Transforming growth factor-beta 1-induced collagen production in cultures of cardiac fibroblasts is the result of the appearance of myofibroblasts

Transforming growth factor-beta 1 (TGF-beta 1), which appears in high concentrations in fibrotic cardiac tissue, is a potent inductor of tissue collagen deposition and of the differentiation of fibroblasts to myofibroblasts. It is accepted that TGF-beta 1 is a potent stimulator of collagen secretion by fibroblasts. The aim of the present study was to determine which type of cells, fibroblasts and/or myofibroblasts are stimulated, in terms of collagen production, by TGF-beta 1. Therefore, using cultures of second-passage rat cardiac fibroblasts, we investigated the dose- (0.003-15 ng/ml) and time-dependence (2-48 h) of the TGF-beta 1-induced effects on collagen production and on the appearance of myofibroblasts, as estimated by the presence of alpha-smooth muscle actin (alpha-SMA; a marker of myofibroblasts). The reversibility of the TGF-beta 1-stimulated effects was also studied. The dose- and time-dependent stimulation of collagen production was closely associated with the induction of alpha-SMA. TGF-beta 1 did not change the cell phenotype or increase collagen production in rat cardiac fibroblasts cultures after a long incubation (24-28 h) at low concentrations (< 1 ng/ml), or after a short incubation (2-4 h) at high concentrations (1-15 ng/ml). However, after a long incubation at high concentrations, TGF-beta 1 changed the cell phenotype and increased collagen production in these cultures through the differentiation of fibroblasts to myofibroblasts. A maximal increase of collagen production (two-fold, p < 0.001) was observed after incubation of fibroblasts with 15 ng/ml TGF-beta 1 for 48 h. Under these conditions, alpha-SMA was increased by 3.5-fold (p < 0.001) and second-passage cultures of fibroblasts and their offspring in the next passage consisted mainly of myofibroblasts. The stimulation of collagen by 15 ng/ml TGF-beta 1 for 48 h was irreversible. In fact, additional incubation of these second-passage TGF-beta 1-stimulated cultures without TGF-beta 1 for 2 days did not decrease the high activity of collagen production. Moreover, the third-passage offspring of these TGF-beta 1-stimulated fibroblasts cultured without TGF-beta 1 also showed a higher production of collagen compared with control fibroblasts. Furthermore, the increased collagen production in the third-passage fibroblast offspring of the second-passage TGF-beta 1-stimulated fibroblasts could not be further stimulated by TGF-beta 1. Thus, the activity of collagen production in TGF-beta 1-stimulated cultures and in their next passage offspring is not sensitive to TGF-beta 1. Our data suggest that TGF-beta 1-stimulated collagen production in cultures of adult rat cardiac ventricular fibroblasts cannot be explained by a direct stimulation of collagen production, either in fibroblasts or in myofibroblasts. Instead, TGF-beta 1 induces differentiation of fibroblasts to myofibroblasts, the latter having a higher activity for collagen production than the former.     

Epithelial-mesenchymal transition as a therapeutic target for prevention of ocular tissue fibrosis

Fibrotic diseases are characterized by the appearance of myofibroblasts, the key cell type involved in the fibrogenic reaction, and by excess accumulation of extracellular matrix with resultant tissue contraction and impaired function. Myofiborblasts are generated by fibroblast-myofibrobalst conversion, and in certain tissues through epithelial-mesenchymal transition (EMT), a process through which an epithelial cell changes its phenotype to become more like a mesenchymal cell. Although inflammatory/fibrogenic growth factors/cytokines produced by injured tissues orchestrate the process of EMT, transforming growth factor beta (TGFbeta) is believed to play a central role in the process. Unlike fibrotic lesions in kidney or other tissues where myofibroblasts are generated from both fibroblasts and epithelial cells, fibrotic lesions in the eye crystalline lens are derived only from lens epithelial cells without contamination of fibroblast-derived myofibroblasts. Thus, this tissue is suitable to investigate detailed mechanisms of EMT and subsequent tissue fibrosis. EMT in retinal pigment epithelium is involved in the development of another ocular fibrotic disease, proliferative vitreoretinopathy, a fibrosis in the retina. EMT-related signal transduction cascades, i. e., TGFbeta/Smad, are a target to prevent or treat unfavorable ocular tissue fibrosis, e. g., fibrotic diseases in the crystalline lens or retina, as well as possibly in other organs.     

