OBJECTIVE: To investigate the effect of TGF-β1 on Shh signaling pathway during the transformation of meningeal fibroblasts into myofibroblasts. METHODS: Primary meningeal fibroblasts were isolated from neonatal (24 h) SD rats and purified using type Ⅳ collagenase. The isolated cells were treated with 10 ng/mL TGF-β1 alone or in combination with 20 μmol/L SB-431542 (a TGF-β1 receptor inhibitor) for 72 h, and the changes in proliferation and migration abilities of the fibroblasts were assessed with CCK-8 assay and cell scratch test. The expression of fibronectin (Fn) was detected with immunofluorescence assay, and Western blotting was performed to examine the expressions of Fn, α-SMA and Shh protein in the cells; the expression of Shh mRNA was detected with real-time fluorescence quantitative PCR. RESULTS: TGF-β1 treatment obviously enhanced the proliferation and migration of primary meningeal fibroblasts (P < 0.05), and promoted the transformation of meningeal fibroblasts into myofibroblasts and the secretion of Fn (P < 0.05). TGF-β1 treatment also upregulated the expression of Shh at both protein and mRNA levels (P < 0.05). Treatment with SB-431542 partially blocked the effect of TGF-β1 on the transformation of meningeal fibroblasts (P < 0.05). CONCLUSION TGF-β1 can induce the transformation of meningeal fibroblasts into myofibroblasts by up-regulating Shh expression in Sonic Hedgehog signaling pathway.
Animals ; Fibroblasts/metabolism* ; Hedgehog Proteins ; Myofibroblasts/metabolism* ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1/metabolism*

OBJECTIVE: To research the relation between the time-dependent appearances of myotibroblasts during the repair of contused skeletal muscle in rat and wound age determination. METHODS: A total of 35 SD male rats were divided into the control and six injured groups according to wound age as follows: 12 h, 1 d, 5 d, 7 d, 10 d and 14 d after injury. The appearances of myofibroblasts were detected by HE staining, immunohistochemistry and confocal laser scanning microscopy. Masson's trichrome staining was utilized to examine collagen accumulation in the contused areas. RESULTS: Immunohistochemical staining showed that α-SMA+ myofibroblasts were initially observed at 5 d post-injury. The average ratio of myofibroblasts was highest at 14 d post-injury, with all samples, ratios more than 50%. In the other five groups, the average of α-SMA positive ratios were less than 50%. The collagen stained areas in the contused zones, concomitant with myofibroblast appearance, were increasingly augmented along with advances of posttraumatic interval. CONCLUSION The immunohistochemical detection of myofibroblasts can be applied to wound age determination. The myofibroblasts might be involved in collagen deposition during the repair of contused skeletal muscle in rat.
Animals ; Collagen/metabolism* ; Contusions/metabolism* ; Immunohistochemistry ; Male ; Microscopy, Confocal ; Muscle, Skeletal/metabolism* ; Myofibroblasts/metabolism* ; Rats ; Time Factors ; Wound Healing

OBJECTIVETo investigate the functions of human periodontal myofibroblast (MFB) in vitro.

METHODSHuman periodontal fibroblast (hPDLFs) was cultured and induced to MFB by transforming growth factor-β1 (TGF-β1). MFB was denoted as the experimental group, whereas the hPDLFs was the control group. The groups were continuously cultured and harvested at 0, 12, 24, 48, and 72 h. The MFB marker α-smooth muscle actin (α-SMA) was examined by immunocytochemistry. The expression of fibronectin (FN) between MFB was examined by immunocytochemistry to detect the MFB contact relationship. The mRNA expression levels of α-SMA, collagen (Col) I, and Col III were measured by reverse transcription-polymerase chain reaction (RT-PCT) to analyze extracellular matrix secretion. The protein expression levels of α-SMA and Col I were also assessed by Western blot.

RESULTSThe experimental group had significantly higher α-SMA expression than the control group at 0 h (P < 0.001). A positive expression of FN was found between MFB. The experimental group had significantly higher expression levels of Col I and Col III than the control group at 24 h (P < 0.001).

CONCLUSIONHuman periodontal MFB presents a continuous, high expression of α-SMA. MFB could interact through FN. MFB is significantly capable of extracellular matrix secretion.

Actins ; Epithelial Cells ; Extracellular Matrix ; Fibroblasts ; Fibronectins ; Humans ; Jaw ; metabolism ; Myofibroblasts ; Transforming Growth Factor beta1

Animals ; Autophagy ; Biomarkers, Tumor ; metabolism ; Caveolin 1 ; deficiency ; metabolism ; Drug Delivery Systems ; Humans ; Myofibroblasts ; pathology ; Neoplasms ; metabolism ; pathology ; Prognosis ; Stromal Cells ; metabolism ; Tumor Microenvironment

AIMTo observe the expression of a-smooth muscle actin(a-SMA) in primary cultural fibroblasts of rats.

