In re-cent 20 years, the development of cell biology technology has promoted the research of keloid. Keloid fibroblasts (KFbs) are the main effector cells in keloid, which are closely related to the occurrence and development of keloid. It is significantly different in terms of biological characteristics and gene expression between KFbs and normal fibroblasts. This articles reviews the characteristics of KFbs from multiple perspectives, describing its biological character- istics in details including microstructures, metabolic character- istics, and proliferation properties, and introducing the main characteristics of heterogeneity and genomics of KFbs. The further research on KFbs will help to elucidate the pathogenesis of keloids and provide valuable strategies for the prevention and treatment of keloids.
Fibroblasts/metabolism* ; Humans ; Keloid/pathology*

Keloids are a common type of pathological scar as a result of skin healing, which are extremely difficult to prevent and treat without recurrence. The pathological mechanism of keloids is the excessive proliferation of fibroblasts, which synthesize more extracellular matrices (ECMs), including type I/III collagen (COL-1/3), mucopolysaccharides, connective tissue growth factor (CTGF, also known as cellular communication network factor 2 (CCN2)), and fibronectin (FN) in scar tissue, mostly through the abnormal activation of transforming growth factor-‍β (TGF-‍β)/Smads pathway (Finnson et al., 2013; Song et al., 2018). Genetic factors, including race and skin tone, are considered to contribute to keloid formation. The reported incidence of keloids in black people is as high as 16%, whereas white people are less affected. The prevalence ratio of colored people to white people is 5:1‍‍-‍‍15:1 (Rockwell et al., 1989; LaRanger et al., 2019). In addition, keloids have not been reported in albinism patients of any race, and those with darker skin in the same race are more likely to develop this disease (LaRanger et al., 2019). Skin melanocyte activity is significantly different among people with different skin tones. The more active the melanocyte function, the more melanin is produced and the darker the skin. Similarly, in the same individual, the incidence of keloids increases during periods when melanocytes are active, such as adolescence and pregnancy. Keloids rarely appear in areas where melanocytes synthesize less melanin, such as in the palms and soles. Thus, the formation of keloids seems to be closely related to melanocyte activity.
Adolescent ; Cells, Cultured ; Exosomes/metabolism* ; Fibroblasts/metabolism* ; Humans ; Keloid/pathology* ; Melanins/metabolism* ; Melanocytes/pathology* ; Pilot Projects ; Skin/metabolism* ; Transforming Growth Factor beta/metabolism*

OBJECTIVETo investigate the effect of Metformin on the proliferation and collagen synthesis of the human keloids fibroblasts as well as the effect on phosphorylation of Akt/FoxO1 signal transduction pathway.

METHODSFibroblasts of keloid were divided into control group treated with medium solution and experimental groups treated with different concentrations of Metformin. 48 h later CCK-8 assay was adopted to evaluate cell survival; Western blot was performed to detect the Akt and FoxO1 phosphorylation; and Hydroxyproline reagent kit was used to detect the collagen synthesis.

RESULTSWith different concentrations (30, 60, 90, 120 mmol/L) of Metformin, the absorbance of cultured keloid fibroblasts detected by CCK8 assay decreased by (13.30 ± 2.04)%, (22.64 ± 4.70)%, (54.00 ± 5.34)% and (63.12 ± 3.48)%. The growth of fibroblasts was suppressed by Metformin in a dose-dependent manner. It showed that the level of phoshpo-akt and phoshpo-foxOl in keloids fibroblasts in experimental groups was lower than that in the control group and the collagen synthesis were also decreased in experimental groups, all in a dose-dependent manner (P < 0.05, P < 0.01).

CONCLUSIONSMetformin can effectively inhibit the proliferation and collagen synthesis of the human keloids fibroblasts in vitro, which may be associated with the suppression of phosphorylation of Akt/FoxO1 signaling pathway

Cell Proliferation ; drug effects ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; metabolism ; Forkhead Box Protein O1 ; Forkhead Transcription Factors ; metabolism ; Humans ; Keloid ; pathology ; Metformin ; pharmacology ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects

OBJECTIVETo construct and characterize the TGF-β1, induced epithelial-mesenchymal transition (EMT) model of keloid epithelial cells in vitro, and to investigate the expression of epithelial stem cells related surface markers in keloid epithelial cells during EMT induction.

