Inactivation of PRMT1 inhibits cardiac fibrosis via transcriptional regulation and perturbation of FBL activity in fibroblast-to-myofibroblast transition
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ABSTRACT Cardiac fibrosis is a recognized cause of morbidity and mortality, yet effective pharmacological therapy that directly targets the fibrotic process remains lacking. Here we surveyed a group of methyltransferases known as protein arginine methyltransferases (PRMT) and demonstrated that PRMT1, which is the most highly expressed PRMT in the heart, was upregulated in activated cardiac fibroblasts, or myofibroblasts, in failing hearts. Deleting Prmt1 specifically in myofibroblasts or treating systemically with the PRMT1 inhibitor MS023 blocked myofibroblast formation, leading to a significant reduction in cardiac fibrosis and improvement in cardiac function in both acute and chronic heart injury models that manifest pervasive cardiac fibrosis. PRMT1 promoted the transition of cardiac fibroblasts to myofibroblasts by regulating transcription and epigenetic status. Additionally, PRMT1 methylated a key nucleolar protein fibrillarin 1 (FBL) and regulated nucleoli morphology and function during fibroblast fate transition. We further demonstrated a previously unrecognized requirement for FBL in myofibroblasts formation, by regulating myofibroblast gene induction and contractile force generation.Keywords:
Myofibroblast
Cardiac Fibrosis
Cardiac fibroblasts play critical role in cardiac remodeling and during development. Cardiac remodeling following myocardial infarction involves the migration, proliferation and differentiation of cardiac fibroblasts to hypersecretory myofibroblasts. Myofibroblasts facilitate wound repair in the myocardium by secreting and organizing extra cellular matrix (ECM) and fibrosis. However, the molecular mechanisms involved in myofibroblast differentiation are not well known. Because the infarcted/ischemic myocardium is known to undergo mechanical stretch and the muscle phenotypic modulator TGF‐β is also increased in ischemia, we hypothesized the mechanotransduction and TGF‐β signaling pathway play active role in the differentiation of cardiac fibroblasts to myofibroblasts. Here, we show that the mechanosensitve ion channel TRPV4 is required for TGF‐β‐induced differentiation of cardiac fibroblasts to myofibroblasts. We found that cardiac fibroblasts functionally express TRPV4 channels and the TRPV4‐specific antagonist, AB159908 significantly inhibited TGFβ‐induced differentiation as measured by incorporation of α‐SMA into actin stress fibers (p < 0.05). Importantly, we demonstrate that TGF‐β‐induced myofibroblast differentiation is regulated by the variations in ECM elasticity in a TRPV4 dependent manner. Further, we found that TGF‐β treatment increased the expression of TRPV4. Finally, calcium imaging experiments with Fluo‐4 revealed that TGF‐β treated fibroblasts exhibit enhanced TRPV4‐dependent calcium influx compared to untreated controls ( p< 0.05). Taken together these results suggest, for the first time, that a mechanosensitive ion channel, TRPV4 regulates cardiac fibroblast differentiation to myofibroblasts which could be used as a novel therapeutic target for the treatment of ischemia and myocardial infarction.
Myofibroblast
TRPV4
Cardiac Fibrosis
Mechanotransduction
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Myofibroblast
Cardiac Fibrosis
Cardiac muscle
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Introduction: Transforming Growth Factor beta (TGFβ) is an important cytokine in mediating cardiac fibrosis. Cardiomyocyte-specific expression of a mutant αB-crystallin (CryAB R120G ) that is responsible for human Desmin Related Myopathy results in significant cardiac fibrosis and cardiac remodeling leading to heart failure. Onset of fibrosis is initiated by the activation of a quiescent fibroblast population to an active, “myofibroblast” state and TGFβ binding is thought to mediate an essential signaling pathway underlying this process. Our central hypothesis is that myofibroblast-based TGFβ signaling can result in significant cardiac fibrosis. Here, we have partially ablated TGFβ signaling in cardiac myofibroblasts to observe if cardiac fibrosis is altered. Objective: To understand the contributions of myofibroblast-based TGFβ signaling to the development of cardiac fibrosis. Methods and Results: To test the hypothesis we partially ablated myofibroblast specific TGFβ signaling by crossing CryAB R120G mice with mice containing a floxed allele of TGFβ’s receptor 1 (TGFβr1). The double transgenic animals were further crossed with activated myofibroblast specific Cre mice in which Cre expression was driven off the periostin promoter so that TGFβr1 would be ablated subsequent to myofibroblast conversion as the periostin promoter became active. Echocardiography, Masson’s Trichome staining, the hydroxyproline assay, PCR arrays, immunohistochemistry and western blots were used to characterize fibrosis and cardiac function in mice lacking TGFβr1 in the myofibroblasts were used to characterize the resultant animals. Conclusion: Myofibroblast-targeted knockdown of Tgfβr1 signaling resulted in reduced fibrosis and improved cardiac function.
