FOXF1 ameliorates angiotensin II-induced cardiac fibrosis in cardiac fibroblasts through inhibiting the TGF-β1/Smad3 signaling pathway

Cardiac fibrosis following myocardial infarction (MI) leads to reduced cardiac function, and contributes to heart failure and mortality. Recent studies shown the extent of adverse remodeling may be mitigated by therapeutic strategies which regulate cardiac fibroblast mediated-remodeling. Since cross-linking by lysyl oxidase (LOX) increases following MI and alters the mechanical properties of the infarct, it is critical to characterize how its expression is regulated by CFs post-MI. While LOX expression is attributable to TGF-β1 signaling, we hypothesize that changes in the stiffness and composition of the ECM can also alter LOX expression via integrin-mediated signaling. To investigate this, we isolated CFs from healthy left ventricle (LV) and infarcted cardiac fibroblasts (ICFs) from 1 week post-MI LV and cultured them on tissue culture plastic (TCP) and collagen I-coated plates (COL) in serum-free media for 48 hours to assess the expression of genes associated with LOX signaling, fibrosis, and myofibroblast activation. Our results show an upregulation of LOX gene expression in both CFs and ICFs when cultured on COL and this is further emphasized with the presence of TGF-β1 (Fig. 1A). Gene expression of col1α1, integrin β1 subunit and αSMA (Fig. 1B-D) also exhibit similar upregulation. Ongoing studies will investigate how altered substrate stiffness and composition affect gene expression of LOX and other genes associated with fibrosis. By understanding the effect of the physical microenvironment on the expression of fibrotic genes including LOX, we aim to develop novel therapeutic strategies to attenuate cardiac fibrosis and thus improve cardiac recovery following MI.

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Bone Morphogenetic Protein 9 Reduces Cardiac Fibrosis and Improves Cardiac Function in Heart Failure

Circulation ◽

10.1161/circulationaha.117.031635 ◽

2018 ◽

Vol 138 (5) ◽

pp. 513-526 ◽

Cited By ~ 19

Author(s):

Kevin J. Morine ◽

Xiaoying Qiao ◽

Sam York ◽

Peter S. Natov ◽

Vikram Paruchuri ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Human Subjects ◽

Lv Function ◽

Transverse Aortic Constriction ◽

Aortic Constriction ◽

Bone Morphogenetic Protein 9 ◽

Human Cardiac Fibroblasts ◽

Tgf Β1

Background: Heart failure is a growing cause of morbidity and mortality worldwide. Transforming growth factor beta (TGF-β1) promotes cardiac fibrosis, but also activates counterregulatory pathways that serve to regulate TGF-β1 activity in heart failure. Bone morphogenetic protein 9 (BMP9) is a member of the TGFβ family of cytokines and signals via the downstream effector protein Smad1. Endoglin is a TGFβ coreceptor that promotes TGF-β1 signaling via Smad3 and binds BMP9 with high affinity. We hypothesized that BMP9 limits cardiac fibrosis by activating Smad1 and attenuating Smad3, and, furthermore, that neutralizing endoglin activity promotes BMP9 activity. Methods: We examined BMP9 expression and signaling in human cardiac fibroblasts and human subjects with heart failure. We used the transverse aortic constriction–induced model of heart failure to evaluate the functional effect of BMP9 signaling on cardiac remodeling. Results: BMP9 expression is increased in the circulation and left ventricle (LV) of human subjects with heart failure and is expressed by cardiac fibroblasts. Next, we observed that BMP9 attenuates type I collagen synthesis in human cardiac fibroblasts using recombinant human BMP9 and a small interfering RNA approach. In BMP9 –/– mice subjected to transverse aortic constriction, loss of BMP9 activity promotes cardiac fibrosis, impairs LV function, and increases LV levels of phosphorylated Smad3 (pSmad3), not pSmad1. In contrast, treatment of wild-type mice subjected to transverse aortic constriction with recombinant BMP9 limits progression of cardiac fibrosis, improves LV function, enhances myocardial capillary density, and increases LV levels of pSmad1, not pSmad3 in comparison with vehicle-treated controls. Because endoglin binds BMP9 with high affinity, we explored the effect of reduced endoglin activity on BMP9 activity. Neutralizing endoglin activity in human cardiac fibroblasts or in wild-type mice subjected to transverse aortic constriction–induced heart failure limits collagen production, increases BMP9 protein levels, and increases levels of pSmad1, not pSmad3. Conclusions: Our results identify a novel functional role for BMP9 as an endogenous inhibitor of cardiac fibrosis attributable to LV pressure overload and further show that treatment with either recombinant BMP9 or disruption of endoglin activity promotes BMP9 activity and limits cardiac fibrosis in heart failure, thereby providing potentially novel therapeutic approaches for patients with heart failure.

