NADPH oxidase inhibition attenuates oxidative stress but not hypertension produced by chronic ET-1

Experiments were conducted to test the hypothesis that hypertension produced by chronic ET-1 infusion is mediated by NADPH oxidase-dependent superoxide production. Mean arterial pressure (MAP) was continuously monitored in male Sprague Dawley rats by telemetry. After baseline measurements, rats were placed on a high-salt diet (8% NaCl) and osmotic minipumps were implanted to infuse ET-1 (5 pmol/kg per minute intravenous) for 12 days. Control rats were maintained on the high-salt diet only. Separate groups of rats were also infused with ET-1 and given the superoxide dismutase mimetic, tempol (1 mmol/L), or the NADPH oxidase inhibitor, apocynin (1.5 mmol/L), in the drinking water. Infusion of ET-1 significantly increased MAP when compared with baseline values (132+/-3 versus 114+/-2 mm Hg, P<0.05). Neither tempol nor apocynin treatment had any effect on the increase in MAP produced by ET-1 when compared with baseline values (127+/-5 versus 113+/-2 and 130+/-3 versus 115+/-2 mm Hg, respectively). Plasma 8-isoprostane, an indicator of oxidative stress, was significantly increased in ET-1-infused rats compared with rats on a high-salt diet alone (128+/-33 versus 51+/-5 pg/mL; P<0.05). Both tempol and apocynin treatment significantly attenuated the ET-1-induced increase in plasma 8-isoprostane (72+/-10 and 61+/-6 pg/mL, respectively). Similarly, ET-1 infusion also significantly increased aortic superoxide production (chemiluminescence and dihydroethidium staining techniques), which was prevented by both tempol and apocynin. These data provide evidence that chronic ET-1 infusion increases vascular NADPH oxidase-dependent superoxide production but does not account for chronic ET-1-induced hypertension.     

Critical role for p47phox in renin-angiotensin system activation and blood pressure regulation

OBJECTIVE: Renin-angiotensin system (RAS) activation leads to increased production of NAD(P)H oxidase-derived reactive oxygen species (ROS), and both have been implicated in the initiation and progression of arterial hypertension, atherosclerosis, and cardiac hypertrophy. The cytosolic subunit p47phox is critically involved in agonist-induced NAD(P)H oxidase activation. Here, we investigated the role of p47phox in blood pressure control, endothelium-dependent relaxation, cardiac hypertrophy, RAS activation, and renal oxidative stress under resting conditions. METHODS AND RESULTS: Mice deficient in p47phox (on C57BL/6 background) developed significantly higher systolic blood pressure levels compared to C57BL/6 wild-type animals (136.0+/-3.0 mmHg vs. 112.2+/-2.6, P<0.01, n=16) as measured by the tail cuff method from week 6 up to week 12 post partum. The increase in blood pressure in p47phox-/- mice was associated with an impaired endothelium-dependent relaxation (P<0.005 vs. wild-type, n=11). At the age of 12 weeks p47phox-/- mice showed increased plasma renin activity as analyzed by radioimmunoassay (14.5+/-1.8 ng/mL/h vs. 9.6+/-1.7 ng/mL/h, P<0.05, n=10) and enhanced angiotensin converting enzyme (ACE) activity in the kidney and aorta as measured by Hip-His-Leu cleavage (7.6+/-0.8 vs. 4.8+/-0.9 nmol/L His-Leu/mg protein, P<0.05, n=5) compared to wild-type mice. No differences in oxygen radical formation was determined in kidney samples by lucigenin- and luminol-enhanced chemiluminescence or by electron spin resonance spectroscopy. Consistently, treatment with the radical scavenger tempol did not lower blood pressure in p47phox-/- mice, whereas ACE and angiotensin II type I receptor inhibition normalized blood pressure. CONCLUSION: Deficiency of the NAD(P)H oxidase subunit p47phox leads to RAS activation, which subsequently contributes to blood pressure increase in a ROS-independent manner.     

