Tzu-Hurng Cheng

Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes

OBJECTIVESnRecent evidence indicates that reactive oxygen species (ROS) may act as second messengers in receptor-mediated signaling pathways. The possible role of ROS during Et-1 stimulation in cardiomyocytes was therefore investigated.nnnMETHODSnIntracellular ROS levels were measured with fluorescence probe 2,7-dichlorofluorescin diacetate by confocal microscopy in cultured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was analyzed by Northern blotting and promoter activity.nnnRESULTSnEt-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The increase of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomyocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) reduced Et-1-induced ROS levels. Et-1 or H2O2 treatment of cardiomyocytes rapidly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (-2.25 kb) and reporter gene chloramphenicol acetyltransferase. The induction of mRNA levels and the promoter activities of c-fos gene by Et-1 or H2O2 were abolished by pretreating cardiomyocytes with catalase or NAC. Cells transiently transfected with the dominant positive mutant of p21ras (RasL61) led to a significant increase in intracellular ROS. Concomitantly, the mRNA levels and the promoter activities of c-fos were also induced. In contrast, cells transfected with the dominant negative mutant of Ras (RasN17) inhibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels and promoter activities by Et-1 were also inhibited.nnnCONCLUSIONSnThese findings clearly indicate that Et-1 treatment to cardiomyocytes can induce ROS via Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second messengers in Et-1-induced responses on cardiomyocytes.

Role of Reactive Oxygen Species-Sensitive Extracellular Signal-Regulated Kinase Pathway in Angiotensin II-Induced Endothelin-1 Gene Expression in Vascular Endothelial Cells

Background: Circulating angiotensin II (Ang II) increases vascular endothelin-1 (ET-1) tissue levels, which in turn mediate a major part of Ang II-stimulated vascular growth and hypertension in vivo. Ang II also stimulates the generation of reactive oxygen species (ROS) within vascular endothelial cells. However, whether ROS are involved in Ang II-induced ET-1 gene expression, and the related intracellular mechanisms occurring within vascular endothelial cells remain unclear. Methods: Cultured endothelial cells were stimulated with Ang II, and the thus elicited ET-1 gene expression was examined by Northern blotting and a promoter activity assay. Antioxidant pretreatment of endothelial cells was performed prior to Ang II-induced extracellular signal-regulated kinase (ERK) phosphorylation in order to elucidate the redox-sensitive pathway for ET-1 gene expression. Results: The ET-1 gene was induced with Ang II, which was inhibited with Ang II type 1 receptor antagonist (irbesartan). Ang II-enhanced intracellular ROS levels were inhibited by irbesartan and several antioxidants, and antioxidants also suppressed Ang II-induced ET-1 gene expression. Further, Ang II-activated ERK phosphorylation was also significantly inhibited by certain antioxidants. An ERK inhibitor, U0126, inhibited Ang II-induced ET-1 expression completely. Cotransfection of the dominant negative mutant of Ras, Raf and MEK1 (ERK kinase) attenuated the Ang II-enhanced ET-1 promoter activity, suggesting that the Ras/Raf/ERK pathway is required for Ang II-induced ET-1 gene expression. Ang II-induced activator protein-1 (AP-1) reporter activities were inhibited by antioxidants. Moreover, mutational analysis of the ET-1 gene promoter showed that the AP-1 binding site was an important cis element in Ang II-induced ET-1 gene expression. Conclusions: Our data suggest that ROS are involved in Ang II-induced ET-1 gene expression within endothelial cells. The redox-sensitive ERK-mediated AP-1 transcriptional pathway plays an important role in Ang II-induced ET-1 gene expression.

Tetramethylpyrazine Inhibits Angiotensin II-Increased NAD(P)H Oxidase Activity and Subsequent Proliferation in Rat Aortic Smooth Muscle Cells

Tetramethylpyrazine (TMP) is the major component extracted from the Chinese herb, Chuanxiong, which is widely used in China for the treatment of cardiovascular problems. The aims of this study were to examine whether TMP may alter angiotenisn II (Ang II)-induced proliferation and to identify the putative underlying signaling pathways in rat aortic smooth muscle cells. Cultured rat aortic smooth muscle cells were preincubated with TMP and then stimulated with Ang II, [3H]-thymidine incorporation and the ET-1 expression was examined. Ang II increased DNA synthesis which was inhibited by TMP (1-100 microM). TMP inhibited the Ang II-induced ET-1 mRNA levels and ET-1 secretion. TMP also inhibited Ang II-increased NAD(P)H oxidase activity, intracellular reactive oxygen species (ROS) levels, and the ERK phosphorylation. Furthermore, TMP and antioxidants such as Trolox and diphenylene iodonium decreased Ang II-induced ERK phosphorylation, and activator protein-1 reporter activity. In summary, we demonstrate for the first time that TMP inhibits Ang II-induced proliferation and ET-1, partially by interfering with the ERK pathway via attenuation of Ang II-increased NAD(P)H oxidase and ROS generation. Thus, this study delivers important new insight in the molecular pathways that may contribute to the proposed beneficial effects of TMP in cardiovascular disease.

