Quercetin downregulates NADPH oxidase, increases eNOS activity and prevents endothelial dysfunction in spontaneously hypertensive rats

BACKGROUND AND OBJECTIVE: Several studies have found that chronic treatment with the dietary flavonoid quercetin lowers blood pressure and restores endothelial dysfunction in hypertensive animal models. We hypothesized that increased endothelial nitric oxide synthase (eNOS) and/or decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase protein expression and activity, and reduced reactive oxygen species might be involved in the improvement of endothelial function induced by quercetin in spontaneously hypertensive rats (SHR). DESIGN AND METHODS: Male SHR and Wistar-Kyoto (WKY) rats (5 weeks old) were treated with quercetin (10 mg/kg) or vehicle for 13 weeks. Changes in vascular expression of eNOS, caveolin-1 and p47 were analysed by Western blot, eNOS activity by conversion of [H]arginine to L-[H]citrulline, and NADPH oxidase activity by NADPH-enhanced chemoluminescence of lucigenin. RESULTS: In SHR, quercetin reduced the increase in blood pressure and heart rate and enhanced the endothelium-dependent aortic vasodilation induced by acetylcholine, but had no effect on the endothelium-independent response induced by nitroprusside. However, quercetin had no effect on endothelium-dependent vasoconstriction and aortic thromboxane B2 production. Compared to WKY, SHR showed upregulated eNOS and p47 protein expression, downregulated caveolin-1 expression, increased NADPH-induced superoxide production but, paradoxically, eNOS activity was reduced. Chronic quercetin treatment prevented all these changes in SHR. In WKY, quercetin had no effect on blood pressure, endothelial function or the expression or activity of the proteins analysed. CONCLUSIONS: Enhanced eNOS activity and decreased NADPH oxidase-mediated superoxide anion (O2) generation associated with reduced p47 expression appear to be essential mechanisms for the improvement of endothelial function and the antihypertensive effects of chronic quercetin.     

Reversal of endothelial nitric oxide synthase uncoupling and up-regulation of endothelial nitric oxide synthase expression lowers blood pressure in hypertensive rats.

OBJECTIVES: We sought to examine the hypothesis that a pharmacologic up-regulation of endothelial nitric oxide synthase (eNOS) combined with a reversal of eNOS uncoupling provides a protective effect against cardiovascular disease. BACKGROUND: Many cardiovascular diseases are associated with oxidant stress involving protein kinase C (PKC) and uncoupling of eNOS. METHODS: Messenger ribonucleic acid (mRNA) expression was analyzed with RNase protection assay or quantitative real-time polymerase chain reaction, vascular nitric oxide (NO) with spin trapping, and reactive oxygen species (ROS) with dihydroethidium fluorescence. RESULTS: Aortas of spontaneously hypertensive rats (SHR) showed an elevated production of ROS when compared with aortas of Wistar-Kyoto rats (WKY). The aortic expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits (Nox1, Nox2, Nox4, and p22phox) was higher in SHR compared with WKY. In SHR, aortic production of ROS was reduced by the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME), indicating eNOS "uncoupling" in hypertension. Oral treatment with the PKC inhibitor midostaurin reduced aortic Nox1 expression, diminished ROS production, and reversed eNOS uncoupling in SHR. Aortic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) were significantly reduced in SHR compared with WKY. Midostaurin normalized BH4 levels in SHR. In both WKY and SHR, midostaurin increased aortic expression of eNOS mRNA and protein, stimulated bioactive NO production, and enhanced relaxation of the aorta to acetylcholine. Midostaurin lowered blood pressure in SHR and, to a lesser extent, in WKY; the compound did not change blood pressure in WKY made hypertensive with L-NAME. CONCLUSIONS: Pharmacologic interventions that combine eNOS up-regulation and reversal of eNOS uncoupling can markedly increase bioactive NO in the vasculature and produce beneficial hemodynamic effects such as a reduction of blood pressure.     

