Acute angiotensin II increases plasma F2-isoprostanes in salt-replete human hypertensives

Angiotensin (Ang) II induces oxidative stress in vitro and in animal models of hypertension. We tested the hypothesis that Ang II increases oxidative stress in human hypertension, as assessed by plasma F2-isoprostane concentrations. Plasma F2-isoprostanes, hemodynamic and endocrine parameters were measured at baseline and following a 55 min infusion of 3 ng/kg/min Ang II in 13 normotensive and 13 hypertensive volunteers ingesting a high- (200 mmol/d) or low- (10 mmol/d) sodium diet. Mean arterial pressure (MAP) and body mass index were higher in hypertensive subjects. Ang II infusion increased MAP (p<.001) and plasma aldosterone concentrations (p<.001) and decreased plasma renin activity (p<.001) and renal plasma flow (p<.001) to a similar extent in both groups. Plasma F2-isoprostane concentrations were similar at baseline. There was no effect of Ang II on F2-isoprostane concentrations during low-salt intake in either group (normotensive 51.7 +/- 7.1 to 53.7 +/- 6.5 pg/ml and hypertensive 52.2 +/- 8.2 to 56.2 +/- 10.0 pg/ml; mean +/- SE). During high-salt intake, Ang II increased F2-isoprostane concentrations in the hypertensive group (52.3 +/- 7.2 to 63.2 +/- 10.4 pg/ml, p=0.010) but not in the normotensive group (54.2 +/- 4.4 to 58.9 +/- 6.6 pg/ml, p=0.83). Acute Ang II infusion increases oxidative stress in vivo in hypertensive humans. The renin-angiotensin system may contribute to oxidative stress in human cardiovascular disease.     

Deferoxamine reduces and nitric oxide synthase inhibition increases neutrophil-mediated myotube injury

We tested the contribution of reactive oxygen species (ROS), reactive nitrogen species (RNS) and the 2 integrin CD18 to neutrophil-mediated myotube injury. Human myotubes were cultured with human neutrophils in the presence or absence of inhibitors directed against ROS, RNS, and CD18. Muscle injury was assessed by a ⁵¹Cr release assay. The inclusion of superoxide dismutase (50–500 U/ml) in the culture medium did not affect myotube injury. A significant protective effect was provided by including catalase (600–2400 U/ml), deferoxamine (1–2 mM), or anti-CD18 antibody (10 g/ml) in the culture medium. S-Ethylisothiourea (500–1000 M), an inhibitor of nitric oxide synthase (NOS), significantly increased myotube injury and reduced nitric oxide (NO) in cultures consisting of only myotubes. In conclusion, neutrophil-mediated skeletal muscle injury appears to be largely dependent on CD18-mediated neutrophil adhesion and iron-dependent hydroxyl radical production. In addition, skeletal muscle NOS activity may protect skeletal muscle against the injury caused by neutrophils.This project was supported by The University of Toledo DeArce Memorial Fund and the National Institutes of Health AR47599-01     

Quantifying F2-isoprostanes in umbilical cord blood of newborn by gas chromatography-mass spectrometry

We attempted to improve the extraction procedures to determine the F(2)-isoprostanes in plasma of umbilical cord arterial and venous blood by gas chromatography mass spectrometry. Plasma samples were deproteinized and hydrolyzed; free and esterified F(2)-isoprostanes were extracted by solid-phase extraction columns with citric acid/methanol/cyclohexane and ammonia solution/methanol and then derivatized by PFBBr and BSTFA. Concentrations of total plasma F(2)-isoprostanes eluted at the retention time of an internal standard of 8-iso-prostaglandin F(2alpha)-D(4) were quantified. The absolute recovery was 83+/-1.9% (95% confidence). Intraassay precision and interassay precision were lower than 1.0%. Analytical accuracy was 99.0+/-0.4% (95% confidence). Linearity, r(2), over the concentration range of 10 to 5000 pg/ml of spiked 8-iso-prostaglandin F(2alpha) in plasma was 0.9985. The method detection limit was 21 pg/ml (99% confidence) and the limit of quantitation was approximately 4 pg/ml. Analysis of 200 neonatal cord blood samples revealed few overlapping peaks causing interference in the elution of the F(2)-isoprostanes. With the use of an autosampler and one technician, 48 samples can be completed within 24h with 6h of actual hands-on work. This method could be potentially employed for routine analysis of plasma F(2)-isoprostanes in clinical laboratories.     