Transforming growth factor-beta 1 promotes contraction of collagen gel by cardiac fibroblasts through their differentiation into myofibroblasts

Myofibroblasts and transforming growth factor-beta 1 (TGF-beta 1) are key elements of cardiac tissue fibrosis development. The aim of this study was to determine whether the ability of TGF-beta 1 to affect the contractile activity of cardiac fibroblasts depends on their differentiation into myofibroblasts. Cardiac fibroblasts (from male adult Wistar rats) from passage 2 were therefore cultured to confluency and incubated on a hydrated collagen gel, both with and without TGF-beta 1 (0, 20, 40, 100, 200, 400 or 600 pmol/l), for 1, 2 and 3 days in a Dulbecco's Modified Eagle's Medium (DMEM) without fetal bovine serum (FBS). Growing cultures of cardiac fibroblasts were obtained by incubating second-passage fibroblasts in DMEM with 10% FBS with or without TGF-beta 1 (0 to 600 pmol/l) for 6 days. These fibroblasts were then further incubated on the collagen gel for 1, 2 and 3 days in DMEM without FBS. TGF-beta 1 dose-dependently increased the contraction of collagen gel mediated by cardiac fibroblasts, either added directly to the gel or after growing of the cardiac fibroblasts in the presence of TGF-beta 1 for 6 days, reaching a maximal effect at 100 pmol/l TGF-beta 1. In both culturing conditions, TGF-beta 1 also stimulated the [3H]-thymidine incorporation and the total protein content in the cardiac fibroblasts in the collagen gel lattice. TGF-beta 1 dose-dependently induced an increase in alpha-smooth muscle actin, a marker of myofibroblasts, in both culturing conditions. The TGF-beta 1-induced reduction of area of the collagen gel was negatively correlated to the TGF-beta 1-evoked appearance of alpha-smooth muscle actin in the collagen gel matrix. TGF-beta 1 increased the contractile activity of adult rat cardiac fibroblasts and their ability to differentiate into myofibroblasts. Because contractile activity was correlated with differentiation, the influence of TGF-beta 1 on cardiac fibroblast-induced collagen gel contraction may depend on the promotion of myofibroblast differentiation.     

Will chymase inhibitors be the next major development for the treatment of cardiovascular disorders?

Chymase is contained in the secretory granules of mast cells. In addition to the synthesis of angiotensin II, chymase is involved in transforming growth factor-β activation and cleaves Type I procollagen to produce collagen. NK301 and BCEAB are orally-active inhibitors of chymase. NK301 was tested in a dog model of vascular intimal hyperplasia after balloon injury and shown to reduce the increased chymase activity in the injured arteries and prevent intimal thickening. In a hamster model of cardiac fibrosis associated with cardiomyopathy, BCEAB reduced the increased cardiac chymase activity in cardiomyopathy and reduced fibrosis. Chymase inhibitors may be an important development for the treatment of cardiovascular injury associated with mast cell degranulation.     

Molecular mechanisms of and possible treatment strategies for idiopathic pulmonary fibrosis

Pulmonary fibrosis is characterized by lung inflammation and abnormal tissue repair, resulting in the replacement of normal functional tissue with an abnormal accumulation of fibroblasts and deposition of collagen in the lung. This process involves cellular interactions via a complex cytokine-signaling mechanism and heightened collagen gene expression, ultimately resulting in its abnormal collagen deposition in the lung. Our current understanding of the pathogenesis of pulmonary fibrosis suggests that in addition to inflammatory cells, the fibroblast and signaling events that mediate fibroblast proliferation and myofibroblasts, play important roles in the diverse processes that constitute fibrosis. Increasing knowledge of cytokine biology, cytokine-signaling and cell matrix interactions have shed some light on the genesis of pulmonary fibrosis; however, the importance of inflammation in pulmonary fibrosis remains controversial. This remains true because the inflammatory component is variable at the time of diagnosis, and the most potent anti-inflammatory drugs that have been widely used in the treatment of pulmonary fibrosis do not seem to interfere with the fibrotic disease progression. Pulmonary fibrosis is a highly lethal disorder, which continues to pose major clinical challenges because an effective therapeutic regimen is yet to be determined. This review summarizes recent progress in understanding the molecular mechanisms of pulmonary fibrosis, and includes a more detailed discussion of the potential points of therapeutic attack in pulmonary fibrosis. In addition, a detailed discussion is presented regarding each of the potential therapies which have emerged from the animal models of pulmonary fibrosis, and which have been developed through advances in cellular and molecular biology.     