Apocynin restores endothelial dysfunction in streptozotocin diabetic rats through regulation of nitric oxide synthase and NADPH oxidase expressions

AimIncreased production of reactive oxygen species (ROS) in the diabetic vasculature results in the impairment of nitric oxide (NO)-mediated relaxations leading to impaired endothelium-dependent vasodilation. An important source of ROS is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the inhibition of this enzyme is an active area of interest. This study aimed to investigate the effects of apocynin, an NADPH oxidase inhibitor, on endothelial dysfunction and on the expression of NO synthase (NOS) and NADPH oxidase in thoracic aorta of diabetic rats.MethodStreptozotocin (STZ)-diabetic rats received apocynin (16 mg/kg per day) for 4 weeks. Endothelium-dependent and -independent relaxations were determined in thoracic aortic rings. Western blotting and RT-PCR analysis were performed for NOSs and NADPH oxidase in the aortic tissue.ResultsAcetylcholine-induced relaxations and l-NAME-induced contractions were decreased in diabetic aorta. The decrease in acetylcholine and l-NAME responses were prevented by apocynin treatment without a significant change in plasma glucose levels. Endothelial NOS (eNOS) protein and mRNA expression exhibited significant decrease in diabetes, while protein and/or mRNA expressions of inducible NOS (iNOS) as well as p22^phox and gp91^phox subunits of NADPH oxidase were increased, and these alterations were markedly prevented by apocynin treatment.ConclusionNADPH oxidase expression is increased in diabetic rat aorta. NADPH oxidase-mediated oxidative stress is accompanied by the decreased eNOS and increased iNOS expressions, contributing to endothelial dysfunction. Apocynin effectively prevents the increased NADPH oxidase expression in diabetic aorta and restores the alterations in NOS expression, blocking the vicious cycle leading to diabetes-associated endothelial dysfunction.     

Impaired activities of antioxidant enzymes elicit endothelial dysfunction in spontaneous hypertensive rats despite enhanced vascular nitric oxide generation

OBJECTIVE: Enhanced oxidative stress is involved in mediating the endothelial dysfunction associated with hypertension. The aim of this study was to investigate the relative contributions of pro-oxidant and anti-oxidant enzymes to the pathogenesis of endothelial dysfunction in genetic hypertension. METHODS: Dilator responses to endothelium-dependent and endothelium-independent agents such as acetylcholine (ACh) and sodium nitroprusside were measured in the thoracic aortas of 28-week-old spontaneously hypertensive rats (SHR) and their matched normotensive counterparts, Wistar Kyoto rats (WKY). The activity and expression (mRNA and protein levels) of endothelial nitric oxide synthase (eNOS), p22-phox, a membrane-bound component of NAD(P)H oxidase, and antioxidant enzymes, namely, superoxide dismutases (CuZn- and Mn-SOD), catalase and glutathione peroxidase (GPx), were also investigated in aortic rings. RESULTS: Relaxant responses to ACh were attenuated in phenylephrine-precontracted SHR aortic rings, despite a 2-fold increase in eNOS expression and activity. Although the activity and/or expression of SODs, NAD(P)H oxidase (p22-phox) and GPx were elevated in SHR aorta, catalase activity and expression remained unchanged compared to WKY. Pretreatment of SHR aortic rings with the inhibitor of xanthine oxidase, allopurinol, and the inhibitor of cyclooxygenase, indomethacin, significantly potentiated ACh-induced relaxation. Pretreatment of SHR rings with catalase and Tiron, a superoxide anion (O(2)(-)) scavenger, increased the relaxant responses to the levels observed in WKY rings whereas pyrogallol, a O(2)(-)-generator, abolished relaxant responses to ACh. CONCLUSION: These data demonstrate that dysregulation of several enzymes, resulting in oxidative stress, contributes to the pathogenesis of endothelial dysfunction in SHR and indicate that the antioxidant enzyme catalase is of particular importance in the reversal of this defect.     

