Inhibition of cyclooxygenase with indomethacin phenethylamide reduces atherosclerosis in apoE-null mice

Non-selective inhibition of cyclooxygenase (COX) has been reported to reduce atherosclerosis in both rabbit and murine models. In contrast, selective inhibition of COX-2 has been shown to suppress early atherosclerosis in LDL-receptor null mice but not more advanced lesions in apoE deficient (apoE(-/-)) mice. We investigated the efficacy of the novel COX inhibitor indomethacin phenethylamide (INDO-PA) on the development of different stages of atherosclerotic lesion formation in female apoE(-/-) mice. INDO-PA, which is highly selective for COX-2 in vitro, reduced platelet thromboxane production by 61% in vivo, indicating partial inhibition of COX-1 in vivo. Treatment of female apoE(-/-) mice with 5mg/kg INDO-PA significantly reduced early to intermediate aortic atherosclerotic lesion formation (44 and 53%, respectively) in both the aortic sinus and aorta en face compared to controls. Interestingly, there was no difference in the extent of atherosclerosis in the proximal aorta in apoE(-/-) mice treated from 11 to 21 weeks of age with INDO-PA, yet there was a striking (76%) reduction in lesion size by en face analysis in these mice. These studies demonstrate the ability of non-selective COX inhibition with INDO-PA to reduce early to intermediate atherosclerotic lesion formation in apoE(-/-) mice, supporting a role for anti-inflammatory approaches in the prevention of atherosclerosis.     

Endothelial dysfunction in normal and prediabetic rats with metabolic syndrome exposed by oral gavage to carbon black nanoparticles

Exposure to nanosized particles may increase the risk of cardiovascular diseases by endothelial dysfunction, particularly in susceptible subjects with metabolic syndrome. We investigated vasomotor dysfunction in aorta from obese and lean Zucker rats after oral exposure to nanosized carbon black (CB). Rats were exposed to 1 or 10 weekly doses of 0, 0.064, 0.64 or 6.4mg/kg bodyweight and sacrificed 24h or 13 weeks later. The exposure to 10 doses of 0.064 or 0.64mg/kg reduced the acetylcholine-induced vasorelaxation in the lean and obese rats. The half maximal effect concentration values increased by twofold (95% CI: 1.1-3.5-fold) and fourfold (95% CI: 2.3-6.9-fold) in the rats exposed to 0.064 and 0.64mg/kg compared with the controls, respectively. The rats exposed to 10 doses of 0.64mg/kg had also 20% (95% CI: 10-29%) lower maximal effect value compared with the controls. However, the nitroglycerin-induced vasorelaxation and phenylephrine-induced vasocontraction was not affected in rats exposed to CB. The endothelial dysfunction was not observed in rats sacrificed 13 weeks after the last CB exposure. There was unaltered expression of Chrm3, Nos3, Nos2, Ccl2, and Hmox1 in aorta tissue of CB-exposed rats. In conclusion, repeated oral exposure to CB was associated with endothelial dysfunction in rats, further aggravating the effect of metabolic syndrome.     

Pulmonary exposure to particles from diesel exhaust,urban dust or single-walled carbon nanotubes and oxidatively damaged DNA and vascular function in apoE-/- mice

This study compared the oxidative stress level and vasomotor dysfunction after exposure to urban dust, diesel exhaust particles (DEP) or single-walled carbon nanotubes (SWCNT). DEP and SWCNT increased the production of reactive oxygen species (ROS) in cultured endothelial cells and acellullarly, whereas the exposure to urban dust did not generate ROS. The apoE^-/- mice, which were exposed twice to 0.5 mg/kg of the particles by intratracheal (i.t.) instillation, had unaltered acetylcholine-elicited vasorelaxation in aorta segments. There was unaltered pulmonary expression level of Vcam-1, Icam-1, Hmox-1 and Ogg1. The levels of oxidatively damaged DNA were unchanged in lung tissue. The exposure to SWCNT significantly increased the expression of Ccl-2 in the lung tissue of the mice. The exposure to DEP and SWCNT was associated with elevated ROS production in cultured cells, whereas i.t. instillation of the same particles had no effect on biomarkers of pulmonary oxidative stress and dilatory dysfunction in the aorta.     

