Myofibroblasts: Trust your heart and let fate decide

Cardiac fibrosis is a substantial problem in managing multiple forms of heart disease. Fibrosis results from an unrestrained tissue repair process orchestrated predominantly by the myofibroblast. These are highly specialized cells characterized by their ability to secrete extracellular matrix (ECM) components and remodel tissue due to their contractile properties. This contractile activity of the myofibroblast is ascribed, in part, to the expression of smooth muscle α-actin (αSMA) and other tension-associated structural genes. Myofibroblasts are a newly generated cell type derived largely from residing mesenchymal cells in response to both mechanical and neurohumoral stimuli. Several cytokines, chemokines, and growth factors are induced in the injured heart, and in conjunction with elevated wall tension, specific signaling pathways and downstream effectors are mobilized to initiate myofibroblast differentiation. Here we will review the cell fates that contribute to the myofibroblast as well as nodal molecular signaling effectors that promote their differentiation and activity. We will discuss canonical versus non-canonical transforming growth factor-β (TGFβ), angiotensin II (AngII), endothelin-1 (ET-1), serum response factor (SRF), transient receptor potential (TRP) channels, mitogen-activated protein kinases (MAPKs) and mechanical signaling pathways that are required for myofibroblast transformation and fibrotic disease. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium ".     

Tissue stiffness, latent TGF-β1 Activation, and mechanical signal transduction: Implications for the pathogenesis and treatment of fibrosis

Tissue stiffening is a predominant feature of fibrosis and it obstructs organs whose mechanical properties are important for their function, such as the heart, lung, skin, and vessels. Stiff scar tissue further modulates the character of the healthy residing cells by driving the differentiation of a variety of precursor cells into fibrogenic myofibroblasts. This mechanical cue for myofibroblast differentiation establishes a vicious cycle because the excessive extracellular matrix-secreting and remodeling activities of myofibroblasts are cause and effect of further connective tissue contracture and stiffening. The second pivotal factor inducing myofibroblast development is transforming growth factor-β1. Recent findings suggest that transforming growth factor-β1 activity is partly controlled by myofibroblast contractile forces and tissue stiffness. This discovery opens new paths to prevent progression of fibrosis by specifically interfering with the stress perception and transmission mechanisms of the myofibroblast.     

Endogenous thrombospondin 1 protects the pressure-overloaded myocardium by modulating fibroblast phenotype and matrix metabolism

The matricellular protein thrombospondin (TSP) 1 is induced after tissue injury and may regulate reparative responses by activating transforming growth factor-β, by suppressing angiogenesis and by modulating inflammation and matrix metabolism. We hypothesized that endogenous TSP-1 may be involved in the pathogenesis of cardiac remodeling in the pressure-overloaded heart. Myocardial TSP-1 expression was increased in a mouse model of pressure overload because of transverse aortic constriction. TSP-1(-/-) mice exhibited increased early hypertrophy and enhanced late dilation in response to pressure overload. Pressure-overloaded TSP-1 null mice had intense degenerative cardiomyocyte changes, exhibiting more extensive sarcomeric loss and sarcolemmal disruption when compared with wild-type hearts. Accentuated hypertrophy and cardiomyocyte injury in TSP-1(-/-) hearts was accompanied by increased myofibroblast density. However, despite a 2-fold higher infiltration of the cardiac interstitium with myofibroblasts, pressure-overloaded TSP-1 null hearts did not exhibit significantly increased collagen content when compared with wild-type hearts. The disproportionately low collagen content in TSP-1 null hearts was attributed to infiltration with abundant, but functionally defective, fibroblasts that exhibited impaired myofibroblast differentiation and reduced collagen expression in comparison with wild-type fibroblasts. Impaired myofibroblast activation in TSP-1 null hearts was associated with reduced Smad2 phosphorylation reflecting defective transforming growth factor-β signaling. Moreover, TSP-1 null hearts had increased myocardial matrix metalloproteinase 3 expression and enhanced matrix metalloproteinase 9 activation after pressure overload. TSP-1 upregulation in the pressure-overloaded heart critically regulates fibroblast phenotype and matrix remodeling by activating transforming growth factor-β signaling and by promoting matrix preservation, thus preventing chamber dilation.     