Orchidectomy, but not ovariectomy, regulates angiotensin II-induced vascular diseases in apolipoprotein E-deficient mice

In humans, the incidence and severity of abdominal aortic aneurysms (AAA) are greater in males than in females. Chronic infusion of angiotensin II (AngII) into apolipoprotein E-deficient (apoE(-/-)) mice promotes atherosclerosis and causes the formation of AAAs. Just as human males are more susceptible to developing AAAs, male mice are more susceptible to AngII-induced AAAs. We hypothesized that sex steroid hormones mediate gender differences in AngII-induced AAA through regulation of the renin-angiotensin system. To define the role of ovarian hormones, female apoE(-/-) mice were subjected to ovariectomy or sham operation and infused with AngII (1000 ng/kg x min) for 28 d. Ovariectomy had no effect on AngII-induced atherosclerosis, nor did it influence the incidence or severity of AAA. To define the role of testicular hormones, male apoE(-/-) mice were subjected to orchidectomy (orx) or sham operation and infused with AngII (1000 ng/kg x min) for 28 d. Orx resulted in a profound reduction in AAA incidence (85% vs. 18%, sham vs. orx; P = 0.003) to the level observed in females (25%). However, orx had no effect on AngII-induced reductions in plasma renin concentration or spleen AngII receptor density. In contrast, orx resulted in an increase in atherosclerosis (0.46 +/- 0.07 vs. 1.20 +/- 0.21 mm(2), sham vs. orx; P = 0.002). These results suggest that estrogen does not mediate gender differences in AngII-induced AAA. In contrast, androgens mediate a higher incidence of AngII- induced AAA, through mechanisms that do not appear to involve circulating renin or angiotensin receptor density.     

Improvement of endothelial dysfunction by selective estrogen receptor-alpha stimulation in ovariectomized SHR

Both known estrogen receptors, ERalpha and ERbeta, are expressed in blood vessels. To gain further insight into the role of ERalpha in a functional setting, we investigated the effect of the novel highly selective ERalpha agonist Cpd1471 on vascular reactivity in ovariectomized spontaneously hypertensive rats (SHR). After ovariectomy or sham operation, 12-week-old female SHR received either 17beta-estradiol (E2, 2 microg/kg body wt per day), the selective ERalpha agonist Cpd1471 (30 microg/kg body wt per day), or placebo. Acetylcholine-induced endothelium-dependent vasorelaxation was significantly blunted in aortas from ovariectomized rats (Rmax, 53%+/-3% versus sham, 79%+/-2%; P<0.001). Treatment with E2 or Cpd1471 significantly augmented acetylcholine-induced relaxation in ovariectomized rats (Rmax, 70%+/-2%; resp, 73%+/-2%). Endothelium-independent relaxation induced by sodium nitroprusside was not different among the four groups. The contractile response induced by the nitric oxide (NO) synthase inhibitor Nomega-nitro-l-arginine, an index of basal NO formation, was significantly lower in ovariectomized rats compared with sham-operated animals (53+/-2% versus 77%+/-5%; P<0.01) and was normalized by both E2 (70%+/-2%) and Cpd1471 (70%+/-3%). Aortic endothelial NO synthase (eNOS) expression and phosphorylation of the vasodilator-stimulated phosphoprotein, an index of NO/cGMP-signaling, was reduced in ovariectomized SHR and normalized by E2 and Cpd1471. In SHR after ovariectomy, endothelium-dependent NO-mediated vasorelaxation and eNOS expression are attenuated. The novel selective ERalpha agonist Cpd1471 prevented these pathophysiological changes to a similar extent as E2. Thus, the pharmacological principle of selective ERalpha activation mediates positive vascular effects.     

Risk factors of atherosclerosis and saphenous vein endothelial function.

BACKGROUND: Impaired vasomotor function has been suggested as playing a role in the pathophysiology of atherosclerosis and it may also affect the late patency of bypass grafts. We evaluated, in vitro, the influence of risk factors of atherosclerosis on saphenous vein endothelial function in patients with cardiovascular diseases. METHODS: Forty-five saphenous vein rings with intact (E+) and denuded endothelium (E-) were studied. The following drugs were used: norepinephrine (NE), acetylcholine (Ach), histamine (H) and serotonine (5-HT). RESULTS: Contraction to norepinephrine (n=15) showed a maximal tension of 783+/-115 percent that was increased in diabetics, smokers, and patients with hypertension. There was a wide range of response to acetylcholine in rings with intact endothelium (n=25), (mean relaxation 16.4+/-1.7 percent, ranging from -22.2 percent to 45 percent) with relaxation (26+/-1.1 percent) and contraction (-11+/-1.2 percent); relaxation was reduced in patients with hypertension and in diabetics (7.4+/-2.6 percent vs non diabetics 24.4+/-1.73 percent; p<0.01). Five of the 12 veins from diabetics exibited contraction (10+/-1.48 percent). Histamine (n=15) caused moderate relaxation at low doses (25+/-2.46 percent) followed by contraction at higher concentrations (184+/-5.7 percent). This was greater in diabetics (193+/-6.8 percent vs non diabetics 157+/-5.3 percent; p=0.045) while in preparations without endothelium (n=10) only relaxation was obtained (45+/-2.89 percent). Contraction (242+/-7.4 percent) was observed in response to serotonine (n=15) that was not affected by endothelial removal. In this study saphenous vein: (1) exhibited a wide range of responses to acetylcholine; (2) evoked marked contraction to norepinephrine and serotonine; (3) elicited contraction in response to histamine that was endothelium-dependent, suggesting the production or the release of an endothelium-derived-contracting-factor (EDCF). CONCLUSIONS: Saphenous vein is able to secrete a contracting factor in patients with risk factors of atherosclerosis and above all diabetes. The mechanisms that regulate the balance between the relaxing and contracting factors and how the endothelial cells become the source of the substances with vasoconstrictor activity remain to be determined.     

