Dietary arginine prevents atherogenesis in the coronary artery of the hypercholesterolemic rabbit

Objectives. This study was designed to test the hypothesis that long-term oral supplementation of dietary l-arginine (to provide a sustained elevation of nitric oxide activity) would inhibit atherogenesis in hypercholesterolemic rabbits, as assessed by histomorphometric measurements.Background. Endothelium-derived nitric oxide inhibits a number of processes that are critical in atherogenesis. Hypercholesterolemia reduces endothelial nitric oxide activity, and we postulate that this may promote atherogenesis. This reduction in nitric oxide activity can be reversed acutely by intravenous infusion of l-arginine, the precursor of nitric oxide. We show that dietary supplementation of l-arginine abrogates the development of coronary atheroma in hypercholesterolemic rabbits.Methods. Male New Zealand White rabbits were fed normal rabbit chow, 1% cholesterol chow or 1% cholesterol chow with dietary arginine or methionine supplementation to increase their intake of these amino acids sixfold. After 1 or 10 weeks of dietary intervention, the left main and left anterior descending coronary arteries were harvested for histologic study. Plasma cholesterol measurements were elevated to the same degree in all groups of rabbits receiving the 1% cholesterol diet, whereas plasma arginine levels were doubled in the arginine-treated group. High density lipoprotein (HDL) cholesterol values were not affected by arginine treatment.Results. In rabbits receiving the 1% cholesterol diet, with or without methionine supplementation, light and electron microscopy revealed a marked increase from 1 to 10 weeks in the intimal accumulation of macrophages, associated with an increase in the intimal area of the left main coronary artery. By contrast, in arginine-treated hypercholesterolemic rabbits, there was a near absence of adherent monocytes and tissue macrophages and no progression of intimal thickness from 1 to 10 weeks.Conclusions. Dietary supplements of l-arginine prevent intimal thickening in the coronary arteries of hypercholesterolemic rabbits. This antiatherogenic effect is not due to an alteration in plasma total cholesterol, HDL cholesterol or caloric or nitrogen balance. The data are consistent with the hypothesis that nitric oxide has antiatherogenic properties.     

No effect of an L-arginine-enriched medical food (HeartBars) on endothelial function and platelet aggregation in subjects with hypercholesterolemia

Background Providing l-arginine as a precursor for nitric oxide has been proposed to improve endothelial function in populations at high risk for cardiovascular events. We studied the effects of dietary l-arginine supplementation with HeartBars (a medical food rich in L-arginine, Cooke Pharma, Belmont, Calif) on flow-mediated dilation and markers of endothelial function in subjects with hypercholesterolemia. Methods We randomly assigned 47 subjects with hypercholesterolemia to receive one HeartBar containing 3.3 g l-arginine each, or a placebo bar, consumed twice daily for 2 weeks. Flow-mediated dilation, platelet aggregation studies, and soluble levels of endothelial and platelet adhesion molecules were obtained before and after the 2-week treatment period. Results Baseline and follow-up levels of l-arginine were 78.5 ± 28.2 μmol/L and 80.7 ± 26.7 μmol/L, respectively (P = .54). The HeartBar group had no improvement in flow-mediated dilation; changes in brachial artery diameter at baseline and follow-up were 5.52% ± 3.32% and 4.96% ± 2.39%, respectively. There were also no changes in the soluble levels of E-selectin and P-selectin by treatment group. Conclusions In our study, 2 weeks of HeartBar supplementation in subjects with hypercholesterolemia showed no favorable effects on endothelial or platelet function. (Am Heart J 2003;145:e15.)     

