Transforming Growth Factor-β1 Induces Transdifferentiation of Fibroblasts into Myofibroblasts in Hypoxic Pulmonary Vascular Remodeling

The muscularization of non-muscular pulmonary arterioles is an important pathological feature of hypoxic pulmonary vascular remodeling. However, the origin of the cells involved in this process is still not well understood. The present study was undertaken to test the hypothesis that transforming growth factor-β1 （TGF-β1） can induce transdifferentiation of fibroblasts into myofibroblasts, which might play a key role in the muscularization of non-muscular pulmonary arterioles. It was found that mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and fight ventricular hypertrophy became evident after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and muscular vessels was significantly different after 7 d of hypoxia. Immunocytochemistry results demonstrated that the expression of α-smooth muscle actin was increased in intra-acinar pulmonary arteries with increasing hypoxic time. TGF-β1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-β1 protein staining was poorly positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments and a well-developed rough endoplasmic reticulum. Taken together, our results suggested that TGF-β1 induces transdifferentiation of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary vascular remodeling.     

Transforming growth factor-beta1 expression in cultured corneal fibroblasts in response to injury

The mechanisms underlying TGF-beta regulation in response to injury are not fully understood. We have developed an in vitro wound model to evaluate the expression and localization of transforming growth factor-beta1 in rabbit corneal fibroblasts in response to injury. Experiments were conducted in the presence or absence of serum so that the effect of the injury could be distinguished from exogenous wound mediators. Cultures were wounded and evaluations conducted over a number of time points. Expression of TGF-beta1 RNA was determined using Northern blot analysis and in situ hybridization, while the TGF-beta receptors were identified by affinity cross-linking. Injury increased the expression of TGF-beta1 mRNA in cells at the wound edge after 30 min; this response was amplified by the addition of serum. TGF-beta1 mRNA expression was observed in a number of cells distal from the wound. After wound closure, TGF-beta1 mRNA was negligible and resembled unwounded cultures. The half-life of TGF-beta1 mRNA was two times greater in the wounded cultures, indicating that the injury itself maintained the expression, while cell migration was present. Analogous to these findings, we found that binding of TGF-beta to its receptors was maximal at the wound edge, decreasing with time and distance from the wound. These results indicate that injury increases the level of expression of TGF-beta1 mRNA and maintains a higher level of receptor binding during events in wound repair and that these might facilitate the migratory and synthetic response of stromal fibroblasts.     

Locally applied GM-CSF induces the accumulation of alpha-smooth muscle actin containing myofibroblasts.

We have examined the histological and cytoskeletal changes in rat connective tissues induced by subcutaneous perfusion with cytokines. Granulocyte macrophage-colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1-alpha (IL-1-alpha), transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) produced a significant fibroblast accumulation, neovascular development and a weak to moderate leukocyte infiltration, while interleukin-2 (IL-2) and gamma-interferon (gamma-IFN) induced intense mononucleated leukocyte infiltration. Immunofluorescence staining showed that accumulated fibroblastic cells were positive for alpha-smooth muscle (SM) actin (but negative for the desmin and muscle myosin) only in GM-CSF-treated tissues. Electron microscopic examination established that a significant proportion of fibroblastic cell in GM-CSF-, IL-1-alpha- or TGF-beta-treated animals were typical myofibroblasts. Only in GM-CSF-treated animals did microfilament bundles of myofibroblasts contain alpha-SM actin, when examined by immuno electron microscopy. Our results suggest that locally applied cytokines induce the formation of distinct granulation tissues. In particular, GM-CSF stimulates alpha-SM actin synthesis in myofibroblasts, illustrating an unexpected extra-hematopoietic in vivo effect of this factor.     

原代培养鼠肺成纤维细胞中α-平滑肌肌动蛋白的表达

目的：观察原代培养大鼠肺成纤维细胞（FB）中α-平滑肌肌动蛋白（α-SMA）的表达情况。方法：24只雌性Wistar大鼠,随机分为正常组和模型组,气管内注入博莱霉素A2（BLM-A2）（5mg／kg）建立大鼠肺间质纤维化（PF）模型,正常组气管内注入等量生理盐水。实验周期第28d腹主动脉放血法处死,肺组织HE染色和透射电镜检查。体外原代培养FB,传3-5代后流式细胞术检测α-SMA表达的细胞阳性率。结果：HE染色28d模型组大片肺泡结构破坏,肺泡腔消失,有成纤维细胞灶形成,电镜观察肺组织超微结构发生改变,肺间质中可见胞浆内有微丝样结构的MF。流式细胞分析结果：正常组α-SMA表达的细胞阳性率为95．5％±4．68％,模型组α-SMA表达的细胞阳性率为96．2％±2．14％,与正常组相比P〉0．05。结论：原代培养的大鼠肺FB中α-SMA的阳性表达率正常组和模型组均显著增高,体外培养的大鼠肺FB传3—5代后可能有部分已经转化为肌纤维母细胞（MF）。     

