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ABSTRACT: We tested the hypothesis that HO-1 (heme oxygenase-1) activity varied between vascular smooth muscle cells (VSMC) in spontaneously
hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. HO-1 levels were measured under baseline and hemin-stimulated
conditions and cell proliferation was monitored. Basal HO-1 levels in untreated cells were lower in SHR compared to WKY rats.
Treatment with hemin increased HO-1 mRNA and protein levels in the cells obtained from WKY rats compared to that of SHR rats.
However, hemin-treatment showed a greater inhibitory effect on VSMC proliferation in SHR rats than in WKY rats. Tin protoporphyrin
IX (SnPPIX) showed a greater reversal of the anti-proliferative effect of hemin on cells from SHR rats than WKY. Similarly,
VSMC proliferation from SHR was significantly inhibited in VSMC transfected with the HO-1 gene. These inhibitory effects were associated with cell cycle arrest in the G1 phase. The level of cyclin D, and cyclin
dependent kinase inhibitor p21 was higher in SHR cells progressing through the G1 phase. Treatment of the cells with hemin
down-regulated the expression of cyclin D and up-regulated that of p21. These results indicate that hemin, an HO-1 inducer,
may play a more critical role in VSMC proliferation in SHR than WKY.
Archives of Pharmacal Research 04/2012; 32(3):375-382. · 1.59 Impact Factor
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ABSTRACT: The aim of the present study is to determine the effects and molecular mechanisms by which activation of LKB1-AMP-activated protein kinase (AMPK) by metformin regulates vascular smooth muscle contraction. The essential ability of vascular smooth muscle cells (VSMCs) to contract and relax in response to an elevation and reduction in intravascular pressure is necessary for appropriate blood flow regulation. Thus, vessel contraction is a critical mechanism for systemic blood flow regulation. In cultured rat VSMCs, AMPK activation through LKB1 by metformin-inhibited phenylephrine-mediated myosin light chain kinase (MLCK) and myosin light chain phosphorylation (p-MLC). Conversely, inhibition of AMPK and LKB1 reversed phenylephrine-induced MLCK and p-MLC phosphorylation. Measurement of the tension trace in rat aortic rings also showed that the effect of AMPK activation by metformin decreased phenylephrine-induced contraction. Metformin inhibited PE-induced p-MLC and α-smooth muscle actin co-localization. Our results suggest that activation of AMPK by LKB1 decreases VSMC contraction by inhibiting MLCK and p-MLC, indicating that induction by the AMPK-LKB1 pathway may be a new therapeutic target to lower high blood pressure.
Biochemical and Biophysical Research Communications 04/2012; 421(3):599-604. · 2.48 Impact Factor
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ABSTRACT: Vascular cells have a limited lifespan with limited cell proliferation and undergo cellular senescence. The functional changes associated with cellular senescence are thought to contribute to age-related vascular disorders. AMP-activated protein kinase (AMPK) has been discussed in terms of beneficial or harmful effects for aging-related diseases. However, the detailed functional mechanisms of AMPK are largely unclear. An aging model was established by stimulating vascular smooth muscle cell (VSMC) with adriamycin. Adriamycin progressively increased the mRNA and protein expressions of AMPK. The phosphorylation levels of LKB1 and acetyl-CoA carboxylase (ACC), the upstream and downstream of AMPK, were dramatically increased by adriamycin stimulation. The expressions of p53 and p21, which contribute to vascular senescence, were also increased. Inhibition of AMPK diminished senescence-associated β-galactosidase (SA-β-gal) staining, and restored VSMC proliferation. Cytosolic translocation of LKB1 by adriamycin could be a mechanism for AMPK activation in senescence. Furthermore, p53 siRNA and p21 siRNA transfection attenuated adriamycin-induced SA-β-gal staining. These results suggest that LKB1 dependent AMPK activation elicits VSMC senescence and p53-p21 pathway is a mediator of LKB1/AMPK-induced senescence.
