Search

Background: Cell Migration Inducing Hyaluronidase 1 (CEMIP) is a protein that plays regulatory functions in a variety of cellular processes in many diseases. Nevertheless, its role and molecular mechanism in keloid hyperplasia are still elusive., Methods: Expressions of CEMIP and Secreted Protein acidic and Rich in Cysteine (SPARC) were detected by qRT-PCR and western blot. CCK-8 assay, along with immunofluorescence staining, was applied for the assessment of cell proliferation. The capabilities of cells to migrate and invade were evaluated utilizing wound healing and Transwell, while Extracellular Matrix (ECM) deposition was measured by immunofluorescence and western blot. The interaction of CEMIP and SPARC was predicted by the Coexpedia and PPA-red databases and verified by co-IP. Western blot was adopted for the estimation of TGF-β/Smad pathway-related proteins., Results: The data demonstrated that CEMIP expression was elevated in Keloid Fibroblasts (KF). CEMIP interference suppressed cell proliferative, migrative and invasive capabilities and ECM deposition in KF. Mechanistically, bioinformatics analysis revealed that CEMIP was co-expressed with SPARC and CEMIP protein could bind to SPARC. SPARC expression was reduced in CEMIP-silenced cells. SPARC overexpression counteracted the impacts of CEMIP silencing on cell proliferative, migrative and invasive capabilities and ECM deposition in KF. In addition, the expressions of TGF-β/Smad signaling-related proteins were decreased by CEMIP silencing via the inhibition of SPARC., Conclusion: In summary, this study revealed that CEMIP modulated KF proliferation, migration, invasion and ECM deposition by TGF-β/Smad signaling through binding to SPARC., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier España, S.L.U.)

Keloid is a skin disease marked by fibroplasia, and fibroblasts viability plays a considerable part in keloid. Our research was devoted to assessing the involvement and mechanism of circPTPN12 in keloid. The level of circPTPN12 and miR-21-5p was estimated by qRT-PCR in keloid tissues and cells. MTT analysis was devoted to evaluating the multiplication of keloid fibroblasts. Additionally, transwell assay was dedicated to verifying cell migration and invasion. Furthermore, keloid fibroblasts apoptosis level was assessed adopting flow cytometry, and the relevancy between miR-21-5p and circPTPN12, miR-21-5p, and SMAD7 was assessed by dual luciferase assay. Similarly, RIP and RNA pull-down assay verified the relevance between genes. Moreover, levels of SMAD7 and proteins concerned in Wnt signaling pathway were appraised by Western blot. The level of circPTPN12 declined in keloid. circPTPN12 knockout could enhance the multiplication, migration, invasion, and decline apoptosis of keloid fibroblasts. Indeed, miR-21-5p could be packed with circPTPN12 sponge, SMAD7 was downstream effect factor of miR-21-5p, and miR-21-5p inhibitors partially reversed the promoting effect of silencing circPTPN12 on keloid formation. Otherwise, the level of SMAD7 was adjusted by circPTPN12 and miR-21-5p. Silencing circPTPN12 targeted miR-21-5p and activated Wnt pathway to accelerate keloid fibroblasts growth. Taken together, silencing circPTPN12 promotes the growth of keloid fibroblasts by activating Wnt pathway targeting miR-21-5p. CircPTPN12 may play a considerable part in keloid formation, which supplies a reference for molecularly targeted therapy keloid.

Keloids are benign skin tumors characterized by aggressive growth. To date, there is no exact treatment because little is known about its pathological mechanism. Therefore, it is important to investigate the mechanism of its occurrence and development to identify therapeutic targets. In this study, the expression of Kindlin-2 was higher in keloid fibroblasts (KFs) than in normal skin fibroblasts (NFs). In vitro experiments showed that knocking down Kindlin-2 in KFs could promote cell apoptosis and inhibit cell proliferation, cell migration and invasion, and contractile capability. Western blot results showed that the phosphorylation of Smad3 in KFs was inhibited after knocking down Kindlin-2, inhibiting the activation of the Smad pathway. Moreover, knocking down Kindlin-2 increased the expression of Fas and FasL in KFs, which demonstrated that knocking down Kindlin-2 promoted the activation of the exogenous apoptotic pathway of KFs and then facilitated apoptosis. The above results revealed that knocking down Kindlin-2 in KFs can inhibit the activation of the Smad pathway and promote the activation of the Fas/FasL exogenous apoptosis pathway, thereby altering the cytological function of KFs. Therefore, Kindlin-2 might play an important role in the occurrence and development of keloids and could become a new target to treat keloids., (Copyright © 2021. Published by Elsevier Inc.)

