BACKGROUND: Fibrosis of the connective tissue in the vaginal wall predominates in pelvic organ prolapse (POP), which is characterized by excessive fibroblast-to-myofibroblast differentiation and abnormal deposition of the extracellular matrix (ECM). Our study aimed to investigate the effect of ECM stiffness on vaginal fibroblasts and to explore the role of methyltransferase 3 (METTL3) in the development of POP. METHODS: Polyacrylamide hydrogels were applied to create an ECM microenvironment with variable stiffness to evaluate the effects of ECM stiffness on the proliferation, differentiation, and expression of ECM components in vaginal fibroblasts. METTL3 small interfering RNA and an overexpression vector were transfected into vaginal fibroblasts to evaluate the effects of METTL3 silencing and overexpression on matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal modulation of the ECM. Both procedures were detected by 5-ethynyl-2'-deoxyuridine (EdU) staining, Western blotting (WB), quantitative real-time polymerase chain reaction (RT-qPCR), and immunofluorescence (IF). RESULTS: Vaginal fibroblasts from POP patients exhibited increased proliferation ability, increased expression of α-smooth muscle actin (α-SMA), decreased expression of collagen I/III, and significantly decreased expression of tissue inhibitors of matrix metalloproteinases (TIMPs) in the stiff matrix ( P <0.05). Compared with those from non-POP patients, vaginal wall tissues from POP patients demonstrated a significant increase in METTL3 content ( P <0.05). However, silencing METTL3 expression in vaginal fibroblasts with high ECM stiffness resulted in decreased proliferation ability, decreased α-SMA expression, an increased ratio of collagen I/III, and increased TIMP1 and TIMP2 expression. Conversely, METTL3 overexpression significantly promoted the process of increased proliferation ability, increased α-SMA expression, decreased ratio of collagen I/III and decreased TIMP1 and TIMP2 expression in the soft matrix ( P <0.05). CONCLUSIONS Elevated ECM stiffness can promote excessive proliferation, differentiation, and abnormal ECM modulation, and the expression of METTL3 plays an important role in alleviating or aggravating matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal ECM modulation.
Humans ; Female ; Extracellular Matrix/metabolism* ; Cell Differentiation/genetics* ; Methyltransferases/metabolism* ; Pelvic Organ Prolapse/pathology* ; Fibroblasts/metabolism* ; Myofibroblasts/metabolism* ; Vagina/metabolism* ; Cell Proliferation/physiology* ; Cells, Cultured ; Middle Aged

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.
Actins/biosynthesis* ; Cell Differentiation/physiology* ; Cell Movement/physiology* ; Electric Stimulation Therapy ; Electricity ; Fibroblasts/physiology* ; Humans ; Myofibroblasts/physiology* ; Proliferating Cell Nuclear Antigen/biosynthesis* ; Skin/cytology*

Objective To investigate the sequential changes of the number of neutrophils and myofibroblasts during diabetic wound healing, and discuss its application value in wound age estimation. Methods Diabetic DB mice and mice of the same age in the normal control group were selected, a wound healing model was established, wound samples were taken at different time points, while the number of neutrophils and myofibroblasts during diabetic wound healing were determined by immunohistochemical staining technique. Results The number of infiltrated neutrophils in the wounds of control and diabetic groups reached the peak respectively at 12 h and 5 d after injury. Compared with the control group, the number of neutrophils in the diabetic group decreased significantly from 6 h to 1 d after injury, but increased markedly from 5 d to 14 d. From 5 d to 10 d after injury, the average number of neutrophils at high magnification in wounds of the diabetic group was over 30, while that of neutrophils in wounds of the control group was less than 20. Myofibroblasts appeared in wounds from 3 d to 14 d after injury in the control group and from 5 d to 14 d after injury in the diabetic group. The difference in the number of myofibroblasts in wounds between control group and diabetic group from 3 to 7 d after injury had statistical significance. Conclusion In comparison with normal wound healing, the number of neutrophils and myofibroblasts during diabetic wound healing shows different sequential changes. The results of this study can provide reference for wound age estimation of patients with severe diabetes.
Animals ; Diabetes Mellitus, Experimental/pathology* ; Mice ; Myofibroblasts ; Neutrophils ; Wound Healing/physiology*

BackgroundRecent research indicates that nerve growth factor (NGF) promotes cardiac repair following myocardial infarction by promoting angiogenesis and cardiomyocyte survival. The purpose of this study was to investigate the effects of NGF on cardiac fibroblasts (CFs) proliferation, cell cycle, migration, and myofibroblast transformation in vitro.