Influence of hypercholesterolemia and endothelin-3 pre-treatment on the effects of shear forces on platelet aggregation and cyclic GMP content

Shear forces induce platelet aggregation and stimulate the endothelial production of anti-aggregatory factors. Among them, endothelin-3 (ET-3) has been reported to reduce aggregation and to increase platelet cyclic GMP (cGMP) content. Since hypercholesterolemia modifies both platelet aggregability and endothelial function, we compared in 14 hypercholesterolemic and 15 normocholesterolemic subjects the influences of shear forces (240 and 650 s(-1)) on platelet aggregation and cGMP content, and their modulation by ET-3. Spontaneous maximal aggregation occurred earlier and at a greater extent in hypercholesterolemic than in normocholesterolemic subjects (63+/-2 vs 46+/-6% P < 0.01). Pre-treatment with ET-3 abolished the shear-induced facilitation of maximal aggregation in platelets of normocholesterolemic (from 70+/-2 to 52+2% at 240 s(-1) and from 73+/-1 to 59+/-2S at 650s(-1); P < 0.05) and hypercholesterolemic (from 78+/-3 to 64+/-2 at 240 s(-1) and from 78+/-2 to 66+/-3 at 650 s(-1); P < 0.05) subjects. cGMP content did not significantly differ between normocholesterolemic and hypercholesterolemic subjects (6.1+/-0.5 vs 6.9+/-0.7 pmol/10(9) platelets). It was reduced in platelets submitted to shear forces (P < 0.05). This shear-dependent reduction was suppressed by ET-3 pre-treatment. These results demonstrate that shear forces enhance platelet aggregation and diminish their cGMP content. ET-3 reduces the pro-aggregating effects of shear, suggesting a rise in cGMP content as a dynamic associated mechanism.     

Investigation of decreased availability of nitric oxide precursor as the mechanism responsible for impaired endothelium-dependent vasodilation in hypercholesterolemic patients

Objective. The purpose of this study was to determine whether the impaired endothelium-dependent vasodllation of hyperchoiesterolemic patients is due to decreased availability of l-arginine, the substrate for nitric oxide.Background. Patients with hypercholesterolemia have impaired endothelium-dependent vasodilation that is related to a defect in the endothelium-derived nitric oxide system. However, the precise location of this abnormality has not been determined.Methods. The study included 12 hypercholesterolemic patients (6men, 6 women; 52 ± 9 years old; serum cholesterol>240mg/dl) and 15 normal volunteers (8 men, 7 women; 50 ± 6 years old; serum cholesterol < 210 mg/dl. The forearm vascular responses to intraaterial infusion of acetylccholine, an endothelium-dependent vasodilator (7.5, 15, 30 μg/min), and sodium nitropruside, a direct smoth muscle dilator (0.8,1.6,3.2 μg/min) were studied before and during Infusion of l- or d-arginine (a stereoisomer of arginine that is not a nitric oxide precursor).Results. The response to acetylcholine was lower in hypercholesterolemic patients than in control sutjects. However, no significant difference was observed with sodium nitroprusside infusion. l-Arginine augmented the response to acetylcholine in normal subjects (maximal blood flow increased from 14.4 ± 7 to 18.9 ± 10 ml/min per 100 ml, p < 0.002). In contrast, in the hypercholesterolemic patients, only a mild but not significant improvement in the response to acetylcholine was observed with the infusion of l-arginine (maximal blood flow increased from 6.8 ± 4 to 8.4 ±5ml/min per 100ml; p = 0.161; however, a similar mild but not significant change was also observed with l-arginine (maximal blood flow increased from 6.8± 4 to 8.3 ± 4 ml/min per 100 ml, p = 0.07). l-Arginine did not modify the response to sodium nitroprusside in either group.Conclusions. The augmentation of endothelium-dependent vasodilation by l-arginine, the nitric oxide precursor, is defective in hypercholesterolemic patients. This supports the concept of an abnormal endothelium-derived nitric oxide system in hypercholesterolemia and indicates that decreased availability of nitric oxide substrate is not responsible for the impaired endothelial function in this condition.     