Apoptosis plays a key role in keloids. Growth arrest and DNA damage-inducible gene 153 (GADD153) is regulated by apoptosis. Botulinum toxin type A (BTXA) can induce apoptosis in keloid fibroblasts. This research aimed to explore the hypothesis that GADD153 mediates apoptosis in keloid fibroblasts exposed to BTXA. BTXA significantly induced GADD153 protein and mRNA expression in keloid fibroblasts. Treatment with c-Jun N-terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA) and tumour necrosis factor-alpha (TNF-α) antibodies reversed the BTXA-induced GADD153 expression. BTXA enhanced the transcriptional activity of GADD153, whereas the GADD153 mutant plasmid, JNK siRNA and anti-TNF-α antibody treatment abolished the BTXA-induced transcriptional activity of GADD153. The addition of TNF-α to keloid fibroblasts markedly increased GADD153 protein expression. The addition of GADD153 siRNA, SP600125 and anti-TNF-α antibodies reversed cell death and caspase 3 and 9 activity induced by BTXA., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Fibroblasts found in keloid tissues are known to present an altered sensitivity to microenvironmental stimuli. However, the impact of changes in extracellular matrix stiffness on phenotypes of normal fibroblasts (NFs) and keloid fibroblasts (KFs) is poorly understood., Objectives: Investigation the impact of matrix stiffness on NFs and KFs mainly via detecting yes-associated protein (YAP) expression., Methods: We used fibronectin-coated polyacrylamide hydrogel substrates with a range from physiological to pathological stiffness values with or without TGF-β (fibrogenic inducer). Atomic force microscopy was used to measure the stiffness of fibroblasts. Cellular mechanoresponses were screened by immunocytochemistry, Western blot and Luminex assay., Results: KFs are stiffer than NFs with greater expression of α-SMA. In NFs, YAP nuclear translocation was induced by increasing matrix stiffness as well as by stimulation with TGF-β. In contrast, KFs showed higher baseline levels of nuclear YAP that was not responsive to matrix stiffness or TGF-β. TGF-β1 induced p-SMAD3 in both KFs and NFs, demonstrating the pathway was functional and not hyperactivated in KFs. Moreover, blebbistatin suppressed α-SMA expression and cellular stiffness in KFs, linking the elevated YAP signaling to keloid phenotype., Conclusions: These data suggest that whilst normal skin fibroblasts respond to matrix stiffness in vitro, keloid fibroblasts have elevated activation of mechanotransduction signaling insensitive to the microenvironment. This elevated signaling appears linked to the expression of α-SMA, suggesting a direct link to disease pathogenesis. These findings suggest changes to keloid fibroblast phenotype related to mechanotransduction contribute to disease and may be a useful therapeutic target., Competing Interests: Declaration of Competing Interest The authors state no conflict of interest., (Copyright © 2021 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Keloid is a benign skin tumor characterized by its cell hyperproliferative activity, invasion into normal skin, uncontrolled growth, overproduction and deposition of extracellular matrices and high recurrence rate after various therapies. Nintedanib is a receptor tyrosine kinase inhibitor targeting VEGF, PDGF, FGF, and TGF-β receptors with proved efficacy in anti-angiogenesis and in treating various types of cancers. In this study, we investigated the effects of nintedanib on keloid fibroblasts in both in vitro and ex vivo models. Keloid fibroblasts were prepared from 54 keloid scar samples in active stages collected from 49 patients. We found that nintedanib (1-4 μM) dose-dependently suppressed cell proliferation, induced G 0 /G 1 cell cycle arrest, and inhibited migration and invasion of keloid fibroblasts. The drug also significantly inhibited the gene and protein expression of collagen I (COL-1) and III (COL-3), fibronectin (FN), and connective growth factor (CTGF), as well as the gene expression of other pathological factors, such as alpha smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), FK506-binding protein 10 (FKBP10), and heat shock protein 47 (HSP47) in keloid fibroblasts. Furthermore, nintedanib treatment significantly suppressed the phosphorylation of p38, JNK, ERK, STAT3, and Smad, enhanced endocytosis of various growth factor receptors. Using an ex vivo tissue explant model, we showed that nintedanib significantly suppressed cell proliferation, migration, and collagen production. The drug also significantly disrupted microvessel structure ex vivo. In summary, our results demonstrate that nintedanib is likely to become a potential targeted drug for keloid systemic therapy.

Background: Keloid is a fibrous tissue proliferative disease in which proliferative scars grow beyond the boundary of the original wound skin. Long non-coding RNAs (lncRNAs), as competing endogenous RNAs (ceRNAs), bind to microRNAs (miRNAs) to regulate various biological processes. The present study was aim to illuminate the mechanism of calcium voltage-gated channel subunit alpha1 G antisense RNA 1 (CACNA1G-AS1) in human keloid fibroblasts., Methods: CACNA1G-AS1 and miR-205 levels were detected using quantitative real-time polymerase chain reaction (qRT-PCR). Cell Counting Kit-8 (CCK-8) assay was used to measure the proliferation and transwell assay was performed to evaluate cell invasion. Furthermore, the apoptosis rates of cells were evaluated by flow cytometry analysis, and the activity of caspase-3 in keloid fibroblasts was tested by Caspase-3 activity assay. Dual luciferase reporter assay was carried out to examine the relationship between CACNA1G-AS1 and miR-205 and RNA immunoprecipitation (RIP) assay was conducted to further confirm the relation., Results: CACNA1G-AS1 level was up-regulated in keloid tissues and keloid fibroblasts. CACNA1G-AS1 overexpression promoted proliferation and invasion and suppressed apoptosis of keloid fibroblasts. Moreover, miR-205 was targeted by CACNA1G-AS1 and miR-205 was markedly decreased in keloid tissues and keloid fibroblasts. Also, miR-205 expression was negatively regulated by CACNA1G-AS1 and miR-205 silencing enhanced proliferation and invasion and inhibited apoptosis. Furthermore, CACNA1G-AS1 and miR-205 played the antagonistic role in miR-205 expression, proliferation, invasion, and apoptosis of keloid fibroblasts., Conclusion: CACNA1G-AS1 suppressed miR-205 expression to promote proliferation and invasion and inhibit apoptosis in human keloid fibroblasts., (© 2020 The Author(s).)

The current experiment was performed to investigate whether luteolin affects the proliferation and apoptosis of keloid fibroblasts by regulating the expression of FRAT1 gene. Keloid fibroblasts were treated with luteolin at different concentrations. MTT method, western blot, flow cytometry, and real-time quantitative PCR (qPCR) were used to detect cell proliferation, cyclin D1 (CyclinD1), p21, B-cell lymphoma / leukemia-2 (Bcl-2), Bcl-2 related X protein (Bax), FRAT1 protein expression, apoptosis and ARHI mRNA expression. Keloid fibroblasts were transfected with si-FRAT1, or pcDNA-FRAT1 and treated with luteolin to observe their roles in cell proliferation and apoptosis. Compared with the control group, luteolin significantly reduced the keloid fibroblast activity, CyclinD1, Bcl-2, and FRAT1 protein levels, and obviously improved the cell apoptosis rate, p21 and Bax protein expression (P<0.05). The expression of FRAT1 mRNA and protein in keloid fibroblasts was greatly increased (P<0.05). Inhibition of FRAT1 expression evidently decreased cell viability at 24 h, 48 h, and 72 h, CyclinD1, and Bcl-2 protein expression of keloid fibroblasts, while-dramatically enhanced cell apoptosis, p21, and Bax protein levels (P<0.05). FRAT1 overexpression reversed the inhibitory effect of luteolin on keloid fibroblast activity, FRAT1, CyclinD1, and Bcl-2 protein expression, and promotion of apoptosis, p21 and Bax protein expression. Luteolin can inhibit the proliferation and induce apoptosis of keloid fibroblasts by regulating the expression of FRAT1 gene.

