J.Gerald Brook

Lovastatin inhibits low-density lipoprotein oxidation and alters its fluidity and uptake by macrophages: In vitro and in vivo studies

Under experimental conditions, oxidized low-density lipoprotein (Ox-LDL) may possess atherogenic properties, as is evidenced by its contribution to cholesterol accumulation in macrophages. LDL was oxidized in a cell-free system by predialysis of the lipoprotein against EDTA-free buffer and the addition of copper ions. Oxidation of LDL in the presence of lovastatin (10 to 1,000 mumol/L) resulted in a time- and dose-dependent reduction in thiobarbituric-acid-reactive substances (TBARS) concentration that was accompanied by increased LDL lysine-amino-group reactivity, in comparison with Ox-LDL produced in the absence of lovastatin. At 100 mumol/L, the drug reduced malondialdehyde concentration and increased amino-group reactivity by 24% and 42%, respectively. However, lovastatins antioxidant effect was limited relative to other antioxidants, such as probucol and vitamin E. The fluidity of Ox-LDL was substantially reduced in comparison with native LDL. However, lovastatin inhibited this reduction in fluidity by 20%. Upon incubation of J-774 macrophage-like cell line with Ox-LDL, the lovastatin-treated Ox-LDL induced a reduction in the cellular cholesterol esterification rate in comparison with the effect of Ox-LDL that was produced in the absence of the drug. In four patients with hypercholesterolemia, the effect of lovastatin therapy (20 mg/d) on the sensitivity of their LDL to in vitro oxidation was studied. In all patients, Ox-LDL prepared from LDL obtained during lovastatin treatment demonstrated a reduced TBARS content, an increased trinitrobenzenesulfonic acid (TNBS) reactivity, an increased fluidity, and an impaired uptake by macrophages. These results were similar to those obtained by adding lovastatin in vitro.

Platelet activation by plasma lipoproteins

Enhanced platelet activity in patients with hypercholesterolemia and moderate hypertriglyceridemia can be attributed to increased LDL and VLDL and/or decreased HDL concentrations. In marked hypertriglyceridemia, where there is an accumulation of chylomicrons, platelet function is reduced. Treatment, whether by diet, medication, or plasmapheresis, that will result in a change in lipoprotein pattern is accompanied by a parallel change in platelet responsiveness. Incubation of lipoproteins with isolated platelets results in enhancement of platelet activation by LDL and VLDL and suppression of activity by HDL and chylomicrons. These findings have in vivo confirmation. They are even more pronounced and sometimes altered when the lipoproteins are derived from hyperlipidemic subjects. The effects of the lipoproteins on platelet activity appear to be dependent on lipoprotein composition and on factors such as cholesterol:protein ratio, apo C-III0:apo C-III2 ratio, apo B concentration, and triglyceride:protein ratio. The lipoproteins interact with platelets at specific receptor sites. Rapid change in platelet composition, particularly with regard to cholesterol, phospholipid, and fatty acid content, might ensue, with consequent alterations in membrane fluidity and enzyme activities and either suppression or activation of platelet function. This review has indicated that lipoproteins have a clear influence on platelet function. This interaction could be well of paramount importance in determining atherogenic risk.

HDL Apolipoprotein A-I Attenuates Oxidative Modification of Low Density Lipoprotein: Studies in Transgenic Mice

Epidemiological evidence suggests that plasma high-density lipoprotein (HDL) is protective against coronary artery disease, whereas oxidatively modified low density lipoprotein is atherogenic. Human apolipoprotein A-I transgenic mice with overexpression of the human apolipoprotein A-I gene have increased plasma levels of apolipoprotein A-I and HDL-cholesterol. We analyzed LDL oxidation by determination of LDL associated malondialdehyde, peroxides and conjugated dienes. The present study demonstrates that HDL from both normal and human apolipoprotein A-I transgenic mice at similar concentrations inhibits LDL (protein concentration 500 mg/l) lipid peroxidation, but the effect of the human apolipoprotein A-I transgenic mice HDL was two-fold greater than that of HDL derived from normal mice. In addition, the electrophoretic mobility of oxidatively modified LDL was reduced about two-fold in the presence of human apolipoprotein A-I transgenic mice HDL than that obtained in the presence of normal mice HDL. We thus suggest that human apolipoprotein A-I possesses antioxidant properties which might neutralize LDL lipid peroxidation. This may underline the mechanism responsible for the lower prevalence of atherosclerosis in subjects with high plasma levels of HDL and apolipoprotein A-I.

