Marina Sirtori

Ultrasonographic measurement of the common carotid artery wall thickness in hypercholesterolemic patients A new model for the quantitation and follow-up of preclinical atherosclerosis in living human subjects

Ultrasound high resolution B-mode imaging of human arteries allows in vivo an accurate and non-invasive determination of the thickness of the intimal-medial complex. A computer assisted procedure to measure this parameter at the level of common carotid arteries was developed. The average difference between duplicate thickness determinations was 4.6%. The thickness of the intimal medial complex of common carotid arteries was then measured in a group of hypercholesterolemic patients. This parameter was significantly greater in these patients as compared to controls (P less than 0.001). The prevalence of small plaques in the carotid arterial tree was also significantly increased in patients. Analysis of data showed that in controls, but not in patients, the thickness of the intimal medial complex increases with age (r = 0.46, P less than 0.05). Within the hypercholesterolemic group, intimal-medial complex values were greater in male patients and in smokers. It is concluded that the common carotid arteries of hypercholesterolemic patients show thickening of the intimal-medial complex. Cigarette smoking, male sex and age increase the extent of this modification. The determination of this parameter using a non-invasive technique may represent an important tool to monitor in vivo the progression and/or the regression of early atherosclerosis in man.

Hypertriglyceridemia and regulation of fibrinolytic activity.

A relation between elevated triglyceride (TG) levels and alterations of the fibrinolytic system has been recognized in studies of patients with coronary heart disease. In this investigation, the total fibrinolytic activity and the levels of specific components of the fibrinolytic system were evaluated in plasma samples from a highly selected group of patients with type IV hyperlipoproteinemia before and after a dietary treatment aimed at reducing TG levels. The fibrinolytic response of type IV patients was comparable to that of normolipidemic subjects, whereas tissue-type plasminogen activator antigen levels before and after venous occlusion (p less than 0.01) and resting plasminogen activator inhibitor-1 (PAI-1) antigen (p less than 0.01) and activity (p less than 0.01) were significantly higher in hypertriglyceridemic subjects compared with controls. After dietary treatment, a 22% reduction in TG levels was attained in type IV patients, with no appreciable modification of fibrinolytic parameters. The analysis of the single-patient data revealed a tendency toward normalization of PAI-1 levels only in those patients who showed a TG reduction greater than or equal to 20%. Very low density lipoproteins (VLDLs) from both normal and type IV patients concentration-dependently stimulated PAI-1 release by endothelial cells and HepG2 cells, with the effect of VLDL from type IV patients being more pronounced on HepG2 cells. The release of PAI-1 induced by VLDL in competent cells may thus account for the elevated levels of this antifibrinolytic protein that occur in hypertriglyceridemic patients.

Increased platelet sensitivity and thromboxane B2 formation in type-II hyperlipoproteinaemic patients

Abstract. Platelet aggregation induced by collagen, ADP and epinephrine, was monitored in 150 type‐II patients (115 type IIA and 35 type IIB) and compared with a reference group of normolipidaemic controls; in addition, malondialdehyde formation and thromboxane B2 were examined in a subsample of the type‐IIA patients. Threshold aggregatory concentrations were significantly lower in the whole group of type‐II patients for all three aggregating agents; no difference in terms of aggregatory response was detected between platelets from type‐IIA and ‐IIB patients. Only 56% of type‐II patients, however, exceeded the 95th percentile of the threshold aggregatory concentrations in controls. The formation of malondialdehyde in platelet‐rich plasma stimulated with thrombin and collagen, was significantly higher in platelets from type‐IIA patients. The production of thromboxane B2 by platelets, from endogenous arachidonic acid in type‐IIA patients, was significantly higher and exceeded the highest level found in controls.

Mechanisms of HDL reduction after probucol. Changes in HDL subfractions and increased reverse cholesteryl ester transfer.

