Aortic composition in HFD rats.

<p>Aortas from rats fed a standard diet (3.5% fat) or a high fat diet (HFD, 33.5% fat) were analyzed. Representative pictures of slides stained for collagen and elastin are presented (magnification 40X) (<b>A</b>). mRNA expression of collagen type I, fibronectin, TGF-β, CTGF in aorta from controls and HFD rats (<b>B</b>). Protein expression of collagen type I, elastin, MMP-2, TIMP-2, TGF-β and CTGF in aorta from controls and HFD rats (<b>C</b>). All conditions were performed at least by triplicate. Scale bar 50 µm. Histogram bars represent the mean ± SEM of 6–7 animals, in arbitrary units normalized to HPRT and β-actin for cDNA and protein respectively. *p<0.05; ***p<0.001 <i>vs</i>. control group.</p

Effects of sST2 on fibrotic parameters in VSMCs.

<p>mRNA levels of collagen type I, fibronectin, TGF-β and CTGF in VSMCs stimulated with sST2 (2 µg/ml) during 6 hours (<b>A</b>). Protein expression of collagen type I, fibronectin and elastin (<b>B</b>). MMP-2, MMP-9, MMP-13 activity in VSMCs stimulated with sST2 (<b>C</b>). All conditions were performed at least by triplicate. Histogram bars represent the mean ± SEM of 4 assays, in arbitrary units normalized to HPRT and β-actin respectively for cDNA and protein. *p<0.05; **p<0.01; ***p<0.001 <i>vs</i>. control.</p

Effect of high fat diet on the IL-33/ST2 pathway in the aorta of rats.

<p>Representative pictures of slides immunostained for IL-33, ST2 and MyD88 are presented (<b>A</b>). Aortic mRNA levels of IL-33, sST2 and ST2L (<b>B</b>). Protein levels of aortic IL-33, ST2L and MyD88 are shown (<b>C</b>). All conditions were performed at least by triplicate. Histogram bars represent the mean ± SEM of 6–7 animals, in arbitrary units normalized to HPRT and β-actin for cDNA and protein respectively. *p<0.05; **p<0.01 <i>vs</i>. control group.</p

Effect of sST2 on the IL-33/ST2 pathway in VSMCs.

<p>Representative pictures of immunocytochemistry for IL-33, ST2, MyD88 and IRAK-1 are presented (<b>A</b>). mRNA levels of IL-33, sST2 and ST2L in VSMCs stimulated with sST2 (2 µg/ml) (<b>B</b>). Protein levels of IL-33, ST2-L, MyD88 and IRAK-1 are shown (<b>C</b>). All conditions were performed at least by triplicate. Histogram bars represent the mean ± SEM of 4 assays, in arbitrary units normalized to HPRT and β-actin respectively for cDNA and protein. *p<0.05 <i>vs</i>. control.</p

Primers used in real time PCR analysis.

<p>Primers used in real time PCR analysis.</p

Parameters measured in aortic tunica media from Controls and HFD rats.

<p>Dry weight and contents in proteins are expressed in mg/cm. Parameters of inflammation were performed at least by triplicate and expressed in arbitrary units normalized to HPRT for cDNA. Values are mean ± SEM.</p>*<p>p<0.05;</p>**<p>p<0.01;</p>***<p>p<0.001 <i>vs</i>. control group.</p

Effect of erythrodiol and uvaol on the fibrotic effect of angiotensin II in cardiac myofibroblasts.

<p>Time course of angiotensin II (Ang II; 1 µM)-stimulated collagen I protein production (A). Effect of erythrodiol (ERY; 5 µM) or uvaol (UVA; 5 µM) on CTGF (B), collagen I (C) and galectin 3 (D) protein expression in angiotensin II-treated cardiac myofibroblasts for 12 hours. Representative immunoblots of 4 experiments. Values are mean ± SEM of four assays. *p<0.05 <i>vs</i> vehicle. †p<0.05 vs angiotensin II. Quantification of band intensities was measured by densitometry and normalized to respective α-tubulin.</p

Efect of erytrodiol and uvaol in cardiac myofibroblasts apoptosis.

<p>Cardiac myofibroblast were treated with different doses of erythrodiol (ERY; A) or uvaol (UVA; B) in the presence or absence of angiotensin II (Ang II, 1 µM). After 24 h of stimulation, cells were labeled with annexin-V PE and analyzed by flow cytometry. Values are mean ± SEM of three experiments. *p<0.05 vs cells in the absence of either erythrodiol or uvaol. Figures C–F show representative immunocytochemistry images of cardiac myofibroblasts treated for 24 hours with erythrodiol (C: 5 µM; D: 25 µM) or uvaol (E: 5 µM; F: 25 µM) examined by fluorescence microscopy. Vimentin staining is shown in green and nuclei staining in blue. Magnification 40X. Scale bar: 50 µm. Figure G: Effect of a specific inhibitor of MEK (PD9805; 25 µM) on the apoptosis in the presence or absence of angiotensin II in cardiac myofibroblasts. Cells obtained after PD98059 treatment in the absence of the inhibitor (open black curve) are compared with cells treated in the presence of the inhibitor (open gray curves). Solid gray curves represent resting control cells.</p

Representative immunocytochemistry images of cardiac myofibroblasts examined by fluorescence microscopy.

<p>Vimentin staining (A), α-smooth muscle actin (α-SMA) staining (B) and vimentin and α-SMA staining merged (C). Magnification 40X. Scale bar: 50 µm.</p

Effect of specific inhibitors on the apoptotic activity of erythrodiol or uvaol in cardiac myofibroblasts.

<p>Effect of a specific inhibitor of either JNK (SP600125) or PPAR-γ, (GW9662) on the apoptosis induced by either erythrodiol (ERY; 25 µM A and C, respectively) or uvaol (UVA; 25 µM; B and D, respectively) in the presence or absence of angiotensin II (Ang II; 1 µM) in cardiac myofibroblasts. Representative of 3 experiments. In all panels, cells obtained after triterpene treatment in the absence of the inhibitor (open black curve) are compared with cells treated in the presence of the inhibitor (open gray curves). Solid gray curves represent control cells.</p