Ticlopidine attenuates progression of atherosclerosis in apolipoprotein E and low density lipoprotein receptor double knockout mice

Platelets are involved in the development of atherothrombosis. However, the anti-atherosclerotic effects of thienopiridines have not been, as yet, proven. We analyzed the effects of ticlopidine on atherogenesis in apolipoprotein E/low density lipoprotein receptor double knockout (apoE/LDLR(-/-)) mice. 2-month-old apoE/LDLR(-/-) mice fed a Western diet (21% fat, 0.15% cholesterol) were treated with ticlopidine (90 mg/kg/day) for a period of 4 months. In 6-month-old apoE/LDLR(-/-) mice treated with ticlopidine and in their non-treated counterparts we analyzed: cholesterol and triglyceride levels, the size of atherosclerotic plaques in aortic roots (oil red-O staining, cross-section method), and in the whole aorta (Sudan IV staining, en face method), the number of macrophages in atherosclerotic plaque (CD68 staining), as well as the endothelial function in the isolated thoracic aorta. Concentrations of total cholesterol and triglycerides in plasma were not altered by treatment with ticlopidine. However, the size of atherosclerotic plaques measured in aortic roots by the cross-section method and the number of macrophages estimated by anti-CD68 staining were significantly reduced by ticlopidine treatment. In contrast, the effect of ticlopidine on the area covered by plaques in the whole aorta (en face analysis) was not statistically significant. Importantly, acetylcholine-induced vasodilation in isolated aorta was improved in ticlopidine-treated apoE/LDLR(-/-) mice as compared to their non-treated counterparts. In conclusion, ticlopidine attenuates the progression of atherosclerosis and improves the endothelial function in apoE/LDLR(-/-) mice.     

Oxidatively damaged DNA and inflammation in the liver of dyslipidemic ApoE-/- mice exposed to diesel exhaust particles

Epidemiological studies have shown that exposure to air pollution particles is associated with cardiovascular diseases, whereas the role in the initiation of atherosclerosis is unresolved. Atherosclerosis is considered to be an inflammatory disease that also involves oxidative stress. Here we investigated effects of oxidative stress elicited by diesel exhaust particles (DEP) in the aorta, liver, and lung of dyslipidemic ApoE(-/-) mice at the age when visual plaques appear in the aorta (11-13 weeks). DEP was administrated by intraperitoneal injection (0, 50, 500 and 5,000 microg DEP/kg bodyweight) in order to omit vascular effects secondary to pulmonary inflammation. The mice were killed either 6 or 24h after the administration. Inflammation was measured as the expression of inducible nitric oxide synthase (iNOS) and serum nitric oxide and DNA damage was measured by the comet assay. The expression of iNOS mRNA was increased in the liver 6h after the administration. The level of oxidized purine bases, determined as formamidopyrimidine DNA glycosylase sites was increased by 67% (95% CI: 11-124%) in the liver after 24h in the mice administrated with only 50 microg/kg bodyweight. However, there was no indication of systemic inflammation determined as the serum concentration of nitric oxide and iNOS expression, and DNA damage was not increased in the aorta. These observations indicate that intraperitoneal DEP injection does not induce inflammation or oxidatively damaged DNA in the lung and aorta, whereas a direct effect in terms of inflammation and oxidized DNA was observed in the liver of dyslipidemic ApoE(-/-) mice.     

Atherosclerosis and vasomotor dysfunction in arteries of animals after exposure to combustion-derived particulate matter or nanomaterials

Exposure to particulate matter (PM) from traffic vehicles is hazardous to the vascular system, leading to clinical manifestations and mortality due to ischemic heart disease. By analogy, nanomaterials may also be associated with the same outcomes. Here, the effects of exposure to PM from ambient air, diesel exhaust and certain nanomaterials on atherosclerosis and vasomotor function in animals have been assessed. The majority of studies have used pulmonary exposure by inhalation or instillation, although there are some studies on non-pulmonary routes such as the gastrointestinal tract. Airway exposure to air pollution particles and nanomaterials is associated with similar effects on atherosclerosis progression, augmented vasoconstriction and blunted vasorelaxation responses in arteries, whereas exposure to diesel exhaust is associated with lower responses. At present, there is no convincing evidence of dose-dependent effects across studies. Oxidative stress and inflammation have been observed in the arterial wall of PM-exposed animals with vasomotor dysfunction or plaque progression. From the data, it is evident that pulmonary and systemic inflammation does not seem to be necessary for these vascular effects to occur. Furthermore, there is inconsistent evidence with regard to altered plasma lipid profile and systemic inflammation as a key step in vasomotor dysfunction and progression of atherosclerosis in PM-exposed animals. In summary, the results show that certain nanomaterials, including TiO₂, carbon black and carbon nanotubes, have similar hazards to the vascular system as combustion-derived PM.     