活性氧簇在低氧诱导的肺纤维化中的作用

活性氧簇是一类氧的衍生分子,因其可引起蛋白质、DNA等大分子物质损伤、脂质过氧化,一直被认为是有害因子.近年来研究表明活性氧簇的产生是受精细调节的,并且它能作为第二信使激活细胞内MAPK(ERK1/2、p38MAPK和JNK)、Akt/PKB、JAK-STAT、核因子κB等多条信号通路,参与转化生长因子β1、血小板衍生生长因子、缺氧诱导因子、结缔组织生长因子等多种致纤维化因子的信号转导,调节细胞的增殖、分化、凋亡等生理功能,并与动脉粥样硬化、肝纤维化、肺纤维化等疾病的发生有关.低氧是一个重要的病理过程,可引起细胞损伤、组织炎细胞浸润,并能促进上皮细胞向间质细胞转化、细胞外基质沉积,上调转化生长因子β1、血小板衍生生长因子、缺氧诱导因子、结缔组织生长因子等多种致纤维化因子,故低氧有诱导肺纤维化形成的可能.而低氧又可引起活性氧簇产生增多.本文就活性氧簇在低氧诱导的肺纤维化中的作用作一综述.     

Glucocorticoid-induced down regulation of transforming growth factor-β1 in adult rat lung fibroblasts

Transforming growth factor-β1 mRNA and transforming growth factor β activity are decreased with exposure of normal adult rat lung fibroblasts to dexamethasone. Dexamethasone caused a decrease in transforming growth factor-β1 mRNA within 2 hours, which was sustained at least over a 24-hour period. The decrease in transforming growth factor-β1 mRNA was dose related. Dexamethasone treatment of rat lung fibroblasts also resulted in a decrease of transforming growth factor β activity as determined by the mink lung cell growth inhibition assay. These data indicate that glucocorticoids may regulate collagen synthesis at least in part through the mediation of transforming growth factor-β1 in rat lung fibroblasts.     

Extracellular matrix alterations in the atria: insights into the mechanisms and perpetuation of atrial fibrillation

Atrial fibrillation is the most common arrhythmia in clinical practice and is associated with increased cardiovascular morbidity and mortality. Atrial fibrosis, a detrimental process that causes imbalance in extracellular matrix deposition and degradation, has been implicated as a substrate for atrial fibrillation, but the precise mechanisms of structural remodelling and the relationship between atrial fibrosis and atrial fibrillation are not completely understood. A large number of experimental and clinical studies have shed light on the mechanisms of atrial fibrosis at the molecular and cellular level, including interactions between matrix metalloproteinases and their endogenous tissue inhibitors, and profibrotic signals through specific molecules and mediators such as angiotensin II, transforming growth factor-β1, connective tissue growth factor, and platelet-derived growth factor. This review focuses on the mechanisms of atrial fibrosis and highlights the relationship between atrial fibrosis and atrial fibrillation.     

转化生长因子β1和结缔组织生长因子在大鼠肺纤维化中的表达

目的观察转化生长因子β1-mRNA（TGFβ1-mRNA）和结缔组织生长因子-mRNA（CTGF-mRNA）在博莱霉素诱导的大鼠肺间质纤维化中的表达。方法48只雌性Wistar大鼠,随机分为正常组、模型组、每组24只,以气管内注入博莱霉素A2（BLM—A2）建立大鼠肺间质纤维化模型,模型组气管内一次性注入BLM-A2（5mg／kg）,正常组气管内注入等量生理盐水。两组大鼠在实验周期第3天、7天、14天和28天用腹主动脉放血法处死进行以下检测：计算肺系数：肺系数=肺湿重（mg）／体重（g）,进行HE和VG染色,原位杂交测定大鼠肺组织中TGFβ1-mRNA和CTGF—mRNA的表达情况。结果TGFβ1-mRNA和CTGF—mRNA在博莱霉素诱导的大鼠肺间质纤维化中的表达明显增强。结论TGFβ1-mRNA和CTGF—mRNA的表达增强表明TGFβ1-mRNA和CTGF—mRNA与肺纤维化的发生、发展有较大关联,为本研究后续实验动物模型的药物干预奠定了基础。     

