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.     

Evidence for a causal role of the renin-angiotensin system in vascular dysfunction associated with insulin resistance

Excess production of superoxide anion in response to angiotensin II plays a central role in the transduction of signal molecules and the regulation of vascular tone. We examined the ability of insulin resistance to stimulate superoxide anion production and investigated the identity of the oxidases responsible for its production. Rats were fed diets containing 60% fructose (fructose-fed rats) or 60% starch (control rats) for 8 weeks. In aortic homogenates from fructose-fed rats, the superoxide anion generated in response to NAD(P)H was more than 2-fold higher than that of control rats. Pretreatment of the aorta from fructose-fed rats with inhibitors of NADPH oxidase significantly reduced superoxide anion production. In the isolated aorta, contraction induced by angiotensin II was more potent in fructose-fed rats compared with control rats. Losartan normalized blood pressure, NAD(P)H oxidase activity, endothelial function, and angiotensin II-induced vasoconstriction in fructose-fed rats. To elucidate the molecular mechanisms of the enhanced constrictor response to angiotensin II, expressions of angiotensin II receptor and subunits of NADPH oxidase were examined with the use of angiotensin II type 1a receptor knockout (AT1a KO) mice. Expression of AT1a receptor mRNA was enhanced in fructose-fed mice, whereas expression of either AT1b or AT2 was unaltered. In addition, protein expression of each subunit of NADPH oxidase was increased in fructose-fed mice, whereas the expression was significantly decreased in fructose-fed AT1a KO mice. The novel observation of insulin resistance-induced upregulation of AT1 receptor expression could explain the association of insulin resistance with endothelial dysfunction and hypertension.     

NADPH oxidase mediates angiotensin II-induced endothelin-1 expression in vascular adventitial fibroblasts

OBJECTIVE: We have recently reported that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (Ang II) stimulation. However, the mechanism by which this occurs in the adventitia remains unclear. As Ang II has been reported to increase oxidant production by NADPH oxidase, we examined the role of this complex in Ang II stimulated ET-1 expression in vascular adventitial fibroblasts. METHODS AND RESULTS: Adventitial fibroblasts were isolated and cultured from mouse aorta. Cells were treated with Ang II (100 nmol/L) in the presence or absence of NADPH oxidase inhibitors, apocynin (100 micromol/L) and diphenyleneiodonium (10 micromol/L), superoxide scavengers, SOD (350 units/mL), tempol (100 micromol/L), tiron (100 micromol/L), and ET-receptor antagonists (10 microM), BQ123 (for ET(A)-) and BQ788 (for ET(B)-). PreproET-1 mRNA and ET-1 level were determined by relative RT-PCR and ELISA, respectively. Type I procollagen-alpha-I (collagen) level was detected by Western blot. Superoxide anion (superoxide) production was determined by coelenterazine or lucigenin chemiluminescence. Ang II-induced collagen expression was inhibited by BQ123, suggesting that adventitial ET-1 plays a significant role in regulating the extracellular matrix. NADPH oxidase inhibitors and superoxide scavengers significantly decreased Ang II-induced ET-1 mRNA and peptide expression, superoxide production as well as collagen expression. Furthermore, deletion of gp91(phox) (a key subunit of NADPH oxidase) and overexpression of SOD1 attenuated Ang II-induced responses. CONCLUSION: Ang II-evoked expression of ET-1 in adventitial fibroblasts appears to be mediated, at least in part, by NADPH oxidase. Functionally, this mechanism stimulates collagen expression thereby implicating the adventitia as a potential contributor to the vascular pathophysiology associated with oxidative stress and vascular remodeling.     

Vascular but not cardiac remodeling is associated with superoxide production in angiotensin II hypertension

