The contribution of Nox4 to NADPH oxidase activity in mouse vascular smooth muscle

OBJECTIVE: NADPH oxidases are important sources of reactive oxygen species (ROS) in the vasculature. In phagocytic cells, the catalytic subunit of NADPH oxidase is a glycoprotein, gp91phox. However, vascular smooth muscle cells (VSMCs), which show prominent NADPH oxidase activity, lack gp91phox. Hence, we examined the role of Nox4, a gp91phox homologue, in superoxide production in mouse-cultured VSMCs. METHODS AND RESULTS: Incubation of VSMCs with NADPH increased ROS production whether detected by lucigenin-enhanced chemiluminescence or dichlorofluorescein. Superoxide production was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium and apocynin, but not by inhibitors of other potential sources of superoxide. In unstimulated VSMCs, phosphorothioate antisense oligonucleotides against Nox4 down-regulated mRNA expression of the subunit by 65% and attenuated superoxide production by 41% without affecting Nox1 expression. Interleukin-1beta (IL-beta) thrombin and platelet-derived growth factor (PDGF) also reduced Nox4 mRNA expression after 3 h without affecting Nox1 levels. Of these stimuli, only IL-beta reduced superoxide, but this effect was more rapid (< or =30 min) than its actions on Nox4. CONCLUSIONS: Under resting conditions, NADPH oxidase activity in VSMCs is largely dependent upon Nox4 expression. Proinflammatory mediators down-regulated Nox4 but did not affect Nox1 expression, so other factors must compensate to regulate superoxide production.     

Redox-sensitive signaling by angiotensin II involves oxidative inactivation and blunted phosphorylation of protein tyrosine phosphatase SHP-2 in vascular smooth muscle cells from SHR

Angiotensin II (Ang II) signaling in vascular smooth muscle cells (VSMCs) involves reactive oxygen species (ROS) through unknown mechanisms. We propose that Ang II induces phosphorylation of growth signaling kinases by redox-sensitive regulation of protein tyrosine phosphatases (PTP) in VSMCs and that augmented Ang II signaling in spontaneously hypertensive rats (SHRs) involves oxidation/inactivation and blunted phosphorylation of the PTP, SHP-2. PTP oxidation was assessed by the in-gel PTP method. SHP-2 expression and activity were evaluated by immunoblotting and by a PTP activity assay, respectively. SHP-2 and Nox1 were downregulated by siRNA. Ang II induced oxidation of multiple PTPs, including SHP-2. Basal SHP-2 content was lower in SHRs versus WKY. Ang II increased SHP-2 phosphorylation and activity with blunted responses in SHRs. Ang II-induced SHP-2 effects were inhibited by valsartan (AT(1)R blocker), apocynin (NAD(P)H oxidase inhibitor), and Nox1 siRNA. Ang II stimulation increased activation of ERK1/2, p38MAPK, and AKT, with enhanced effects in SHR. SHP-2 knockdown resulted in increased AKT phosphorylation, without effect on ERK1/2 or p38MAPK. Nox1 downregulation attenuated Ang II-mediated AKT activation in SHRs. Hence, Ang II regulates PTP/SHP-2 in VSMCs through AT(1)R and Nox1-based NAD(P)H oxidase via two mechanisms, oxidation and phosphorylation. In SHR Ang II-stimulated PTP oxidation/inactivation is enhanced, basal SHP-2 expression is reduced, and Ang II-induced PTP/SHP-2 phosphorylation is blunted. These SHP-2 actions are associated with augmented AKT signaling. We identify a novel redox-sensitive SHP-2-dependent pathway for Ang II in VSMCs. SHP-2 dysregulation by increased Nox1-derived ROS in SHR is associated with altered Ang II-AKT signaling.     

