting glycolysis, reduces KFb proliferation, migration, invasion, and type I collagen expression. Targeting PGK1 to inhibit the Warburg effect may be a new therapeutic strategy for keloids. This article may provide new suggestions into the pathogenesis and treatment of keloids. Therapeutic, V.

Melatonin influences the biological characteristics of keloid fibroblasts through the Erk and Smad signalling pathways.

Keloids are abnormal fibrous hyperplasias that are difficult to treat. Melatonin can be used to inhibit the development of certain fibrotic diseases but has never been used to treat keloids. We aimed to discover the effects and mechanisms of melatonin in keloid fibroblasts (KFs). Flow cytometry, CCK-8 assays, western blotting, wound-healing assays, transwell assays, collagen gel contraction assays and immunofluorescence assays were applied to demonstrate the effects and mechanisms of melatonin in fibroblasts derived from normal skin, hypertrophic scars and keloids. The therapeutic potential of the combination of melatonin and 5-fluorouracil (5-FU) was investigated in KFs. Melatonin significantly promoted cell apoptosis and inhibited cell proliferation, migration and invasion, contractile capability and collagen production in KFs. Further mechanistic studies demonstrated that melatonin could inhibit the cAMP/PKA/Erk and Smad pathways through the membrane receptor MT2 to alter the biological characteristics of KFs. Moreover, the combination of melatonin and 5-FU remarkably promoted cell apoptosis and inhibited cell migration and invasion, contractile capability and collagen production in KFs. Furthermore, 5-FU suppressed the phosphorylation of Akt, mTOR, Smad3 and Erk, and melatonin in combination with 5-FU markedly suppressed the activation of the Akt, Erk and Smad pathways. Collectively, melatonin may inhibit the Erk and Smad pathways through the membrane receptor MT2 to alter the cell functions of KFs, while combination with 5-FU could exert even more inhibitory effects in KFs through simultaneous suppression of multiple signalling pathways.

SPARC promotes fibroblast proliferation, migration, and collagen production in keloids by inactivation of p53

BackgroundKeloid, an aggressive fibroproliferative disease of the skin, is usually caused by infectious skin diseases, burns, and trauma. ObjectiveThis study aimed to assess the effect of SPARC on the keloid pathogenesis. MethodsIn normal skin and keloid scar tissues, changes in SPARC expression were analysed by qRT-PCR, western blotting, and immunohistochemistry. Keloid fibroblasts were isolated from human keloid tissue. GSEA was performed to investigate the signalling pathways related to SPARC. Cell Counting Kit-8, 5-Ethynyl-2′-deoxyuridine, transwell assay, and scratching assays were used to assess fibroblast proliferation and migration. Changes in α-SMA, fibronectin, collagen I, and collagen III levels were examined in fibroblasts by western blotting. ResultsSPARC expression was upregulated in keloid scar tissues. In fibroblasts, cell proliferation, migration, collagen production, and extracellular matrix (ECM) synthesis were promoted by SPARC overexpression, whereas SPARC knockdown resulted a converse result. GSEA showed that SPARC regulates the p53 pathway. In keloid scar tissues, there was a negative correlation between SPARC and p53 expression. p53 expression was decreased by SPARC overexpression, whereas SPARC knockdown increased p53 expression. Furthermore, the effects of SPARC on the fibroblast phenotype were reversed by p53 overexpression. ConclusionsFibroblast proliferation, migration, and ECM synthesis were promoted by SPARC overexpression, which was achieved by regulating the p53 pathway. Our findings provide new therapeutic targets for keloids.