MethodsCFs were obtained from ventricles of neonatal Sprague-Dawley rats and incubated with various concentrations of NGF (0, 0.01, 0.1, 1, 10, and 100 ng/ml; 0 ng/ml was designated as the control group). Cell proliferation and cell cycle of the CFs were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry (FCM), respectively. A cell scratch wound model and transwell were carried out to observe effects of NGF on migration of CFs after 24 h of culture. Real-time polymerase chain reaction (RT-PCR) and Western blotting were used to measure α-smooth muscle actin (α-SMA) at mRNA and protein levels after CFs were incubated with various concentrations of NGF.

ResultsExpression of α-SMA measured by RT-PCR and Western blotting significantly increased in the 1 and 10 ng/ml NGF groups (P < 0.05). Absorbance values of CFs showed that NGF did not influence the proliferation of CFs (The Avalues were 0.178 ± 0.038, 0.182 ± 0.011, 0.189 ± 0.005, 0.178 ± 0.010, 0.185 ± 0.025, and 0.177 ± 0.033, respectively, in the 0, 0.01, 0.1, 1, 10, and 100 ng/ml NGF groups [P = 0.800, 0.428, 0.981, 0.596, and 0.913, respectively, compared with control group]), and FCM analysis showed that the percentage of CFs in G0/G1, S, and G2/M phases was not changed (P > 0.05). The cell scratch wound model and transwell showed that CFs migration was not significantly different (P > 0.05).

ConclusionNGF induces myofibroblast transformation but does not influence proliferation, cell cycle, or migration of CFs in vitro.

Actins ; metabolism ; Animals ; Cell Cycle ; drug effects ; physiology ; Cell Movement ; drug effects ; physiology ; Cell Proliferation ; physiology ; Cells, Cultured ; Myofibroblasts ; cytology ; drug effects ; Nerve Growth Factor ; metabolism ; pharmacology ; Rats ; Rats, Sprague-Dawley

Mesothelial cells (MCs) cover the surface of visceral organs and the parietal walls of cavities, and they synthesize lubricating fluids to create a slippery surface that facilitates movement between organs without friction. Recent studies have indicated that MCs play active roles in liver development, fibrosis, and regeneration. During liver development, the mesoderm produces MCs that form a single epithelial layer of the mesothelium. MCs exhibit an intermediate phenotype between epithelial cells and mesenchymal cells. Lineage tracing studies have indicated that during liver development, MCs act as mesenchymal progenitor cells that produce hepatic stellate cells, fibroblasts around blood vessels, and smooth muscle cells. Upon liver injury, MCs migrate inward from the liver surface and produce hepatic stellate cells or myofibroblast depending on the etiology, suggesting that MCs are the source of myofibroblasts in capsular fibrosis. Similar to the activation of hepatic stellate cells, transforming growth factor β induces the conversion of MCs into myofibroblasts. Further elucidation of the biological and molecular changes involved in MC activation and fibrogenesis will contribute to the development of novel approaches for the prevention and therapy of liver fibrosis.
Epithelial Cells/*physiology ; Epithelium/metabolism ; Hepatic Stellate Cells/*physiology ; Humans ; Liver/*cytology/injuries/*physiology ; Liver Cirrhosis/etiology/prevention & control ; Liver Regeneration/*physiology ; Mesenchymal Stromal Cells/physiology ; Myofibroblasts/physiology