Evidence for an estradiol-agonistic action of nebivolol in spontaneously hypertensive rats

OBJECTIVES: Unlike classical beta1-selective blockers, nebivolol (NEB) has vasodilatory properties due to the release of nitric oxide (NO) by a mechanism that is, so far, unknown. We hypothesized that NEB stimulates NO release by binding to estrogen receptors (ER) and subsequent activation of endothelial NO synthase (eNOS). The aim of this study was to elucidate the underlying mechanism of NEB action by investigating estradiol-dependent effects of NEB on the NO system in spontaneously hypertensive rats (SHR). METHODS: The effects of NEB on the NO system were determined by measuring urinary nitrate/nitrite (NOx) as well as eNOS and caveolin-1 protein expression in aortae. RESULTS: NEB did not influence NOx excretion in sham-operated (SO) female rats during proestrus. In male and ovariectomized female (OVX) rats, NEB increased NOx excretion significantly, whereas N(G)-nitro-L-arginine methyl ester (L-NAME) inhibited the NEB-induced increase in NOx. ER blockade with ICI182,780 prevented NEB-induced NOx excretion in OVX rats. In the aortae of SO females, NEB treatment did not alter eNOS expression. In OVX rats eNOS expression was increased two-fold after NEB application and this could be prevented by pretreatment with ICI182,780. In contrast to eNOS, NEB did not influence caveolin-1 expression in either group. CONCLUSION: The ability of NEB to up-regulate NOx excretion in male and OVX SHR and the inhibitory effect of ICI182,780 on NEB-induced NOx excretion suggests that NEB has an estradiol-agonistic action in vivo. NEB provokes NO generation by up-regulation of eNOS protein expression, whereas the expression of the negative eNOS regulator caveolin-1 remains unaffected.     

Tea polyphenols regulate nicotinamide adenine dinucleotide phosphate oxidase subunit expression and ameliorate angiotensin II-induced hyperpermeability in endothelial cells.

Out-of-control reactive oxygen species (ROS) signaling is one of the key events in the pathogenesis of endothelial dysfunction and essential hypertension. We observed that tea polyphenols decreased the production of ROS via regulation of the protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in bovine carotid artery endothelial cells (BCAECs). Both green tea polyphenols (GTP) and black tea polyphenols (BTP) down-regulated the expression of NADPH oxidase subunits p22phox and p67phox while up-regulating catalase expression (p < 0.05, respectively). Pre-treatment with GTP or BTP for 24 h significantly decreased the superoxide anion level (p < 0.05) and permeable fluorescence intensities in Ang II-stimulated BCAECs. A decrease in cell permeability was also observed by pre-treatment with diphenylene iodonium chloride (DPI) or vitamin E (p < 0.05, respectively). The result demonstrates that tea polyphenols alleviate angiotensin (Ang) II-induced hyperpermeability mainly by decreasing ROS production. Our results suggest that tea polyphenols regulate ROS-related protein expression and may be beneficial in preventing endothelial cell dysfunction and development of cardiovascular diseases, including hypertension.     

Activation of protein kinase C and nicotinamide adenine dinucleotide phosphate oxidase in leukocytes of spontaneously hypertensive rats.

The involvement of oxidative stress in polymorphonuclear leukocytes (PMN) in the pathogenesis of hypertension remains to be elucidated. We analyzed the generation of reactive oxygen species (ROS) by the circulating and peritoneally infiltrating PMN from spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). Flow cytometric analysis revealed that ROS generation by PMN from SHR was higher than that from WKY before (at 6 weeks of age) and after (at 16 weeks of age) the onset of hypertension. In vivo, ROS generation by PMN from SHR, but not that by PMN from WKY, was significantly suppressed by 10-week treatment with 50 mg/kg/day carvedilol, and this treatment did not affect blood pressure. Western blotting analysis revealed that protein kinase C alpha (PKCalpha), but not PKCbetaI or betaII, was activated more strongly in PMN from SHR than in PMN from WKY. Furthermore, expression of p47phox of nicotinamide adenine dinucleotide phosphate oxidase, but not of p67phox, in PMN from SHR was higher than that in PMN from WKY. These results suggest that ROS generation by PMN is principally enhanced in SHR through activation of PKCalpha and p47phox.     