Transforming growth factor-beta induces the expression of ANF and hypertrophic growth in cultured cardiomyoblast cells through ZAK

Transforming growth factor-beta (TGF-beta) has been associated with the onset of cardiac cell hypertrophy, but the mechanisms underlying this dissociation are not completely understood. By a previous study, we investigated the involvement of a MAP3K, ZAK, which in cultured H9c2 cardiac cells is a positive mediator of cell hypertrophy. Our results showed that expression of a dominant-negative form of ZAK inhibited the characteristic TGF-beta-induced features of cardiac hypertrophy, including increased cell size, elevated expression of atrial natriuretic factor (ANF), and increased organization of actin fibers. Furthermore, dominant-negative MKK7 effectively blocked both TGF-beta-and ZAK-induced ANF expression. In contrast, a JNK/SAPK specific inhibitor, sp600125, had little effect on TGF-beta- or ZAK-induced ANF expression. Our findings suggest that a ZAK mediates TGF-beta-induced cardiac hypertrophic growth via a novel TGF-beta signaling pathway that can be summarized as TGF-beta>ZAK>MKK7>ANF.     

Transforming growth factor-beta does not alter interleukin-1 expression in cultured human macrophages

Transforming growth factor-beta (TGF beta) is a growth modulator that stimulates the growth of fibroblastic cells but inhibits the growth of cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins, and of proteases and the type 1 plasminogen activator inhibitor (PAI-1) by cultured cells. TGF beta appears also to have various immunoregulatory effects, suppressing both T- and B-cell activities. It has been proposed that it might increase the expression of interleukin-1 (IL-1) mRNA in cultured human monocytes, thus potentiating immune functions. To analyze the role of TGF beta in IL-1 production we have now quantitated the effect of this factor on the production of biologically active IL-1 as well as IL-1 beta mRNA expression. The effect of TGF beta on IL-1 production optimally activated with bacterial lipopolysaccharide (LPS) was also studied. It was found that IL-1 activity and mRNA levels were rapidly elevated by LPS but not by TGF beta. Culture fluids from monocytes treated with TGF beta alone or with TGF beta plus LPS inhibited the proliferation of the test thymocytes. After gel filtration, the media from TGF beta-treated cultures showed no activity in the molecular weight area of IL-1 (approx. 15 kD), while the supernatants from TGF beta plus LPS-induced cells contained IL-1 activity in these fractions, the magnitude of which was, however, at the same level as in the culture fluids derived from cells stimulated with LPS alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of alpha-smooth muscle actin gene expression in myofibroblast differentiation from rat lung fibroblasts

Myofibroblasts express alpha-smooth muscle actin and have a phenotype intermediate between fibroblasts and smooth muscle cells. Their emergence can be induced by cytokines such as transforming growth factor beta; but the regulatory mechanism for induction of alpha-smooth muscle actin gene expression in myofibroblast differentiation has not been determined. To examine this mechanism at the level of the alpha-smooth muscle actin promoter, rat lung fibroblasts were transfected with varying lengths of the alpha-smooth muscle actin promoter linked to the chloramphenicol acetyl transferase reporter gene and treated with transforming growth factor beta1. The results show that the shortest inducible promoter was 150 base pairs long, suggesting the presence in this region of cis-elements of potential importance in transforming growth factor beta1 induced myofibroblast differentiation. Transfection of "decoy" oligonucleotides corresponding to sequences for four suspected regulatory factors demonstrated that only the transforming growth factor beta control element is involved in the regulation of transforming growth factor beta1-induced alpha-smooth muscle actin expression in myofibroblast differentiation. Consistent with this conclusion is the finding that a mutation in the transforming growth factor beta control element caused a significant reduction in promoter activity. These observations taken together show that alpha-smooth muscle actin promoter regulation during myofibroblast differentiation is uniquely different from that in smooth muscle cells and other cell lines. Since myofibroblasts play a key role in wound contraction and synthesis of extracellular matrix, clarification of this differentiation mechanism should provide new insight into fibrogenesis and suggest future novel strategies for modulation of wound healing and controlling fibrosis.     