Vascular effects of ambient particulate matter instillation in spontaneous hypertensive rats

Exposure to ambient particulate matter (PM) is associated with increased mortality and morbidity among those people with cardiovascular impairment. We have studied the effects of exposure to PM or lypopolysaccharide (LPS) on ex vivo vascular function of spontaneous hypertensive rats (SHR) at 4 and 24 h post-instillation. Receptor-dependent and -independent relaxation was studied by using acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. We have used phenylephrine (Phe) and KCl for receptor-dependent and -independent contraction. The role of the endothelium was investigated using denuded aorta rings. Exposure to PM (EHC-93, 10 mg/kg) or LPS (350 EU/animal) caused maximal pulmonary inflammation at 24 h post-instillation. PM and LPS elicited a significant increase in receptor-dependent vasorelaxation of aorta compared to saline-instilled rats. The largest effect was seen with PM at 4 h post-instillation (EC50 ACh = 2.3 vs. 5 nM control), while at 24 h effects were much smaller (EC50 ACh = 5.6 vs. 5 nM control). SNP-induced vasorelaxation was increased only in EHC-93-treated rats (EC50 = 71.9 vs. 95.7 nM) at 4 h, and this response was higher than that observed at 24 h. Phe induced a dose-dependent vasoconstriction, but no difference was seen between treatments in the presence or absence of endothelium at 4 h. However, at 24 h after instillation of LPS, a right shift of contraction curve was seen (EC50 = 65.3 vs. 43.3 nM). No change was seen in receptor-independent vasoconstriction induced by KCl, except in the LPS group at 24 h. A direct relaxation was also observed upon in vitro exposure of aorta rings to PM, and model particles coated with metals. Blood metal analysis showed an increase of zinc and vanadium concentration at 1 and 4 h post-instillation. In conclusion, our data show that PM and LPS instillation has a transient effect on the vasorelaxation of rat aorta that is maximal at 4 h. On the other hand, pulmonary inflammation reaches a maximum at 24 h and coincides with impairment of vasorelaxation. Current data do not allow discriminating among the potential mechanisms, but suggest that both a direct effect of metals and inflammation play a role.     

Carbon black nanoparticles and vascular dysfunction in cultured endothelial cells and artery segments

Exposure to small size particulates is regarded as a risk factor for cardiovascular disease. We investigated effects of exposure to nanosized carbon black (CB) in human umbilical vein endothelial cells (HUVECs) and segments of arteries from rodents. The CB exposure was associated with increased surface expression of intercellular cell adhesion molecule 1 (ICAM-1) and vascular adhesion molecule 1 (VCAM-1) in HUVECs at 100μg/ml. CB exposure was also associated with increased reactive oxygen species production and damage to the cell membranes in the form of increased lactate dehydrogenase leakage, whereas it did not alter the mitochondrial enzyme activity (WST-1) or the nitric oxide level in HUVECs. Incubation of aorta segments with 10μg/ml of CB increased the endothelial-dependent vasorelaxation, induced by acetylcholine, and shifted the endothelium-independent vasorelaxation, induced by sodium nitroprusside, towards a decreased sensitivity. In mesenteric arteries, the exposure to 10μg/ml was associated with a reduced pressure-diameter relationship. Incubation with 100μg/ml CB significantly decreased both acetylcholine and sodium nitroprusside responses as well as decreased the receptor-dependent vasoconstriction caused by phenylephrine. In conclusion, nanosized CB exposure activates endothelial cells and generates oxidative stress, which is associated with vasomotor dysfunction.     