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.     

Increased oxidative stress impairs endothelial modulation of contractions in arteries from spontaneously hypertensive rats

OBJECTIVES: The endothelium modulates vascular contractions. We investigated the effects of oxidative stress on endothelial modulation of contractions in hypertension. METHODS: Changes in isometric tension of femoral arterial rings from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats were recorded. RESULTS: The contractile response to norepinephrine of arteries with endothelium was greater in SHR than in WKY rats (P < 0.0001). Endothelium removal augmented the norepinephrine-induced contraction (P < 0.05). The augmentation was more pronounced in WKY than in SHR, which resulted in comparable contraction of arteries without endothelium in both strains. Nomega-nitro-L-arginine methyl ester (100 micromol/l) mimicked the effect of endothelium removal. Production of nitric oxide (NO, assessed by measuring nitrite/nitrate concentrations) during the contraction was not different between SHR and WKY. Vitamin C suppressed the contraction of arteries with endothelium from SHR but not from WKY (P < 0.05). Diphenyleneiodonium and apocynin, inhibitors of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase, attenuated the contraction of arteries with endothelium from SHR (P < 0.001) but not WKY, but did not affect contractions induced by serotonin. Superoxide generated by xanthine oxidase/hypoxanthine enhanced the norepinephrine-induced contraction of arteries with endothelium from WKY (P < 0.0001), and this effect was reversed by vitamin C. CONCLUSIONS: In rat femoral arteries, NO released from the endothelium modulates vascular contraction. In SHR, production of superoxide by NADH/NADPH oxidase, which may be activated by norepinephrine, is enhanced, resulting in the inactivation of NO and impairment of endothelial modulation of vascular contractions. Vascular oxidative stress may contribute to the altered circulation in hypertension by impairing endothelial modulation of vascular contractions.     

Long-term administration of rho-kinase inhibitor ameliorates renal damage in malignant hypertensive rats

We have shown recently that fasudil, a Rho-kinase inhibitor, has renoprotective effects in salt-sensitive hypertensive rats. We hypothesized that activation of Rho-kinase is involved in the pathogenesis of glomerulosclerosis in malignant hypertensive rats. To test this hypothesis, we studied the following 4 groups: control Wistar-Kyoto rats, untreated deoxycorticosterone-acetate salt spontaneously hypertensive rats (DOCA-SHR), low-dose fasudil-treated DOCA-SHR, and high-dose fasudil-treated DOCA-SHR. After 3 weeks of treatment, the effects of fasudil were examined. DOCA-SHR was characterized by increased blood pressure (BP); increased kidney weight; decreased renal function; increased proteinuria; abnormal histological findings; increased monocyte/macrophage infiltration; increased urinary 8-isoprostran levels; increased gene expression of collagen I, collagen III, transforming growth factor-beta, and reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits (p40phox, p47phox, and p67phox); and decreased gene expression of endothelial NO synthase (eNOS) in the renal cortex as compared with Wistar-Kyoto rats. Long-term high-dose fasudil treatment significantly improved renal function and histological findings without changing BP, as compared with untreated DOCA-SHR. Interestingly, long-term fasudil treatment significantly decreased monocyte/macrophage infiltration and urinary 8-isoprostran excretion, in association with decreased mRNA levels of transforming growth factor-beta, collagen I, collagen III, and NADPH oxidase subunits (p40phox, p47phox, and p67phox), and increased mRNA levels of eNOS in the renal cortex. Long-term low-dose fasudil treatment tended to improve these variables slightly but did not affect most of them significantly. Our results suggest that long-term fasudil treatment provides renoprotective effects independent of BP-lowering activity. These renoprotective effects are associated with inhibition of extracellular matrix gene expression, monocyte/macrophage infiltration, oxidative stress, and upregulation of eNOS gene expression.     

