Hypertrophic scar (HS) affects the function and beauty of patients, and brings a heavy psychological burden to patients. However, the specific pathogenesis mechanism of HS in molecular biology level is not yet clear, and this disease is still one of the clinical diseases difficult to prevent and cure. MicroRNA (miR) is a family of single-stranded endogenous noncoding RNAs that can regulate gene expression. The abnormal transcription of miR in hypertrophic scar fibroblasts can affect the transduction and expression of downstream signal pathway or protein, and the exploration of miR and its downstream signal pathway and protein helps deeply understand the occurrence and development mechanism of scar hyperplasia. This article summarized and analyzed how miR and multiple signal pathways involve in the formation and development of HS in recent years, and further outlined the interaction between miR and target genes in HS.
Humans ; MicroRNAs/genetics* ; Cicatrix, Hypertrophic/genetics* ; Fibroblasts ; Hyperplasia

Objective: To explore the effect of microRNA(miR)-222 on cell proliferation and apoptosis of fibroblasts in hypertrophic scar (HS) and the underlying mechanisms. Methods: The expression of miR-222 in the HS and the normal skin tissues was detected by real-time RT-PCR. The HS fibroblasts were transfected with miR-222 mimic and miR-222 inhibitor respectively. The cell viability was tested with MTT assay, cell cycle distribution and apoptosis were detected with flow cytometry and the expression levels of proliferation, apoptosis and cell cycle related proteins were determined with Western blot. Direct target of miR-222 was evaluated by dual-luciferase reporter assay. Results: miR-222 was significantly up-regulated in HS tissues compared with normal skin tissues(P<0.05). Overexpression of miR-222 enhanced the cell viability of HS fibroblasts; increased mRNA and protein expressions of proliferating cell nuclear antigen (PCNA), collagen alpha-1 (Ⅰ) chain (Col1A1) and collagen alpha-1 (Ⅲ) chain (Col3A1); increased cell population in S phase and protein expressions of cyclin D1, cyclin E1 and cyclin-dependent kinases 1 (CDK1); inhibited cell apoptosis and reduced protein expressions of caspase-3/9. Overexpression of MMP1 attenuated the effects of miR-222 on the cell viability and apoptosis in fibroblasts, reduced expression levels of PCNA, cyclin D1 and the expression of caspase-3 was increased. Conclusion: miR-222 enhances cell proliferation and inhibits cell apoptosis of HS fibroblasts through negative regulation of MMP1, which suggests that miR-222 and MMP1 might be used as novel biomarkers and targets in diagnostic and therapeutic approaches for HS.
Apoptosis ; genetics ; Cell Proliferation ; genetics ; Cicatrix, Hypertrophic ; Fibroblasts ; Humans ; Matrix Metalloproteinase 1 ; metabolism ; MicroRNAs ; metabolism

BACKGROUNDHypertrophic scar is one of the most common complications and often causes the disfigurement or deformity in burn or trauma patients. Therapeutic methods on hypertrophic scar treatment have limitations due to the poor understanding of mechanisms of hypertrophic scar formation. To throw light on the molecular mechanism of hypertrophic scar formation will definitely improve the outcome of the treatment. This study aimed to illustrate the negative role of eukaryotic initiation factor 6 (eIF6) in the process of human hypertrophic scar formation, and provide a possible indicator of hypertrophic scar treatment and a potential target molecule for hypertrophic scar.

METHODSIn the present study, we investigated the protein expression of eIF6 in the human hypertrophic scar of different periods by immunohistochemistry and Western blot analysis.

RESULTSIn the hypertrophic scar tissue, eIF6 expression was significantly decreased and absent in the basal layer of epidermis in the early period, and increased slowly and began to appear in the basal layer of epidermis by the scar formation time.

CONCLUSIONSThis study confirmed that eIF6 expression was significantly related to the development of hypertrophic scar, and the eIF6 may be a target molecule for hypertrophic scar control or could be an indicator of the outcomes for other treatment modalities.

Adult ; Blotting, Western ; Cicatrix, Hypertrophic ; metabolism ; Female ; Gene Expression Regulation ; genetics ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Peptide Initiation Factors ; metabolism ; Pregnancy ; Retrospective Studies ; Young Adult

OBJECTIVETo study the role of integrin-linked kinase (ILK) on the proliferation and differentiation of human fibroblast in hypertrophic scar and its effect on the scar formation.