Statins attenuate the development of atherosclerosis and endothelial dysfunction induced by exposure to urban particulate matter (PM10)

Exposure to ambient air particulate matter (particles less than 10 μm or PM₁₀) has been shown to be an independent risk factor for the development and progression of atherosclerosis. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have well-established anti-inflammatory properties. The aim of this study was to determine the impact of statins on the adverse functional and morphological changes in blood vessels induced by PM₁₀. New Zealand White rabbits fed with a high fat diet were subjected to balloon injury to their abdominal aorta followed by PM₁₀/saline exposure for 4 weeks ± lovastatin (5 mg/kg/day) treatment. PM₁₀ exposure accelerated balloon catheter induced plaque formation and increased intimal macrophages and lipid accumulation while lovastatin attenuated these changes and promoted smooth muscle cell recruitment into plaques. PM₁₀ impaired vascular acetylcholine (Ach) responses and increased vasoconstriction induced by phenylephrine as assessed by wire myograph. Supplementation of nitric oxide improved the impaired Ach responses. PM₁₀ increased the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in blood vessels and increased the plasma levels of endothelin-1 (ET-1). Incubation with specific inhibitors for iNOS, COX-2 or ET-1 in the myograph chambers significantly improved the impaired vascular function. Lovastatin decreased the expression of these mediators in atherosclerotic lesions and improved endothelial dysfunction. However, lovastatin was unable to reduce blood lipid levels to the baseline level in rabbits exposed to PM₁₀. Taken together, statins protect against PM₁₀-induced cardiovascular disease by reducing atherosclerosis and improving endothelial function via their anti-inflammatory properties.     

Potassium- and acetylcholine-induced vasorelaxation in mice lacking endothelial nitric oxide synthase

1. The contribution of an endothelium-derived hyperpolarizing factor (EDHF) was investigated in saphenous and mesenteric arteries from endothelial nitric oxide synthase (eNOS) (-/-) and (+/+) mice. 2. Acetylcholine-induced endothelium-dependent relaxation of saphenous arteries of eNOS(-/-) was resistant to N(omega)-nitro-L-arginine (L-NNA) and indomethacin, as well as the guanylyl cyclase inhibitor, 1H-(1,2,4)oxadiazolo(4,3-a) quinoxalin-1-one(ODQ). 3. Potassium (K(+)) induced a dose-dependent vasorelaxation which was endothelium-independent and unaffected by either L-NNA or indomethacin in both saphenous and mesenteric arteries from eNOS(-/-) or (+/+) mice. 4. Thirty microM barium (Ba(2+)) and 10 microM ouabain partially blocked potassium-induced, but had no effect on acetylcholine-induced vasorelaxation in saphenous arteries. 5. Acetylcholine-induced relaxation was blocked by a combination of charybdotoxin (ChTX) and apamin which had no effect on K(+)-induced relaxation, however, iberiotoxin (IbTX) was ineffective against either acetylcholine- or K(+)-induced relaxation. 6. Thirty microM Ba(2+) partially blocked both K(+)- and acetylcholine-induced relaxation of mesenteric arteries, and K(+), but not acetylcholine-induced relaxation was totally blocked by the combination of Ba(2+) and ouabain. 7. These data indicate that acetylcholine-induced relaxation cannot be mimicked by elevating extracellular K(+) in saphenous arteries from either eNOS(-/-) or (+/+) mice, but K(+) may contribute to EDHF-mediated relaxation of mesenteric arteries.     

Exposure to nitrogen dioxide is not associated with vascular dysfunction in man

Background: Exposure to air pollution is associated with increased cardiorespiratory morbidity and mortality. It is unclear whether these effects are mediated through combustion-derived particulate matter or gaseous components, such as nitrogen dioxide.

Objectives: To investigate the effect of nitrogen dioxide exposure on vascular vasomotor and six fibrinolytic functions.

Methods: Ten healthy male volunteers were exposed to nitrogen dioxide at 4?ppm or filtered air for 1?h during intermittent exercise in a randomized double-blind crossover study. Bilateral forearm blood flow and fibrinolytic markers were measured before and during unilateral intrabrachial infusion of bradykinin (100–1000?pmol/min), acetylcholine (5–20?μg/min), sodium nitroprusside (2–8?μg/min), and verapamil (10–100?μg/min) 4?h after the exposure. Lung function was determined before and after the exposure, and exhaled nitric oxide at baseline and 1 and 4?h after the exposure.

