Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species.

Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.

The NAD(P)H oxidase inhibitor apocynin improves endothelial NO/superoxide balance and lowers effectively blood pressure in spontaneously hypertensive rats: comparison to calcium channel blockade.

The vascular NAD(P)H oxidase contributes to endothelial dysfunction and high blood pressure in the spontaneously hypertensive rat by enhancing superoxide production. We investigated the effects of apocynin, a NAD(P)H oxidase inhibitor, on blood pressure and vascular radical and nitric oxide formation in SHR and compared its effects to the calcium channel blocker nifedipine. Apocynin (over four weeks) lowered systolic blood pressure significantly and as effectively as nifedipine. Both apocynin and nifedipine significantly reduced superoxide production. In parallel, vascular nitric oxide production and ecNOS activity was significantly increased by apocynin treatment. Therefore, apocynin may be an effective antihypertensive drug in essential hypertension.

Novel Nox inhibitor VAS2870 attenuates PDGF-dependent smooth muscle cell chemotaxis, but not proliferation.

OBJECTIVE: Reactive oxygen species (ROS) produced by NAD(P)H oxidases (Nox) play a significant role in the pathophysiology of cardiovascular diseases. Expression and activity of NAD(P)H oxidases are regulated by growth factors such as angiotensin II and platelet-derived growth factor (PDGF). We characterized the effects of the novel Nox inhibitor VAS2870 on PDGF-dependent ROS liberation and cellular events in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: PDGF-BB increased NAD(P)H oxidase activity (lucigenin-enhanced chemiluminescence) and intracellular ROS levels (detected by confocal laserscanning microscopy using 2,7-DCF) to 229+/-9% and 362+/-54% at 1 and 2 h, respectively. Preincubation with VAS2870 (10 and 20 microM) completely abolished PDGF-mediated NAD(P)H oxidase activation and ROS production. Since ROS are involved in various growth factor-induced cellular functions, the influence of VAS2870 on PDGF-induced DNA synthesis and chemotaxis was determined. PDGF promoted a 4.2+/-0.2-fold increase of VSMC migration (modified Boyden chamber, p<0.01) and increased DNA synthesis by maximally 3.2+/-0.4-fold (BrdU incorporation, p<0.01) in a concentration-dependent manner. Preincubation with VAS2870 (0.1-20 microM) did not affect PDGF-induced cell cycle progression. However, it abolished PDGF-dependent chemotaxis of VSMC in a concentration-dependent manner (100% inhibition at 10 microM). These findings were related to PDGF-dependent signaling events. Western blot analyses using phospho-specific antibodies revealed that the downstream signaling molecules Akt, Erk, and Src were activated by PDGF. However, VAS2870 blocked PDGF-dependent activation of Src, but not of Akt and Erk, in a concentration-dependent manner. CONCLUSIONS: VAS2870 effectively suppresses growth factor-mediated ROS liberation in VSMC. Furthermore, it completely inhibits PDGF-dependent VSMC migration, whereas it does not affect DNA synthesis. These divergent effects reflect the critical role of Src activity, which-in contrast to Akt and Erk-appears to be redox-sensitive and is absolutely required for PDGF-induced chemotaxis, but not cell cycle progression.

17Beta-estradiol attenuates PDGF signaling in vascular smooth muscle cells at the postreceptor level.

Estrogens are known to display significant vasoprotective effects in premenopausal women. PDGF is an important mediator of vascular smooth muscle cell (VSMC) migration and proliferation, and thus atherogenesis. We analyzed the effects of 17beta-estradiol (E2) on beta-PDGF receptor (beta-PDGFR) expression/activation and PDGF-dependent VSMC proliferation, migration, and downstream signaling events. Pretreatment of VSMCs with E2 (0.3 microM-0.1 mM) for 24 h concentration-dependently inhibited PDGF-induced proliferation and migration up to 85.5 +/- 15.8% and 79.4 +/- 9.8%, respectively (both P < 0.05). These effects were prevented by coincubation with the ER antagonist ICI-182780. E2 did not alter beta-PDGFR expression, nor did it impair the ligand-induced tyrosine phosphorylation of the beta-PDGFR and consecutive binding of the receptor-associated signaling molecules Src homology region 2-containing phosphatase-2, PLC-gamma, phosphatidylinositol 3-kinase, and RasGAP. Thus estrogens inhibited PDGF-induced cellular responses at the postreceptor level. Although stimulation of VSMCs with PDGF-BB led to a transient increase of rac-1 activity, pretreatment with E2 for 24 h concentration-dependently inhibited PDGF-induced rac-1 activation. Furthermore, inhibition of rac-1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative rac-1 (rac-N17) significantly inhibited PDGF-induced VSMC migration, indicating that rac-1 activity is essential for PDGF-dependent cellular responses. E2 did not further reduce PDGF-induced migration in rac-N17-overexpressing cells, suggesting that it diminishes VSMC migration by altering rac-1 activity. We conclude that E2 attenuates PDGF-dependent cellular functions of VSMCs downstream of the beta-PDGFR via inhibition of rac-1. These observations offer a molecular explanation for the vasoprotective effects of estrogens.