Myofibroblast
Cardiac Fibrosis
Periostin
Desmin
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Cardiac fibrosis is the excess deposition of extracellular matrix (ECM), such as collagen. Myofibroblasts are major players in the production of collagen, and are differentiated primarily from resident fibroblasts. Collagen can compensate for the dead cells produced by injury. The appropriate production of collagen is beneficial for preserving the structural integrity of the heart, and protects the heart from cardiac rupture. However, excessive deposition of collagen causes cardiac dysfunction. Recent studies have demonstrated that myofibroblasts can change their phenotypes. In addition, myofibroblasts are found to have functions other than ECM production. Myofibroblasts have macrophage-like functions, in which they engulf dead cells and secrete anti-inflammatory cytokines. Research into fibroblasts has been delayed due to the lack of selective markers for the identification of fibroblasts. In recent years, it has become possible to genetically label fibroblasts and perform sequencing at single-cell levels. Based on new technologies, the origins of fibroblasts and myofibroblasts, time-dependent changes in fibroblast states after injury, and fibroblast heterogeneity have been demonstrated. In this paper, recent advances in fibroblast and myofibroblast research are reviewed.
Myofibroblast
Cardiac Fibrosis
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Introduction: Transforming Growth Factor beta (TGFβ) is an important cytokine in mediating cardiac fibrosis. Cardiomyocyte-specific expression of a mutant αB-crystallin (CryAB R120G ) that is responsible for human Desmin Related Myopathy results in significant cardiac fibrosis and cardiac remodeling leading to heart failure. Onset of fibrosis is initiated by the activation of a quiescent fibroblast population to an active, “myofibroblast” state and TGFβ binding is thought to mediate an essential signaling pathway underlying this process. Our central hypothesis is that myofibroblast-based TGFβ signaling can result in significant cardiac fibrosis. Here, we have partially ablated TGFβ signaling in cardiac myofibroblasts to observe if cardiac fibrosis is altered. Objective: To understand the contributions of myofibroblast-based TGFβ signaling to the development of cardiac fibrosis. Methods and Results: To test the hypothesis we partially ablated myofibroblast specific TGFβ signaling by crossing CryAB R120G mice with mice containing a floxed allele of TGFβ’s receptor 1 (TGFβr1). The double transgenic animals were further crossed with activated myofibroblast specific Cre mice in which Cre expression was driven off the periostin promoter so that TGFβr1 would be ablated subsequent to myofibroblast conversion as the periostin promoter became active. Echocardiography, Masson’s Trichome staining, the hydroxyproline assay, PCR arrays, immunohistochemistry and western blots were used to characterize fibrosis and cardiac function in mice lacking TGFβr1 in the myofibroblasts were used to characterize the resultant animals. Conclusion: Myofibroblast-targeted knockdown of Tgfβr1 signaling resulted in reduced fibrosis and improved cardiac function.
Myofibroblast
Periostin
Cardiac Fibrosis
Desmin
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Review of the literature shows that numerous authors admit the existence during various inflammatory reactions, and especially in granulation and cicatricial tissues, of peculiar connective tissue cells e.g. modified fibroblast or myofibroblast, with intermediate features between a fibroblast and a smooth muscle cell. Some of the physical and structural characteristics of these tissues would depend on these cells. The ultrastructural features of the cells in human and experimental pathology as well as their connection with the extracellular matrix are recalled.
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Granulation tissue
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Idiopathic pulmonary fibrosis (IPF) is a chronic disease of the lung caused by a rampant inflammatory response that results in the deposition of excessive extracellular matrix (ECM). IPF patient lungs also develop fibroblastic foci that consist of activated fibroblasts and myofibroblasts. In concert with ECM deposition, the increased cell density within fibroblastic foci imposes confining forces on lung fibroblasts. In this work, we observed that increased cell density increases the incidence of the fibroblast-to-myofibroblast transition (FMT), but mechanical confinement imposed by micropillars has no effect on FMT incidence. We found that human lung fibroblasts (HLFs) express more α-SMA and deposit more collagen matrix, which are both characteristics of myofibroblasts, in response to TGF-β1 when cells are seeded at a high density compared with a medium or a low density. These results support the hypothesis that HLFs undergo FMT more readily in response to TGF-β1 when cells are densely packed, and this effect could be dependent on increased OB-cadherin expression. This work demonstrates that cell density is an important factor to consider when modelling IPF in vitro, and it may suggest decreasing cell density within fibroblastic foci as a strategy to reduce IPF burden.