养阴活血汤早期干预对大鼠肺间质纤维化的影响

目的 观察养阴活血汤对博莱霉素诱导的大鼠肺间质纤维化疗效以及对转化生长因子β1(transforming growth factor-β1,TGF-β1)和结缔组织生长因子(connective tissue growth factor,CTGF)mRNA表达的影响.方法 96只雌性Wistar大鼠,随机分为正常组、模型组、中药组和干扰素β1a(interferon-β1a,IFN-β1a)组.气管内注入博莱霉素A2建立大鼠肺间质纤维化模型.中药组自造模次日始,每只大鼠灌胃浓缩中药每天20 ml/kg,IFN-β1a组皮下注射重组人IFN-β1a 4.6μg/kg,每周3次.正常组和模型组等体积生理盐水灌胃.各组大鼠在实验周期第3天、第7天、第14天和第28天腹主动脉放血法处死,进行肺系数检测,HE和VG染色.原位杂交测定大鼠肺组织中TGF-β1和CTGF的mRNA表达情况.结果 养阴活血汤能明显降低肺系数,使肺组织的病理改变减轻,可以减弱大鼠肺组织和原代培养肺成纤维细胞中TGF-β1 mRNA和CTGF mRNA的表达强度.结论 养阴活血汤可以减少TGF-β1 mRNA和CTGF mRNA的表达量,减弱细胞因子对肺间质纤维化的促进作用,从而起到抑制肺间质纤维化的作用.     

转化生长因子-β1对心房结构重构及细胞骨架蛋白的影响

心房颤动是临床上最常见的持续性心律失常。大量研究发现,心房颤动的发生发展与心房结构重构密切相关,而心房纤维化是最主要的结构重构改变。转化生长因子-β1是心房颤动纤维化重构中重要的致纤维化因子,不仅能引起细胞间质重构,还能影响心肌细胞骨架重塑及相关骨架蛋白表达异常。特别是使心脏特异性肌动蛋白交联蛋白α-actinin-2表达增加。细胞骨架蛋白参与了结构重构改变。现对转化生长因子-β1对心房结构重构及心房肌细胞骨架蛋白的影响进行综述。     

特发性肺纤维化中Smurf2对TGF-β1/Smad通路调控机制

特发性肺纤维化是肺间质纤维化的主要原因,为最常见的间质性肺疾病.其主要病理特点为大量成纤维细胞、肌成纤维细胞集聚、增殖.近年来研究表明转化生长因子β1(transforming growth factor-β1,TGF-β1)在肺纤维化的形成与发展中起关键性作用.Smad蛋白是参与TGF-β1信号细胞内传导的一类信号蛋白,是TGF-β1信号传导重要通路.近年来发现Smad泛素调节因子2(Smad ubiquitination regulatory factor 2,Smurf2)可选择性作用于Smad蛋白,进而调控TGF-β信号传导.本文就特发性肺纤维化中Smurf2对TGF-β1/Smad通路的调控机制作一综述.     

Negative regulation of myofibroblast differentiation by PTEN (Phosphatase and Tensin Homolog Deleted on chromosome 10)

