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 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*

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*

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

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

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*

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

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

BACKGROUNDUrotensin II (UII) is a new vasoconstrictive peptide that may activate the adventitial fibroblasts. Transforming growth factor-β1 (TGF-β1) is an important factor that could induce the phenotypical transdifferentiation of adventitial fibroblasts. This study aimed to explore whether TGF-β1 is involved in UII-induced phenotypic differentiation of adventitial fibroblasts from rat aorta.

METHODSAdventitial fibroblasts were prepared by the explant culture method. TGF-β1 protein secretion from the cells was determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression of α-smooth nuscle actin (α-SM-actin), the marker of phenotypic differentiation from fibroblasts to myofibroblasts, were determined using real-time quantitative RT-PCR (real-time RT-PCR) and Western blotting, respectively.

RESULTSUII stimulated the secretion of TGF-β1 in cultured adventitial fibroblasts in a time-dependent manner. The secretion reached a peak at 24 hours, was higher by 69.8% (P < 0.01), than the control group. This effect was also concentration dependent. Maximal stimulation was reached at 10(-8) mol/L of UII (P < 0.01), which was increased by 59.9%, compared with in the control group (P < 0.01). The secretion of TGF-β1 induced by UII was significantly blocked by SB-710411 (10(-7) mol/L), a specific antagonist of UII receptor. In addition, both UII (10(-8) mol/L) and TGF-β1 significantly stimulated α-SM-actin mRNA and protein expression. Moreover, the α-SM-actin induced by UII was inhibited by the specific neutralizing antibody (20 µg/ml) of TGF-β1, while the α-SM-actin expression stimulated by TGF-β1 (20 ng/ml) was inhibited by SB-710411 (10(-7) mol/L), the UII receptor antagonist.

CONCLUSIONThis study suggests that UII could induce TGF-β1 secretion in adventitial fibroblasts via UT activation, and TGF-β1 might be involved in phenotypic differentiation from adventitial fibroblasts into myofibroblasts induced by UII, and TGF-β1 signaling might be one of the important pathways by which UII is involved in vascular fibrosis.

Actins ; analysis ; genetics ; Animals ; Aorta ; cytology ; Cell Transdifferentiation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; drug effects ; Male ; Myofibroblasts ; cytology ; Phenotype ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Signal Transduction ; Transforming Growth Factor beta1 ; physiology ; Urotensins ; antagonists & inhibitors ; pharmacology