NO-dependent vasorelaxation is impaired after gene transfer of inducible NO-synthase

Proinflammatory stimuli produce expression of inducible NO-synthase (iNOS) within blood vessels and are associated with impaired endothelium-dependent relaxation. Gene transfer of iNOS was used to test the hypothesis that expression of iNOS in blood vessels produces impairment of NO-dependent relaxation as well as contraction. An adenoviral vector containing cDNA for murine iNOS, AdCMViNOS, and a control virus, AdCMVBglII, were used for gene transfer to rabbit carotid arteries in vitro and in vivo. After gene transfer of iNOS in vitro, contractile responses to KCl, phenylephrine, and U46619 were impaired. Relaxation in response to acetylcholine, ADP, A23187, and nitroprusside was also impaired. For example, maximum relaxation of vessels to acetylcholine (10 micromol/L) was 78+/-4% (mean+/-SE) after AdBglII (10(10.5) plaque-forming units) and 34+/-5% after AdiNOS (10(10.5) plaque-forming units, P<0.05). NO-independent relaxation in response to 8-bromo-cGMP and papaverine was not impaired after AdiNOS. Contraction and relaxation were improved in carotid arteries expressing iNOS by aminoguanidine and L-N-iminoethyl lysine, inhibitors of iNOS. After intraluminal gene transfer of iNOS in vivo, contraction of vessels in vitro was normal, but responses to acetylcholine were impaired. In summary, the major finding is that NO-dependent relaxation is impaired in arteries after gene transfer of iNOS in vitro and in vivo. Thus, expression of iNOS per se impairs NO-dependent relaxation.     

Role of increased production of superoxide anions by NAD(P)H oxidase and xanthine oxidase in prolonged endotoxemia.

Superoxide anions (O2-) are supposedly involved in the pathogenesis of endothelial dysfunction. We investigated whether the enhanced formation of O2- is involved in the attenuation of endothelium-dependent relaxation induced by lipopolysaccharide (LPS). Rats were injected with LPS (10 mg/kg IP), the aorta was removed after 12 or 30 hours, and generation of O2-, H2O2, and ONOO- was measured using chemiluminescence assays. Protein tyrosine nitration and expression of xanthine oxidase (XO), NAD(P)H oxidase, and manganese superoxide dismutase were determined by Western or Northern blotting, and endothelium-dependent relaxation in aortic rings was studied. LPS treatment increased vascular O2- (from 35+/-2 cpm/ring at baseline to 166+/-21 cpm/ring at 12 hours and 225+/-16 cpm/ring at 30 hours) and H2O2 formation, which was partially sensitive to the NAD(P)H oxidase inhibitor diphenylene iodonium at both time points studied and to the XO inhibitor oxypurinol only 30 hours after LPS treatment. Expression of XO and NAD(P)H oxidase (p22phox, p67phox, and gp91phox) were increased by LPS in a time-dependent manner, as were protein tyrosine nitration and ONOO- formation. LPS also induced expression of the oxidative stress-sensitive protein manganese superoxide dismutase. Endothelium-dependent relaxation was impaired after LPS treatment and could not be restored by inhibition of inducible NO synthase. Inhibition of O2- with superoxide dismutase, oxypurinol, tiron, or the superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid)porphyrin chloride did not restore but further deteriorated the relaxation of LPS-treated rings. In summary, treatment of rats with LPS enhances vascular expression of XO and NAD(P)H oxidase and increases formation of O2- and ONOO-. Because removal of O2- compromised rather than restored endothelium-dependent relaxation, a direct role of O2- in the induction of endothelial dysfunction is unlikely. Other mechanisms, such as prolonged protein tyrosine nitration by peroxynitrite (which is formed from NO and O2-) or downregulation of the NO effector pathway, are more likely to be involved.     

