Transforming growth factor-beta and Forkhead box O transcription factors as cardiac fibroblast regulators

Fibroblasts play several homeostatic roles, including electrical coupling, paracrine signaling and tissue repair after injury. Fibroblasts have low secretory activity. However, in response to injury, they differentiate to myofibroblasts. These cells have an increased extracellular matrix synthesis and secretion, including collagen fibers, providing stiffness to the tissue. In pathological conditions myofibroblasts became resistant to apoptosis, remaining in the tissue, causing excessive extracellular matrix secretion and deposition, which contributes to the progressive tissue remodeling. Therefore, increased myofibroblast content within damaged tissue is a characteristic hallmark of heart, lung, kidney and liver fibrosis. Recently, it was described that cardiac fibroblast to myofibroblast differentiation is triggered by the transforming growth factor β1 (TGF-β1) through a Smad-independent activation of Forkhead box O (FoxO). FoxO proteins are a transcription factor family that includes FoxO1, FoxO3, FoxO4 and FoxO6. In several cells types, they play an important role in cell cycle arrest, oxidative stress resistance, cell survival, energy metabolism, and cell death. Here, we review the role of FoxO family members on the regulation of cardiac fibroblast proliferation and differentiation.Comision Nacional de Ciencia y Tecnologia (CONICYT), Chile, FONDECYT 1140329, FONDECYT 11160531, FONDECYT 113030

Legislative Documents

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Angiotensin II-regulated autophagy is required for vascular smooth muscle cell hypertrophy

Copyright © 2019 Mondaca-Ruff, Riquelme, Quiroga, Norambuena-Soto, Sanhueza-Olivares, Villar-Fincheira, Hernández-Díaz, Cancino-Arenas, San Martin, García, Lavandero and Chiong.Hypertension is a disease associated to increased plasma levels of angiotensin II (Ang II). Ang II can regulate proliferation, migration, ROS production and hypertrophy of vascular smooth muscle cells (VSMCs). However, the mechanisms by which Ang II can affect VSMCs remain to be fully elucidated. In this context, autophagy, a process involved in self-digestion of proteins and organelles, has been described to regulate vascular remodeling. Therefore, we sought to investigate if Ang II regulates VSMC hypertrophy through an autophagy-dependent mechanism. To test this, we stimulated A7r5 cell line and primary rat aortic smooth muscle cells with Ang II 100 nM and measured autophagic markers at 24 h by Western blot. Autophagosomes were quantified by visualizing fluorescently labeled LC3 using confocal microscopy. The results showed that treatment with Ang II increases Beclin-1, Vps34, Atg-12–Atg5, Atg4 and Atg7 protein levels, Beclin-1 phosphorylation, as well as the number of autophagic vesicles, suggesting that this peptide induces autophagy by activating phagophore initiation and elongation. These findings were confirmed by the assessment of autophagic flux by co-administering Ang II together with chloroquine (30 μM). Pharmacological antagonism of the angiotensin type 1 receptor (AT1R) with losartan and RhoA/Rho Kinase inhibition prevented Ang II-induced autophagy. Moreover, Ang II-induced A7r5 hypertrophy, evaluated by α-SMA expression and cell size, was prevented upon autophagy inhibition. Taking together, our results suggest that the induction of autophagy by an AT1R/RhoA/Rho Kinase-dependent mechanism contributes to Ang II-induced hypertrophy in VSMC

Angiotensin-(1-9) prevents vascular remodeling by decreasing vascular smooth muscle cell dedifferentiation through a FoxO1-dependent mechanism

