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Connective tissue growth factor expression after angiotensin II exposure is dependent on transforming growth factor-? signaling via the canonical Smad-dependent pathway in hypertensive induced myocardial fibrosis.

ABSTRACT: Transforming growth factor-? (TGF-?) and connective tissue growth factor (CTGF) are often described as the initial pro-fibrotic mediators upregulated early in fibrosis models dependent on angiotensin II (Ang-II). In the present study, we explore the mechanistic link between TGF-? and CTGF expression by using a novel TGF-? trap.NIH/3T3 fibroblasts were subjected to TGF-? with or without TGF-? trap or 1D11 antibody, CTGF or CTGF plus TGF-? for six or 24 hours, and then used for quantitative real-time polymerase chain reaction (qRT-PCR) or immunocytochemistry. Male C57BL/6 mice were infused with Ang-II and randomly assigned TGF-? trap for six or 24 hours. Hearts were harvested for histological analyses, qRT-PCR and western blotting.Exogenous TGF-?-induced fibroblasts resulted in significant upregulation of CTGF, TGF-? and type I collagen transcript levels in vitro. Additionally, TGF-? promoted the differentiation of fibroblasts into ?-SMA+ myofibroblasts. CTGF expression was reduced by the addition of TGF-? trap or neutralizing antibody, confirming that its expression is dependent on TGF-? signaling. In contrast, exogenous CTGF did not appear to have an effect on fibroblast production of pro-fibrotic transcripts or fibroblast differentiation. Ang-II infusion in vivo led to a significant increase in TGF-? and CTGF mRNA expression at six and 24 hours with corresponding changes in Smad2 phosphorylation (pSmad2), indicative of increased TGF-? signaling. Ang-II animals that received the TGF-? trap demonstrated reduced CTGF mRNA levels and pSmad2 at six hours, suggesting that early CTGF expression is dependent on TGF-? signaling.We demonstrated that CTGF expression is dependent on TGF-? signaling both in vitro and in vivo in a model of myocardial fibrosis. This also suggests that early myocardial CTGF mRNA expression (six hours) after Ang-II exposure is likely dependent on latent TGF-? activation via the canonical Smad-dependent pathway in resident cardiac cells.

SUBMITTER: Wong CKS

PROVIDER: S-EPMC5888824 | biostudies-other | 2018 Jan-Mar

REPOSITORIES: biostudies-other

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Connective tissue growth factor expression after angiotensin II exposure is dependent on transforming growth factor-β signaling via the canonical Smad-dependent pathway in hypertensive induced myocardial fibrosis.

Wong Chloe Kok Sum CKS Falkenham Alec A Myers Tanya T Légaré Jean-Francois JF

Journal of the renin-angiotensin-aldosterone system : JRAAS 20180101 1

<h4>Introduction</h4>Transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) are often described as the initial pro-fibrotic mediators upregulated early in fibrosis models dependent on angiotensin II (Ang-II). In the present study, we explore the mechanistic link between TGF-β and CTGF expression by using a novel TGF-β trap.<h4>Materials and methods</h4>NIH/3T3 fibroblasts were subjected to TGF-β with or without TGF-β trap or 1D11 antibody, CTGF or CTGF plus TGF-β for six ...[more]