OBJECTIVE: Angiotensin (Ang) II increases reactive oxygen species (ROS), decreases nitric oxide (NO) bioavailability and promotes cardiovascular remodeling. ROS have been identified as critical second messengers of the trophic responses by Ang II. In rats with Ang II-induced hypertension, we investigated the role of ROS in cardiac hypertrophy as well as the remodeling of aortas and mesenteric (resistance) arteries. METHODS: Sprague-Dawley rats received Ang II (0.7 mg/kg per day by mini-pump, n = 7) or vehicle (n = 7) for 5 days. Endothelium-dependent relaxation to acetylcholine (EDR) in aortas was determined in organ baths and in mesenteric resistance vessels in a pressurized myograph. Superoxide (O2) production was measured by lucigenin chemiluminescence, laser-confocal fluorescence microscopy (LCM) and NADPH oxidase assay. RESULTS: Ang II-treated rats developed hypertension (183 +/- 3 versus 138 +/- 4 mmHg, P < 0.05), increased aortic O2 (50%), aortic hypertrophy (12%) and impaired EDR. Mesenteric arteries manifested impaired EDR, increased NADPH oxidase activity (356%) and eutrophic inward remodeling (decreased lumen diameter and increased wall/lumen ratio). However, although Ang II-treated rats developed cardiac hypertrophy (13%), this was not accompanied by an increase in cardiac O2, as measured by lucigenin, LCM or NADPH oxidase assay. On the other hand, cardiac calcineurin, a molecule that promotes cardiac hypertrophy linked to Ang II, was increased by 40% (52 +/- 8 versus 33 +/- 5 pmol/min per mg protein, P < 0.05). CONCLUSION: These studies demonstrate that the role of ROS in Ang II-induced vascular remodeling differ across vascular territories. Although in conduit and resistance vessels, vascular hypertrophy and endothelial dysfunction are linked to increased ROS production, cardiac hypertrophy is not. Instead, cardiac hypertrophy is associated, at least in part, with an increase in calcineurin. These studies unveil novel mechanisms that may play an important role in the pathogenesis of cardiac and vascular injury in hypertension.     

Molecular mechanisms of angiotensin II-mediated mitochondrial dysfunction: linking mitochondrial oxidative damage and vascular endothelial dysfunction

Mitochondrial dysfunction is a prominent feature of most cardiovascular diseases. Angiotensin (Ang) II is an important stimulus for atherogenesis and hypertension; however, its effects on mitochondrial function remain unknown. We hypothesized that Ang II could induce mitochondrial oxidative damage that in turn might decrease endothelial nitric oxide (NO.) bioavailability and promote vascular oxidative stress. The effect of Ang II on mitochondrial ROS, mitochondrial respiration, membrane potential, glutathione, and endothelial NO. was studied in isolated mitochondria and intact bovine aortic endothelial cells using electron spin resonance, dihydroethidium high-performance liquid chromatography -based assay, Amplex Red and cationic dye fluorescence. Ang II significantly increased mitochondrial H2O2 production. This increase was blocked by preincubation of intact cells with apocynin (NADPH oxidase inhibitor), uric acid (scavenger of peroxynitrite), chelerythrine (protein kinase C inhibitor), N(G)-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor), 5-hydroxydecanoate (mitochondrial ATP-sensitive potassium channels inhibitor), or glibenclamide. Depletion of p22(phox) subunit of NADPH oxidase with small interfering RNA also inhibited Ang II-mediated mitochondrial ROS production. Ang II depleted mitochondrial glutathione, increased state 4 and decreased state 3 respirations, and diminished mitochondrial respiratory control ratio. These responses were attenuated by apocynin, 5-hydroxydecanoate, and glibenclamide. In addition, 5-hydroxydecanoate prevented the Ang II-induced decrease in endothelial NO. and mitochondrial membrane potential. Therefore, Ang II induces mitochondrial dysfunction via a protein kinase C-dependent pathway by activating the endothelial cell NADPH oxidase and formation of peroxynitrite. Furthermore, mitochondrial dysfunction in response to Ang II modulates endothelial NO. and generation, which in turn has ramifications for development of endothelial dysfunction.     

Supplementation with tetrahydrobiopterin prevents the cardiovascular effects of angiotensin II-induced oxidative and nitrosative stress