Using forward and reverse genetics and global gene expression analyses, we explored the crosstalk between the IκB kinase β (IKKβ) and the transforming growth factor β (TGFβ) signaling pathways. We show that in vitro ablation of Ikkβ in fibroblasts led to progressive ROS accumulation and TGFβ activation, and ultimately accelerated cell migration, fibroblast-myofibroblast transformation and senescence. Mechanistically, the basal IKKβ activity was required for anti-oxidant gene expression and redox homeostasis. Lacking this activity, IKKβ-null cells showed ROS accumulation and activation of stress-sensitive transcription factor AP-1/c-Jun. AP-1/c-Jun activation led to up-regulation of the Tgfβ2 promoter, which in turn further potentiated intracellular ROS through the induction of NADPH oxidase (NOX). These data suggest that by blocking the autocrine amplification of a ROS-TGFβ loop IKKβ plays a crucial role in the prevention of fibroblast-myofibroblast transformation and senescence.
Adenoviridae ; genetics ; Animals ; Autocrine Communication ; physiology ; Cell Line ; Cell Movement ; Cellular Senescence ; Genetic Vectors ; genetics ; metabolism ; I-kappa B Kinase ; deficiency ; genetics ; metabolism ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Mice ; Myofibroblasts ; cytology ; metabolism ; NADPH Oxidases ; metabolism ; Oxidative Stress ; Promoter Regions, Genetic ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Superoxide Dismutase ; genetics ; metabolism ; Transcription Factor AP-1 ; metabolism ; Transforming Growth Factor beta ; genetics ; metabolism ; Up-Regulation

In order to investigate the roles of Wnt signal pathway in transformation of cardiac valvular myofibroblasts to the osteoblast-like phenotype, the primary cultured porcine aortic valve myofibroblasts were incubated with oxidized low density lipoprotein (ox-LDL, 50 mg/L), and divided into four groups according to the ox-LDL treatment time: control group, ox-LDL 24-h group, ox-LDL 48-h group, and ox-LDL 72-h group. Wnt signal pathway blocker Dickkopf-1 (DDK-1, 100 μg/L) was added in ox-LDL 72-h group. The expression of a-smooth muscle actin (α-SMA), bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), and osteogenic transcription factor Cbfa-1 was detected by Western blotting, and that of β-catenin, a key mediator of Wnt signal pathway by immunocytochemical staining method. The Wnt/β-catenin was observed and the transformation of myofibroblasts to the osteoblast-like phenotype was examined. The expression of α-SMA, BMP2, ALP and Cbfa-1 proteins in the control group was weaker than in the ox-LDL-treated groups. In ox-LDL-treated groups, the protein expression of a-SMA, BMP2, ALP, and Cbfa-1 was significantly increased in a time-dependent manner as compared with the control group, and there was significant difference among the three ox-LDL-treated groups (P<0.05 for all); β-catenin protein was also up-regulated in the ox-LDL-treated groups in a time-dependent manner as compared with the control group (P<0.05), and its transfer from cytoplasm to nucleus and accumulation in the nucleus were increased in the same fashion (P<0.05). After addition of DKK-1, the expression of α-SMA, bone-related proteins and β-catenin protein was significantly reduced as compared with ox-LDL 72-h group (P<0.05). The Wnt/ β-catenin signaling pathway may play an important role in transformation of valvular myofibroblasts to the osteoblast-like phenotype.
Actins ; metabolism ; Animals ; Aortic Valve ; cytology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Gene Expression Regulation ; drug effects ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Lipoproteins, LDL ; pharmacology ; Myofibroblasts ; drug effects ; Osteoblasts ; physiology ; Phenotype ; Swine ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

