P2Y1 Receptor Agonist Attenuates Cardiac Fibroblasts Activation Triggered by TGF-β1

Cardiac fibroblasts (CFs) activation is a hallmark feature of cardiac fibrosis caused by cardiac remodeling. The purinergic signaling molecules have been proven to participate in the activation of CFs. In this study, we explored the expression pattern of P2Y receptor family in the cardiac fibrosis mice model induced by the transverse aortic constriction (TAC) operation and in the activation of CFs triggered by transforming growth factor β1 (TGF-β1) stimulation. We then investigated the role of P2Y1receptor (P2Y1R) in activated CFs. The results showed that among P2Y family members, only P2Y1R was downregulated in the heart tissues of TAC mice. Consistent with our in vivo results, the level of P2Y1R was decreased in the activated CFs, when CFs were treated with TGF-β1. Silencing P2Y1R expression with siP2Y1R accelerated the effects of TGF-β1 on CFs activation. Moreover, the P2Y1R selective antagonist BPTU increased the levels of mRNA and protein of profibrogenic markers, such as connective tissue growth factor (CTGF), periostin (POSTN). periostin (POSTN), and α-smooth muscle actin(α-SMA). Further, MRS2365, the agonist of P2Y1R, ameliorated the activation of CFs and activated the p38 MAPK and ERK signaling pathways. In conclusion , our findings revealed that upregulating of P2Y1R may attenuate the abnormal activation of CFs via the p38 MAPK and ERK signaling pathway.

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Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00095.2018 ◽

2019 ◽

Vol 316 (3) ◽

pp. H596-H608 ◽

Cited By ~ 4

Author(s):

Rachel C. Childers ◽

Ian Sunyecz ◽

T. Aaron West ◽

Mary J. Cismowski ◽

Pamela A. Lucchesi ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Pressure Overload ◽

Volume Overload ◽

Transforming Growth Factor Β ◽

Collagen Type I ◽

Tissue Growth ◽

Type I

Hemodynamic load regulates cardiac remodeling. In contrast to pressure overload (increased afterload), hearts subjected to volume overload (VO; preload) undergo a distinct pattern of eccentric remodeling, chamber dilation, and decreased extracellular matrix content. Critical profibrotic roles of cardiac fibroblasts (CFs) in postinfarct remodeling and in response to pressure overload have been well established. Little is known about the CF phenotype in response to VO. The present study characterized the phenotype of primary cultures of CFs isolated from hearts subjected to 4 wk of VO induced by an aortocaval fistula. Compared with CFs isolated from sham hearts, VO CFs displayed a “hypofibrotic” phenotype, characterized by a ~50% decrease in the profibrotic phenotypic markers α-smooth muscle actin, connective tissue growth factor, and collagen type I, despite increased levels of profibrotic transforming growth factor-β1 and an intact canonical transforming growth factor-β signaling pathway. Actin filament dynamics were characterized, which regulate the CF phenotype in response to biomechanical signals. Actin polymerization was determined by the relative amounts of G-actin monomers versus F-actin. Compared with sham CFs, VO CFs displayed ~78% less F-actin and an increased G-actin-to-F-actin ratio (G/F ratio). In sham CFs, treatment with the Rho kinase inhibitor Y-27632 to increase the G/F ratio resulted in recapitulation of the hypofibrotic CF phenotype observed in VO CFs. Conversely, treatment of VO CFs with jasplakinolide to decrease the G/F ratio restored a more profibrotic response (>2.5-fold increase in α-smooth muscle actin, connective tissue growth factor, and collagen type I). NEW & NOTEWORTHY The present study is the first to describe a “hypofibrotic” phenotype of cardiac fibroblasts isolated from a volume overload model. Our results suggest that biomechanical regulation of actin microfilament stability and assembly is a critical mediator of cardiac fibroblast phenotypic modulation.

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Latent adenoviral infection induces production of growth factors relevant to airway remodeling in COPD

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00315.2002 ◽

2004 ◽

Vol 286 (1) ◽

pp. L189-L197 ◽

Cited By ~ 22

Author(s):

Emiko Ogawa ◽

W. Mark Elliott ◽

Fiona Hughes ◽

Thomas J. Eichholtz ◽

James C. Hogg ◽

...