OBJECTIVE: The pteridine cofactor tetrahydrobiopterin (BH4) has emerged as a critical determinant of endothelial nitric oxide synthase (eNOS) activity. When BH4 availability is limited, eNOS does not produce nitric oxide (NO) but instead generates superoxide. BH4 may reverse endothelial dysfunction due to cardiovascular disease, including atherosclerosis, coronary artery disease and hypertension. In this study, the influence of BH4 on cardiovascular parameters and the production of free radicals following angiotensin II (Ang II) infusion was assessed. METHODS: BH4 (20 mg/kg per day in drinking water) was administered with Ang II (300 ng/kg per min subcutaneously, osmotic pump) for 7 days in Sprague-Dawley rats. In addition, BH4 was also given in vehicle-infused rats. RESULTS: Treatment with BH4 significantly prevented some of the effects of Ang II, such as impaired vascular responses to acetylcholine, hypertension and increases in heart weight index values. Treatment with BH4 also significantly reduced Ang II-induced increases in inducible NO synthase expression, nitrotyrosine immunostaining, NO production and superoxide anion formation in rats. CONCLUSION: These results indicate that BH4 might prevent the development of hypertension and myocardial hypertrophy, as well as the Ang II-induced production of superoxide and NO, thereby reducing the production of peroxynitrite. Therefore, BH4 may protect against the cardiovascular manifestations of oxidative and nitrosative stress in this experimental model of Ang II-mediated hypertension.     

Wine polyphenols improve endothelial function in large vessels of female spontaneously hypertensive rats

Red wine polyphenols (RWPs) have been reported to prevent hypertension and endothelial dysfunction. Several individual RWPs exert estrogenic effects. We analyzed the possible in vivo protective effects on blood pressure and endothelial function of RWPs in female spontaneously hypertensive rats (SHR) and its relationship with ovarian function. RWPs (40 mg/kg by gavage) were orally administered for 5 weeks. Ovariectomized rats showed both increased isoprostaglandin F(2alpha) excretion and aortic superoxide production and reduced relaxant response to acetylcholine and contraction to the endothelial nitric oxide synthase (eNOS) inhibitor l-NAME measured in the aorta but similar blood pressure, as compared with sham-operated rats. Moreover, in ovariectomized rats aortic eNOS expression was unchanged, whereas caveolin-1, angiotensin II receptor (AT)-1, and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22(phox) and p47(phox) expression was increased compared with sham-operated rats. In both ovariectomized and sham-operated SHR, RWPs reduced systolic blood pressure, urinary isoprostaglandin F(2alpha) excretion, and aortic O(2)(-) production, improving the endothelium-dependent relaxant response to acetylcholine in SHR. These changes were associated with unchanged aortic eNOS expression, whereas caveolin-1 was increased and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22(phox) and p47(phox) expression was reduced. RWPs had no effect on the AT-1 overexpression found in ovariectomized animals. All these results suggest that a chronic treatment with RWPs reduces hypertension and vascular dysfunction through reduction in vascular oxidative stress in female SHR in a manner independent of the ovarian function.     

Nebivolol inhibits superoxide formation by NADPH oxidase and endothelial dysfunction in angiotensin II-treated rats

Nebivolol is a beta(1)-receptor antagonist with vasodilator and antioxidant properties. Because the vascular NADPH oxidase is an important superoxide source, we studied the effect of nebivolol on endothelial function and NADPH oxidase activity and expression in the well-characterized model of angiotensin II-induced hypertension. Angiotensin II infusion (1 mg/kg per day for 7 days) caused endothelial dysfunction in male Wistar rats and increased vascular superoxide as detected by lucigenin-derived chemiluminescence, as well as dihydroethidine staining. Vascular NADPH oxidase activity, as well as expression at the mRNA and protein level, were markedly upregulated, as well as NOS III uncoupled, as evidenced by NO synthase III inhibitor experiments and dihydroethidine staining and by markedly decreased hemoglobin-NO concentrations. Treatment with the beta-receptor blocker nebivolol but not metoprolol (10 mg/kg per day for each drug) normalized endothelial function, reduced superoxide formation, increased NO bioavailability, and inhibited upregulation of the activity and expression of the vascular NADPH oxidase, as well as membrane association of NADPH oxidase subunits (Rac1 and p67(phox)). In addition, NOS III uncoupling was prevented. In vitro treatment with nebivolol but not atenolol or metoprolol induced a dissociation of p67(phox) and Rac1, as well as an inhibition of NADPH oxidase activity assessed in heart membranes from angiotensin II-infused animals, as well as in homogenates of Nox1 and cytosolic subunit-transfected and phorbol ester-stimulated HEK293 cells. These findings indicate that nebivolol interferes with the assembly of NADPH oxidase. Thus, inhibitory effects of this beta-blocker on vascular NADPH oxidase may explain, at least in part, its beneficial effect on endothelial function in angiotensin II-induced hypertension.     

