Specific cellular features of atheroma associated with development of neointima after carotid endarterectomy: the carotid atherosclerosis and restenosis study.

BACKGROUND: The purpose of this study was to investigate whether some cellular and molecular features of tissue retrieved at carotid endarterectomy are associated with the extent of neointima formation at ultrasound follow-up. METHODS AND RESULTS: One hundred fifty patients were studied. Endarterectomy specimens were tested by immunocytochemistry with the use of (1) monoclonal antibodies that identify smooth muscle cells (SMCs) and fetal-type SMCs on the basis of smooth muscle and nonmuscle myosin content, (2) the anti-macrophage HAM 56, and (3) the anti-lymphocyte CD45RO. The maximum intima-media thickness (M-IMT) of the revascularized vessel was assessed by the use of B-mode ultrasonography 6 months after surgery. The M-IMT values were related positively to the number of SMCs (r=0.534, P<0.0005) and negatively to that of macrophages and lymphocytes (r=-0.428, P<0.0005, and -0.538, P=0.001, respectively). Patients were classified as class 1 (M-IMT 1.3 mm). An abundance of SMCs, mostly of fetal type, was found in the plaque of class 3 patients, whereas lesions from class 1 patients were rich in macrophages and lymphocytes. In the multivariate analysis, factors related to M-IMT were the number of SMCs and the percentage of fetal-type SMCs present in the plaque. CONCLUSIONS: Although the classic risk factors did not play a role, an abundance of SMCs and a scarcity of macrophages characterized the primary lesion of patients in whom neointima developed after surgery. In patients in whom neointima did not develop, lesions were rich in macrophages and lymphocytes. This approach can be useful in defining patients at risk of restenosis.

Effects of vitamin E supplementation on F(2)-isoprostane and thromboxane biosynthesis in healthy cigarette smokers.

BACKGROUND: Increased formation of 8-iso-prostaglandin (PG) F(2alpha) and thromboxane (TX) A(2), potent agonists of platelet and vascular thromboxane (TH)/PGH(2) receptors, has been detected in cigarette smokers. We performed a randomized, double-blind, placebo-controlled study of the effects of vitamin E (300, 600, and 1200 mg/d, each dose for 3 consecutive weeks) on 8-iso-PGF(2alpha) and TXA(2) biosynthesis in 46 moderate cigarette smokers. METHODS AND RESULTS: Urinary immunoreactive 8-iso-PGF(2alpha) and 11-dehydro-TXB(2), plasma vitamin E, and serum TXB(2) were measured by previously validated techniques. Baseline urinary 8-iso-PGF(2alpha) and 11-dehydro-TXB(2) excretion averaged 241+/-78 and 430+/-293 pg/mg creatinine, respectively. Urinary 8-iso-PGF(2alpha) was significantly correlated with 11-dehydro-TXB(2) (r=0.360, n=138, P<0.0001). Baseline plasma vitamin E levels averaged 20.6+/-4.9 micromol/L and were inversely correlated with urinary 11-dehydro-TXB(2) (r=-0.304, P=0.039) but not with 8-iso-PGF(2alpha) (r=-0.227, P=0.129). Vitamin E supplementation caused a dose-dependent increase in its plasma levels that reached a plateau at 600 mg (42.3+/-11.2 micromol/L, P<0. 001). This was not associated with any statistically significant change in urinary 8-iso-PGF(2alpha) or 11-dehydro-TXB(2) excretion. CONCLUSIONS: Supplementation with pharmacological doses of vitamin E has no detectable effects on lipid peroxidation and thromboxane biosynthesis in vivo in healthy subjects with a mild degree of oxidant stress. These findings are consistent with the hypothesis that the basal rate of lipid peroxidation is a major determinant of the response to vitamin E supplementation and have implications for the use of vitamin E in healthy subjects as well as for the design and interpretation of clinical trials of antioxidant intervention.

Is glycation of low density lipoproteins in patients with Type 2 diabetes mellitus a LDL pre-oxidative condition?

