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Objective To investigate the effects of Smad7 knock down by lentivirus on rat cardiac fibroblasts proliferation, migration, cell differentiation and collagen secretion in vitro. Methods The primary cardiac fibroblasts were separated from the hearts of ten SD rats and identified by immunohistochemical method. The lentivirus transfection knocked down the expresson of Smad7 in cardiac fibroblasts, Western blotting was used to detect the efficiency of Smad7 knock down by lentivirus. The proliferation of cardiac fibroblasts was quantified by real-time unlabeled cell analyzer. Cell migration was evaluted by cell wound scratch assay. Western blotting was used to detect expression of α-smooth muscle actin（α-SMA） and collagen Ⅰ（Col Ⅰ）. Results Myocardial fibroblasts were successfully cultured and identified by immunocytochemical methods. The multiplicity of infection（MOI） that lentivirus transduction of myocardial fibroblasts was 100. After lentivirus transduction, 88.33% myocardial fibroblasts expressed green fluorescent protein, showed that the lentivirus could significantly reduce the protein expression of Smad7. Smad7 deficiency decreased the proliferation and migration of cardiac fibroblasts, increased the protein expression of α-SMA and decreased collagen secretion. The results indicated that Smad7 deficiency significantly down-regulated the proliferation and migration of cardiac fibroblasts, increased α-SMA protein expression and reduced ColⅠ protein expression. Conclusion Smad7 deficiency can significantly change the cardiac fibroblasts function, that is related to the pathological mechanism that lead to myocardial fibrosis
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Objective To observe the effect of urantide on the expression of osteopontin (OPN) and α-smooth muscle actin (a-SMA) in the heart tissue of atherosclerosis (AS) rats, and to explore its mechanism of prevention and treatment of myocardial fibrosis injury in rats. Methods Totally 120 3-week-old healthy male Wistar rats in SPF grade were randomly divided into six groups; control group, model group, simvastatin group, urantide (3 days, 7 days, 14 days). HE and Masson trichrome staining were used to observe the morphology of rat heart and the expression of collagen fibers. Immunohistochemistry and Western blotting were used to detect the expression of OPN and α-SMA protein. Results In AS model group, cardiomyocyte hypertrophy or atrophy, a large number of inflammatory cell infiltration and a small amount of foam cells were observed in the heart tissue of rats. The increase of collagen fibers and the expression of OPN and α-SMA protein in cardiac tissue were significantly higher than those in the control group. Compared with the AS model group, after urantide treatment, cardiac injury was significantly improved, and the expression of collagen fiber, OPN and α-SMA protein was decreased. Conclusion Urantide can inhibit the expression of OPN and α-SMA protein in the heart tissue of AS rats to alleviate myocardial fibrosis and play a protective role in the heart tissue of AS rats.
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Objective To investigate the effect of fibroblast growth factor (FGF) on the proliferation and transdifferentiation of cardiac fibroblasts ( CFs ) into myofibroblasts ( MFs ). Methods Rat CFs were isolated and cultured, and then induced by FGF. CCK-8 was used to detect the cell activity and proliferation. Immunofluorescence and Western blotting were used to detect the expression of a smooth muscle actin ( α-SMA ) and collagen I ( Col I ). Results The expression and activation of α-SMA and Col I increased with the increase of CFs culture generation. The number of CFs induced by FGF did not increased significantly; the expression of α-SMA in CFs induced by FGF1 and FGF2 decreased, and the number of activated MFs decreased. Conclusion FGF family has no effect on the proliferation of CFs, but FGF1 and FGF2 can inhibit the activation of CFs and reduce the differentiation into MFs.
