Jye Hong

Antiproliferative Effect of Isosteviol on Angiotensin-II-Treated Rat Aortic Smooth Muscle Cells

Isosteviol is a derivative of stevioside, a constituent of Stevia rebaudiana, which is commonly used as a noncaloric sugar substitute in Japan and Brazil. The aims of this study were to examine whether isosteviol alters angiotensin-II-induced cell proliferation in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with isosteviol, then stimulated with angiotensin II, after which [3H]thymidine incorporation and endothelin-1 secretion were examined. Isosteviol (1–100 µmol/l) inhibits angiotensin-II-induced DNA synthesis and endothelin-1 secretion. Measurements of 2′7′-dichlorofluorescin diacetate, a redox-sensitive fluorescent dye, showed an isosteviol-mediated inhibition of intracellular reactive oxygen species generated by the effects of angiotensin II. The inductive properties of angiotensin II on extracellular signal-regulated kinase (ERK) phosphorylation were found reversed with isosteviol and antioxidants such as N-acetylcysteine. In summary, we speculate that isosteviol inhibits angiotensin-II-induced cell proliferation and endothelin-1 secretion via attenuation of reactive oxygen species generation. Thus, this study provides important insights that may contribute to the effects of isosteviol on the cardiovascular system.

17β-Estradiol Downregulates Angiotensin-II-Induced Endothelin-1 Gene Expression in Rat Aortic Smooth Muscle Cells

It is well documented that 17β-estradiol (E2) exerts a cardiovascular protective effect. A possible role of E2 in the regulation of endothelin-1 (ET-1) production has been reported. However, the complex mechanisms by which E2 inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E2 may alter angiotensin II (Ang II)-induced cell proliferation and ET-1 gene expression and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with E2, then stimulated with Ang II, and [3H]thymidine incorporation and ET-1 gene expression were examined. The effect of E2 on Ang-II-induced extracellular signal-regulated kinase (ERK) phosphorylation was tested to elucidate the intracellular mechanism of E2 in proliferation and ET-1 gene expression. Ang II increased DNA synthesis which was inhibited with E2 (1– 100 nM). E2, but not 17α-estradiol, inhibited the Ang-II-induced ET-1 gene expression as revealed by Northern blotting and promoter activity assay. This effect was prevented by coincubation with the estrogen receptor antagonist ICI 182,780 (1 µM). E2 also inhibited Ang-II-increased intracellular reactive oxygen species (ROS) as measured by a redox-sensitive fluorescent dye, 2′,7′-dichlorofluorescin diacetate, and ERK phosphorylation. Furthermore, E2 and antioxidants, such as N-acetyl cysteine and diphenylene iodonium, decreased Ang-II-induced cell proliferation, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1-mediated reporter activity. In summary, our results suggest that E2 inhibits Ang-II-induced cell proliferation and ET-1 gene expression, partially by interfering with the ERK pathway via attenuation of ROS generation. Thus, this study provides important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system.

Tanshinone IIA Inhibits Angiotensin II-Induced Cell Proliferation in Rat Cardiac Fibroblasts

Tanshinone IIA extracted from danshen, a popular medicinal herb used in traditional Chinese medicine, exhibits cardio-protective effects. However, the mechanism of its cardioprotective effect is not well established. The aims of this study were to examine whether tanshinone IIA may alter angiotensin II (Ang II)-induced cell proliferation and to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Cultured rat cardiac fibroblasts were pre-treated with tanshinone IIA and stimulated with Ang II, cell proliferation and endothelin-1 (ET-1) expression were examined. The effect of tanshinone IIA on Ang II-induced reactive oxygen species (ROS) formation, and extracellular signal-regulated kinase (ERK) phosphorylation were also examined. In addition, the effect of tanshinone IIA on nitric oxide (NO) production, and endothelial nitric oxide synthase (eNOS) phosphorylation were tested to elucidate the intracellular mechanism. The increased cell proliferation and ET-1 expression by Ang II (100 nM) were partially inhibited by tanshinone IIA. Tanshinone IIA also inhibited Ang II-increased ROS formation, and ERK phosphorylation. In addition, tanshinone IIA was found to increase the NO generation, and eNOS phosphorylation. N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, and the short interfering RNA transfection for eNOS markedly attenuated the inhibitory effect of tanshinone IIA on Ang II-induced cell proliferation. The results suggest that tanshinone IIA prevents cardiac fibroblast proliferation by interfering with the generation of ROS and involves the activation of the eNOS-NO pathway.