METHODSThe human scar fibroblasts were isolated and cultured in vitro. The cells were divided into 4 groups. (1) control group: only contains DMEM; (2) jetPRIME group: DMEM with 200 microl jetPRIME buffer and 4 microl jetPRIME; (3) ILK siRNA group: DMEM and ILK siRNA; (4) ILK cDNA group: DMEM and ILK cDNA. The cell proliferation was detected by XTT assay and the mRNA and protein expressions of ILK and alpha-SMA were detected by Real-time qPCR and Western blot.

RESULTS(1) XTT results showed that the cellular proliferation level after 48 h in four groups were 0.820 +/- 0.065, 0.873 +/- 0.041, 0.554 +/- 0.013 and 1.296 +/- 0.094, respectively. The cellular proliferation curve showed that the cellular proliferation level was very flat in ILK siRNA group while the cellular proliferation level gradually increased from 12 h. 48 h after transfection, the cellular proliferation level in ILK siRNA group was significant lower than those in other groups (P value were 0.021, 0.034, 0), while the cellular proliferation level in ILK cDNA group was the highest among all 4 groups (P value were 0.017, 0.009, 0). (2) The Real-time qPCR showed that the expressions of ILK mRNA and alpha-SMA mRNA were 0.693 +/- 0.412 and 0.422 +/- 0.037 in control group, were 0.621 +/- 0.183 and 0.388 +/- 0.005 in jetPRIME group, were 0.052 +/- 0.019 and 0.073 +/- 0.023 in ILK siRNA group, were 240.193 +/- 35.170 and 138.056 +/- 24.060 in ILK cDNA group. The expressions of ILK mRNA and alpha-SMA mRNA in ILK siRNA group were significantly lower than those in other three groups (P < 0.05). And the expressions of ILK mRNA and alpha-SMA mRNA in ILK cDNA group were significantly higher than those in other three groups (P < 0.05). (3) The Western blot also showed that the expression of ILK and alpha-SMA proteins were decreased in ILK siRNA group and increased in ILK cDNA group.

CONCLUSIONILK may promote the proliferation and differentiation of human scar fibroblast. It may play an important role in scar formation and contracture.

Actins ; metabolism ; Adolescent ; Adult ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; Female ; Fibroblasts ; cytology ; drug effects ; metabolism ; Humans ; Male ; Middle Aged ; Protein-Serine-Threonine Kinases ; pharmacology ; RNA, Messenger ; genetics ; Transfection ; Young Adult

OBJECTIVETo investigate the effects and regulatory mechanism of ILK on angiogenesis in hypertrophic scar.

METHODSThe human scar microvascular endothelial cells (HSMECs) were isolated from 6 patients' hypertrophic scar in vitro. The HSMECs with good condition in 2nd to 4th generation were selected as experimental objectives. (1) HSMECs were divided into the blank control group (treated with routine culture), negative control group (treated with only Lipofectamine 2000), LY294002 group (incubated with 50 nmol/L LY294002), ILK siRNA group (incubated with 20 nmol/L ILK siRNA). RT-PCR and Western Blot were used to detect the expression of ILK mRNA and its protein after transfecion for 48 h. (2) The digested HSMECs of four groups were resuspended with DMEM without serum and then seeded onto the upper compartment of transwell insert which contained complete medium in its lower compartment. The cell migration experiment was stopped in 10 h and then the migrated cells were counted to analyze the effects of different interventions on the migration ability of HSMECs. (3) The thawed ECMatrix was put into each well of pre-colled 48-well tissue culture plate, and then the plate was put into the incubator at 37 degrees C to make it to become gel. The HSMECs of four groups were seeded onto the surface of the ECMatrix gel and were put into incubator. Eight random view-fields per well should be valued by the sheet of pattern recognition about angiogenesis after 8 hours to evaluate the ability of angiogenesis in vitro between four groups.

RESULTS(1) The expression of ILK mRNA (ILK mRNA = 0.829 +/- 0.109, t = 13.151, P = 0.006) and protein (ILK protein = 0.096 +/- 0.049, t = 36.656, P = 0.000) were both inhibited obviously in ILK siRNA group compared with the blank control group (ILK mRNA = 0.829 +/- 0.109, ILK protein = 1). And, the expression of ILK in LY294002 group was slightly lower than that of black control group, but there was no statistical difference. (2) The number of migrated cells in ILK siRNA group (88.111 +/- 3.079) and LY294002 group (138. 667 +/- 2.404) were respectively lower than that in blank control group (322.333 +/- 3.712, P < 0. 05) in 10th hour. (3) Compared to blank control group (4.333 +/- 0.191), the ability of angiogenesis in vitro decreased significantly ILK siRNA group (2.625 +/- 0.125) and LY294002 group (3.125 +/- 0.250), in which, the vascular network structures were not formed perfectly in 8th hour (P < 0.05).