Pivotal role of pericytes in kidney fibrosis

1. Kidney pericytes were recently identified as collagen Iα1-producing cells in healthy kidney, but the developmental, physiological and pathological roles of kidney pericytes remain poorly understood. Pericytes are stromal-derived cells that envelop and have intimate connections with adjacent capillary endothelial cells (EC). Recent studies in the eye and brain have revealed that pericytes are crucial for angiogenesis, vascular stability and vessel integrity. 2. In response to kidney injury, pericytes promptly migrate away from the capillary wall into the interstitial space. Here, pericytes are activated and differentiate into scar-forming myofibroblasts. In the absence of pericytes, peritubular capillaries are destabilized, leading to vascular regression. Consequently, capillary loss and fibrosis following kidney injury are intimately linked and hinge centrally around pericyte detachment from EC. 3. Kinetic mathematical modelling has demonstrated that pericytes are the major source of myofibroblasts in the fibrotic kidney. Comprehensive genetic fate mapping studies of nephron epithelia or kidney stroma has demonstrated that epithelial cells do not migrate outside of the epithelial compartment to become myofibroblasts; rather, interstitial pericytes are progenitors of scar-forming myofibroblasts. Bidirectional signalling between pericytes and EC is necessary for pericyte detachment from peritubular capillaries. 4. In the present review, we summarize the pathologically vital roles of kidney pericytes in fibrosis, including our new findings. The study of kidney pericytes and endothelial-pericyte cross-talk will identify novel therapeutic targets for currently incurable chronic kidney diseases.     

Transforming growth factor-beta receptor type 1 (TGFbetaRI) kinase activity but not p38 activation is required for TGFbetaRI-induced myofibroblast differentiation and profibrotic gene expression

Transforming growth factor-beta (TGFbeta) is a major mediator of normal wound healing and of pathological conditions involving fibrosis, such as idiopathic pulmonary fibrosis. TGFbeta also stimulates the differentiation of myofibroblasts, a hallmark of fibrotic diseases. In this study, we examined the underlying processes of TGFbetaRI kinase activity in myofibroblast conversion of human lung fibroblasts using specific inhibitors of TGFbetaRI (SD-208) and p38 mitogen-activated kinase (SD-282). We demonstrated that SD-208, but not SD-282, inhibited TGFbeta-induced SMAD signaling, myofibroblast transformation, and collagen gel contraction. Furthermore, we extended our findings to a rat bleomycin-induced lung fibrosis model, demonstrating a significant decrease in the number of myofibroblasts at fibroblastic foci in animals treated with SD-208 but not those treated with SD-282. SD-208 also reduced collagen deposition in this in vivo model. Microarray analysis of human lung fibroblasts identified molecular fingerprints of these processes and showed that SD-208 had global effects on reversing TGFbeta-induced genes involved in fibrosis, inflammation, cell proliferation, cytoskeletal organization, and apoptosis. These studies also revealed that although the p38 pathway may not be needed for appearance or disappearance of the myofibroblast, it can mediate a subset of inflammatory and fibrogenic events of the myofibroblast during the process of tissue repair and fibrosis. Our findings suggest that inhibitors such as SD-208 may be therapeutically useful in human interstitial lung diseases and pulmonary fibrosis.     

EPAC expression and function in cardiac fibroblasts and myofibroblasts

In the heart, cardiac fibroblasts (CF) and cardiac myofibroblasts (CMF) are the main cells responsible for wound healing after cardiac insult. Exchange protein activated by cAMP (EPAC) is a downstream effector of cAMP, and it has been not completely studied on CF. Moreover, in CMF, which are the main cells responsible for cardiac healing, EPAC expression and function are unknown. We evaluated in both CF and CMF the effect of transforming growth factor β1 (TGF-β1) on EPAC-1 expression. We also studied the EPAC involvement on collagen synthesis, adhesion, migration and collagen gel contraction.     

