Complex interactions between plasma lipoproteins, platelets, and arterial-wall monocyte-derived macrophages are of major importance in atherogenesis. The formation of cholesterol-loaded macrophage foam cells, which constitute the early atherosclerotic lesion, is probably a product of such interactions. Macrophage cholesterol accumulation and foam-cell formation can result from increased cellular cholesterol synthesis, increased low-density lipoprotein (LDL)-receptor activity on arterial-wall cell surface, or enhanced uptake of modified forms of LDL (1,2).
Platelets are responsible for the maintenance of hemostasis, and their activation state can be measured by determination of platelet adhesion, degranulation, and aggregation. The observation of aggregated platelets and cholesterol-rich lipoproteins in the atherosclerotic plaque suggests that these lipoproteins and activated platelets participate in the pathogenesis of atherosclerosis (3-8).
Atherogenic plasma lipoproteins, LDL, and very low density lipoprotein (VLDL) have been shown to enhance platelet activity, whereas plasma high-density lipoprotein (HDL) showed the opposite effect (9,10). The increased concentrations of plasma LDL or VLDL and decreased plasma HDL concentration could account for the enhanced platelet activity observed in hypercholesterolemic patients (8,11).
Platelets have been shown to contribute to cholesterol accumulation in arterial-wall cells (including macrophages) either directly or through their effect on LDL-receptor activity (12-21). Platelets can also affect macrophage uptake of oxidized LDL through changes in cellular lipoprotein-receptor activities. Platelet-derived growth factor released from the platelet alpha granules has been shown to increase the uptake of oxidized LDL by macrophages, leading to cholesterol loading of these cells (22-25). Platelet secretory products also have been shown to increase the susceptibility of LDL to oxidation, through the involvement of platelet-released oxidative agents (26).
Whereas increased cellular uptake of LDL can lead to cellular cholesterol accumulation in arterial-wall macrophages, the enhanced uptake of LDL by liver cells, resulting in the reduction of plasma LDL concentration, does not cause cellular cholesterol accumulation, because the liver can efficiently metabolize cholesterol and excrete it as bile acids. Macrophage cholesterol accumulation and foam cell formation, as well as macrophage activation, are important features of the early atherosclerotic lesion. Macrophage activation during atherogenesis is associated with the production and release of cytokines, the secretion of reactive oxygen species and metalloproteinases (which contribute to the weakening of the lesion fibrous cap), the expression of tissue factor (which contribute to the lesion thrombogenicity), and the modulation of nitric oxide (which plays an important role in endothelium relaxation and oxidative stress). Statins affect macrophage atherogenicity, as shown by the potency of fluvastatin to inhibit human and mouse macrophage-secretion of metalloproteinases-9 (gelatinase B), the synthesis of tissue factor, and the production and release of reactive oxygen species (ROS). Furthermore, preliminary studies also suggest beneficial roles for statins in nitric oxide synthesis and in the prevention of lipoprotein-induced cellular cholesterol accumulation.
The key enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is inhibited by potent hypocholesterolemic agents such as lovastatin, pravastatin, and fluvastatin. As a result, these HMG-CoA reductase inhibitors increase LDL-receptor activity on the cell surface, leading to the removal of LDL from plasma. Because HMG-CoA reductase inhibitors act mainly on the liver, they can efficiently reduce plasma LDL concentration without causing cellular cholesterol accumulation.
Hypercholesterolemia is a major risk factor for atherosclerosis; in hypercholesterolemic patients, the reduced LDL-receptor activity in the liver (27) contributes to increased plasma LDL concentration. Plasma LDL in these patients is more "aged" and thus more susceptible to oxidative modifications than is LDL derived from healthy individuals (28).
The purpose of our study was to analyze the effect of HMG-CoA reductase inhibitor therapy in hypercholesterolemic patients on platelet aggregation and LDL susceptibility to oxidation and to elucidate the mechanisms involved in these processes.
This study was approved by the Israeli Ministry of Health Helsinki Committee, and all patients signed an informed consent before entry into the study.
The hypercholesterolemic patients recruited for this study were aged 40-70 years, with plasma cholesterol concentrations of 240-350 mg/dl despite dietary therapy. All patients had plasma triglyceride concentrations of <350 mg/dl, no chronic or metabolic diseases, no acute coronary events, and no previous lipid-lowering therapy.
The apolipoprotein (apo) E-deficient mice were kindly provided by Dr. Jan Breslow, the Rockefeller University (New York, NY, U.S.A.). In apo E-deficient mice, accelerated atherosclerosis is associated with increased plasma LDL concentrations and with enhanced lipoprotein susceptibility to oxidation (5).
