Effect of SRSF2 on the biological characteristics of keloid fibroblasts

Objective To observe the effects of IFNα-2b on keloid fibroblasts in cell prolifera-tion, apoptosis, expression of hTERT and bcl-2 mRNA and to explore its anti-keloid mechanism. Methods Primary cultures of dermal fibroblasts derived from 8 keloid and 8 normal skin samples were established, strains of fibroblasts at passages 3 to 4 were used in this study. Keloid and normal skin fibroblasts in culture medium in vitro were given IFNα-2b and were obsevered in different time. The proliferation of the fibroblasts was measured by MTT assay, the apoptosis was analysed by flow cytometry(FCM), and the expression of hTERT and bcl-2 mRNA were obsevered by semi-qnantitativere verse transcriptase-polymerase chain reaction (RT-PCR). The data were analyzed by statistical software (SPSS11. 5). Results IFNα-2b could inhibit the growth of keloid and nomal skin fibroblasts. The suppression of keloid and nomal skin fibroblasts was time-dependent. After the effect of 10 000 U/ml INFα-2b on cultured fibroblast of keloid and normal skin,the fibroblasts apoptosis was induced and the expression of hTERT and bcl-2 mRNA was lower than that of controlled group . The result was significantly different between control group and treatment group and was related with the treatment time of INFα-2b (P<0.01). Conclusions As a negative regulatory factor,interferon α-2b can suppress growth and proliferation of keloid fibroblasts and induce apoptosis. Decreasing the telomerase activity of keloid fibroblasts may be one of the most important mechamisms. That IFNa-2b inhibited telomerase activity in keloid fibroblasts is an important pathway that may play a key role in the anti- keloid therapy. Key words: Keloid; hTERT; Apoptosis; IFNα-2b

Objective To detect the expression and content of decorin in fibroblasts of keloid to deeply reveal the mechenism and the role of decorin plays in scar formation.Methods Fibroblasts of keloid,normal scar and normal skin were cultured in vitro,and the morphology,activity,apoptosis of fibroblast were observed under light microscope and electron microscope; the mRNAs of decorin and TGF-β1 were detected and analyzed with real-time fluorescent quantitative-PCR (FQ-PCR).Results Fibroblasts of keloid showed irregular morphology,larger size and disorder arrangement.There were a large number of mitochondria,swelling rough endoplasmic reticulum,and euchromatin-rich in nucleus of fibroblasts,suggesting the protein synthesis of keloid fibroblast was very active.Compared with normal skin,the expression of decorin was significantly lower in keloid fibroblast; On the contrary,the expression of TGF-β1 was significantly higher in keloid fibroblast than in normal scar and normal skin.Conclusions Compared with normal skin,the expression of decorin in keloid fibroblast is significantly lower.Lower content of decorin in early stage of wound healing may induce weakly suppression of proliferation and synthesis of fibroblast,and up-regulate the activity of TGF-β1,which promotes the proliferation,migration and excessive collagen synthesis of the fibroblast of keloid.Thus,decorinis an suppressor factor of keloid formation. Key words: Keloid; Decorin; β1-transforming growth factor (TGF-β1) ; Fibroblast; fluorescent quantitative-PCR (FQ-PCR)

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

To verify the existence and significance of calcium/calmodulin dependent serine protein kinase/inhibitors of differentiation 1 (CASK/Id1) pathway in fibroblasts of human keloid. Immunofluorescence 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. CASK 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. CASK/Id1 signal pathway may be existed and involved in the proliferation of keloid fibroblasts, which is related with the occurrence of keloid.