Strain-dependent variation in vascular responses to nitric oxide in the isolated murine heart

Numerous studies in the literature have employed gene-modified mice to investigate vascular function. However, only very limited information exists on baseline murine vascular physiology or on potential variations between different strains. We therefore compared coronary and aortic vascular responses to endothelium-derived vasodilators and exogenous nitric oxide (NO) in three commonly used mouse strains and correlated these data with expression of eNOS, NADPH oxidase subunits, gp91(phox) and p67(phox), and superoxide production. Isolated perfused hearts from MF1, 129sv and C57BL/6J mice were subjected to: (a) increasing doses of bradykinin, acetylcholine and sodium nitroprusside, and (b) bolus doses of adenosine and the NO synthase inhibitor, N(G)-monomethyl- L -arginine. Vascular responses of thoracic aortic rings were assessed for comparison. Expression of eNOS and NADPH oxidase subunits was assessed by immunoblotting, and superoxide production by lucigenin-enhanced chemiluminescence. Coronary vasodilator responses to bradykinin, acetylcholine and sodium nitroprusside were significantly attenuated in MF1 compared with C57BL/6J and 129sv hearts. Similarly, aortic relaxation to acetylcholine was significantly impaired in MF1 aortic rings compared with in C57BL/6J aortae; these differences were reversed by Tiron. N(G)-monomethyl- L -arginine induced significantly less vasoconstriction in MF1 and 129sv hearts compared with C57BL/6J. No differences in aortic relaxation to A23187 or sodium nitroprusside were observed. Cardiac and aortic superoxide production and cardiac expression of p67(phox) and gp91(phox) were significantly greater in MF1 mice compared with the other strains. There is significant strain-dependent variation in coronary and aortic vascular responsiveness in mice, which may reflect differences in the balance between NO and superoxide generation.     

Albuminuria, expression of nicotinamide adenine dinucleotide phosphate oxidase and monocyte chemoattractant protein-1 in the renal tubules of hypertensive Dahl salt-sensitive rats.

In chronic renal diseases, experimental and human data suggest that excess albumin filtered through the glomerular capillary barrier is over-reabsorbed by proximal tubular cells, thereby activating these cells and upregulating the expression of chemokines. On the other hand, a high-salt diet has been shown to induce proteinuria in hypertensive Dahl salt-sensitive (DSS) rats, accompanied with the expression of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the kidney. In the current study, we therefore examined albuminuria and the expressions of NADPH oxidase and monocyte chemoattractant protein-1 (MCP-1) in the renal tubular cells in hypertensive DSS rats, as well as the effects of the antioxidant N-acetylcysteine (NAC) on each of these parameters. DSS rats were fed a normal-salt diet (0.24% NaCl), a high-salt diet (8% NaCl), or a high-salt diet plus NAC supplementation (15 mg/mL drinking water) for 4 weeks. The high-salt diet provoked an increase in glomerular injuries accompanied with albuminuria and in urinary H2O2 and MCP-1 excretion. Immunohistochemical analysis showed the prominent expression of MCP-1 in the dilated tubular cells, where the NADPH oxidase subunit p47phox was also expressed. The current results suggest that albuminuria caused expression of NADPH oxidase and MCP-1 in the dilated renal tubules, resulting in interstitial inflammation and migration of mononuclear cells in DSS rats, because blockade of albuminuria by NAC counteracted the p47phox and MCP-1 expression.     