AIMS: The study aimed to evaluate whether low density lipoprotein (LDL) in diabetic patients is more glycated and susceptible to oxidation than in non-diabetic subjects and investigated the hypothesis that LDL glycation is associated with an increased plasma concentration of LDL- (a circulating electronegatively charged LDL), proposed as an index of in vivo oxidation. METHODS: LDL glycation was measured by a competitive enzyme immunoadsorbent assay, using a monoclonal antibody against glycated apoB in 24 Type 2 diabetic patients and 12 healthy controls. LDL- was separated by ion-exchange HPLC in LDL samples obtained after sequential preparative ultracentrifugation (density range 1.019-1.063). In vitro LDL susceptibility to oxidation was evaluated by following the kinetics of conjugated diene formation and by measuring the lag-phase time in the presence of copper (Cu2+) ions. RESULTS: The percentages of glycated apoB (3.33+/-2.54% vs. 1.24+/-0.71%) and of LDL- (3.88+/-1.49% vs. 2.34+/-1.03%) in total LDL were significantly higher in diabetic patients (P<0.01 for both). LDL- was positively correlated with glycated apoB (r = 0.68, P<0.001). LDL isolated from Type 2 diabetic patients showed a significant decrease (P<0.001) in the resistance to oxidative stress, as indicated by the shorter lag-phase time (91+/-12.6 vs. 120+/-24.5 min). The lag-phase time was inversely correlated with glycated apoB (r = -0.65, P<0.001) and LDL- concentrations (r = -0.69, P<0.001). CONCLUSIONS: In this population of Type 2 diabetic patients, LDL were more glycated, more susceptible to in vitro oxidation and had a higher percentage of electronegative LDL. The glycation of apoB is proposed to be associated with a significative increase of in vivo and in vitro LDL oxidation.

Postprandial plasma lipid hydroperoxides: a possible link between diet and atherosclerosis.

There is increasing evidence implicating a dietary source of plasma lipid peroxides that become elevated in the postprandial state. This phenomenon may be a contributing factor to the correlation found between postprandial hyperlipidemia and increased risk of cardiovascular disease. Using a newly developed method for measuring lipid hydroperoxides directly in plasma, a pilot study was performed which revealed that lipid hydroperoxides are indeed elevated following a fatty meal. Lipid hydroperoxides increased within 2-4 h after the meal and returned to basal levels, corresponding to the usual postprandial hyperlipidemia. A marked suppression of postprandial hydroperoxides was found when a meal was consumed with wine, suggesting that these hydroperoxides can be formed and then absorbed during the digestive process.

In vivo formation of 8-Epi-prostaglandin F2 alpha is increased in hypercholesterolemia.

F2-isoprostanes are bioactive prostaglandin (PG)-like compounds that are produced from arachidonic acid through a nonenzymatic process of lipid peroxidation catalyzed by oxygen free-radicals. 8-Epi-PGF2 alpha may amplify the platelet response to agonists, circulates in plasma, and is excreted in urine. We examined the hypothesis that the formation of 8-epi-PGF2 alpha is altered in patients with hypercholesterolemia and contributes to platelet activation in this setting. Urine samples were obtained from 40 hypercholesterolemic patients and 40 age- and sex-matched control subjects for measurement of immunoreactive 8-epi-PGF2 alpha. Urinary excretion of 11-dehydro-thromboxane (TX) B2, a major metabolite of TXA2, was measured as an in vivo index of platelet activation. Low-dose aspirin, indobufen, and vitamin E were used to investigate the mechanism of formation and effects of 8-epi-PGF2 alpha on platelet activation. Urinary 8-epi-PGF2 alpha was significantly (P = .0001) higher in hypercholesterolemic patients than in control subjects: 473 +/- 305 versus 205 +/- 95 pg/mg creatinine. Its rate of excretion was inversely related to the vitamin E content of LDL and showed a positive correlation with urinary 11-dehydro-TXB2. Urinary 8-epi-PGF2 alpha was unchanged after 2-week dosing with aspirin and indobufen despite complete suppression of TX metabolite excretion. Vitamin E supplementation was associated with dose-dependent reductions in both urinary 8-epi-PGF2 alpha and 11-dehydro-TXB2 by 34% to 36% and 47% to 58% at 100 and 600 mg daily, respectively. We conclude that the in vivo formation of the F2-isoprostane 8-epi-PGF2 alpha is enhanced in the vast majority of patients with hypercholesterolemia. This provides an aspirin-insensitive mechanism possibly linking lipid peroxidation to amplification of platelet activation in the setting of hypercholesterolemia. Dose-dependent suppression of enhanced 8-epi-PGF2 alpha formation by vitamin E supplementation may contribute to the beneficial effects of antioxidant treatment.