Role of Menkes ATPase in angiotensin II-induced hypertension: a key modulator for extracellular superoxide dismutase function

The extracellular superoxide dismutase (SOD3), a secretory copper-containing enzyme, regulates angiotensin II (Ang II)-induced hypertension by modulating levels of extracellular superoxide anion. The present study was designed to determine the role of the copper transporter Menkes ATPase (MNK) in Ang II-induced SOD3 activity and hypertension in vivo. Here we show that chronic Ang II infusion enhanced systolic blood pressure and vascular superoxide anion production in MNK mutant (MNK(mut)) mice as compared with those in wild-type mice, which are associated with impaired acetylcholine-induced endothelium-dependent vasorelaxation in MNK(mut) mice. These effects in MNK(mut) mice are rescued by infusion of the SOD mimetic Tempol. By contrast, norepinephrine-induced hypertension, which is not associated with an increase in vascular superoxide anion production, is not affected in MNK(mut) mice. Mechanistically, basal and Ang II infusion-induced increase in vascular SOD3-specific activity is significantly inhibited in MNK(mut) mice. Coimmunoprecipitation analysis reveals that Ang II stimulation promotes association of MNK with SOD3 in cultured vascular smooth muscle cell and in mouse aortas, which may contribute to SOD3-specific activity by increasing copper delivery to SOD3 through MNK. In summary, MNK plays an important role in modulating Ang II-induced hypertension and endothelial function by regulating SOD3 activity and vascular superoxide anion production and becomes a potential therapeutic target for oxidant stress-dependent cardiovascular diseases.     

Angiotensin II and nitric oxide interaction

Nitric oxide degradation linked to endothelial dysfunction plays a central role in cardiovascular diseases. Superoxide producing enzymes such as NADPH oxidase and xanthine oxidase are responsible for NO degradation as they generate a variety of reactive oxygen species (ROS). Moreover, superoxide is rapidly degraded by superoxide dismutase to produce hydrogen peroxide leading to the uncoupling of NO synthase and production of increased amount of superoxide.Angiotensin II is an important stimulus of NADPH oxidase. Through its AT₁ receptor, Ang II stimulates the long-term increase of several membrane component of NADPH oxidase such as P₂₂ phox or nox-1 and causes an increased activity of NADPH oxidase with inactivation of NO leading to impaired endothelium-dependent vasorelaxation, vascular smooth muscle cell hypertrophy, proliferation and migration, extracellular matrix formation, thrombosis, cellular infiltration and inflammatory reaction. Several preclinical and clinical studies have now confirmed the involvement of the AT₁ receptor in endothelial dysfunction. It is proposed that the AT₂ receptor counterbalances the deleterious effect of the Ang II-induced AT₁ receptor stimulation through bradykinin and NOS stimulation. This mechanism could be especially relevant in pathological cases when the NADPH oxidase activity is blocked with an AT₁ receptor antagonist.     

Uncoupled endothelial nitric oxide synthase and oxidative stress in a rat model of pregnancy-induced hypertension

BACKGROUND: Preeclampsia is a human pregnancy-associated syndrome associated with hypertension, proteinuria, and endothelial dysfunction. We tested whether increased reactive oxygen species (superoxide and peroxynitrite) production and decreased bioavailability of the endothelial nitric oxide (NO) synthase (eNOS) cofactor tetrahydrobiopterin (BH4) contributes to maternal endothelial dysfunction in rats with pregnancy-induced hypertension and several characteristics of preeclampsia. METHODS: Nonpregnant (DS) and pregnant (PDS) rats were treated with deoxycorticosterone acetate and 0.9% saline for approximately 3 weeks and nonpregnant (Con) and pregnant (P) rats received tap water. Blood pressure, urinary protein levels, mesenteric vascular reactivity, aortic protein expression, and aortic reactive oxygen species levels were compared between the four groups. RESULTS: The PDS rats had significantly decreased mesenteric endothelium-dependent relaxation responses and aortic NO production compared to Con, DS, and P rats despite increased aortic eNOS expression. Aortic superoxide and peroxynitrite levels were increased in PDS rats compared with Con, DS, and P rats. Scavenging of reactive oxygen species or increasing tetrahydrobiopterin levels normalized mesenteric endothelium-dependent relaxation responses, aortic NO production, and aortic superoxide and peroxynitrite levels in PDS rats. CONCLUSIONS: These data suggest that increased superoxide production by NADPH oxidase, peroxynitrite degradation of BH4, and uncoupled eNOS contribute to endothelial dysfunction in a rat model of pregnancy-induced hypertension.     