Nicorandil inhibits angiotensin-II-induced proliferation of cultured rat cardiac fibroblasts

Background/Aims: Nicorandil, an ATP-sensitive potassium (KATP) channel opener, nitric oxide (NO) donor and antioxidant, was shown to exert a variety of pharmacological effects including cardioprotective properties. However, its mechanisms of action are not completely understood. The aims of this study were to examine whether nicorandil may alter angiotensin-II (Ang II)-induced cell proliferation and to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Methods: Cultured rat cardiac fibroblasts were pretreated with nicorandil, then stimulated with Ang II, and cell proliferation and endothelin-1 (ET-1) expression were examined. The effects of nicorandil on Ang-II-induced reactive oxygen species (ROS) formation and extracellular signal-regulated kinase (ERK) phosphorylation were also examined. In addition, the effects of nicorandil on NO production and endothelial nitric oxide synthase (eNOS) phosphorylation were tested to elucidate the intracellular mechanism. Results: Nicorandil (0.1–10 µmol/l) caused a concentration-dependent inhibition of Ang-II-increased cell proliferation and ET-1 expression which were prevented by the KATP channel blocker glibenclamide (1 µmol/l). Nicorandil also inhibited Ang-II-increased ROS and ERK phosphorylation. In addition, nicorandil was found to increase the NO and eNOS phosphorylation. N-nitro-L-arginine methyl ester, an inhibitor of NOS, and the short interfering RNA transfection for eNOS markedly attenuated the inhibitory effect of nicorandil on Ang-II-induced cell proliferation. Conclusion: Our results suggest that nicorandil prevents cardiac fibroblast proliferation, and the inhibitory effect might be associated with the opening KATP channels, by interfering with the generation of ROS, and the activation of the eNOS-NO pathway.

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.

Lycopene inhibits cyclic strain‐induced endothelin‐1 expression through the suppression of reactive oxygen species generation and induction of heme oxygenase‐1 in human umbilical vein endothelial cells

Lycopene is the most potent active antioxidant among the major carotenoids, and its use has been associated with a reduced risk for cardiovascular disease (CVD). Endothelin‐1 (ET‐1) is a powerful vasopressor synthesized by endothelial cells and plays a crucial role in the pathophysiology of CVD. However, the direct effects of lycopene on vascular endothelial cells have not been fully described. This study investigated the effects of lycopene on cyclic strain‐induced ET‐1 gene expression in human umbilical vein endothelial cells (HUVECs) and identified the signal transduction pathways that are involved in this process. Cultured HUVECs were exposed to cyclic strain (20% in length, 1 Hz) in the presence or absence of lycopene. Lycopene inhibited strain‐induced ET‐1 expression through the suppression of reactive oxygen species (ROS) generation through attenuation of p22phox mRNA expression and NAD(P)H oxidase activity. Furthermore, lycopene inhibited strain‐induced ET‐1 secretion by reducing ROS‐mediated extrace‐llular signal‐regulated kinase (ERK) phosphorylation. Conversely, lycopene treatment enhanced heme oxygenase‐1 (HO‐1) gene expression through the activation of phosphoinositide 3‐kinase (PI3K)/Akt pathway, followed by induction of the nuclear factor erythroid 2‐related factor 2 (Nrf2) nuclear translocation; in addition, HO‐1 silencing partially inhibited the repressive effects of lycopene on strain‐induced ET‐1 expression. In summary, our study showed, for the first time, that lycopene inhibits cyclic strain‐induced ET‐1 gene expression through the suppression of ROS generation and induction of HO‐1 in HUVECs. Therefore, this study provides new valuable insight into the molecular pathways that may contribute to the proposed beneficial effects of lycopene on the cardiovascular system.

Tanshinone IIA attenuates cyclic strain-induced endothelin-1 expression in human umbilical vein endothelial cells

1. Tanshinone IIA, one of the active components of the Radix of Salvia miltiorrhiza, is used in traditional Chinese medicine to treat cardiovascular diseases. However, the intracellular mechanism of action of tanshinone IIA remain to be determined. The aims of the present study were to test the hypothesis that tanshinone IIA alters strain‐induced endothelin (ET)‐1 expression and nitric oxide (NO) production, as well as to identify the putative signalling pathways involved, in human umbilical vein endothelial cells (HUVEC).