METHODS12 female Wistar rats were randomly assigned into two groups, the normal group and the model group. The model group was filled with bleomycin A2 (5 mg/kg) once into the trachea. The normal group was filled with equal saline into the trachea. The rats were sacrificed under drugged state at 28 days of feeding, then Hematoxylin-Eosin staining and electron microscopy were used to evaluate the foundation of the model. The isolated fibroblasts from the rats were cultured in vitro. Flow cytometry was used in the test to observe the expression of alpha-SMA in fibroblast in vitro in rats.

RESULTSThe formation of fibroblast foci was observed in the model group by optical microscope. The ultrastructure in pulmonary tissue of the model group rats were changed and proliferated myofibroblasts with filaments were found in the alveolar septa by electron microscopy. The expression of alpha-SMA was positive in the normal and model group. There was no difference between the two groups in the rates of positive cells (P > 0.05).

CONCLUSIONBoth the normal and model groups had the phenotype conversion in lung fibroblasts in vitro.

Actins ; metabolism ; Animals ; Bleomycin ; Cells, Cultured ; Female ; Fibroblasts ; metabolism ; pathology ; Lung ; pathology ; Myofibroblasts ; pathology ; Pulmonary Fibrosis ; chemically induced ; pathology ; Rats ; Rats, Wistar

OBJECTIVETo explore the role of the fibroblast transdifferentiation into myofibroblasts (MFBs) in the pathogenesis of systemic sclerosis (SSc) and investigate the influence of transforming growth factor β(1) (TGF-β(1)) and blocking of its signal transduction on fibroblast transdifferentiation.

METHODSFibroblasts derived from the skin lesions of SSc patients and normal skin tissue were cultured in vitro. The proportion of MFBs in the fibroblast culture was analyzed qualitatively using immunocytochemistry and quantitatively with ELISA for α-smooth muscle actin (α-SMA). The changes in fibroblast transdifferentiation were observed after addition of TGF-β(1) in the cell culture and after blocking the signal transduction of TGF-β(1).

RESULTSThe fibroblasts isolated from SSc patients and control subjects showed a similar morphology. The mean number of cells positive for α-SMA in SSc group was significantly higher than that in the control group (P<0.01). As culture time extended, α-SMA levels of the two groups both increased gradually (P<0.01), but the increments were significantly greater in SSc group than in the control group at 24, 48, and 72 h (P<0.05 all). Addition of TGF-β(1) resulted in significantly increased α-SMA levels in both groups (P<0.05), and SSc group showed significantly higher α-SMA levels than the control group at 24, 48, and 72 h (P<0.01). In the presence of TGF-β(1), blocking of Smads, ERK/MAPK, and p38MAPK pathways, but not JNK/MAPK pathway, caused an obvious decrease in α-SMA levels in the fibroblasts in both groups.

CONCLUSIONThe fibroblasts in the skin lesion of SSc patients have strong potential of transdifferentiation into MFBs, and TGF-β(1) can promote this transdifferentiation process possibly involving Smads, and ERK/MAPK, and p38MAPK signalling pathways.

Actins ; metabolism ; Adult ; Cell Transdifferentiation ; physiology ; Cells, Cultured ; Female ; Fibroblasts ; pathology ; Humans ; Male ; Myofibroblasts ; pathology ; Scleroderma, Systemic ; pathology ; Signal Transduction ; Skin ; pathology ; Transforming Growth Factor beta1 ; metabolism

In the kidney, pericyte is the major source of myofibroblast (MyoF) in renal interstitium. It is reported that pericyte-myofibroblast transition（PMT）is one of the important pathomechanisms of renal interstitial fibrosis（RIF）. Among them, the main reasons for promoting RIF formation include pericyte recruitment, activation and isolation, as well as the lack of pericyte-derived erythropoietin. During the PMT startup process, pericyte activation and its separation from microvessels are controlled by multiple signal transduction pathways, such as transforming growth factor-β（TGF-β）pathway, vascular endothelial growth factor receptor (VEGFR) pathway and platelet derived growth factor receptor (PDGFR) pathway;Blocking of these signaling pathways can not only inhibit PMT, but also suppress renal capillaries reduction and further alleviate RIF. In clinic, many traditional Chinese medicine compound prescriptions, single traditional Chinese herbal medicine (CHM) and their extracts have the clear effects in alleviating RIF, and some of their intervention actions may be related to pericyte and its PMT. Therefore, the studies on PMT and its drug intervention will become the main development direction in the research field of anti-organ fibrosis by CHM.
Drugs, Chinese Herbal ; pharmacology ; Fibrosis ; Humans ; Kidney ; cytology ; drug effects ; pathology ; Myofibroblasts ; cytology ; Pericytes ; cytology ; Receptors, Platelet-Derived Growth Factor ; metabolism ; Signal Transduction ; Vascular Endothelial Growth Factor A ; metabolism