罗格列酮对血管紧张素Ⅱ诱导高血压大鼠的血压影响及其机制

目的：探讨罗格列酮（RSG）对血管紧张素Ⅱ（AngⅡ）诱导高血压大鼠血压的影响及机制。方法：选择24只SD大鼠随机分为4组：正常对照组、RSG组、AngⅡ组及AngⅡ＋RSG组。每组6只大鼠（n=6）。采用alzet渗透泵持续皮下泵入AngⅡ[300ng／（kg·min）×7d]建立高血压大鼠模型,RSG组和AngⅡ＋RSG组给予RSG灌胃[5mg／（kg·d）]7d,7d后观察各组大鼠的血压、心脏质量指数、空腹血糖变化,测定大鼠主动脉NADPH氧化酶的活性及超氧阴离子的含量。结果：与AngⅡ组对比,AngⅡ＋RSG组血压下降[（136±6）mmHgw．（166±6）mmHg,P〈0．01]及心脏质量指数下降[（3．54±0．04）mg,／kg绑．（3．85±0．08）mg／kg,P〈0．01];NADPH氧化酶活性及血管超氧阴离子含量下降[（288．49±36．19）cpm／μg vs．（584．04±69．67）cpm／μg,P〈0．01;（2792．82．7±726．76）cpm／mg vs．（4765．50±597．34）cpm/mg,P〈0．01]。结论：RSG抑制NADPH氧化酶的活性,降低血管超氧阴离子的含量,拮抗血管AngⅡ诱导的血压升高及心肌肥厚,发挥保护心血管的作用。     

PPARalpha activator effects on Ang II-induced vascular oxidative stress and inflammation

Docosahexaenoic acid (DHA), a peroxisome proliferator-activated receptor-alpha (PPARalpha) activator, reduces blood pressure (BP) in some hypertensive models by unclear mechanisms. We tested the hypothesis that DHA would prevent BP elevation and improve vascular dysfunction in angiotensin (Ang) II-infused rats by modulating of NADPH oxidase activity and inflammation in vascular wall. Sprague-Dawley rats received Ang II (120 ng/kg per minute SC) with or without DHA (2.5 mL of oil containing 40% DHA/d PO) for 7 days. Systolic BP (mm Hg), elevated in Ang II-infused rats (172+/-3) versus controls (108+/-2, P<0.01), was reduced by DHA (112+/-4). In mesenteric small arteries studied in a pressurized myograph, media/lumen ratio was increased (P<0.05) and acetylcholine-induced relaxation impaired in Ang II-infused rats (P<0.05); both were normalized by DHA. In blood vessels of Ang II-infused rats, NADPH oxidase activity measured by chemiluminescence and expression of adhesion molecules intercellular adhesion molecule and vascular cell adhesion molecule-1 were significantly increased. These changes were abrogated by DHA. PPARalpha activator DHA attenuated the development of hypertension, corrected structural abnormalities, and improved endothelial dysfunction induced by Ang II. These effects are associated with decreased oxidative stress and inflammation in the vascular wall.     

Early life stress enhances angiotensin II-mediated vasoconstriction by reduced endothelial nitric oxide buffering capacity

We reported previously that maternal separation (MS) sensitizes adult rats to angiotensin II (Ang II)-induced hypertension. The aim of this study was to investigate the vascular reactivity to Ang II and the role of renin-angiotensin system components, reactive oxygen species production, and NO synthase (NOS) buffering capacity mediating the exacerbated Ang II-induced responses. MS rats were separated from their mothers for 3 h/d from days 2 to 14 of life. Controls were nonhandled littermates. At 12 weeks of age, aortic Ang II-induced constriction was greater from MS rats compared with controls (P<0.05); moreover, endothelial denudation abolished this difference. The response to other constrictors was unchanged. Angiotensin type 2 receptor function was reduced in aortic Ang II-induced constriction from MS rats compared with controls. Angiotensin type 1 receptor function was similarly abolished in both groups. However, protein expressions of angiotensin type 1 and angiotensin type 2 receptors were similar in aortic rings from MS and control rats. Preincubation with superoxide inhibitor or scavenger attenuated the Ang II-induced vasoconstriction in control but not in MS rats. However, acute preincubation with an NOS inhibitor enhanced aortic Ang II-induced constriction in aorta from control rats, but this effect was significantly reduced in MS rats compared with control rats. Accordingly, a further increase in Ang II-induced hypertension attributed to chronic NOS inhibition (days 10 to 13) was blunted in MS rats compared with control rats. Similar NOS expression and activity were observed in control and MS rats. In conclusion, MS induces a phenotype with reduced endothelial NOS buffering capacity leading to dysfunctional endothelial Ang II-mediated signaling and sensitization to Ang II-induced vasoconstriction.     