Addition of spironolactone to angiotensin-converting enzyme inhibition in heart failure improves endothelial vasomotor dysfunction: role of vascular superoxide anion formation and endothelial nitric oxide synthase expression.

OBJECTIVES: We sought to investigate the effects of adding spironolactone (SP) to angiotensin-converting enzyme (ACE) inhibition on endothelium-dependent vasodilation in rats with chronic heart failure (CHF). BACKGROUND: Adding SP to ACE inhibitors reduces mortality and morbidity in CHF. Endothelial vasomotor dysfunction contributes to increased peripheral vascular resistance and reduced myocardial perfusion in CHF. METHODS: Seven days after extensive myocardial infarction (CHF) or sham operation, Wistar rats were treated with placebo, the ACE inhibitor trandolapril (TR, 0.3 mg/kg body weight per day), SP (10 mg/kg per day) or a combination of both for 11 weeks. RESULTS: Maximal acetylcholine-induced, nitric oxide (NO)-dependent relaxation was significantly attenuated in aortic rings from rats with CHF as compared with sham-operated animals (R(max) 44 +/- 3% vs. 63 +/- 3%). Spironolactone alone had no influence (46 +/- 5%) and TR improved NO-mediated relaxation (55 +/- 4%), whereas treatment with both completely restored endothelium-dependent vasorelaxation (64 +/- 4%). Aortic superoxide formation was significantly increased in rats with CHF as compared with sham-operated animals, but was normalized by treatment with SP or SP plus TR. In addition, aortic messenger ribonucleic acid expression of the oxidase subunit p22(phox) in rats with CHF was significantly reduced by SP or TR plus SP. Endothelial NO synthase expression was increased in TR-treated animals. Incubation of isolated porcine coronary arteries with SP dose-dependently attenuated superoxide formation. CONCLUSIONS: Spironolactone added to an ACE inhibitor normalizes NO-mediated relaxation in experimental CHF by beneficially modulating the balance of NO and superoxide anion formation.     

Exaggerated coronary vasoreactivity to endothelin-1 in aged rats: role of protein kinase C

OBJECTIVE: The interaction between advanced age and increased susceptibility to ischemic insult is well documented. Age-related increases in coronary vascular resistance, in part due to impaired dilator responses, have been reported. Our aim was to determine the role of endothelin-1 (ET-1) on enhanced constrictor responses in aged coronary arteries (CAs) and whether protein kinase C (PKC) signaling mechanisms impact ET-1 responses. METHODS: Vasoreactivity was assessed in CAs isolated from aged (24 months; n=16) and adult (4 months; n=21) male F344 rats following ET-1 (10(-10)-10(-8)) with and without specific ETA/ETB receptor antagonists (BQ-123, 1 microM; BQ-788, 30 nM) or the PKC inhibitor bisindolylmaleimide (Bis; 10(-6) M). Constrictor responses to KCl (80 mM) were also measured and voltage-gated Ca2+ channel (VGCC) determined in isolated coronary smooth muscle cells. Dilator responses to acetylcholine (ACH) and sodium nitroprusside (SNP) were assessed. RESULTS: Passive diameter was greater (357+/-19 vs. 309+/-9; p<0.02) while spontaneous tone was similar in 24 months vs. 4 months. ET-1 resulted in greater constriction in 24 months vs. 4 months (79% vs. 67%; p<0.01). Group differences persisted following selective ETB inhibition with BQ-788 (p<0.02), while BQ-123 abolished contractile responses to ET-1. Importantly, inhibition of ET-1 constriction by Bis occurred in 24 months but not 4 months (p<0.01). Constrictor responses to KCl and peak VGCC current density were similar in 24 months vs. 4 months (48% vs. 50%). No age-related differences were observed in ACH- or SNP-mediated dilation. Western blotting revealed increases in Ca2+-sensitive PKCalpha, -betaI, and -betaII levels with age, while eNOS and ETA receptor protein levels were unchanged. CONCLUSION: Aberrant ETA constrictor responses and directional changes in PKC are likely to contribute to coronary vascular pathology with advanced age.     

