Role of sphingosine 1-phosphate signalling in tissue fibrosis

Fibrosis is characterized by the excessive accumulation of extracellular matrix components, leading to loss of tissue function in affected organs. Although the majority of fibrotic diseases have different origins, they have in common a persistent inflammatory stimulus and lymphocyte-monocyte interactions that determine the production of numerous fibrogenic cytokines. Treatment to contrast fibrosis is urgently needed, since some fibrotic diseases lead to systemic fibrosis and represent a major cause of death. In this article, the role of the bioactive sphingolipid sphingosine 1-phosphate (S1P) and its signalling pathway in the fibrosis of different tissue contexts is extensively reviewed, highlighting that it may represent an innovative and promising pharmacological therapeutic target for treating this devastating multifaceted disease. In multiple tissues S1P influences different aspects of fibrosis modulating the recruitment of inflammatory cells, as well as cell proliferation, migration and transdifferentiation into myofibroblasts, the cell type mainly involved in fibrosis development. Moreover, at the level of fibrotic lesions, S1P metabolism is profoundly influenced by multiple cross-talk with profibrotic mediators, such as transforming growth factor β, thus finely regulating the development of fibrosis.This article is part of a Special Issue entitled "Physiological and pathological roles of bioactive sphingolipids".     

Matrix stiffness controls cardiac fibroblast activation through regulating YAP via AT1R

Cardiac fibrosis is a common pathway leading to heart failure and involves continued activation of cardiac fibroblasts (CFs) into myofibroblasts during myocardium damage, causing excessive deposition of the extracellular matrix (ECM) and thus increases matrix stiffness. Increasing evidence has shown that stiffened matrix plays an important role in promoting CF activation and cardiac fibrosis, and several signaling factors mediating CF mechanotransduction have been identified. However, the key molecules that perceive matrix stiffness to regulate CF activation remain to be further explored. Here, we detected significantly increased expression and nuclear localization of Yes-associated protein (YAP) in native fibrotic cardiac tissues. By using mechanically regulated in vitro cell culture models, we found that a stiff matrix-induced high expression and nuclear localization of YAP in CFs, accompanied by enhanced cell activation. We also demonstrated that YAP knockdown decreased fibrogenic response of CFs and that YAP overexpression promoted CF activation, indicating that YAP plays an important role in mediating matrix stiffness-induced CF activation. Further mechanistic studies revealed that the YAP pathway is an important signaling branch downstream of angiotensin II type 1 receptor in CF mechanotransduction. The findings help elucidate the mechanism of fibrotic mechanotransduction and may contribute to the development of new approaches for treating fibrotic diseases.     

Endothelial to mesenchymal transition (EndoMT) in the pathogenesis of Systemic Sclerosis-associated pulmonary fibrosis and pulmonary arterial hypertension. Myth or reality?

Systemic Sclerosis (SSc) is a systemic autoimmune disease characterized by progressive fibrosis of skin and multiple internal organs and severe functional and structural microvascular alterations. SSc is considered to be the prototypic systemic fibrotic disorder. Despite currently available therapeutic approaches SSc has a high mortality rate owing to the development of SSc-associated interstitial lung disease (ILD) and pulmonary arterial hypertension (PAH), complications that have emerged as the most frequent causes of disability and mortality in SSc. The pathogenesis of the fibrotic process in SSc is complex and despite extensive investigation the exact mechanisms have remained elusive. Myofibroblasts are the cells ultimately responsible for tissue fibrosis and fibroproliferative vasculopathy in SSc. Tissue myofibroblasts in SSc originate from several sources including expansion of quiescent tissue fibroblasts and tissue accumulation of CD34 + fibrocytes. Besides these sources, myofibroblasts in SSc may result from the phenotypic conversion of endothelial cells into activated myofibroblasts, a process known as endothelial to mesenchymal transition (EndoMT). Recently, it has been postulated that EndoMT may play a role in the development of SSc-associated ILD and PAH. However, although several studies have described the occurrence of EndoMT in experimentally induced cardiac, renal, and pulmonary fibrosis and in several human disorders, the contribution of EndoMT to SSc-associated ILD and PAH has not been generally accepted. Here, the experimental evidence supporting the concept that EndoMT plays a role in the pathogenesis of SSc-associated ILD and PAH will be reviewed.     

微小RNA介导心肌纤维化信号通路的研究进展

心肌纤维化以细胞外基质沉积为主要特征,是许多心血管疾病发展到一定阶段的共同病理变化.心肌纤维化过程中的一些微小RNA(microRNAs,miRNAs)表达异常,并通过对多种信号通路的调控,参与心肌成纤维细胞的活化和增殖过程,从而介导心肌纤维化的发生和发展.本文将综述这些miRNAs在心肌纤维化中的作用和机制,为心肌纤...     