A gp91phox containing NADPH oxidase selectively expressed in endothelial cells is a major source of oxygen radical generation in the arterial wall

Reactive oxygen species (ROS) play an important role in regulating vascular tone and intracellular signaling; the enzymes producing ROS in the vascular wall are, however, poorly characterized. We investigated whether a functionally active NADPH oxidase similar to the leukocyte enzyme, ie, containing the subunits p22phox and gp91phox, is expressed in endothelial cells (ECs) and smooth muscle cells (SMCs). Phorbol 12-myristate 13-acetate (PMA), a stimulus for leukocyte NADPH oxidase, increased ROS generation in cultured ECs and endothelium-intact rat aortic segments, but not in SMCs or endothelium-denuded arteries. NADPH enhanced chemiluminescence in all preparations. p22phox mRNA and protein was detected in ECs and SMCs, whereas the expression of gp91phox was confined to ECs. Endothelial gp91phox was identical to the leukocyte form as determined by sequence analysis. In contrast, mitogenic oxidase-1 (mox1) was expressed in SMCs, but not in ECs. To determine the functional relevance of gp91phox expression, experiments were performed in aortic segments from wild-type, gp91phox(-/-), and endothelial NO synthase (eNOS)(-/-) mice. PMA-induced ROS generation was comparable in aortae from wild-type and eNOS(-/-) mice, but was attenuated in segments from gp91phox(-/-) mice. Endothelium-dependent relaxation was greater in aortae from gp91phox(-/-) than from wild-type mice. The ROS scavenger tiron increased endothelium-dependent relaxation in segments from wild-type, but not from gp91phox(-/-) mice. These data demonstrate that ECs, in contrast to SMCs, express a gp91phox-containing leukocyte-type NADPH oxidase. This enzyme is a major source for arterial ROS generation and affects the bioavailability of endothelium-derived NO.     

Phagocytosis-Coupled Activation of the Superoxide-Producing Phagocyte Oxidase, a Member of the NADPH Oxidase (Nox) Family

The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a crucial role in host defense by neutrophils and macrophages. When cells ingest invading microbes, this enzyme becomes activated to reduce molecular oxygen to superoxide, a precursor of microbicidal oxidants, in the phagosome. The catalytic core of the oxidase is membrane-bound cytochrome b558, which comprises gp91phox and p22phox. gp91phox belongs to the NADPH oxidase (Nox) family, which contains the entire electron-transporting apparatus from NADPH to molecular oxygen. In resting neutrophils, cytochrome b558 is mainly present in the membrane of the specific granule, an intracellular component, and is targeted to the phagosomal membrane during phagocytosis. Activation of gp91phox involves the integrated function of cytoplasmic proteins such as p47phox, p67phox, p40phox, and the small guanosine triphosphatase Rac; these proteins translocate to the phagosomal membrane to interact with cytochrome b558, leading to superoxide production. Here we describe a current molecular model for phagocytosis-coupled activation of the NADPH oxidase.     

The ligands/activators for peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma increase Cu2+,Zn2+-superoxide dismutase and decrease p22phox message expressions in primary endothelial cells

We examined the effects of a variety of ligands/activators of the peroxisome proliferator-activated receptor (PPAR) on the expression of the superoxide scavenger enzyme, Cu2+,Zn2+-superoxide dismutase (CuZn-SOD), and the superoxide generating enzyme nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase in primary cultures of human umbilical vein endothelial cells (HUVEC) and human aorta endothelial cells (HAEC). Our data show that 3 types of PPARs, PPARalpha, PPARbeta/delta/Nuc1, and PPARgamma are expressed in endothelial cells. Bezafibrate, which is a ligand/activator for PPARalpha, increased the CuZn-SOD gene expression and protein levels in endothelial cells. Troglitazone and pioglitazone, which are ligands/activators for PPARgamma, also induced PPARalpha gene and protein expression and increased CuZn-SOD gene expression and protein levels in addition to increasing PPARgamma gene and protein expression in endothelial cells. Moreover, with treatment of monounsaturated and polyunsaturated fatty acids (PUFA), the CuZn-SOD mRNA levels were positively correlated with PPARalpha mRNA levels (r = .872, P < .0001) in primary endothelial cells. In addition, the phorbol myristate acetate (PMA)-stimulated or PMA-nonstimulated 22-kd a-subunit (p22phox) mRNA levels and 47-kd a-subunit (p47phox) protein levels in NADPH oxidase were decreased by treatment with PPARalpha and PPARgamma ligands/activators. These results suggest that PPARalpha and PPARgamma gene and protein expression in endothelial cells may play a physiologic role as radical scavengers, although the details of these mechanisms remain to be established.     