At 0, 4, 12, and 24 weeks, venous blood (40 ml) was collected through a siliconized syringe into 3.8% sodium citrate (volume ratio, 9:1) for platelet-rich plasma preparation or into a solution of 1.4% citric acid, 2.5% sodium citrate, and 2% glucose (volume ratio, 9:1) for washed-platelet preparation.
Platelet-rich plasma was prepared by low-speed centrifugation (100 g for 10 min) at 25°C; the remaining sample was recentrifuged at 1,000 g for 10 min to obtain platelet-poor plasma. Platelets in platelet-rich plasma were counted and diluted with platelet-poor plasma to achieve a uniform concentration of 3 × 108 cells/ml.
Washed platelets were prepared from platelet-rich plasma by centrifugation at 240 g for 20 min. The platelet pellet was washed twice in 5 mM HEPES buffer (pH 7.4; NaCl, 140 mM; KCl, 2 mM; MgCl2, 1 mM; NaHCO3, 12 mM; and glucose, 5.5 mM).
Collagen, 4 μg/ml (Nycomed Arzneimittel, Munich, Germany), was used as the aggregating agent (this concentration caused aggregation amplitudes of ∼75% in platelet-rich plasma and ∼60% in washed platelets). Platelet aggregation was performed at 37°C in a computerized aggregometer model PAP-4 (Bio Data Corporation, Hatboro, PA, U.S.A.) by using plateletpoor plasma as a reference for platelet-rich plasma or HEPES as a reference for washed platelets (29). Increasing concentrations of HMG-CoA reductase inhibitors (0.01, 0.1, 1, and 10 μg/ml) were incubated for 30 min with platelet-rich plasma, 1 ml, or washed platelets, 1 ml, before platelet aggregation was tested. Results were expressed as the extent of maximal aggregation (percentage of maximal amplitude) or the slope of the aggregation curve (cm/min).
Platelet cholesterol and phospholipid content
Platelet lipids were extracted with hexane-isopropanol (volume ratio, 3:2). The cholesterol content was measured in the dried hexane phase by using the Chiamori method (30). The platelet lipid extract was separated by thin-layer chromatography and developed in hexane-diethylether-acetic acid (volume ratio, 130:30:1.5). Phospholipid spots were visualized by iodine vapor and scraped, and their phospholipid content was determined by the Rouser method (31).
Platelet lipid peroxidation
To induce oxidative stress, washed platelets from human platelet-rich plasma or from apo E-deficient mice were incubated for 2 h at 37°C with 100 μM iron sulfate or with 10−7M angiotensin II. After incubation, cellular oxidative state was assayed by determination of the peroxide and conjugated diene content in platelet extracts and of peroxides released into the medium.
Plasma lipid peroxidation
Blood was drawn into 1 mM sodium EDTA and centrifuged at 1,000 g for 10 min at room temperature. Before oxidation, plasma was diluted (×2) with phosphate-buffered saline. The diluted plasma was incubated for 4 h at 37°C with no addition or with 100 mM 2,2′-azobis,2-amidinopropane hydrochloride (AAPH; Polysciences, Worthington, PA, U.S.A.). AAPH is a water-soluble azo compound that thermally decomposes and generates water-soluble peroxyl radicals at a constant rate (32).
The oxidation state of the plasma samples was analyzed after the incubation period by using the thiobarbituric acid-reactive substances (TBARS) assay, which is used to measure malondialdehyde equivalents (33).
Plasma lipid peroxidation was calculated by subtracting the values obtained without AAPH from those obtained with AAPH.
LDL was separated from plasma by discontinuous density-gradient ultracentrifugation (34) and dialyzed against 1 mM saline-sodium EDTA. Before oxidation, LDL was dialyzed overnight at 4°C against phosphate-buffered saline. The LDL protein concentration was determined by the Lowry method (35).
LDL, 100 μg/ml, was incubated with 10 μM copper sulfate at 37°C. The kinetics of LDL oxidation were continuously monitored by measuring conjugated diene formation as the increase in absorbency at 234 nm (36). LDL also was oxidized by incubation with 5 mM AAPH for 4 h at 37°C. After incubation, LDL oxidation was measured by the TBARS assay (33).
Cellular lipid peroxidation and platelet activation
Atherosclerosis was shown to be associated with lipid peroxidation and platelet activation in areas of the atherosclerotic lesions. Incubation of 100 μg/ml LDL with washed platelets resulted in enhanced platelet aggregation by 25% (from 60 ± 5% to 75 ± 6%). Oxidized LDL at a similar concentration further increased platelet aggregation by 47% ± 5% (n = 15).