Mechanisms of fibrosis: therapeutic translation for fibrotic disease

Fibrosis is a pathological feature of most chronic inflammatory diseases. Fibrosis, or scarring, is defined by the accumulation of excess extracellular matrix components. If highly progressive, the fibrotic process eventually leads to organ malfunction and death. Fibrosis affects nearly every tissue in the body. Here we discuss how key components of the innate and adaptive immune response contribute to the pathogenesis of fibrosis. We also describe how cell-intrinsic changes in important structural cells can perpetuate the fibrotic response by regulating the differentiation, recruitment, proliferation and activation of extracellular matrix-producing myofibroblasts. Finally, we highlight some of the key mechanisms and pathways of fibrosis that are being targeted as potential therapies for a variety of important human diseases.     

Global gene expression analysis reveals a role for the alpha 1 integrin in renal pathogenesis.

Kidney fibrosis is the hallmark of most types of progressive kidney disease, including the genetic disorder Alport's syndrome. We undertook gene expression analysis in Alport's syndrome mouse kidneys using microchip arrays to characterize the development of fibrosis. In addition to matrix and matrix-remodeling genes, consistent with interstitial fibrosis, macrophage-related genes show elevated expression levels in Alport's syndrome kidneys. Immunohistochemical analysis of kidney sections illustrated that macrophages as well as myofibroblasts accumulate in the tubular interstitium. Deletion of alpha(1) integrin results in decreased accumulation of both myofibroblasts and macrophages in the tubular interstitium in Alport's syndrome mice and delays disease progression. Transforming growth factor beta antagonism, although reducing interstitial fibrosis, does not limit macrophage accumulation in the tubular interstitium and disease progression. In this study, we identified previously overlooked inflammatory events that occur in the tubulointerstitial region. We propose that in addition to the previously suggested role for the alpha(1)beta(1) integrin in mesangial expansion and abnormal laminin deposition, this integrin may be critical for monocyte accumulation that, in turn, may lead directly to renal failure. Our gene expression and immunohistochemical data indicate that macrophage accumulation is dependent on alpha(1) integrin expression on the macrophage cell surface and that anti-alpha(1) integrin strategies may be employed as therapeutics in the treatment of chronic inflammatory and fibrotic diseases.     

Enhanced deposition of cartilage oligomeric matrix protein is a common feature in fibrotic skin pathologies

Skin fibrosis is characterized by activated fibroblasts and an altered architecture of the extracellular matrix. Excessive deposition of extracellular matrix proteins and altered cytokine levels in the dermal collagen matrix are common to several pathological situations such as localized scleroderma and systemic sclerosis, keloids, dermatosclerosis associated with venous ulcers and the fibroproliferative tissue surrounding invasively growing tumors. Which factors contribute to altered organization of dermal collagen matrix in skin fibrosis is not well understood. We recently demonstrated that cartilage oligomeric matrix protein (COMP) functions as organizer of the dermal collagen I network in healthy human skin (Agarwal et al., 2012). Here we show that COMP deposition is enhanced in the dermis in various fibrotic conditions. COMP levels were significantly increased in fibrotic lesions derived from patients with localized scleroderma, in wound tissue and exudates of patients with venous leg ulcers and in the fibrotic stroma of biopsies from patients with basal cell carcinoma. We postulate enhanced deposition of COMP as one of the common factors altering the supramolecular architecture of collagen matrix in fibrotic skin pathologies. Interestingly, COMP remained nearly undetectable in normally healing wounds where myofibroblasts transiently accumulate in the granulation tissue. We conclude that COMP expression is restricted to a fibroblast differentiation state not identical to myofibroblasts which is induced by TGFβ and biomechanical forces.     

Conditioned media from lung cancer cell line A549 and PC9 inactivate pulmonary fibroblasts by regulating protein phosphorylation

Pulmonary fibrosis is a devastating condition resulting from excess extracellular matrix deposition that leads to progressive lung destruction and scarring. In the pathogenesis of fibrotic diseases, activation of myofibroblasts by transforming growth factor-β (TGF-β) plays a crucial role. Since no effective therapy for pulmonary fibrosis is currently recognized, finding an effective antifibrotic agent is an important objective. One approach might be through identification of agents that inactivate myofibroblasts. In the current study we examined the potential of conditioned medium obtained from several types of cells to exhibit myofibroblast inactivating activity. Conditioned media from lung cancer cell lines A549 and PC9 were found to have this action, as shown by its ability to decrease α-smooth muscle actin expression in MRC-5 cells. Subsequently the inhibitory factor was purified from the medium and identified as 5'-deoxy-5'-methylthioadenosine (MTA), and its mechanism of action elucidated. Activation of protein kinase A and cAMP responsive element binding protein (CREB) were detected. MTA inhibited TGF-β-induced mitogen-activated protein kinase activation. Furthermore, the gain-of-function mutant CREB caused inactivation of myofibroblasts. These results show that A549 and PC9 conditioned media have the ability to inactivate myofibroblasts, and that CREB-phosphorylation plays a central role in this process.     