Intracellular Acid-extruding regulators and the effect of lipopolysaccharide in cultured human renal artery smooth muscle cells.

Homeostasis of the intracellular pH (pHi) in mammalian cells plays a pivotal role in maintaining cell function. Thus far, the housekeeping Na+-H+ exchanger (NHE) and the Na+-HCO3 − co-transporter (NBC) have been confirmed in many mammalian cells as major acid extruders. However, the role of acid-extruding regulators in human renal artery smooth muscle cells (HRASMCs) remains unclear. It has been demonstrated that lipopolysaccharide (LPS)-induced vascular occlusion is associated with the apoptosis, activating calpain and increased [Ca2+]i that are related to NHE1 activity in endothelia cells. This study determines the acid-extruding mechanisms and the effect of LPS on the resting pHi and active acid extruders in cultured HRASMCs. The mechanism of pHi recovery from intracellular acidosis (induced by NH4Cl-prepulse) is determined using BCECF-fluorescence in cultured HRASMCs. It is seen that (a) the resting pHi is 7.19±0.03 and 7.10±0.02 for HEPES- and CO2/HCO3 −- buffered solution, respectively; (b) apart from the housekeeping NHE1, another Na+-coupled HCO3 − transporter i.e. NBC, functionally co-exists to achieve acid-equivalent extrusion; (c) three different isoforms of NBC: NBCn1 (SLC4A7; electroneutral), NBCe1 (SLC4A4; electrogenic) and NBCe2 (SLC4A5), are detected in protein/mRNA level; and (d) pHi and NHE protein expression/activity are significantly increased by LPS, in both a dose- and time- dependent manner, but NBCs protein expression is not. In conclusion, it is demonstrated, for the first time, that four pHi acid-extruding regulators: NHE1, NBCn1, NBCe1 and NBCe2, co-exist in cultured HRASMCs. LPS also increases cellular growth, pHi and NHE in a dose- and time-dependent manner.

Functional characterization of intracellular pH regulators responsible for acid extrusion in human radial artery smooth muscle cells.

Intracellular pH (pHi) is a critical factor influencing many important cellular functions. Acid extrusion carriers such as an Na⁺/H⁺ exchanger (NHE) Na⁺/HCO₃⁻ cotransporter (NBC) and monocarboxylate transporters (MCT) can be activated when cells are in an acidic condition (pHi < 7.1). Human radial artery smooth muscle cells (HRASMC) is an important conduit in coronary artery bypass graft surgery. However, such far, the pHi regulators have not been characterized in HRASMCs. We therefore investigated the mechanism of pHi recovery from intracellular acidosis and alkalosis, induced by NH₄Cl-prepulse and Na-acetate-prepulse, respectively, using intracellular 2,7-bis(2-carboxethyl)-5(6)- carboxy-fluorescein (BCECF)-fluorescence in HRASMCs. Cultured HRASMCs were derived from the segments of human radial artery that were obtained from patients undergoing bypass grafting. The resting pHi is 7.22 ± 0.03 and 7.17 ± 0.02 for HEPES- (nominally HCO₃⁻-free) and CO₂/HCO₃⁻- buffered solution, respectively. In HEPES-buffered solution, a pHi recovery from induced intracellular acidosis could be blocked completely by 30 μM HOE 694 (3-methylsulfonyl-4-piperidinobenzoyl, guanidine hydrochloride) a specific NHE inhibitor, or by removing [Na⁺]₀. In 3% CO₂/HCO₃⁻-buffered solution, HOE 694 slowed the pHi recovery from the induced intracellular acidosis only, while adding together with DIDS (a specific NBC inhibitor) or removal of [Na⁺]₀ entirely inhibited the acid extrusion. Moreover, α-cyano-4-hydroxycinnamate (CHC; a specific blocker of MCT) blocked the lactate-induced pHi changes. In conclusion, we demonstrate, for the first time, that 3 different pHi regulators responsible for acid extruding, i.e. NHE and NBC, and MCT, are functionally co-existed in cultured HRASMCs.

945 TANSHINONE IIA REDUCES UROTENSIN II SIGNALING AND COMPROMISED MIGRATION IN CULTURED VASCULAR SMOOTH MUSCLE CELLS

Background: Urotensin II (UII) is a potent stimulator of growth and motility of vascular smooth muscle cells (VSMCs). Tanshinone IIA is the main effective component of Salvia miltiorrhiza known as ’Danshen’ in traditional Chinese medicine for treating cardiovascular disorders, but the mechanism by which it exerts the protective effect is not well established. In this study we examined the effect of tanshinone IIA on UII signaling and migration in VSMCs. Design and methods: VSMCs were treated with tanshinone IIA in the presence/absence of UII. Western blotting was performed to examine UII signaling. Flowcytometry was used to examine intracellular reactive oxygen species (ROS) production. Modified Boyden chamber system (Transwell apparatus) was used for migration assay. Results: Tanshinone IIA reduced UII signaling, including phosphorylation of epidermal growth factor receptor (EGFR) and extracelluar signal-regulated kinases (ERK). Tanshinone IIA also reduced UII-induced intracellular reactive oxygen species (ROS) production and attenuated ROS- (H2O2-) induced ERK activation. A further analysis indicated tanshinone IIA could inhibit UII-induced EGFR signaling, which is known to act through an oxidative inhibition of protein tyrosine phosphatase. Finally, we showed that tanshinone IIA inhibited UII-induced VSMC migration. Conclusions: Our study shows that tanshinone IIA can reduce UII signaling and migration in VSMCs, suggesting a possible beneficial effect of tanshinone IIA on prevention of vascular diseases.