Angiotensin-converting enzyme inhibitors reveal non-NO-, non-prostacycline-mediated endothelium-dependent relaxation in internal thoracic artery of hypertensive patients

BACKGROUND: We have shown that treatment of hypertension with ACE inhibitors (ACE-I) enhances relaxation to acetylcholine in human internal thoracic artery (ITA) above this in nonhypertensive patients receiving no ACE-I. Present study assesses the endothelium-dependent responses mediated by neither NO nor prostacyclin in human ITA. METHODS: We compared isolated ITA rings from hypertensive patients treated with ACE-I (ACE-I group) with those from normotensive patients on no ACE-I (control group). Relaxation to acetylcholine was assessed before and after inhibition of NO synthase and cyclooxygenase with L-NMMA and indomethacin, respectively. RESULTS: The maximal relaxation in ACE-I group was 79+/-3.3% and was depressed by incubation with L-NMMA and indomethacin to 41+/-2.7% (p<0.001); pD(2)=7.7+/-0.1 vs. 7.4+/-0.8 (p=0.265). The maximal relaxation to acetylcholine was lower in the control group: 65+/-3.3% (p=0.01); pD(2)=7.5+/-0.1 (p=0.07). Incubation with L-NMMA and indomethacin produced contraction to acetylcholine with a maximum of 43+/-7% (p<0.001); pD(2)=5.3+/-0.3 (p<0.001). The area under the concentration-response curve for acetylcholine-induced relaxation in ACE-I group equaled [arbitrary units] 596+/-71 and after incubation with L-NMMA and indomethacin 281+/-40 (p=0.002). Estimated LNMMA- and indomethacin-resistant relaxation, absent in control group, accounted for 47+/-4% of relaxation to acetylcholine in ACE-I group. Estimated NO- and prostacyclin-mediated relaxation was higher in control group than ACE-I group: 628+/-74 vs. 315+/-47 (p=0.009). CONCLUSIONS: The results suggest that therapy with ACE-I improves endothelial function of hypertensive patients mainly by enhancing the endothelium-derived hyperpolarizing factor (EDHF) (and not NO)-mediated responses. It seems that it reveals measurable non-NO- non-PGI-mediated endothelium-dependent relaxation otherwise absent in conduit arteries.     

Impaired renal vascular endothelial function in vitro in experimental hypercholesterolemia

Hypercholesterolemia (HC) induces alterations in systemic vascular reactivity, which can manifest as an attenuated endothelium-dependent relaxation, partly consequent to an impairment in nitric oxide (NO) activity. To determine whether experimental HC has a similar effect on renal vascular function, renal artery segments obtained from pigs fed a HC (n=5) or normal (n=5) diet were studied in vitro. Endothelium-dependent relaxation was examined using increasing concentrations of acetylcholine (Ach), calcium ionophore A23187, and Ach following pre-incubation with N(G)-monomethyl-L-arginine or L-arginine (L-ARG). The NO-donor diethylamine (DEA) was used to examine smooth muscle relaxation response and cyclic GMP generation in endothelium-denuded vessels. The expression of endothelial NO synthase (eNOS) in the renal arteries was examined using Western blotting. Endothelium-dependent relaxation to Ach was significantly attenuated in the HC group compared to normal (53.3+/-9.1 vs. 98.8+/-3.7%, P<0.005), but normalized after pre-incubation with L-ARG (82.3+/-13.8%, P=0.21). Receptor-independent endothelium-dependent relaxation to A23187 was also significantly blunted in HC (75.2+/-10.5 vs. 115.5+/-4.2%, P<0. 017). Smooth muscle relaxation and cyclic GMP generation in response to DEA were greater in denuded HC vessels, while relaxation of intact vessels to nitroprusside was unaltered. In the HC vessels eNOS was almost undetectable. In conclusion, experimental HC attenuates in vitro endothelium-dependent relaxation of the porcine renal artery, possibly due to low bioavailability of NO. These vascular alterations in HC could play a role in the pathogenesis of renal disease or hypertension, supporting a role for HC as a risk factor for renovascular disease.     

NADPH oxidase-derived superoxide augments endothelin-1-induced venoconstriction in mineralocorticoid hypertension