CONCLUSIONSThe ability of HSMECs' migration and angiogenesis in vitro are inhibited significantly when the expression of ILK is down-regulated. It reveals that ILK may play an role in the regulation of scar angiogenesis.

Cell Movement ; Cell Proliferation ; Chromones ; pharmacology ; Cicatrix, Hypertrophic ; enzymology ; pathology ; Endothelial Cells ; cytology ; drug effects ; Humans ; Lipids ; pharmacology ; Morpholines ; pharmacology ; Neovascularization, Pathologic ; etiology ; pathology ; Protein-Serine-Threonine Kinases ; genetics ; physiology ; RNA, Messenger ; analysis ; RNA, Small Interfering ; metabolism

OBJECTIVETo observe the effect of tetrandine on gene expression of collagen type I, collagen type III, transformation growth factor-beta1 and to investigate the inhibitory effect of tetrandine on the scar tissue hyperplasia in rabbits' ears.

METHODSAfter the scar model was formed on the rabbits' ears, the rabbits were divided into 4 groups to receive intro-lesion injection with saline, or prednisolone (Pre) or tetrandrine in low concentration (L-Tet, 1.0 mg/ml) or tetrandrine in high concentration (H-Tet, 7.5 mg/ml). The morphological changes of scar tissue were observed. The changes of fibroblasts quantity and collagen expression were observed with HE and Masson staining. Immunohistochemical study was used to observe the expression level of collagen type I and collagen type III and TGF-beta1. Collagen type I and collagen type III and TGF-beta1, and signal factor Smad 3 mRNA were detected with RT-PCR.

RESULTS(1) 24 days after injury, all the wounds healed completely with formation of red, tough and hypertrophic scar. HE and Masson staining showed significant increase of fibroblasts and collagen density with irregularly arrangement. (2) Compared with that in saline group, the scar in other groups became softer, lighter and thinner, especially in H-Tet group. (3) HE and Masson staining shows the scar in Tet and Pre groups contained less fibroblasts and lower collagen dentsity with comparatively regular arrangement than that in saline group (P < 0.01), especially in H-Tet group. (4) According to the immunohistochemical study, the expression of collage type I and III and TGF-beta was positive in all the groups, but the positive rate and the ratio of collagen density I to III decreased in the order of saline, L-Tet, H-Tet and Pre groups (P < 0.01). (5) PT-PCR detection results showed that the amplification bands brightness of collagen type I and III and TGF-beta1 and signal molecular Smad 3 mRNA in scar tissue were obviously different. Compared with that in saline group, the expression of collagen type I and III and TGF-beta1 and Smad 3 mRNA decreased in Tet and Pre groups (P < 0.01). H-Tet group showed the most obvious reduce in the expression of type I collagen and TGF-beta1 and Smad 3 mRNA. Conclusions Tetrandine can significantly suppress the expression of collagen type I and collagen type III and TGF-beta1 on hypertrophic scar of rabbit ears, and reduce signal factor Smad 3 mRNA' s expression. It may be one of the important mechanism for its inhibitory effect on scar hyperplasia.

Animals ; Benzylisoquinolines ; pharmacology ; Cicatrix, Hypertrophic ; drug therapy ; genetics ; pathology ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Ear ; Fibroblasts ; Gene Expression ; Male ; RNA, Messenger ; metabolism ; Rabbits ; Smad3 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism

BACKGROUNDPressure therapy improves hypertrophic scar healing, but the mechanisms for this process are not well understood. We sought to investigate the differential expression of matrix metalloproteinases (Mmps) and collagen in posttraumatic hypertrophic scar tissue with mechanical pressure and delineate the molecular mechanisms of pressure therapy for hypertrophic scars.

METHODSFibroblast lines of normal skin and scar tissue were established and a mechanical pressure system was devised to simulate pressure therapy. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting assays were used to compare differences in the mRNA and protein expression of Mmps and collagen in scar fibroblasts before and after pressure therapy.