Treatment with probucol, a widely used lipid-lowering agent, is associated with a significant reduction of high density lipoprotein (HDL) cholesterol levels, but with an apparently improved removal of cholesteryl esters from tissues (e.g., from tendon xanthomas). The effects of probucol (500 mg twice daily) on HDL subfraction distribution and cholesteryl ester transfer activity were tested in 12 patients with stable type II hyperlipidemia [low density lipoprotein (LDL) cholesterol greater than 180 mg/dl] after a placebo-controlled cross-over trial. Probucol significantly lowered total cholesterol (-13.8%), LDL cholesterol (-9.1%), and HDL cholesterol (-30%). By rate zonal ultracentrifugation, a marked reduction of HDL2 cholesterol (-68%) was shown, whereas changes in HDL3 were less significant (-21%). These findings were confirmed by polyacrylamide gradient gel electrophoresis, typically showing a reduction or disappearance of HDL2b particles and the prevalence of particles in the HDL3a range. Cholesteryl ester transfer from HDL to lower density lipoproteins was significantly increased (30%) in all patients. These findings suggest that, in addition to the well-documented in vitro changes (prevention of LDL peroxidation and macrophage uptake), probucol characteristically modifies HDL particle distribution in vivo, and is associated with a significant increase of cholesteryl ester transfer activity.

Pravastatin effectively lowers LDL cholesterol in familial combined hyperlipidemia without changing LDL subclass pattern.

Familial combined hyperlipidemia (FCHL) is the most common genetic lipid disorder among young survivors of myocardial infarction. Elevations of plasma total and low-density lipoprotein (LDL) cholesterol and the prevalence of small, dense LDL particles are both involved in the high coronary risk of FCHL patients. We investigated the ability of pravastatin to favorably correct plasma lipid and lipoprotein levels and LDL structure in FCHL patients. Twelve patients with FCHL, documented by studies of first-degree relatives, received pravastatin (40 mg/d) for 12 weeks. Pravastatin significantly lowered plasma total and LDL cholesterol levels by 21% and 32%, respectively. Triglyceride levels did not change, and apolipoprotein B (apoB) concentrations decreased by 9% (P = NS). High-density lipoprotein (HDL) cholesterol increased by 6% because of a significant 73% rise of HDL2 cholesterol. LDL were smaller (diameter, 24.5 +/- 0.5 nm), less buoyant, and apoB-rich (cholesteryl ester-apoB ratio, 1.64 +/- 0.46) in the selected patients compared with patients with familial hypercholesterolemia or healthy control subjects. LDL became even smaller (23.8 +/- 0.6 nm) and richer in apoB (cholesteryl ester-apoB ratio, 1.27 +/- 0.52) after pravastatin treatment. Although pravastatin favorably altered plasma lipid and lipoprotein levels in FCHL patients, the abnormal LDL particle distribution and composition were not affected. Because of the apparent resistance of the small, dense LDL to drug-induced modifications, a maximal lipid-lowering effect is needed to reduce coronary risk in FCHL patients.

Metformin Improves Peripheral Vascular Flow in Nonhyperlipidemic Patients with Arterial Disease

The clinical activity of metformin (N,N-dimethyl biguanide), a widely used antidiabetic agent, on arterial blood flow was evaluated in 15 patients with peripheral atherosclerosis. Flow was determined by quantitative strain-gauge plethysmography; plasma lipid, lipoprotein, and apoprotein levels were repeatedly tested during the cross-over trial, comparing 6 months of drug and placebo administration. Metformin (850 mg tid) significantly increased arterial flow after a standardized ischemia (+ 17.3% after 3 months and + 40.0% after 6 months). The increase in arterial flow was reversible after the switch to placebo was made. The drug was similarly effective, although to a lesser extent (+ 18.6% after 6 months), when given after the placebo. A highly significant effect of drug treatment, as well as of the sequence of administration, could be established by analysis of variance. In spite of the minimal changes of plasma lipid levels during metformin, a highly significant increase of high density lipoprotein cholesterol (+ 8.3% during the whole treatment) was demonstrated; plasma levels of isoprotein AI-1 were also raised during the metformin period. This controlled experiment confirms data from previous open studies, as well as from a longstanding clinical experience. Although the mechanism of the metformin effect cannot, at present, be defined, the reported results indicate that treatments not markedly affecting plasma lipid-lipoprotein levels may improve vascular function in selected arterial districts.