FcgammaRIIa mediates C-reactive protein-induced inflammatory responses of human vascular smooth muscle cells by activating NADPH oxidase 4

OBJECTIVES: We investigated the mechanism by which C-reactive protein (CRP) affects pro-inflammatory activities of vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: RT-PCR, flow cytometry, and immunoblotting assays consistently showed the expression of FcgammaRIIa by cultured VSMCs isolated from human coronary arteries. Immunofluorescence staining of human coronary artery plaque showed the co-localization of FcgammaRIIa with alpha-actin(+) VSMCs in atheromatous regions. Confocal microscopic image analysis of H(2)DCFDA-labeled cells showed that CRP induced intracellular reactive oxygen species (ROS) generation by FcgammaRIIa(+) HEK293T cells. Moreover, CRP time- and dose-dependently generated ROS in VSMCs through FcgammaRIIa activation. VSMCs mainly express NADPH oxidase 4 isoform (Nox4), the suppression of which using a specific siRNA completely abolished CRP-induced ROS generation by VSMCs. The downregulation of p22(phox), a component of the active Nox4 complex, by transfecting with specific decoy oligomers and functional blocking of FcgammaRIIa not only inhibited the CRP-induced ROS generation but also reduced the degree of AP-1 and NF-kappaB activation, the production of MCP-1, IL-6, and ET-1, and the apoptotic changes of VSMCs in response to CRP. CONCLUSIONS: CRP-induced ROS generation by VSMCs, which requires functional activation of FcgammaRIIa and NADPH oxidase 4, orchestrates pro-inflammatory activities of VSMCs and may eventually promote atherogenesis and plaque rupture.     

Activation of apocynin-sensitive NADPH oxidase (Nox2) activity in INS-1 832/13 cells under glucotoxic conditions

Several lines of recent evidence provided compelling evidence to suggest increased generation of reactive oxygen species (ROS) as causal for mitochondrial dysregulation and apoptosis in islet β-cells exposed to noxious stimuli including high glucose, lipids and proinflammatory cytokines. Studies along these lines are also suggestive of a significant contributory role for NADPH oxidase in the generation of ROS under the above conditions. We have recently reported a marked increase in the expression and activation of cytosolic components of NADPH oxidase (p47phox, Rac1) in cell culture models of glucotoxicity and in islets from T2DM animals (Zucker Diabetic Fatty rat) and humans. In this communication, we provide further evidence indicating significant activation of NADPH activity (~2-fold) in INS-1 832/13 cells exposed to chronic hyperglycemic conditions (20 mM; 48 h). We also report marked attenuation of this activity, by apocynin, a selective inhibitor of phagocyte-like NADPH oxidase (Nox2) activity. Together, our findings implicate Nox2 as a source for ROS generation in β-cells exposed to glucotoxic conditions.     

Angiotensin II and endothelin-1 regulate MAP kinases through different redox-dependent mechanisms in human vascular smooth muscle cells

OBJECTIVE: The role of reactive oxygen species (ROS) in mitogen-activated protein kinase (MAPK) signaling by angiotensin (Ang) II and endothelin-1 (ET-1) in human vascular smooth muscle cells (VSMC) was investigated. DESIGN: VSMCs were derived from resistance arteries from healthy subjects. MAPK activity was assessed using phospho-specific antibodies. ROS generation was measured by CMH2DCFDA fluorescence and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity by lucigenin chemiluminescence. RESULTS: Ang II and ET-1 increased MAPK phosphorylation (P < 0.01). Pre-treatment with Tiron and Tempol, *O2 scavengers, attenuated agonist-stimulated phosphorylation of p38MAPK, c-Jun N-terminal kinases (JNK) and ERK5, but not of ERK1/2 (extracellular signal-regulated kinases). Apocynin and diphenylene iodinium (DPI), NAD(P)H oxidase inhibitors, decreased Ang II-induced responses 60-70%. ET-1-mediated MAPK phosphorylation was unaffected by apocynin but was reduced (> 50%) by thenoyltrifluoroacetone (TIFT) and carboxyl cyanide-m-chlorophenylhydrazone (CCCP), mitochondrial inhibitors. Allopurinol and N-nitro-l-arginine methyl ester (l-NAME), xanthine oxidase and nitric oxide synthase (NOS) inhibitors, respectively, did not influence MAPK activation. Intracellular ROS generation, was increased by Ang II and ET-1 (P < 0.01). DPI inhibited Ang II- but not ET-1-mediated ROS production. Expression of p22phox and p47phox and activation of NAD(P)H oxidase were increased by Ang II but not by ET-1. CCCP and TIFT significantly attenuated ET-1-mediated ROS formation (P < 0.05), without influencing Ang II effects. CONCLUSIONS: Ang II activates p38MAPK, JNK and ERK5 primarily through NAD(P)H oxidase-generated ROS. ET-1 stimulates these kinases via redox-sensitive processes that involve mitochondrial-derived ROS. These data suggest that redox-dependent activation of MAPKs by Ang II and ET-1 occur through distinct ROS-generating systems that could contribute to differential signaling by these agonists in VSMCs.     

Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src

Increasing evidence indicates that aldosterone elicits vascular effects through nongenomic signaling pathways. We tested the hypothesis that aldosterone induces activation of vascular mitogen-activated protein (MAP) kinases and NADPH oxidase via c-Src-dependent mechanisms in vascular smooth muscle cells (VSMCs). Aldosterone effects on activation of c-Src, p38MAP kinase, and NADPH oxidase, and incorporation of [3H]proline, an index of collagen synthesis, were assessed in cultured rat VSMCs. Studies were performed in the absence and presence of eplerenone, a selective mineralocorticoid receptor blocker, PP2, a selective Src inhibitor, and SB212190, a selective p38MAPK inhibitor. Phosphorylation of c-Src was dose-dependently increased by aldosterone, with maximal responses obtained at 10(-7) mol/L. Aldosterone increased p38MAP kinase phosphorylation, NAD(P)H oxidase activation, and [3H]proline incorporation. These responses were abrogated by eplerenone and almost abolished by PP2. Aldosterone-stimulated incorporation of [3H]proline was significantly reduced by SB212190, indicating that p38MAP kinase plays a role in profibrotic actions of aldosterone. To unambiguously demonstrate the importance of aldosterone in c-Src signaling, VSMCs from c-Src+/+ and c-Src+/- mice were also studied. Aldosterone increased phosphorylation of c-Src, p38MAP kinase, and cortactin, a Src-specific substrate, in c-Src+/+ VSMCs, but not in c-Src-deficient cells. Taken together, our findings demonstrate that nongenomic signaling by aldosterone occurs through c-Src-dependent pathways. These processes may play an important role in profibrotic actions of aldosterone.     

Redox signaling in angiogenesis: role of NADPH oxidase

Angiogenesis, a process of new blood vessel formation, is a key process involved in normal development and wound repair as well as in the various pathophysiologies such as ischemic heart and limb diseases and atherosclerosis. Reactive oxygen species (ROS) such as superoxide and H(2)O(2) function as signaling molecules in many aspects of growth factor-mediated responses including angiogenesis. Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor and stimulates proliferation, migration, and tube formation of endothelial cells (ECs) primarily through the VEGF receptor type2 (VEGR2, KDR/Flk1). VEGF binding initiates autophosphorylation of VEGFR2, which results in activation of downstream signaling enzymes including ERK1/2, Akt, and eNOS in ECs, thereby stimulating angiogenesis. The major source of ROS in EC is a NADPH oxidase which consists of Nox1, Nox2 (gp91phox), Nox4, p22phox, p47phox, p67phox and the small G protein Rac1. The endothelial NADPH oxidase is activated by angiogenic factors including VEGF and angiopoietin-1. ROS derived from this enzyme stimulate diverse redox signaling pathways leading to angiogenesis-related gene induction as well as EC migration and proliferation, which may contribute to postnatal angiogenesis in vivo. The aim of this review is to provide an overview of the recent progress on the emerging area of the role of ROS derived from NADPH oxidase and redox signaling in angiogenesis. Understanding these mechanisms may provide insight into the NADPH oxidase and redox signaling components as potential therapeutic targets for treatment of angiogenesis-dependent cardiovascular diseases and for promoting angiogenesis in ischemic limb and heart diseases.     