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Cardiomyocyte Derived miR-328 Promotes Cardiac Fibrosis by Paracrinely Regulating Adjacent Fibroblasts

Cellular Physiology and Biochemistry ◽

10.1159/000489201 ◽

2018 ◽

Vol 46 (4) ◽

pp. 1555-1565 ◽

Cited By ~ 8

Author(s):

Dandan Zhao ◽

Cui Li ◽

He Yan ◽

Tianyu Li ◽

Ming Qian ◽

...

Keyword(s):

Myocardial Infarction ◽

Left Coronary Artery ◽

Cardiac Fibrosis ◽

Cardiac Fibroblasts ◽

Collagen Deposition ◽

Paracrine Signaling ◽

Potential Therapeutic Target ◽

Transmission Electron ◽

Elevated Expression ◽

Tgf Β1

Background/Aims: In our previous study, we demonstrated that elevated expression of miR-328 is a potent determinant of cardiac fibrosis during myocardial infarction (MI). In the present study, histological examination revealed progressive fibrosis in transgenic mice overexpressing cardiomyocyte-specific miR-328. This study investigated whether the transfer of miR-328 from cardiomyocytes (CMs) to cardiac fibroblasts (CFs) in a paracrine manner contributes to myocardial fibrosis. Methods: Myocardial infarction was established by the occlusion of the left coronary artery. Masson’s trichrome staining and collagen assays were used to evaluate the progression of fibrosis. The vesicles and translocation of miR-328 in a co-culture assay system were respectively observed using transmission electron microscopy (TEM) and immunofluorescence staining (IF). Real-time PCR was employed to detect the level of miR-328, Col1α1 and Col3α1. The protein expression of Col1α1, TGF-βRIII, p-smad2/3 (phosphorylated-smad2/3) and TGF-β1 were probed using western blot analysis. Results: Cardiomyocyte-specific miR-328 overexpressing transgenic (TG) mice showed enhanced collagen deposition and provoked cardiac fibrosis by the activation of the TGF-β1 pathway, and this effect was abrogated after knockdown of endogenous miR-328 in mice. Correspondingly, the expression of miR-328 was increased in CFs co-cultured with CMs transfected with miR-328 mimics, likely in a paracrine manner. The cardiomyocyte-mediated augmentation of miR-328 contributes to fibrogenesis in CFs, and this pro-fibrotic effect was reversed after the transfection of miR-328 inhibitor in CFs. Conclusion: A novel molecular mechanism for miR-328 derived from CMs as a paracrine signaling mediator of cardiac fibrogenesis further demonstrates that miR-328 is a potential therapeutic target.

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Upregulation of α8β1-integrin in cardiac fibroblast by angiotensin II and transforming growth factor-β1

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.5.c1457 ◽

2001 ◽

Vol 281 (5) ◽

pp. C1457-C1467 ◽

Cited By ~ 32

Author(s):

Gaétan Thibault ◽

Marie-Josée Lacombe ◽

Lynn M. Schnapp ◽

Alexandre Lacasse ◽

Fatiha Bouzeghrane ◽

...