Keyword(s):

Growth Factors ◽

Growth Factor ◽

Transforming Growth Factor ◽

Airway Remodeling ◽

Smooth Muscle Actin ◽

Tissue Growth ◽

Chronic Obstructive ◽

Adenoviral Infection ◽

E1a Gene ◽

Tgf Β1

Previous studies showed an association between latent adenoviral infection with expression of the adenoviral E1A gene and chronic obstructive pulmonary disease (COPD). The present study focuses on how the adenoviral E1A gene could alter expression of growth factors by human bronchial epithelial (HBE) cells. The data show that connective tissue growth factor (CTGF) and transforming growth factor (TGF)-β1 mRNA and protein expression were upregulated in E1A-positive HBE cells. Upregulation of CTGF in this in vitro model was independent of TGF-β secreted into the growth medium. Comparison of E1A-positive with E1A-negative HBE cells showed that both expressed cytokeratin but only E1A-positive cells expressed the mesenchymal markers vimentin and α-smooth muscle actin. We conclude that latent infection of epithelial cells by adenovirus E1A could contribute to airway remodeling in COPD by the viral E1A gene, inducing TGF-β1 and CTGF expression and shifting cells to a more mesenchymal phenotype.

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JNK and p38 Inhibitors Prevent Transforming Growth Factor-β1-Induced Myofibroblasts Transdifferentiation in Graves' Orbital Fibroblasts

10.21203/rs.3.rs-134877/v1 ◽

2021 ◽

Author(s):

Tzu-Yu Hou ◽

Shi-Bei Wu ◽

Hui-Chuan Kau ◽

Chieh-Chih Tsai

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β1 ◽

Tissue Growth ◽

Mitogen Activated Protein Kinase ◽

Tissue Inhibitors Of Metalloproteinases ◽

Mapk Pathways ◽

Orbital Fibroblasts ◽

Tgf Β1

Abstract Transforming growth factor-β1 (TGF-β1)-induced myofibroblasts transdifferentiation from orbital fibroblasts is known to dominate tissue remodeling and fibrosis in Graves’ ophthalmopathy (GO). However, the signaling pathway through which TGF-β1 activates Graves’ orbital fibroblasts is unclear. This study investigated the role of mitogen-activated protein kinase (MAPK) pathways in TGF-β1-induced myofibroblasts transdifferentiation of Graves’ orbital fibroblasts. MAPK pathways were assessed by measuring the phosphorylation levels of p38, c-Jun N-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) using Western blot analysis. The expression levels of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), fibronectin, and tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-3) representing fibrogenesis processes were analysed. Specific pharmacologic kinase inhibitors were used to confirm the involvement of MAPK pathways. After treating Graves’ orbital fibroblasts with TGF-β1, the phosphorylation levels of p38 and JNK but not ERK were increased. Meanwhile, CTGF, α-SMA and fibronectin were overexpressed. After pre-incubation with p38, JNK and ERK inhibitors respectively, the TGF-β1-induced expression of CTGF, α-SMA, fibronectin, TIMP-1 and TIMP3 was abolished by p38 and JNK inhibitors but not ERK inhibitors. This study confirmed TGF-β1-induced myofibroblasts transdifferentiation in Graves' orbital fibroblasts via p38 and JNK signaling. Thus, MAPK pathways may be potential targets for the management of GO.

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Overexpression of Calreticulin Promotes Cardiac Fibroblasts Activation Via Regulating IRE1 Pathway

10.21203/rs.3.rs-749805/v1 ◽

2021 ◽

Author(s):

liu zhiyue ◽

Zhiyue Liu ◽

Wen Zhang ◽

Junli Li ◽

Lei Xiao ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

High Titer ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β ◽

Tissue Growth ◽

Migration Rates ◽

And Migration ◽

Proliferation And Migration

Abstract Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone involved in cardiac fibroblasts (CFs) activation. It has been reported that the expression of CRT increased in the process of CFs activation. However, the role of CRT in CFs activation and the mechanism is not yet fully elucidated. Therefore, we aimed to verify whether CRT was involved in CFs activation and the possible mechanism underlying this process. We found that CRT protein level was elevated in AngⅡ-induced CFs activation. Knocking down CRT by its siRNA could decrease the protein expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and transforming growth factor-β (TGF-β), and meanwhile attenuate proliferation and migration ratio of CFs. Moreover, the proliferation and migration rates of CFs were promoted and the expression of CTGF, α-SMA and TGF-β were increased when transfection with high-titer adenovirus of CRT. In AngⅡ-induced CFs, inositol-requiring enzyme 1(IRE-1), one of the main ER pathways, was inhibited through CRT silence and activated through CRT overexpression. Overall, this study demonstrates that CRT overexpression could promote AngⅡ induced-CFs activation by activating IRE1 pathway, which could be a potential target for CFs activation.