Catecholamine-induced vascular wall growth is dependent on generation of reactive oxygen species

Alpha1-adrenoceptor-dependent proliferation of vascular smooth muscle cells (VSMCs) is strongly augmented by vascular injury, and may contribute to intimal growth and lumen loss. Because reactive oxygen species (ROS) are increased by injury and have been implicated as second messengers in proliferation of VSMCs, we investigated the role of ROS in catecholamine-induced VSMC growth. Rat aortae were isolated 4 days after balloon injury, maintained in organ culture under circumferential wall tension, and exposed to agents for 48 hours. The antioxidants N-acetylcysteine (NAC, 10 mmol/L) and Tiron (5 mmol/L) and the flavin-inhibitor diphenylene iodonium (DPI, 20 micromol/L) abolished norepinephrine-induced increases in protein synthesis and DNA content in media. In aortic sections, norepinephrine augmented ROS production (dihydroethidium confocal microscopy), which was dose-dependently inhibited by NAC, Tiron, and DPI. In cultured VSMCs, phenylephrine caused time- and dose-dependent ROS generation (aconitase activity), had similar efficacy to thrombin (1 U/mL), and was eliminated by the superoxide dismutase (SOD) mimetic Mn-(III)-tetrakis-(4-benzoic-acid)-porphyrin-chloride (200 micromol/L) and Tiron. Phenylephrine-induced ROS production and increases in DNA and protein content were blocked by prazosin (0.3 micromol/L) and abolished in p47phox-/- cells. PEG-SOD (25 U/mL) had little effect, whereas PEG-catalase (50 U/mL) eliminated phenylephrine-induced proliferation in VSMCs. DPI (10 micromol/L) and apocynin (30 micromol/L) abolished phenylephrine-stimulated mitogenesis, whereas inhibitors of other intracellular ROS sources had not effect. Furthermore, PE increased p47phox expression (RT-PCR). These data demonstrate that the trophic effect of catecholamines on vascular wall cells is dependent on a ROS-sensitive step that we hypothesize consists of activation of the NAD(P)H-dependent vascular oxidase.     

Transactivation of the insulin-like growth factor-I receptor by angiotensin II mediates downstream signaling from the angiotensin II type 1 receptor to phosphatidylinositol 3-kinase

Angiotensin II (AngII) activates phosphatidylinositol 3-kinase (PI3-kinase), a known effector of receptor tyrosine kinases. Treatment of smooth muscle cells with AngII has also been shown to promote phosphorylation of various tyrosine kinase receptors. We therefore investigated the relationship between AngII and IGF-I receptor activation in smooth muscle cells with a phosphorylation-specific antibody. Our experiments showed that IGF-I receptor phosphorylation was maximally stimulated within 10 min by AngII. Inclusion of an IGF-I-neutralizing antibody in the culture media did not prevent IGF-I receptor phosphorylation after AngII treatment, which argues that a paracrine/autocrine loop is not required. Furthermore, this process was blocked by losartan and 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP-1), indicating stimulation of IGF-I receptor phosphorylation occurs via AngII type 1 receptor-dependent activation of Src kinase. The functional significance of IGF-I receptor transactivation was examined with selective inhibitors of the IGF-I receptor kinase (AG1024, AG538). When AngII-treated cells were incubated with AG1024 or AG538, phosphorylation of the regulatory p85 subunit of PI3-kinase was blocked. Furthermore, phosphorylation of the downstream factor p70(S6K) did not occur. In contrast, AG1024 did not prevent MAPK or Src kinase activation by AngII. AG1024 also did not inhibit AngII-dependent cell migration, although this process was blocked by inhibitors of the epidermal growth factor and platelet-derived growth factor receptors. Transactivation of the IGF-I receptor is therefore a critical mediator of PI3-kinase activation by AngII but is not required for stimulation of the MAPK cascade.     