A current perspective of canstatin,a fragment of type IV collagen alpha 2 chain

Type IV collagen is a main component of basement membrane extracellular matrix. Canstatin, a non-collagenous C-terminal fragment of type IV collagen α2 chain, was firstly identified as an endogenous anti-angiogenic and anti-tumor factor, which also has an anti-lymphangiogenic effect. Then, canstatin has been widely investigated as a novel target molecule for cancer therapy. The anti-angiogenic effect of canstatin may be also useful for the treatment of ocular neovascularization. Recently, we have demonstrated that canstatin, which is abundantly expressed in the heart tissue, exerts various biological activities in cardiac cells. In rat H9c2 cardiomyoblasts, canstatin inhibits isoproterenol- or hypoxia-induced apoptosis. Canstatin plays an important role in modulating voltage-dependent calcium channel activity in rat cardiomyocytes. Canstatin also regulates various biological functions in rat cardiac fibroblasts and myofibroblasts. The expression of canstatin decreases in the infarcted area after myocardial infarction. This review focuses on a current perspective for the roles of canstatin in tumorigenesis, ocular neovascularization and cardiac pathology.     

Glabridin inhibits the activation of myofibroblasts in human fibrotic buccal mucosal fibroblasts through TGF‐β/smad signaling

Oral submucous fibrosis (OSF) has been recognized as one of the oral potentially malignant disorders. Areca nut chewing is implicated in this pathological fibrosis, and it causes chronic inflammation and persistent activation of myofibroblasts. As yet, existing treatments only provide temporary symptomatic relief and there is a lack of an effective intervention to cure OSF. Therefore, development of approaches to ameliorate myofibroblast activities becomes a crucial objective to prevent the malignant progression of OSF. In this study, we examined the inhibitory effect of glabridin, an isoflavane extracted from licorice root, on the myofibroblast characteristics in human fibrotic buccal mucosal fibroblasts (fBMFs). Our results showed that myofibroblast activities, including collagen gel contractility, migration, invasion and wound healing abilities were reduced after exposure of glabridin in a dose‐dependent manner. Most importantly, we demonstrated that the arecoline‐induced myofiroblast activities were abolished by glabridin treatment. Additionally, the expression of the myofibroblast marker α‐smooth muscle actin and other fibrogenic marker, type I collagen, in fBMFs were dose‐dependently downregulated. Moreover, we showed that the production of TGF‐β was suppressed by glabridin in fBMFs and the protein expression of phospho‐Smad2 was decreased as well. In summary, our data suggested that glabridin repressed the myofibroblast features in fBMFs via TGF‐β/Smad2 signaling pathway. Glabridin also prevented the arecoline‐increased myofibroblast activities, and could serve as a natural anti‐fibrosis compound for OSF.     

Will chymase inhibitors be the next major development for the treatment of cardiovascular disorders?

Chymase is contained in the secretory granules of mast cells. In addition to the synthesis of angiotensin II, chymase is involved in transforming growth factor-beta activation and cleaves Type I procollagen to produce collagen. NK301 and BCEAB are orally-active inhibitors of chymase. NK301 was tested in a dog model of vascular intimal hyperplasia after balloon injury and shown to reduce the increased chymase activity in the injured arteries and prevent intimal thickening. In a hamster model of cardiac fibrosis associated with cardiomyopathy, BCEAB reduced the increased cardiac chymase activity in cardiomyopathy and reduced fibrosis. Chymase inhibitors may be an important development for the treatment of cardiovascular injury associated with mast cell degranulation.     