METHODSThe epithelial cells from 3 keloid samples of ears were cultured in vitro and induced by transforming growth factor betal (TGF-β1, 1 ng/ml) for 5 days, which was the experimental group, the same cells untreated were considered as the negative control group. The expressions of EMT-associated markers and regulative genes were detected using immunofluorescence staining, real-time PCR and western blot analysis. Then the surface markers of epithelial stem cells were detected using real-time PCR. Statistical significance was determined using Independent-Samples t Test, a p value less than 0. 05 was considered statistically significant.

RESULTSThe mRNA expression of transcription factor snail2 and mesenchymal-specific marker vimentin increased significantly in TGF-β1, induced keloid epithelial cells (P < 0. 05), in which snail2 increasing from 0. 91 ± 0. 23 to 1. 69 ± 0. 10, and vimentin from 5. 86 ± 2. 07 to 24. 29 ± 5. 39. Whereas the mRNA expression of epithelial-specific marker E-cadherin decreased from 1. 06 ± 0. 19 to 0. 65 ± 0. 09. The mRNA expression of CD29 and Lgr6, two surface markers of epithelial stem cells, significantly increased after induction of the TGF-β1, (P < 0. 05), from 0. 55 ± 0. 14 and 1. 61 ± 0. 31 to 1. 19 ± 0. 12 and 3. 84 t 0. 62 respectively. In induced cells, the immunofluorescence results showed staining of E- cadherin became faint, but the number of positive staining cells of vimentin increased. Western blot confirmed the protein expression of E-cadherin weakened, and the vimentin and p-Smad3 enhanced (P < 0. 05).

CONCLUSIONSTGF-β1, initiated EMT in keloid epithelial cells by inducing the up-regulation of snail2, and TGF-β1,/Smad3 signaling pathway was involved in EMT. EMT could change the phenotype of epithelial stem cells in keloid.

Biomarkers ; metabolism ; Cadherins ; genetics ; metabolism ; Epithelial Cells ; drug effects ; physiology ; Epithelial-Mesenchymal Transition ; drug effects ; physiology ; Humans ; In Vitro Techniques ; Keloid ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction ; Smad3 Protein ; genetics ; metabolism ; Snail Family Transcription Factors ; Transcription Factors ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; pharmacology ; Up-Regulation ; Vimentin ; genetics ; metabolism

OBJECTIVETo investigate the effect of hIL-24 gene on proliferation, migration and invasion activity of human keloid fibroblasts (KFs).

METHODShIL-24 gene was cloned into lentivirus vector, then the lentivirus particles expressing hlL-24 were infected into KF cells. Real-time PCR and Western blot were performed to examine the expression of hIL-24 in lentivirus infected cells. The growth ability was detected by MTT assay. The cell cycle was analyzed by flow cytometry, The invasion and migration were detected by matrigel invasion assay and wound healing assay.

RESULTSComparing to controls group and KF-NC group, the expression levels of hIL-24 mRNA and protein were both significantly up-regulated after 4 days of hIL-24 lentivims infection. Comparing with the KF-NC group, MTT assay showed that the A490 of KF-hlL-24 group was down-regulated after lentivims infection ( P < 0. 05 ). Comparing with the KF-NC group, Cell cycle test revealed hlL-24 gene could block KF cells in G1 [(75. 40 ±2. 10)% ] , the proportion of KF cells was decreased in S phase [(4. 96 ± 1. 60)% ] and G2 phase [(0.01 ± 0.01)% ]. After KF cells were infected(P <0.01). Transfection of hlL-24 lentivirus inhibited the migration and invasion activity of KF cells.

CONCLUSIONLentivirus-mediated hlL-24 gene efficiently inhibits proliferation, cell cycle progression, migration and invasion activity of KF cells.