Dietary soya lecithin decreases plasma triglyceride levels and inhibits collagen- and ADP-induced platelet aggregation

Elevated plasma lipid concentrations and increased platelet activation are risk factors in the development of atherosclerosis. Nine patients with type IIa hyperlipoproteinemia and nine patients with type IV hyperlipoproteinemia were given soya lecithin, 12 g/day, for 3 months. Plasma cholesterol and triglycerides were reduced by 15 and 23%, respectively, and HDL-cholesterol increased by 16% in the hypercholesterolemic patients. Platelet function was unchanged. In the hypertriglyceridemic patients, total cholesterol fell by 18%, triglycerides by 36%, and HDL-cholesterol increased by 14%. There was a 27% reduction in platelet aggregation (P less than 0.01). Seventeen hypertriglyceridemic patients then received increasing doses of soya lecithin for 1-month periods (6, 12, and 18 g/day). The optimal lipoprotein-lowering effect was achieved with a daily dose of 12 g soya lecithin per day. Both low-density lipoprotein and very-low-density lipoprotein levels were reduced, and HDL-cholesterol and apolipoprotein levels were reduced, and HDL-cholesterol and apolipoprotein A-I concentrations were increased. Platelet aggregation in response to collagen and ADP was significantly reduced, parallel with the reduction in triglyceride level. Soya lecithin supplementing the diet may be useful in the management of the hypertriglyceridemic patient.

Chylomicrons from patients with type V hyperlipoproteinemia inhibit platelet function.

Platelet aggregation and [14C]serotonin release induced by collagen and also by ADP and thrombin were significantly decreased in patients with primary Type V hyperlipoproteinemia. Platelets derived from these patients lost their hyporesponsiveness to thrombin and ADP (but not to collagen) after washings and isolation from their plasma environment. On incubation of platelets derived from normolipidemic controls with plasma derived from patients, platelet aggregation and [14C]serotonin release were lowered by 20% and 30%, respectively. On incubation of these platelets with 100 mg/dl of chylomicron triglyceride, a 40% reduction in both platelet aggregation and [14C]serotonin release was observed. The inhibition of platelet activity was positively correlated with chylomicron concentration up to a concentration of 225 mg/dl by chylomicron triglyceride. In 2 patients, bezafibrate administration (600 mg/day) resulted in marked reduction of plasma triglyceride concentration and a parallel improvement in platelet function. The platelet hyporesponsiveness in patients with Type V hyperlipoproteinemia appears to be a consequence of platelet-chylomicron interaction. This depressed platelet function may be responsible for the absence of overt atherosclerosis noted in these patients.

The D allele of the angiotensin-converting enzyme gene contributes towards blood LDL-cholesterol levels and the presence of hypertension

Coronary artery disease is a polygenic disease whose phenotypic manifestation depends on the interaction of the genetic background with a number of environmental factors. Recently, the gene coding for the angiotensin-converting enzyme (ACE) has been characterized and a deletion/insertion (D/I) polymorphism was defined. The prevalence of the three genotypes and their association with coronary artery disease (CAD) differ in different population groups. Mostly, the D allele was found as a significant risk factor for CAD, independently from other risk factors. In the present study, we determined the distribution of ACE alleles (D or I) in a cohort of healthy Israeli men and examined the correlation of the different genotypes with various CAD risk factors. We found LDL cholesterol levels to be highest in the DD genotype group, intermediate in the DI genotype group and lowest in the II genotype group. We also found higher blood pressure levels in subjects bearing the D allele compared to II homozygous subjects. In conclusion, it appears that the genetic influence of the D/I polymorphism on CAD manifests primarily through traditional risk factors.