Cardioprotective effect of angiotensin II type 1 receptor antagonist associated with bradykinin-endothelial nitric oxide synthase and oxidative stress in Dahl salt-sensitive hypertensive rats

OBJECTIVES: The interactions between eNOS or oxidative stress and bradykinin under long-term treatment of angiotensin II type 1 receptor antagonists (ATRA) remain unknown. To elucidate the molecular mechanisms of the cardioprotective effect of ATRA, we evaluated whether valsartan affects the bradykinin-eNOS and nicotinamide adenine dinucleotide (NAD(P)H) oxidase pathway. METHODS: After 5 weeks of feeding an 8% NaCl diet to 6-week-old Dahl salt-sensitive hypertensive (DS) rats, a distinct stage of concentric left ventricular hypertrophy (LVH) was noted. Six-week-old DS rats were treated with one of the following drug combinations for 5 weeks until the onset of LVH: vehicle; bradykinin B2 receptor antagonist FR172,357 alone; high-dose hydralazine; low-dose hydralazine; high-dose valsartan; low-dose valsartan; high and low-dose valsartan plus FR172,357. Age-matched Dahl salt-resistant rats fed the same diet served as controls. RESULTS: eNOS expression and activity, which was decreased in hypertrophy, was increased by high or low-dose valsartan, but not by high and low-dose valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. The increased expression of NAD(P)H oxidase p22phox, p47phox, p67phox, and gp91phox in DS rats was suppressed by high or low-dose valsartan, but not by high or low-dose valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. Valsartan effectively inhibited vascular lesion formation and suppressed the expression of transforming growth factor-beta1, connective tissue growth factor, and type I collagen, but not valsartan plus FR172,357 or FR172,357 alone or high and low-dose hydralazine. CONCLUSION: These findings suggest that valsartan may have cardioprotective effects in this model, partly associated with the bradykinin-eNOS and oxidative stress pathway.     

Creation of a genetic system for analysis of the phagocyte respiratory burst: high-level reconstitution of the NADPH oxidase in a nonhematopoietic system

The phagocyte nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase was functionally reconstituted in monkey kidney COS-7 cells by transfection of essential subunits, gp91(phox), p22(phox), p47(phox), and p67(phox). COS-7 cells express the essential small guanosine 5'-triphosphatase, Rac1. Transgenic COS-phox cells were capable of arachidonic acid-induced NADPH oxidase activity up to 80% of that of human neutrophils, and of phorbol myristate acetate (PMA)-induced activity up to 20% of that of neutrophils. Expression of all 4 phox components was required for enzyme activity, and enzyme activation was associated with membrane translocation of p47(phox), p67(phox), and Rac1. Expression of p47(phox) Ser303Ala/Ser304Ala or Ser379Ala phosphorylation-deficient mutants resulted in significantly impaired NAPDH oxidase activity, compared with expression of wild-type p47(phox) or the p47(phox) Ser303Glu/Ser304Glu phosphorylation mimic, suggesting that p47(phox) phosphorylation contributes to enzyme activity in the COS system, as is the case in neutrophils. Hence, COS-phox cells should be useful as a new whole-cell model that is both capable of high-level superoxide production and readily amenable to genetic manipulation for investigation of NADPH oxidase function. PMA-elicited superoxide production in COS-phox cells was regulated by activation of protein kinase C (PKC) and Rac. Although COS-7 cells differ from human neutrophils in PKC isoform expression, transient expression of major neutrophil isoforms in COS-phox cells did not increase PMA-induced superoxide production, suggesting that endogenous isoforms were not rate limiting. Val204 in p67(phox), previously shown to be required for NADPH oxidase activity under cell-free conditions, was found to be essential for superoxide production by intact COS-phox cells, on the basis of transfection studies using a p67(phox) (Val204Ala) mutant.