Cell Cycle ; Cell Line, Tumor ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Down-Regulation ; Fibroblasts ; physiology ; virology ; Genetic Vectors ; Humans ; Interleukins ; genetics ; physiology ; Keloid ; genetics ; pathology ; Lentivirus ; RNA, Messenger ; metabolism ; Transfection ; methods

OBJECTIVETo study the expressions of Notch1-4 gene in human keloid-derived mesenchymal-like stem cells, and to explore the Notch signaling pathway's role in the formation of keloid.

METHODSKeloid samples were collected to harvest human keloid-derived mesenchymal-like stem cells through two-step enzymatic dissociation method. By flow cytometry, cell phenotype of primary and P3 generation were analyzed. By immunocytochemistry, the expressions of Oct4, vimentin and CK19 were examined. Keloid-derived mesenchymal-like stem cells were induced into osteoblasts in vitro and calcium deposition was detected by Alizarin red S stain. Realtime polymerase chain reaction (RT-PCR) was used to detect the expressions of Notch1-4 mRNA in keloid-derived mesenchymal-like stem cells.

RESULTSFlow cytometry showed that keloid-derived mesenchymal-like stem cells of primary and P3 generation highly expressed CD29, CD44, CD90 from the typical MSC phenotype marker, but they failed to express HSC phenotype markers, such as CD34 and CD45. The results of immunocytochemistry showed that Oct4 from pluripotent stem cell markers and vimentin from mesenchymal cell markers was positive and CK19 from epithelial cell markers was negative. After induced differentiation into osteoblasts in vitro after 21 day, calcium nodules could be seen clearly; Notch1-4 gene were expressed in keloid-derived mesenchymal-like stem cells through RT-PCR. The relative quantitative of Notch2, Notch3 gene were higher than Notch1, Notch4 gene (P < 0.05).

CONCLUSIONSThe expression difference of different subtypes from Notch gene in human keloid-derived mesenchymal-like stem ceils may be related to self-renewal, proliferation, differentiation, and participate in the formation of keloid.

Adolescent ; Cells, Cultured ; Child ; Female ; Humans ; Keloid ; metabolism ; pathology ; Male ; Mesenchymal Stromal Cells ; metabolism ; Receptors, Notch ; metabolism

OBJECTIVETo verify the existence and significance of calcium/calmodulin dependent serine protein kinase/inhibitors of differentiation 1 (CASK/Id1) pathway in fibroblasts of human keloid.

METHODSImmunofluorescence laser was used to confirm CASK and Id1 protein expression and localization in fibroblasts of the keloid and normal skin. RT-PCR and Western-blot were adopted to analysis the CASK and Id1 expression and differences between keloid and normal skin fibroblasts. The natural combination of CASK and Id1 protein of keloid fibroblasts was tested by immunoprecipitation.

RESULTSCASK and Id1 protein expression were both found in fibroblast cells of keloid and normal skin under normal circumstances. Most of CASK and Id1 were distributed in the cytoplasm and nucleus of fibroblasts. The results of RT-PCR showed that the expression of CASK mRNA in the keloid group was 0.658 +/- 0.024, which was lower than that in the normal control group (1.076 +/- 0.008, t = 11.159, P < 0.05). The expression of Id1 mRNA was 0.497 +/- 0.014, which was higher than that in the normal control group (0.307 +/- 0.017, t = 15.148, P < 0.05). The results of Western-blot showed that the expression level for CASK protein in the keloid group was 0.057 +/- 0.006, which was lower than that in the normal control group (0.168 +/- 0.012, t = 13.524, P < 0.05); the expression of Id1 protein was 0.812 +/- 0.035, which was higher than that in the normal control group (0.368 +/- 0.031, t = 16.356, P < 0.05). The results of immunoprecipitation showed that Id1 could be detected in the CASK precipitate, while CASK also could be detected in the Id1 precipitate. There was a natural binding of CASK and Id1 in keloid fibroblasts.

CONCLUSIONCASK/Id1 signal pathway may be existed and involved in the proliferation of keloid fibroblasts, which is related with the occurrence of keloid.

Cell Proliferation ; genetics ; Cyclin-Dependent Kinase Inhibitor Proteins ; genetics ; metabolism ; Fibroblasts ; metabolism ; Humans ; Inhibitor of Differentiation Protein 1 ; genetics ; metabolism ; Keloid ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction

OBJECTIVETo investigate the effect of 5-aza-2-deoxycytidine on the TGF-beta/smad signal transduction pathway in human keloid fibroblasts (KFSs).