Deoxycorticosterone acetate (DOCA)-salt hypertension is characterized by low renin/angiotensin but increased arterial superoxide levels. We have recently reported that the arterial endothelin-1 (ET-1) level is increased, resulting in NADPH oxidase activation and superoxide generation. However, the effect of ET-1 on venous superoxide production and its relation to venoconstriction are unknown. The present study tested the hypotheses that ET-1 stimulates venous NADPH oxidase and superoxide via its ET(A) receptors, resulting in enhanced venoconstriction in DOCA-salt hypertensive rats. Treatment with ET-1 (0.01 to 1 nmol/L), but not the selective ET(B) receptor agonist sarafotoxin s6c, of vena cavas of normal rats concentration-dependently increased superoxide levels, an effect that was abolished by the selective ET(A) receptor antagonist ABT-627. Although the ET-1 level was not increased in the vena cava and plasma, both venous NADPH oxidase activity and superoxide levels were significantly higher in DOCA-salt compared with sham rats. Moreover, ET-1 treatment (10(-9) mol/L, 10 minutes) of isolated vena cavas further elevated superoxide levels in DOCA-salt rats only but not sham rats, an effect that was abrogated by the superoxide scavenger tempol. Similarly, ET-1-induced contractions of isolated vena cavas of DOCA-salt but not sham rats were significantly inhibited by tempol. The NADPH oxidase inhibitor apocynin significantly reduced superoxide levels in vena cavas of DOCA-salt rats and in ET-1-treated vena cavas of normal rats. Finally, in vivo ET(A) receptor blockade by ABT-627 significantly lowered venous superoxide levels and blood pressure in DOCA-salt but not sham rats. These results suggest that superoxide contributes to ET-1-induced venoconstriction through an elevated venous NADPH oxidase activity in mineralocorticoid hypertension.     

Vitamin C blocks vascular dysfunction and release of interleukin-6 induced by endothelin-1 in humans in vivo

OBJECTIVE: Inflammation of the vessel wall is of importance in atherosclerosis. Endothelin-1 (ET-1) exerts pro-inflammatory effects and contributes to endothelial dysfunction. The objective was to test whether ET-1 impairs vascular function by increasing oxidative stress and release of pro-inflammatory cytokines in humans. METHODS: Forearm blood flow (FBF) was determined in 12 young healthy males with venous occlusion plethysmography. RESULTS: Intra-brachial infusion of ET-1 (20 pmol/min) decreased both endothelium-dependent and -independent vasodilatation (P<0.001). ET-1 also increased venous IL-6 levels (0.96+/-0.14-1.40+/-0.15 ng/ml; P<0.001). Administration of Vitamin C (24 mg/min) following the ET-1 infusion did not restore vascular function. However, pre-treatment with Vitamin C before ET-1 prevented the decrease in endothelium-dependent and -independent vasodilatation as well as the increase in IL-6 levels (1.20+/-0.28 versus 1.29+/-0.27 ng/ml; P=0.57). Infusion of a control vasoconstrictor substance, noradrenaline (80 ng/min) for 30 min did not affect IL-6 levels. CONCLUSIONS: ET-1 impairs endothelium-dependent and -independent vasodilatation and stimulates release of IL-6 in humans in vivo. These effects are inhibited by pre-treatment with the antioxidant Vitamin C. This suggests that the mechanism by which ET-1 impairs vascular function and stimulates release of IL-6 involves increased oxidative stress.     

Enhanced endothelin-1-induced contractions in mesenteric arteries from rats with congestive heart failure: role of ET(B) receptors

Studies of congestive heart failure (CHF) in man and in experimental CHF have demonstrated elevated circulating levels of endothelin (ET). In order to examine whether there are concomitant ET receptor alterations, the vasomotor effects of endothelin-1 (ET-1) and sarafotoxin 6c (S6c) were examined in endothelium-intact and -denuded isolated mesenteric arteries from rats with CHF. CHF was induced by ligation of the left anterior descending coronary artery. Vasomotor responses were studied using small mesenteric arteries (approx. 250 microm in diameter, determined after normalisation). The antagonists IRL2500 and FR139317 were used in order to characterise the ET-1-induced response. In mesenteric arteries with intact endothelium, ET-1-induced contractions were more potent in CHF as compared to sham (pEC(50) 9.6+/-0.2 and 9.1+/-0.1, respectively, P<0.01). In endothelium-denuded arteries, there was no difference in potency of ET-1 between CHF and sham arteries, or in maximum contraction. In the presence of IRL2500, a selective ET(B)-receptor antagonist, ET-1 was more potent in endothelium-denuded arteries of CHF rats, while this difference was not seen in sham arteries. S6c had no consistent contractile or dilatory effect in CHF and sham rats. The results indicate that the enhanced contractile effects of ET-1 noted in CHF might be due to an attenuated endothelial function and that inhibition of smooth muscle cell ET(B) receptors increase the effects of contractile ET(A) receptors in CHF rats.     