Nicorandil attenuates cyclic strain-induced endothelin-1 expression via the induction of activating transcription factor 3 in human umbilical vein endothelial cells.

Nicorandil is an adenosine triphosphate-sensitive potassium channel opener that combines an organic nitrate and a nicotinamide group which respectively confer to nicorandil the additional properties of being a nitric oxide (NO) donor and antioxidant; it also induces vasodilation, decreases the blood pressure, and protects the heart. However, the intracellular mechanism of nicorandil remains to be delineated. The aims of this study were to test the hypothesis that nicorandil alters strain-induced endothelin-1 secretion and NO production, and to identify the putative underlying signaling pathways in human umbilical vein endothelial cells (HUVECs). Cultured HUVECs were exposed to cyclic strain in the presence of nicorandil; endothelin-1 expression was examined by reverse-transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay. Activation of extracellular signal-regulated protein kinase (ERK), endothelial NO synthase (eNOS), and activating transcription factor (ATF)-3 was assessed by Western blot analysis. We show that nicorandil inhibited strain-induced endothelin-1 expression. Nicorandil also inhibited strain-increased reactive oxygen species formation and ERK phosphorylation. On the contrary, NO production, eNOS phosphorylation, and ATF3 expression were enhanced by nicorandil; however, L-NAME (an inhibitor of eNOS) and LY294002 (an inhibitor of phosphatidylinositol 3-kinase) inhibited nicorandil-increased ATF3 expression. Moreover, treatment of HUVECs with either an NO donor (NOC18; 3,3-bis[aminoethyl]-1-hydroxy-2-oxo-1-triazene) or an ATF3 activator (MG-132; carbobenzoxy-L-leucyl-L-leucyl-L-leucinal) resulted in repression of strain-induced endothelin-1 expression. Furthermore, L-NAME, and small interfering RNA transfection of eNOS also partially attenuated the inhibitory effect of nicorandil on strain-induced endothelin-1 expression. We demonstrate for the first time that nicorandil inhibits strain-induced endothelin-1 secretion via an increase in NO and upregulation of ATF3 in HUVECs. This study provides important new insights into the molecular pathways that may contribute to the beneficial effects of nicorandil in the cardiovascular system.

Nicorandil Inhibits Cyclic Strain-Induced Interleukin-8 Expression in Human Umbilical Vein Endothelial Cells

Background: Nicorandil, a mitochondrial adenosine triphosphate-sensitive potassium (mitoKATP) channel opener, exerts protective effects on the cardiovascular system. This study examined the effect of nicorandil on cyclic strain-induced interleukin-8 (IL-8) expression in human umbilical vein endothelial cells (HUVECs). Methods: Cultured HUVECs were exposed to cyclic strain in the presence or absence of nicorandil (1-10 μmol/l); we then analyzed IL-8 expression. We also assessed the effects of nicorandil on heme oxygenase-1 (HO-1) expression and cyclic strain-modulated IL-8 expression after HO-1 silencing in HUVECs. Summary: HUVECs exposed to cyclic strain showed increased IL-8 messenger RNA expression and protein secretion. Nicorandil (1-10 μmol/l) inhibited cyclic strain-induced IL-8 expression, whereas 5-hydroxydecanoate (100 μmol/l), a selective inhibitor of the mitoKATP channel, completely reversed the inhibitory effects of nicorandil on cyclic strain-induced IL-8 expression. We demonstrated that nicorandil increased HO-1 expression in HUVECs. In addition, cobalt protoporphyrin (10 μmol/l), an inducer of HO-1 expression, mimicked the effects of nicorandil and inhibited IL-8 expression under cyclic strain, whereas zinc protoporphyrin IX (10 μmol/l), an inhibitor of HO-1 expression, antagonized the effect of nicorandil. HO-1 silencing significantly abrogated the inhibitory effects of nicorandil on cyclic strain-induced IL-8 expression, suggesting that HO-1 plays a role in the mechanism of action of nicorandil. Key Messages: This study is the first to report that nicorandil inhibits cyclic strain-induced IL-8 expression through the induction of HO-1 expression in HUVECs. This finding provides valuable new insight into the molecular pathways contributing to the vasoprotective effects of nicorandil.