Endogenous nitric oxide activation protects against cigarette smoking induced apoptosis in endothelial cells

Cigarette-induced endothelial dysfunction could be an early mediator of atherosclerosis. In this study, we explored the mechanisms of cigarette smoke extract (CSE)-induced human aortic endothelial cells (HAEC) apoptosis. We found that 10-65% of HAECs underwent apoptotic changes when HAECs were exposed to 0.001-0.02 cigarette equivalent unit of CSE for 4 h. CSE activated the caspases-3 and 8, the p38 MAP kinase and stress activated protein kinase/c-Jun N-terminal protein kinase (SAPK/JNK). Specific inhibitors of p38 MAP or SAPK/JNK reduced CSE-induced caspase activation. We further showed that eNOS pre-activation by L-arginine reduced endothelial apoptosis from 65% to 5%; and eNOS inhibition by N-omega-nitro-L-arginine methyl ester accentuated CSE-induced endothelial apoptosis. We suggest that appropriate endogenous NO production may be an important protective mechanism against smoking-induced endothelial damage.     

'Normal' cigarette smoking increases free cortisol in habitual smokers.

In habitual smokers salivary cortisol responses to cigarette smoking were investigated. In the first study, 31 adults assigned to two experimental groups smoked either one or two cigarettes of their preferred brand. Mean salivary cortisol levels were significantly elevated after smoking of two cigarettes. In the second study, 10 smokers and 10 nonsmokers provided saliva samples at 20 min intervals over a 12-hr period. While environmental stimuli were paralleled in both groups overall cortisol output was significantly elevated in the smokers. These data suggest that 'normal' cigarette smoking can increase free cortisol levels.     

Elevated F2-isoprostanes in thalassemic patients

This study was aimed at investigating oxidative stress in thalassemic patients by measurement of the oxidative damage biomarker, F₂-isoprostanes (F₂-IsoPs), using gas chromatography-mass spectrometry. The results showed that the mean value of urinary F₂-IsoPs, normalized with creatinine, in the thalassemic group was significantly higher than that from healthy subjects (3.38 ± 2.15 ng/mg creatinine vs 0.86 ± 0.55 ng/mg creatinine, respectively), and the mean value of plasma total F₂-IsoPs in the thalassemic group was also significantly higher than that from healthy subjects (0.39 ± 0.15 ng/ml vs 0.18 ± 0.03 ng/ml, respectively). Serum ferritin, erythrocyte superoxide dismutase (SOD), glutathione peroxidase, glutathione, and TBARS levels after treatment of erythrocytes with H₂O₂ were also investigated, and serum ferritin and erythrocyte SOD levels were significantly higher in thalassemic patients. Our findings are consistent with oxidative stress in thalassemia patients.     

Functional and cellular interactions between nitric oxide and prostacyclin

Nitric oxide (NO) and prostacyclin (PGI(2)) can be released by vascular agents to synergize their effects on vascular relaxation. In the present study we assess whether NO could affect PGI(2) production. We evaluated the effect of NO on PGI(2)-mediated arachidonic acid (AA)-induced relaxation in the perfused heart. We used cultured endothelial cells to characterize the mechanism involved in the NO effect on PGI(2) synthesis. AA-induced PGI(2) synthesis was enhanced when NO synthesis was inhibited. NO inhibited AA-induced relaxation and PGI(2) release in the coronary circulation. S-Nitroso-acetyl-DL-penicillamine (SNAP) decreased PGI(2) production in cultured endothelial cells. The SNAP effect was blunted by the inhibitor of soluble guanylate cyclase (LY-83,583) and the blocker of cGMP-dependent protein kinases (H-9). Specific cyclooxygenase-1 (COX-1) immunoprecipitation was associated to co-precipitation of four proteins. COX-1 showed neither serine nor threonine phosphorylation. One of the proteins that co-precipitated with COX-1 presented increased serine phosphorylation in the presence of SNAP. This effect was inhibited by the H-9. We suggest that NO, through cGMP-dependent protein kinases, produces the phosphorylation of a 104-kDa protein that is associated with inhibition in the activity of the COX-1, decreasing PGI(2) synthesis and thereby decreasing coronary PGI(2)-mediated vasodilatation.     

Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase.

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.     

Maternal cigarette smoking, Down syndrome in live births, and infant race.

Previous studies have suggested that maternal smoking is negatively associated with a Down syndrome live birth. We analyzed the data of the U.S. Perinatal Collaborative Study in a search for racial variation in Down syndrome risk factors. There were 22 cases in 25,346 live births to smoking mothers (4/10,780 blacks, 18/13,320 whites, and 0/1,246 other races) and 42/29,130 live births to nonsmoking mothers (24/14,665 blacks, 14/11,694 whites, and 4/2,771 others). The crude overall rates per 1,000 live births were 0.4 in black smokers and 1.6 in black nonsmokers but 1.4 in white smokers and 1.2 in white non-smokers. Adjusted for maternal age, the summary relative risk for a Down syndrome live birth to a smoking mother was 0.2 in blacks (95% interval 0.1-0.7) but 1.2 in whites (95% interval 0.6-2.5). Stratification on variables associated with socioeconomic status or gestational age at time of entry into the study did not alter the racial difference. A comparison of smokers with those who never smoked revealed essentially the same trends. Among all nonsmokers the ratio of the maternal age-adjusted risks for a Down syndrome live birth in whites compared with blacks was 0.7 (95% interval 0.3-1.3), and among all smokers this ratio was 3.6 (95% interval 1.3-9.9). If the results are not attributable to statistical fluctuation or undetected confounding, then differences in the probability of intrauterine survival of the Down syndrome fetus would appear to be one plausible explanation for the difference.     

Effect of cigarette smoking on nitric oxide, structural, and mechanical properties of mouse arteries

Cigarette smoking (CS) is a major risk factor for vascular disease. The aim of this study was to quantitatively assess the influence of CS on mouse arteries. We studied the effect of short-term (6 wk) and long-term (16 wk) CS exposure on structural and mechanical properties of coronary arteries compared with that of control mice. We also examined the reversibility of the deleterious effects of CS on structural [e.g., wall thickness (WT)], mechanical (e.g., stiffness), and biochemical [e.g., nitric oxide (NO) by-products] properties with the cessation of CS. The left and right coronary arteries were cannulated in situ and mechanically distended. The stress, strain, elastic modulus, and WT of coronary arteries were determined. Western blot analysis was used to analyze endothelial NO synthase (eNOS) in the femoral and carotid arteries of the same mice, and NO by-products were determined by measuring the levels of nitrite. Our results show that the mean arterial pressure was increased by CS. Furthermore, CS significantly increased the elastic modulus, decreased stress and strain, and increased the WT and WT-to-radius ratio compared with those of control mice. The reduction of eNOS protein expression was found only after long-term CS exposure. Moreover, the NO metabolite was markedly decreased in CS mice after short- and long-term exposure of CS. These findings suggest that 16 wk of CS exposure can cause an irreversible deterioration of structural and elastic properties of mouse coronary arteries. The decrease in endothelium-derived NO in CS mice was seen to significantly correlate with the remodeling of arterial wall.     