α-smooth muscle actin (α-SMA) and tenascin-C are stress-induced phenotypic features of myofibroblasts. The expression levels of these two proteins closely correlate with the extracellular mechanical microenvironment. We investigated how the expression of α-SMA and tenascin-C was altered in the periodontal ligament (PDL) under orthodontic loading to indirectly reveal the intrinsic mechanical microenvironment in the PDL. In this study, we demonstrated the synergistic effects of transforming growth factor-β1 (TGF-β1) and mechanical tensile or compressive stress on myofibroblast differentiation from human periodontal ligament cells (hPDLCs). The hPDLCs under higher tensile or compressive stress significantly increased their levels of α-SMA and tenascin-C compared with those under lower tensile or compressive stress. A similar trend was observed in the tension and compression areas of the PDL under continuous light or heavy orthodontic load in rats. During the time-course analysis of expression, we observed that an increase in α-SMA levels was matched by an increase in tenascin-C levels in the PDL under orthodontic load in vivo. The time-dependent variation of α-SMA and tenascin-C expression in the PDL may indicate the time-dependent variation of intrinsic stress under constant extrinsic loading.
Actins ; analysis ; drug effects ; Adult ; Animals ; Biomechanical Phenomena ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cells, Cultured ; Cellular Microenvironment ; physiology ; Humans ; Male ; Myofibroblasts ; physiology ; Orthodontic Wires ; Periodontal Ligament ; chemistry ; cytology ; Pressure ; Rats ; Rats, Sprague-Dawley ; Stress, Mechanical ; Tenascin ; analysis ; drug effects ; Time Factors ; Tooth Movement Techniques ; instrumentation ; Transforming Growth Factor beta1 ; pharmacology

The myofibroblasts have dual characteristics of smooth muscle cells and fibroblasts. In repairing tissular wound, myofibroblasts are involved in fibrogenesis and remodeling the extracellular matrix of the fibrotic cascades reaction. The review describes the morphological characteristics and biological behaviors of myofibroblasts and the application of skin wound age determination, which may provide reference for research in forensic medicine.
Actins/metabolism* ; Animals ; Cell Differentiation ; Cells, Cultured ; Extracellular Matrix/metabolism* ; Fibroblasts/physiology* ; Forensic Pathology/methods* ; Humans ; Muscle, Smooth/physiology* ; Myofibroblasts/physiology* ; Skin/injuries* ; Time Factors ; Wound Healing ; Wounds and Injuries/pathology*

BACKGROUNDTransforming growth factor-β1 (TGF-β1) is known to be a key fibrogenic cytokine in a number of chronic fibrotic diseases, including chronic allograft nephropathy. We examined the effects of inhibition of TGF-β1 expression by RNA interference on renal allograft fibrosis, and explored the mechanisms responsible for these effects.

METHODSA Sprague-Dawley-to-Wistar rat model of accelerated kidney transplant fibrosis was used. Sixty recipient adult Wistar rats were randomly divided into four groups: group J (sham-operated group), group T (plasmid-transfected group), group H (control plasmid group), and group Y (transplant only group). Rats in group T were transfected with 200 µg of TGF-β1 short hairpin RNA (shRNA). Reverse transcription-polymerase chain reaction and Western blotting were used to examine the expression of TGF-β1, Smad3/7, E-cadherin, and type I collagen. The distribution of type I collagen was measured by immunohistochemistry. The pathologic changes and extent of fibrosis were assessed by hematoxylin and eosin and Masson staining. E-cadherin and α-smooth muscle actin immunohistochemical staining were used to label tubular epithelial cells and fibroblasts, respectively.

RESULTSPlasmid transfection significantly inhibited the expression of TGF-β1, as well as that of its target gene, type I collagen (P < 0.05 and P < 0.01, respectively). In addition, the degree of fibrosis was mild, and its development was delayed in plasmid-transfected rats. In contrast, TGF-β1-shRNA transfection maintained the expression of E-cadherin in tubular epithelial cells while it inhibited the transformation from epithelial cells to fibroblasts. Blood urea nitrogen and serum creatinine were lower in the plasmid group than in the control groups (P < 0.05 and P < 0.01, respectively).

CONCLUSIONSThis study suggests that transfection of a TGF-β1-shRNA plasmid could inhibit the fibrosis of renal allografts. The mechanism may be associated with the downregulation of Smad3 and upregulation of Smad7, resulting in suppressed epithelial-myofibroblast transdifferentiation and extracellular matrix synthesis.

Animals ; Blotting, Western ; Cell Transdifferentiation ; genetics ; physiology ; Epithelial Cells ; cytology ; Fibrosis ; prevention & control ; Kidney ; metabolism ; pathology ; Kidney Transplantation ; methods ; Myofibroblasts ; cytology ; RNA, Small Interfering ; genetics ; physiology ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Transplantation, Homologous