RATIONALE: Myofibroblasts are primary effector cells in idiopathic pulmonary fibrosis (IPF). Defining mechanisms of myofibroblast differentiation may be critical to the development of novel therapeutic agents. OBJECTIVE: To show that myofibroblast differentiation is regulated by phosphatase and tensin homolog deleted on chromosome 10 (PTEN) activity in vivo, and to identify a potential mechanism by which this occurs. METHODS: We used tissue sections of surgical lung biopsies from patients with IPF to localize expression of PTEN and alpha-smooth muscle actin (alpha-SMA). We used cell culture of pten(-/-) and wild-type fibroblasts, as well as adenoviral strategies and pharmacologic inhibitors, to determine the mechanism by which PTEN inhibits alpha-SMA, fibroblast proliferation, and collagen production. RESULTS: In human lung specimens of IPF, myofibroblasts within fibroblastic foci demonstrated diminished PTEN expression. Furthermore, inhibition of PTEN in mice worsened bleomycin-induced fibrosis. In pten(-/-) fibroblasts, and in normal fibroblasts in which PTEN was inhibited, alpha-SMA, proliferation, and collagen production was upregulated. Addition of transforming growth factor-beta to wild-type cells, but not pten(-/-) cells, resulted in increased alpha-SMA expression in a time-dependent fashion. In pten(-/-) cells, reconstitution of PTEN decreased alpha-SMA expression, proliferation, and collagen production, whereas overexpression of PTEN in wild-type cells inhibited transforming growth factor-beta-induced myofibroblast differentiation. It was observed that both the protein and lipid phosphatase actions of PTEN were capable of modulating the myofibroblast phenotype. CONCLUSIONS: The results indicate that in IPF, myofibroblasts have diminished PTEN expression. Inhibition of PTEN in vivo promotes fibrosis, and PTEN inhibits myofibroblast differentiation in vitro.     

Pellino1-mediated TGF-β1 synthesis contributes to mechanical stress induced cardiac fibroblast activation

Activation of cardiac fibroblasts is a key event in the progression of cardiac fibrosis that leads to heart failure. However, the molecular mechanisms underlying mechanical stress-induced cardiac fibroblast activation are complex and poorly understood. This study demonstrates that Pellino1, an E3 ubiquitin ligase, was activated in vivo in pressure overloaded rat hearts and in cultured neonatal rat cardiac fibroblasts (NRCFs) exposed to mechanical stretch in vitro. Suppression of the expression and activity of Pellino1 by adenovirus-mediated delivery of shPellino1 (adv-shpeli1) attenuated pressure overload-induced cardiac dysfunction and cardiac hypertrophy and decreased cardiac fibrosis in rat hearts. Transfection of adv-shpeli1 also significantly attenuated mechanical stress-induced proliferation, differentiation and collagen synthesis in NRCFs. Pellino1 silencing also abrogated mechanical stretch-induced polyubiquitination of tumor necrosis factor-alpha receptor association factor-6 (TRAF6) and receptor-interacting protein 1 (RIP1) and consequently decreased the DNA binding activity of nuclear factor-kappa B (NF-κB) in NRCFs. In addition, Pellino1 silencing prevented stretch-induced activation of p38 and activator protein 1 (AP-1) binding activity in NRCFs. Chromatin Immunoprecipitation (ChIP) and luciferase reporter assays showed that Pellino1 silencing prevented the binding of NF-κB and AP-1 to the promoter region of transforming growth factor-β1 (TGF-β1) thus dampening TGF-β1 transactivation. Our data reveal a previously unrecognized role of Pellino1 in extracellular matrix deposition and cardiac fibroblast activation in response to mechanical stress and provides a novel target for treatment of cardiac fibrosis and heart failure.     

Different Modulation of Decorin Production by Lung Fibroblasts from Patients with Mild and Severe Emphysema

We have previously reported diminished immunohistochemical staining of decorin in lung tissue from patients with severe emphysema. The aim of this study is to investigate whether this diminished staining is due to a quantitative abnormal production of decorin by pulmonary fibroblasts in vitro. Therefore, we measured decorin (Western blot), collagen type I (ELISA), and fibronectin (ELISA) production by fibroblasts obtained from lung tissue of patients with severe and mild emphysema at basal culture conditions and after modulation with transforming growth factor-β₁, basic fibroblast growth factor, and interferon-γ. Decorin production at basal culture conditions was significantly higher in fibroblast cultures from patients with severe emphysema compared to fibroblasts from mild emphysema. After stimulation with transforming growth factor-β₁ and basic fibroblast growth factor, decorin production was significantly more reduced in fibroblast cultures from patients with severe emphysema whereas collagen type I and fibronectin production were not affected. We conclude that decorin production by lung fibroblasts of patients with severe emphysema is dysregulated after modulation with cytokines known to be important in smoking associated inflammation. This dysregulation of decorin production may contribute to the impaired lung tissue repair, present in patients with emphysema, since these alterations in the extracellular matrix may cause diminished cytokine binding and neutralization.     