To investigate the effect of rapamycin on biological characteristics and autophagy of keloid fibroblasts, and the regulation of rapamycin in mTOR (mammalian target of rapamycin) signaling pathway and autophagy-related non-coding RNAs in keloid fibroblasts. After Keloid fibroblasts were treated with rapamycin (10、50、100 nmol/L), and MTS assay was used to test the cell proliferation. The apoptosis of cells was tested by the flow cytometry analysis. The formation of autophagy was observed by TEM, and the Western Blot was used to detect the expression of autophagy-related protein LC3.Real-time PCR was used to detect the expression of genes of involued in mTOR pathway and autophagy-related non-coding RNAs. Statistical significance was determined using Paired-Samples t Test,P value less than 0.05 was considered statistically significant. The ratio of 490nm was decreased significantly in rapamycin-treated keloid fibroblasts compared with that in untreated cells (P ＜ 0.05).Meanwhile the mRNA expressions of extracellular matrix (ECM) genes, including collagen-1 、α-SMA and Fibronectin, were inhibited by rapamycin (P ＜ 0.05).The flow cytometry analysis showed that the percent of apoptosis cells was not increased in rapamycin-induced cells (P ＞ O.05). The double-layer membrane structure of autophagosomes could be observed under the TEM in rapamycin-treated fibroblasts, accompanied by the increased expression of autophagy-related protein LC3.The mRNA expressions of downstream genes of mTOR pathway,4EBP1 and p70S6K,were down-regulated in rapamycin-treated fibroblasts, while the expressions of autophagy-related miRNAs, including miR-885-3p,miR-204,miR-101,miR-376b and lncRNA FLJ11812 were enhanced, and miR-30a,lncRNA HULC5 was decreased in rapamycin-treated fibroblasts (P ＜ 0.05). Rapamycin could inhibit the proliferation of keloid fibroblasts, and could not affect the apoptosis of cells.However, rapamycin induced the autophagy of keloid fibroblasts through regulating the expression of autophagy-related non-coding RNAs and genes in the mTOR signaling pathway.

To isolate the transforming growth factor-beta 1 (TGF-β1) phage model peptides from phage 12-mer display peptide library to inhibit the proliferation of keloid fibroblasts. The phage display 12-mer peptide library was screened for 4 rounds with monoclonal anti-human TGF-β1 as the target to yield the specific phage model peptides. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used for the quantitative determination of cellular proliferation. Apoptosis was detected by the Annexin V-FITC/PI apoptosis detection kit and the cells were analyzed with flow cytometry. Immunofluorescent assay was employed to show the binding affinity of model peptides for keloid fibroblasts. Quantitative real-time polymerase chain reaction (PCR) was performed to detect the expressions of nuclear factor kappa B (NF-κB) and connective tissue growth factor (CTGF). Ten phage model peptides were obtained and they were similar to TGF-β1, TGF-β2, TGF-β receptor II (TβRII), TGF-β-induced factor, NF-κB or mitogen-activated protein kinase (MAPK). The results of MTT showed that four phage model peptides (No. 7 - 10) could inhibit the proliferation of keloid fibroblasts (P < 0.05). The results of apoptotic assessment showed that phage model peptides (No. 7 - 10) could slightly trigger the late apoptotic stage of keloid fibroblasts. The data of immunofluorescence assay revealed that the model peptides on phages rather than phages could bind to keloid fibroblasts. The findings of quantitative real-time PCR analysis suggested that the relative expression of NF-κB decreased in phage model peptides groups (No. 7 - 10). The quantitative expression was 0.28, 0.26, 0.46 and 0.30 respectively versus the negative control group. The relative expression of CTGF decreased in phage model peptides groups (No. 7 - 10). The quantitative expression was 0.26, 0.60, 0.34 and 0.17 respectively versus the negative control group. Four phage model peptides (No. 7 - 10) isolated from phage display 12-mer peptide library can inhibit the proliferation of keloid fibroblasts via regulating the expressions of NF-κB and CTGF.

To explore the effect of simvastatin on the proliferation, apoptosis and protein expressions of keloid fibroblasts under normoxia,hypoxia or TGF-β1 treatment. Keloid fibroblasts (KFs) were isolated by explants culture method. KFs were treated with different concentrations of simvastatin under normoxia or hypoxia (2％ O2) for 24 h and 48 h. The effects of simvastatin on cell proliferation were detected by CCK-8.Flow cytometer was used to detect the apoptosis of KFs treated with 10 μ mol/L simvastatin for 24 h or 48 h under normoxia, hypoxia or 10 ng/ml TGF-β1 treatment. Then the expressions of keloid-related proteins were analyzed by Western Blot. It showed that simvastatin could inhibit the proliferation of KFs in a concentration-and time-dependent manner with the concentration range of 10-500 μ mol/L for 24 h and 0.1-500 μ mol/L for 48 h. This inhibitory effect could be significantly enhanced when cells were incubated under hypoxia for 48h with 10-500 μ mol/L simvastatin.10 μ mol/L simvastatin could not influence the apoptosis of KFs under normoxia or TGF-β1 treatment, neither incubated for 24 h nor 48 h.When incubated under hypoxia,10 μ mol/L simvastatin could significantly induce the apoptosis of KFs, with the rate of 155.6％ for 24 h and 478.8％ for 48 h, compared with no-drug control. There are no significant influences on the expression of type Ⅰ collagen, CTGF or TIMP-1 when KFs were treated with 10 μ mol/L simvastatin under normoxia for 48 h. When incubated with 10 ng/ml TGF-β1 together with 10 μmol/L simvastatin for 48 h, the expression of CTGF was significantly inhibited. KFs treated with 10 μ mol/L simvastatin under hypoxia for 48 h showed a significant decrease of type Ⅰ collagen and CTGF, and a significant increase of TIMP-1. Simvastatin has different effects on the proliferation, apoptosis and protein expressions of KFs in a dosedependent manner under different conditions. The effects are enhanced under hypoxia.

