Role of scleraxis in mechanical stretch-mediated regulation of cardiac myofibroblast phenotype

The phenotype conversion of fibroblasts to myofibroblasts plays a key role in the pathogenesis of cardiac fibrosis. Numerous triggers of this conversion process have been identified, including plating of cells on solid substrates, cytokines such as transforming growth factor-β, and mechanical stretch; however, the underlying mechanisms remain incompletely defined. Recent studies from our laboratory revealed that the transcription factor scleraxis is a key regulator of cardiac fibroblast phenotype and extracellular matrix expression. Here we report that mechanical stretch induces type I collagen expression and morphological changes indicative of cardiac myofibroblast conversion, as well as scleraxis expression via activation of the scleraxis promoter. Scleraxis causes phenotypic changes similar to stretch, and the effect of stretch is attenuated in scleraxis null cells. Scleraxis was also sufficient to upregulate expression of vinculin and F-actin, to induce stress fiber and focal adhesion formation, and to attenuate both cell migration and proliferation, further evidence of scleraxis-mediated regulation of fibroblast to myofibroblast conversion. Together, these data confirm that scleraxis is sufficient to promote the myofibroblast phenotype and is a required effector of stretch-mediated conversion. Scleraxis may thus represent a potential target for the development of novel antifibrotic therapies aimed at inhibiting myofibroblast formation.

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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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Abstract 14664: Reduced Activin Like Kinase 1 Activity Promotes Cardiac Fibrosis in Heart Failure

Circulation ◽

10.1161/circ.132.suppl_3.14664 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Kevin Morine ◽

Vikram Paruchuri ◽

Xiaoying Qiao ◽

Emily Mackey ◽

Mark Aronovitz ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Remodeling ◽

Cardiac Fibrosis ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Left Ventricular ◽

Lv Function ◽

Type I ◽

Mrna Levels ◽

Protein Levels

Introduction: Activin receptor like kinase 1 (ALK1) mediates signaling via transforming growth factor beta-1 (TGFb1), a pro-fibrogenic cytokine. No studies have defined a role for ALK1 in heart failure. We tested the hypothesis that reduced ALK1 expression promotes maladaptive cardiac remodeling in heart failure. Methods and Results: ALK1 mRNA expression was quantified by RT-PCR in left ventricular (LV) tissue from patients with end-stage heart failure and compared to control LV tissue obtained from the National Disease Research Interchange (n=8/group). Compared to controls, LV ALK1 mRNA levels were reduced by 85% in patients with heart failure. Next, using an siRNA approach, we tested whether reduced ALK1 levels promote TGFb1-mediated collagen production in human cardiac fibroblasts. Treatment with an ALK1 siRNA reduced ALK1 mRNA levels by 75%. Compared to control, TGFb1-mediated Type I collagen and pSmad-3 protein levels were 2.5-fold and 1.7-fold higher, respectively, after ALK1 depletion. To explore a role for ALK1 in heart failure, ALK1 haploinsufficient (ALK1) and wild-type mice (WT; n=8/group) were studied 2 weeks after thoracic aortic constriction (TAC). Compared to WT, baseline LV ALK1 mRNA levels were 50% lower in ALK1 mice. Both LV and lung weights were higher in ALK1 mice after TAC. Cardiomyocyte area and LV mRNA levels of BNP, RCAN, and b-MHC were increased similarly, while SERCa levels were reduced in both ALK1 and WT mice after TAC. Compared to WT, LV fibrosis (Figure) and Type 1 Collagen mRNA and protein levels were higher among ALK1 mice. Compared to WT, LV fractional shortening (48±12 vs 26±10%, p=0.01) and survival (Figure) were lower in ALK1 mice after TAC. Conclusions: Reduced LV expression of ALK1 is associated with advanced heart failure in humans and promotes early mortality, impaired LV function, and cardiac fibrosis in a murine model of heart failure. Further studies examining the role of ALK1 and ALK1 inhibitors on cardiac remodeling are required.