Transforming growth factor-beta 1 expression in irradiated liver

The expression of transforming growth factor-beta 1 (TGF-beta 1) in the liver of irradiated rats was increased in a dose-dependent fashion 9 months after irradiation. Expression of TGF-beta 1 was confined primarily to hepatocytes in the pericentral region of the liver, and the percentage of hepatocytes strongly positive for TGF-beta 1 was significantly correlated with the extent of fibrosis. We further showed that a localized injection of TGF-beta 1 into normal rat liver elicited a strong fibrotic reaction at the injection site. These results suggest that the increased hepatic concentration of TGF-beta 1 in response to radiation injury may be important in the pathogenesis of radiation hepatitis. TGF-beta 1 was also found to be present at a significantly higher concentration in unirradiated human hepatocytes than in normal rat hepatocytes, implying that the propensity for humans to develop radiation hepatitis may result in part from the elevated levels of TGF-beta 1 normally found in human liver.     

Transforming growth factor-beta 1 up-regulates type IV collagenase expression in cultured human keratinocytes.

During the wound healing process lysis of basement membranes precedes keratinocyte migration into the wound bed. We studied, in vitro, whether this degradation of basement membranes could be regulated by transforming growth factor-beta 1 (TGF-beta 1), which is known to accelerate wound healing in vivo. Transforming growth factor-beta 1 was found to increase the expression of both 92- and 72-kDa type IV collagenases (gelatinases) in cultured human mucosal and dermal keratinocytes. The 92-kDa enzyme predominated in both unstimulated and stimulated cultures. The 92-kDa form was stimulated over 5-fold, and the other form by a factor of 2-3. This increase in the synthesis of type IV collagenases was associated with a marked increase in the mRNA levels of these enzymes as well. The induction of the 92-kDa enzyme was similar in culture medium containing either 0.15 or 1.2 mM calcium chloride. Rat mucosal keratinocytes secreted only 92-kDa type IV collagenase, the secretion of which was not regulated by TGF-beta 1. Also, TGF-beta 1 did not cause any significant induction (maximum about 1.2-fold) of either type IV collagenase in human gingival fibroblasts. The induction levels of both collagenases in human keratinocytes were independent of the type of the extracellular matrix the cells were grown on. However, the basement membrane matrix (Matrigel) activated about half of the 92-kDa type to its 84-kDa active form. The data suggest that TGF-beta 1 has a specific function in up-regulating the expression of type IV collagenases in human keratinocytes, offering a possible explanation of how keratinocytes detach from basement membranes prior to the migration over the wound bed.     

Transforming growth factor-beta induces collagenase-3 expression by human gingival fibroblasts via p38 mitogen-activated protein kinase

Human collagenase-3 (matrix metalloproteinase 13 (MMP-13)) is characterized by exceptionally wide substrate specificity and restricted tissue specific expression. Human skin fibroblasts in culture express MMP-13 only when they are in three-dimensional collagen (Ravanti, L., Heino, J., López-Otín, C., and K?h?ri. V.-M. (1999) J. Biol. Chem. 274, 2446-2455). Here we show that MMP-13 is expressed by fibroblasts during normal human gingival wound repair. Expression of MMP-13 by human gingival fibroblasts cultured in monolayer or in collagen gel was induced by transforming growth factor-beta1 (TGF-beta1). Treatment of gingival fibroblasts with TGF-beta1 activated two distinct mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinase 1/2 (ERK1/2) in 15 min and p38 MAPK in 1 and 2 h. Induction of MMP-13 expression by TGF-beta1 was blocked by SB203580, a specific inhibitor of p38 MAPK, but not by PD98059, a selective inhibitor of ERK1/2 activation. Adenovirus-mediated expression of dominant negative p38alpha and c-Jun potently inhibited induction of MMP-13 expression in gingival fibroblasts by TGF-beta1. Infection of gingival fibroblasts with adenovirus for constitutively active MEK1 resulted in activation of ERK1/2 and JNK1 and up-regulation of collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) production but did not induce MMP-13 expression. In addition, activation of p38 MAPK by constitutively active MKK6b or MKK3b was not sufficient to induce MMP-13 expression. These results show that TGF-beta-elicited induction of MMP-13 expression by gingival fibroblasts is dependent on the activity of p38 MAPK and the presence of functional AP-1 dimers. These observations demonstrate a fundamental difference in the regulation of collagenolytic capacity between gingival and dermal fibroblasts and suggest a role for MMP-13 in rapid turnover of collagenous matrix during repair of gingival wounds, which heal with minimal scarring.     