Emerging evidence has revealed that microRNAs (miRNAs) play critical roles in keloid pathogenesis. However, potential molecular mechanism of keloid formation remains unclear. In the present study, our findings showed that miR-152-3p mRNA expression level was notably up-regulated in keloid tissues and keloid fibroblasts compared with that of normal skin tissues and normal skin fibroblasts, respectively. Furthermore, miR-152-3p inhibition remarkably suppressed cell proliferation, which was increased by miR-152-3p overexpression. Cell invasion was also significantly decreased by miR-152-3p inhibition, whereas was increased by miR-152-3p overexpression. The mRNA and protein expression levels of extracellular matrix components including type I collagen, type III collagen and fibronectin were decreased by miR-152-3p inhibition, but were increased by miR-152-3p overexpression. In addition, results of dual-luciferase reporter assay indicated that FOXF1 is a direct target of miR-152-3p. FOXF1 overexpression significantly inhibits cell proliferation, invasion, and extracellular matrix in keloid fibroblasts, and the suppressive effects of miR-152-3p mimic on these functions were notably partly reversed by FOXF1 overexpression. Taken together, these findings indicated that miR-152-3p regulates cell proliferation, invasion and extracellular matrix expression through targeting FOXF1 in keloid fibroblasts, suggesting that miR-152-3p is a novel and promising molecular target for keloid treatment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Topography modified cell behavior remains less explored in diseased cells. This study investigated the reversing effect on the pathological phenotype of keloid fibroblasts via culturing cells on a parallel microgrooved surface. The results showed that this particular topography with 3 μm groove depth and 10 μm width could significantly elongate and align the cultured cells with reduced cell (nucleus) area and increased cell (nucleus) body aspect ratio and cell (nucleus) body major axis (p < 0.05). Importantly, the elongated cells gradually lost their fibrotic phenotype with inhibited cell proliferation and cell cycle arrest in S-phase (p < 0.05), reduced expression of fibrotic markers such as collagen, fibronectin, connective tissue growth factor, α-smooth muscle actin, transforming growth factor-β1 (p < 0.05), and increased matrix metalloproteinases/tissue inhibitor-1 ratio (p < 0.05) along with attenuated Smad and Erk phosphorylation level. All these indicate that this parallel topography is powerful enough to modify keloid cell phenotype, a benign skin tumor with excessive cell proliferation and matrix production. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2019., (© 2019 Wiley Periodicals, Inc.)

Keloid formation is characterized by hyperproliferation of secretory and responsive keloid fibroblasts (KFs) and overproduction of extracellular matrix (ECM). Eukaryotic translation initiation factor 3 subunit A (eIF3a) one of the core subunits of the translation initiation complex, eIF3, has previously been reported to possess an anti‑fibrogenic effect. However, the role of eIF3a in keloid formation has not yet been investigated. Therefore, the present study examined the effect of eIF3a on transforming growth factor‑β1 (TGF‑β1)‑mediated ECM expression in KFs. The expression levels of eIF3a in human keloid tissues was evaluated using reverse transcription‑quantitative polymerase chain reaction and western blotting. KFs were incubated with siRNA‑eIF3a or siRNA‑mock for 48 h. The cells were then treated with TGF‑β1 (10 ng/ml) for 72 h. Cell proliferation was evaluated using the CCK‑8 assay. The expression levels of α‑SMA, collagen type I, TGF‑β receptor I (RI), TGF‑β RII, phosphorylated (p)‑mothers against decapentaplegic homolog (Smad2), Smad2, p‑Smad3 and Smad3 were detected western blotting. The present study identified significant upregulation of eIF3a mRNA and protein and in human keloid tissues compared with in normal tissues. Knockdown of eIF3a inhibited KF proliferation induced by TGF‑β1. In addition, eIF3a silencing significantly suppressed the TGF‑β1‑induced expression of α‑smooth muscle actin, collagen I, TGF‑β RI and TGF‑β RII in KFs. Furthermore, eIF3a silencing inhibited the phosphorylation levels of Smad2 and Smad3 in TGF‑β1‑induced KFs. To the best of our knowledge, the current study is the first to demonstrate that siRNA‑eIF3a inhibits the expression ECM proteins via the TGF‑β1/Smad signaling pathway in KFs. Therefore, eIF3a may be a potential, novel target for treatment of keloids.

Background: A keloid is a fibroproliferative disorder occurring in wounds characterized by an exaggerated response to injury. To date, no effective cure has been identified. As multipotent stem cells, human adipose-derived stem cells (ADSCs) may show the possibility for curing diseases such as fibrosis. This study sought to explore the potential role of human ADSCs in curing keloids., Methods: After culture in conditioned medium, gene and protein expression of keloid fibroblasts was examined using real-time polymerase chain reaction (RT-PCR) and Western blotting, while analysis of the cell cycle was used to measure the proliferative properties of the cells. Furthermore, ex vivo explant cultures were used to test the effects of ADSC-conditioned medium (ADSC-CM) on CD31 + and CD34 + expression in keloid tissue., Results: Our experimental results show that ADSC-CM was able to attenuate extracellular matrix-related gene expression as well as decrease protein expression. Cell proliferation was significantly suppressed in our study. CD31 + and CD34 + vessels in ex vivo explants were reduced by 55% and 57% in treatment groups compared with control groups., Conclusions: Human ADSC-CM significantly inhibited keloid fibroblast-related bioactivities.

It has been reported that Dickkopf-3 (DKK3) down-regulation was examined in keloid fibroblasts, but the biological functions of DKK3 have not yet been investigated. In this study, we examined the expression of DKK3 in human keloid tissues, further evaluated the biological function of DKK3 and explored its potential molecular mechanism in transforming growth factor-β1 (TGF-β1)-induced keloid fibroblasts. Our results showed that DKK3 mRNA expression in human keloid tissues is down-regulated. DKK3 overexpression inhibited cell proliferation in TGF-β1-induced keloid fibroblasts transfected with pcDNA3.1-DKK3. Furthermore, DKK3 overexpression remarkably upregulated the protein expression levels of Bax and caspase-3, but decreased the protein expression of Bcl-2. In addition, DKK3 overexpression dramatically inhibited the protein and mRNA levels of collagen I (Col-I), collagen III (Col-III) and α-smooth muscle actin (α-SMA). Moreover, the protein expression of TGF-β receptor I (TGF-β RI), TGF-β receptor II (TGF-β RII), the phosphorylation of Smad2 (p-Smad2) and Smad3 (p-Smad3) was dramatically inhibited by pcDNA3.1-DKK3. LY2109761, a TGF-β receptor inhibitor, also suppressed cell proliferation, apoptosis and collagen synthesis in TGF-β1-induced keloid fibroblasts. Taken together, DKK3 overexpression could inhibit cell proliferation, induced cell apoptosis, and suppressed collagen synthesis through TGF-β1/Smad signaling in TGF-β1-induced keloid fibroblasts. Our findings suggest that DKK3 is a novel and promising molecular target for keloid treatment., (Copyright © 2017. Published by Elsevier Masson SAS.)