Systematic evaluation of anti-apoptotic growth factor signaling in vascular smooth muscle cells. Only phosphatidylinositol 3'-kinase is important.

Peptide growth factors contribute to the pathogenesis of cardiovascular diseases by inducing a variety of cellular responses including anti-apoptotic effects. Several of the signaling molecules that are activated by growth factor receptors such as Src family kinases (Src), phosphatidylinositol 3'-kinase (PI3K), phospholipase Cgamma (PLCgamma), Ras, and SHP-2 were shown to mediate survival signals. We systematically investigated the relative contribution of each signaling molecule for growth factor-dependent cell survival in vascular smooth muscle cells (VSMC). Our approach was the use of mutated plateletderived growth factor (PDGF) beta-receptors (betaPDGFR) in which the tyrosine residues required for binding of each signaling molecule were individually mutated to phenylalanine. To bypass endogenous PDGFR in VSMC we used chimeric receptors (ChiRs), containing the extracellular domain of the macrophage colony-stimulating factor (M-CSF) receptor and the cytoplasmic domain of the wild type (WT) or mutated betaPDGFR. Selective activation of the ChiR-WT with M-CSF significantly reduced apoptosis to the same extent as PDGF-BB in non-transfected cells. Deletion of the binding site for PI3K, but not for Src, RasGAP, SHP-2, or PLCgamma, completely abolished the anti-apoptotic effect. Consistently, a ChiR mutant that only binds PI3K was fully able to mediate cell survival as efficiently as the ChiR-WT. Furthermore, the PDGF-dependent anti-apoptotic effect in non-transfected cells was completely abolished by the PI3K inhibitor wortmannin, whereas inhibitors of Src, PLCgamma, ERK, or p38 MAP kinase had no effect. The exploration of downstream signaling events revealed that PDGF-BB activates the anti-apoptotic Akt signaling pathway in a PI3K-dependent manner. Moreover, Akt phosphorylates and thus inactivates the pro-apoptotic proteins BAD and Forkhead transcription factors (FKHR, FKHRL1). We conclude that growth factor-dependent cell survival in VSMC is mediated only by activation of the PI3K/Akt pathway, whereas all other receptor-associated signaling molecules do not play a significant role.

Ritonavir exhibits anti-atherogenic properties on vascular smooth muscle cells.

OBJECTIVES: HIV protease inhibitors (PI) such as ritonavir have dramatically decreased HIV-related morbidity and mortality. However they exhibit significant side-effects such as hyperlipidemia, hyperglycemia with or without lipodystrophy, which may increase patients' risk for atherosclerosis. Direct effects of PI on the vascular wall have not been investigated. Platelet-derived growth factor (PDGF) is a major contributor to atherogenesis. DESIGN: In the present study the effects of ritonavir on PDGF-BB-induced responses of vascular smooth muscle cells (VSMCs) were evaluated. METHODS: PDGF-induced proliferation of VSMCs was measured by BrdU-incorporation, and chemotaxis was assessed by utilizing modified Boyden chambers. Cytotoxicity and apoptosis were quantified using LDH-release- and apoptosis-kits. Immunoprecipitation and Western blot analyses were performed to evaluate betaPDGF receptor (betaPDGFR) expression and phosphorylation, and to monitor intracellular signaling. RESULTS: Pretreatment of VSMCs with ritonavir resulted in a significant concentration-dependent inhibition of PDGF-BB-induced cellular responses. At a therapeutic concentration (10 microg/ml), ritonavir significantly reduced PDGF-induced DNA synthesis and chemotaxis by 46.8 +/- 5.5% and 37.2 +/- 3.3%, respectively (P < 0.05 each). In addition it significantly inhibited PDGF-dependent downstream signaling, such as Erk activation. These inhibitory effects were not due to cytotoxicity or apoptosis. Instead, ritonavir inhibited the ligand-induced tyrosine phosphorylation of the betaPDGFR, whereas it did not alter betaPDGFR expression. CONCLUSIONS: Ritonavir has direct effects on VSMCs at clinically relevant concentrations in vitro, as it inhibits betaPDGFR activation and PDGF-dependent proliferation and migration of VSMCs. Although ritonavir may increase the risk of vascular disease by its metabolic side effects, it may exhibit anti-atherogenic properties on the cellular level.