Apoptosis modulated by oxidative stress and inflammation during obstructive nephropathy

Kidney apoptosis and fibrosis are an inevitable outcome of progressive chronic kidney diseases where congenital obstructive nephropathy is the primary cause of the end-stage renal disease in children, and is also a major cause of renal failure in adults. The injured tubular cells linked to interstitial macrophages, and myofibroblasts produce cytokines and growth factors that promote an inflammatory state in the kidney, induce tubular cell apoptosis, and facilitate the accumulation of extracellular matrix. Angiotensin II plays a central role in the renal fibrogenesis at a very early stage leading to a rapid progression in chronic kidney disease. The increasing levels of angiotensin II induce pro-inflammatory cytokines, NF-κB activation, adhesion molecules, chemokines, growth factors, and oxidative stress. Furthermore, growing evidence reports that angiotensin II (a pro-inflammatory hormone) increases the mitochondrial oxidative stress regulating apoptosis induction. This review summarizes our understanding about possible mechanisms that contribute to apoptosis modulated by inflammation and/or oxidative stress during obstructive nephropathy. The new concept of antiinflammatory tools regulating mitochondrial oxidative stress will directly affect the inflammatory process and apoptosis. This idea could have attractive consequences in the treatment of renal and other inflammatory pathologies.     

Butylidenephthalide abrogates the myofibroblasts activation and mesenchymal transdifferentiation in oral submucous fibrosis

Oral submucous fibrosis (OSF) is a premalignant disorder in the oral cavity, and areca nut chewing habit has been implicated in the persistent activation of myofibroblasts and the subsequent fibrosis. Therefore, it is critical to ameliorate the excessive activities of myofibroblasts prior to the malignant transformation of OSF. In the current study, we evaluated the cytotoxicity of butylidenephthalide (BP), a major phthalide ingredient of Angelica sinensis, in fibrotic buccal mucosal fibroblasts (fBMFs) as well as various myofibroblast hallmarks, including the phenotypical characteristics and fibrosis‐related markers. Our results demonstrated that myofibroblast activities, including collagen gel contraction, migration, invasion and wound healing abilities were inhibited in response to BP. The expression levels of myofibroblast marker, α‐smooth muscle actin (α‐SMA), fibronectin and type 1 collagen A1 were decreased after exposure of BP. Moreover, we found that the EMT‐related markers, Twist, Snail and ZEB1 were all downregulated after BP treatment. Most importantly, our findings demonstrated that BP impeded the binding of Snail to the E‐box region in the α‐SMA promoter, which may lead to inhibition of the arecoline‐induced myofibroblast activities. Collectively, our data indicated that BP reduced numerous myofibroblast features in fBMFs and hindered the binding of Snail to α‐SMA, thereby may function as an effective and natural antifibrosis compound.     

Hematopoietic colony stimulating factors in cardiovascular and pulmonary remodeling: promoters or inhibitors?

Hemopoietic colony stimulating factors (HCSFs) are naturally occurred substances that are released in response to infection or inflammation and regulate the proliferation and differentiation of hemopoietic progenitor cells. Some representative members of this peptide family induce atherogenesis through the mediation of monocyte-endothelial cell adhesive interaction and promotion of angiogenesis within the atherosclerotic plaques. HCSFs, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), also promote post-infarction cardiac remodeling though the enhanced activation and infiltration of monocytes into injured myocardial tissue and through altered equilibrium of collagen deposition/degradation. On the other hand, exogenous administration of granulocyte colony-stimulating factor (G-CSF) or eythropoietin (EPO) in patients with chronic ischemic disease or recent myocardial infarction have lead to beneficial arteriogenesis or myocardial cell regeneration, thus preventing adverse cardiac remodeling. While GM-CSF may hold therapeutic potential as an inhibitor of lung fibrogenesis, G-CSF appears to promote fibrosis in the lungs. The pathophysiological role of HCSFs also depends on the timing of their action on cardiovascular remodeling, as well as on the target progenitor hematopoietic cell. This article summarizes current knowledge about the clinical and therapeutic implications of these factors in chronic artery disease, post-infarction cardiac remodeling, chronic heart failure and in pulmonary fibrosis.     

Fibroblasts, myofibroblasts, and fibrosis: fact, fiction, and the future

Fibroblasts and their activated phenotype known as myofibroblasts are nonexcitable cells found in all organs of the body. In the heart, fibroblasts, along with the endothelial and smooth muscle cells of the blood vessels, make up approximately 30% of tissue mass. In vitro, myofibroblasts cocultured with cardiac myocytes can propagate electrical signals down cellular strands indicating that under these conditions myofibroblasts are capable of depolarizing enough to maintain electrical propagation. This has obvious implications for cardiac biology if heterocellular coupling between fibroblasts and myocytes were to occur in the intact heart either under normal conditions or during cellular stress. The purpose of this review series is to highlight the newest information on cardiac fibroblasts and myofibroblasts and to review the data on their interactions with cardiac myocytes.     

