Liying Chen

Preservation of Endogenous Antioxidant Activity and Inhibition of Lipid Peroxidation as Common Mechanisms of Antiatherosclerotic Effects of Vitamin E, Lovastatin and Amlodipine☆

OBJECTIVES We sought to document the common mechanisms of the antiatherogenic effects of the cholesterol-lowering hydroxy-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin, the dihydropyridine Ca2+ blocker amlodipine and the antioxidant vitamin E. BACKGROUND Vitamin E, HMG-CoA reductase inhibitors and Ca2+ blockers each inhibit atherosclerosis in hypercholesterolemic animals. METHODS New Zealand White rabbits were fed regular chow (Group A), chow with 1% cholesterol (Group B), 1% cholesterol diet plus lovastatin (Group C), 1% cholesterol diet plus vitamin E (Group D) or 1% cholesterol diet plus amlodipine (Group E) for 12 weeks. The extent of aortic atherosclerosis was measured by planimetry of the sudanophilic area. Malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured as indexes of lipid peroxidation and antioxidant activity, respectively. RESULTS Group A rabbits showed no atherosclerosis, whereas Group B rabbits had 17.4 +/- 9.3% (mean +/- SD) of the aorta covered with atherosclerosis, and Groups C, D and E rabbits had significantly less atherosclerosis. Plasma SOD activity was lower in Group B than in Group A (6.9 +/- 1.1 vs. 12.8 +/- 1.5 U/ml, p < 0.01) and was preserved in the groups given lovastatin, vitamin E or amlodipine with a high cholesterol diet. The serum MDA level was higher in Group B rabbits than Group A rabbits (12.1 +/- 2.6 vs. 1.2 +/- 0.1 nmol/ml, p < 0.01) and increased minimally in rabbits given lovastatin, vitamin E or amlodipine with a high cholesterol diet. In in vitro experiments, both lovastatin and amlodipine preserved SOD activity and reduced the oxidizability of low density lipoproteins by rabbit leukocytes. CONCLUSIONS This study suggests that a reduction in lipid peroxidation and preservation of SOD may be common mechanisms of antiatherosclerotic effects of lovastatin, vitamin E and amlodipine.

Alterations in nitric oxide synthase activity, superoxide anion generation, and platelet aggregation in systemic hypertension, and effects of celiprolol.

A decrease in endothelium-derived relaxing factor or nitric oxide has been proposed as a potential mechanism of increased vascular resistance in hypertension. An increase in the generation of superoxide anions, which degrade nitric oxide and induce platelet aggregation, may also compromise regional blood flow in hypertension. Recent studies show that human neutrophils generate nitric oxide, which has a similar biologic profile to the endothelium-derived relaxing factor. This study measured nitric oxide synthase activity and superoxide generation in human neutrophils and platelet aggregation in patients with essential hypertension. Nitric oxide synthase activity, measured as conversion of 3H-L-arginine to 3H-L-citrulline, in peripheral blood neutrophils was decreased in hypertensive subjects (percent conversion of 3H-L-arginine: 4.2 +/- 0.5 vs 9.0 +/- 3.0 in control subjects; p < 0.01). Neutrophil superoxide anion generation, measured as conversion of ferricytochrome C to ferrocytochrome C, in response to phorbol-12-myristate 13-acetate (100 ng/ml) was higher in hypertensive subjects (17.5 +/- 8.1 vs 13.2 +/- 3.0 nmoles/10(6) cells/10 minutes in control subjects; p < 0.05). Patients were treated with a selective beta blocker, celiprolol, for 8 weeks. Supine blood pressure decreased from 177/103 mm Hg (mean +/- SD 18/7) to 160/92 mm Hg (mean +/- 10/5; p < 0.02), while heart rate was unchanged (73 +/- 11 vs 69 +/- 10 beats/min). Epinphrine and adenosine diphosphate-induced platelet aggregation was also increased in hypertensive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction in human neutrophil superoxide anion generation by n-3 polyunsaturated fatty acids: Role of cyclooxygenase products and endothelium-derived relaxing factor