No effect of taurine on platelet aggregation in men with a predisposition to type 2 diabetes mellitus

Any diet therapy that potentially could affect platelet function would also influence the initiation of atherosclerotic plaque formation which is an important complication of diabetes mellitus eventually resulting in myocardial infarction and stroke. Blood platelets are rich in taurine, and it has been shown that taurine inhibits platelet aggregation in healthy subjects. The purpose was to examine the effect of taurine supplementation on platelet aggregation in high-risk subjects with a positive family history of T2DM. Twenty healthy men were included in a double-blinded, randomized, crossover study, receiving daily supplementation of 1.5 g taurine or placebo for two 8-week periods. Subjects were overweight and first-degree relatives of T2DM patients. At the end of each treatment, fasting blood samples for assessment of platelet aggregation was drawn. Platelet aggregation was induced by ADP. Plasma taurine concentration was significantly greater after taurine intervention compared to placebo (131.4+/-61.7 vs. 38.9+/-6.7 micromol/l, P<0.0001). There was no difference in the threshold level for complete platelet aggregation induced by ADP in vivo between placebo and taurine intervention (placebo 3.86+/-2.21 vs. taurine 3.86+/-3.25 micromol/l). Supplementation with 1.5 g of taurine for 8 weeks had no effect on platelet aggregation in overweight prediabetic men.     

Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice

The pathogenic mechanisms by which physical exercise influences atherosclerotic lesion formation remain poorly understood. Because vigorous physical training increases oxidative stress, this study tested the hypothesis that graduated and moderate physical exercise together with metabolic intervention (l-arginine and antioxidants) may contribute to increased vascular protection. Exercise training in mice was induced by graduated swimming. In hypercholesterolemic male mice on an atherogenic high-cholesterol diet, graduated and moderate exercise lowered plasma cholesterol and decreased atherosclerotic lesions compared with sedentary control mice. Antioxidants (1.0% vitamin E added to the chow and 0.05% vitamin C added to the drinking water) and l-arginine (6% in drinking water) supplementation to exercising hypercholesterolemic mice further and synergistically reduced atherosclerosis compared with untreated exercised mice. Arterial oxidation-specific epitopes and systemic oxidative stress were reduced by metabolic intervention. Graduated chronic exercise elicited an increase in production of nitric oxide through increased endothelial nitric oxide synthase expression and ameliorated scavenger activities. Thus, metabolic intervention with l-arginine and antioxidants together with graduated and moderate exercise training reduce atherosclerotic lesion formation.     

Nitric oxide-dependent inhibition of platelet aggregation in elderly patients with hypercholesterolemia

Hypercholesterolemia reduces production of nitric oxide (NO), a potent inhibitor of platelet aggregation, in endothelial cells. Recently platelet has been found to have NO synthase. Hypercholesterolemia may influence platelet NO production. We investigated NO-dependent inhibition of platelet aggregation in elderly hypercholesterolemic patients with total cholesterol (Tchol) of 240 mg/dl or more (n = 21). In elderly controls with Tchol less than 240 mg/dl (n = 61), L-arginine (5-50 mM) inhibited ADP-induced platelet aggregation in a dose-dependent manner (42.4% inhibition at 50 mM). However, L-arginine did not inhibit platelet aggregation in elderly hypercholesterolemic patients. L-arginine increased cyclic GMP production in elderly controls, but not in hypercholesterolemic patients (p < 0.02). Hypercholesterolemic patients showed increased platelet aggregation compared with elderly controls(p = 0.018). L-nitro-arginine methyl ester 12.5-50 uM increased platelet aggregation in both groups. Superoxide dismutase improved L-arginine inhibition of platelet aggregation in elderly hypercholesterolemic patients (p = 0.02). LDL cholesterol of 160 mg/dl or more was an independent predictor for loss of L-arginine inhibition of platelet aggregation (relative risk 3.9, p = 0.0098). This result suggests that hypercholesterolemia causes decreased NO-dependent inhibition of platelet aggregation due to reduced NO utilization. NO-dependent platelet aggregation may be a powerful tool for detection of vascular injury.     