Up‐regulated HMGB1 in EAM directly led to collagen deposition by a PKCβ/Erk1/2‐dependent pathway: cardiac fibroblast/myofibroblast might be another source of HMGB1

High mobility group box 1 (HMGB1), an important inflammatory mediator, is actively secreted by immune cells and some non‐immune cells or passively released by necrotic cells. HMGB1 has been implicated in many inflammatory diseases. Our previous published data demonstrated that HMGB1 was up‐regulated in heart tissue or serum in experimental autoimmune myocarditis (EAM); HMGB1 blockade could ameliorate cardiac fibrosis at the last stage of EAM. And yet, until now, no data directly showed that HMGB1 was associated with cardiac fibrosis. Therefore, the aims of the present work were to assess whether (1) up‐regulated HMGB1 could directly lead to cardiac fibrosis in EAM; (2) cardiac fibroblast/myofibroblasts could secrete HMGB1 as another source of high‐level HMGB1 in EAM; and (3) HMGB1 blockade could effectively prevent cardiac fibrosis at the last stage of EAM. Our results clearly demonstrated that HMGB1 could directly lead to cardiac collagen deposition, which was associated with PKCβ/Erk1/2 signalling pathway; furthermore, cardiac fibroblast/myofibroblasts could actively secrete HMGB1 under external stress; and HMGB1 secreted by cardiac fibroblasts/myofibroblasts led to cardiac fibrosis via PKCβ activation by autocrine means; HMGB1 blockade could efficiently ameliorate cardiac fibrosis in EAM mice.     

Cadherin-11 contributes to pulmonary fibrosis: potential role in TGF-β production and epithelial to mesenchymal transition

Pulmonary fibrosis, characterized by excess deposition of extracellular matrix by myofibroblasts, is a serious component of chronic lung diseases. Cadherin-11 (CDH11) is increased in wound healing and fibrotic skin. We hypothesized that CDH11 is increased in pulmonary fibrosis and contributes its development. CDH11 expression was assessed in lung tissue from idiopathic pulmonary fibrosis patients. The role of CDH11 in lung fibrosis was determined using the bleomycin model of pulmonary fibrosis, and in vitro analyses were performed on A549 cells during the process of epithelial to mesenchymal transition (EMT). Immunohistochemical studies demonstrated CDH11 expression on fibroblasts, epithelial cells, and alveolar macrophages of patients with pulmonary fibrosis and mice given bleomycin. Interestingly, CDH11-deficient mice had decreased fibrotic endpoints in the bleomycin model of pulmonary fibrosis compared to wild-type mice. Furthermore, anti-CDH11-neutralizing monoclonal antibodies successfully treated established pulmonary fibrosis induced by bleomycin. TGF-β levels were reduced in bronchoalveolar lavage (BAL) fluid, BAL cells, and primary alveolar macrophages from CDH11-deficient mice. Mechanistic studies demonstrated that TGF-β up-regulated CDH11 expression on A549 cells, and inhibition of CDH11 expression using siRNA reduced TGF-β-induced EMT. Together, these results identify CDH11 as a novel therapeutic target for pulmonary fibrosis.     

Mitochondrial uncoupling protein‐2 reprograms metabolism to induce oxidative stress and myofibroblast senescence in age‐associated lung fibrosis

Mitochondrial dysfunction has been associated with age‐related diseases, including idiopathic pulmonary fibrosis (IPF). We provide evidence that implicates chronic elevation of the mitochondrial anion carrier protein, uncoupling protein‐2 (UCP2), in increased generation of reactive oxygen species, altered redox state and cellular bioenergetics, impaired fatty acid oxidation, and induction of myofibroblast senescence. This pro‐oxidant senescence reprogramming occurs in concert with conventional actions of UCP2 as an uncoupler of oxidative phosphorylation with dissipation of the mitochondrial membrane potential. UCP2 is highly expressed in human IPF lung myofibroblasts and in aged fibroblasts. In an aging murine model of lung fibrosis, the in vivo silencing of UCP2 induces fibrosis regression. These studies indicate a pro‐fibrotic function of UCP2 in chronic lung disease and support its therapeutic targeting in age‐related diseases associated with impaired tissue regeneration and organ fibrosis.     