Platelet Aggregation and Malondialdehyde Formation in Type IIA Hypercholesterolemic Patients

Platelet aggregation induced by threshold concentrations of ADP, adrenaline, collagen and Thrombofax was evaluated in 25 type IIA hypercholesterolemic patients in comparison with 15 control subjects. Malondialdehyde (MDA) formation induced by collagen and thrombin was also studied in a subgroup of 8 patients. In the patient group, irreversible platelet aggregation was induced by significantly lower concentrations of both adrenaline (p is less than 0.05) and Thrombofax (p is less than 0.01). MDA formation induced by both aggregating agents was markedly increased (p is less than 0.01) in type IIA hypercholesterolemic patients.

Atherogenic lipoproteins and release of plasminogen activator inhibitor-1 (PAI-1) by endothelial cells

The effects of atherogenic lipoproteins on the release of tissue plasminogen activator inhibitor (PAI-1) by endothelial cells were evaluated. Incubation of cultured human umbilical vein endothelial cells (EC) with very low density (VLDL) and low density (LDL) lipoproteins induced a two fold increase of PAI-1 release by EC. Similarly acetylated LDL (acetyl-LDL) stimulated PAI-1 release by EC, whereas oxidized LDL (ox-LDL) reduced it. It is concluded that atherogenic lipoproteins profoundly influence the capacity of EC to release proteins with thrombogenic properties.

Changes in high-density lipoprotein subfraction distribution and increased cholesteryl ester transfer after probucol

The effects of probucol (500 mg twice daily) on high-density lipoprotein (HDL) subfractions and cholesteryl ester transfer from HDL to lower density lipoproteins were tested in a series of patients with Type II hypercholesterolemia. In this placebo-controlled crossover trial, patients received probucol or placebo for 8 weeks, then switched to the other agent for another 8 weeks. Probucol significantly lowered total, low-density lipoprotein and HDL cholesterol levels. HDL subfractions, separated by rate zonal ultracentrifugation, showed a dramatic reduction in HDL2, whereas changes in HDL3 were not significant. Both subfractions eluted at a characteristically lower volume, indicating a reduced flotation rate. These findings were confirmed by gradient gel electrophoretic separation, which showed a typical reduction or disappearance of HDL2b particles and the prevalence of particles in the HDL3a-HDL3b electrophoretic range in almost all patients. After treatment, cholesteryl ester transfer from HDL to lower density lipoproteins was significantly increased in all patients. These data suggest that probucol may accelerate HDL particle conversion, leading to improvement in reverse cholesterol transport from the periphery to the liver, through HDL and very low density lipoprotein.

Increased apoprotein B in very low density lipoproteins of patients with peripheral vascular disease.

Lipoproteln compositional studies were carried out in 20 patients with atherosclerotic peripheral vascular disease. Twelve of these patients were normollpldemlc, the other eight, hypertriglycerldemic. Ten normollpldemlc and 10 hypertriglyceridemlc age-matched subjects were used as controls. High density lipoprotein cholesterol levels were markedly reduced in the hypertriglyceridemlc subjects, both with (35.1 ± 5.0 mg/dl) and without (36.2 ±11.7 mg/dl) peripheral vascular disease, as compared to the normolipldemic patients (47.0 ± 6.3 mg/dl) and controls (48.1 ± 10.0 mg/dl). A decreased relative content of apo C-ll in very low density lipoproteins in the hypertriglyceridemic subjects, as compared to the normolipidemics, was detected by isoelectric focusing. Hypertriglyceridemla in patients with peripheral vascular disease shows a typical Type IV lipoprotein and apoprotein profile. Apoprotein B levels In very low and low density lipoproteins were determined by electroimmunodiffusion and selective precipitation with tetramethylurea (r = 0.981 between the two methods). All the patients with peripheral vascular disease showed an increased apo B content in very low density lipoproteins (VLDL) as compared to controls (apo B cholesterol In VLDL = 0.431 ± 0.124 for peripheral vascular disease patients and 0.236 ± 0.086 for controls, p < 0.001). A significant correlation between VLDL cholesterol and apo B levels was detected both in peripheral vascular disease patients and in controls; however, two distinct populations could be clearly separated (slopes of the regression lines: peripheral vascular disease patients = 0.350; controls = 0.215, p < 0.001). The data suggest a possible discriminatory power of VLDL-apo B levels in patients with peripheral vascular disease independent from other lipoprotein and lipid parameters.