Early endothelial dysfunction in adults at risk from atherosclerosis: different responses to l-arginine

Objectives. We sought to examine endothelial responses to l-arginine in three groups with isolated risk factors: hypercholesterolemia, smoking and insulin-dependent diabetes mellitus (IDDM).Background. Endothelial dysfunction occurs early in atherosclerosis, predating clinical disease. We hypothesized that the nature of endothelial injury associated with individual cardiovascular risk factors might be different and that this might affect the response to l-arginine, the substrate for endothelial nitric oxide synthase.Methods. We studied the effects of intravenous l-arginine on brachial artery flow-mediated dilation (FMD) and glyceryl trinitrate (GTN)–mediated dilation in 36 young subjects (18 to 40 years old) without clinical atherosclerosis: 9 each of normal control subjects, hypercholesterolemic subjects, cigarette smokers and subjects with IDDM.Results. Baseline FMD was significantly impaired in hypercholesterolemic subjects (mean ± SD 1.7 ± 2.3%), smokers (1.6 ± 1.8%) and diabetic subjects (1.8 ± 1.5%) compared with that in control subjects (6.9 ± 3.3%, p = 0.001). The response to GTN was not significantly different between the subjects with risk factors and control subjects, apart from those with IDDM, in whom it was significantly impaired (p = 0.026). After infusion of l-arginine, there was no change in FMD in control or diabetic subjects. In hypercholesterolemic subjects and smokers, FMD improved from 1.9 ± 1.9% to 4.1 ± 2.1% (p = 0.01) and from 2.0 ± 1.71% to 3.1 ± 2.5% (p = 0.02), respectively.Conclusions. FMD was impaired in all three risk factor groups; however, they responded differently to l-arginine, FMD being improved in hypercholesterolemic subjects and smokers but unchanged in diabetic subjects. These results indicate differing underlying pathophysiologies that may facilitate the design of treatment strategies for subjects with different risk factors.     

Increase of erythrocyte aggregation in hypertensive men with dyslipidemia]

We have already reported that erythrocyte aggregation (EA) is increased in hypertensive subjects. To study the influence of other risk factors in EA, we have measured aggregation index IA and disaggregation shear rate threshold (gamma c) by a laser technique and the biochemical parameters in 16 normotensive normocholesterolemic subjects (NT/NCT) and 45 hypertensive subjects where 17 were normocholesterolemic (HT/NCT), 18 were pure hypercholesterolemic (HT/HCTIIa) and 10 were hypercholesterolemic hypertriglyceridemic (HT/HCTIIb). The results show that IA and gamma c are more important in HT/NCT than NT/NCT patients and much more in patients with hypercholesterolemia and hypertriglyceridemia. The cumulative effects of hypertension and hyperlipidemia merit take into consideration in pathophysiology of cardiovascular complications and could help to new strategy therapeutic developments for treatment of these complications.     

Statins have an early antiplatelet effect in patients with acute myocardial infarction

Statins confer an antiplatelet effect in hypercholesterolemic subjects and in stable coronary artery disease patients. We explored the antiplatelet effects of statins in ST-elevation myocardial infarction (STEMI) patients undergoing primary angioplasty. Of 120 STEMI patients, 80 (67%) received statins while 40 (33%) did not. Ex vivo platelet reactivity was studied on admission and 72 hours later by conventional aggregometry and under flow conditions (Impact R). Measures of platelet reactivity under flow conditions included aggregate size and surface coverage, signifying platelet aggregation and adhesion respectively. The effect of statins on platelet function under flow conditions and platelet aggregation was studied in?vitro in platelets from 10 STEMI patients. Platelets from each patient were incubated in?vitro with lovastatin or PBS as a control. The effect of lovastatin in the presence of a nitric oxide synthase inhibitor (L-NMMA) was also studied. Patients treated with statins were compared with those who did not have significantly lower ADP-induced platelet aggregation on the 4th day (56 ± 18% vs. 64 ± 17%, p=0.02). Platelet deposition under flow conditions as measured by surface coverage was reduced from admission to 72 hours later among statin-treated patients (19 ± 28% reduction, p<0.01), but was unchanged in non-treated patients (for comparison p<0.01). The extent of platelet inhibition was unrelated to patient characteristics, including lipid profile and type of statin administered (lipophylic vs. hydrophilic). In the in vitro study platelet incubation with statin compared with PBS resulted in a lower aggregate-size (29 ± 9 μm(2) vs. 39 ± 15 μm(2), p<0.01), and lower surface coverage (8.5 ± 4% vs. 12 ± 4%, p<0.01). The effect of the statin on both parameters was significantly blunted by L-NMMA. Incubation with statin also resulted in a reduction in collagen-induced platelet aggregation (31 ± 20% vs. 54 ± 25%, p<0.01). We concluded that in acute myocardial infarction patients, statins have an early antiplatelet effect, in addition to that afforded by standard antiplatelet therapy.     