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Context: Sub-epithelial myofibroblasts are known to influence the biology (proliferation, differentiation and apoptosis) of overlying epithelia. In the intestine, myofibroblasts have been demonstrated to be essential for epithelial differentiation. It is therefore hypothesized that myofibroblasts may also be involved in intestinal metaplasia that is characteristic of Barrett esophagus. Objective: This study endeavors to immunohistologically evaluate epithelial-myofibroblast interaction in Barrett's metaplasia. Materials and Methods: Nineteen archival esophageal endoscopic biopsies of Barrett's metaplasia were immune-phenotyped for the following epithelial and myofibroblast antigens - cytokeratins (CK) 8, 13, 18, CDX2 (Caudal type homeobox 2), a-smooth muscle actin (SMA). Results: α-SMA immunostaining revealed close association between myofibroblasts and metaplastic Barrett's epithelium but not with normal esophageal squamous epithelium. Myofibroblasts were more prominent in dysplastic than in non-dysplastic Barrett metaplasia. CDX2 and CK 8/18, indicators of intestinal differentiation were expressed in Barrett metaplasia but not normal esophageal squamous epithelium, while the reverse was the case for CK 13, which only stained normal esophageal squamous epithelium. Conclusion: Although their precise role is yet to be clearly defined, sub-epithelial myofibroblasts are very likely involved in the pathogenesis of Barrett's metaplasia.
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Objective To investigate the effect of 15d-PGJ2 on the expression of collagen type,CTGF and a-SMA in the hypertrophic scar in the rabbit ear,and the possibility of hypertrophic scar treated by 15d-PGJ2.Methods 18 New Zealand white rabbits were used to establish a hypertrophic scar model on the rabbit ear.The wounds were established as follows:2 cm × 3 cm wounds with total skin loses on the ventral side,2 wounds for each ear,totally 72 wounds.The wounds were randomly divided into the 15d-PGJ2 treatment group and NS control group.20μl 15d-PGJ2 or NS was injected into the ear scar once a day for 7 days.At 7,14 and 21 days after the injection,12 scars of each group were harvested.The expression of collagen type Ⅰ,CTGF and a-SMA was detected by immunohistochemical method.Results Excessive dermal scars on rabbit ears that were similar to human hypertrophic scar appeared in the two groups.Compared with the NS-treated scars group,the 15d-PGJ2-treated scars appeared to be smaller,softer,flatter and lighter in color.The expression of collagen type Ⅰ,CTGF and a-SMA in the 15d-PGJ2 group was significantly decreased as compared with that in the control group at different time points(P＜0.05).Conclusion 15d-PGJ2,the ligand of PPAR-r,can reduce the expression of collagen type Ⅰ,CTGF and a-SMA of hypertrophic scar in the rabbit ears and plays an important role in the prevention and treatment of hypertrophic scar.It may provide a new approach for the treatment of hypertrophic scar in clinical setting.
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[Objective]To examine the effects of TGF-?1 on the production of ?-SMA and extracellular matrix in flexor tendon sheath fibroblasts. [Methods]Sheath fibroblasts were obtained from rabbit flexor tendons.Cell culture was supplemented with 5ng/ml of TGF-?1.After 48 hours incubation,the production of a-SMA was assayed by Western-Blot.The productions of collagen I and fibronectin in supernatants culture were examined using ELISA.[Results]Treatment with TGF-?1 significantly stimulated a-SMA production in flexor tendon sheath fibroblasts (P
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Los miofibroblastos representan una subpoblación de fibroblastos con un fenotipo similar al de las células del músculo liso, debido a que expresan a-actina de músculo liso en su citoesqueleto, aunque también como subpoblación exhiben diferencias fenotípicas entre sí en diferentes órganos, su fisiología es semejante en los diferentes tejidos y órganos en que se encuentren. Con base en su amplio espectro de síntesis y secreción de moléculas, tales como citocinas, interleucinas, quimiocinas, factores del crecimiento, lípidos, diversos mediadores fisiológicos, moléculas de la matriz extracelular, MMPs y TIMPs, desempeñan una participación muy importante durante la embriogénesis, organogénesis, inflamación, reparación y cicatrización, siendo además fundamentales en los diferentes procesos de regeneración y reparación (fibrosis) que ocurren en los distintos órganos. En el caso del sistema respiratorio, los miofibroblastos son importantes, tanto en las vías aéreas como en el pulmón, participando fundamentalmente en los diversos procesos patogénicos, ya sea en enfermedades con un patrón degradativo como el enfisema, o bien, con un patrón reparativo con depósito excesivo de los diversos componentes de la matriz extracelular, tal como ocurre en la fibrosis pulmonar y el asma. Son especialmente importantes en las diferentes formas de fibrosis pulmonar ya sea de causa conocida o idiopática. Esta última, a semejanza del asma, parece restringirse fundamentalmente a zonas del tejido adyacente a epitelios alveolares dañado, donde se da una relación fisiopatogénica neumocito tipo II-miofibroblasto-fibroblasto. Los miofibroblastos se originan principalmente por transdiferenciación de fibroblastos y principalmente por estimulación del TGF-β1.