Clinical Challenge and Call for Research on Keloid Disorder: Meeting Report from The 3rd International Keloid Research Foundation Symposium, Beijing 2019

To compare the differences of mitochondrial functions between keloid fibroblasts and normal skin fibroblasts and explore its relationship with cell proliferation. Keloid fibroblasts (KFb) and normal skin fibroblasts (NFb) were isolated by explants culture method. KFb and NFb were cultured under normoxia or hypoxia (2％ O2).Differences of cell proliferation were detected by CCK-8.Flow cytometer was used to detect the content of mitochondria and reactive oxygen species (ROS) in KFb and NFb. Ultra-structures of mitochondria in KFb and NFb were observed by transmission electron microscope (TEM).Mitochondria fusion/fission related genes MFN1,MFN2 and FIS1 were detected by RT-PCR. Oxygen consumption rate, lactate production and ATP contents were determined by spectrophotometry. KFb showed a higher proliferation rate compared with NFb, especially under hypoxia. The oxygen consumption rate, ATP content, lactate production and ROS of KFb were lower than NFb under normoxia. After incubated under hypoxia, there was a significant increase in oxygen consumption, ATP content, lactate production and ROS in KFb, while NFb showed less increase compared with KFb. KFb had 15.33％ more mitochondrion than NFb, and expressions of MFN1, MFN2, FIS1 in KFb were 33.27％,113.39％ and 20.34％ higher compared with NFb. Under TEM, KFb showed an increase of enlarged mitochondrion, with disrupted inner membrane and loss of cristae. KFb may have dysfunctions of mitochondrion which lead to changes of cell metabolism and continuous proliferation of KFb.

Objective To investigate the correlation of HIF-1α and hypoxia in keloids fibroblasts, and to investigate the mechanism that hypoxia promotes abnormal scarring by HIF-1α pathway. Methods Keloid fibroblasts cultured in vitro were placed in an incubator with different O2 concentrations. After 24 h, the keloid fibroblasts were collected for further study. Western blotting was performed to detect the expression of HIF-1α in the keloid fibroblasts. Results Relative amounts of HIF-1α in keloid fibroblasts cultured under O2 concentrations at 20 %, 10 %, 5 % and 1 % were 0. 007 ±0. 006, 0. 133 ±0. 006, 0. 537±0. 015 and 0. 903±0. 021, respectively. It indicated that hypoxia could increase the expression of HIF-lα in keloid fibroblasts. Conclusions Hypoxia can induce the expression of HIF-1α in fibroblasts of keloids. Moreover, there still is a positive relation between hypoxia and the expression of HIF-1α. Therefore, a close relationship exists between abnormal scarring and HIF-1α pathway by hypoxia. Key words: Keloid; Fibroblast; Hypoxia inducible factor, HIF-1α; Hypoxia

636 Mechano-sensing and inflammatory signalling in normal and keloid dermal fibroblasts

Monoclonal antibodies were used to demonstrate proliferating cell nuclear antigen (PCNA) and Ki-67 antigen of dermal fibroblasts in formalin-fixed, paraffin-embedded tissue sections of keloids, hypertrophic scars and normal skin. PCNA-stained fibroblasts were more pronounced than Ki-67, which showed only scanty Ki-67-positive fibroblasts. The mean density of dermal fibroblasts was significantly higher in keloids and hypertrophic scars than in normal skin (p < 0.01). The proliferating activity of fibroblasts detected by PCNA was significantly higher in keloids than in hypertrophic scars or normal skin (p < 0.01). The mean densities and the proliferating activities were apparently not correlated with the age of the patient. This indicates that keloids, with higher density and proliferating activity of dermal fibroblasts, continue to increase their volume and invade the surrounding tissue, while hypertrophic scars, with higher density and lower proliferating activity, show a tendency towards spontaneous regression.