Endothelial Nox2 overexpression potentiates vascular oxidative stress and hemodynamic response to angiotensin II: studies in endothelial-targeted Nox2 transgenic mice

Vascular disease states are associated with endothelial dysfunction and increased production of reactive oxygen species (ROS) derived from vascular NADPH oxidases in both vascular smooth muscle cells (VSMCs) and endothelial cells. Recent evidence suggests an important role for VSMC NADPH oxidases in vascular ROS production. However, it is unclear whether increased NADPH oxidase activity in endothelial cells alone is sufficient to alter overall vascular ROS production and hemodynamics. We sought to address these questions using transgenic mice with endothelial-targeted overexpression of the catalytic subunit of NADPH oxidase, Nox2. Aortas of Nox2 transgenic (Nox2-Tg) mice had increased total Nox2 mRNA and protein levels compared with wild-type littermates. Both p22phox mRNA and protein levels were also significantly elevated in Nox2-Tg aortas. Aortic superoxide production was significantly increased in Nox2-Tg mice compared with wild-type, but this difference was abolished by endothelial removal. Superoxide dismutase inhibition increased superoxide release and levels of Mn superoxide dismutase protein were significantly elevated in aortas from Nox2-Tg mice compared with wild type. Increased ROS production from endothelial Nox2 overexpression led to increased endothelial nitric oxide synthase protein and extracellular signal-regulated kinase 1/2 phosphorylation in transgenic aortas. Basal blood pressure was similar, however the pressor responses to both acute and chronic angiotensin II administration were significantly increased in Nox2-Tg mice compared with wild type. These results demonstrate that endothelial-targeted Nox2 overexpression is sufficient to increase vascular NADPH oxidase activity, activate downstream signaling pathways, and potentiate the hemodynamic response to angiotensin II, despite compensatory increases in vascular antioxidant enzymes. Endothelial cell Nox2-containing NADPH oxidase plays an important functional role in vascular redox signaling.     

Lysophosphatidylcholine activates extracellular signal-regulated kinases 1/2 through reactive oxygen species in rat vascular smooth muscle cells

Lysophosphatidylcholine (lysoPC) acts on vascular smooth muscle cells (VSMCs) to produce a mitogenic response through the activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we examined the importance of reactive oxygen species (ROS) in lysoPC-stimulated ERK1/2 activation in cultured rat VSMCs. Treatment with lysoPC for 3 minutes caused a 2-fold increase in intracellular ROS that was blocked by the NADH/NADPH oxidase inhibitor, diphenylene iodonium (DPI). Antioxidants, N-acetyl-L-cysteine, glutathione monoester, or alpha -tocopherol, inhibited ERK1/2 activation by lysoPC. Almost identical results were obtained in the VSMC line A10. Pretreatment of VSMCs with DPI but not allopurinol or potassium cyanide (KCN) abrogated the activation of ERK1/2. The Flag-tagged p47phox expressed in A10 cells was translocated from the cytosol to the membrane after 2 minutes of stimulation with lysoPC. The overexpression of dominant-negative p47phox in A10 cells suppressed lysoPC-induced ERK activation. The ROS-dependent ERK activation by lysoPC seems to involve protein kinase C- and Ras-dependent raf-1 activation. Induction of c-fos expression and enhanced AP-1 binding activity by lysoPC were also inhibited by DPI and NAC. Taken together, these data suggest that ROS generated by NADH/NADPH oxidase contribute to lysoPC-induced activation of ERK1/2 and subsequent growth promotion in VSMCs.     