转化生长因子β在特发性肺纤维化发病中的作用及机制研究进展

特发性肺纤维化(IPF)是一种病因不明、慢性纤维化性的间质性肺炎,以间质纤维化、肌成纤维细胞过度分化增殖以及细胞外基质沉积为主要病理特征,最终导致肺结构破坏,呼吸功能衰竭.IPF预后差,治疗手段十分有限.目前认为,IPF发生的始动因素是不明原因的肺泡微损伤,继而导致转化生长因子β (TGF-β)的激活和肺泡基底膜的破坏.激活的TGF-β可促进上皮细胞的凋亡、上皮间质转化、成纤维细胞分化为肌成纤维细胞及细胞外基质的沉积,最终导致肺组织纤维化的形成及肺功能的丧失.本文主要对TGF-β在IPF的发生、发展过程中的作用作一简要阐述.     

Cytokine Regulation of Hepatic Stellate Cells in Liver Fibrosis

Cytokines constitute a major class of mediators responsible for “activation” of hepatic stellate cells (HSCs) in vitro and in vivo. They are largely divided into mitogenic (transforming growth factor-α, platelet-derived growth factor, interleukin-1, tumor necrosis factor-α, and insulin-like growth factor) and fibrogenic (transforming growth factor-β and interleukin-6) cytokines. In addition to their mitogenic (stimulation of cell proliferation) and fibrogenic (induction of matrix proteins) properties, they are also shown to confer in vitro unique cellular changes known to be the key features of HSC “activation,” including loss of vitamin A, stimulation of migration, enhanced cellular contractility, and matrix metalloproteinase and tissue inhibitor of metalloproteinase induction. Potential cellular sources of the cytokines consist of hepatic macrophages, endothelial cells, biliary epithelial cells, lymphocytes, platelets, hepatocytes, and activated HSCs. To better understand the mode of actions and the pathogenetic significance of cytokineskhemokines involved in “activation” of HSCs, the following four questions need to be addressed: (1) What other cytokines are expressed by HSCs to establish critical autocrine stimulation? (2) What are endogenous or exogenous priming factors for HSC stimulation? (3) What is the mechanism of activation for transforming growth factor-β, the pivotal fibrogenic cytokine? (4) How important are HSC-derived proinflammatory mediators in liver fibrosis? This review will discuss these questions, along with the current understanding of the role of cytokines in HSC activation.     

Selective expression of connective tissue growth factor in fibroblasts in vivo promotes systemic tissue fibrosis

Objective

Connective tissue growth factor (CTGF) is a cysteine‐rich secreted matricellular protein involved in wound healing and tissue repair. Enhanced and prolonged expression of CTGF has been associated with tissue fibrosis in humans. However, questions remain as to whether CTGF expression alone is sufficient to drive fibrosis. This study was undertaken to investigate whether CTGF alone is sufficient to cause fibrosis in intact animals and whether its effects are mediated through activation of transforming growth factor β (TGFβ) signaling or through distinct signal transduction pathways.

Methods

We generated mice overexpressing CTGF in fibroblasts under the control of the fibroblast‐specific collagen α2(I) promoter enhancer. Tissues such as skin, lung, and kidney were harvested for histologic analysis. Mouse embryonic fibroblasts were prepared from embryos (14.5 days postcoitum) for biochemical analysis.

Results

Mice overexpressing CTGF in fibroblasts were susceptible to accelerated tissue fibrosis affecting the skin, lung, kidney, and vasculature, most notably the small arteries. We identified a marked expansion of the myofibroblast cell population in the dermis. RNA analysis of transgenic dermal fibroblasts revealed elevated expression of key matrix genes, consistent with a fibrogenic response. CTGF induced phosphorylation of p38, ERK‐1/2, JNK, and Akt, but not Smad3, in transgenic mouse fibroblasts compared with wild‐type mouse fibroblasts. Transfection experiments showed significantly increased basal activity of the CTGF and serum response element promoters, and enhanced induction of the CTGF promoter in the presence of TGFβ.