The renin-angiotensin system, one of the main regulators of vascular function, controls vasoconstriction, inflammation and vascular remodeling. Antagonistic actions of the counter-regulatory renin-angiotensin system, which include vasodilation, anti-proliferative, anti-inflammatory and anti-remodeling effects, have also been described. However, little is known about the direct effects of angiotensin-(1-9), a peptide of the counter-regulatory renin-angiotensin system, on vascular smooth muscle cells. Here, we studied the anti-vascular remodeling effects of angiotensin-(1-9), with special focus on the control of vascular smooth muscle cell phenotype. Angiotensin-(1-9) decreased blood pressure and aorta media thickness in spontaneously hypertensive rats. Reduction of media thickness was associated with decreased vascular smooth muscle cell proliferation. In the A7r5 VSMC cell line and in primary cultures of rat aorta smooth muscle cells, angiotensin-(1-9) did not modify basal proliferation. However, angiotensin-(1-9) inhibited proliferation, migration and contractile protein decrease induced by platelet derived growth factor-BB. Moreover, angiotensin-(1-9) reduced Akt and FoxO1 phosphorylation at 30 min, followed by an increase of total FoxO1 protein content. Angiotensin-(1-9) effects were blocked by the AT2R antagonist PD123319, Akt-Myr overexpression and FoxO1 siRNA. These data suggest that angiotensin-(1-9) inhibits vascular smooth muscle cell dedifferentiation by an AT2R/Akt/FoxO1-dependent mechanism.Agencia Nacional de Investigacion y Desarrollo (ANID, Chile): Fondecyt 1140329 1180157 FONDAP 15130011 Puente Pontificia Universidad Catolica de Chile P1705/2017 Bayer AG 2017-08-2260 Comision Nacional de Investigacion Cientifica y Tecnologica (CONICYT) CONICYT PIA/ANILLOS ACT192144 ANID PhD fellowship

Autophagy mediates TNF-α-induced secretion of IL-6 in A7r5 cells.

<p>(<b>A</b>) Determination of IL-6 mRNA using RT-qPCR in A7r5 cells treated with TNF-α (100 ng/mL) for 30 min, 1 and 6 h (n = 6; *<i>p</i><0.05, **<i>p</i><0.01 vs 0 h). (<b>B</b>) Determination of IL-6 using ELISA assay in A7r5 cells treated with TNF-α (100 ng/mL) for 24 and 48 h (n = 4; **<i>p</i><0.01, ***<i>p</i><0.001 vs 0 h) or (<b>C</b>) co-administered with or without chloroquine (CQ, 20 μmol/L) during the last 4 h of the 24 h stimulus with TNF-α (n = 4; ***<i>p</i><0.001 vs control). Data are expressed as mean ± SEM and analyzed by one-way ANOVA, followed by Dunnett and Tukey post-tests.</p

TNF-α requires autophagy to induce migration in A7r5 cells.

<p>(<b>A</b>) Assessment of migration by the wound healing and transwell assays in A7r5 cells stimulated with TNF-α (100 ng/mL) for 24 h in the presence or absence of chloroquine (CQ, 20 μmol/L) during the last 4 h of TNF-α stimulus (n = 4; **<i>p</i><0.01 vs control) or (<b>C</b>) siScramble and siBeclin1 for 24 h (n = 4; **<i>p</i><0.01 vs control). Migration was visualized using a phase contrast microscope (upper panels of <b>A</b> and <b>C</b>). The results of the wound healing and transwell assays were quantified by measuring wound width and the number of cells that migrated through the Boyden chamber, respectively (lower panels of <b>A</b> and <b>C</b>). (<b>B</b>) Zymography analysis of matrix metalloproteinase 9 (MMP-9) in A7r5 cells stimulated with TNF-α (100 ng/mL) for 24 h (n = 3; *<i>p</i><0.05 vs control). Data are expressed as mean ± SEM and analyzed by one-way ANOVA, followed by Holm Sidak (<b>A</b> and <b>C</b>) and Dunnett (<b>B</b>) post-tests.</p

TNF-α induces dedifferentiation of A7r5 cells by an autophagy-dependent pathway.

<p>(<b>A</b>) Western blot analysis of α-SMA and SM22 (n = 3–4; *<i>p</i><0.05, **<i>p</i><0.01 vs control) or (<b>B</b>) collagen type I and osteopontin (n = 3–4; **p<0.01 vs control) in A7r5 cells incubated with TNF-α (100 ng/mL) for 48 h in the presence or absence of siScramble and siBeclin1. GAPDH was used as loading control. (<b>C</b>) Visualization of actin filaments in A7r5 cells stained with rhodamine-phalloidin after treatment with TNF-α (100 ng/mL) for 48 h in the presence or absence of chloroquine (CQ, 5 μmol/L) during the last 24 h of TNF-α stimulus. Lower panel represent a fluorescence intensity profile of the lines depicted on the images. Data are expressed as mean ± SEM and analyzed by two-way ANOVA, followed by Holm Sidak post-test.</p