Human platelet-rich plasma treated with ferrous ions or angiotensin II showed substantial platelet lipid peroxidation. In response to collagen, 4 μg/ml, the aggregation amplitudes of the peroxidized platelets were increased by 54% with ferrous ions (100 μM) and by 59% with angiotensin II (10−7M), compared with control nonoxidized platelets. In similar incubation conditions but with the antioxidant butylated hydroxytoluene (BHT), platelet aggregation was not affected, suggesting that platelet lipid peroxidation caused the platelet activation.
In washed-platelet preparations derived from apo E-deficient transgenic mice, platelet aggregation was 35% greater after stimulation with collagen than in control platelets (37 ± 3% vs. 27 ± 4%, respectively). This increment could be related to increased platelet cholesterol content because the cholesterol/phospholipid (C/PL) ratio was 57% higher in platelets from the apo E-deficient mice than from the control mice. Although resting platelet lipid peroxidation was similar in apo E-deficient and control mice, after platelet incubation for 2 h with 100 μM iron sulfate, the susceptibility to lipid peroxidation of platelets derived from the apo E-deficient mice was twofold higher than that of control platelets, determined by analysis of platelet peroxides (3.1 ± 0.3 nmol/108 vs. 1.5 ± 0.3 nmol/108 platelets, respectively). Glutathione and superoxide dismutase content were found to be 40-60% lower in the apo E-deficient mice platelets than in controls. The addition of plasma from apo E-deficient mice to washed platelets increased platelet aggregation by 115% ± 11% (n = 5).
Inhibition of platelet activation
Platelets from hypercholesterolemic patients, compared with those from normal individuals, had higher molar C/PL ratio (0.86 ± 0.15 vs. 0.57 ± 0.06, respectively) and phosphatidylcholine/sphingomyelin (PC/SM) ratio (2.64 ± 0.87 vs. 2.00 ± 0.15, respectively) and a greater tendency to aggregate (aggregation amplitude, 84% ± 6% vs. 78% ± 7%, respectively). After therapy with 40 mg lovastatin orally daily for 24 weeks, plasma cholesterol concentrations in hypercholesterolemic patients decreased by 37%. Lovastatin therapy normalized platelet lipid composition (C/PL ratio, 0.58 ± 0.13; PC/SM ratio, 1.84 ± 0.60) and aggregation (amplitude, 76% ± 10%), suggesting that lovastatin treatment may attenuate the contribution of platelets to the pathogenesis of atherosclerosis (37). The variability between the different patients ranged from 5 to 12%. In a similar study, after 24 weeks of fluvastatin therapy, the platelet C/PL ratio was reduced by 30%, and platelet-aggregation amplitude was reduced by 13% (Fig. 1). Fluvastatin thus may reduce platelet activation in vivo because of the reduction in platelet cholesterol content.
Fluvastatin and lovastatin also significantly reduced in vitro platelet aggregation in platelet-rich plasma after incubation for 30 min (p < 0.01), from 54 ± 4% to 37 ± 4% (fluvastatin) and 40 ± 5% (lovastatin). Similarly in washed platelets without plasma constituents, fluvastatin and lovastatin reduced in vitro platelet aggregation by 69 ± 7% and 44 ± 6%, respectively.
Inhibition of LDL oxidation
Fluvastatin at low and pharmacologic concentration (1 μM) was found to be a potent inhibitor of LDL oxidation induced by copper sulfate ex vivo in hypercholesterolemic patients (Fig. 2). By using 3[H]-labeled fluvastatin or 3[H]-labeled lovastatin, we could demonstrate the binding of these drugs to the LDL phospholipid fraction (Table 1). Furthermore, the binding of each of these drugs to LDL significantly reduced the LDL electrophoretic mobility (Table 1). This may indicate that HMG-CoA reductase inhibitors caused structural changes to the lipoprotein surface.
Comparative analyses of the effects of statins
Table 2 summarizes the in vivo effects of the statins on LDL oxidation (part A) and on platelet aggregation (part B) in comparison to known potent inhibitory agents, such as vitamin E and probucol, against LDL oxidation (part A) and aspirin against platelet activation (part B).
The results show that although the statins were not as potent as the specific targeted agents, they still demonstrated significant inhibitory effects in both systems (Table 2A and B), and these phenomena are in addition to their potent hypocholesterolemic effects.
Figure 3 summarizes the antiatherogenic properties of statins along the atherogenic pathway, including monocyte macrophage differentiation, macrophage activation with the release of metalloproteinases (MMP) and ROS, the expression of tissue factor (TF), and the production of nitric oxide (NO). In addition, statins can affect lipoprotein retention and oxidation, macrophage cholesterol accumulation and foam cell formation, and platelet aggregation with thrombus generation.