Biochemical and Biophysical Research Communications 08/2011; 413(1):143-8. · 2.48 Impact Factor
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ABSTRACT: Cilostazol is a selective inhibitor of phosphodiesterase 3 that increases intracellular cAMP levels and activates protein kinase A, thereby inhibiting vascular smooth muscle cell (VSMC) proliferation. We investigated whether AMP-activated protein kinase (AMPK) activation induced by heme oxygenase-1 (HO-1) is a mediator of the beneficial effects of cilostazol and whether cilostazol may prevent cell proliferation and reactive oxygen species (ROS) production by activating AMPK in VSMC. In the present study, we investigated VSMC with various concentrations of cilostazol. Treatment with cilostazol increased HO-1 expression and phosphorylation of AMPK in a dose- and time-dependent manner. Cilostazol also significantly decreased platelet-derived growth factor (PDGF)-induced VSMC proliferation and ROS production by activating AMPK induced by HO-1. Pharmacological and genetic inhibition of HO-1 and AMPK blocked the cilostazol-induced inhibition of cell proliferation and ROS production.These data suggest that cilostazol-induced HO-1 expression and AMPK activation might attenuate PDGF-induced VSMC proliferation and ROS production.
Korean Journal of Physiology and Pharmacology 08/2011; 15(4):203-10. · 0.96 Impact Factor
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ABSTRACT: The dihydropyridine calcium channel blocker nifedipine induces specific pharmacological effects by binding to L-type calcium channels, which results in a reduced calcium influx in vascular smooth muscle cells (VSMCs) and is currently employed in antihypertensive drug. Dihydropyridine calcium channel blocker is reported to reduce oxidative stress and exhibits anti-proliferative effect in VSMCs. VSMCs are useful in the study of atherosclerosis because they show cell proliferation and reactive oxygen species (ROS) production with growth factor. To determine the mechanisms involved in these effects, we investigated the influence of nifedipine-induced AMP-activated protein kinase (AMPK) activation on VSMC proliferation and ROS production by using rat aortic VSMCs in vitro and in vivo. Nifedipine induced phosphorylation of AMPK in a dose-and time-dependent manner, and inhibited rat VSMC proliferation and ROS production following stimulation with 15% fetal bovine serum (FBS). Nifedipine also blocked the FBS-stimulated cell cycle progression through the G0/G1 arrest. Compound C, a specific inhibitor of AMPK, or AMPK siRNA reduced the nifedipine-mediated inhibition of VSMC proliferation. As an upstream kinase, LKB1 is required for nifedipine-induced AMPK activation in VSMCs. 7 days oral administration of 1 mg/kg nifedipine resulted in activation of LKB1 and AMPK in vivo. These data suggest that nifedipine suppress the VSMC proliferation and ROS production via activating LKB1-AMPK pathway.
Vascular Pharmacology 06/2011; 56(1-2):1-8. · 1.99 Impact Factor
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ABSTRACT: Acetylsalicylic acid (aspirin), used to reduce risk of cardiovascular disease, plays an important role in the regulation of cellular proliferation. However, mechanisms responsible for aspirin-induced growth inhibition are not fully understood. Here, we investigated whether aspirin may exert therapeutic effects via AMP-activated protein kinase (AMPK) activation in vascular smooth muscle cells (VSMC) from wistar kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Aspirin increased AMPK and acetyl-CoA carboxylase phosphorylation in a time- and dose-dependent manner in VSMCs from WKY and SHR, but with greater efficacy in SHR. In SHR, a low basal phosphorylation status of AMPK resulted in increased VSMC proliferation and aspirin-induced AMPK phosphorylation inhibited proliferation of VSMCs. Compound C, an AMPK inhibitor, and AMPK siRNA reduced the aspirin-mediated inhibition of VSMC proliferation, this effect was more pronounced in SHR than in WKY. In VSMCs from SHR, aspirin increased p53 and p21 expression and inhibited the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. These results indicate that in SHR VSMCs aspirin exerts anti-proliferative effects through the induction of AMPK phosphorylation.
Biochemical and Biophysical Research Communications 05/2011; 408(2):312-7. · 2.48 Impact Factor
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ABSTRACT: Reparixin, an inhibitor of CXCL8 receptor CXCR1 and CXCR2 activation, has been shown to attenuate inflammatory responses in various injury models. In the present study, the hypertension-related functional roles of reparixin were examined in hypertensive animals. Spontaneously hypertensive rats (SHR) at the age of 18 weeks were administered a subcutaneous injection of reparixin (5 mg/kg) daily for 3 weeks (SHR-R, n=5). Control groups consisted of normal saline-treated SHR (SHR-N, n=5) and normotensive Wistar-Kyoto rats (WKY-N, n=5). Reparixin effectively decreased systolic blood pressure and increased the blood flow. The thoracic aorta wall thickness was significantly decreased in SHR-R compared to SHR-N. Expressions of CXCL8, CCL2, 12-lipoxygenase (LO) and endothelin (ET)-1 were significantly decreased in SHR-R thoracic aorta tissues compared to SHR-N. Furthermore, expression of angiotensin II subtype I receptor (AT(1)R) protein was decreased in SHR-R thoracic aorta tissues compared to SHR-N. In addition, the plasma levels of nitric oxide were slightly elevated in SHR-R compared to the levels in SHR-N. These findings indicate that inhibition of hypertension-related mediators by reparixin results in the reduction of blood pressure in SHR. Therefore, these results suggest that reparixin-mediated blockade of CXCL8 receptor activation attenuates vascular hypertension in SHR.
Biological & Pharmaceutical Bulletin 01/2011; 34(1):120-7. · 1.66 Impact Factor
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ABSTRACT: Losartan is a selective angiotensin II (Ang II) type 1 (AT(1)) receptor antagonist which inhibits vascular smooth muscle cells (VSMCs) contraction and proliferation. We hypothesized that losartan may prevent cell proliferation by activating AMP-activated protein kinase (AMPK) in VSMCs. VSMCs were treated with various concentrations of losartan. AMPK activation was measured by Western blot analysis and cell proliferation was measured by MTT assay and flowcytometry. Losartan dose- and time-dependently increased the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase (ACC) in VSMCs. Losartan also significantly decreased the Ang II- or 15% FBS-induced VSMC proliferation by inhibiting the expression of cell cycle associated proteins, such as p-Rb, cyclin D, and cyclin E. Compound C, a specific inhibitor of AMPK, or AMPK siRNA blocked the losartan-induced inhibition of cell proliferation and the G(0)/G(1) cell cycle arrest. These data suggest that losartan-induced AMPK activation might attenuate Ang II-induced VSMC proliferation through the inhibition of cell cycle progression.
Korean Journal of Physiology and Pharmacology 10/2010; 14(5):299-304. · 0.96 Impact Factor
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ABSTRACT: The oxidation of LDLs is considered a key step in the development of atherosclerosis. How LDL oxidation contributes to atherosclerosis remains poorly defined. Here we report that oxidized and glycated LDL (HOG-LDL) causes aberrant endoplasmic reticulum (ER) stress and that the AMP-activated protein kinase (AMPK) suppressed HOG-LDL-triggered ER stress in vivo.
ER stress markers, sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) activity and oxidation, and AMPK activity were monitored in cultured bovine aortic endothelial cells (BAECs) exposed to HOG-LDL or in isolated aortae from mice fed an atherogenic diet.
Exposure of BAECs to clinically relevant concentrations of HOG-LDL induced prolonged ER stress and reduced SERCA activity but increased SERCA oxidation. Chronic administration of Tempol (a potent antioxidant) attenuated both SERCA oxidation and aberrant ER stress in mice fed a high-fat diet in vivo. Likewise, AMPK activation by pharmacological (5'-aminoimidazole-4-carboxymide-1-beta-d-ribofuranoside, metformin, and statin) or genetic means (adenoviral overexpression of constitutively active AMPK mutants) significantly mitigated ER stress and SERCA oxidation and improved the endothelium-dependent relaxation in isolated mouse aortae. Finally, Tempol administration markedly attenuated impaired endothelium-dependent vasorelaxation, SERCA oxidation, ER stress, and atherosclerosis in ApoE(-/-) and ApoE(-/-)/AMPKalpha2(-/-) fed a high-fat diet.
We conclude that HOG-LDL, via enhanced SERCA oxidation, causes aberrant ER stress, endothelial dysfunction, and atherosclerosis in vivo, all of which are inhibited by AMPK activation.
Diabetes 03/2010; 59(6):1386-96. · 8.29 Impact Factor
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ABSTRACT: Prostacyclin synthase (PGIS) is tyrosine nitrated in diseased animals. Whether PGIS nitration occurs in human diabetic atherosclerotic arteries has not been reported. The present study was designed to determine PGIS nitration and its association with the inflammatory response in atherosclerotic carotid arteries from patients with or without type 2 diabetes, and carotid plaques were obtained from patients who underwent carotid endarterectomy. PGIS nitration, nitric oxide synthases, adhesion molecules, myeloperoxidase, osteopontin, and matrix metalloproteinase (MMP) were measured by using immunohistochemistry and Western blotting. In low stenosis areas, diabetes enhanced reactive nitrogen species production, as evidenced by increases in 3-nitrotyrosine and PGIS nitration. In parallel, diabetes dramatically increased inflammatory markers including intracellular adhesion molecule-1, vascular adhesion molecule-1, and osteopontin. In both diabetic and nondiabetic patients, MMP-2 and MMP-9 protein levels were significantly increased in the arteries with high stenosis as compared with those with low stenosis. Moreover, diabetes enhanced inducible nitric oxide synthase expression in the plaques from low stenosis areas and up-regulated myeloperoxidase expression in the plaques from both high and low stenosis areas. These data demonstrate that diabetes preferentially increases PGIS nitration that is associated with excessive vascular inflammation in atherosclerotic carotid arteries from patients with type 2 diabetes, suggesting a possible role of tyrosine nitration of PGIS in the development of atherosclerosis in patients with diabetes.
American Journal Of Pathology 03/2010; 176(5):2542-9. · 4.89 Impact Factor
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ABSTRACT: Aberrant endoplasmic reticulum (ER) stress is associated with several cardiovascular diseases, including atherosclerosis. The mechanism by which aberrant ER stress develops is poorly understood. This study investigated whether dysfunction of AMP-activated protein kinase (AMPK) causes aberrant ER stress and atherosclerosis in vivo.
Human umbilical vein endothelial cells and mouse aortic endothelial cells from AMPK-deficient mice were used to assess the level of ER stress with Western blotting. Reduction of AMPKalpha2 expression significantly increased the level of ER stress in human umbilical vein endothelial cells. In addition, mouse aortic endothelial cells from AMPKalpha2 knockout (AMPKalpha2(-/-)) mice had higher expression of markers of ER stress and increased levels of intracellular Ca2+. These phenotypes were abolished by adenovirally overexpressing constitutively active AMPK mutants (Ad-AMPK-CA) or by transfecting sarcoendoplasmic reticulum calcium ATPase (SERCA). Inhibition of SERCA induced ER stress in endothelial cells. Furthermore, reduction of AMPKalpha expression suppressed SERCA activity. In addition, SERCA activity was significantly reduced concomitantly with increased oxidation of SERCA in mouse aortic endothelial cells from AMPKalpha2(-/-) mice. Both of these phenotypes were abolished by adenovirally overexpressing Ad-AMPK-CA. Furthermore, Tempol, which restored SERCA activity and decreased oxidized SERCA levels, markedly reduced the level of ER stress in mouse aortic endothelial cells from AMPKalpha2(-/-) mice. Finally, oral administration of tauroursodeoxycholic acid, a chemical chaperone that inhibits ER stress, significantly reduced both ER stress and aortic lesion development in low-density lipoprotein receptor- and AMPKalpha2-deficient mice.
These results suggest that AMPK functions as a physiological suppressor of ER stress by maintaining SERCA activity and intracellular Ca2+ homeostasis.
Circulation 02/2010; 121(6):792-803. · 14.74 Impact Factor
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ABSTRACT: Spontaneous hypertensive rats (SHR) are an established model of genetic hypertension. Vascular smooth muscle cells (VSMC) from SHR proliferate faster than those of control rats (Wistar-Kyoto rats; WKY). We tested the hypothesis that induction of heme oxygenase (HO)-1 induced by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats. Aprotinin treatment inhibited VSMC proliferation in SHR more than in normotensive rats. These inhibitory effects were associated with cell cycle arrest in the G1 phase. Tin protoporphyrin IX (SnPPIX) reversed the anti-proliferative effect of aprotinin in VSMC from SHR. The level of cyclin D was higher in VSMC of SHR than those of WKY. Aprotinin treatment downregulated the cell cycle regulator, cyclin D, but upregulated the cyclin-dependent kinase inhibitor, p21, in VSMC of SHR. Aprotinin induced HO-1 in VSMC of SHR, but not in those of control rats. Furthermore, aprotinin-induced HO-1 inhibited VSMC proliferation of SHR. Consistently, VSMC proliferation in SHR was significantly inhibited by transfection with the HO-1 gene. These results indicate that induction of HO-1 by aprotinin inhibits VSMC proliferation through cell cycle arrest in hypertensive rats.
Korean Journal of Physiology and Pharmacology 08/2009; 13(4):309-13. · 0.96 Impact Factor
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ABSTRACT: Heme oxygenase (HO)-1 is a well-known cytoprotectant against oxidative stress and exhibits an antiproliferative effect in vascular smooth muscle cells (VSMCs). The purpose of the present study was to test whether isoproterenol, one of the synthetic catecholamines having beta-adrenergic activity, affected angiotensin II (Ang II)-induced cell proliferation and reactive oxygen species (ROS) production. Also, the presumptive underlying signaling pathways in VSMCs were studied. Aortic VSMCs from 11-week-old male Sprague-Dawley rats were used. Isoproterenol dose-dependently increased HO-1 expression through beta(2)-adrenoceptor (AR) and protein kinase A (PKA) pathway, and isoproterenol concentration-dependently increased beta(2)-AR mRNA expression. Isoproterenol attenuated Ang II-induced cell proliferation, as evidenced by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay. This effect of isoproterenol was inhibited by pretreatment of the cells with beta(2)-AR antagonist butoxamine, PKA inhibitor H-89 and HO inhibitor Tin Protoporphyrin IX (SnPP IX), respectively. Isoproterenol inhibited phosphorylation level of Ang II-induced extracellular signal-regulated kinase (ERK1/2). Isoproterenol significantly inhibited Ang II-induced ROS production through the ERK1/2 pathway. These findings suggest that isoproterenol, via induction of HO-1, inhibits Ang II-stimulated proliferation and ROS production in cultured VSMCs.
Biological & Pharmaceutical Bulletin 07/2009; 32(6):1047-52. · 1.66 Impact Factor
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ABSTRACT: This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A(2) mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser(1177) and Akt at Ser(473). These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser(380)/Thr(382/383) PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G(i) family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser(380)/Thr(382/383) phosphorylation, and dephosphorylation of both Akt (at Ser(473)) and eNOS (at Ser(1177)). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.
Journal of Biological Chemistry 05/2009; 284(25):17120-8. · 4.77 Impact Factor
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ABSTRACT: Aprotinin is used clinically in cardiopulmonary bypass surgery to reduce transfusion requirements and the inflammatory response. The mechanism of action for the anti-inflammatory effects of aprotinin is still unclear. We examined our hypothesis whether inhibitory effects of aprotinin on cytokine-induced inducible nitric oxide synthase (iNOS) expression (IL-1beta plus TNF-alpha), reactive oxygen species (ROS) generation, and vascular smooth muscle cell (VSMC) proliferation were due to HO-1 induction in rat VSMCs. Aprotinin induced HO-1 protein expression in a dose-dependent manner, which was potentiated during inflammatory condition. Aprotinin reduced cytokine mixture (CM)-induced iNOS expression in a dose dependent manner. Furthermore, aprotinin reduced CM-induced ROS generation, cell proliferation, and phosphorylation of JNK but not of P38 and ERK1/2 kinases. Aprotinin effects were reversed by pre-treatment with the HO-1 inhibitor, tin protoporphyrin IX (SnPPIX). HO-1 is therefore closely involved in inflammatory-stimulated VSMC proliferation through the regulation of ROS generation and JNK phosphorylation. Our results suggest a new molecular basis for aprotinin anti-inflammatory properties.
Korean Journal of Physiology and Pharmacology 04/2009; 13(2):123-9. · 0.96 Impact Factor
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ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists of the thiazolidinedione class are widely used for the treatment of type 2 diabetes subjects due to their ability to improve insulin resistance. Troglitazone and ciglitazone belong to the PPARgamma agonists of thiazolidinediones. We report here that troglitazone but not ciglitazone increased IL-1beta induced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in vascular smooth muscle cell (VSMC) from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Potentiated expression of COX-2 and iNOS by troglitazone was inhibited by MG-132, a specific inhibitor of inhibitory factor kappaB (IkappaB) activation. Troglitazone treatment of these cells also resulted in a dose-dependent increase in IL-1beta induced IkappaBalpha phosphorylation. These data suggest that troglitazone is capable of increasing IL-1beta induced COX-2 and iNOS expression through an IkappaBalpha dependent mechanism in VSMC from WKY and SHR.
Biological & Pharmaceutical Bulletin 11/2008; 31(10):1955-8. · 1.66 Impact Factor
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ABSTRACT: We investigated whether NS-398, a selective inhibitor of COX-2, induces HO-1 in IL-1beta-stimulated vascular smooth muscle cells (VSMC). NS-398 reduced the production of PGE(2) without modulation of expression of COX-2 in IL-1beta-stimulated VSMC. NS-398 increased HO-1 mRNA and protein in a dose-dependent manner, but inhibited proliferation of IL-1beta-stimulated VSMC. Furthermore, SnPPIX, a HO-1 inhibitor, reversed the effects of NS-398 on PGE(2) production, suggesting that COX-2 activity can be affected by HO-1. Hemin, a HO-1 inducer, also reduced the production of PGE(2) and proliferation of IL-1beta-stimulated VSMC. CORM-2, a CO-releasing molecule, but not bilirubin inhibited proliferation of IL-1beta-stimulated VSMC. NS-398 inhibited proliferation of IL-1beta-stimulated VSMC in a HbO(2)-sensitive manner. In conclusion, NS-398 inhibits proliferation of IL-1beta-stimulated VSMC by HO-1-derived CO. Thus, NS-398 may facilitate the healing process of vessels in vascular inflammatory disorders such as atherosclerosis.
Biochemical and Biophysical Research Communications 10/2008; 376(4):753-7. · 2.48 Impact Factor
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ABSTRACT: A brief ischemic insult induces significant protection against subsequent massive ischemic events. The molecular mechanisms known as preconditioning (PC)-induced ischemic tolerance are not completely understood. We investigated whether kinetic changes of cyclooxygenase (COX)-2 during reperfusion time-periods after PC were related to ischemic tolerance. Rats were given PC by occlusion of middle cerebral artery (MCAO) for 10 min and sacrificed after the indicated time-periods of reperfusion (1, 2, 4, 8, 12, 18 or 24 h). In PC-treated rats, focal ischemia was induced by occlusion of MCA for 24 h and brain infarct volume was then studied to determine whether different reperfusion time influenced the damage. We report that the most significant protection against focal ischemia was obtained in rats with 8 h reperfusion after PC. Administration of indomethacin (10 mg/kg, oral) or rofecoxib (5 mg/kg, oral) 48 h prior to PC counteracted the effect of PC. Immunohistochemical analysis showed that COX-2 and HO-1 protein were induced in PC-treated rat brain, which was significantly inhibited by rofecoxib. Taken together, we concluded that the kinetic changes of COX-2 expression during the reperfusion period after PC might be partly responsible for ischemic tolerance.