Neuronal nitric oxide synthase is up-regulated by angiotensin II and attenuates NADPH oxidase activity and facilitates relaxation in murine left ventricular myocytes

Angiotensin II (Ang II) is critical in myocardial pathogenesis, mostly via stimulating NADPH oxidase. Neuronal nitric oxide synthase (nNOS) has recently been shown to play important roles in modulating myocardial oxidative stress and contractility. Here, we examine whether nNOS is regulated by Ang II and affects NADPH oxidase production of intracellular reactive oxygen species (ROS(i)) and contractile function in left ventricular (LV) myocytes. Our results showed that Ang II induced biphasic effects on ROS(i) and LV myocyte relaxation (TR(50)) without affecting the amplitude of sarcomere shortening and L-type Ca(2+) current density: TR(50) was prolonged at 30 min but was shortened after 3h (or after Ang II treatment in vivo). Correspondingly, ROS(i) was increased, followed by a reduction to control level. Quantitative RT-PCR and immunoblotting experiments showed that Ang II (3h) increased the mRNA and protein expression of nNOS and increased NO production (nitrite assay) in LV myocyte homogenates, suggesting that nNOS activity may be enhanced and involved in mediating the effects of Ang II. Indeed, n(omega)-nitro-l-arginine methyl ester (l-NAME) or a selective nNOS inhibitor, S-methyl-l-thiocitrulline (SMTC) increased NADPH oxidase production of superoxide/ROS(i) and abolished faster myocyte relaxation induced by Ang II. The positive lusitropic effect of Ang II was not mediated by PKA-, CaMKII-dependent signaling or peroxynitrite. Conversely, inhibition of cGMP/PKG pathway abolished the Ang II-induced faster relaxation by reducing phospholamban (PLN) Ser(16) phosphorylation. Taken together, these results clearly demonstrate that myocardial nNOS is up-regulated by Ang II and functions as an early adaptive mechanism to attenuate NADPH oxidase activity and facilitate myocardial relaxation.     

Increased aortic NADPH oxidase activity in rats with genetically high angiotensin-converting enzyme levels

In humans and rats, angiotensin I-converting enzyme activity is significantly determined by a gene polymorphism. Homozygous Brown Norway rats have higher plasma angiotensin I-converting enzyme activity and circulating angiotensin II (Ang II) levels than Lewis rats. Because Ang II induces NAD(P)H oxidase activation, we hypothesized here that Brown Norway rats have higher vascular NAD(P)H oxidase activity and superoxide anion production than Lewis rats. Homozygous Brown Norway (n=15) and Lewis (n=13) male rats were used. Plasma angiotensin I-converting enzyme activity (by fluorimetry), Ang II levels (by high-performance liquid chromatography and radioimmunoassay), and aortic NAD(P)H oxidase activity, as well as superoxide anion production (by chemiluminescence with lucigenin) were measured. Plasma angiotensin I-converting enzyme activity and Ang II levels were 100% higher in Brown Norway rats than in Lewis rats (P<0.05). Aortic angiotensin I- converting enzyme, but not Ang II, was elevated (P<0.05). Aortic superoxide anion production and NAD(P)H oxidase activity were 300% and 260% higher in Brown Norway than in Lewis rats, respectively (P<0.05), which was not observed in Brown Norway rats treated with candesartan (10 mg/kg per day for 7 days). Endothelial NO synthase activity in the aorta from Brown Norway rats was significantly lower than in Lewis rats. However, inducible NO synthase activity and both endothelial NO synthase and inducible NO synthase mRNA and protein levels were similar in both genotypes. In summary, Brown Norway rats have higher vascular NAD(P)H oxidase activity and superoxide anion production than Lewis rats, suggesting the presence of a higher level of vascular oxidative stress in rats with genetically higher angiotensin I-converting enzyme levels. This effect is mediated through the angiotensin I receptor.     

Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling

Angiotensin II infusion causes endothelial dysfunction by increasing NAD(P)H oxidase-mediated vascular superoxide production. However, it remains to be elucidated how in vivo angiotensin II treatment may alter the expression of the gp91(phox) isoforms and the endothelial nitric oxide synthase (NOS III) and subsequent signaling events and whether, in addition to the NAD(P)H oxidase, NOS III contributes to vascular superoxide formation. We therefore studied the influence of in vivo angiotensin II treatment (7 days) in rats on endothelial function and on the expression of the NAD(P)H oxidase subunits p22(phox), nox1, nox4, and gp91(phox) and NOS III. Further analysis included the expression of NO-downstream targets, the soluble guanylyl cyclase (sGC), the cGMP-dependent protein kinase I (cGK-I), and the expression and phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) at Ser239 (P-VASP). Angiotensin II caused endothelial dysfunction and increased vascular superoxide. Likewise, we found an increase in vascular protein kinase C (PKC) activity, in the expression of nox1 (6- to 7-fold), gp91(phox) (3-fold), p22(phox) (3-fold), NOS III mRNA, and protein. NOS-inhibition with N(G)-nitro-L-arginine decreased superoxide in vessels from angiotensin II-treated animals, compatible with NOS-uncoupling. Vascular NO assessed with electron paramagnetic resonance was markedly reduced. Likewise, a decrease in sGC-expression and P-VASP levels was found. In vivo PKC-inhibition with chelerythrine reduced angiotensin II-induced superoxide production and markedly inhibited upregulation of NAD(P)H oxidase subunits. We therefore conclude that angiotensin II-induced increases in the activity and the expression of NAD(P)H oxidase are at least in part PKC-dependent. NADPH oxidase-induced superoxide production may trigger NOS III uncoupling, leading to impaired NO/cGMP signaling and to endothelial dysfunction in this animal model. The full text of this article is available at http://www.circresaha.org.     

Requirement for Rac1-dependent NADPH oxidase in the cardiovascular and dipsogenic actions of angiotensin II in the brain

We have shown that intracellular superoxide (O(2)(*-)) production in CNS neurons plays a key role in the pressor, bradycardic, and dipsogenic actions of Ang II in the brain. In this study, we tested the hypothesis that a Rac1-dependent NADPH oxidase is a key source of O(2)(*-) in Ang II-sensitive neurons and is involved in these central Ang II-dependent effects. We performed both in vitro and in vivo studies using adenoviral (Ad)-mediated expression of dominant-negative Rac1 (AdN17Rac1) to inhibit Ang II-stimulated Rac1 activation, an obligatory step in NADPH oxidase activation. Ang II induced a time-dependent increase in Rac1 activation and O(2)(*-) production in Neuro-2A cells, and this was abolished by pretreatment with AdN17Rac1 or the NADPH oxidase inhibitors apocynin or diphenylene iodonium. AdN17Rac1 also inhibited Ang II-induced increases in NADPH oxidase activity in primary neurons cultured from central cardiovascular control regions. In contrast, overexpression of wild-type Rac1 (AdwtRac1) caused more robust NADPH oxidase-dependent O(2)(*-) production to Ang II. To extend the in vitro studies, the pressor, bradycardic, and drinking responses to intracerebroventricularly (ICV) injected Ang II were measured in mice that had undergone gene transfer of AdN17Rac1 or AdwtRac1 to the brain. AdN17Rac1 abolished the increase in blood pressure, decrease in heart rate, and drinking response induced by ICV injection of Ang II, whereas AdwtRac1 enhanced these physiological effects. The exaggerated physiological responses in AdwtRac1-treated mice were abolished by O(2)(*-) scavenging. These results, for the first time, identify a Rac1-dependent NADPH oxidase as the source of central Ang II-induced O(2)(*-) production, and implicate this oxidase in cardiovascular diseases associated with dysregulation of brain Ang II signaling, including hypertension.