Physical training and metabolic supplementation reduce spontaneous atherosclerotic plaque rupture and prolong survival in hypercholesterolemic mice

Moderate physical exercise (PE) combined with metabolic treatment (MT) (antioxidants and l-arginine) are well known to reduce atherosclerotic lesion formation in hypercholesterolemic mice. However, the long-term beneficial effects on unstable atheroma remain poorly understood. We started early PE training in large groups of 6-week-old hypercholesterolemic mice (by graduated swimming) alone or in combination with nutritional supplementation (1.0% vitamin E added to the chow and 0.05% vitamin C and 6% l-arginine added to the drinking water). Inactive controls did not receive PE. The spontaneous development of atherosclerotic plaque rupture (associated with advanced atherosclerosis) and survival rates were evaluated. Moderate PE elicited an increase in plasma levels of nitric oxide. Early combined treatment with PE and MT in the hypercholesterolemic mice significantly reduced lesions (also detected noninvasively at 10 months) and spontaneous atherosclerotic plaque rupture and prolonged survival more effectively than each intervention alone. Thus, early concerted actions of MT and PE improve the natural history of atherosclerotic lesions and reduce the plaque instability in hypercholesterolemic mice.     

Homocysteine-induced inhibition of nitric oxide production in platelets: a study on healthy and diabetic subjects

Aims/hypothesis: The molecular mechanisms involved in the platelet activation observed in hyperhomocysteinemia are not known. We aimed to discover if homocysteine concentrations are associated with abnormal platelet nitric oxide production in healthy and diabetic subjects. Methods: The study cohort included 28 patients with Type I (insulin-dependent) diabetes mellitus, 30 patients with Type II (non-insulin-dependent) diabetes mellitus, and 34 healthy subjects. Homocysteine plasma concentrations were measured by high-performance liquid chromatography. Platelet nitric oxide production was measured using a nitric oxide meter before and after a 3-h incubation with 100 μmol/l homocysteine. Stimulation experiments were done in vitro by the addition of α-thrombin (0.2 U/ml). Results: Basal platelet nitric oxide production was lower in diabetic patients than in healthy subjects. Nitric oxide release was reduced by in vitro homocysteine incubation, being lower in platelets from diabetic patients than in platelets from control subjects. Thrombin increased nitric oxide synthesis in platelets from healthy subjects both in the presence and absence of homocysteine. In diabetic subjects thrombin increased nitric oxide release in the absence of homocysteine. But in the presence of homocysteine the response was reduced. An inverse relation was found between plasma homocysteine levels and basal platelet nitric oxide release in diabetic and healthy subjects. Conclusion/interpretation: Homocysteine could exert its atherogenic action in healthy and diabetic subjects partly by inhibiting platelet nitric oxide production with the subsequent increased platelet activation and aggregation. [Diabetologia (2001) 44: 979–982] Received: 29 November 2000 and in revised form: 4 April 2001     

Chronic l-arginine treatment reduces vascular smooth muscle cell hypertrophy through cell cycle modifications in spontaneously hypertensive rats

