Yen Ling Chen

Inhibitory effect of resveratrol on angiotensin II-induced cardiomyocyte hypertrophy

Resveratrol is proposed to account in part for the protective effect of red wine on the cardiovascular system. Angiotensin II (Ang II) is a potent hypertrophic stimulus in cardiomyocytes. In this study, we determined the effect of resveratrol on Ang II-induced cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were stimulated with Ang II, and [3H]leucine incorporation and β-myosin heavy chain (β-MyHC) promoter activity were examined. Intracellular reactive oxygen species (ROS) were measured by a redox-sensitive fluorescent dye, 2’ 7’-dichlorofluorescin diacetate, and the extracellular signal-regulated kinase (ERK) phosphorylation was examined by Western blotting. Resveratrol inhibited Ang II-increased intracellular ROS levels. Furthermore, resveratrol, as well as the antioxidant N-acetyl-cysteine, decreased Ang II- or H2O2-increased protein synthesis, β-MyHC promoter activity, and ERK phosphorylation. In summary, we demonstrate for the first time that resveratrol inhibits Ang II-induced cardiomyocyte hypertrophy via attenuation of ROS generation.

Uric acid activates extracellular signal-regulated kinases and thereafter endothelin-1 expression in rat cardiac fibroblasts

BACKGROUND The association between hyperuricemia and cardiovascular diseases has long been recognized. Elevated levels of uric acid may have a causal role in hypertension and cardiovascular diseases. However, the direct effect of uric acid on cardiac cells remains unclear. Therefore, this study was aimed to examine the effect of uric acid in rat cardiac fibroblasts and to identify the putative underlying signaling pathways. METHODS Cultured rat cardiac fibroblasts were stimulated with uric acid; cell proliferation and endothelin-1 (ET-1) gene expression were examined. The effect of uric acid on NADPH oxidase activity, reactive oxygen species (ROS) formation, and extracellular signal-regulated kinases (ERK) phosphorylation were tested to elucidate the intracellular mechanism of uric acid in ET-1 gene expression. RESULTS Uric acid-increased cell proliferation and ET-1 gene expression. Uric acid also increased NADPH oxidase activity, ROS formation, ERK phosphorylation, and activator protein-1 (AP-1)-mediated reporter activity. Antioxidants suppressed uric acid-induced ET-1 gene expression, and ERK phosphorylation, and AP-1 reporter activities. Mutational analysis of the ET-1 gene promoter showed that AP-1 binding site was an important cis-element in uric acid-induced ET-1 gene expression. CONCLUSIONS These results suggest that uric acid-induced ET-1 gene expression, partially by the activation of ERK pathway via ROS generation in cardiac fibroblasts.

Involvement of reactive oxygen species in urotensin II-induced proliferation of cardiac fibroblasts

Urotensin II, a cyclic dodecapeptide, has recently been demonstrated to play an important role in cardiac remodeling and fibrosis. Cardiac fibroblast is the cell type known to proliferate during cardiac fibrosis and to produce the excess matrix proteins characteristic of cardiac remodeling. However, the effect of urotensin II on cardiac fibroblast proliferation and the intracellular mechanisms remain to be clarified. Cultured neonatal rat cardiac fibroblasts were stimulated with urotensin II, cell proliferation and the reactive oxygen species generation were examined. We also examined the effects of antioxidant pretreatment on urotensin II-induced cell proliferation, extracellular signal-regulated kinase phosphorylation, and the tyrosine phosphorylation of epidermal growth factor receptor, to elucidate the redox-sensitive pathway in urotensin II-induced cell proliferation. Urotensin II-increased cell proliferation and intracellular reactive oxygen species levels which were inhibited by antioxidants N-acetylcysteine, and the flavin inhibitor diphenyleneiodonium. Urotensin II potently activated the tyrosine phosphorylation of epidermal growth factor receptors and extracellular signal-regulated kinase. Pretreatment of cells with U0126, an inhibitor of the upstream activator of mitogen-activated protein kinase kinase, or with AG1478, a selective epidermal growth factor receptor kinase inhibitor, reduced the urotensin II-increased extracellular signal-regulated kinase phosphorylation. Antioxidants, U0126, and AG1478, all significantly inhibited urotensin II-increased cell proliferation in cardiac fibroblasts. Our data suggest that the redox-sensitive intracellular signaling pathway plays a role in urotensin II-induced proliferation in rat cardiac fibroblasts.

Inhibition of angiotensin II induced endothelin-1 gene expression by 17-β-oestradiol in rat cardiac fibroblasts

Objective: To examine whether 17-β-oestradiol (E2) may alter angiotensin II (Ang II) induced cell proliferation and to identify the putative underlying signalling pathways in rat cardiac fibroblasts. Design: Cultured rat cardiac fibroblasts were preincubated with E2 then stimulated with Ang II. [3H]Thymidine incorporation and endothelin-1 (ET-1) gene expression were examined. The effect of E2 on Ang II induced NADPH oxidase activity, reactive oxygen species (ROS) formation, and extracellular signal regulated kinase (ERK) phosphorylation were tested to elucidate the intracellular mechanism of E2 in proliferation and ET-1 gene expression. Results: Ang II increased DNA synthesis, which was inhibited with E2 (1–100 nmol/l). E2, but not 17-α-oestradiol, inhibited Ang II induced ET-1 gene expression as shown by northern blotting and promoter activity assay. This effect was prevented by co-incubation with the oestrogen receptor antagonist ICI 182 780 (1 µmol/l). E2 also inhibited Ang II increased NADPH oxidase activity, ROS formation, ERK phosphorylation, and activator protein-1 mediated reporter activity. Conclusions: The results suggest that E2 inhibits Ang II induced cell proliferation and ET-1 gene expression, partially by interfering with the ERK pathway through attenuation of ROS generation. Thus, this study provides important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of oestrogen on the cardiovascular system.