Angiotensin II infusion induces site-specific intra-laminar hemorrhage in macrophage colony-stimulating factor-deficient mice

Angiotensin II (AngII) infusion promotes macrophage infiltration into the aortic wall resulting in several forms of vascular pathology. To determine the causal role of macrophages in these vascular diseases, we used osteopetrotic (op) male mice in which a natural mutation ablates production of M-CSF and results in severe depletion of monocytes. AngII infusion into apoE-/- mice resulted in increased atherosclerosis that was attenuated in op mice. AngII infusion in op mice unexpectedly produced grossly discernable thickening of the proximal thoracic aorta characterized by intra-mural hematoma. This pathology was also observed in apoE+/+ x op male mice, and therefore, independent of hyper-lipidemia. No perceptible structural properties of aortas from op mice could be discerned prior to AngII infusion. Regional effects in the contractile response to phenylephrine were noted in aortic rings with enhanced responsivity in the upper thoracic aortas of op mice compared to those from +/+ mice. No differences in contractile response were noted in aortic rings from the lower thorax. In conclusion, deficiency of M-CSF attenuated AngII-induced atherosclerosis but led to an unanticipated pathology of intra-laminar hemorrhage in the upper aortic regions.     

Enhanced angiotensin II-mediated effects in fibroblasts of patients with familial hypercholesterolemia

OBJECTIVE: Familial hypercholesterolemia (FH) is characterized by a high incidence of coronary heart disease. Evidence suggests an important role for angiotensin II (AngII) in the fibrotic response to tissue injury, and in promoting myocardial hypertrophy via paracrine mechanisms mediated by fibroblasts. We sought to determine whether AngII promotes proliferative and pro-atherogenic responses in FH patients. METHODS: We used primary fibroblasts -- from five patients with heterozygous FH and five control subjects (C) -- to study AngII-induced cell growth, intracellular calcium fluxes, and expression/release of matrix components and pro-inflammatory peptides [transforming growth factor-beta1 (TGFbeta1) and endothelin-1 (ET-1)] and metalloproteinases involved in plaque remodeling and vulnerability. RESULTS: AngII stimulated cell replication (5.1 +/- 0.03 versus 3.2 +/- 0.04 cells/50 cells per well, P < 0.001), and induced a larger increase in intracellular calcium content in FH cells than in C cells, in a dose-dependent fashion (mean difference = 76 nmol/l, P < 0.001). Similarly, TGFbeta1 and ET-1 expression and release were potentiated (after 24-h incubation with 1 micromol/l AngII: TGFbeta1 was 190 +/- 12 in C and 376 +/- 9 pg/ml per 10(6) cells in FH, and ET-1 was 93 +/- 5 in C and 192 +/- 7 pmol/ml per 10(6) cells in FH; P < 0.001 for both). AngII-induced release of the metalloproteinases MMP-1 and MMP-2 was also increased in FH versus C cells (0.52 +/- 0.04 versus 0.36 +/- 0.05 and 24 +/- 4 versus 13 +/- 3 ng/mg protein with 1 micromol/l AngII). These enhanced responses were likely due to an increased angiotensin receptor 1 (AT1) expression in cells from FH patients induced by AngII, and were prevented by pretreating cells with the selective AT1 antagonist irbesartan. CONCLUSIONS: These findings show that some AngII-mediated pathways are enhanced in FH subjects irrespective of the presence of low-density lipoprotein (LDL), thus contributing to the development and progression of atherosclerosis in these patients.     

Enhanced contractility of renal afferent arterioles from angiotensin-infused rabbits: roles of oxidative stress, thromboxane prostanoid receptors, and endothelium

We tested the hypothesis that cyclooxygenase (COX), thromboxane A2 synthase (TxA2-S), thromboxane prostanoid receptors (TP-Rs), or superoxide anion (O2-) mediates enhanced contractions of renal afferent arterioles (Aff) of angiotensin II (Ang II)-infused rabbits. Rabbits were infused with vehicle (sham), Ang II 60 ng x kg(-1) x min(-1) (Ang II 60) or 200 ng x kg(-1) x min(-1) (Ang II 200). There was a selective enhanced vasoconstriction of Affs from Ang II 60 rabbits to Ang II (Deltadiameter-78+/-8% versus -43+/-9%; P<0.01) that was normalized by a TP-R antagonist but not by a superoxide dismutase (SOD) mimetic. Affs from Ang II 200 rabbits had increased (P<0.01) mRNA for COX-2 and enhanced vasoconstriction to Ang II, U-46 619 (TP-R mimetic), and endothelin-1 that was normalized by ifetroban plus tempol together. Endothelium removal enhanced Ang II responses of Affs from sham rabbits but blunted responses from Ang II 200 rabbits and abolished responses to ifetroban. Affs from Ang II 200 rabbits had an endothelium-dependent contraction factor (EDCF) response to that was blunted (P<0.001) by a SOD mimetic or antagonists of COX-1 or TxA2-S but normalized by antagonists of COX-2 or TP-R. Thus, enhanced Ang II responses in Affs from rabbits infused with slow pressor Ang II are mediated independently by O2- in the vascular smooth muscle cells and by an EDCF that is principally a vasoconstrictor prostaglandin generated by COX-2 >-1 activating TP-Rs, whereas enhanced responses in rabbits infused with a lower Ang II dose are dependent on TP-R but not O2-.     