Keloid is a common and refractory disease characterized by abnormal fibroblast proliferation and excessive deposition of extracellular matrix components. Hypocrellin B (HB) is a natural perylene quinone photosensitizer. In this experiment, we studied the effects of photodynamic therapy (PDT) using yellow light from light-emitting diode (LED) combined with HB on keloid fibroblasts (KFB) in vitro . Our results showed that HB-LED PDT treatment induced significant KFB apoptosis and decreased KFB cell viability. HB-LED PDT treatment lead to significant BAX upregulation and BCL-2 downregulation in KFB cells, which led to elevation of intracellular free Ca 2+ and activation of caspase-3. Our data provides preliminary evidence for the potential of HB-LED PDT as a therapeutic strategy for keloid., Competing Interests: Competing Interests: The authors have no conflicts of interest to declare.

Keloid is an overgrowth of scar tissue that develops around a wound. The mechanisms of keloid formation and development still remain unknown, and no effective treatment is available. Searching for active natural resources may develop better prevention and treatment approaches for keloids. Aspidin PB is a natural resource with lower toxicity. We explored its effect on the regulation of TGF-β1-induced expression of type I collagen, CTGF, and α-SMA in keloid fibroblasts (KFs). Western blotting was used to detect the expression levels of type I collagen, CTGF, α-SMA, PI-3K/Akt and Smad-dependent and Smad-independent signaling pathway. The effect of aspidin PB on cell viability in human keloid fibroblasts was measured by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide). The percentage of the apoptotic cells was studied by flow cytometry. Based on our results, we revealed that aspidin PB inhibited the production of type I collagen, CTGF, and α-SMA in TGF-β1-induced KFs by blocking PI-3K/Akt signaling pathway. The TGF-β1-mediated phosphorylated levels of Smad2/3 were inhibited by aspidin PB pretreatment. Conclusively, our study suggests that aspidin PB has an inhibitory effect on fibrogenesis in TGF-β1-induced KFs. Our findings imply that aspidin PB has a therapeutic potential to intervene and prevent keloids and other fibrotic diseases., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Micro-ribonucleic acids (miR) are small, noncoding RNA molecules 19 to 25 nucleotides in length that typically function as negative regulators of expression for many target genes involved in cell proliferation, differentiation, and apoptosis. However, the effects of miR-21 on keloid fibroblasts are currently unknown., Objectives: The authors investigate whether miR-21, a specific miR implicated in multiple aspects of keloid fibroblasts, affects the expression of Fas ligand (FasL) in the presence of transforming growth factor (TGF)-β1., Methods: The relationship between TGF-β1 and miR-21 expression was investigated by TaqMan quantitative real-time polymerase chain reaction (Life Technologies, Grand Island, New York). FasL protein was determined by Western blotting, and regulation of cell proliferation/migration/apoptosis ability by TGF-β1 inhibitor or plasmid was evaluated respectively by EdU incorporation, Transwell assay, and flow cytometry analysis., Results: Fibroblasts from keloid tissue were confirmed to express high levels of TGF-β1 and miR-21 compared with normal skin fibroblasts. Expression of TGF-β1 and miR-21 was positively correlated in fibroblasts. In addition, cells transfected with TGF-β1 inhibitor or miR-21 inhibitor showed significant increases in FasL protein levels and number of apoptotic cells compared with control cells, whereas cell growth and migration significantly decreased. The opposite results could also be confirmed when TGF-β1 was upregulated in normal skin fibroblasts., Conclusions: TGF-β1 could effectively influence cell proliferation, apoptosis, and migration via its control of miR-21. These findings also identify a novel mechanism of interaction between TGF-β1 and miR-21 in the regulation of FasL protein, which is involved in keloid formation.

This work prepared and investigated the impact of carboxymethyl chitosan nanoparticles (MC-NPs) on the proliferative capability of keloid fibroblasts (KFBs) while analyzing the mechanistic roles of miR-214 and adenosine A2A receptor (A2AR) in fibroblasts within hypertrophic scars. MC-NPs were synthesized through ion cross-linking, were characterized using transmission electron microscopy (TEM) and laser particle size scattering. The influence of MC-NPs on the proliferation capacity of KFBs was assessed using the MTT method. Changes in the expression levels of miR-214 and A2AR in KFBs, normal skin fibroblasts (NFBs), hypertrophic scar tissue, and normal skin tissue were analyzed. KFBs were categorized into anti-miR-214, anti-miR-NC, miR-214 mimics, miR-NC, si-A2AR, si-con, anti-miR-214+ si-con, and anti-miR-214+ si-A2AR groups. Bioinformatics target prediction was conducted to explore the interaction between miR-214 and A2AR. Real-time quantitative PCR and immunoblotting (WB) were employed to detect the expression levels of miR-214, A2AR, apoptotic protein Bax, and TGF-β in different cells. Cell counting kit-8 (CCK8) and flow cytometry were employed to assess cell proliferation activity and apoptosis. The results indicated that MC-NPs exhibited spherical particles with an average diameter of 236.47 ± 4.98 nm. The cell OD value in the MC-NPs group was lower than that in KFBs (P < 0.05). The mRNA levels of miR-214 in KFBs and hypertrophic scar tissue were lower than those in NFBs and normal tissue (P < 0.001), while the mRNA and protein levels of A2AR were significantly elevated (P < 0.05). Compared to the control group and anti-miR-NC, the anti-miR-214 group showed significantly increased cell OD values and Bcl-2 protein expression (P < 0.001), decreased levels of apoptotic gene Bax protein, TGF-β gene mRNA, and protein expression (P < 0.001). Continuous complementary binding sites were identified between miR-214 and A2AR. Compared to the control group, the si-A2AR group exhibited a significant decrease in A2AR gene mRNA and protein expression levels (P < 0.001), reduced cell viability (P < 0.001), increased apoptosis rate (P < 0.001), and a significant elevation in TGF-β protein expression (P < 0.001). miR-214 targetedly regulated the expression of A2AR, inducing changes in TGF-β content, promoting the proliferation of keloid fibroblasts, and inhibiting cell apoptosis. [ABSTRACT FROM AUTHOR]