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Curcumin Suppresses TGF-β1-Induced Myofibroblast Differentiation and Attenuates Angiogenic Activity of Orbital Fibroblasts

International Journal of Molecular Sciences ◽

10.3390/ijms22136829 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6829

Author(s):

Wei-Kuang Yu ◽

Wei-Lun Hwang ◽

Yi-Chuan Wang ◽

Chieh-Chih Tsai ◽

Yau-Huei Wei

Keyword(s):

Endothelial Cells ◽

Growth Factor ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Tissue Growth ◽

Myofibroblast Differentiation ◽

Differentiation Markers ◽

Angiogenic Activity ◽

Orbital Fibroblasts ◽

Tgf Β1

Orbital fibrosis, a hallmark of tissue remodeling in Graves’ ophthalmopathy (GO), is a chronic, progressive orbitopathy with few effective treatments. Orbital fibroblasts are effector cells, and transforming growth factor β1 (TGF-β1) acts as a critical inducer to promote myofibroblast differentiation and subsequent tissue fibrosis. Curcumin is a natural compound with anti-fibrotic activity. This study aims to investigate the effects of curcumin on TGF-β1-induced myofibroblast differentiation and on the pro-angiogenic activities of orbital fibroblasts. Orbital fibroblasts from one healthy donor and three patients with GO were collected for primary cell culture and subjected to myofibroblast differentiation under the administration of 1 or 5 ng/mL TGF-β1 for 24 h. The effects of curcumin on TGF-β1-induced orbital fibroblasts were assessed by measuring the cellular viability and detecting the expression of myofibroblast differentiation markers, including connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA). The pro-angiogenic potential of curcumin-treated orbital fibroblasts was evaluated by examining the transwell migration and tube-forming capacities of fibroblast-conditioned EA.hy926 and HMEC-1 endothelial cells. Treatment of orbital fibroblasts with curcumin inhibited the TGF-β1 signaling pathway and attenuated the expression of CTGF and α-SMA induced by TGF-β1. Curcumin, at the concentration of 5 μg/mL, suppressed 5 ng/mL TGF-β1-induced pro-angiogenic activities of orbital fibroblast-conditioned EA hy926 and HMEC-1 endothelial cells. Our findings suggest that curcumin reduces the TGF-β1-induced myofibroblast differentiation and pro-angiogenic activity in orbital fibroblasts. The results support the potential application of curcumin for the treatment of GO.

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JNK and p38 Inhibitors Prevent Transforming Growth Factor-β1-Induced Myofibroblast Transdifferentiation in Human Graves’ Orbital Fibroblasts

International Journal of Molecular Sciences ◽

10.3390/ijms22062952 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2952

Author(s):

Tzu-Yu Hou ◽

Shi-Bei Wu ◽

Hui-Chuan Kau ◽

Chieh-Chih Tsai

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Mapk Pathway ◽

Transforming Growth Factor Β1 ◽

Tissue Growth ◽

Mitogen Activated Protein Kinase ◽

Tissue Inhibitors Of Metalloproteinases ◽

Western Blots ◽

Orbital Fibroblasts ◽

Tgf Β1

Transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation from orbital fibroblasts is known to dominate tissue remodeling and fibrosis in Graves’ ophthalmopathy (GO). However, the signaling pathways through which TGF-β1 activates Graves’ orbital fibroblasts remain unclear. This study investigated the role of the mitogen-activated protein kinase (MAPK) pathway in TGF-β1-induced myofibroblast transdifferentiation in human Graves’ orbital fibroblasts. The MAPK pathway was assessed by measuring the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase (ERK) by Western blots. The expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and fibronectin representing fibrogenesis was estimated. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for extracellular matrix (ECM) metabolism were analyzed. Specific pharmacologic kinase inhibitors were used to confirm the involvement of the MAPK pathway. After treatment with TGF-β1, the phosphorylation levels of p38 and JNK, but not ERK, were increased. CTGF, α-SMA, and fibronectin, as well as TIMP-1 and TIMP-3, were upregulated, whereas the activities of MMP-2/-9 were inhibited. The effects of TGF-β1 on the expression of these factors were eliminated by p38 and JNK inhibitors. The results suggested that TGF-β1 could induce myofibroblast transdifferentiation in human Graves’ orbital fibroblasts through the p38 and JNK pathways.

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The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis

International Journal of Molecular Sciences ◽

10.3390/ijms22041861 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1861

Author(s):

Jemima Seidenberg ◽

Mara Stellato ◽

Amela Hukara ◽

Burkhard Ludewig ◽

Karin Klingel ◽

...

Keyword(s):

Transcription Factor ◽

Molecular Mechanisms ◽

Cardiac Fibrosis ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Beclin 1 ◽

Type I ◽

Alpha Smooth Muscle Actin

Background: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. Methods and Results: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-β. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-β-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. Conclusion: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-β-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.