Dietary supplementation with n-3 polyunsaturated acids (PUFAs) results in augumented vasorelaxation and reduction in superoxide anion generation. Augmented vasorelaxation may be mediated by enhanced generation of vasodilator prostaglandins and/or endothelium-derived relaxing factor (EDRF), now thought to be nitric oxide (NO). To determine the importance of enhanced vasodilator prostaglandins or EDRF-NO in reduction in superoxide anion generation during n-3 PUFAs intake, human polymorphonuclear leukocytes (PMNs) were incubated with n-6 PUFA arachidonic acid (AA), or n-3 PUFAs eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (each 10(-7) M) for 1 hr at 37 degrees C. Parallel sets of PMNs were treated with the cyclooxygenase inhibitors indomethacin (10(-5) M), or aspirin (10(-5) M), or the EDRF-NO synthase inhibitor L-NMMA (10(-3) M) prior to incubation with PUFAs. Superoxide anion generation by PMNs was determined by measuring the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome C. PMNs incubated with EPA or DHA, but not AA, demonstrated marked reduction in superoxide anion generation. This reduction in superoxide anion generation by n-3 PUFAs was abolished by treatment of PMNs with indomethacin or aspirin, but not by L-NMMA. These observations suggest that n-3 PUFAs decrease superoxide anion generation primarily by a prostaglandin-dependent pathway.

Melagatran, an oral active-site inhibitor of thrombin, prevents or delays formation of electrically induced occlusive thrombus in the canine coronary artery.

Intravenous administration of thrombin inhibitors, such as hirudin, has been shown to decrease the frequency of coronary artery reocclusion after thrombolysis. However, recent findings in large clinical trials in patients with unstable angina and myocardial infarction have failed to demonstrate a sustained antithrombotic effect after cessation of drug treatment. These findings indicate a need for a prolonged antithrombotic regimen, preferably an orally active thrombin inhibitor. To test the hypothesis that a regimen consisting of oral thrombin inhibitor will delay or prevent the formation of occlusive clot, anesthetized dogs were given saline (n = 9) or a single dose of a novel active site low-molecular-weight thrombin inhibitor melagatran by nasogastric tube (1.5 mg/kg, n = 6; 2.5 mg/kg, n = 6), and 15 min later, a potent thrombogenic stimulus in the form of anodal current (100 microA) was applied to the intimal surface of the narrowed left anterior descending coronary artery (LAD). All saline-treated dogs developed stable thrombus, indicated by zero flow at 34 +/- 7 min after initiation of direct current. On the other hand, one of the six dogs given high-dose melagatran did not develop thrombotic occlusion of the LAD during the entire 4 h of observation. Mean time to occlusive thrombus formation in 11 other dogs was prolonged 4-5 times as compared with that in the saline-treated dogs (p < 0.001). Spontaneous thrombolysis was observed in three of 11 dogs after initial clot formation. Overall, the coronary artery was patent for 68% (low dose) and 75% (high dose) of the observation period in melagatran-treated dogs (vs. 14% of observation period in saline-treated dogs). Peak plasma concentration was 0.87 +/- 0.22 microM in dogs given low-dose and 1.38 +/- 0.30 microM in dogs given high-dose melagatran. The activated partial thromboplastin time (aPTT) increased 1.5-fold at peak plasma concentration of melagatran. These observations imply (a) thrombin generation plays a critical role in thrombus formation in narrowed coronary arteries, (b) oral melagatran prevents or delays thrombus formation, whereas the aPTT is only modestly prolonged, and (c) the thrombus formed in the presence of melagatran is prone to spontaneous lysis in this canine model of coronary thrombosis.

Effect of stable fish oil on arterial thrombogenesis, platelet aggregation, and superoxide dismutase activity

We examined the influence of dietary stable fish oil on aortic thrombosis, platelet aggregation, and superoxide dismutase (SOD) activity in a rat model. Twenty-nine Sprague-Dawley rats were fed regular chow supplemented with stable fish oil preparation (for 1 or 3 weeks), and 37 rats fed regular chow served as controls. The abdominal cavity was opened, and the abdominal aorta isolated. Whatman paper impregnated with 35% FeCl3 was wrapped around the surface of the aorta, and aortic flow was continuously recorded. In control rats, an occlusive platelet-fibrin-rich thrombus was formed in 21 +/- 3 min. Dietary fish oil in a time-dependent fashion delayed time to thrombus formation (24 +/- 2 min in rats fed fish oil for 1 week and 31 +/- 2 min in rats fed fish oil for 3 weeks), inhibited platelet aggregation (21 +/- 5% vs. 45 +/- 6%; p < 0.01) and increased SOD activity (p < 0.01). We conclude that dietary supplementation with stable fish oil delays formation of arterial thrombus, probably by reducing platelet aggregation and oxidative stress-associated arterial injury.