Aberrant 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. RT-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. In 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. Circ_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. All data generated or analyzed during this study are included in this article. • Enhancement of circ_0000129 expression in keloid. • Inhibited metastatic burden by circ_0000129 silence. • Circ_0000129 targets miR-485–3p. • MiR-485–3p targets SGMS2. [ABSTRACT FROM AUTHOR]

Purpose: Accumulating evidence has reported that microRNAs (miRNAs) play a critical role in the mechanism of keloid formation, and recent research found that miR-23b-3p was upregulated in keloid fibroblasts (KFs). Herein, we explored the potential effect of miR-23b-3p on fibroblasts in keloid. Materials and Methods: Clinical tissues, primary KFs and KEL FIB cells were used to detect the expression of miR-23b-3p by performing qRT-PCR. Gene knockdown was carried out to evaluate the molecular and biological changes of primary KFs and KEL FIB cells by conducting CCK-8 assay, flow cytometry and Western blot. The online databases and luciferase reporter assay were utilized to screen and identify the potential target of miR-23b-3p. Results: Upregulation of miR-23b-3p was detected in keloid tissues, primary KFs and KF cell line KEL FIB cells, and inhibition of miR-23b-3p promoted apoptosis and suppressed proliferation and the expression of collagen I, collagen III and fibronectin of primary KFs and KEL FIB cells. Further investigation revealed that TNFAIP3, the ubiquitin-editing enzyme A20, was the direct target of miR-23b-3p, and inhibition of miR-23b-3p promoted the expression of A20 in primary KFs and KEL FIB cells. The in vitro assays indicated that A20 suppression inhibited apoptosis and facilitated proliferation and the expression of collagen I, collagen III and fibronectin of miR-23b-3p inhibitor-transfected primary KFs and KEL FIB cells. Finally, we found that miR-23b-3p inhibitor reduced the expression of receptor interacting serine/threonine protein kinase 1 (RIPK1), which was partially reversed by A20 inhibition. Conclusion: These findings suggested that inhibition of miR-23b-3p/A20/RIPK1 axis induced apoptosis, limited proliferation and decreased extracellular matrix of KFs, providing a potential therapeutic target for treatment of keloid. [ABSTRACT FROM AUTHOR]

Keloid is a fibroproliferative disorder resulting from trauma, characterized by abnormal activation of keloid fibroblasts and excessive deposition of extracellular matrix (ECM). It affects life quality of patients and lacks of effective therapeutic targets. Protein tyrosine phosphatase 1B (PTP1B) belongs to the protein tyrosine phosphatases and participates in many cellular processes such as metabolism, proliferation and motility. It has been reported that PTP1B negatively regulated diabetic wound healing and tumor progression. However, its effects in keloid remain unclear. Here, we aimed to evaluate the effects of PTP1B on keloid fibroblasts which play essential roles in keloids pathogenesis. Our results revealed that PTP1B expression was decreased both in keloid tissues and in keloid fibroblasts compared to healthy controls. Keloid fibroblasts (KFs) showed higher cell proliferation, motility, ECM production and ERK activity than normal fibroblasts (NFs). Overexpression of PTP1B in KFs and NFs inhibited cell proliferation, motility, ECM synthesis and the MAPK/ERK signalling pathway while knockdown of PTP1B showed converse effects. The rescue experiments with ERK inhibitor further verified that MAPK/ERK signalling pathway involved in PTP1B regulatory network. Taken together, our findings indicated that overexpression of PTP1B suppressed keloid fibroblasts bio‐behaviours and promoted their phenotype switch to normal cells via inhibiting the MAPK/ERK signalling pathway, suggesting it may be a potential anti‐keloid therapy. [ABSTRACT FROM AUTHOR]

Long non-coding RNA (lncRNA) homeobox (HOX) A11 antisense (HOXA11-AS) mediates cell-biological phenotypes of keloid fibroblasts and influence the keloid progression, yet the underlying mechanism need to be further understood. HOXA11-AS, microRNA miR-148b-3p and Insulin like growth factor binding protein 5 (IGFBP5) expression were detected by RT-qPCR or western blot. CCK-8 and colony formation assays were applied to examine the cell proliferation. The cell migration was determined via Transwell migration assays. The cell apoptosis was determined by western blots with anti-Bax antibodies and anti-Cleaved Caspase-3 antibodies. The interplay between miR-148b-3p , HOXA11-AS and IGFBP5 was confirmed by luciferase reporter or RNA immunoprecipitation assay. The amplification of HOXA11-AS and IGFBP5 was detected in keloid and keloid fibroblasts, while miR-148b-3p expression was reduced. Moreover, downregulation of HOXA11-AS in keloid fibroblasts inhibited cell proliferation, migration and triggered apoptosis. Mechanically, HOXA11-AS was proved to sponge miR-148b-3p and abrogate the inhibition on miR-148b-3p target, IGFBP5 mRNA, thus promoting keloid fibroblasts proliferation, migration and inhibiting apoptosis. These results find that HOXA11-AS promotes keloid progression by miR-148b-3p/IGFBP5 axis, suggesting the potential of targeting HOXA11-AS/miR-148b-3p/IGFBP5 axis to combat keloid. • The IGFBP5 and miR-148b-3p were identified as the key regulators in keloid. • Silencing of HOXA11-AS significantly inhibited the proliferation and migration of KEL FIB. • HOXA11-AS specifically binds to miR-148b-3p. • HOXA11-AS silence upregulats miR-148b-3p leading to repressed proliferation and migration in KEL FIB. • Verification of HOXA11-AS/miR-148b-3p/IGFBP5 axis in KEL FIB. [ABSTRACT FROM AUTHOR]