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Regulation of Cellular Senescence Is Independent from Profibrotic Fibroblast-Deposited ECM

Cells ◽

10.3390/cells10071628 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1628

Author(s):

Kaj E. C. Blokland ◽

Habibie Habibie ◽

Theo Borghuis ◽

Greta J. Teitsma ◽

Michael Schuliga ◽

...

Keyword(s):

Growth Factor ◽

Cellular Senescence ◽

Transforming Growth Factor ◽

Cell Function ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β1 ◽

Tissue Growth ◽

Alveolar Epithelial Cells ◽

Lung Fibroblasts ◽

Alpha Smooth Muscle Actin

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor survival. Age is a major risk factor, and both alveolar epithelial cells and lung fibroblasts in this disease exhibit features of cellular senescence, a hallmark of ageing. Accumulation of fibrotic extracellular matrix (ECM) is a core feature of IPF and is likely to affect cell function. We hypothesize that aberrant ECM deposition augments fibroblast senescence, creating a perpetuating cycle favouring disease progression. In this study, primary lung fibroblasts were cultured on control and IPF-derived ECM from fibroblasts pretreated with or without profibrotic and prosenescent stimuli, and markers of senescence, fibrosis-associated gene expression and secretion of cytokines were measured. Untreated ECM derived from control or IPF fibroblasts had no effect on the main marker of senescence p16Ink4a and p21Waf1/Cip1. However, the expression of alpha smooth muscle actin (ACTA2) and proteoglycan decorin (DCN) increased in response to IPF-derived ECM. Production of the proinflammatory cytokines C-X-C Motif Chemokine Ligand 8 (CXCL8) by lung fibroblasts was upregulated in response to senescent and profibrotic-derived ECM. Finally, the profibrotic cytokines transforming growth factor β1 (TGF-β1) and connective tissue growth factor (CTGF) were upregulated in response to both senescent- and profibrotic-derived ECM. In summary, ECM deposited by IPF fibroblasts does not induce cellular senescence, while there is upregulation of proinflammatory and profibrotic cytokines and differentiation into a myofibroblast phenotype in response to senescent- and profibrotic-derived ECM, which may contribute to progression of fibrosis in IPF.

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Aucubin Protects against TGFβ1-Induced Cardiac Fibroblasts Activation by Mediating the AMPKα/mTOR Signaling Pathway

Planta Medica ◽

10.1055/s-0043-118663 ◽

2017 ◽

Vol 84 (02) ◽

pp. 91-99 ◽

Cited By ~ 6

Author(s):

Yang Xiao ◽

Wei Chang ◽

Qing-Qing Wu ◽

Xiao-Han Jiang ◽

Ming-Xia Duan ◽

...

Keyword(s):

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Smooth Muscle Actin ◽

Mammalian Target Of Rapamycin ◽

Transforming Growth Factor Β1 ◽

Neonatal Rat ◽

Cell Counting ◽

Compound C ◽

Underlying Mechanisms

AbstractFibrosis is a key feature of various cardiovascular diseases and compromises cardiac systolic and diastolic performance. The lack of effective anti-fibrosis drugs is a major contributor to the increasing prevalence of heart failure. The present study was performed to investigate whether the iridoid aucubin alleviates cardiac fibroblast activation and its underlying mechanisms. Neonatal rat cardiac fibroblasts were incubated with aucubin (1, 10, 20, 50 µM) followed by transforming growth factor β1 (TGFβ1, 10 ng/mL) stimulation for 24 h. Fibrosis proliferation was measured by cell counting kit-8 assay. The differentiation of fibroblasts into myofibroblasts was determined by measuring the expression of α-smooth muscle actin. Then, the expressions levels of cardiac fibrosis-related proteins in myofibroblasts were analyzed by western blot and real-time PCR to confirm the anti-fibrosis effect of aucubin. As a result, aucubin suppressed TGFβ1-induced proliferation in fibroblasts and inhibited the TGFβ1-induced activation of fibroblasts to myofibroblasts. In addition, aucubin further attenuated fibrosis-related protein expression in myofibroblasts. Furthermore, this protective effect was related to increased adenosine 5′-monophosphate-activated protein kinase (AMPK) phosphorylation and decreased mammalian target of rapamycin (mTOR) phosphorylation, which was confirmed by an mTOR inhibitor (rapamycin), an AMPK agonist (AICAR) and an AMPKα inhibitor compound C. Collectively, our findings suggest that aucubin protects against TGFβ1-induced fibroblast proliferation, activation and function by regulating the AMPKα/mTOR signal axis.

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