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Targeted inhibition of TGF-β results in an initial improvement but long-term deficit in force production after contraction-induced skeletal muscle injury

Journal of Applied Physiology ◽

10.1152/japplphysiol.00374.2013 ◽

2013 ◽

Vol 115 (4) ◽

pp. 539-545 ◽

Cited By ~ 30

Author(s):

Jonathan P. Gumucio ◽

Michael D. Flood ◽

Anthony C. Phan ◽

Susan V. Brooks ◽

Christopher L. Mendias

Keyword(s):

Type I Collagen ◽

Transforming Growth Factor ◽

Force Production ◽

Transforming Growth Factor Β ◽

Control Group ◽

Type I ◽

Muscle Injuries ◽

Early Recovery ◽

Initial Improvement ◽

Fiber Atrophy

Transforming growth factor-β (TGF-β) is a proinflammatory cytokine that regulates the response of many tissues following injury. Previous studies in our lab have shown that treating muscles with TGF-β results in a dramatic accumulation of type I collagen, substantial fiber atrophy, and a marked decrease in force production. Because TGF-β promotes atrophy and fibrosis, our objective was to investigate whether the inhibition of TGF-β after injury would enhance the recovery of muscle following injury. We hypothesized that inhibiting TGF-β after contraction-induced injury would improve the functional recovery of muscles by preventing muscle fiber atrophy and weakness, and by limiting the accumulation of fibrotic scar tissue. To test this hypothesis, we induced an injury using a series of in situ lengthening contractions to extensor digitorum longus muscles of mice treated with either a bioneutralizing antibody against TGF-β or a sham antibody. Compared with controls, muscles from mice receiving TGF-β inhibitor showed a greater recovery in force 3 days and 7 days after injury but had a decrease in force compared with controls at the 21-day time point. The early enhancement in force in the TGF-β inhibitor group was associated with an initial improvement in tissue morphology, but, at 21 days, while the control group was fully recovered, the TGF-β inhibitor group displayed an irregular extracellular matrix and an increase in atrogin-1 gene expression. These results indicate that the inhibition of TGF-β promotes the early recovery of muscle function but is detrimental overall to full muscle recovery following moderate to severe muscle injuries.

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Gonadotropin Releasing Hormone and Transforming Growth Factor β Activate Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase and Differentially Regulate Fibronectin, Type I Collagen, and Plasminogen Activator Inhibitor-1 Expression in Leiomyoma and Myometrial Smooth Muscle Cells

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2004-0161 ◽

2004 ◽

Vol 89 (11) ◽

pp. 5549-5557 ◽

Cited By ~ 54

Author(s):

Li Ding ◽

Jingxia Xu ◽

Xiaoping Luo ◽

Nasser Chegini

Keyword(s):

Type I Collagen ◽

Transforming Growth Factor ◽

Plasminogen Activator Inhibitor ◽

Transforming Growth Factor Β ◽

Mitogen Activated Protein Kinase ◽

Type I ◽

Plasminogen Activator Inhibitor 1 ◽

Mitogen Activated Protein ◽

Activator Inhibitor ◽

Fibronectin Type

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High doses of TGF-β potently suppress type I collagen via the transcription factor CUX1

Molecular Biology of the Cell ◽

10.1091/mbc.e10-08-0669 ◽

2011 ◽

Vol 22 (11) ◽

pp. 1836-1844 ◽

Cited By ~ 26

Author(s):

Maria Fragiadaki ◽

Tetsurou Ikeda ◽

Abigail Witherden ◽

Roger M Mason ◽

David Abraham ◽

...

Keyword(s):

Type I Collagen ◽

Transforming Growth Factor ◽

Interstitial Fibrosis ◽

Transforming Growth Factor Β ◽

Negative Regulator ◽

Mitogen Activated Protein Kinase ◽

Type I ◽

Aberrant Expression

Transforming growth factor-β (TGF-β) is an inducer of type I collagen, and uncontrolled collagen production leads to tissue scarring and organ failure. Here we hypothesize that uncovering a molecular mechanism that enables us to switch off type I collagen may prove beneficial in treating fibrosis. For the first time, to our knowledge, we provide evidence that CUX1 acts as a negative regulator of TGF-β and potent inhibitor of type I collagen transcription. We show that CUX1, a CCAAT displacement protein, is associated with reduced expression of type I collagen both in vivo and in vitro. We show that enhancing the expression of CUX1 results in effective suppression of type I collagen. We demonstrate that the mechanism by which CUX1 suppresses type I collagen is through interfering with gene transcription. In addition, using an in vivo murine model of aristolochic acid (AA)-induced interstitial fibrosis and human AA nephropathy, we observe that CUX1 expression was significantly reduced in fibrotic tissue when compared to control samples. Moreover, silencing of CUX1 in fibroblasts from kidneys of patients with renal fibrosis resulted in increased type I collagen expression. Furthermore, the abnormal CUX1 expression was restored by addition of TGF-β via the p38 mitogen-activated protein kinase pathway. Collectively, our study demonstrates that modifications of CUX1 expression lead to aberrant expression of type I collagen, which may provide a molecular basis for fibrogenesis.

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