Aortic Carboxypeptidase-like Protein (ACLP) Enhances Lung Myofibroblast Differentiation through Transforming Growth Factor β Receptor-dependent and -independent Pathways

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal lung disease characterized by the overgrowth, hardening, and scarring of lung tissue. The exact mechanisms of how IPF develops and progresses are unknown. IPF is characterized by extracellular matrix remodeling and accumulation of active TGFβ, which promotes collagen expression and the differentiation of smooth muscle α-actin (SMA)-positive myofibroblasts. Aortic carboxypeptidase-like protein (ACLP) is an extracellular matrix protein secreted by fibroblasts and myofibroblasts and is expressed in fibrotic human lung tissue and in mice with bleomycin-induced fibrosis. Importantly, ACLP knockout mice are significantly protected from bleomycin-induced fibrosis. The goal of this study was to identify the mechanisms of ACLP action on fibroblast differentiation. As primary lung fibroblasts differentiated into myofibroblasts, ACLP expression preceded SMA and collagen expression. Recombinant ACLP induced SMA and collagen expression in mouse and human lung fibroblasts. Knockdown of ACLP slowed the fibroblast-to-myofibroblast transition and partially reverted differentiated myofibroblasts by reducing SMA expression. We hypothesized that ACLP stimulates myofibroblast formation partly through activating TGFβ signaling. Treatment of fibroblasts with recombinant ACLP induced phosphorylation and nuclear translocation of Smad3. This phosphorylation and induction of SMA was dependent on TGFβ receptor binding and kinase activity. ACLP-induced collagen expression was independent of interaction with the TGFβ receptor. These findings indicate that ACLP stimulates the fibroblast-to-myofibroblast transition by promoting SMA expression via TGFβ signaling and promoting collagen expression through a TGFβ receptor-independent pathway.     

Controlling cardiac fibrosis through fibroblast state space modulation

The transdifferentiation of cardiac fibroblasts into myofibroblasts after cardiac injury has traditionally been defined by a unidirectional continuum from quiescent fibroblasts, through activated fibroblasts, and finally to fibrotic-matrix producing myofibroblasts. However, recent lineage tracing and single cell RNA sequencing experiments have demonstrated that fibroblast transdifferentiation is much more complex. Growing evidence suggests that fibroblasts are more heterogenous than previously thought, and many new cell states have recently been identified. This review reexamines conventional fibroblast transdifferentiation paradigms with a dynamic state space lens, which could enable a more complex understanding of how fibroblast state dynamics alters fibrotic remodeling of the heart. This review will define cellular state space, how it relates to fibroblast state transitions, and how the canonical and non-canonical fibrotic signaling pathways modulate fibroblast cell state and cardiac fibrosis. Finally, this review explores the therapeutic potential of fibroblast state space modulation by p38 inhibition, yes-associated protein (YAP) inhibition, and fibroblast reprogramming.     

A-kinase anchoring protein-Lbc promotes pro-fibrotic signaling in cardiac fibroblasts

In response to stress or injury the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy and fibrosis. Transformation of cardiac fibroblasts to myofibroblasts is a crucial event initiating the fibrotic process. Cardiac myofibroblasts invade the myocardium and secrete excess amounts of extracellular matrix proteins, which cause myocardial stiffening, cardiac dysfunctions and progression to heart failure. While several studies indicate that the small GTPase RhoA can promote profibrotic responses, the exchange factors that modulate its activity in cardiac fibroblasts are yet to be identified. In the present study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor (GEF) activity, is critical for activating RhoA and transducing profibrotic signals downstream of type I angiotensin II receptors (AT₁Rs) in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly reduces the ability of angiotensin II to promote RhoA activation, differentiation of cardiac fibroblasts to myofibroblasts, collagen deposition as well as myofibroblast migration. Interestingly, AT₁Rs promote AKAP-Lbc activation via a pathway that requires the α subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as a key Rho-guanine nucleotide exchange factor modulating profibrotic responses in cardiac fibroblasts.     

Myofibroblasts. I. Paracrine cells important in health and disease

Myofibroblasts are aunique group of smooth-muscle-like fibroblasts that have a similarappearance and function regardless of their tissue of residence.Through the secretion of inflammatory and anti-inflammatory cytokines,chemokines, growth factors, both lipid and gaseous inflammatorymediators, as well as extracellular matrix proteins and proteases, theyplay an important role in organogenesis and oncogenesis, inflammation,repair, and fibrosis in most organs and tissues. Platelet-derivedgrowth factor (PDGF) and stem cell factor are two secreted proteinsresponsible for differentiating myofibroblasts from embryological stemcells. These and other growth factors cause proliferation ofmyofibroblasts, and myofibroblast secretion of extracellular matrix(ECM) molecules and various cytokines and growth factors causesmobility, proliferation, and differentiation of epithelial orparenchymal cells. Repeated cycles of injury and repair lead to organor tissue fibrosis through secretion of ECM by the myofibroblasts.Transforming growth factor- and the PDGF family of growth factorsare the key factors in the fibrotic response. Because of theirubiquitous presence in all tissues, myofibroblasts play important rolesin various organ diseases and perhaps in multisystem diseases as well.