METHODSFirstly, immunohistochemical method was used to detect the positive expression rate of phospho-smad2 and phospho-smad3 in the specimens of 15 cases of keloid and 15 cases of normal skin. The keloid fibroblasts were cultured in vitro with 5-aza-2-deoxycytidine(experimental group) or with DMEM (control group). The effect of 5-aza-2-deoxycytidine on the cell cycle and apoptosis of fibroblasts was analysed with flow cytometry ( FCM). Transforming growth factor (TGF)-beta1, Smad7, phospho-smad2 and phospho-smad3 were analyzed by Western Blot, and Immunofluorescence.

RESULTSIt was found that the positive expression of phospho-smad2 and phospho-smad3 in keloid were higher than those in normal skin. The FCM showed that the proportion of cells in G0/G1 stage was increased, and so does the proportion of apoptosis cells in keloid fibroblasts intervened by 5-aza-2-deoxycytidine. The expression of TGF-beta1, phospho-smad2 and phospho-smad3 protein were significantly suppressed while the expression of smad7 protein increased in keloid fibroblasts with 5-aza-2-deoxycytidine. In addition, 5-aza-2-deoxycytidine reversed phosphorylation and nuclear translocation of smad2 and smad3.

CONCLUSIONS5-aza-2-deoxycytidine, methylase inhibitors, inhibits cell proliferation and promotes apoptosis of KFSs, which may be associated with the suppression of TGF-beta/smad signal pathway.

Apoptosis ; drug effects ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Enzyme Inhibitors ; pharmacology ; Female ; Fibroblasts ; drug effects ; metabolism ; Humans ; Keloid ; metabolism ; pathology ; Male ; Signal Transduction ; drug effects ; Smad2 Protein ; metabolism ; Smad3 Protein ; metabolism ; Smad7 Protein ; metabolism ; Transforming Growth Factor beta ; metabolism

OBJECTIVETo screen out related microRNAs in keloid tissue, and identify their effect on the proliferation of keloid fibroblasts.

METHODS8 cases of keloid tissue and 8 cases of normal skin tissue were collected as specimens. The differently expressed miRNA in keloid tissue from normal skin tissue were screened out with gene chip( Exiqon company), which was validated with quantitative real-time PCR. Then miRNA mimics was transfected into keloid fibroblasts line to stimulate high expression of mature miRNA in cells. The effect on the proliferation of fibroblasts in keloid was tested by Edu.

RESULTS(1) A total of 17 differently expressed microRNAs were found, including miR-199a-5p. (2) The expression of miR-199a-5p had been verified by qRT-PCR to be down-regulated in keloid, which was consistent with the result of array. (3) The positive rate of EdU in miR-199a-5p mimics transfected group and negative control group was (20.72 +/- 2.50)% and (27.68 +/- 4.92)%, respectively. The proliferative rate of keloid fibroblasts turned down in miR-199a-5p-transfected group (t = 2.183, P = 0.047). Besides that, the cell cycle changed after transfection. The percentage of S and G2/M phase in miR-199a-5p mimics transfected group was 33.93 +/- 1.30 and 10.87 +/- 0.80, respectively, while it was 31.39 +/- 0.79 and 9.27 +/- 0.46 in negative control group, and the difference was statistically significant.

CONCLUSIONS(1) The miRNA expression profile is different between keloid and normal skin; (2) The expression of miR-199a-5p is down-regulated in keloid and miR-199a-5p can affect the cell cycle and suppress proliferation of keloid fibroblasts. It indicateds that miR-199a-5p may be involved in regulating fibroblastic proliferation.

Cell Proliferation ; Cells, Cultured ; Down-Regulation ; Female ; Fibroblasts ; metabolism ; Gene Expression Profiling ; Humans ; Keloid ; genetics ; metabolism ; pathology ; Male ; MicroRNAs ; genetics ; metabolism

OBJECTIVETo verify whether abnormal expression of calcium/calmodulin dependent serine protein kinase (CASK) and inhibitors of differentiation 1 (ID1) exist in Fb of keloid, and to observe the effect of artesunate on two genes.