In this study, we showed that HMG-CoA reductase inhibitor therapy, in addition to its hypocholesterolemic effect on plasma LDL, markedly reduced platelet aggregation in hypercholesterolemic patients. Both lovastatin and fluvastatin may be considered antiatherogenic in vivo because of their ability to reduce plasma LDL concentrations and their inhibitory effect on platelet aggregation.
The inhibitory effect of lovastatin and fluvastatin on platelet aggregation could be associated with a decrease in platelet C/PL ratio, which paralleled the reduction in plasma cholesterol concentration. Increased platelet cholesterol content contributes to platelet activation in hypercholesterolemic patients (7-11). This phenomenon may be related to the effect of platelet cholesterol on the interaction of the aggregating agents with the platelets. HMG-CoA reductase inhibitors may reduce platelet activation in hypercholesterolemic patients as a result of their hypocholesterolemic effect on platelet cholesterol content and the direct effect of the drugs binding to the platelets (38).
In our study, we also showed that lovastatin or fluvastatin therapy in hypercholesterolemic patients markedly reduced the susceptibility of LDL to oxidation. We previously showed that both lovastatin and pravastatin reduced the ex vivo susceptibility of LDL from hypercholesterolemic patients to oxidation (39,40). In one of those studies (39) in vitro oxidation of LDL after lovastatin therapy resulted in a time- and dose-dependent reduction in the TBARS concentration, compared with oxidized LDL produced without lovastatin; however, the inhibitory effect of lovastatin on LDL oxidation required a high drug concentration (1 mM), and this inhibition was limited relative to other antioxidants such as probucol or vitamin E. In that study, in four patients with hypercholesterolemia, lovastatin, 20 mg daily for 8 weeks, markedly reduced susceptibility of their LDL to oxidation.
A possible explanation for the ex vivo effect of HMG-CoA reductase inhibitors on susceptibility of LDL to oxidation is that, because HMG-CoA reductase inhibitors increase cellular LDL receptor synthesis, the LDL obtained after drug treatment contained fewer aged particles than could be found before drug administration. By removal of the aged plasma LDL, the plasma is enriched with newly formed lipoproteins (as a result of the response of the liver to plasma LDL reduction) by the secretion of new VLDL (the source of plasma LDL). Because aged plasma LDL is more prone to oxidative modification during its long presence in the circulation, removal of aged plasma LDL after therapy with HMG-CoA reductase inhibitors can contribute to the reduction in susceptibility of the new LDL to oxidation (40). The substantial decrease in plasma and LDL susceptibilities to lipid peroxidation may be the result of the hypocholesterolemic effect of the HMG-CoA reductase inhibitors.
HMG-CoA reductase inhibitors such as lovastatin or fluvastatin bind to LDL, mainly to the phospholipid fraction, thereby preventing the diffusion of free radicals, generated under oxidative stress, into the lipoprotein core. This mechanism is suggested because the oxidation of the major LDL lipid component (cholesteryl ester) involves transport of oxidants through the hydrophilic areas of the LDL coat (phospholipids, protein) into the core lipids. The HMG-CoA reductase inhibitors reduce the electrophoretic mobility of LDL on cellulose acetate, with no effect on LDL size or apo B-100 integrity. The altered LDL electrophoretic mobility may be the result of conformational changes in the lipoprotein phospholipid region, which could contribute to inhibition of subsequent oxidation of the LDL cholesteryl ester (39,41). The inhibitory effect of fluvastatin on LDL oxidation did not involve binding of copper ions or free-radical scavenging (because of its inability to reduce linoleic acid peroxidation). It may, however, exert its inhibitory effect on LDL oxidation as a result of binding to LDL (42).
Recently we analyzed the effect of fluvastatin in atherosclerotic apo E-deficient transgenic mice that are under oxidative stress (43,44) on the susceptibility of their LDL to oxidation. Fluvastatin (2.5 mg/week/mouse) was given in drinking water for 12 weeks and resulted in a 36% reduction in LDL oxidation induced by copper ions.
HMG-CoA reductase inhibitors may possess antiatherogenic properties involving not only their hypocholesterolemic effects. The antiatherogenicity of HMG-CoA reductase inhibitors may involve both their hypocholesterolemic capabilities (on plasma, arterial cell walls, and platelet's cholesterol) and the effects of their direct binding to LDL, arterial cells, and platelets on LDL oxidation, macrophage foam cells, platelet activation, and lesion formation (Fig. 4).
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