scholarly journals Autophagy inhibition of hsa-miR-19a-3p/19b-3p by targeting TGF-β R II during TGF-β1-induced fibrogenesis in human cardiac fibroblasts

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Meijuan Zou ◽  
Fang Wang ◽  
Rui Gao ◽  
Jingjing Wu ◽  
Yingwei Ou ◽  
...  
Keyword(s):  
Cardiac Fibroblasts ◽  
Human Cardiac Fibroblasts ◽  
Autophagy Inhibition ◽  
Tgf Β1

scholarly journals Featured Article: TGF-β1 dominates extracellular matrix rigidity for inducing differentiation of human cardiac fibroblasts to myofibroblasts

2018 ◽  
Vol 243 (7) ◽  
pp. 601-612 ◽  
Author(s):  
Nathan Cho ◽  
Shadi E Razipour ◽  
Megan L McCain
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Matrix Rigidity ◽  
Growth Factor Beta ◽  
Human Cardiac Fibroblasts ◽  
Tgf Β1

Cardiac fibroblasts and their activated derivatives, myofibroblasts, play a critical role in wound healing after myocardial injury and often contribute to long-term pathological outcomes, such as excessive fibrosis. Thus, defining the microenvironmental factors that regulate the phenotype of cardiac fibroblasts and myofibroblasts could lead to new therapeutic strategies. Both chemical and biomechanical cues have previously been shown to induce myofibroblast differentiation in many organs and species. For example, transforming growth factor beta 1, a cytokine secreted by neutrophils, and rigid extracellular matrix environments have both been shown to promote differentiation. However, the relative contributions of transforming growth factor beta 1 and extracellular matrix rigidity, two hallmark cues in many pathological myocardial microenvironments, to the phenotype of human cardiac fibroblasts are unclear. We hypothesized that transforming growth factor beta 1 and rigid extracellular matrix environments would potentially have a synergistic effect on the differentiation of human cardiac fibroblasts to myofibroblasts. To test this, we seeded primary human adult cardiac fibroblasts onto coverslips coated with polydimethylsiloxane of various elastic moduli, introduced transforming growth factor beta 1, and longitudinally quantified cell phenotype by measuring expression of α-smooth muscle actin, the most robust indicator of myofibroblasts. Our data indicate that, although extracellular matrix rigidity influenced differentiation after one day of transforming growth factor beta 1 treatment, ultimately transforming growth factor beta 1 superseded extracellular matrix rigidity as the primary regulator of myofibroblast differentiation. We also measured expression of POSTN, FAP, and FSP1, proposed secondary indicators of fibroblast/myofibroblast phenotypes. Although these genes partially trended with α-smooth muscle actin expression, they were relatively inconsistent. Finally, we demonstrated that activated myofibroblasts incompletely revert to a fibroblast phenotype after they are re-plated onto new surfaces without transforming growth factor beta 1, suggesting differentiation is partially reversible. Our results provide new insights into how microenvironmental cues affect human cardiac fibroblast differentiation in the context of myocardial pathology, which is important for identifying effective therapeutic targets and dictating supporting cell phenotypes for engineered human cardiac disease models. Impact statement Heart disease is the leading cause of death worldwide. Many forms of heart disease are associated with fibrosis, which increases extracellular matrix (ECM) rigidity and compromises cardiac output. Fibrotic tissue is synthesized primarily by myofibroblasts differentiated from fibroblasts. Thus, defining the cues that regulate myofibroblast differentiation is important for understanding the mechanisms of fibrosis. However, previous studies have focused on non-human cardiac fibroblasts and have not tested combinations of chemical and mechanical cues. We tested the effects of TGF-β1, a cytokine secreted by immune cells after injury, and ECM rigidity on the differentiation of human cardiac fibroblasts to myofibroblasts. Our results indicate that differentiation is initially influenced by ECM rigidity, but is ultimately superseded by TGF-β1. This suggests that targeting TGF-β signaling pathways in cardiac fibroblasts may have therapeutic potential for attenuating fibrosis, even in rigid microenvironments. Additionally, our approach can be leveraged to engineer more precise multi-cellular human cardiac tissue models.

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

Circulation ◽  
2018 ◽  
Vol 138 (5) ◽  
pp. 513-526 ◽  
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.

scholarly journals MiRNA-1202 promotes the TGF-β1-induced proliferation, differentiation and collagen production of cardiac fibroblasts by targeting nNOS

PLoS ONE ◽  
2021 ◽  
Vol 16 (8) ◽  
pp. e0256066
Author(s):  
Jingwen Xiao ◽  
Yan Zhang ◽  
Yuan Tang ◽  
Hengfen Dai ◽  
Yu OuYang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Myocardial Fibrosis ◽  
Cardiac Fibroblasts ◽  
Collagen I ◽  
Luciferase Reporter ◽  
Control Group ◽  
Protein Levels ◽  
Collagen Iii ◽  
Human Cardiac Fibroblasts ◽  
Tgf Β1