Results: There were no differences in resting forearm blood flow after either exposure. There was a dose-dependent increase in forearm blood flow with all vasodilators but this was similar after either exposure for all vasodilators (p?>?.05 for all). Bradykinin caused a dose-dependent increase in plasma tissue-plasminogen activator, but again there was no difference between the exposures. There were no changes in lung function or exhaled nitric oxide following either exposure.

Conclusion: Inhalation of nitrogen dioxide does not impair vascular vasomotor or fibrinolytic function. Nitrogen dioxide does not appear to be a major arbiter of the adverse cardiovascular effects of air pollution.     

Targeted mutation of CCK2 receptor gene modifies the behavioural effects of diazepam in female mice

Rationale Evidence suggests that GABA and CCK have opposite roles in the regulation of anxiety. Objective The aim of the present work was to study diazepam-induced anxiolytic-like action and impairment of motor co-ordination, and the parameters of benzodiazepine receptors in mice lacking CCK₂ receptors. Methods The action of diazepam (0.5–3 mg/kg IP) was studied in the elevated plus-maze model of anxiety and rotarod test using mice lacking CCK₂ receptors. The parameters of benzodiazepine receptors were analysed using [³H]-flunitrazepam binding. Results In the plus-maze test, the exploratory activity of the homozygous (−/−) mice was significantly higher compared to their wild-type (+/+) littermates. However, the wild-type (+/+) mice displayed higher sensitivity to the anxiolytic-like action of diazepam. Even the lowest dose of diazepam (0.5 mg/kg) induced a significant increase of open arm entries in the wild-type (+/+) mice. A similar effect in the homozygous (−/−) mice was established after the administration of diazepam 1 mg/kg. The highest dose of diazepam (3 mg/kg) caused a prominent anxiolytic-like effect in the wild-type (+/+) mice, whereas in the homozygous (−/−) animals suppression of locomotor activity was evident. The performance of the homozygous (−/−) mice in the rotarod test did not differ from that of the wild-type (+/+) littermates. However, a difference between the wild-type (+/+) and homozygous (−/−) animals became evident after treatment with diazepam. Diazepam (0.5 and 3 mg/kg) induced significantly stronger impairment of motor co-ordination in the homozygous (−/−) mice compared to their wild-type (+/+) littermates. The density of benzodiazepine binding sites was increased in the cerebellum, but not in the cerebral cortex and hippocampus, of the homozygous (−/−) mice. Conclusions Female mice lacking CCK₂ receptors are less anxious than their wild-type (+/+) littermates. The reduced anxiety in homozygous (−/−) mice probably explains why the administration of a higher dose of diazepam is necessary to induce an anxiolytic-like action in these animals. The highest dose of diazepam (3 mg/kg) induced significantly stronger suppression of locomotor activity and impairment of motor co-ordination in the homozygous (−/−) mice compared to the wild-type (+/+) littermates. The increase in the action of diazepam is probably related to the elevated density of benzodiazepine receptors in the cerebellum of homozygous (−/−) mice. The present study seems to be in favour of increased tone of the GABAergic system in mice without CCK₂ receptors.     

Vascular effects of delta 9-tetrahydrocannabinol (THC), anandamide and N-arachidonoyldopamine (NADA) in the rat isolated aorta