Myofibroblasts are a fibroblast subpopulation with a phenotype similar to smooth muscle cells, since they express the cytoskeletal a-smooth muscle actin (α-SMA); however, in different organs, they show some phenotypical differences. Their physiology is similar in the different tissues and organs. Based on their extensive spectrum of synthesis and secretion of molecules such as cytokines, interleukins, chemokines, growth factors, lipids, diverse physiological mediators, molecules of the extracelullar matrix, MMPs and TIMPs, they play a very important role during embryogenesis, organogenesis, inflammation, repair and wound healing, besides being fundamental in the processes of regeneration and repair (fibrosis) that occur in the different organs. In the case of the respiratory system, the myofibroblasts are as important in the air ways as in the lung, mainly participating in the diverse pathogenic processes; whether in pathologies with a derivative pattern such as emphysema, or in diseases with a fibrogenic pattern with excessive synthesis of the diverse components of the extracellular matrix, as occurs in pulmonary fibrosis and asthma. Myofibroblasts are especially important in the different forms of pulmonary fibrosis whether idiopathic or of known cause. Idiopathic fibrosis, as asthma, seems to be essentially restricted to areas of tissue adjacent to damaged alveolar-epithelial areas, where a physiopathogenic relation of type II neumocyte-myofibroblast-fibroblast exists. On the other hand, myofibroblasts are mostly derived from fibroblast transdifferentiation by TGF-β1 stimulation.
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The purpose of this study is to investigate that the biological effect of mitomycin C(MMC) on inhibition of cellular proliferation, extracellular synthesis of type_I coolagen, lamini, and study of myofibroblast is derived directly from the primary and recurrent pterygial mesenchymal cell by MMC concentration and duration of exposure time used clinically. Human pterygial mesenchymal cells were exposed for 3 minutes, 5 minutes, and 10 minutes to MMC 0.01%, 0.03%, 0.05%, and DMEM(control). After cells were incubated for 24 hours, [H3] thymidine proliferative assay, immunoassay of type_I collagen and laminin, immunohistochemical and ultrastructual study of -smooth muscle actin were perfromed in vitro. Recurrent pterygal mesenchymal cells were more proliferated and stronger than primary pterygial cells in proliferation and inhibition of cellular proliferation assay. In immunoassay of extracellular matrix, the higher the concentration of MMC and longer the duration of exposure time, the inhibition of laminin are strong. However, there was a little effect of inhibition of synthesis of type-I collagen. Also the results of positive responsed immunohistochemical and ultrastructual finding such as a few pinocytosis, microfilaments, microtendon, and basal lamina like materal by TEM of myofibroblast were revealed. We think that the reccurent pterygial tissue have more effect on inhibition of cellular proliferation and laminin synthesis than primary pterygium. Therefore, reconsideration of MMC concentreation and duration time should be need in case of recurrent tissue, further experimental and clinical research on the myofibroblast and inhibition of type-I collagen also should be need.