Phenotype-dependent expression of alpha-smooth muscle actin in visceral smooth muscle cells

Alpha-Smooth muscle actin is one of the molecular markers for a phenotype of vascular smooth muscle cells, because the actin is a major isoform expressed in vascular smooth muscle cells and its expression is upregulated during differentiation. Here, we first demonstrate that the phenotype-dependent expression of this actin in visceral smooth muscles is quite opposite to that in vascular smooth muscles. This actin isoform is not expressed in adult chicken visceral smooth muscles including gizzard, trachea, and intestine except for the inner layer of intestinal muscle layers, whereas its expression is clearly detected in these visceral smooth muscles at early stages of the embryo (10-day-old embryo) and is developmentally downregulated. In cultured gizzard smooth muscle cells maintaining a differentiated phenotype, alpha-smooth muscle actin is not detected while its expression dramatically increases during serum-induced dedifferentiation. Promoter analysis reveals that a sequence (-238 to -219) in the promoter region of this actin gene acts as a novel negative cis-element. In conclusion, the phenotype-dependent expression of alpha-smooth muscle actin would be regulated by the sum of the cooperative contributions of the negative element and well-characterized positive elements, purine-rich motif, and CArG boxes and their respective transacting factors.     

Reexpression of alpha-smooth muscle actin isoform in cultured adult rat cardiomyocytes

Expression of alpha-smooth muscle (sm) actin in regenerating adult cardiomyocytes in culture was investigated. No alpha-sm-actin could be detected in adult ventricular tissue or in newly dissociated rod-shaped cells, whereas a fraction of the polymorphic flattened out adult cardiac cells in culture did express the protein. Immunofluorescence studies revealed a characteristic staining pattern, suggesting the preferential presence of alpha-sm-actin in stress fiber-like structures, while newly formed myofibrils contained only little alpha-sm-actin isoprotein. Cell-cell contacts were resumed, but formation of new gap junctions, as revealed by microinjecting Lucifer yellow, was not dependent on alpha-sm-actin expression. The behavior corresponds to fetal cardiomyocytes either in tissue or as single cells in culture where expression of alpha-sm-actin can be observed. Such immunofluorescence staining patterns with corresponding immunoblot data can be expected when a return to a less differentiated, more fetal state of the adult cardiomyocyte in culture is assumed. The possible role of the alpha-sm-actin and alpha-sarcomeric actin isoforms during reformation of myofibrillar sarcomeres is discussed.     

Transforming growth factor-beta 1 induces angiogenesis in vitro via VEGF production in human airway smooth muscle cells

Increase in size and number of bronchial blood vessels as well as hyperaemia are factors that contribute to airway wall remodelling in patients with chronic airway diseases, such as asthma and chronic obstructive pulmonary diseases (COPD). Expression of transforming growth factor beta 1 (TGF-beta 1), a multifunctional cytokine as well as vascular endothelial growth factor (VEGF), a key angiogenic molecule, has been shown in the inflammed airways in patients with chronic airway diseases. TGF-beta 1 has been implicated in the regulation of extracellular matrix, leading to airway remodelling in patients with chronic airway diseases. However, the role of TGF-beta 1 in regulating VEGF expression in patients with chronic airway diseases, as well as the underlying mechanisms are not yet well established. We investigated whether TGF-beta 1 stimulates VEGF expression in vitro and hence could influence vascular remodelling. Cultured human airway smooth muscle cells (HASMC) were serum deprived for 60 h before incubation with 5ng/ml of TGF-beta 1 for different time points. Control cells received serum-free culture medium. TGF-beta 1 treatment resulted in time dependent HASMC cell proliferation with maximal values for DNA biosynthesis at 24 h and cell number at 48 h. Northern blot analysis of VEGF mRNA expression showed increased levels in cells treated with TGF-beta 1 for 4 to 8 h. TGF-beta 1 also induced a time-dependent release of VEGF proteins in the conditioned medium after 48 h of treatment. Furthermore, the ability of HASMC-released VEGF proteins to induce human umbilical vein endothelial cells proliferation was inhibited by VEGF receptor antagonist, confirming that TGF-beta 1 induced VEGF was biologically active. We conclude that TGF-beta 1 in addition to an extracellular matrix regulator also could play a key role in bronchial angiogenesis and vascular remodelling via VEGF pathway in asthma.