The vascular effects of cannabinoids have been compared in the rat isolated aorta. Delta9-Tetrahydrocannabinol (THC), anandamide and N-arachidonoyl-dopamine (NADA) all caused vasorelaxation to similar degrees in pre-constricted aortae. Vasorelaxation to THC was inhibited by in vivo pre-treatment with pertussis toxin (10 microg/kg) or with the synthetic cannabinoid CP55,940 (((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), acutely or chronically), exposure to capsaicin in vitro (10 microM for 1 h), and de-endothelialisation. Vasorelaxation to anandamide was only inhibited by pertussis toxin and chronic CP55,940 pre-treatment (0.4 mg/kg for 11 days). Vasorelaxation to NADA was inhibited by pertussis toxin and chronic CP55,940 pre-treatment, and by de-endothelialisation. The vasorelaxant effects of the cannabinoids were not inhibited by cannabinoid CB1 receptor antagonism; however, vasorelaxation to both CP55,940 and THC was inhibited by cannabinoid CB2 receptor antagonism. Vasorelaxation to all cannabinoids was enhanced in the presence of indomethacin (10 microM). THC also caused vasoconstriction of the aorta while anandamide, NADA, CP55,940 and WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4benzoxazin-yl]-(1-naphthalenyl)methanone mesylate) did not. The vasoconstrictor effects of THC were inhibited by in vivo pre-treatment with pertussis toxin or CP55,940, acute exposure to CP55,940, cannabinoid CB1 receptor antagonism and cyclooxygenase inhibition. These results demonstrate the opposing vascular effects of cannabinoids in the rat aorta, and although vasorelaxation to each of the cannabinoids is of similar magnitude, it is mediated through different pathways. This gives further indication of the different vascular actions of cannabinoid compounds.     

Lack of critical involvement of endothelial nitric oxide synthase in vascular nitrate tolerance in mice

We examined the direct involvement of endothelial nitric oxide (eNOS) in nitrate tolerance using eNOS knockout (eNOS (-/-)) and wild-type (eNOS (+/+)) mice. Animals were treated with either nitroglycerin (NTG, 20 mg kg(-1)s.c. 3 x daily for 3 days) or vehicle (5% dextrose, D5W), and nitrate tolerance was assessed ex vivo in isolated aorta by vascular relaxation studies and cyclic GMP accumulation. Western blot was performed to determine NOS expression after NTG treatment. In both the eNOS (-/-) and (+/+) mice, the EC(50) from NTG concentration-response curve was increased by approximately 3 fold, and vascular cyclic GMP accumulation was similarly decreased after NTG pretreatment. Vascular tolerance did not lead to changes in eNOS protein expression in eNOS (+/+) mice. These results indicate that vascular nitrate tolerance was similarly induced in eNOS (-/-) and (+/+) mice, suggesting that eNOS may not be critically involved in nitrate tolerance development in mice.     

Effects of organic chemicals derived from ambient particulate matter on lung inflammation related to lipopolysaccharide

The effects of components of ambient particulate matter (PM) on individuals with predisposing respiratory disorders are not well defined. We have previously demonstrated that airway exposure to diesel exhaust particles (DEP) or organic chemicals (OC) extracted from DEP (DEP–OC) enhances lung inflammation related to bacterial endotoxin (lipopolysaccharide, LPS). The present study aimed to examine the effects of airway exposure to OC extracted from urban PM (PM–OC) on lung inflammation related to LPS. ICR mice were divided into four experimental groups that intratracheally received vehicle, LPS (2.5 mg/kg), PM–OC (4 mg/kg), or PM–OC + LPS. Lung inflammation, lung water content, and lung expression of cytokines were evaluated 24 h after intratracheal administration. LPS challenge elicited lung inflammation evidenced by cellular profiles of bronchoalveolar lavage fluid and lung histology, which was further aggravated by the combined challenge with PM–OC. The combination with PM–OC and LPS did not significantly exaggerate LPS-elicited pulmonary edema. LPS instillation induced elevated lung expression of interleukin-1β, macrophage inflammatory protein-1α, macrophage chemoattractant protein-1, and keratinocyte chemoattractant, whereas the combined challenge with PM–OC did not influence these levels. All the results were consistent with our previous reports on DEP–OC. These results suggest that the extracted organic chemicals from PM exacerbate infectious lung inflammation. The mechanisms underlying the enhancing effects are not mediated via the enhanced local expression of proinflammatory cytokines.     

Inhibitory effects of azelastine on substance P-induced itch-associated response in mice

The anti-pruritic mechanisms of azelastine were studied in mice. Scratching induced by intradermal histamine was inhibited by azelastine (30 mg/kg) and chlorpheniramine (30 mg/kg). Substance P-induced scratching was dose dependently suppressed by azelastine (3-30 mg/kg), but not by chlorpheniramine (10 and 30 mg/kg). Azelastine (30 mg/kg) inhibited the substance P-induced production of leukotriene B4, but not prostaglandin E2, in the skin. Azelastine (3-30 mg/kg) suppressed scratching induced by intradermal injection of leukotriene B4. The results suggest that inhibition of the production and action of leukotriene B4, as well as an anti-histamine action, is involved in the anti-pruritic action of azelastine.     