RESULTSThe expression differed between the hypertrophic scar cell line and the normal cell line. RT-PCR assays showed that Collagen I, highly expressed in the hypertrophic scar cell line, decreased significantly after pressure therapy. Mmp2, Mmp9, and Mmp12 expression in the hypertrophic scar tissue increased significantly after pressure therapy (P < 0.05). Western blotting assays further revealed that Mmp9 and Mmp12 expression increased significantly in the hypertrophic scar tissue after pressure therapy (P < 0.05) but not Mmp2 expression (P > 0.05).

CONCLUSIONMechanical pressure induces degradation of Collagen I in hypertrophic scar tissue by affecting the expression of Mmp9 and Mmp12.

Cell Line ; Cicatrix, Hypertrophic ; enzymology ; metabolism ; Collagen Type I ; genetics ; metabolism ; Humans ; Matrix Metalloproteinase 12 ; genetics ; metabolism ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism

OBJECTIVETo explore the binding domain of hydroxypyruvate isomerase homologues (HYI) in the interaction with protein P311 in hypertrophic scar fibroblasts (Fb).

METHODS(1) P 311 was amplified by PCR using plasmid pMD18-T-P 311 as template. The total RNA of hypertrophic scar Fb was extracted by Trizol to amplify HYI with RT-PCR. Recombinant vectors pGADT7-P 311 and pGBKT7-HYI were constructed by double-enzyme digestion, and they were verified by PCR and sequencing. The secondary structure of protein HYI was analyzed with software Prot Seale and HNN. Fragments of HYI-1 (1-447 bp), HYI-2 (247-447 bp), HYI-3 (1-279 bp), and HYI-4 (247-654 bp) were amplified based on the result of software analysis. And then the recombinant vectors pGBKT7-HYI-1, 2, 3, and 4 were constructed by double-enzyme digestion and verified by PCR and sequencing. (2) AH109 yeast cells were transformed into competent cells by lithium acetate method and divided into 7 groups roughly in the same amount, including HYI full length, HYI-1, HYI-2, HYI-3, and HYI-4 hybrid groups, positive control group, and negative control group. Cells in the first five groups were respectively transformed with recombinant vector pGBKT7-HYI full length, pGBKT7-HYI-1, pGBKT7-HYI-2, pGBKT7-HYI-3, pGBKT7-HYI-4 and recombinant vector pGADT7-P 311, and that in the rest two groups were transformed with recombinant vectors pGBKT7-53 and pGADT7-RecT, pGADT7-RecT and pGBKT7-Lam by polyethyleneglycol/lithium acetate method. Immediately after transformation, a part of the transformed cells in each group was spread onto the medium lacking leucine, tryptophan, adenine, and histidine (briefly called four-factor lacking medium), and another portion of the cells was spread onto the medium lacking leucine and tryptophan (briefly called two-factor lacking medium). After 3 to 6 days' culture, the growth of yeast was observed, and the expression of β-galactosidase of yeast was detected by color reaction with 5-bromo-4-chloro-indolyl-β-D-galactopyranoside.

RESULTS(1) Cloned P 311 and the reported P 311 (GenBank ID hsu36189) had the same sequence. The A base at 496 bp in reported HYI (GenBank ID AY775560) was replaced by G base as found in cloned HYI. It was verified that the insert segment of each recombinant vector was correct. (2) Among those 216 amino acids which composed the protein HYI, 101 amino acids might form α helices, 90 amino acids might form random coils, 25 amino acids might form extended-chains as revealed in the simulated structure analysis by computer. (3) Cloned segments HYI-1, 2, 3, 4 showed expected lengths. It was verified that the insert segment of each recombinant vector was correct. (4) Except for strains in negative control group which did not show growth on four-factor lacking medium, all strains in other groups grew on both kinds of media, and growth of colonies was less in HYI-2 (with the fewest number of α helices) and HYI-3 hybrid groups. (5) Positive expression of β-galactosidase was observed in strains of all groups growing on four-factor lacking medium except for the HYI-2 hybrid group. No expression of β-galactosidase was observed in strains of negative control group which grew on two-factor lacking medium.

CONCLUSIONSProtein HYI may closely bind with protein P311 by α helix, which plays an important role in fibroblast-to-myofibroblast transdifferentiation in hypertrophic scar.