NAD(P)H oxidase inhibition improves endothelial function in rat and human blood vessels

The NO/superoxide (O2-) balance is a key regulator of endothelial function. O2- levels are elevated in many forms of cardiovascular disease; therefore, decreasing O2- should improve endothelial function. To explore this hypothesis, internal mammary arteries and saphenous veins, obtained from patients undergoing coronary artery revascularization, and aortic and carotid arteries from Wistar-Kyoto and spontaneously hypertensive stroke-prone rats were incubated with O2- dismutase or NAD(P)H oxidase inhibitors. O2- levels were measured using lucigenin chemiluminescence; NO bioavailability was assessed in organ chambers; and mRNA expression of NAD(P)H oxidase components was quantified by use of a Light Cycler. In rat arteries, phenylarsine oxide, 4-(2-aminoethyl)-benzenesulfanyl fluoride, and apocynin all decreased NADH-stimulated O2- production, but only apocynin increased NO bioavailability. In human internal mammary arteries and saphenous veins, apocynin decreased NAD(P)H-stimulated O2- generation and caused vasorelaxation that was endothelium dependent and reversed on addition of the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester. In addition, it increased NO production from cultured human endothelial saphenous vein cells. Polyethylene-glycolated O2- dismutase also increased NO bioavailability in rat carotid arteries and human blood vessels, but the effects were smaller than those observed with apocynin. NADH-generated O2- and mRNA expression of p22(phox), gp91(phox), and nox-1 were comparable between the 2 strains of rat. This is the first study to demonstrate pharmacological effects of apocynin in human blood vessels. The increases in NO bioavailability shown here suggest that the NAD(P)H oxidase pathway may be a novel target for drug intervention in cardiovascular disease.     

Angiotensin II-dependent chronic hypertension and cardiac hypertrophy are unaffected by gp91phox-containing NADPH oxidase

The gp91phox-containing NADPH oxidase is the major source of reactive oxygen species (ROS) in the cardiovascular system and inactivation of gp91phox has been reported to blunt hypertension and cardiac hypertrophy seen in angiotensin (Ang) II-infused animals. In the current study, we sought to determine the role of gp91phox-derived ROS on cardiovascular outcomes of chronic exposure to Ang II. The gp91phox-deficient mice were crossed with transgenic mice expressing active human renin in the liver (TTRhRen). TTRhRen mice exhibit chronic Ang II-dependent hypertension and frank cardiac hypertrophy by age 10 to 12 weeks. Four genotypes of mice were generated: control, TTRhRen trangenics (TTRhRen), gp91phox-deficient (gp91-), and TTRhRen transgenic gp91phox-deficient (TTRhRen/gp91-). Eight to 10 mice/group were studied. ROS levels were significantly reduced (P<0.05) in the heart and aorta of TTRhRen/gp91- and gp91-mice compared with control counterparts, and this was associated with reduced cardiac, aortic, and renal NADPH oxidase activity (P<0.05). Systolic blood pressure (SBP), cardiac mass, and cardiac fibrosis were increased in TTRhRen versus controls. In contrast to its action on ROS generation, gp91phox inactivation had no effect on development of hypertension or cardiac hypertrophy in TTRhRen mice, although interstitial fibrosis was reduced. Cardiac and renal expression of gp91phox homologues, Nox1 and Nox4, was not different between groups. Thus, although eliminating gp91phox-associated ROS production may be important in cardiovascular consequences in acute insult models, it does not prevent the development of hypertension and cardiac hypertrophy in a model in which the endogenous renin-angiotensin system is chronically upregulated.     