Background: A keloid is a benign skin tumour characterised by excessive proliferation of fibroblasts, a process that requires a sufficient amount of energy. The energy needs are associated with adequate oxygen (O2) flow and well-functioning mitochondria. It is known that cytoglobin (CYGB) has a function in O2 distribution. The aim of the present study was to explore whether the inhibition of CYGB expression caused impaired mitochondrial function of keloid fibroblasts. Methods: An in vitro study was conducted on a keloid fibroblast derived from our previous study. The study was carried out in the laboratory of the Biochemistry & Molecular Biology Department, Faculty of Medicine, Universitas Indonesia (FMUI), from July to December 2018. CYGB expression was inhibited by small interfering ribonucleic acid (siRNA) and CYGB. Analysis of mitochondrial function was observed through peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), a mitochondrial biogenesis marker and the activity of the succinate dehydrogenase (SDH) enzyme in mitochondria. Results: The CYGB gene and protein were downregulated after treatment with CYGB siRNA. Inhibition of CYGB expression with siRNA also tended to decrease the levels of PGC-1α messenger ribonucleic acid (mRNA) and protein, as well as SDH enzyme activity. Conclusion: Inhibition of CYGB expression with siRNA tended to decrease mitochondrial biogenesis and function. This may be useful for understanding the excessive proliferation of fibroblasts in keloids and for development of treatment for keloids. [ABSTRACT FROM AUTHOR]

Thanks to their multi-potency and secretory functions, mesenchymal stem cells (MSCs) have long been established as an ideal cell type for skin wound healing and a candidate therapeutic strategy for excessive pathological scarring in the meantime. This study focuses on the effect of placental MSCs (PMSCs) on the activity of keloid fibroblasts (KFs) and the potential involvement of proenkephalin (PENK). Secretory protein of PMSC that are lowly expressed in KFs were predicted by bioinformatics analyses. The expression of PENK in KFs was detected by RT-qPCR and western blot analysis. PMSCs were co-cultured with KFs and dermal fibroblasts (DFs) to examine their effect on proliferation, migration, invasion, and apoptosis of the distinct cell types. PENK secretion by PMSCs and its uptake by KFs were examined by ELISA, WB, and immunofluorescence staining. Loss-of-functions of PENK and p38-MAPK were induced to examine the activity of KFs in vitro and in mice. PENK, a secretory protein of PMSCs, was conspicuously downregulated in KFs compared to normal DFs. PMSC stimulation suppressed proliferation, migration, invasion, and resistance to apoptosis of the co-cultured KFs but not DFs, which was ascribed to the upregulation of PENK protein in KFs. PMSCs-secreted PENK suppressed p38 phosphorylation in KFs. The proliferative and aggressive properties of KFs in vitro and the nodule-forming capacity of KFs in vivo were promoted upon PENK downregulation but suppressed by the p38 MAPK inhibitor SB202190. This work unravels that PMSCs-secreted PENK suppresses the p38 MAPK signaling to block hyperproliferation of KFs. • PENK is significantly downregulated in KFs. • Co-culturing with PMSCs inhibits activity of KFs. • PMSC-secreted PENK is taken up by KFs and suppresses the p38 MAPK signaling. • PENK knockdown promotes but p38 MAPK inhibition inhibits activity of KFs. • PENK knockdown promotes but p38 MAPK inhibition suppresses keloid genesis in mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background/Aims: Keloids are fibrous overgrowths induced by cutaneous injury. MicroRNAs (miRNAs) have recently emerged as post-transcriptional gene repressors and participants in a diverse array of pathophysiological processes leading to skin disease. The purpose of the current study was to explore the precise functions of miR-181a in human keloid development and the underlying mechanisms. Methods: A miRNA microarray analysis was performed to compare expression profiles between keloid and normal skin tissues. Quantitative real-time PCR was conducted to estimate miR-181a expression. Cell proliferation was determined using the cell counting kit-8 (CCK-8) and 5-ethynyl-2-deoxyuridine (EdU) assays, and cell cycle and apoptosis were detected with flow cytometry. Direct targets of miR-181a were identified using the luciferase reporter assay. Results: miR-181a was significantly upregulated in human keloid tissues and fibroblasts, compared with their control counterparts. Overexpression of miR-181a enhanced keloid fibroblast DNA synthesis and proliferation and inhibited apoptosis, whereas miR-181a suppression triggered the opposite effects. Moreover, miR-181a suppressed the expression of PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2) through direct interactions with its 3′UTR region and subsequently enhanced AKT activation. Overexpression of PHLPP2 without its 3′UTR attenuated the effects of miR-181a on cell proliferation and apoptosis in keloid fibroblast cells. Furthermore, miR-181a mimics increased normal skin fibroblast proliferation. Conclusions: Our results highlight a novel pathway mediated by miR-181a, which may be effectively used as a therapeutic target for treatment of keloids. [ABSTRACT FROM AUTHOR]

Summary: The mechanisms of keloid resistance to therapy and high recurrence rate remain undefined. This study explored the difference in drug resistance between keloid and normal fibroblasts. Fibroblasts derived from 9 patients with keloid and 9 skin donors were randomly combined into 3 keloid cell pools (3 cases per pool) and 3 normal cell pools (3 cases per pool) and were compared for their resistance to vincristine and mitoxantrone and related molecule expression. The results revealed stronger resistance to both vincristine and mitoxantrone with a higher survival rate in keloid cells than in normal cells (P < .05). The resistance of keloid fibroblasts could be largely abrogated by verapamil treatment. In addition, messenger RNA expression levels of multiple-drug resistance-1, ABCB5 (P-glycoprotein family member), and cytochrome P450 3A4 but not ABCG2 (ATP-binding cassette transporter) were significantly higher in passage 1 keloid fibroblasts than in passage 1 normal fibroblasts; no significant difference was found in latter passages. Immunohistochemistry staining revealed significantly more multiple-drug resistance-1–positive cells (round) in keloid tissue than in normal skin (mostly spindle shaped) (P < .05) but no significant difference in the percentage of positive cells in the 2 groups. Enhanced expression of membrane transporters and increased resistance to chemotherapy agents may contribute to the keloid''s resistance to therapy and the high posttherapy recurrence rate. [Copyright &y& Elsevier]

Abstract: SMAD3 is a key player in the TGFβ signaling pathway as a primary inducer of fibrosis. The inhibition of SMAD3 production is one strategy to alleviate fibrosis in keloid fibroblasts. In the present study, antisense oligonucleotides (ASOs) against SMAD3 were designed to specifically block the expression of SMAD3. The cationic lipid nanoparticles (cLNs) were formulated to enhance an intracellular activity of SMAD3 ASOs in keloid fibroblasts. This formulation was prepared using melt-homogenization method, composed of 3-[N-(N′,N′-dimethylaminoethane)-carbamol] cholesterol (DC-Chol), dioleoylphosphatidylethanolamine (DOPE), Tween20, and trimyristin as a lipid core (1:1:1:1.3, w/w). The size and zeta potential of cLNs and cLN/ASO complexes were measured using light scattering. AFM was used to confirm the morphology and the size distribution of cLNs and cLN/ASO complexes. The prepared cLNs had a nano-scale sized spherical shape with highly positive charge, which were physically stable without aggregation during the storage. The cLN/SMAD3 ASO complexes were successfully generated and internalized onto keloid fibroblasts without toxicity. After the treatment with cLN/ASO complexes, SMAD3 was inhibited and collagen type I was also significantly suppressed in keloid fibroblasts. These results suggest that SMAD3 ASOs complexed with cLNs have a therapeutic potential to suppress collagen deposition in fibrotic diseases. Therefore, this strategy might be developed to lead to anti-fibrotic therapies. [Copyright &y& Elsevier]

