Donate Help Contact The AHA Sign In Home
American Heart Association
Arteriosclerosis, Thrombosis, and Vascular Biology
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:979-988

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lesnik, P.
Right arrow Articles by Chapman, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lesnik, P.
Right arrow Articles by Chapman, M. J.
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:979-988.)
© 1997 American Heart Association, Inc.

Articles

Impact of a Combination of a Calcium Antagonist and a ß-Blocker on Cell- and Copper-Mediated Oxidation of LDL and on the Accumulation and Efflux of Cholesterol in Human Macrophages and Murine J774 Cells

Philippe Lesnik; Christiane Dachet; Laure Petit; Martine Moreau; Sabine Griglio; Philippe Brudi; ; M. John Chapman

From the Institut National de la Santé et de la Recherche Médicale (INSERM), Unité de Recherches sur Les Lipoprotéines et l'Athérogénèse, Hôpital de la Pitié, Paris; and Zeneca-Pharma, Cergy (P.B.), France.

Correspondence to Philippe Lesnik or M. John Chapman, Unité de Recherches sur Les Lipoprotéines et l'Athérogénèse, U-321, Pavillon Benjamin Delessert, Hôpital de la Pitié, 83, Bd de l'Hôpital, 75651 Paris Cedex 13, France.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Abstract Calcium antagonists and ß-blockers may retard or inhibit atherogenesis. In the absence of data pertaining to the potential cardioprotective action of an association of such agents, we have investigated the impact of nifedipine and atenolol, alone or in combination, on the capacity of monocyte-macrophages (ex vivo) and copper ions (in vitro) to oxidize LDL and on intracellular metabolism and efflux of free and esterified forms of cholesterol in human macrophages and foam cells. At concentrations up to 100 µmol/L, atenolol had no effect on the oxidative resistance of LDL; on the contrary, nifedipine displayed a significant dose-dependent capacity to protect LDL during copper-mediated oxidation (100 µmol/L; P<.001). Using a DPPH radical generating system, nifedipine was shown to exert free radical–trapping activity (molar ratio of scavenging activity, nifedipine:{alpha}-tocopherol, 1:114). The addition of atenolol to nifedipine was without effect on the antioxidant activity of the calcium antagonist. In experiments in which oxidative modification was mediated by monocyte-macrophages, nifedipine but not atenolol conserved its antioxidant capacity. Furthermore, we demonstrated that association of atenolol with nifedipine did not modify the antioxidant properties of nifedipine itself. Using a human monocyte-derived macrophage culture system, nifedipine, atenolol, or a combination of the two drugs was ineffective in inhibiting foam cell formation induced by acetylated LDL or oxidized LDL. However, atenolol (100 µmol/L) increased cellular accumulation of cholesteryl ester (+17%; P<.05), whereas nifedipine (100 µmol/L) decreased total cholesterol (-37.4%; P<.05) accumulation induced by acetylated LDL in the mouse macrophage cell line J774. A combination of the two drugs neutralized these antagonistic effects. None of these results were reproduced during the oxidized LDL–induced transformation of murine J774 cells into foam cells. Furthermore, cholesterol efflux from preloaded human macrophages was equally unaffected by the addition of the drugs alone or in combination. It therefore seems unlikely that the beneficial effect of atenolol on coronary heart disease is mediated by changes in either LDL oxidizability or cholesterol metabolism in human macrophages and foam cells. Our findings with nifedipine suggest, however, that this calcium antagonist may potentially exert antiatherosclerotic properties via a reduction of the oxidative modification of LDL, thereby affecting a reduction in foam cell formation and in the pathophysiological cellular activities of oxidized lipids, rather than by inducing a direct reduction in cholesterol accumulation in human foam cells of macrophage origin.


Key Words: atenolo1 • nifedipine • monocytes • LDL • macrophages • oxidation


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
It is now established that hyperlipidemia represents a major independent risk factor for the premature development of cardiovascular disease, the leading cause of mortality and morbidity in industrialized countries. To date, intervention studies involving lipid-lowering therapy have suggested that this approach may lead to reduced rates of morbidity.1 2 However, favorable modifications of plasma lipid profile alone do not correct the problem entirely,3 and alternative and eventually complementary therapeutic approaches to reduction of cardiovascular risk are required.

Earlier studies have demonstrated that calcium blockers, which selectively inhibit transmembrane calcium influx into the cell, may exert antiatherogenic effects (for review, see References 33 and 44 ). Indeed, it has been clearly demonstrated that these drugs not only inhibit smooth muscle cell proliferation and migration but also diminish accumulation of CE in macrophages, thereby reducing foam cell formation. Equally, calcium channel blockers inhibit platelet aggregation in addition to the accumulation of calcium and extracellular matrix components in the vessel wall.4 Furthermore, these agents can exert protective effects on the cellular- and copper-mediated oxidation of LDL.4 It is, however, of considerable relevance that many of these studies have been conducted in vitro at concentrations that would not be tolerated in human subjects. Their relevance to clinical practice is therefore dubious.