The antioxidant tempol inhibits cardiac hypertrophy in the insulin-resistant GLUT4-deficient mouse in vivo

Reactive oxygen species such as superoxide are implicated in cardiac hypertrophy, but their contribution to the cardiac complications of insulin resistance is unresolved. We tested the hypothesis that the antioxidant tempol attenuates cardiac hypertrophy in insulin-resistant mice. Mice with cardiac GLUT4 deletion (GLUT4-knockout), superimposed on global GLUT4 suppression (GLUT4-knockdown) were administered tempol for 4 weeks. Age-matched GLUT4-knockdown littermates were used as controls (14 mice/group). GLUT4-knockout mice exhibited marked cardiac hypertrophy: heart to body weight ratio was increased 61+/-7% and expression of the hypertrophic genes beta-myosin heavy chain and B-type natriuretic peptide (BNP) were elevated 5.5+/-0.7- and 6.2+/-1.5-fold relative to control, respectively. Pro-fibrotic pro-collagen III expression was also higher (3.8+/-0.7-fold) in the GLUT4-knockout myocardium (all p<0.001). Both gp91(phox) and Nox1 subunits of NADPH oxidase were also upregulated, 4.9+/-1.2- and 9.3+/-2.8-fold (both p<0.01). Tempol treatment significantly attenuated all of these abnormalities in GLUT4-knockout mice. Heart to body weight ratio was decreased, as was fold expression of beta-myosin heavy chain (to 3.8+/-0.8), BNP (to 2.5+/-0.5), pro-collagen III (to 1.9+/-0.4), gp91(phox) (to 0.9+/-0.3) and Nox1 (to 2.3+/-0.1, all p<0.05 versus untreated GLUT4-knockout mice). In addition, tempol upregulated ventricular expression of both thioredoxin-2 (confirming an antioxidant action) and glycogen synthase kinase-3beta (GSK-3beta). Tempol did not elicit any other significant changes in control mice. Cardiac superoxide generation, however, was not altered by GLUT4-knockout or tempol. In conclusion, tempol treatment reduced morphological and molecular evidence of cardiac hypertrophy in the GLUT4-knockout insulin-resistant mouse in vivo, even at doses insufficient to lower cardiac superoxide. Parallel reductions in pro-collagen III and NADPH oxidase have important implications for our understanding of the molecular basis of cardiac hypertrophy in the setting of insulin resistance. Antioxidants may offer new alternatives in this disorder.     

Detection and Characterization of a Novel Reassortant Mammalian Orthoreovirus in Bats in Europe

A renewed interest in mammalian orthoreoviruses (MRVs) has emerged since new viruses related to bat MRV type 3, detected in Europe, were identified in humans and pigs with gastroenteritis. This study reports the isolation and characterization of a novel reassortant MRV from the lesser horseshoe bat (Rhinolophus hipposideros). The isolate, here designated BatMRV1-IT2011, was first identified by electron microscopy and confirmed using PCR and virus-neutralization tests. The full genome sequence was obtained by next-generation sequencing. Molecular and antigenic characterizations revealed that BatMRV1-IT2011 belonged to serotype 1, which had not previously been identified in bats. Phylogenetic and recombination detection program analyses suggested that BatMRV1-IT2011 was a reassortant strain containing an S1 genome segment similar to those of MRV T1/bovine/Maryland/Clone23/59 and C/bovine/Indiana/MRV00304/2014, while other segments were more similar to MRVs of different hosts, origins and serotypes. The presence of neutralizing antibodies against MRVs has also been investigated in animals (dogs, pigs, bovines and horses). Preliminary results suggested that MRVs are widespread in animals and that infections containing multiple serotypes, including MRVs of serotype 1 with an S1 gene similar to BatMRV1-IT2011, are common. This paper extends the current knowledge of MRVs and stresses the importance to continue and improve MRV surveillance in bats and other mammals through the development and standardization of specific diagnostic tools.