Keyword(s):

Angiotensin Ii ◽

Growth Factor ◽

Matrix Protein ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Transforming Growth Factor Β1 ◽

Cardiac Fibroblast ◽

Extracellular Matrix Protein ◽

Ang Ii ◽

Tgf Β1

Using a novel pharmacological tool with125I-echistatin to detect integrins on the cell, we have observed that cardiac fibroblasts harbor five different RGD-binding integrins: α8β1, α3β1, α5β1, αvβ1, and αvβ3. Stimulation of cardiac fibroblasts by angiotensin II (ANG II) or transforming growth factor-β1 (TGF-β1) resulted in an increase of protein and heightening by 50% of the receptor density of α8β1-integrin. The effect of ANG II was blocked by an AT1, but not an AT2, receptor antagonist, or by an anti-TGF-β1 antibody. ANG II and TGF-β1 increased fibronectin secretion, smooth muscle α-actin synthesis, and formation of actin stress fibers and enhanced attachment of fibroblasts to a fibronectin matrix. The α8- and β1-subunits were colocalized by immunocytochemistry with vinculin or β3-integrin at focal adhesion sites. These results indicate that α8β1-integrin is an abundant integrin on rat cardiac fibroblasts. Its positive modulation by ANG II and TGF-β1 in a myofibroblast-like phenotype suggests the involvement of α8β1-integrin in extracellular matrix protein deposition and cardiac fibroblast adhesion.

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Angiotensin II increases periostin expression via Ras/p38 MAPK/CREB and ERK1/2/TGF-β1 pathways in cardiac fibroblasts

Cardiovascular Research ◽

10.1093/cvr/cvr067 ◽

2011 ◽

Vol 91 (1) ◽

pp. 80-89 ◽

Cited By ~ 99

Author(s):

Li Li ◽

Dong Fan ◽

Cheng Wang ◽

Jin-Yu Wang ◽

Xiao-Bing Cui ◽

...

Keyword(s):

Angiotensin Ii ◽

P38 Mapk ◽

Cardiac Fibroblasts ◽

Tgf Β1

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MicroRNA-101a Inhibits Cardiac Fibrosis Induced by Hypoxia via Targeting TGFβRI on Cardiac Fibroblasts

Cellular Physiology and Biochemistry ◽

10.1159/000369689 ◽

2015 ◽

Vol 35 (1) ◽

pp. 213-226 ◽

Cited By ~ 49

Author(s):

Xin Zhao ◽

Kejing Wang ◽

Yuhua Liao ◽

Qiutang Zeng ◽

Yushu Li ◽

...

Keyword(s):

Signaling Pathway ◽

Transforming Growth Factor Beta ◽

Cardiac Remodeling ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Cardiac Fibroblasts ◽

Coronary Artery Occlusion ◽

Artery Occlusion ◽

Luciferase Reporter ◽

Migration Ability

Background/Aims: Hypoxia is a basic pathological challenge that is associated with numerous cardiovascular disorders including aberrant cardiac remodeling. Transforming growth factor beta (TGF-β) signaling pathway plays a pivotal role in mediating cardiac fibroblast (CF) function and cardiac fibrosis. Recent data suggested that microRNA-101a (miR-101a) exerted anti-fibrotic effects in post-infarct cardiac remodeling and improved cardiac function. This study aimed to investigate the potential relationship between hypoxia, miR-101a and TGF-β signaling pathway in CFs. Methods and Results: Two weeks following coronary artery occlusion in rats, the expression levels of both TGFβ1 and TGFβRI were increased, but the expression of miR-101a was decreased at the site of the infarct and along its border. Cultured rat neonatal CFs treated with hypoxia were characterized by the up-regulation of TGFβ1 and TGFβRI and the down-regulation of miR-101a. Delivery of miR-101a mimics significantly suppressed the expression of TGFβRI and p-Smad 3, CF differentiation and collagen content of CFs. These anti-fibrotic effects were abrogated by co-transfection with AMO-miR-101a, an antisense inhibitor of miR-101a. The repression of TGFβRI, a target of miR-101a, was validated by luciferase reporter assays targeting the 3'UTR of TGFβRI. Additionally, we found that overexpression of miR-101a reversed the improved migration ability of CFs and further reduced CF proliferation caused by hypoxia. Conclusion: Our study illustrates that miR-101a exerts anti-fibrotic effects by targeting TGFβRI, suggesting that miR-101a plays a multi-faceted role in modulating TGF-β signaling pathway and cardiac fibrosis.

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