David Fontaine

Acute Effects of Passive Smoking on Peripheral Vascular Function

Environmental tobacco smoke (ETS) acutely affects peripheral and coronary vascular tone. Whether ETS exerts specific deleterious effects on aortic wave reflection through nicotine exposure, whether they persist after ETS cessation, and whether the smoke environment impairs microvascular function and increases asymmetrical dimethyl-arginine levels are not known. We tested these hypotheses in a randomized, crossover study design in 11 healthy male nonsmokers. The effects of 1 hour of exposure to ETS, as compared with a nontobacco smoke and normal air, on augmentation index corrected for heart rate and skin microvascular hyperemia to local heating were examined. Augmentation index increased both during (P=0.01) and after (P<0.01) the ETS session but remained unchanged in the nontobacco smoke session when compared with normal air. Nicotine levels after the exposure were related to the peak rise in augmentation index (r=0.84; P<0.01), denoting a predominant role of nicotine in ETS vascular effects. This was confirmed in a second set of experiments (n=14), where the sublingual administration of nicotine was associated with an acute impairment in wave reflection as compared with placebo (P=0.001). Both ETS and nontobacco smokes increased plasma asymmetrical dimethyl-arginine levels (P<0.001), but only ETS reduced the late rise in skin blood flow in response to heating (P=0.03). In conclusion, passive smoking specifically increases aortic wave reflection through a nicotine-dependent pathway and impairs microvascular function, even after the end of the exposure. However, both tobacco and nontobacco passive smoking inhalation increase plasma asymmetrical dimethyl-arginine levels.

Absence of nitrate tolerance after long-term treatment with ramipril: an endothelium-dependent mechanism.

To determine whether nitrate tolerance is attenuated on aortas isolated from rats treated in the long term with an angiotensin-converting enzyme (ACE) inhibitor, five groups of rats were studied in parallel. Group 1 received ramipril, 1 mg/ kg/day, p.o., for 6 weeks; group 2 received ramipril at the same dose for 4 weeks, and the last 2 weeks, a cotreatment with ramipril plus HOE 140 (a bradykinin B2 antagonist, 500 microg/ kg/day, s.c. injections); group 3 received losartan, 2 mg/kg/day, p.o., for 6 weeks; group 4 received losartan at the same dose, and the last 2 weeks, a cotreatment with losartan plus HOE 140; and group 5 served as control. Rings of thoracic aorta from these groups were studied in organ baths. After nitroglycerin preincubation (10 microM for 30 min) in vitro, the dose-response curves to nitroglycerin were significantly shifted to the right in the control group but not in group 1. This protective effect was partially present in group 3; it was completely abolished in groups 2 and 4. In groups 1 and 3, it also was abolished after nitric oxide synthase (cNOS) inhibition (L-NMMA incubation) or removal of the endothelium. Superoxide anion accumulation (assessed by lucigenin chemiluminescence) was increased by nitroglycerin incubation in the control group but not in groups 1 and 3. After in vivo exposure to nitroglycerin (50 mg/kg subcutaneously twice daily for 4 days), this protection against nitrate tolerance also was observed in groups 1 and 3. Thus long-term ACE inhibition prevents nitrate tolerance by an endothelium-dependent mechanism involving mainly an enhanced NO availability via B2-kinin receptor. This effect on the cNOS pathway seems to attenuate the superoxide anion accumulation induced by nitroglycerin exposure (probably via a downregulation of oxidative enzyme).

Rosuvastatin treatment protects against nitrate-induced oxidative stress.