Low density lipoprotein-apheresis decreases oxidized low density lipoproteins and monocyte adhesion to endothelial cells.

The mutual interaction between monocytes and low density lipoprotein (LDL) in atherogenesis prompted a test of the hypothesis that LDL-apheresis could reduce the adhesive properties of monocytes to endothelium; and therefore interfere with a key mechanism in atheroma formation. Five patients affected by heterozygous familial hypercholesterolemia were studied. All patients received LDL-apheresis treatment with selective adsorption of LDL-cholesterol on dextran-sulphate columns. Low density lipoprotein particles were isolated by sequential preparative ultracentrifugation and subfractionated by ion exchange high performance liquid chromatography. Thiobarbituric acid reacting products of lipid peroxidation were measured fluorometrically. Vitamin E was estimated by high performance liquid chromatographic technique. Monocytes were isolated from patients blood before and 1 day after LDL-apheresis by Percoll gradient. The blood samples for monocyte adhesion were drawn from control subjects for 2 consecutive days. The adhesion of monocytes to an endothelial monolayer was evaluated by assaying the peroxidase content of the adherent monocytes. Low density lipoprotein-apheresis reduced total cholesterol (-65%; p < 0.01), LDL-cholesterol (-75%; p < 0.01), triglycerides (-51%; p < 0.05), and fibrinogen (-28%; p < 0.01). With LDL-apheresis treatment, a reduction of 54% in oxidized LDLs was observed; vitamin E concentration significantly increased in LDLs (+ 14.2%; p < 0.05). The monocyte adhesion decreased by approximately 61% after apheresis; the variation became statistically significant (-65%; p < 0.01) when endothelial cells were stimulated by lipopolysaccaride.

Efficacy and safety of atorvastatin compared to pravastatin in patients with hypercholesterolemia.

Plasma cholesterol and other lipoproteins play a significant role in the development of atherosclerosis and subsequent coronary heart disease (CHD). This 1 year study was designed to confirm the efficacy and safety of atorvastatin (Lipitor) compared to pravastatin, a marketed agent for low density lipoprotein cholesterol (LDL-C) reduction in hypercholesterolemic patients. Patients were recruited at 26 centers in six European countries. After a 6 week placebo baseline phase, patients were randomized to receive atorvastatin 10 mg or pravastatin 20 mg daily. The dose could be doubled at week 16, if LDL-C levels remained > or = 3.4 mmol/l (135 mg/dl). Atorvastatin significantly lowered LDL-C from baseline by 35% compared with 23% for pravastatin (P < 0.05). A total of 72% of atorvastatin patients attained the LDL-C target level of < 3.4 mmol/l, compared to 26% of pravastatin patients. Atorvastatin also significantly reduced TC, TG and apo B (P < 0.05). Safety was assessed by recording adverse events and measuring clinical laboratory parameters. The adverse event profile was similar for both treatment groups and neither treatment caused clinically relevant laboratory abnormalities. Atorvastatin 10 and 20 mg once daily is superior to pravastatin 20 and 40 mg once daily in treating patients with hypercholesterolemia.

Regulatory function of glucose and insulin on high-density lipoprotein cholesterol in normolipidemic subjects.