Background Atrial fibrillation (AF) is a clinically common arrhythmia that affects human health. Myocardial fibrosis serves as an important contributor to AF. Recently, miRNA-1202 have been reported to be up-regulated in AF. However, the role of miRNA-1202 and its mechanism in myocardial fibrosis remain unclear. Methods Human cardiac fibroblasts (HCFs) were used to construct a fibrosis model by TGF-β1 induction. The expression of miR-1202 was measured by qRT-PCR. Cell proliferation was assessed by CCK-8 assays. Protein expression levels were measured by western blot. Collagen accumulation was measured by ELISA. The relationship between miR-1202 and nNOS was investigated by luciferase reporter assays. Results MiR-1202 expression was obviously increased in HCFs and was both time- and dose-independent. MiR-1202 could increase the proliferation and collagen I, collagen III, and α-SMA levels with or without TGF-β1. MiR-1202 could also increase TGF-β1 and p-Smad2/3 protein levels in comparison to the control group. However, they were obviously decreased after inhibitor transfection. MiR-1202 targets nNOS for negative regulation of HCFs fibrosis by decreasing cell differentiation, collagen deposition and the activity of the TGF-β1/Smad2/3 pathway. Co-transfection of miR-1202 inhibitor and siRNA of nNOS inhibited nNOS protein expression, thereby enhancing the HCFs proliferation. Furthermore, co-transfection of the miR-1202 inhibitor and siRNA of nNOS significantly promoted collagen I, collagen III, TGF-β1, Smad2/3 and α-SMA protein expression and Smad2/3 protein phosphorylation. These findings suggested that miR-1202 promotes HCFs transformation to a pro-fibrotic phenotype by targeting nNOS through activating the TGF-β1/Smad2/3 pathway.

scholarly journals Ac-SDKP inhibits transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts

2010 ◽  
Vol 298 (5) ◽  
pp. H1357-H1364 ◽  
Author(s):  
Hongmei Peng ◽  
Oscar A. Carretero ◽  
Edward L. Peterson ◽  
Nour-Eddine Rhaleb
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Cardiac Fibroblasts ◽  
Transforming Growth Factor Β1 ◽  
Collagen Production ◽  
Smad7 Expression ◽  
Human Cardiac Fibroblasts ◽  
Tgf Β1

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibits collagen production and cell proliferation in cultured rat cardiac fibroblasts, but its effect on differentiation of fibroblasts into myofibroblasts is not known. High amounts of transforming growth factor-β1 (TGF-β1) have been found in fibrotic cardiac tissue. TGF-β1 converts fibroblasts into myofibroblasts, which produce more extracellular matrix proteins than fibroblasts. We hypothesized that 1) Ac-SDKP inhibits TGF-β1-induced differentiation of fibroblasts into myofibroblasts; and 2) this effect is mediated in part by blocking phosphorylation of small-mothers-against-decapentaplegic (Smad) 2 and extracellular signal-regulated kinase (ERK) 1/2. For this study, we used human fetal cardiac fibroblasts (HCFs), which do not spontaneously become myofibroblasts when cultured at low passages. We investigated the effect of Ac-SDKP on TGF-β1-induced HCF transformation into myofibroblasts, Smad2 and ERK1/2 phosphorylation, Smad7 expression, cell proliferation, and collagen production. We also investigated TGF-β1 production by HCFs stimulated with endothelin-1 (ET-1). As expected, HCFs treated with TGF-β1 transformed into myofibroblasts as indicated by increased expression of α-smooth muscle actin and a higher proportion of the embryonic isoform of smooth muscle myosin compared with untreated cells. TGF-β1 also increased Smad2 and ERK1/2 phosphorylation but did not affect Smad7 expression. In addition, TGF-β1 stimulated HCF proliferation as indicated by an increase in mitochondrial dehydrogenase activity and collagen production (hydroxyproline assay). Ac-SDKP significantly inhibited all of the effects of TGF-β1. It also inhibited ET-1-stimulated TGF-β1 production. We concluded that Ac-SDKP markedly suppresses differentiation of human cardiac fibroblasts into myofibroblasts, probably by inhibiting the TGF-β/Smad/ERK1/2 signaling pathway, and thus mediating its anti-fibrotic effects.