Conclusion

These results demonstrate that selective expression of CTGF in fibroblasts alone causes tissue fibrosis in vivo through specific signaling pathways, integrating cues from the extracellular matrix into signal transduction pathways to orchestrate pivotal biologic responses relevant to tissue repair and fibrosis.

     

Matrix Stiffness: the Conductor of Organ Fibrosis

Purpose of Review

Organ fibrosis is a lethal component of scleroderma. The hallmark of scleroderma fibrosis is extensive extracellular matrix (ECM) deposition by activated myofibroblasts, specialized hyper-contractile cells that promote ECM remodeling and matrix stiffening. The purpose of this review is to discuss novel mechanistic insight into myofibroblast activation in scleroderma.

Recent Findings

Matrix stiffness, traditionally viewed as an end point of organ fibrosis, is now recognized as a critical regulator of tissue fibrogenesis that hijacks the normal physiologic wound-healing program to promote organ fibrosis. Here, we discuss how matrix stiffness orchestrates fibrosis by controlling three fundamental pro-fibrotic mechanisms: (a) mechanoactivation of myofibroblasts, (b) integrin-mediated latent transforming growth factor beta 1 (TGF-β1) activation, and (c) activation of non-canonical TGF-β1 signaling pathways. We also summarize novel therapeutic targets for anti-fibrotic therapy based on the mechanobiology of scleroderma.

Summary

Future research on mechanobiology of scleroderma may lead to important clinical applications such as improved diagnosis and treatment of patients with scleroderma and other fibrotic-related diseases.

     

Hepatocyte growth factor reduces cardiac fibrosis by inhibiting endothelial-mesenchymal transition

The purpose of this study was to investigate the effect of hepatocyte growth factor (HGF) on the pathogenesis of cardiac fibrosis induced by pressure overload in mice. Although cardiac fibrosis is attributed to excess pathological deposition of extracellular matrix components, the mechanism remains unclear. Recent reports revealed that α-smooth muscle actin-expressing myofibroblasts are primarily responsible for fibrosis. It is believed that myofibroblasts are differentiated from resident fibroblasts, whereas the transformation of vascular endothelial cells into myofibroblasts, known as endothelial-mesenchymal transition, has been suggested to be intimately associated with perivascular fibrosis. Thus, we hypothesized that HGF prevents cardiac fibrosis by blocking these pathways. We analyzed the pressure-overloaded HGF-transgenic mouse model made by transverse aortic constriction. Human coronary artery endothelial cells and human cardiac fibroblasts were examined in vitro after being treated with transforming growth factor-β1 or angiotensin II with or without HGF. The amount of cardiac fibrosis significantly decreased in pressure-overloaded HGF-transgenic mice compared with pressure-overloaded nontransgenic controls, particularly in the perivascular region. This was accompanied by a reduction in the expression levels of fibrosis-related genes and by significant preservation of echocardiographic measurements of cardiac function in the HGF-transgenic mice (P<0.05). The survival rate 2 months after transverse aortic constriction was higher by 45% (P<0.05). HGF inhibited the differentiation of human coronary artery endothelial cells into myofibroblasts induced by transforming growth factor-β1 and the phenotypic conversion of human cardiac fibroblasts into myofibroblasts. We conclude that HGF reduced cardiac fibrosis by inhibiting endothelial-mesenchymal transition and the transformation of fibroblasts into myofibroblasts.     

The pathogenesis of idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with an appalling prognosis. The failure of anti-inflammatory therapies coupled with the observation that deranged epithelium overlies proliferative myofibroblasts to form the fibroblastic focus has lead to the emerging concept that IPF is a disease of deregulated epithelial-mesenchymal crosstalk. IPF is triggered by an as yet unidentified alveolar injury that leads to activation of transforming growth factor-β (TGF-β) and alveolar basement membrane disruption. In the presence of persisting injurious pathways, or disrupted repair pathways, activated TGF-β can lead to enhanced epithelial apoptosis and epithelial-to-mesenchymal transition (EMT) as well as fibroblast, and fibrocyte, transformation into myofibroblasts which are resistant to apoptosis. The resulting deposition of excess disrupted matrix by these myofibroblasts leads to the development of IPF.