A decreased plasma concentration of high-density lipoprotein (HDL) cholesterol is associated with a higher incidence of coronary artery disease in populations. Therefore, there is intense investigation into the mechanisms responsible for the regulation of HDL cholesterol concentration in plasma. Insulin has a potent effect on HDL cholesterol, but it is unclear whether this is mediated by the primary effect insulin has on plasma triglycerides (TG). In this study, the question of the relationship between glucose, insulin, and HDL cholesterol has been addressed by investigating a cohort of nondiabetic normolipidemic men living in the Venice, Italy, area. One hundred twenty-eight men aged 30 to 69 years were initially recruited. The following parameters were measured: fasting plasma cholesterol, TG, HDL cholesterol, glucose, and insulin. One hundred seventeen of these subjects underwent an oral glucose tolerance test (OGTT), and the glucose and insulin responses were assessed. The final statistical analysis was performed on 98 nondiabetic individuals with plasma lipid levels within the 75th percentile for cholesterol and TG concentrations of the general population of the same age. The insulin response was a positive independent variable for plasma TG (P < .005) and HDL cholesterol (P < .005). On the other hand, HDL cholesterol was negatively associated with plasma TG. This relationship remained significant (P < .0001) also after controlling for age, body mass index (BMI), and glucose- and insulin-related measurements. Consistent with these results, both a stepwise variable selection analysis and a stratification analysis of the data indicated that the plasma TG concentration is the major determinant of HDL cholesterol level.(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of a modified low density lipoprotein in humans.

Low density lipoproteins (LDL) collected from 18 fasting humans were subjected to ion exchange chromatography on DEAE Sepharose. By this procedure, a LDL subfraction was isolated with an electric charge more negative than the LDL bulk. This LDL appeared to be mainly characterized by low phospholipid content, high free cholesterol and protein content, low esterified/free cholesterol ratio, and a high content of conjugated dienes, particularly of cholesterol esters. This subfraction, in an amount ranging from 5% to 20% of total LDL, was characterized by the presence of apo B-100 and protein aggregates that were reactive to anti-apo B monoclonal antibodies. Electron microscopy showed the more electronegative LDL to be heterogeneous in size with a tendency to aggregate. This LDL had low binding capacity with high affinity receptors of fibroblasts and low immunoreactivity with the monoclonal antibodies that recognize the receptor binding domain of apo B. Finally, the incubation of this LDL subfraction with cultured macrophages led to a higher increase in cellular cholesterol in spite of a lower rate of uptake as compared to the LDL bulk and to acetyl-LDL. The more electronegative LDL subfraction that we isolated for chemico-physical behavior and conjugated diene content may represent the peroxidized aliquot of human LDL.

Plasma malondialdehyde in coronary patients and in "normal" people at rest and after exercise.

The behavior of plasma lipid peroxides, expressed as plasma malondialdehyde, was studied in 27 patients with documented coronary artery disease and 17 volunteers without coronary artery disease (henceforth designated "normal"), after a standardized exercise on a bicycle ergometer. In the control group, the basal values of malondialdehyde were significantly lower than in coronary patients. Persons in the control group did not show any significant variation of malondialdehyde after exercise and recovery, whereas patients with coronary artery disease showed malondialdehyde levels significantly higher than the baseline, both after exercise and after recovery. In the control group, a significant inverse correlation between the malondialdehyde variations during the exercise and the total work produced was ten times lower than in coronary patients. It seems probable that the higher levels of lipid peroxides in these patients may leave some long-term unwanted effects. Furthermore, the increased values of lipid peroxides after exercise may be regarded as a possible trigger of fatal myocardial malfunction occurring during physical activity.

Effects of pantethine on in-vitro peroxidation of low density lipoproteins.

The effects of pantethine on LDL peroxidation in vitro are reported. LDL isolation by density gradient ultracentrifugation from 12 normal subjects were dialyzed 48 h under conditions allowing oxidation. The LDL peroxides were assayed for the presence of malondialdehyde (MDA) on the lipoprotein. The effect of peroxidation on the LDL protein moiety (apo B) was studied by SDS-gel electrophoresis. The presence in the dialysis buffer of 1 mM reduced glutathione or of an equimolar concentration of pantethine markedly inhibited the MDA formation in LDL. Less effective were 0.5 and 2 mM pantethine, while 10 mM pantethine did not prevent the LDL peroxidation. Both glutathione and pantethine (1 or 2 mM) preserved the original LDL electrophoretic mobility. The electronegative charge of LDL was correlated to the MDA production during the dialysis procedures. Freshly prepared LDL showed a single apo B band by SDS-gel electrophoresis (apo B-100). Following peroxidation 2 or 3 bands with higher molecular weight appeared. Both glutathione and pantethine (1 or 2 mM) strongly inhibited the appearance of higher molecular weight peptides. In appropriate concentrations therefore pantethine inhibits the LDL peroxidation in vitro, thus preserving the molecular integrity of apo B.