Effects of Exercise-Induced Oxidative Stress on Nitric Oxide Release and Antioxidant Activity☆

This study shows that acute exercise in healthy subjects is a modest oxidative stress, which may be related to an increase in antioxidant activity and down-regulation of nitric oxide formation.

Reperfusion injury in the endotoxin-treated rat heart: reevaluation of the role of nitric oxide.

The role of nitric oxide (NO) in ischaemia‐reperfusion injury to the heart continues to be debated. The role of NO released during endotoxemia on myocardial reperfusion injury was examined in rats given saline or lipopolysaccharide (LPS, 10 mg kg−1). Aortic rings from LPS‐treated rats showed a markedly decreased contractile response to both noradrenaline (NA) and U46619, and a diminished relaxation response to acetylcholine, thrombin and aggregating platelets. Treatment of rat aortic rings from LPS‐treated rats with the NO synthesis inhibitor Nω‐nitro‐l‐arginine (l‐NOARG) reversed the diminished contractile response to NE and U46619. Before ischaemia‐reperfusion, baseline force of cardiac contraction (FCC) and coronary perfusion pressure (CPP) were lower and coronary flow was higher in hearts from LPS‐treated rats (all P<0.05 vs. saline‐treated group). Treatment of hearts from LPS‐treated rats with l‐NOARG increased baseline FCC and CPP. After ischaemia‐reperfusion, hearts from saline‐treated rats showed a 36±5% fall in FCC, a 38±6% rise in CPP and a 38±5% fall in coronary flow, whereas hearts from LPS‐treated rats revealed only a 16±9% fall in FCC, a 10±3% rise in CPP and a 20±4% fall in coronary flow (all P<0.05 vs. changes in saline‐treated group). Fewer hearts from LPS‐treated rats developed reperfusion arrhythmias (6% vs. 60% hearts from saline‐treated rats, P<0.02). Myocardial superoxide dismutase activity was higher in the LPS‐treated group (P<0.05). NO synthesis, measured as formation of nitrite, was higher (P<0.05) in cardiac and aortic tissues from LPS‐treated rats. Prostacyclin (PGI2) release in coronary effluent was greater in LPS‐treated rat hearts (P<0.05 vs. saline‐treated rats). Thus LPS‐treated hearts demonstrate a basal decrease in FCC and coronary vascular resistance. These hearts demonstrate a modest protection from reperfusion injury. Induction of NO synthesis, and possibly PGI2 release, may underlie cardioprotection from ischaemia‐reperfusion.

Aspirin does not potentiate effect of suboptimal dose of the thrombin inhibitor inogatran during coronary thrombolysis.

OBJECTIVES Coronary artery often reoccludes after thrombolytic therapy with recombinant tissue-plasminogen activator (rt-PA). This reocclusion is thought to be due to in situ platelet activation mediated by thromboxane (Tx) A2 and thrombin; hence, aspirin and thrombin inhibitors are often used in patients with acute myocardial infarction. This study was designed to examine the modulation of coronary artery reocclusion by a novel low molecular weight direct thrombin inhibitor inogatran with or without aspirin. METHODS 22 dogs with electrically-induced occlusive intracoronary thrombus were treated with saline (n = 7, group A), or high dose inogatran (0.25 mg/kg bolus followed by 0.6 mg/kg per h for 2 h, n = 5, group B), or low dose inogatran (0.125 mg/kg bolus followed by 0.3 mg/kg per h for 2 h, n = 5, group C), or aspirin+low dose inogatran (n = 5, Group D). Recombinant tissue-plasminogen activator (rt-PA) was infused for 20 min starting 2 min after the bolus in all dogs. Coronary artery blood flow was monitored for 120 min after rt-PA administration. RESULTS Reperfusion rates were similar in all groups, but the time to reperfusion was shortest in group B dogs (18 +/- 2 min vs. 32 +/- 7 min in group A dogs, P < 0.05). Reocclusion rates were 80%, 0%, 50%, and 60% in groups A, B, C, and D dogs, respectively. The restored blood flow persisted for 19 +/- 10, > 120 min, 71 +/- 30 and 54 +/- 26 min in groups A, B, C, and D dogs, respectively. At the end of rt-PA infusion, prothrombin time (PT) and activated partial thromboplastin time (APTT) were increased 1.3-2 times the control value, and the changes in PT and APTT were similar in all groups. Thrombin generation and activity, assessed by rise in thrombin-antithrombin complex and fibrinopeptide A levels, and decrease in fibrinogen levels were most marked in group A dogs, and less so in group B, C and D dogs. CONCLUSIONS These data show that high dose of direct thrombin inhibitor inogatran shortens time to reflow and abolishes coronary artery reocclusion. However, aspirin does not potentiate the effect of suboptimal doses of inogatran.