Treatment of keloid disease (KD) is ill-defined and remains challenging. We previously reported successful clinical application of photodynamic therapy (PDT) in KD. The aim here was to evaluate cytotoxic effect of PDT using methyl aminolevulinate (M-ALA) and 5-aminolevulinic acid (5-ALA) on keloid fibroblasts (KF) ( n = 8) from different lesional sites (top, middle and margin) as compared to normal skin fibroblasts ( n = 3). The effect of protoporphyrin IX (PpIX) precursors was evaluated by fluorescence emission, LDH and WST-1 assay, reactive oxygen species (ROS) generation and qRT-PCR analysis. Apoptosis/necrosis differentiation and senescence were studied by fluorometric staining with Hoechst 33258/propidium iodide and β-galactosidase activity, respectively. Three hours post incubation with 4 mM precursors of photosensitisers, PpIX accumulation was site specific and higher with M-ALA. Cytotoxicity was also site specific (higher in fibroblasts from middle of the keloid as compared to cells from other sites) and increased proportionately to fluence rates post-PDT. Additionally, cytoproliferation was significantly decreased post-PDT depending on the light energy. Fluorescence analysis revealed that M-ALA instigated higher KF cytotoxicity at lower fluence (≤20 J/cm) while 5-ALA instigated higher KF cytotoxicity at higher fluence, except in cells derived from middle of the keloid. ROS-mediated cytotoxicity was light energy dependent. Senescence was not observed at higher light energies (>10 J/cm). Compared to other sites, fibroblasts from the middle were more prone to cell death post 5-ALA treatment. We conclude that cytotoxicity post-PDT in KD fibroblasts is dependent on the lesional site, precursor of intracellular photosensitiser and fluence. Thus, PDT may be used for site-targeted therapy of KD. [ABSTRACT FROM AUTHOR]

Abstract: Background: Current evidence suggests the potential role of Wnt signalling in keloids pathogenesis but such literature remains scanty. We hypothesize that Wnt signalling is upregulated in keloid fibroblasts (KFs) and this promotes cellular growth, migration and extracellular matrix (ECM) production in such fibroblasts. Objectives: To verify the downregulation of secreted frizzled-related protein 1 (SFRP1), a Wnt inhibitor and test KFs sensitivity to Wnt3a treatment compared to NFs in terms of activation of Wnt/β-catenin, cellular growth, migration and ECM expressions. Next, to investigate if ectopic expression of SFRP1 and treatment of quercetin in KFs can reverse their phenotypes. Methods: Quantitative Real-time PCR and western blotting were used to verify SFRP1 expression in NFs and KFs. The fibroblasts were tested with Wnt3a conditioned media and its effects were tested for (1) the cells’ sensitivity to direct Wnt signalling via the activation of TCF reporter assay and protein expression of β-catenin, (2) cellular growth, (3) cell migration and (4) expressions of ECM components. Finally KFs were stably transduced with SFRP1 and treated with 2 doses of quercetin. Results: Lower levels of SFRP1 were confirmed at mRNA and protein levels in KFs which partly explained their sensitivity to Wnt3a treatment in terms of higher Wnt activation, cellular growth and fibronectin expression. Interestingly, Wnt3a did not promote higher cell migration rate and increase in collagen I expression. Ectopic expression of SFRP1 and quercetin treatment was able to mitigate Wnt3a-mediated phenotype of KFs. Conclusions: Using SFRP1 or inhibitors of Wnt signalling might be one of the therapeutic solutions to treat keloid scarring. [Copyright &y& Elsevier]

Abstract: Background: We recently reported use of photodynamic therapy (PDT) for treating keloid disease (KD). However, in view of high recurrence rates post any treatment modality, adjuvant therapies should be considered. Additionally, we previously demonstrated the effect of a novel electrical waveform, the degenerate wave (DW) on differential gene expression in keloid fibroblasts. Objective: In this study, we evaluated the in vitro cytotoxic effect of PDT at 5J/cm2 and 10J/cm2 of red light (633±3nm) using 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) with and without DW, on keloid fibroblasts compared to normal skin fibroblasts. Methods: The rate of intracellular photosensitizer (protoporphyrin IX, PPIX) generation and disintegration, reactive oxygen species (ROS) generation, LDH cytotoxicity, WST-1 cytoproliferation, apoptosis by Caspase-3 activation, mitochondrial membrane potential assessment by JC-1 aggregates, qRT-PCR, flow cytometry and In-Cell Western Blotting were performed. Results: PPIX accumulation and disintegration rate was higher in keloid than normal fibroblasts after incubation with MAL compared to ALA. Increased cytotoxicity and decreased cytoproliferation were observed for keloid fibroblasts after PDT+DW treatment compared to PDT alone. ROS generation, mitochondrial membrane depolarization, apoptosis (Caspase-3 activation) and collagens I and III gene down-regulation were higher in keloid compared to normal skin fibroblasts after MAL–PDT+DW treatment. An increase in the number of cells entering apoptosis and necrosis was observed after PDT+DW treatment by flow cytometry analysis. All positive findings were statistically significant (P <0.05). Conclusion: The cytotoxic effect of PDT on keloid fibroblasts can be enhanced significantly with addition of DW stimulation, indicating for the first time the utility of this potential combinational therapy. [Copyright &y& Elsevier]