Nitrate tolerance is associated with an enhanced superoxide anion production and can be attenuated by statins, which interact with the 2 main [eNOS and NAD(P)H oxidase] pathways involved in producing this oxidative stress. Three groups of normocholesterolemic rats were treated: group 1 received rosuvastatin (10 mg/kg/d PO) for 5 weeks and in the last 3 days cotreatment with nitroglycerin (NTG 50 mg/kg/d, subcutaneous injections BID); group 2 received only NTG (50 mg/kg/d BID for the last 3 days); and group 3 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O2− production (RLU/10 s/mg dry weight) was assessed by lucigenin and the luminol analogue (L-012) chemiluminescence technique. In group 2 (NTG), the concentration-response curves to NTG were significantly shifted to the right: the pD2 (−log NTG concentration evoking a half-maximal relaxation) was 6.75 ± 0.06 (n = 7) versus 7.75 ± 0.07 (n = 7) in group 3 (not exposed to NTG, P < 0.05); O2− production was enhanced (10,060 ± 1,205, n = 7 versus 5,235 ± 1,052, n = 7; P < 0.05). In contrast, in group 1, the rightward shift was attenuated: pD2 value was 7.20 ± 0.10 (n = 8), P < 0.05 versus group 2; O2− production was decreased (5911 ± 663; n = 9, P < 0.05 versus group 2). In addition, before NTG exposure, rosuvastatin treatment decreased p22phox [the essential NAD(P)H oxidase subunit] abundance in the aortic wall and decreased NAD(P)H oxidase activity. In contrast, this treatment did not alter either eNOS abundance or the basal release of endothelium-derived NO. Interestingly, in vivo treatment with apocynin, an NAD(P)H oxidase inhibitor, produced a protection similar to that with rosuvastatin. Long-term rosuvastatin treatment protects against nitrate tolerance in the rat aorta by counteracting NTG-induced increase in O2− production. This protection seems to involve a direct interaction with the NAD(P)H oxidase pathway rather than an up-regulation of the eNOS pathway.

Prevention of Nitrate Tolerance by Long-Term Treatment with Statins

AbstractRecent studies have shown that statins seem to upregulate the endothelial NO synthase pathway (eNOS) and may, therefore, enhance NO availability, a direct scavenger of O2− and an inhibitor of oxidative enzymes. Methods. To assess whether the oxidative stress produced by an in vivo exposure to nitroglycerin (NTG) is attenuated by statins, 4 groups of normocholesterolemic rats were treated; group 1 received pravastatin (20 mg/kg/d p.o) and group 2 atorvastatin (10 mg/kg/d) both for 5 weeks and the last 3 days, a cotreatment with the statin plus NTG (50 mg/kg/d, s.c. injections b.i.d.); group 3 (NTG) received only NTG (50 mg/kg/d, b.i.d. for 3 days) and group 4 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O2− production (counts/10 s/mg) was assessed by lucigenin chemiluminescence technique. Results. In vivo NTG exposure induced a rightward shift of the concentration-response curves to NTG: the pD2 (−log NTG concentration evoking a half maximal relaxation) was 5.8 ± 0.3 (n = 7) vs. 7.2 ± 0.2 in the control group (not exposed to NTG, n = 7) and O2− production was enhanced (1259 ± 71 vs. 787 ± 76, (n = 5) P < .05). In contrast, groups 1 (n = 7) and 2 (n = 7) behaved as the control group (pD2 values were 7.4 ± 0.1 (n = 7) and 6.9 ± 0.1 (n = 7); O2− production was 721 ± 109 and 647 ± 121). The protective effect on nitrate tolerance disappeared when L-NAME (an eNOS inhibitor, 100 mg/kg/d) was co-administered with NTG in groups 1 and 2. Incubation of aortic rings with NAD(P)H (100 μM) also impaired the protective effect of both statins. Moreover, before NTG exposure, aortic cGMP content, reflecting EDNO availability, was significantly enhanced in group 1 (P < .05 vs. control). Conclusion. Long-term statin treatment protects against nitrate tolerance by counteracting NTG-induced increase in O2− production. Both eNOS pathway and NAD(P)H oxidases seem to be involved in this protective mechanism.

Acute effect of sidestream cigarette smoke extract on vascular endothelial function.