Frataxin promotes antioxidant defense in a thiol-dependent manner resulting in diminished malignant transformation in vitro.

Friedreich ataxia is an inherited disorder caused by decreased expression of frataxin protein. Increasing evidence suggests that this protein might detoxify reactive oxygen species (ROS) by an unknown mechanism. Here we demonstrate that transgenic overexpression of human frataxin increases cellular antioxidant defense via activation of glutathione peroxidase and elevation of reduced thiols, thereby reducing the incidence of malignant transformation induced by ROS, as observed by soft agar assays and tumour formation in nude mice. These findings expand the understanding of antioxidant properties of frataxin, and tentatively suggest a role in the early induction of cancer.

Gene expression of adrenomedullin in failing myocardium: comparison to atrial natriuretic peptide.

The expression of adrenomedullin (AM) and atrial natriuretic factor (ANF) were investigated in the myocardium of a rat model of chronic ischemic heart failure (CHF) compared with sham-operated controls. In addition, human myocardium of patients with end-stage heart failure due to idiopathic dilated cardiomyopathy compared with myocardium of normal subjects (NF) was studied. In CHF, similar AM levels but increased ANF expression were observed in left ventricular myocardium, as assessed by semiquantitative PCR. Functional experiments with freshly excised papillary muscles showed no influence of AM on myocardial contractility. In NF human myocardium, the expression of AM mRNA was threefold higher in atrial compared with ventricular tissue. In analogy, ANF mRNA was increased by approximately 15-fold in atrial tissue. In dilated cardiomyopathy, the expression of AM was significantly increased in right and left ventricles compared with NF. In parallel, ventricular ANF expression was enhanced.

Cellular antioxidant effects of atorvastatin in vitro and in vivo.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert direct effects on vascular cells and beneficially influence endothelial dysfunction. Because reactive oxygen species (ROS) may lead to vascular damage and dysfunction, we investigated the effect of atorvastatin on ROS production and the underlying mechanisms in vitro and in vivo. Cultured rat aortic vascular smooth muscle cells were incubated with 10 micromol/L atorvastatin. Angiotensin II-induced and epidermal growth factor-induced ROS production were significantly reduced by atorvastatin (dichlorofluorescein fluorescence laser microscopy). Atorvastatin downregulated mRNA expression of the NAD(P)H oxidase subunit nox1, whereas p22phox mRNA expression was not significantly altered (reverse transcription-polymerase chain reaction, Northern analysis). Membrane translocation of rac1 GTPase, which is required for the activation of NAD(P)H oxidase, was inhibited by atorvastatin (Western blot). mRNA expression of superoxide dismutase isoforms and glutathione peroxidase was not modified by atorvastatin, whereas catalase expression was upregulated at mRNA and protein levels, resulting in an increased enzymatic activity. Effects of atorvastatin on ROS production and nox1, rac1, and catalase expression were inhibited by L-mevalonate but not by 25-hydroxycholesterol. In addition, spontaneously hypertensive rats were treated with atorvastatin for 30 days. ROS production in aortic segments was significantly reduced in statin-treated rats (lucigenin chemiluminescence). Treatment with atorvastatin reduced vascular mRNA expression of p22phox and nox1 and increased aortic catalase expression. mRNA expression of superoxide dismutases, glutathione peroxidase, and NAD(P)H oxidase subunits gp91phox, p40phox, p47phox, and p67phox remained unchanged. Translocation of rac1 from the cytosol to the cell membrane was also reduced in vivo. Thus, atorvastatin exerts cellular antioxidant effects in cultured rat vascular smooth muscle cells and in the vasculature of spontaneously hypertensive rats mediated by decreased expression of essential NAD(P)H oxidase subunits and by upregulation of catalase expression. These effects of atorvastatin may contribute to the vasoprotective effects of statins.