METHODSFifteen samples of keloid and 12 samples of normal skin tissue (discarded) excised from patients admitted to our hospital were collected. Tissue particle adherent method was used in the primary culture of Fb, and cells from the third to the eighth passage were used for test. Expressions of CASK and ID1 in Fb harvested from both sources were observed with immunofluorescence staining. Fb of keloid were stimulated with artesunate in various concentration for different time, and the median inhibitory concentration (IC50) was determined with the MTT colorimetric assay, which served as the intervention concentration of artesunate. Fb of normal skin were set as normal control group (NC, treated with medium solution). Fb of keloid were divided into scar control group (SC, treated with medium solution) and scar administration group (SA, treated with artesunate in IC50). The cycle and apoptosis of Fb were detected with flow cytometric assay, and the nucleic acid and protein expressions of CASK and ID1 of Fb in each group were determined with RT-PCR and Western blotting. Data were processed with one-way analysis of variance and LSD-t test.

RESULTSExpressions of CASK and ID1 were detected in two kinds of Fb. The concentration of 75 mg/L was selected as the intervention concentration of artesunate. (1) There were statistically significant differences among the three groups in the percentages of cells in G0/G1 phase and G2/M phase (with F values respectively 118.064 and 163.840, P values all below 0.01). The percentage of cells in G0/G1 phase of group SA was (91.4 ± 1.4)%, which was significantly higher than that of group SC and group NC [respectively (80.7 ± 0.3)% and (82.4 ± 0.6)%, with t values respectively 12.740 and 9.872, P values all below 0.05]. The percentage of cells in G2/M phase of group SA was (6.9 ± 0.3)%, which was significantly lower than that of group SC and group NC [respectively (13.7 ± 0.3)% and (12.7 ± 0.8)%, with t values respectively 43.702 and 12.276, P values all below 0.05]. (2) There were statistically significant differences among the three groups in the early and late apoptotic rates (with F values respectively 61.879 and 4710.862, P values all below 0.01). The early and late apoptotic rates of group SA were respectively (7.1 ± 1.0)% and (14.9 ± 0.3)%, which were significantly higher than those of group SC and group NC [with early apoptotic rate respectively (2.6 ± 0.4)% and (2.7 ± 0.3)%, t values respectively 7.974 and 7.767, P values all below 0.05; with late apoptotic rate respectively (2.3 ± 0.3)% and (2.5 ± 0.4)%, t values respectively 72.882 and 69.792, P values all below 0.05]. (3) The mRNA expression of CASK in group SC was 0.658 ± 0.024, and it was lower than that of group NC (1.076 ± 0.008, t = 28.997, P < 0.01) and group SA (0.855 ± 0.008, t = 13.549, P < 0.01). The protein expression of CASK in group SC was 0.067 ± 0.007, and it was lower than that of group NC (0.179 ± 0.015, t = 12.042, P < 0.01) and group SA (0.132 ± 0.010, t = 9.498, P < 0.01). (4) The mRNA expression of ID1 in group SC was 0.416 ± 0.006, which was higher than that of group NC (0.317 ± 0.020, t = 8.299, P < 0.01) and group SA (0.217 ± 0.009, t = 32.417, P < 0.01). The protein expression of ID1 in group SC was 0.789 ± 0.034, and it was higher than that of group NC (0.366 ± 0.029, t = 16.341, P < 0.01) and group SA (0.114 ± 0.006, t = 33.978, P < 0.01).

CONCLUSIONSIt is speculated that CASK and ID1 participate in the proliferation of Fb in keloid. The mechanism of artesunate in inhibiting the proliferation of Fb in keloid may be related to the up-regulation of CASK and down-regulation of ID1.

Adolescent ; Adult ; Apoptosis ; drug effects ; Artemisinins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Fibroblasts ; metabolism ; Gene Expression Regulation ; Guanylate Kinases ; genetics ; metabolism ; Humans ; Inhibitor of Differentiation Protein 1 ; genetics ; metabolism ; Keloid ; metabolism ; pathology ; Male ; Middle Aged ; Young Adult