DIOL triterpenes block profibrotic effects of angiotensin II and protect from cardiac hypertrophy.

BACKGROUND: The natural triterpenes, erythrodiol and uvaol, exert anti-inflammatory, vasorelaxing and anti-proliferative effects. Angiotensin II is a well-known profibrotic and proliferative agent that participates in the cardiac remodeling associated with different pathological situations through the stimulation and proliferation of cardiac fibroblasts. Therefore, the aim of the study was to investigate the preventive effects of the natural triterpenes erythrodiol and uvaol on the proliferation and collagen production induced by angiotensin II in cardiac myofibroblasts. Their actions on cardiac hypertrophy triggered by angiotensin II were also studied. METHODOLOGY/PRINCIPAL FINDINGS: The effect of erythrodiol and uvaol on angiotensin II-induced proliferation was evaluated in cardiac myofibroblasts from adult rats in the presence or the absence of the inhibitors of PPAR-γ, GW9662 or JNK, SP600125. The effect on collagen levels induced by angiotensin II was evaluated in cardiac myofibroblasts and mouse heart. The presence of low doses of both triterpenes reduced the proliferation of cardiac myofibroblasts induced by angiotensin II. Pretreatment with GW9662 reversed the effect elicited by both triterpenes while SP600125 did not modify it. Both triterpenes at high doses produced an increase in annexing-V binding in the presence or absence of angiotensin II, which was reduced by either SP600125 or GW9662. Erythrodiol and uvaol decreased collagen I and galectin 3 levels induced by angiotensin II in cardiac myofribroblasts. Finally, cardiac hypertrophy, ventricular remodeling, fibrosis, and increases in myocyte area and brain natriuretic peptide levels observed in angiotensin II-infused mice were reduced in triterpene-treated animals. CONCLUSIONS/SIGNIFICANCE: Erythrodiol and uvaol reduce cardiac hypertrophy and left ventricle remodeling induced by angiotensin II in mice by diminishing fibrosis and myocyte area. They also modulate growth and survival of cardiac myofibroblasts. They inhibit the angiotensin II-induced proliferation in a PPAR-γ-dependent manner, while at high doses they activate pathways of programmed cell death that are dependent on JNK and PPAR-γ

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

Effect of erythrodiol and uvaol on the fibrotic effects of angiotensin II on mice.

<p>Mice infused with angiotensin II (Ang II; 1.44 mg Kg⁻¹ day⁻¹) were treated with erythrodiol (ERY; 50 mg Kg⁻¹ day⁻¹) or uvaol UVA (50 mg Kg⁻¹ day⁻¹) for two weeks. Fibrosis was assessed by Picro-sirius red staining procedure. Interstitial (A) and perivascular (F) collagen quantification. Representative microphotographs of myocardial sections showing interstitial and perivascular fibrosis from control (B, G), angiotensin II-infused animals treated with vehicle (C, H), erythrodiol (D, I), or uvaol (E, J). Magnification 40X. Scale bar: 100 µm. Values are mean ± SEM of 5–6 animals. *p<0.05 <i>vs</i> control. †p<0.05 vs angiotensin II.</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 erythrodiol and uvaol on the hypertrophyc effects of angiotensin II on mice.

<p>Mice infused with angiotensin II (Ang II; 1.44 mg Kg⁻¹ day ⁻¹) were treated with erythrodiol (ERY; 50 mg Kg⁻¹ day⁻¹) or uvaol (UVA; 50 mg Kg⁻¹ day ⁻¹) for two weeks. (A) relative heart weight and (B) cardiac myocyte area. Representative microphotographs of myocardial sections from control (C, CT), angiotensin II-infused animals treated with vehicle (D), erythrodiol (E), or uvaol (F). Magnification 40X. Samples were stained with Masson’s trichrome technique. Scale bar: 100 µm. Values are mean ± SEM of 5–6 animals. *p<0.05 <i>vs</i> control. †p<0.05 vs angiotensin II.</p

Effect of specific inhibitors on the antiproliferative activity of erythrodiol or uvaol in angiotensin II-treated cardiac myofibroblasts.

<p>Cells pretreated for 30 min with the specific inhibitors of either MEK (A and B; PD98059) or PPAR-γ (C and D; GW9662) were stimulated with 1 µM of angiotensin II (Ang II) for 24 hours in the presence of 5 µM of either erythrodiol (ERY; A and C) or uvaol (UVA; B and D). Proliferation was determined by an MTT assay. Data are expressed as percent of unstimulated cells. Values are mean ± SEM of three assays. Panel E represents the effect of either ERY (5 µM) or UVA (5 µM) on nuclear PPAR-γ protein levels in Ang II-treated cardiac myofibroblasts. Nuclear proteins from cells stimulated with 1 µM of Ang II from 12 hours in the presence or absence of the indicated triterpene were analysed by western blotting a specific antibody against PPAR-γ. Representative immunoblots of 4 experiments. Quantification of band intensities was measured by densitometry and normalized to respective α-tubulin. *p<0.05 <i>vs</i> vehicle. †p<0.05 vs angiotensin II. #p<0.05 vs erythrodiol or uvaol.</p

Effect of erythrodiol and uvaol on and left ventricle remodeling and serum BNP levels induced by angiotensin II on mice.

<p>Mice infused with angiotensin II (Ang II; 1.44 mg Kg⁻¹ day⁻¹) were treated with erythrodiol (ERY; 50 mg Kg⁻¹ day⁻¹) or uvaol (UVA; 50 mg Kg⁻¹ day⁻¹) for two weeks. Left ventricular cross sectional area (LVCSA, A) and left ventricular wall thickness (LVWT, B) were measured in Masson’s trichrome stained sections. Two-three sections for each animal at the midregion area were analysed. Magnification 5X. Serum BNP levels were assessed by using a mouse BNP-specific ELISA (C). Values are mean ± SEM of 5–6 animals. *p<0.05 <i>vs</i> control. †p<0.05 vs angiotensin II.</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

Effect of erythrodiol or uvaol on the proliferation and ERK 1/2 phosphorylation induced by angiotensin II.

<p>Cardiac myofibroblasts were stimulated with different doses of angiotensin II (Ang II; A) or with 1 µM of Ang II at different times (B). Cells in the absence or presence of different concentrations of a specific MEK inhibitor (PD98059; C and D), erythrodiol (ERY; E and G) or uvaol (UVA; F and H). After 24 h of incubation at 37°C (A, C, E and F), cell proliferation was determined by an MTT assay and expressed as percent of controls (vehicle, v). (B, D, G and H), after 15 min stimulation, whole cell lysates were extracted and protein phosphorylation was assessed by Western blotting using phospho-ERK and total ERK antibodies. Membranes always were stained with Ponceau S as a loading control. Representative immunoblots of 3 experiments. Values are mean ± SEM of three assays; *p<0.05 <i>vs</i> vehicle (V). †p<0.05 vs either 0 (absence of ERY or UVA) or Ang II.</p