90 TANSHINONE IIA ATTENUATES DOXORUBICIN-INDUCED INJURY IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

Background: The injury of endothelial cell is the critical event of vascular disease. Tanshinone IIA is the main effective component of Salvia miltiorrhiza known as ’Danshen’ in traditional Chinese medicine for treating cardiovascular disorders, but the mechanism by which it exerts the protective effect is not well established. The present study was designed to test the hypothesis that tanshinone IIA can inhibit doxorubicin-induced injury and unravel its intracellular mechanism in human umbilical vein endothelial cells (HUVECs). Design and methods: HUVECs were treated with tanshinone IIA in the presence/absence of doxorubicin. The protective effects of tanshinone IIA against doxorubicin were evaluated. Results: HUVECs incubated with doxorubicin had significantly decreased the viability of endothelial cells, which was accompanied with apparent cells apoptosis, the activation of caspase-3 and the upregulation of p53 expression, which was known to play a key role in doxorubicin-induced cell apoptosis. However, pretreatment with tanshinone IIA (3-10 &mgr;M) resulted in a significant recovery from doxorubicin-induced cell apoptosis. Furthermore, pretreatment with tanshinone IIA decreased the activity of caspase-3 and p53 expression. Tanshinone IIA also induced activating transcription factor (ATF) 3 expression; while knockdown of ATF-3 with ATF-3 siRNA significantly reduced tanshinone IIAs protective effect. Conclusions: The present study shows that tanshinone IIA can protect endothelial cells against oxidative injury induced by doxorubicin, suggesting that this compound may constitute a promising intervention against cardiovascular disorders and ATF-3 may play an important role in this process.

M-011 TANSHINONE IIA ATTENUATES H2O2-INDUCED INJURY IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS

Background The injury of endothelial cell is the critical event of hypertension. In endothelial cell, oxidative stress is regarded as critical pathogenic factors in endothelial cell injury and apoptosis. Tanshinone IIA is the main effective component of Salvia miltiorrhiza known as ’Danshen’ in traditional Chinese medicine for treating cardiovascular disorders, but the mechanism by which it exerts the protective effect is not well established. The present study was designed to test the hypothesis whether tanshinone IIA can inhibit H2O2-induced injury in human umbilical vein endothelial cells. Methods Human umbilical vein endothelial cells (HUVECs) were treated with tanshinone IIA in the presence/absence of hydrogen peroxide (H2O2). The protective effects of tanshinone IIA against H2O2 were evaluated. Results HUVECs incubated with 200u200a&mgr;M H2O2 had significantly decreased the viability of endothelial cells, which was accompanied with apparent cells apoptosis, the activation of caspase-3 and the upregulation of p53 expression. However, pretreatment with tanshinone IIA(3–10u200a&mgr;M) resulted in a significant recovery from H2O2-induced cell apoptosis. Furthermore, pretreatment with tanshinone IIA decreased the activity of caspase-3 and p53 expression, which was known to play a key role in H2O2-induced cell apoptosis. Tanshinone IIA also induced activating transcription factor (ATF) 3 at both mRNA and protein level; while knockdown of ATF3 with ATF3 siRNA significantly reduced ATF3 protein levels and tanshinone IIAs protective effect. Conclusions The present study shows that tanshinone IIA can protect endothelial cells against oxidative injury induced by H2O2, suggesting that this compound may constitute a promising intervention against cardiovascular disorders and ATF3 may play an important role in this process.

P-260 Urotensin II-Induced Cell Proliferation Via Epidermal Growth Factor Receptor Transactivation in Rat Aortic Smooth Muscle Cells

signal-regulated protein kinase (ERK), endothelial nitric oxide synthase (eNOS), and Akt were assessed by Western blot analysis. Results: We show that nicorandil inhibits strain-induced ET-1 expression. Nicorandil also inhibits strain-increased reactive oxygen species (ROS) formation and ERK phosphorylation. On the contrary, NO production, eNOS activity, and Akt phosphorylation were enhanced by nicorandil treatment in HUVECs. Furthermore, L-NAME, an inhibitor of NO synthase, and the siRNA transfection for Akt or eNOS markedly attenuated the inhibitory effect of nicorandil on straininduced ET-1 expression. Conclusions: In summary, we demonstrate for the first time that nicorandil inhibits strain-induced ET-1 secretion and enhances strain-increased NO production in HUVECs. Thus, this study delivers important new insight in the molecular pathways that may contribute to the proposed hypotensive effects of nicorandil in the cardiovascular system.