The Inhibitory Effect of Trilinolein on Norepinephrine-Induced β-Myosin Heavy Chain Promoter Activity, Reactive Oxygen Species Generation, and Extracellular Signal-Regulated Kinase Phosphorylation in Neonatal Rat Cardiomyocytes

The myocardial protective effects of trilinolein, isolated from the traditional Chinese herb Sanchi (Panax notoginseng), are thought to be related to its antioxidant activity. However, the intracellular mechanism underlying the protective effect of trilinolein in the heart remains unclear. In the present study, we investigated the effect of trilinolein on norepinephrine (NE)-induced protein synthesis in cardiomyocytes. Cultured neonatal rat cardiomyocytes were stimulated with NE, then protein content, [3H]-leucine incorporation, and β-myosin heavy chain (β-MyHC) promoter activity were examined. The effect of trilinolein on NE-induced intracellular reactive oxygen species (ROS) generation was measured with a redox- sensitive fluorescent dye (2′,7′-dichlorofluorescin diacetate) and extracellular signal-regulated kinase (ERK) phosphorylation by Western blotting. Trilinolein inhibited NE-increased protein synthesis, β-MyHC promoter activity, and intracellular ROS. Both trilinolein and the antioxidant, N-acetyl-cysteine, decreased NE- and H2O2-induced protein synthesis, β-MyHC promoter activity, and ERK phosphorylation. These data indicate that trilinolein inhibits NE-induced protein synthesis via attenuation of ROS generation in cardiomyocytes.

Magnolol depresses urotensin-II-induced cell proliferation in rat cardiac fibroblasts.

1 Accumulating evidence suggests that oxidative stress plays a key role in the development of cardiac fibrosis. Urotensin‐II (U‐II) has been reported to play an important role in cardiac remodelling and fibrosis. Recently, we demonstrated the involvement of reactive oxygen species (ROS) production in U‐II‐induced cardiac fibroblast proliferation. Magnolol is an anti‐oxidant compound extracted from the cortices of Magnolia officinalis. Thus, it is feasible that magnolol may attenuate cardiac fibroblast proliferation by inhibiting ROS production. Therefore, the aims of the present study were to determine whether magnolol alters U‐II‐induced cell proliferation and to identify the putative underlying signalling pathways in rat cardiac fibroblasts. 2 Cultured rat cardiac fibroblasts were pretreated with magnolol (1, 3 and 10 µmol/L) for 30 min, followed by exposure to U‐II (30 nmol/L) for 24 h, after which cell proliferation and endothelin‐1 (ET‐1) protein secretion was examined. The effects of magnolol on U‐II‐induced ROS formation and extracellular signal‐regulated kinase (ERK) phosphorylation were examined to elucidate the intracellular mechanisms by which magnolol affects cell proliferation and ET‐1 expression. 3 Urotensin‐II (30 nmol/L) stimulated cell proliferation, ET‐1 protein secretion and ERK phosphorylation, all of which were inhibited by magnolol (10 µmol/L). Pretreatment of cardiac fibroblasts with N‐acetylcysteine (5 mmol/L) for 30 min prior to exposure to U‐II resulted in inhibition of U‐II increased ROS formation. Similar effects were observed with 10 µmol/L magnolol. 4 In conclusion, the results suggest that magnolol inhibits cardiac fibroblast proliferation by interfering with ROS generation. Thus, the present study provides important new insights into the molecular pathways involved, which may contribute to our understanding of the effects of magnolol on the cardiovascular system.

MECHANICAL STRETCH INDUCES ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE EXPRESSION IN NEONATAL RAT CARDIOMYOCYTES

1 Mechanical stretch leads to cardiac hypertrophy and may ultimately cause heart failure. However, the effect of mechanical stretch on gene induction in cardiomyocytes remains to be determined. 2 In the present study, we compared transcript profiles of mechanically stretched neonatal rat cardiomyocytes with those of unstretched cells using cDNA microarrays. The microarrays contained probes for 480 known genes, including those involved in signal transduction, cell cycle regulation, the cytoskeleton and cell motility. Eighteen genes, including the eNOS gene, were identified as having significantly differential expression in response to mechanical stretch in cardiomyocytes. 3 Northern and western blot analysis further quantified the expression of the eNOS gene. Mechanical stretch increased constitutive NOS activity and nitric oxide (NO) production. The NO donor s‐nitroso‐N‐acetylpenicillamine (SNAP) inhibited mechanical stretch‐stimulated protein synthesis, as measured by [3H]‐leucine uptake. In addition, cardiomyocytes were infected with adenoviral vectors encoding cDNA for eNOS (Ad‐eNOS) and a phosphoglycerate kinase (PGK) empty vector (Ad‐PGK). In contrast with Ad‐PGK‐infected cells, in cardiomyocytes infected with Ad‐eNOS, there was increased calcium‐dependent NOS activity and nitrite production. Cardiomyocytes infected with Ad‐eNOS exhibited diminished mechanical stretch‐stimulated protein synthesis. In contrast, in eNOS‐knockdown cells, the increased eNOS protein levels and NOS activity induced by mechanical stretch were abolished, but protein synthesis was enhanced. 4 The results of the present study indicate that eNOS gene expression is induced by mechanical stretch, leading to increased constitutive NOS activity and NO production, which may be a negative regulator in cardiomyocyte hypertrophy.