Meso-2,3-dimercaptosuccinic acid in the diagnosis and treatment of lead poisoning

Lead poisoning remains one of the hazards of industrialized civilization. CaNa2 EDTA and dimercaprol, the usual therapeutic measures, have many side effects and can be given by parenteral route alone. The authors present a case of chronic lead poisoning caused by ingestion of contaminated flour ground in a primitive flour mill. The diagnosis was confirmed by the CaNa2 EDTA provocative test. Dimercaptosuccinic acid (DMSA) was given orally as a further provocation and resulted in an 11-fold increase in urinary lead excretion. A 5-day course of treatment with DMSA was instituted, during which symptoms abated, urinary lead excretion increased and the blood lead level decreased. No side effects were noticed. There has been no relapse over several months of follow-up. The authors conclude that the oral use of DMSA is effective, safe and convenient both as a provocative test in establishing the diagnosis of lead poisoning and as a therapeutic tool.

Platelet function in a case with abetalipoproteinemia

Platelet aggregation, [14C]serotonin release and platelet malondialdehyde production were determined in a patient with abetalipoproteinemia (ABL) and found to be normal. The patient demonstrated complete absence of apolipoprotein (apo) B and decreased apo A-I concentration in plasma. The high density lipoprotein (HDL) composition of plasma was abnormal, with an increased cholesterol/protein ratio, increased apo E levels and reduced apo C concentration. The patients platelets, like platelets derived from a normolipidemic control, possessed receptors capable of binding lipoproteins. On incubating washed platelets derived from a control subject with HDL obtained from the ABL patient, an enhancement in platelet function was observed. A similar concentration of HDL derived from the control had the opposite effect, a depression of platelet function. Lipoprotein-deficient plasma (LPDP) derived from the patient, on the other hand, decreased platelet aggregation and [14C]serotonin release in comparison to the LPDP obtained from the control. The normal platelet function observed in the patient appears to be the result of the platelet-enhancing effect of an abnormal HDL, thus compensating for the absence of low and very low density lipoproteins from the patients plasma which, when present, stimulate platelet function.

Secretory products from human monocyte-derived macrophages enhance platelet aggregation.

Both macrophages and platelets play an important role in atherogenesis. We studied the effect of conditioned medium obtained from human monocyte-derived macrophages on in vitro platelet aggregation. Incubation of macrophage-conditioned medium (MCM) with platelets resulted in enhanced platelet aggregation (up to 35% difference between basal and MCM-stimulated activity), which was time dependent. This MCM effect on platelet function was increased both with time of mononuclear cell culturing (up to 10 days) and with the time of macrophage incubation in serum-free medium (up to 24 hours) prior to MCM collection. MCM from either cholesterol-loaded macrophages or from macrophages obtained from patients with familial hypercholesterolemia demonstrated a 37% and 20% increased effect, respectively, in comparison to MCM derived from normal subjects. Macrophage activation with lipopolysaccharide resulted in the harvesting of a MCM that enhanced platelet activity 60% more than MCM obtained from nonactivated cells. The active component of MCM was inhibited fivefold following heating at 100 degrees C for 10 minutes or after treatment with trypsin or protease, but was not affected by antioxidants. MCM activation of blood platelets may be of importance in atherogenesis. Understanding the mechanisms involved may contribute to an improved appreciation of the role of both platelets and macrophages in atherosclerosis.

Low-density lipoprotein derived from atherosclerotic patients enhances macrophage cholesterol accumulation and in vitro platelet aggregation.

The aims of our study were to assess the differences between plasma lipoproteins separated from five angiographically normal subjects and five patients with proven CHD. The patients with CHD had significantly higher levels of LDL-cholesterol and apo-B, and reduced levels of HDL-cholesterol and apo-Al. The biological characteristics of LDL and HDL from both groups of patients demonstrated that the LDL from the CHD patients enhanced platelet aggregation and increased cholesterol content and cholesterol esterification in MPM compared to the normal patients. HDL had no significant effect on MPM; however, there was an increased platelet aggregation with HDL derived from the CHD patients, while the HDL from the normal group decreased platelet aggregation. The data suggest that lipoproteins isolated from CHD patients are more atherogenic than lipoproteins from normal patients.