Pulsed electric fields-processed orange juice consumption increases plasma vitamin C and decreases F2-isoprostanes in healthy humans

Orange juice, a rich source of vitamin C, accounts for 60% of all fruit juices and juice-based drinks consumed in western Europe. Orange juice preservation is currently accomplished by traditional pasteurization. Pulsed electric fields (PEF) have been studied as a nonthermal food preservation method. Food technology needs in the area of processing are driven by nutrition. Therefore, the objectives of this study were to assess the bioavailability of vitamin C from pulsed electric fields-treated orange juice in comparison with freshly squeezed orange juice and its impact on 8-epiPGF(2alpha) concentrations (biomarker of lipid peroxidation) in a healthy human population. Six subjects consumed 500 mL/day of pulsed electric fields-treated orange juice and six subjects consumed 500 mL/day of freshly squeezed orange juice for 14 days, corresponding to an intake of about 185 mg/day of ascorbic acid. On the first day of the study, subjects drank the juice in one dose, and on days 2-14 they consumed 250 mL in the morning and 250 mL in the afternoon. Blood was collected every hour for 6 hours on the first day and again on days 7 and 14. In the dose-response study, the maximum increase in plasma vitamin C occurred 4 hours postdose. Vitamin C remained significantly higher on days 7 and 14 in both orange juice groups. Plasma 8-epiPGF(2alpha) concentrations was lower at the end of the study (P < 0.001) in both groups. Plasma levels of vitamin C and 8-epiPGF(2alpha) were inversely correlated. Pulsed electric fields-preservation of orange juice retains the vitamin C bioavailability and antioxidant properties of fresh juice with a longer shelf-life.     

Endostatin induces acute endothelial nitric oxide and prostacyclin release

Chronic exposure to endostatin (ES) blocks endothelial cell (EC) proliferation, and migration and induces EC apoptosis thereby inhibiting angiogenesis. Nitric oxide (NO) and prostacyclin (PGI(2)), in contrast, play important roles in promoting angiogenesis. In this study, we examined the acute effects of ES on endothelial NO and PGI(2) production. Unexpectedly, a cGMP reporter cell assay showed that ES-induced acute endothelial NO release in cultured bovine aortic endothelial cells (BAECs). Enzyme immunoassay showed that ES also induced an acute increase in PGI(2) production in BAECs. These results were confirmed by ex vivo vascular ring studies that showed vascular relaxation in response to ES. Immunoblot analysis showed that ES stimulated acute phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser116, Ser617, Ser635, and Ser1179, and dephosphorylation at Thr497 in BAECs, events associated with eNOS activation. Short-term exposure of EC to ES, therefore, unlike long-term exposure which is anti-angiogenic, may be pro-angiogenic.     

Maternal aggression in endothelial nitric oxide synthase-deficient mice

Lactating female rodents protect their pups by expressing fierce aggression, termed maternal aggression, toward intruders. Mice lacking the neuronal nitric oxide synthase gene (nNOS-/-) exhibit significantly impaired maternal aggression, but increased male aggression, suggesting that nitric oxide (NO) produced by nNOS has opposite actions in maternal and male aggression. In contrast, mice lacking the endothelial nitric oxide synthase gene (eNOS-/-) exhibit almost no male aggression, suggesting that NO produced by eNOS facilitates male aggression. In the present study, maternal aggression in eNOS-/- mice was examined and found to be normal relative to wild-type (WT) mice in terms of the percentage displaying aggression, the average number of attacks against a male intruder, and the total amount of time spent attacking the male intruder. The eNOS-/- females also displayed normal pup retrieval behavior. Because a significant elevation of citrulline, an indirect marker of NO synthesis, occurs in neurons of the hypothalamus of lactating WT mice in association with maternal aggression, we examined the brains of eNOS-/- females for citrulline immunoreactivity following an aggressive encounter. The aggressive eNOS-/- females exhibited a significant elevation of citrulline in the medial preoptic nucleus and the subparaventricular zone of the hypothalamus relative to unstimulated lactating eNOS-/- females. Taken together, these results suggest that NO produced by eNOS neither facilitates nor inhibits maternal aggression and that NO produced by eNOS has a different role in maternal and male aggression.     