Acute exposure to passive smoking adversely affects vascular function by promoting oxidative stress and endothelial dysfunction. However, it is not known whether tobacco sidestream (SS) smoke has a greater deleterious effect on the endothelium than non-tobacco SS smoke and whether these effects are related to nicotinic endothelial stimulation. To test these hypotheses, endothelial-dependent relaxation and superoxide anion production were assessed in isolated rat aortas incubated with tobacco SS smoke, non-tobacco SS smoke, or pure nicotine. Tobacco SS smoke decreased the maximal relaxation to acetylcholine (Ach) from 79 ± 6% to 57 ± 7.3% (% inhibition of phenylephrine-induced plateau, P < 0.001) and increased superoxide anion production from 31 ± 9.7 to 116 ± 24 count/10sec/mg (P < 0.01, lucigenin-enhanced chemiluminescence technique). The non-tobacco SS smoke extract had no significant effect on the response to Ach but increased superoxide anion production in the aortic wall to 133 ± 2 count/10sec/mg (P < 0.001). Furthermore, concentration-response curves to Ach and superoxide production remained unaltered with nicotine (0.001, 0.01, or 0.1 mM). In conclusion, despite similar increases in vascular wall superoxide production with tobacco and non-tobacco SS smoke, only the tobacco SS smoke extracts affected endothelium-dependent vasorelaxation. Nicotine alone does not reproduce the effects seen with tobacco SS smoke, suggesting that the acute endothelial toxicity of passive smoking cannot simply be ascribed to a nicotine-dependent mechanism.

Vitamin D deficiency-induced hypertension is associated with vascular oxidative stress and altered heart gene expression.

Vitamin D deficiency (VDD) is associated with an increased cardiovascular risk. We investigated the effect of VDD on the cardiovascular system of growing male rats fed with a vitamin D-deficient diet. Using isolated rat aorta, we assessed both superoxide anion and endothelial-dependent relaxations. Microarray technology was used to identify changes induced by VDD in cardiac gene expression. Compared with control, VDD increased systolic blood pressure (P < 0.05) and superoxide anion production in the aortic wall (P < 0.05) and tended to increase serum levels of angiotensin II and atrial natriuretic peptide (P < 0.15). However, VDD slightly improved maximal relaxation to acetylcholine from 75 % ± 3% to 83% ± 2% (P < 0.05). Incubation of aortic rings either with nitro-l-arginine methyl ester (l-NAME) or catalase did not eliminate the enhancement of endothelial-mediated relaxation observed in vitamin D-deficient rats. Only incubation with indometacin or calcium-activated potassium channels blockers suppressed this difference. Compared with control, the expression of 51 genes showed different expression, including several genes involved in the regulation of oxidative stress and myocardial hypertrophy. In conclusion, VDD in early life increases arterial blood pressure, promotes vascular oxidative stress, and induces changes in cardiac gene expression. However, the endothelial-mediated regulation of vasomotor tone is maintained throughout the enhancement of an NO-independent compensatory pathway.

Rosuvastatin treatment protects against nitrate‐induced oxidative stress in eNOS knockout mice: implication of the NAD(P)H oxidase pathway

1 Nitrate tolerance is associated with an enhanced superoxide anion (O2−) production and may be attenuated by statins as they interact with the two main endothelial NO synthase (eNOS) and NAD(P)H oxidase pathways involved in this oxidative stress. 2 Groups of wild‐type (wt, C57Bl/6J) and eNOS knock‐out mice (eNOS−/−) received rosuvastatin (20 mg kg−1 day−1 p.o.) for 5 weeks and a cotreatment with the statin plus nitroglycerin (NTG; 30 mg kg−1 day−1, subcutaneous injections b.i.d.) for the last 4 days. Another group received only NTG (30 mg kg−1 d−1, b.i.d. for 4 days) and finally control mice from both strains received no treatment. 3 Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM–0.1 mM) were determined on thromboxane analogue (U44619)‐precontracted rings and O2− production (RLU 5 s−1 mg−1 of total protein content) was assessed in aorta homogenates with the lucigenin‐enhanced chemiluminescence technique. Reverse transcriptase–polymerase chain reaction analysis was performed on aortas from both mice strains. 4 In vivo NTG treatment induced a significant rightward shift of the concentration–effect curve to NTG compared to control group. There was, however, no cross‐tolerance with non‐nitrate sources of NO (unaltered response to acetylcholine in wt group). The rosuvastatin+NTG cotreatment was able to protect against the development of nitrate tolerance in both mice strains and L‐mevalonate abolished this protective effect of rosuvastatin. 5 In vivo treatment with apocynin, a purported NAD(P)H oxidase inhibitor, also produced a similar protection to that observed with rosuvastatin in both strains. 6 Superoxide anion formation was increased after NTG treatment in both mice strains and the rosuvastatin+NTG cotreatment was able to reduce that production. 7 Moreover, rosuvastatin treatment abolished the increase in gp91phox mRNA (an endothelial membrane NAD(P)H oxidase subunit) expression induced by in vivo exposure to NTG. 8 These findings suggest that long‐term rosuvastatin treatment protects against nitrate tolerance by counteracting NTG‐induced increase in O2− production, probably via a direct interaction with the NAD(P)H oxidase pathway.