Lys-and Glu-plasminogen potentiate the inhibitory effect of recombinant tissue plasminogen activator on human platelet aggregation

To examine the basis of enhanced thrombolytic effect of tissue-type plasminogen activator (t-PA) in the presence of lys- or glu-plasminogen, studies were performed with human platelet-rich plasma (PRP) and washed platelets (WP). t-PA inhibited platelet aggregation in PRP and this effect was potentiated by lys-plasminogen as well as glu-plasminogen. t-PA inhibited WP aggregation only in the presence of lys- or glu-plasminogen. The potentiation of the effects of t-PA was greater (P < 0.05) with lys-plasminogen than with glu-plasminogen. t-PA alone also decreased 14C-serotonin release from WP, and lys- as well as glu-plasminogen reversed this effect of low concentrations of t-PA in WP. Aggregation of WP was also inhibited by plasmin, a proteolytic product of plasminogen. Low, but not high concentrations, of plasmin increased the release of 14C-serotonin. Anti-aggregatory effects of plasmin and lys-plasminogen plus t-PA on platelets were attenuated by preincubation of PRP or WP suspension with aprotinin. These observations suggest enhanced inhibitory effect of t-PA on platelet function in the presence of lys-plasminogen as potential basis of salutary interaction in models of arterial thrombosis.

Recombinant lys-plasminogen, but not glu-plasminogen, improves recombinant tissue-type plasminogen activator-induced coronary thrombolysis in dogs

OBJECTIVES This study examined the modification of recombinant tissue-type plasminogen activator (rt-PA)-induced thrombolysis by recombinant lys-plasminogen. BACKGROUND Recombinant tissue-type plasminogen activator restores flow in the thrombosed coronary artery, but the artery often reoccludes. The rt-PA-induced thrombolysis is a result of activation of plasminogen bound to fibrin in the thrombus and results in generation of the fibrinolytic enzyme plasmin. Small amounts of lys-plasminogen are formed when rt-PA is used. Lys-plasminogen binds to fibrin with a 10-fold greater affinity than the predominant native glu-plasminogen, leading to a loose fibrin structure. METHODS Dogs with electrically induced occlusive intracoronary thrombus were treated with saline solution (n = 9), glu-plasminogen (2 mg/kg body weight, n = 5) or lys-plasminogen (2 mg/kg, n = 5), followed by infusion of rt-PA (1 mg/kg over 20 min) 10 min later. RESULTS Reperfusion rates were similar in all groups of dogs, but the time to reflow was lowest in dogs given lys-plasminogen compared with those given saline solution or glu-plasminogen before rt-PA (mean [+/- SE] 14 +/- 2 vs. 22 +/- 2 and 23 +/- 3 min, respectively, p < 0.05). None of the reperfused coronary arteries reoccluded in the lys-plasminogen plus rt-PA group, whereas 75% reoccluded in dogs given saline solution plus rt-PA, and 50% reoccluded in those given glu-plasminogen plus rt-PA. Accordingly, duration of reflow was greater in the lys-plasminogen plus rt-PA group (> 120 vs. 39 +/- 7 and 82 +/- 21 min, respectively, p < 0.05). Plasminogen activator inhibitor-1 activity decreased during rt-PA infusion and thereafter increased in all dogs, but less so in dogs given lys-plasminogen (p < 0.05 vs. those given saline solution before rt-PA). CONCLUSIONS Treatment with recombinant lys-plasminogen before rt-PA reduces time to reflow and sustains reflow after thrombolysis, whereas glu-plasminogen has no such effect.