Reduced perivascular PO2 increases nitric oxide release from endothelial cells

Many studies have suggested that endothelial cells can act as "oxygen sensors" to large reductions in oxygen availability by increasing nitric oxide (NO) production. This study determined whether small reductions in oxygen availability enhanced NO production from in vivo intestinal arterioles, venules, and parenchymal cells. In vivo measurements of perivascular NO concentration ([NO]) were made with NO-sensitive microelectrodes during normoxic and reduced oxygen availability. During normoxia, intestinal first-order arteriolar [NO] was 397 +/- 26 nM (n = 5), paired venular [NO] was 298 +/- 34 nM (n = 5), and parenchymal cell [NO] was 138 +/- 36 nM (n = 3). During reduced oxygen availability, arteriolar and venular [NO] significantly increased to 695 +/- 79 nM (n = 5) and 534 +/- 66 nM (n = 5), respectively, whereas parenchymal [NO] remained unchanged at 144 +/- 34 nM (n = 4). During reduced oxygenation, arteriolar and venular diameters increased by 15 +/- 3% and 14 +/- 5%, respectively: NG-nitro-L-arginine methyl ester strongly suppressed the dilation to lower periarteriolar Po2. Micropipette injection of a CO2 embolus into arterioles significantly attenuated arteriolar dilation and suppressed NO release in response to reduced oxygen availability. These results indicated that in rat intestine, reduced oxygen availability increased both arteriolar and venular NO and that the main site of NO release under these conditions was from endothelial cells.     

Maternal smoking and twinning.

In order to investigate a possible association between maternal smoking during pregnancy and twinning, information on 1,096,330 single births and 12,342 twin births in 1983-95 was obtained from the Swedish Medical Birth Registry (MBR). All odds ratios (OR) were estimated after stratification for year of birth and maternal age, parity, and educational level. Smoking women, compared with non-smoking women, were at increased risk of having dizygotic (DZ) twins, but the risk increase was only evident among multiparas. A strong association between previous involuntary childlessness and dizygotic (DZ) twinning (especially in primiparas) was found. The strongest association between maternal smoking and DZ twinning was found among multiparas without any history of involuntary childlessness (OR: 1.35, 95%CI:1.22-1.49), whereas among women who had experienced involuntary childlessness, the opposite was seen (OR: 0.82, 95%CI:0.66-1.00, no difference between parity strata). Weinberg's differential method was used to estimate the number of monozygotic (MZ) twins, and a method of estimating stratified ORs among mothers of MZ twins was presented. No association was found between MZ twinning and maternal smoking (OR: 0.96, 95%CI:0.86-1.07), and no confounding by parity or previous involuntary childlessness was indicated. Several non-causal explanations to the positive association between DZ twinning and maternal smoking among multiparas were discussed, but homogeneity over strata indicated that maternal smoking may be a true risk factor for double ovulation.     

Analysis of nitric oxide donor effectiveness in resistance vessels

Decreased nitric oxide (NO) bioavailability is associated with a number of pathological conditions. Administration of a supplemental source of NO can counter the pathological effects arising from decreased NO bioavailability. A class of NO-nucleophile adducts that spontaneously release NO (NONOates) has been developed, and its members show promise as therapeutic sources of NO. Because the NONOates release NO spontaneously, a significant portion of the NO may be consumed by the myriad of NO reactive species present in the body. Here we develop a model to analyze the efficacy of NO delivery, by membrane-impermeable NONOates, in the resistance arterioles. Our model identifies three features of blood vessels that will enhance NONOate efficacy: 1) the amount of NO delivered to the abluminal region increases with lumen radius; 2) the presence of a flow-induced red blood cell-free zone will augment NO delivery; and 3) extravasation of the NONOate into the interstitial space will increase abluminal NO delivery. These results suggest that NONOates may be more effective in larger vessels and that NONOate efficacy can be altered by modifying permeability to the interstitial space.     

Malignant hypertension and cigarette smoking.

The smoking habits of 48 patients with malignant hypertension were compared with those of 92 consecutive patients with non-malignant hypertension. Thirty-three of the patients with malignant and 34 of the patients with non-malignant hypertension were smokers when first diagnosed. This difference was significant, and remained so when only men or black and white patients were considered separately. Results suggest that malignant hypertension is yet another disease related to cigarette smoking.