OBJECTIVE: To investigate the effect of long-term l-arginine supplementation on phenotype and proliferative status of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) as well as the possible changes in nitric oxide (NO) availability. METHODS: Male SHR, 22 weeks of age, received l-arginine (660 mg/kg per day) in their drinking water for 12 weeks. VSMCs from untreated (C-VSMC) and l-arginine-treated (l-Arg-VSMC) SHR were isolated from the common carotid artery, cultured and used until passage five. Size, protein content, cell proliferation and ploidy were evaluated in carotid VSMCs in culture, as well as the possible association of NO in these changes. RESULTS: Relative cell size, total protein content per cell, and number of polyploid cells were significantly lower in l-Arg-VSMC compared to C-VSMC. Fetal calf serum stimulation (10% FCS) increased cell number only in l-Arg-VSMC. DNA synthesis, assessed by [H]methylthymidine incorporation after 10% FCS stimulation, was higher in l-Arg-VSMC than in C-VSMC. Cell cycle analysis revealed a significant increase of the number of l-Arg-VSMC at the G1 phase, together with a reduction at the G2 + M phase. In contrast, C-VSMC were arrested at the G2 + M phase of the cell cycle. Nitrite/nitrate levels, as well as intracellular cyclic guanosine monophosphate (cGMP) content, were significantly higher in l-Arg-VSMC. This was accompanied by enhanced inducible nitric oxide synthase (iNOS) expression and activity and a decreased constitutive nitric oxide synthase (cNOS) activity in these cells. CONCLUSIONS: The results suggest that chronic treatment with l-arginine induces changes in VSMC size, ploidy and cell cycle. These changes are accompanied by iNOS induction and stimulation of the NO-cGMP pathway.     

No effect of taurine on platelet aggregation in men with a predisposition to type 2 diabetes mellitus

Abbreviations

ADP–denosine diphosphate; BMI–body mass index; FFA–free fatty acids; FFM–fat-free mass; IGT–impaired glucose tolerance; OGTT–oral glucose tolerance test; PPP–platelet-poor plasma; PRP–platelet-rich plasma; T2DM–type 2 diabetes mellitus; TG–triglyceride.

Contributed equally to this study.

Any diet therapy that potentially could affect platelet function would also influence the initiation of atherosclerotic plaque formation which is an important complication of diabetes mellitus eventually resulting in myocardial infarction and stroke. Blood platelets are rich in taurine, and it has been shown that taurine inhibits platelet aggregation in healthy subjects. The purpose was to examine the effect of taurine supplementation on platelet aggregation in high-risk subjects with a positive family history of T2DM. Twenty healthy men were included in a double-blinded, randomized, crossover study, receiving daily supplementation of 1.5?g taurine or placebo for two 8-week periods. Subjects were overweight and first-degree relatives of T2DM patients. At the end of each treatment, fasting blood samples for assessment of platelet aggregation was drawn. Platelet aggregation was induced by ADP. Plasma taurine concentration was significantly greater after taurine intervention compared to placebo (131.4?±?61.7 vs. 38.9?±?6.7?µmol/l, P?<?0.0001). There was no difference in the threshold level for complete platelet aggregation induced by ADP in vivo between placebo and taurine intervention (placebo 3.86?±?2.21 vs. taurine 3.86?±?3.25?µmol/l). Supplementation with 1.5?g of taurine for 8 weeks had no effect on platelet aggregation in overweight prediabetic men.     

急性冠状动脉综合征患者血小板一氧化氮合酶活性及表达改变

观察急性冠状动脉综合征患者血小板一氧化氮合酶活性及表达改变情况。急性冠状动脉综合征患者32例 ,以健康成人 2 2例作对照 ,取外周静脉血后 ,利用凝胶色谱柱法收集血小板 ,用同位素两步色谱法测定一氧化氮合酶活性 ;蛋白免疫印迹增强化学发光法检测内皮型一氧化氮合酶表达水平。结果发现 ,基础状态下血小板一氧化氮合酶活性正常人为 95 6 .0± 4 8.2pmol 10 8血小板 ,急性冠状动脉综合征患者为 5 2 5 .5± 6 0 .8pmol 10 8血小板 ,明显低于正常人 (P <0 .0 1) ,组胺刺激后血小板一氧化氮合酶活性仍低于正常对照组 (P <0 .0 1) ;与正常人相比 ,急性冠状动脉综合征患者血小板内皮型一氧化氮合酶表达无明显改变。结果提示 ,急性冠状动脉综合征患者血小板一氧化氮合酶活性降低 ,一氧化氮释放减少可能是其血小板活化的机制之一。由于血小板粘附 ,聚集白色血栓形成是急性冠状动脉综合征早期事件 ,这对我们认识冠心病发病机理、开发临床新药提供新的思路。     

Effect of ciprofibrate on platelet aggregation in patients with combined hyperlipidemia

This study was designed to investigate the effect of ciprofibrate on platelet aggregation in patients with combined hyperlipidaemia. Platelet aggregation measurements in platelet-rich plasma were carried out in 30 patients using the Biola 230 LA platelet aggregation analyser before treatment, 4 weeks and 8 weeks after ciprofibrate (100 mg/ day) therapy. A control group consisted of 37 healthy subjects. We found that spontaneous and 0.25 mu M ADPinduced platelet aggregation were significantly decreased after 4-weeks of therapy, from 4.6 to 3.2% and from 11.3 to 7.6%, respectively. However, there was no difference 8 weeks after the treatment onset. Platelet aggregation induced by adrenaline was unchanged during the ciprofibrate therapy.     

Hormone replacement effects on endothelial function measured in the forearm resistance artery in normocholesterolemic and hypercholesterolemic postmenopausal women

We investigated whether forearm resistance artery endothelial function differed between hypercholesterolemic postmenopausal women (n = 41) and normocholesterolemic postmenopausal women (n = 37), both generally and in terms of effects of long-term hormone replacement therapy (HRT) on endothelial function. Both menopause and hypercholesterolemia are associated with endothelial dysfunction and increased coronary risk. Forearm blood flow (FBF) during reactive hyperemia and after sublingual nitroglycerin (NTG) administration was measured by strain-gauge plethysmography. Treated women received conjugated equine estrogen (0.625 mg) plus medroxyprogesterone acetate (2.5 mg) daily for 6 months. Nitrite/nitrate, angiotensin-converting enzyme, and lipids were measured in serum. FBF during reactive hyperemia as well as serum nitrite/nitrate concentrations were significantly lower in hypercholesterolemic than normocholesterolemic subjects. Increases in the FBF induced by NTG were similar in the two groups. HRT significantly increased estradiol, high-density lipoprotein cholesterol, and serum nitrite/nitrate, while decreasing circulating angiotensin-converting enzyme activity in both groups. Reduction in total and low-density lipoprotein cholesterol was seen only in hypercholesterolemic subjects. After 6 months of HRT, maximal FBF response during reactive hyperemia increased in both groups. Augmentation of this response was greater in hypercholesterolemic than in normocholesterolemic subjects (maximal FBF, 55.4 +/- 11.2 vs. 25.9 +/- 11.5%; P < 0.05). Changes in the FBF with NTG were not altered by HRT in either group. Long-term HRT augments endothelial function in forearm resistance artery. This beneficial effect is greater in patients with hypercholesterolemia.     

Effect of ciprofibrate on platelet aggregation in patients with combined hyperlipidemia

This study was designed to investigate the effect of ciprofibrate on platelet aggregation in patients with combined hyperlipidaemia. Platelet aggregation measurements in platelet-rich plasma were carried out in 30 patients using the Biola 230 LA platelet aggregation analyser before treatment, 4 weeks and 8 weeks after ciprofibrate (100 mg/day) therapy. A control group consisted of 37 healthy subjects. We found that spontaneous and 0.25 microM ADP-induced platelet aggregation were significantly decreased after 4-weeks of therapy, from 4.6 to 3.2% and from 11.3 to 7.6% , respectively. However, there was no difference 8 weeks after the treatment onset. Platelet aggregation induced by adrenaline was unchanged during the ciprofibrate therapy.     

Isolation and study of insulin activated nitric oxide synthase inhibitory protein in acute myocardial infarction subjects

Insulin inhibits platelet aggregation through nitric oxide synthesis by stimulating platelet insulin activated nitric oxide synthase. Impaired platelet insulin activated nitric oxide synthase in acute myocardial infarction (AMI) patients had been reported and thus our aim was to identify and isolate the factors impairing insulin activated nitric oxide in acute myocardial infarction patients’ plasma and study its effect on platelets aggregation in vitro. The insulin activated nitric oxide synthase inhibitor was identified as a protein and was purified from the plasma of AMI subjects using DEAE cellulose and Sephadex G-50 column, molecular weight determined by SDS-PAGE, nitric oxide quantified by methaemoglobin method, inhibitor protein quantified in plasma by immunoblot and ELISA, platelet aggregation studies done using an aggregometer, thromboxane-A2 in the platelets determined by radioimmunoassay, ¹²⁵I-insulin radioligand binding studies done using normal subject platelets. The purified nitric oxide synthase inhibitor protein was ~66 kDa, concentration in AMI subjects’ plasma varied from 114 to 9,090 μM and was undetected in normal subjects’ plasma. The inhibitor protein competes with insulin for insulin receptor binding sites. The Incubation of the normal subject PRP with 5.0 μM inhibitor for 30 min followed by 0.4 μM ADP addition caused platelet aggregation in vitro, 130 μM aspirin or 400 μU insulin/ml addition was able to abrogate 0.4 μM ADP induced platelet aggregation even in the presence of 5.0 μM inhibitor. A potent inhibitory protein against insulin activated nitric oxide synthase in platelets appears in circulation of AMI subjects impairing nitric oxide production, potentiating ADP induced platelet aggregation and increasing the thromboxane-A2 level in platelets.     

Secondhand tobacco smoke impairs rabbit pulmonary artery endothelium-dependent relaxation.

OBJECTIVES: To determine whether secondhand smoke (SHS) induces pulmonary artery endothelial dysfunction, and whether dietary L-arginine supplementation is preventive. BACKGROUND: SHS causes coronary and peripheral arterial endothelial dysfunction. METHODS: The effects of L-arginine supplementation (2.25% solution) and SHS (10 weeks) on pulmonary vascular reactivity were examined in 32 rabbits fed a normal diet. Endothelium-dependent relaxation of precontracted pulmonary artery segments was studied using acetylcholine and calcium ionophore. Endothelium-independent relaxation was studied using nitroglycerin. Endothelial and serum L-arginine levels were measured by chromatography. In eight SHS-exposed and in eight control rats, pulmonary artery nitric oxide synthase (NOS) activity and arginase activity were studied using the titrated arginine to citrulline conversion assay. RESULTS: SHS reduced maximal acetylcholine-induced (p = 0.04) and calcium ionophore-induced (p = 0.02) relaxation. L-Arginine increased maximal acetylcholine-induced (p = 0.047) vasodilation. SHS and L-arginine did not influence nitroglycerin-induced relaxation. SHS reduced endothelial L-arginine (p = 0.04) but not serum L-arginine. L-Arginine supplementation increased endothelial (p = 0.007) and serum L-arginine (p < 0.0005). Endothelium-dependent relaxation induced by acetylcholine and calcium ionophore varied directly with endothelial (r = 0.67, r = 0.67) and serum L-arginine (r = 0.43, r = 0.45), respectively. SHS reduced constitutive NOS activity (p = 0.03). CONCLUSIONS: SHS reduces pulmonary artery endothelium-dependent relaxation by decreasing NOS activity and possibly by decreasing endothelial arginine content. L-Arginine supplementation increases serum and endothelial L-arginine stores and prevents SHS-induced endothelial dysfunction. L-Arginine may offset the deleterious effect of SHS on pulmonary arteries by substrate loading of the nitric oxide pathway.