Relationship between apolipoproteins and chemical components of lipoproteins in survivors of myocardial infarction.

The relationship of VLDL-, LDL-, HDL-cholesterol, VLDL-triglycerides and of apolipoproteins A-I, A-II and apo-D has been studied in 89 survivors of myocardial infarction and in 80 age- and sex-matched controls. The two groups as a whole were analysed as well as sub-groups of subjects divided according to their lipid phenotype (normolipidaemic, IIA, IIB and IV). The data support the view that plasma levels of apo-B and apoA-I as well as their ratio and the TC/apo-B and LDL-C/apo-B ratios are better indicators of lipid derangement in survivors of myocardial infarction than the levels of LDL-cholesterol and HDL-cholesterol. Total cholesterol, total triglycerides, VLDL-cholesterol and VLDL-triglycerides are of no help in discriminating between the two series. The VLDL-C/VLDL-TG ratio in survivors is, however, very close to the ratio thought to be peculiar to type III hyperlipidaemia.

Determination of high-density lipoproteins: screening methods compared.

Factors reflecting the concentration of high-density lipoproteins in serum were assessed for 108 men and 106 women participating in a Venetian screening program for hyperlipoproteinemia. The methods applied, optimized in our laboratory, were: (a) cholesterol in high-density lipoproteins, determined in the supernate after sedimentation of the very-low-density lipoproteins + low-density lipoproteins with dextran sulfate or sodium phosphotungstate; and (b) immunochemical quantitation of apolipoprotein A-I and apolipoprotein A-II by Laurell's "rocket" technique. The latter determinations were performed with total serum before and after delipidation with diispropyl ether/n-butanol (6/4 by vol). The dextran sulfate method gave about 5% higher values than did the phosphotungstate method, but the correlation between the two was excellent (r = 0.95). Results of the immunochemical quantitation indicate that delipidation of lipoproteins before Laurell electrophoresis may not be necessary if only freshly drawn sera are used.

Are apolipoproteins better discriminators than lipids for atherosclerosis?

Plasma-levels of major lipids (cholesterol, triglyceride, high-density-lipoprotein cholesterol), two major apolipoproteins (apo-B and apo-A1), and two ratios (total-cholesterol/apo-B and apo-A1/apo-B) were studied in 218 survivors of myocardial infarction and 160 controls. Apolipoproteins were as good as lipids as discriminators between the populations under the age of 50 and better in the sixth to eighth decades.. Furthermore, values of total-cholesterol/apo-B and apo-A1/apo-B obtained from controls and normolipaemic survivors of myocardial infarction gave a bimodal distribution. The protein moiety of lipoproteins is a better discriminator than lipids between atherosclerotic subjects and controls.

Variations in apolipoproteins B and A1 during the course of myocardial infarction.

The plasma apolipoproteins B and A1, and plasma lipids and lipoproteins, were studied in fifteen patients with acute myocardial infarction. In the days immediately after acute infarction there was a decrease in total cholesterol, low density lipoprotein-cholesterol, total apolipoprotein-B, low density lipoprotein apolipoprotein-B and high density lipoprotein apolipoprotein A1. High density lipoprotein-cholesterol remained unchanged. In the same period the total triglycerides, very low density lipoprotein-protein, very low density lipoprotein-cholesterol, very low density lipoprotein apolipoprotein-B and very low density lipoprotein apolipoprotein A1 were increased. A reduction of the apolipoprotein ratio CII/CIII occurred after the acute phase. After 25--30 days all these values regained their baseline values.