Abstract: Transforming growth factor-beta receptor II (TGF-βRII) is an attractive target for anti-scarring therapy in wound healing because it attenuates excessive TGF-β which has pleiotropic effects on the immune system. In the present study, the cDNA of rabbit TGF-βRII (rTGF-βRII) was amplified from rabbit peripheral blood by RT-PCR. The open reading frame of rTGF-βRII encodes a protein consisting of 567 amino acids, which contains a predicted transmembrane domain and a Serine/Threonine protein kinase domain similar to other identified mammalian TGF-βRIIs. The amino acid sequences of the biologically active, soluble rTGF-βRII and mouse, rat, human and chicken counterparts are 81%, 81%, 89% and 61%, respectively, identical. Recombinant soluble rTGF-βRII (rsTGF-βRII) fused with His tag was efficiently expressed in Escherichia coli BL21 (DE3) expression host strain. This fusion protein''s molecular weight of ∼19kDa was identified by SDS-PAGE and Western blotting. In vitro, purified rsTGF-βRII was able to inhibit the proliferation of keloid rabbit fibroblasts and decrease the level of collagen. These findings indicate that rTGF-βRII plays an important role in inhibiting the proliferation of keloid rabbit fibroblasts and provides the basis for investigations on the role of TGF-βRII in this important domestic species. [Copyright &y& Elsevier]

Background Keloids are recognized as benign tumours characterized by fibroblastic proliferation and accumulation of extracellular matrix, especially collagen deposition. The transforming growth factor (TGF)-β1/Smad pathway plays an important role in keloid pathogenesis; however the underlying mechanisms are not fully understood. Objectives To define further the mechanisms of TGF-β1/Smad signal transduction mediated by mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 pathways, in keloid fibroblasts. Methods In the absence or presence of three MAPK (ERK, JNK and p38)-specific inhibitors, keloid fibroblasts were stimulated with exogenous TGF-β1 to activate Smad signalling. Smad protein expression was measured by immunoprecipitation/immunoblotting and immunofluorescence; plasminogen activator inhibitor (PAI)-1 transcriptional activity was measured by real-time reverse transcriptase-polymerase chain reaction analysis. Results TGF-β1 induced Smad2/3 phosphorylation at both the C-terminal and the linker region, thus promoting formation of the Smad2/3/4 complex and nuclear translocation, and PAI-1 mRNA expression in keloid fibroblasts; in addition, TGF-β1 decreased inhibitory Smad7 expression. Meanwhile, the p38 inhibitor significantly inhibited Smad2/3 phosphorylation, especially at the linker region, and furthermore blocked Smad2/3/4 complex formation, and thus decreased PAI-1 mRNA expression; decreased Smad7 expression induced by TGF-β1 was also reversed by the p38 inhibitor. The ERK and JNK inhibitors interrupted Smad2/3/4 complex translocation into the nucleus and consequently decreased PAI-1 mRNA expression. Conclusions These results suggested that the ERK, JNK and p38 pathways mediate TGF-β1/Smad signal transduction and might be considered as specific targets of drug therapy for keloids. [ABSTRACT FROM AUTHOR]

Hypertrophic scars and keloid are dermal proliferative disorders in wound healing. Transforming growth factor β (TGF-β) has been implicated in scar formation through the activation of fibroblasts and the acceleration of collagen deposition. Our study aimed to design a novel truncated (27-123 residues) type II TGF-β receptor (tTGFβRII) and to determine its effects on the proliferation of keloid fibroblasts and the collagen synthesis as well as TGF-β I expression of the cells. The coding sequences of TGF-β I and tTGFβRII were amplified using RT-PCR and then cloned into pGBKT7 and pGADT7 vectors. A yeast two-hybrid experiment and a glutathione S-transferase (GST)-pull down assay were performed to verify the affinity of tTGFβRII to TGF-β I. Our results indicated that treatment with tTGFβRII inhibited the growth of keloid fibroblasts and suppressed the synthesis of type I collagen in keloid fibroblasts in a concentration-dependent manner. Moreover, northern and western blot analysis revealed a decline of the TGF-β I expression at both mRNA and protein levels after exposure to 5, 10 or 20 μg/ml of tTGFβRII. Together, our data suggested that the exogenous tTGFβRII can efficiently trap TGF-β I from access to wild-type receptors and can suppress TGF-β I triggered signals. Thus it may potentially be clinically applied to scar therapy. [ABSTRACT FROM AUTHOR]

Abstract: Keloids represent a prolonged inflammatory fibrotic state with areas that display distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells, and a milieu of enriched cytokines. Previous studies from our laboratory demonstrated an intrinsic higher level of HIF-1α and VEGF protein expression in keloid tissues compared with their adjacent unremarkable skins. To further investigate the mechanisms underlying the elevated expression of HIF-1α and VEGF in keloids, we exposed a co-culture of keloid fibroblasts and mast cells (HMC-1) to hypoxic conditions and studied the expression of HIF-1α and its target gene, VEGF. Our results showed that hypoxia-dependent HIF-1α protein accumulation and VEGF expression is augmented in keloid fibroblasts when co-cultured with HMC-1 cells under the condition where direct cell–cell contact is allowed. But such augmentation is not observed in the transwell co-culture system whereas fibroblasts and HMC-1 cells were separated by a porous membrane. Our results also indicated that the enhancement of hypoxia-mediated activation of ERK1/2 and Akt requires direct cell–cell interaction between mast cells and keloid fibroblasts, and activation of both ERK1/2 and Akt is involved in the hypoxia-dependent HIF-1α protein accumulation and VEGF expression in the co-culture system. These findings suggest that under hypoxic conditions mast cells may contribute, at least in part, to an elevated expression of HIF-1α and VEGF protein in keloids via direct cell–cell interaction with fibroblasts. [Copyright &y& Elsevier]

Keloids are characterised by the excessive accumulation of extracellular matrix ( ECM), especially overabundant collagen formation. In keloid fibroblasts ( KFs), transforming growth factor ( TGF)-β-dependent signalling is closely associated with a variety of keloid pathologic responses such as ECM production and fibroblast overgrowth. Thus, inhibition of TGF-β signalling would be a potential therapeutic approach to prevent keloid scar formation. Thereby, we aimed to identify a novel TGF-β signalling blocker among natural products using a simplified screening approach. We discovered that the extract of Aneilema keisak ( A. K- Ex) lowered TGF-β-dependent signalling by reducing Smad2 protein levels. Given that KFs showed altered dependency on TGF-β for survival and proliferation, A. K- Ex-mediated reduction in Smad2 protein levels significantly inhibited the major characteristics of KFs such as cell growth, migration and collagen synthesis, suggesting that A. K- Ex exhibits possible therapeutic activity on keloids. [ABSTRACT FROM AUTHOR]