Aldose-Ketose Isomerases ; genetics ; Cicatrix, Hypertrophic ; enzymology ; genetics ; Cloning, Molecular ; Fibroblasts ; enzymology ; Genetic Vectors ; Humans ; Molecular Sequence Data ; Nerve Tissue Proteins ; metabolism ; Oncogene Proteins ; metabolism ; Plasmids ; Protein Binding ; Protein Interaction Domains and Motifs

OBJECTIVETo explore the pathogenesis mechanism of hypertrophic scar (HS) and the effective means for its clinical treatment, the difference of the gene expressions between HS and normal skin was compared.

METHODSThe differentially expressed genes between HS and normal skin were obtained by mining PubMed. The dysregulated genes in HS were analyzed by a series of bioinformatics methods, including protein-protein interaction networks, pathways, Gene Ontology and functional annotation clustering analysis.

RESULTSA total of 55 dysregulated genes in HS was identified (46 up-regulated genes and 9 down-regulated genes). Fifty-one genes were found to encode proteins with interaction network, including up-regulated genes TGFB1, FN1, JUN, COL1A1, CTGF, VEGFA, FOS, COL3A1, IGF1, IL4, PELO, SMAD2, TIMP1, PCNA, and ITGA4 and down-regulated genes ITGB1 and DCN as the central nodes for this network. The dysregulated genes in HS involved in a variety of biological pathways, such as focal adhesion formation, integrin signal transduction, and tumor formation. Furthermore, the dysregulated genes in HS played the important roles in biological processes of cell surface receptor linked signal transduction, tissue development, cell proliferation and apoptosis, and macromolecule biosynthetic process, as well as in molecular function of calcium ion binding, double-stranded DNA binding, heparin binding, promoter binding and MAP kinase activity. The results of functional annotation clustering analysis revealed that the dysregulated genes in HS involved in epidermis development, angiogenesis, and apoptosis.

CONCLUSIONSuch key genes as TGFB1, FN1, and JUN, along with the pathways, biological processes and molecular functions involving epidermis development, angiogenesis, and extracellular matrix-integrin-focal adhesion signal transduction may play the important roles in the development of HS. The investigations of the dysregulated genes in HS could provide the new targets for clinical treatment.

Cicatrix, Hypertrophic ; genetics ; Cluster Analysis ; Computational Biology ; Data Mining ; Gene Expression ; Gene Expression Profiling ; Gene Regulatory Networks ; Humans

OBJECTIVETo explore the expression of integrin-linked kinase (ILK) and its effect on VEGF expression in fibroblasts from human hypertrophic scar.

METHODSFibroblasts were isolated from hypertrophic scar of 8 patients and cultured in vitro. Then the cells were divided into three groups: (1) Cells were cultured only in DMEM containing 10% FCS in the control group; (2) Cells were transfected with empty plasmid in the empty plasmid group; (3) Cells were transfected with plasmid expressing ILKcDNA in the ILK cDNA plasmid transfection group. First, the expression of ILK and VEGF was observed by immunocytochemistry before and after ILK cDNA transfection. Second, ILK and VEGF mRNA expression was investigated by real-time PCR (RT-PCR). Third, the protein expression of ILK and VEGF was detected by Western blot. Finally, the protein level of VEGF in supernatant of fibroblasts was measured by ELISA.

RESULTSBefore ILK cDNA transfection, the expression of ILK was positive and the VEGF expression was weak in cytoplasm of fibroblasts . After ILK cDNA transfection, both the expression of ILK and VEGF was enhanced. The level of VEGF mRNA was significantly higher in ILK cDNA transfection group (0.338 +/- 0.060) than that in control group (0.022 +/- 0.001) and empty plasmid group (0.028 +/- 0.005, P < 0.05). The level of VEGF protein was significantly higher in ILK cDNA transfection group (0.819 +/- 0.019) than that in control group (0.607 +/- 0.033) and empty plasmid group (0. 591 +/- 0.024, P<0. 05). Secretion of VEGF increased remarkably in ILK cDNA transfection group comparing with the other two groups (P < 0.05).

CONCLUSIONSILK could up-regulate the VEGF mRNA and protein level in human scar fibroblasts. It may play an important role in the angiogenesis in hypertrophic scar.

Cells, Cultured ; Cicatrix, Hypertrophic ; genetics ; metabolism ; pathology ; Fibroblasts ; secretion ; Humans ; Plasmids ; Protein-Serine-Threonine Kinases ; genetics ; RNA, Messenger ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism