Publications (10) View all
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Article: Ascorbate stimulates endothelial nitric oxide synthase enzyme activity by rapid modulation of its phosphorylation status.
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ABSTRACT: Long-term exposure to ascorbate is known to enhance endothelial nitric oxide synthase (eNOS) activity by stabilizing the eNOS cofactor tetrahydrobiopterin (BH4). We investigated acute effects of ascorbate on eNOS function in primary (HUVEC) and immortalized human endothelial cells (EA.hy926), aiming to provide a molecular explanation for the rapid vasodilatation seen in vivo upon administration of ascorbate. Enzymatic activity of eNOS and intracellular BH4 levels were assessed by means of an arginine-citrulline conversion assay and HPLC analysis, respectively. Over a period of 4h, ascorbate steadily increased eNOS activity, although endothelial BH4 levels remained unchanged compared to untreated control cells. Immunoblot analyses revealed that as early as 5 min after treatment ascorbate dose-dependently increased phosphorylation at eNOS-Ser1177 and concomitantly decreased phosphorylation at eNOS-Thr495, a phosphorylation pattern indicative of increased eNOS activity. By employing pharmacological inhibitors, siRNA-mediated knockdown approaches, and overexpression of the catalytic subunit of protein phosphatase 2A (PP2A), we show that this effect was at least partly owing to reduction of PP2A activity and subsequent activation of AMP-activated kinase. In this report, we unravel a novel mechanism for how ascorbate rapidly activates eNOS independent of its effects on BH4 stabilization.Free radical biology & medicine 04/2012; 52(10):2082-90. · 5.42 Impact Factor -
Article: Resveratrol inhibits migration and Rac1 activation in EGF- but not PDGF-activated vascular smooth muscle cells.
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ABSTRACT: Migration of vascular smooth muscle cells (VSMC) reflects one of the initial steps in atherosclerosis. Resveratrol (RV) is suggested to mediate putative vasoprotective properties of red wine leading to the hypothesis that RV interferes with growth factor-induced migration of VSMC. We show here that RV (50 μM) strongly reduces epidermal growth factor (EGF)- but not platelet-derived growth factor (PDGF)-induced VSMC migration using the wound-healing technique. Accordingly, RV inhibited Rac1 activation and lamellipodia formation in response to EGF but not PDGF as shown by pull-down assays and fluorescence microscopy after actin staining with phalloidin-FITC, respectively. Since Src-family kinases and the phosphatidylinositol-3 kinase (PI3K) are reported to be crucial upstream mediators of Rac1 activation we examined the PI3K inhibitor wortmannin and the src kinase inhibitor SU6656 side-by-side with RV for their anti-migratory potential. Whereas src inhibition abrogated both EGF- and PDGF-triggered migration, wortmannin, like RV, was more effective in EGF- than PDGF-activated cells, suggesting that PI3K inhibition, previously shown for RV in growth factor-activated VSMC, contributes to the anti-migratory effect of RV in EGF-stimulated VSMC. This study is the first to discover an anti-migratory potential of RV in EGF-activated VSMC that is most likely mediated via Rac1 inhibition.Molecular Nutrition & Food Research 08/2011; 55(8):1230-6. · 4.30 Impact Factor -
Article: Caffeic acid phenethyl ester inhibits PDGF-induced proliferation of vascular smooth muscle cells via activation of p38 MAPK, HIF-1α, and heme oxygenase-1.
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ABSTRACT: Hyperproliferation of vascular smooth muscle cells (VSMCs) is critically involved in the onset of atherosclerosis and restenosis. Although caffeic acid phenethyl ester (CAPE, 1), one of the main constituents of honeybee propolis, has been shown to exert a beneficial effect in models of vascular injury in vivo, detailed mechanistic investigations in vascular cells are scarce. This study has examined the antiproliferative activity of 1 in platelet-derived growth factor (PDGF)-stimulated primary rat aortic VSMCs and aimed to shed light on underlying molecular mechanisms. Compound 1 inhibited the proliferation of VSMCs upon exposure to PDGF in a dose-dependent manner by interfering with cell cycle progression from the G0/1- to the S-phase. Enhanced phosphorylation of p38 mitogen-activated protein kinase (MAPK) as well as stabilization of hypoxia-inducible factor (HIF)-1α and subsequent induction of heme oxygenase-1 (HO-1) could be identified as molecular events contributing to the observed growth arrest in PDGF-activated VSMCs upon exposure to 1.Journal of Natural Products 01/2011; 74(3):352-6. · 3.13 Impact Factor -
Article: Indirubin-3'-monoxime blocks vascular smooth muscle cell proliferation by inhibition of signal transducer and activator of transcription 3 signaling and reduces neointima formation in vivo.
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ABSTRACT: Our goal was to examine the influence of indirubin-3'-monoxime (I3MO), a natural product-derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation by arresting cells in the G(0)/G(1) phase of the cell cycle as assessed by 5-bromo-2'-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38(MAPK) was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-γ-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [(14)C]l-arginine/[(14)C]l-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells. I3MO represses PDGF- and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis.Arteriosclerosis Thrombosis and Vascular Biology 12/2010; 30(12):2475-81. · 6.37 Impact Factor -
Article: Resveratrol blocks Akt activation in angiotensin II- or EGF-stimulated vascular smooth muscle cells in a redox-independent manner.
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ABSTRACT: Resveratrol (RV), an antioxidant, inhibits angiotensin II (Ang II)-induced hypertrophy and Ang II- or epidermal growth factor (EGF)-induced Akt phosphorylation in rat vascular smooth muscle cells (VSMCs). Both signalling pathways are reported to utilize reactive oxygen species (ROS). The aim of this study was to show whether RV reduces the ROS level in Ang II- or EGF-activated VSMCs and whether reduction of ROS causes the impeded signalling towards Akt in the presence of RV. We show here that RV reduces intracellular ROS and extracellular H₂O₂ release from VSMCs as measured using 2',7'-dichlorodihydrofluorescein-diacetate and Amplex Red™. Since NADPH oxidases (Nox) 1 and 4 are major ROS sources in VSMCs, we examined their need for Akt phosphorylation in response to Ang II or EGF. Experiments using the blocking peptide gp91ds-tat verified a role for Nox1 in Ang II signalling towards Akt, but excluded a role for Nox1 in the respective EGF signalling. A small interfering RNA-mediated knock-down of Nox4 showed that Nox4 was not required for Ang II- or EGF-induced Akt phosphorylation. Use of the flavoprotein inhibitor diphenyleneiodonium, N-acetyl-cysteine, and non-antioxidant RV derivatives revealed that the antioxidant capacity of RV is not required for the inhibition of Akt phosphorylation, in both rat and human VSMCs. Thus, although RV acts as an antioxidant, the antihypertrophic response of RV in VSMCs and the signalling downstream of the EGF receptor towards Akt seem to be largely redox independent.Cardiovascular research 11/2010; 90(1):140-7. · 5.80 Impact Factor