Vascular adventitial fibroblasts (AF) differentiation to myofibroblasts (MF) is the critical physiopathologic feature of vascular remodeling. This study was to investigate the role of RhoA-Rho kinase signaling pathway in AF differentiation to MF induced by transforming growth factor β1 (TGF-β1). The results showed that TGF-β1 up-regulated total RhoA protein expression and RhoA activity in cultured AF by Western blotting and Rho pull-down assay, respectively. TGF-β1 up-regulated phospho-Myosin phosphatase target subunit (MYPT1, a downstream substrate of Rho kinase) expression without altering Rho kinase protein expression, indicating TGF-β1 induced the enhancement of activity of Rho kinase. Ad-N19RhoA-hrGFP virus infection and Y27632, a specific inhibitor of Rho kinase, dose-dependently inhibited TGF-β1-induced α-SM-actin and Calponin expression, as markers of MF differentiation. In conclusion, the RhoA-Rho kinase pathway is involved in AF differentiation to MF induced by TGF-β1.
Actins ; metabolism ; Adventitia ; cytology ; Calcium-Binding Proteins ; metabolism ; Cell Differentiation ; Cells, Cultured ; Fibroblasts ; cytology ; Microfilament Proteins ; metabolism ; Myofibroblasts ; cytology ; Signal Transduction ; Transforming Growth Factor beta1 ; pharmacology ; Up-Regulation ; rho-Associated Kinases ; metabolism ; rhoA GTP-Binding Protein ; metabolism

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

Objective To investigate the effects and mechanism of Interleukin-33 (IL-33) mediated proliferation and differentiation of pulmonary myofibroblasts (MFbs) in pulmonary fibrosis (PF). Methods C57BL/6 mice were randomly divided into four groups: a control group, a bleomycin (BLM) group, a BLM combined with IL-33 group and a BLM combined with anti-IL-33 antibody group, 12 mice in each group. The PF model was induced by intratracheal injection of BLM (5000 U/kg). The degrees of fibrosis were examined using HE and Masson staining. ELISA was used to measure the plasma levels of IL-33. Immunohistochemical staining was used to measure the expression of alpha smooth muscle actin (α-SMA) in lung tissue. Primary pulmonary fibroblasts were isolated and cultured from lung tissues of mice. The cells were divided into four groups: a control group, an IL-33 group, an IL-33 combined with dimethyl sulfoxide (DMSO) group and an IL-33 combined with pyrrolidine dithiocarbamate (PDTC) group. The cells were treated with DMSO or PDTC for 1 hour and then with IL-33 for 48 hours. Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay and cell cycle was measured by flow cytometry. Transwell^TM assay was used to analyze cell migration. Real-time quantitative PCR was used to measure the expression of collagen type I (Col1), Col3 and α-SMA mRNA. The protein levels of IL-33, Col1, Col3, α-SMA, eukaryotic initiation factor 3a (eIF3a), phosphorylated IκBα (p-IκBα) (total lysate), p-NF-κB p65(total lysate) and NF-κB p65 (nucleus) were measured by Western blot analysis. Results In vivo, compared with the control group, the expressions of IL-33, p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65(nucleus), eIF3a, α-SMA, Col1 and Col3 in the BLM group significantly increased. Compared with the BLM group, the expressions of p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3 in the IL-33 group increased further and the PF was further aggravated. But the effect of anti-IL-33 antibody was just opposite to that of IL-33. In vitro, IL-33 markedly induced the proliferation and migration of pulmonary fibroblasts, and significantly up-regulated the expression of p-IκBα (total lysate), p-NF-κB p65(total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3. But all these effects of IL-33 were reversed by pyrrolidine dithiocarbamate. Conclusion The results suggest that IL-33 may promote the expression of eIF3a by activating NF-κB signaling pathway, thus inducing the proliferation and differentiation of MFbs and promoting the occurrence and development of PF.
Animals ; Mice ; Bleomycin/metabolism* ; Cell Differentiation ; Cell Proliferation ; Dimethyl Sulfoxide/pharmacology* ; Fibroblasts ; Interleukin-33/pharmacology* ; Mice, Inbred C57BL ; Myofibroblasts/metabolism* ; NF-kappa B/metabolism* ; NF-KappaB Inhibitor alpha/metabolism* ; Pulmonary Fibrosis ; Signal Transduction