PMID: 29575960

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Project description:Fibrosis is an ominous pathological process in failing myocardium, but its pathogenesis is poorly understood. We recently reported that loss of an extracellular matrix (ECM) protein, fibulin-2, protected against ventricular dysfunction after myocardial infarction (MI) in association with absence of activation of transforming growth factor (TGF)-β signaling and suppressed upregulation of ECM protein expression during myocardial remodeling. Here we investigated the role of fibulin-2 in the development of myocardial hypertrophy and fibrosis induced by continuous pressor-dosage of angiotensin II (Ang II) infusion. Both wild type (WT) and fibulin-2 null (Fbln2KO) mice developed comparable hypertension and myocardial hypertrophy by Ang II infusion. However, myocardial fibrosis with significant upregulation of collagen type I and III mRNA was only seen in WT but not in Fbln2KO mice.Transforming growth factor (TGF)-β1 mRNA and its downstream signal, Smad2, were significantly upregulated in WT by Ang II, whereas there were no Ang II-induced changes in Flbn2KO, suggesting fibulin-2 is necessary for Ang II-induced TGF-β signaling that induces myocardial fibrosis. To test whether fibulin-2 is sufficient for Ang II-induced TGF-β upregulation, isolated Flbn2KO cardiac fibroblasts were treated with Ang II after transfecting with fibulin-2 expression vector or pretreating with recombinant fibulin-2 protein. Ang II-induced TGF-β signaling in Fbln2KO cells was partially rescued by exogenous fibulin-2, suggesting that fibulin-2 is required and probably sufficient for Ang II-induced TGF-β activation. Smad2 phosphorylation was induced just by adding recombinant fibulin-2 to KO cells, suggesting that extracellular interaction between fibulin-2 and latent TGF-β triggered initial TGF-β activation. Our study indicates that Ang II cannot induce TGF-β activation without fibulin-2 and that fibulin-2 has an essential role in Ang II-induced TGF-β signaling and subsequent myocardial fibrosis. Fibulin-2 can be considered as a critical regulator of TGF-β that induces myocardial fibrosis.

Project description:BACKGROUND:Adenomyosis is a medical condition defined by the abnormal presence of endometrial tissue within the myometrium, in which fibrosis occurs with new collagen deposition and myofibroblast differentiation. In this study, the effect of several mediators and growth factors on collagen expression was investigated on human endometrial stromal cells (fibroblasts) derived from adenomyotic endometrium. EXPERIMENTAL APPROACH:RT-PCR, Western blot analysis, pharmacological interventions and siRNA interference were applied to primary cultured human endometrial stromal cells (fibroblasts). Immunohistochemistry was used to analyze protein expression in adenomyotic endometrium tissue specimens. RESULTS:Of the tested mediators, transforming growth factor β1 (TGFβ1) and its isoforms were effective to induce collagen and connective tissue growth factor (CTGF) expression. Collagen and CTGF induction by TGFβ1 could be reduced by the inhibitors targeting DNA transcription, protein translation, and Smad2/3 signaling. Interestingly, TGFβ1 induced Smad2/3 phosphorylation and CTGF mRNA expression, but not collagen mRNA expression, suggesting that TGFβ1 mediates collagen expression through CTGF induction and Smad2/3 activation. In parallel, TGFβ1 and CTGF also induced expression of heat shock protein (HSP) 47, a protein required for the synthesis of several types of collagens. However, only CTGF siRNA knockdown, could compromise TGFβ1-induced collagen expression. Finally, the immunohistochemistry revealed vimentin- and α-SMA-positive staining for (myo)fibroblasts, TGFβ1, collagen, and CTGF in the subepithelial stroma region of human adenomyotic endometria. CONCLUSION AND IMPLICATIONS:We reveal here that TGFβ1, collagen, and CTGF are expressed in the stroma of adenomyotic endometria and demonstrate that TGFβ1 can induce collagen production in endometrium-derived fibroblasts through cellular Smad2/3-dependent signaling pathway and CTGF expression, suggesting that endometrial TGFβ may take part in the pathogenesis of adenomyosis and ectopic endometrium may participate in uterine adenomyosis.

Dataset Information

Connective tissue growth factor expression after angiotensin II exposure is dependent on transforming growth factor-? signaling via the canonical Smad-dependent pathway in hypertensive induced myocardial fibrosis.

Publications

Connective tissue growth factor expression after angiotensin II exposure is dependent on transforming growth factor-β signaling via the canonical Smad-dependent pathway in hypertensive induced myocardial fibrosis.

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