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Purpose: To investigate the effect of β3-adrenoceptors (β3-AR) up-regulation on fibrosis in cardiac fibroblast cells in rats and its potential mechanism.Methods: Cardiac fibroblast cells (CFB) were isolated and identified from rats’ hearts. The β3-ARupregulated cardiac fibroblast cells were constructed by lentiviral transfection technology. Thereafter, Ang II was used to induce fibrosis in cardiac fibroblast cells, and subsequently, Western blot assay was performed to investigate fibrosis related marker proteins (TGF-β, Smad-2, p-Smad-2, Col-I and Col-III) in cardiac fibroblast cells.Results: β3-AR up-regulated cardiac fibroblast cells were successfully constructed. Furthermore, the results show that up-regulation of β3-AR increased the expressions of TGF-β, p-Smad-2, Col-I and Col- III proteins in Ang II treated cardiac fibroblast cells.Conclusion: The results suggest that up-regulation of β3-AR aggravates fibrosis of cardiac fibroblast cells. In other words, inhibition of β3-AR expressions in cardiac tissues would be beneficial for treating cardiac fibrosis and its related cardiac diseases.Keywords: Cardiac fibrosis, β3-AR, TGF/Smads, Col-I/III, Cardiac fibroblast cells
Cardiac Fibrosis
Myocardial fibrosis
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Abstract Fibrosis is a broad pathology of excessive scarring with substantial medical implications. The fibrotic scar is produced by myofibroblasts that interact with macrophages. Fibrosis is a complex process involving thousands of factors, therefore, to better understand fibrosis and develop new therapeutic approaches, it is necessary to simplify and clarify the underlying concepts. Recently, we described a mathematical model for a macrophage-myofibroblast cell circuit, predicting two types of fibrosis - hot fibrosis with abundant macrophages and myofibroblasts, and cold fibrosis dominated by myofibroblasts alone. To test these concepts and intervention strategies in a medically relevant system, we use a widely studied in-vivo injury model for fibrosis, myocardial infarction (MI). We show that cold fibrosis is the final outcome of MI in both mice and pigs and demonstrate that fibrosis can shift toward healing in regenerative settings. MI begind with an increase of myofibroblasts and macrophages, followed by macrophage decline leading to persistent cold fibrosis (only myofibroblasts). During this process, fibroblasts, unlike macrophages, acquire distinct fate changes. Using mathematical modeling we predict that targeting of the autocrine signal for myofibroblast division could block cold fibrosis. We identify TIMP1 as an autocrine cardiac myofibroblast growth factor in-vitro . Treatment of adult mice after MI with anti-TIMP1 antibodies reduces fibrosis in-vivo . This study shows the utility of the concepts of hot and cold fibrosis and the feasibility of our circuit-to-target approach to reduce fibrosis after acute cardiac injury by inhibiting the myofibroblast autocrine loop.
Myofibroblast
Cardiac Fibrosis
Transdifferentiation
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Long noncoding RNAs (lncRNAs) have been reported to play a key role in diversity cardiovascular diseases, including cardiac fibrosis. The present study aims to investigate the biological role of lncRNA SRA1 in the activation of cardiac myofibroblasts and the underlying mechanism. Results showed that the expression of SRA1 was upregulated accompanied by cardiac fibrosis in an abdominal aortic banding-treated rat model. Ang-II treatment increased the SRA1 expression of cardiac myofibroblasts, whereas SRA1 knockdown by siRNA inhibited the proliferation, myofibroblast conversion, and collagen production of cardiac myofibroblasts induced by Ang-II. SRA1 overexpression by pcDNA3.1 SRA1-stimulated cardiac myofibroblast activation. To further investigate the underlying mechanism, miR-148b was predicted to be a targeted microRNA of SRA1. Different methods, including sequence alignment, luciferase activity, and MS2 RNA immunoprecipitation were performed to detect the interaction between SRA1 and miR-148b, which suggested that SRA1 negatively regulated miR-148b in cardiac myofibroblasts. Moreover, miR-148b knockdown stimulated cardiac myofibroblast activation, and miR-148b mediated promoting effect of SRA1 on cardiac myofibroblast activation. Collectively, our study suggested that SRA1 promoted cardiac myofibroblast activation by acting as a competitive endogenous RNAs for miR-148b. SRA1 may be a novel potential target for the prevention or therapy of cardiac fibrosis.
Myofibroblast
Cardiac Fibrosis
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