Abstract 348: Phorbol 12-myristate 13-acetate Induces Dedifferentiation of Cardiac Myofibroblasts to Fibroblasts and Other Unidentified Type of Cells

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Vy Tran Luu ◽  
Sang Phan ◽  
Zhuqiu Jin
Keyword(s):  
Cardiac Fibrosis ◽  
De Novo ◽  
Cardiac Fibroblasts ◽  
Collagen Gel ◽  
Collagen Gel Contraction ◽  
Human Cardiac Fibroblasts ◽  
Multiple Cells ◽  
Tgf Β1 ◽  
Cardiac Myofibroblasts ◽  
Shape And Size

Cardiac fibrosis plays an essential role in cardiac pathogenic processes that occur as a result of myocardial infarction or hypertrophic cardiomyopathy. The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGFβ is one of the essential molecules that promote transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. Phorbol 12-Myristate 13-Acetate (PMA) regulates metabolism and functions of multiple cells via PKC activation mostly. To study effects of PMA on differentiation of de novo formed cardiac myofibroblasts, human cardiac fibroblasts were utilized. Human cardiac fibroblasts (HCF) cultured in fibroblast medium (FM)-2 were converted into myofibroblasts in the presence of 2 ng/mL of TGF-β1 for 48 hours. Expression of α-SMA, the biomarker of myofibroblasts, and FSP1, the biomarker of fibroblasts, was detected using Western blot and immunofluorescence. Collagen gel contraction induced by fibroblasts was determined as well. TGF-β1 increased the expression of α-SMA and reduced the expression of FSP1. Distinct cellular morphology changes in the shape and size of HCF were observed after incubation with TGF-β1 for 48 hours. To investigate effect of PMA on dedifferentiation of formed myofibroblasts, these TGF-β1-pretreated cells were divided into four groups for additional 48 hours incubation: PMA groups (10, 50, and 100 ng/mL) or DMSO (vehicle control). Both 50 and 100 ng/mL of PMA reduced the expression of α-SMA but only 100 ng/mL of PMA increased the expression of FSP1. The shape and size of cells changed after treatment with PMA. PMA also reduced TGF-β1-induced collagen gel contraction (P<0.05, compared to DMSO group). These data indicated that PMA can reverse the differentiation of de novo formed human cardiac myofibroblasts induced by TGF-β1 to fibroblasts and other unidentified type of cells. Although the mechanism of dedifferentiation remains to be identified, the novel finding of this study shed light on future development of agents to treat fibrotic diseases.

Abstract 17416: miR-1468-3p Regulates Development of Cardiac Fibrosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Ruizhu Lin ◽  
Lea Rahtu-Korpela ◽  
Johanna Magga ◽  
Lasse Pakanen ◽  
Katja Porvari ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Collagen I ◽  
Loss Of Function ◽  
Collagen Iii ◽  
Approaches To Study ◽  
Human Cardiac Fibroblasts ◽  
Tgf Β1

Background: Accumulation of extracellular matrix disturbs the electrical conduction and stiffens myocardium, leading to higher risk for arrhythmias and diastolic dysfunction. MicroRNAs (miRNAs) function in post-translational gene regulation, and aberrant alteration of miRNAs level has been implicated in cardiac pathologies. Methods and Results: RNA sequencing of RNA samples of sudden cardiac death (SCD) victims with idiopathic myocardial fibrosis (IMF) for differentially expressed miRNAs identified miR-1468-3p. qPCR analysis validated that expression of miR-1468-3p is upregulated in hearts of SCD victims with IMF comparing to control subjects. However, the role and molecular function of miR-1468-3p in cardiac fibrosis are not known. We utilized human cardiac fibroblasts (hCFs) and gain- and loss-of-function approaches to study the role of miR-1468-3p in modulating fibroblast function. Overexpressing miR-1468-3p in hCFs resulted in an increase in expression of several fibrotic genes compared with hCFs transfected with control mir-mimic. Western blot analysis showed that miR-1468-3p mimic was sufficient to drive expression of collagen I and CTGF protein expression. Treatment of hCFs with miR-1468-3p antagomir did not alter expression of fibrosis-related gene at basal level, whereas miR-1468-3p inhibition significantly attenuated TGF-β1-induced collagen I and collagen III expression. Treatment of hCFs with miR-1468-3p antagomir blunted TGF-β1-induced collagen I and CTGF protein expression, but not TGF-β1-induced αSMA expression. Employing Sirius Red/Fast Green assay, we validated that depletion of miR-1468-3p antagonized both TGF-β1-triggered collagen and non-collagen protein production. Finally, we found that miR-1468-3p antagomir downregulated TGF-β1-induced collagen expression partially through the interference of TGF-β1/MAPK signals (p38 and JNK) and Integrin signaling. Conclusions: Our data indicate a pro-fibrotic role of miR-1468-3p in modulating cardiac fibrosis, and manipulating the expression of miR-1468-3p may provide a therapeutic strategy for treatment of cardiac fibrosis.

scholarly journals A protocol for transdifferentiation of human cardiac fibroblasts into endothelial cells via activation of innate immunity

2021 ◽  
Vol 2 (2) ◽  
pp. 100556
Author(s):  
Chun Liu ◽  
Pedro Medina ◽  
Dilip Thomas ◽  
Ian Y. Chen ◽  
Karim Sallam ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Endothelial Cells ◽  
Cardiac Fibroblasts ◽  
Human Cardiac Fibroblasts
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