Objective To observe the effects of secreted protein, acidic and rich in cysteine （SPARC） on the expression of TGF-β1 and collagen type I in cultured human keloid fibroblasts by real-time fluorescence quantitative RT-PCR. Methods In vitro keloid fibroblasts were stimulated by different concentrations of recombinant human SPARC, and with the control group for comparison, real-time fluorescence quantitative RT-PCR to detect expression of TGF-β1 and collagen type I . Results Compared with the control group, the expression of TGF-β1 and collagen type I was significantly increased in the experimental group. Conclusions SPARC could enhance the expression of TGF-β1 and collagen type I in keloid fibroblasts significantly. Key words: Keloid; Secreted protein, acidi and rich in cysteine; quantitative reverse transcription-PCR; Transforming growth factor-β1 ; Real-time fluorescence Collagen type

To observe the effects of conditioned medium from keloid fibroblasts under hypoxia on angiogenesis, and to investigate the role of hypoxic microenvironment in invasive growth of keloid. Primary keloid fibroblasts and human umbilical endothelial cells (HUVEC) were cultured as conventional method. Keloid fibroblasts were cultured either in a hypoxic incubator (2% O2) for 48 h or in a normoxic incubator (20% O2) as control. Then those cell culture mediums were collected and mixed with endothelial cell medium by the proportion of 1:1 as conditioned medium. The mRNA and secreted protein of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and periostin of keloid fibroblasts under hypoxia were detected by real time PCR and ELISA. The proliferation, migration and invasion, tube formation of HUVEC cultured with conditioned medium were evaluated by CCK-8 assay, Transwell assay and matrigel tube formation assay, respectively. Hypoxia increased the expression of VEGF, Ang-1 and periostin in both mRNA (increased by 75%, 43% and 118% respectively, P < 0.05) and secreted protein (increased by 30.2%, 14.2% and 19.5% respectively, P < 0.05) levels; the proliferations of HUVEC in hypoxic conditioned medium in 1, 2 and 3 d were 0.67 +/- 0.07, 0.84 +/- 0.09 and 1.08 +/- 0.10 respectively, which were higher compared to those in control group (0.52 +/- 0.08, 0.72 +/- 0.10 and 0.91 + 0.14, P < 0.05); the numbers of migration, invasion and tube formation of HUVEC were (73.2 +/- 8.9), (56.3 +/- 12.5), (9.66 +/- 1.96) cells/HP, which were higher compared to those in control group [(59.0 +/- 8.0), 35.5 +/- 8.5), (6.5 +/- 1.87) cells/HP, P < 0.05]. Hypoxia increases the expression of pro-angiogenic factors of keloid fibroblasts, and its conditioned medium under hypoxia could promote angiogenesis. The results suggest hypoxic microenvironment may play a significant role in the invasive growth of keloid by inducing angiogenesis.

Objective To test whether nicotine induces the secretion of transforming growth factor-β1 (TGF-β1)and fibroblast growth factor 2 (FGF-2) in HBECs in vitro via the high mobility group protein B1 (HMGB1)/receptor for advanced glycation end products (RAGE) signaling pathway. Methods Cultured HBECs were exposed to nicotine (6×10-6 mol/L) for 24 h. The secretion of HMGB1, TGF-β1 and FGF-2 was assessed by ELISA, the expression of RAGE was assessed by Western blotting.HBECs were either transfected with a small interfering RNA (siRNA) targeting HMGB1 for 48 h or incubated with anti-RAGE antibodies for 1 h and subsequently stimulated with nicotine for 24 h, then the secretion of HMGB1, TGF-β1 and FGF-2 was assessed by ELISA, the expression of RAGE was assessed by Western blotting. Results We showed that cells exposed to nicotine for 24 h exhibited significantly increased HMGB1, TGF-β1 and FGF-2 secretion and RAGE expression.The HMGB1 siRNA prevented these effects.Furthermore, anti-RAGE antibodies significantly decreased the secretion of TGF-β1 and FGF-2 from HBECs. Conclusions These results suggest that nicotine induces the secretion of TGF-β1 and FGF-2 in HBECs via the HMGB1/RAGE signaling pathway. Key words: Nicotine; Transforming growth factor beta1; Fibroblast growth factor 2; Human bronchial epithelial cells; High mobility group protein B1/Receptor for advanced glycation end products