Chronic hydroxymethylglutaryl coenzyme a reductase inhibition and endothelial function of the normocholesterolemic rat: comparison with angiotensin-converting enzyme inhibition.

Hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors seem to have clinical benefits beyond those predicted by their lipid-lowering action. The objective was to evaluate the vascular effect of long-term treatment with statins on isolated rat aorta and their ability to prevent the acute toxicity of oxidized low-density lipoproteins (oxLDLs) compared with angiotensin-converting enzyme (ACE) inhibitors. Four groups of Wistar rats were treated for 5 weeks. Group 1 received pravastatin 20 mg/kg/d orally; group 2 received atorvastatin 10 mg/kg/d; group 3 received ciprofibrate 25 mg/kg/d; and group 4 served as control. Total cholesterol and triglyceride plasma levels were not altered, except in group 3, in which both parameters were decreased. The inhibitory effect of the endothelium on serotonin-induced contractions was significantly increased in group 1. A significant leftward shift of the concentration-response curves to acetylcholine (1 n M–0.1 m M) was observed in group 1 but the maximal relaxation to acetylcholine was similar in the four groups. In contrast, in the presence of human Cu2+-oxLDL (300 &mgr;g/ml, 30 min of preincubation), the maximal relaxation to acetylcholine was markedly decreased (p < 0.02) in groups 3 and 4 versus that of groups 1 and 2. No difference in superoxide accumulation was observed by the chemiluminescence technique. Cyclic guanosine monophosphate (cGMP), measured by enzyme immunoassay in aortic tissues, was increased in group 1 in the presence of superoxide dismutase. Endothelial nitric oxide synthase (eNOS) expression was not altered (Western blot and enzyme-linked immunosorbent assay). In aortas isolated from a fifth group of rats treated with an ACE inhibitor (ramipril 10 mg/kg/d for 6 weeks), similar results to those of group 1 were observed except that the eNOS abundance was significantly enhanced. Thus, long-term statin treatment upregulates the eNOS pathway and attenuates the acute toxicity of human oxLDL. In contrast to chronic ACE inhibition, the eNOS abundance is not increased.

Oxidative stress produced by circulating microparticles in on-pump but not in off-pump coronary surgery.

Objective — This study was undertaken to assess whether plasmas isolated during off-pump coronary surgery trigger less oxidative stress than those isolated during on-pump surgery. Methods and results — Plasmas were sampled from patients before (T0), just after (T1) and 24 hours after (T2) cardiac surgery (n = 24 on-pump and n = 10 off-pump). Rings of rat thoracic aortas were incubated for 20 hours with these different plasmas (100 μl + 4 ml medium) or saline (control). Thereafter, superoxide anion production was assessed by chemiluminescence and the mean signal was expressed as percent of that in the control ring. In rat aorta exposed to plasmas from on-pump CABG patients (n = 6), the signal was enhanced by 210 ± 29% at T1 (P < 0.05) and by 174 ± 29% at T2 (P < 0.05) versus 53 ± 12% at T0. Moreover, at T1 and T2, there was an upregulation of p22phox , the key subunit of NADPH oxidase, the main enzyme involved in oxidative stress of the vascular wall. In contrast, off-pump plasmas did not induce this superoxide production. Incubation with microparticles obtained by ultracentrifugation also markedly enhanced the signal at T1 and T2 (vs. T0) in the on-pump group (but not in the off-pump group). Selective removal of CD34, CD105, CD59, CD146, CD42 microparticles using flow cytometry did not abolish the signal. CRP and SAA plasma levels were enhanced only at T2 in both groups. Conclusions — Plasmas isolated after on-pump but not off-pump coronary bypass surgery can induce superoxide generation by the vascular wall which seems related to circulating microparticles remaining present at least 24 hours after the procedure that might be of endothelial origin.