Korean Journal of Physiology and Pharmacology 10/2008; 12(5):275-80. · 0.96 Impact Factor
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ABSTRACT: GTP cyclohydrolase 1 (GTPCH1) is the rate-limiting enzyme in de novo synthesis of tetrahydrobiopterin (BH4), an essential cofactor for endothelial NO synthase (eNOS) dictating, at least partly, the balance of NO and superoxide produced by this enzyme. The aim of this study was to determine the effect of acute inhibition of GTPCH1 on BH4, eNOS function, and blood pressure (BP) in vivo. Exposure of bovine or mouse aortic endothelial cells to GTPCH1 inhibitors (2,4-diamino-6-hydroxypyrimidine or N-acetyl-serotonin) or GTPCH1 small-interference RNA (siRNA) significantly reduced BH4 and NO levels but increased superoxide levels. This increase was abolished by sepiapterin (BH4 precursor) or N(G)-nitro-L-arginine methyl ester (nonselective NOS inhibitor). Incubation of isolated murine aortas with 2,4-diamino-6-hydroxypyrimidine or N-acetyl-serotonin impaired acetylcholine-induced endothelium-dependent relaxation but not endothelium-independent relaxation. Aortas from GTPCH1 siRNA-injected mice, but not their control-siRNA injected counterparts, also exhibited impaired endothelium-dependent relaxation. BH4 reduction induced by GTPCH1 siRNA injection was associated with increased aortic levels of superoxide, 3-nitrotyrosine, and adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1), as well as a significantly elevated systolic, diastolic, and mean BP in C57BL6 mice. GTPCH1 siRNA was unable to elicit these effects in eNOS(-/-) mice. Sepiapterin supplementation, which had no effect on high BP in eNOS(-/-) mice, partially reversed GTPCH1 siRNA-induced elevation of BP in wild-type mice. In conclusion, GTPCH1 via BH4 maintains normal BP and endothelial function in vivo by preserving NO synthesis by eNOS.
Hypertension 09/2008; 52(3):484-90. · 6.21 Impact Factor
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ABSTRACT: The AMP-activated protein kinase (AMPK) is reported to mediate the beneficial effects of statin on the vascular functions, but the biochemical mechanisms are incompletely understood. The aim of the study was to determine how statin activates AMPK. Exposure of confluent bovine aortic endothelial cells to simvastatin (statin) dose-dependently increased phosphorylation of AMPK at Thr(172) and activities of AMPK, which was in parallel with increased detection of both LKB1 phosphorylation at Ser(428) and LKB1 nuclear export. Furthermore, statin treatment was shown to increase protein kinase C (PKC)-zeta activity and PKC-zeta phosphorylation at Thr(410)/Thr(403). Consistently, inhibition of PKC-zeta either by pharmacological or genetic manipulations abolished statin-enhanced LKB1 phosphorylation at Ser(428), blocked LKB1 nucleus export, and prevented the subsequent activation of AMPK. Similarly, in vivo transfection of PKC-zeta-specific small interfering RNA in C57BL/6J mice significantly attenuated statin-enhanced phosphorylation of AMPK-Thr(172), acetyl-CoA carboxylase (ACC)-Ser(79), and LKB1-Ser(428). In addition, statin significantly increased reactive oxygen species, whereas preincubation of mito-TEMPOL, a superoxide dismutase mimetic, abolished statin-enhanced phosphorylation of both AMPK-Thr(172) and ACC-Ser(79). Finally, in vivo administration of statin increased 3-nitrotyrosine and the phosphorylation of AMPK and ACC in C57BL/6J mice but not in mice deficient in endothelial nitric-oxide synthase. Taken together, our data suggest that AMPK activation by statin is peroxynitrite-mediated but PKC-zeta-dependent.
Journal of Biological Chemistry 08/2008; 283(29):20186-97. · 4.77 Impact Factor