Activation of nicotinamide adenine dinucleotide phosphate (reduced form) oxidase by advanced glycation end products links oxidative stress to altered retinal vascular endothelial growth factor expression

Increasing evidence indicates that advanced glycation end products (AGEs) promote retinal alterations through oxidative stress. However, the pathways involved in AGE-induced generation of reactive oxygen species (ROS) in retinal cells are poorly defined. In the present study, we investigated the role of nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase in AGE-induced ROS intracellular generation and vascular endothelial growth factor (VEGF) expression in bovine retinal endothelial cells (BRECs). Incubation of BRECs with 100 microg/mL AGEs increased ROS generation and VEGF expression in these cells. Treatment of the cells with the NADPH oxidase inhibitors, apocynin and diphenylene iodonium, inhibited these effects. In retinal endothelial cells exposed to AGEs, translocation of protein kinase C (PKC)-beta2 and p47phox was observed. Inhibition of PKC by treatment of the cells with calphostin C, GF10923X, and LY379196 totally suppressed AGE-mediated p47phox translocation and ROS generation. Incubation of BRECs with gliclazide inhibited AGE-induced PKC-beta2 and p47phox translocation and totally abrogated AGE-mediated ROS generation and VEGF expression. Overall, these results demonstrate that AGEs induce intracellular ROS generation and VEGF expression in retinal endothelial cells through a PKC-dependent activation of NADPH oxidase. Inhibition of retinal NADPH oxidase expression and ROS generated by this system provides a new potential mechanism by which gliclazide may affect retinal VEGF expression and exert a beneficial effect on diabetic retinopathy.     

Phospholipase C expression and activity in smooth muscle cells of renal arterioles and aorta of young, spontaneously hypertensive rats during culture

BACKGROUND: Phospholipase C (PLC)-beta(1) and -delta(1), but not -gamma(1), protein expressions in fresh renal arterioles and aorta are greater in 6-week-old, spontaneously hypertensive rats (SHRs) versus normotensive Wistar-Kyoto rats (WKYs). This PLC activity is also greater in both vessels of SHRs. In the present study, we tested whether cultured vascular smooth muscle cells (VSMCs) of preglomerular arterioles and aorta accurately reflect strain differences observed in fresh vessels, with VSMCs of SHRs predicted to have higher levels of PLC isozymes and enzyme activity. We assessed the stability of variables over passages 3 to 11. METHODS: The VSMCs were isolated and cultured using standard techniques. The PLC-isozyme protein levels and catalytic activity were determined by Western blot analysis and inositol 1,4,5-trisphosphate (IP(3)) production, respectively. RESULTS: Immunoblots showed expression of PLC-gamma(1) and -delta(1), but not PLC-beta(1), in VSMCs from both vessels. Arteriolar VSMCs of SHRs had three-to-fivefold higher levels of PLC-gamma(1) and -delta(1) during passages 3 to 8. Enzymatic activity in these VSMCs was higher in SHRs versus WKYs, especially during passages 6 to 11. In contrast, cultured aortic VSMCs of SHRs had two-to-threefold lower densities of PLC-gamma(1) and -delta(1) protein. CONCLUSIONS: Compared with fresh resistance arterioles and aorta, cultured VSMCs exhibit changes in PLC-isozyme protein levels and enzyme activity that vary with passage. The differences between cultured VSMCs of SHRs and WKYs do not accurately reflect those in fresh resistance and conduit vessels, either qualitatively or quantitatively. The results of VSMC culture studies should be interpreted with caution and should ideally be compared with more physiologically relevant fresh preparations.     

Lipid rafts keep NADPH oxidase in the inactive state in human renal proximal tubule cells

Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22(phox) and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D(1)-like receptor agonist, fenoldopam (1 micromol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7+/-3.3%). In contrast, cholesterol depletion (2% methyl-beta-cyclodextrin [beta CD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0+/-10.5% versus control, 103.1+/-3.4%), which was reversed by cholesterol repletion (118.9+/-9.9%). Moreover, NADPH oxidase activation by beta CD (145.5+/-9.0%; control: 98.6+/-1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4+/-3.2%) and diphenylene iodonium (9.5+/-3.3%). Furthermore, beta CD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0+/-5.1% versus beta CD: 162.0+/-2.0%) or Nox4 (108.0+/-10.8% versus beta CD: 152.0+/-9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state.