肝内肌成纤维细胞的来源及其在肝纤维化中作用的研究

肝脏慢性损伤,包括病毒性肝炎、酗酒、药物作用、代谢性疾病等各种致病因子导致肝内结缔组织异常增生,肝内细胞外基质过度沉淀形成肝纤维化,在此过程中分泌胶原能力更强且具有收缩能力的肌成纤维细胞的大量生成被认为是肝纤维化发生与发展的关键环节。目前对于肝纤维化时肌成纤维细胞来源的认识尚没有完全明了,在肝纤维化发生时肝星状细胞活化为肌成纤维细胞的主要来源;而骨髓间充质干细胞、上皮细胞经上皮间质转化及一些仍具有分化能力的外周血细胞在一定条件下也可转变为肌成纤维细胞并发挥作用。该文就近年来此方面的研究作一综述。     

Protein kinase inhibitors in the treatment of pulmonary fibrosis

Fibrosis of the lung and other organ systems is an increasing cause of morbidity and mortality worldwide. Effective anti-fibrotic agents for such disorders are currently lacking. Injury to epithelium-lined tissues in mammals is typically associated with a mesenchymal response, including the activation of myofibroblasts. Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease that results from effacement of the normal alveolar architecture of the lung. Loss of lung capacity for gas-exchange and increased work of breathing eventually leads to respiratory failure and death. In cutaneous wound models, apoptosis of myofibroblasts are essential to scar-less wound healing. Recent studies indicate that acquisition of an apoptosis-resistant myofibroblast phenotype in the injured lung is associated with non-resolving and persistent fibrosis. The acquired resistance to apoptosis in myofibroblasts is mediated, at least in part, by the sustained activation of two critical pro-survival protein kinases, focal adhesion kinase (FAK) and protein kinase B (PKB/AKT). Therapeutic interventions that modulate the activity of these protein kinases with resultant alterations in the phenotype of myofibroblasts may prove to be effective anti-fibrotic therapeutic strategies. We discuss the potential roles for protein kinase inhibitors as novel drugs for fibrotic disorders. Progress in pre-clinical and clinical development of small molecule inhibitors targeting pro-survival protein kinases is reviewed.     

Long-Term Angiotensin II Blockade May Improve Not Only Hyperglycemia but Also Age-Associated Cardiac Fibrosis

In the present study, the effects of long-term angiotensin (Ang) II antagonism on the development of cardiac and endothelial disorders were examined in Spontaneously Diabetic Torii (SDT) rats. Blood glucose concentration started to increase markedly in the untreated SDT rats from 20 weeks of age, while the blood glucose concentrations of candesartan cilexetil–treated SDT rats were significantly lower until 30 weeks of age. Cardiac function deteriorated in SDT rats and was accompanied by severe cardiac fibrosis, cardiac hypertrophy, and microstructural pathologic change in cardiomyocytes. Cardiac function was very well preserved in the age-matched Sprague Dawley (SD) rats, but cardiac fibrosis developed with aging. Candesartan cilexetil treatment improved cardiac structural remodeling and cardiac function in SDT rats. Surprisingly, the degree of cardiac fibrosis in candesartan cilexetil–treated SDT rats was less than that of SD rats. Immunohistological staining confirmed that in addition to collagen deposition, fibroblasts and myofibroblasts were the main cellular components in the cardiac fibrotic areas. The diabetic hearts showed positive staining for ACE, Ang II, and AT₁ receptors. SDT rats also showed decreased endothelial function, which was improved with candesartan cilexetil treatment. These findings indicate that Ang II is involved in the development of cardiac dysfunction by accelerating cardiac remodeling and cardiomyocyte damage in the presence of hyperglycemia. On the other hand, although the mechanisms responsible for the cardiac fibrosis that occurs under normal conditions may differ greatly from those responsible for cardiac fibrosis with hyperglycemia, Ang II seems to play an important role in both.