Imbalanced synthesis of cyclooxygenase-derived thromboxane A2 and prostacyclin compromises vasomotor function of the thoracic aorta in Marfan syndrome

BACKGROUND AND PURPOSE: Thoracic aortic dissection is a life-threatening complication of Marfan syndrome, a connective tissue disorder caused by mutations in the gene encoding fibrillin-1. We have demonstrated that nitric oxide-mediated endothelial-dependent relaxation is impaired in the thoracic aorta in Marfan syndrome. In the present study, we determined whether the cyclooxygenase (COX)-pathway is involved in the compromised aortic vasomotor function. EXPERIMENTAL APPROACH: Thoracic aortae from mice at 3, 6 and 9 months of age, heterozygous for the Fbn1 allele encoding a cysteine substitution (Fbn1 (C1039G/+), 'Marfan', n=35), were compared with those from age-matched controls (n=35). KEY RESULTS: Isometric force measurement revealed that preincubation with indomethacin, a non-specific COX inhibitor, but not valeryl salicylate, a specific COX-1 inhibitor, improved the phenylephrine-induced contractions (at 6 months, EC(50) and E(max) were increased 4.5-fold and by 45%, respectively) in Marfan aortae. Sensitivity to acetylcholine-induced relaxation was improved 10-fold. Blockade of the thromboxane-endoperoxide receptor by SQ-29548 did not affect phenylephrine-mediated contractions in Marfan aortae, although they did respond to the thromboxane analogue, U46619. From 6 months on, phenylephrine-induced secretion of prostacyclin and thromboxane A(2) in Marfan aortae was 200% and 40%, respectively, of those in controls. Reduced COX-1 expression was detected in Marfan aortae at 3 and 9 months, whilst COX-2 expression was increased from 3 months on. CONCLUSIONS AND IMPLICATIONS: The compromised vasomotor function in Marfan thoracic aortae is associated with an imbalanced synthesis of thromboxane A(2) and prostacyclin resulting from the differential protein expression of COX-1 and COX-2.     

Poly(ADP-ribose) polymerase inhibition improves endothelial dysfunction induced by reactive oxidant hydrogen peroxide in vitro

Reactive oxygen species, such as hydrogen peroxide (H(2)O(2)) induce oxidative stress and DNA-injury. The subsequent activation of poly(ADP-ribose) polymerase (PARP) has been implicated in the pathogenesis of various cardiovascular diseases including ischaemia-reperfusion injury, circulatory shock, diabetic complications and atherosclerosis. We investigated the effect of PARP-inhibition on endothelial dysfunction induced by H(2)O(2). In vascular reactivity measurements on isolated rat aortic rings we investigated the phenylephrine-induced contraction, and endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine and sodium nitroprusside. Endothelial dysfunction was induced by exposing the rings to H(2)O(2) (200 and 400 muM) for 30 min. In the treatment group, rings were preincubated with the potent PARP-inhibitor INO-1001. DNA strand breaks were assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Immunohistochemical analysis was performed for poly(ADP-ribose) (the enzymatic product of PARP) and for apoptosis inducing factor (a pro-apoptotic factor regulated by PARP). Exposure to H(2)O(2) resulted in reduced contraction forces and a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings (maximal relaxation to acetylcholine: 86.21+/-1.574% control vs. 72.55+/-1.984% H(2)O(2) 200 muM vs. 66.86+/-1.961% H(2)O(2) 400 muM; P<0.05). PARP-inhibition significantly improved the acetylcholine-induced vasorelaxation (77.75+/-3.019% vs. 66.86+/-1.961%; P<0.05), while the contractility remained unaffected. The dose-response curves of endothelium-independent vasorelaxation to sodium nitroprusside did not differ in any groups studied. In the H(2)O(2) groups immunohistochemical analysis showed enhanced PARP-activation and nuclear translocation of apoptosis inducing factor, which were prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of H(2)O(2)-induced endothelial dysfunction, which can be prevented by PARP inhibitors.