A second family of drugs, the ß-adrenergic blockers (ß-blockers), are effective cardiovascular drugs that are widely used in the treatment of hypertension, an independent risk factor for premature cardiovascular disease (for review, see References 33 and 55 ). Hypertension increases the permeability of endothelial cells to lipoproteins and promotes the accumulation of LDL in the subintimal space of the arterial wall.6 The ß-blockers display several antiatherogenic properties,3 5 which include (1) reduction in endothelial cell permeability to lipoproteins and (2) inhibition of acyl-CoA:cholesterol acyltransferase, the intracellular enzyme that esterifies FC. The inhibition of such enzyme activity results in an increase in intracellular FC content and inhibition of the formation of CE-loaded foam cells. Indeed, FC is readily mobilized by HDL for cellular efflux; this pathway is also termed "reverse cholesterol transport"; and (3) reduction in the affinity of LDL for proteoglycans.3 5 Indeed, proteoglycans can trap these cholesterol-rich particles in the subendothelial space, where they may subsequently undergo oxidative modification. In addition, animal data indicate that in spite of apparently adverse alterations in plasma lipoprotein profile, ß-blockers retard atheromatous plaque formation.3

Inhibition of LDL oxidation and macrophage foam cell formation resulting from uptake of modified LDL has been suggested as a novel therapeutic approach by which atherogenesis may be impeded. The present study was therefore undertaken to assess the impact of the association of a ß-blocking agent, atenolol, with another potential antiatherogenic drug, the calcium channel blocker nifedipine, on the capacity of both monocyte-macrophages (ex vivo) and copper ions (in vitro) to oxidize LDL and on the intracellular metabolism and efflux of cholesterol in human macrophages and foam cells. The rationale for the choice of these drugs was as follows. First, it has been reported that several ß-blockers, including atenolol, may exert potentially atherogenic effects on plasma lipid profile7 and on cholesterol homeostasis in macrophages8 9 ; by contrast, the effects of these drugs on the oxidizability of LDL are essentially neutral.10 11 We postulated that the association of nifedipine, a calcium antagonist known to exert antiatherogenic effects on both macrophage lipid metabolism4 12 13 and LDL oxidizability14 15 16 but with neutral impact on circulating lipid levels,17 might counterbalance the potentially adverse properties of atenolol. On the other hand, clinical studies with nifedipine have failed to date to reveal significant reduction in cardiovascular mortality3 18 ; by contrast, ß-blockers have an established place in reducing myocardial infarction and mortality despite adverse effects on circulating triglyceride levels.3 The association of a ß-blocker and a calcium channel blocker might therefore prove of considerable therapeutic interest in the treatment of cardiovascular disease.


*    Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
CHOD iodide was obtained from Merck. BHT, nifedipine, CuCl2, and bovine serum albumin were purchased from Sigma Chemical Co. dl-{alpha}-Tocopherol was from ICN. Ham's F-10 and RPMI-1640 media were from Eurobio. Atenolol was provided by Zeneca-Pharma (Cergy, France). The U937 human monocyte-like cell line and the J774 murine monocyte-macrophage–like cell line were from American Type Culture Collection. Nutridoma HU was supplied by Boehringer Mannheim. Contact of nifedipine with the daylight was avoided at all times.

Purification of Lipoproteins
The most abundant subspecies of LDL (d=1.024 to 1.050 g/mL) in normolipidemic human plasma was isolated by sequential preparative ultracentrifugation.19 All density solutions contained EDTA (final concentration, 0.03 mmol/L), gentamicin (0.005%), and a serine-protease inhibitor, PMSF, at a final concentration of 1 mmol/L, at pH 7.4. To avoid any contribution of lipoprotein(a) to LDL preparations and because significant amounts of lipoprotein(a) may be present over the density interval corresponding to dense LDL (d=1.050 to 1.063 g/mL),20 we chose an upper-limiting density interval of 1.050 g/mL for LDL isolation. The purity and integrity of LDL and apolipoprotein B were established on the basis of criteria described earlier.21 In this way, the potential contamination of LDL with other lipoproteins or plasma proteins was excluded. HDL3 was isolated by ultracentrifugation from normolipidemic human plasma in the density interval 1.125 to 1.210 g/mL. The isolated LDL and HDL3 were dialyzed at 4°C in Spectrapor membrane tubing (Spectrum Medical Industries, exclusion limit 12 000 to 14 000) against 0.01 mol/L PBS containing 0.3 mmol/L EDTA (pH 7.4) and subsequently filtered through a 0.22-µm filter (Costar) and stored at 4°C. The protein content of lipoprotein fractions was determined by the procedure of Lowry et al22 using bovine serum albumin (Sigma) as the working standard.

Chemical Modification of Lipoproteins
Ac-LDL was prepared according to the procedure of Basu et al.23 Before oxidation, LDL was dialyzed in Spectrapor tubing against 0.01 mol/L PBS (pH 7.4) at 4°C to remove EDTA. Copper-oxidized LDL was prepared by incubating 0.5 mg of LDL protein per mL with 2.5 µmol/L aqueous CuCl2 in PBS at 37°C for 48 hours. In control experiments, BHT was added at a final concentration of 60 µmol/L. Oxidized and modified LDL was extensively dialyzed at 4°C against PBS to remove unreacted chemical products.

Monocyte Isolation and Culture
Monocytic U937 cells and macrophage-like J774 cells were cultured in RPMI-1640 (Eurobio) containing 10% fetal calf serum (Eurobio) and 40 µg/mL gentamycin. Monolayers of U937 cells were obtained by incubating cells for 4 days with PMA (final concentration, 50 ng/mL). J774 cells were seeded in 35-mm dishes. All experiments were performed on subconfluent cultures. Cell viability was assessed by the trypan blue exclusion method. Buffy-coat cells were separated from the fresh anticoagulated blood of healthy normolipidemic volunteers and the subsequent isolation of monocytes was performed as described by Rouis et al.24 Cell viability was typically >95% by the trypan blue exclusion method.

Cell-Induced Modification
U937 and freshly isolated human monocytes were washed three times with Ham's F-10 medium to remove serum. Cells were plated at 106 cells per 15-mm dish (Primaria) in 1.0 mL Ham's F-10 supplemented with gentamycin (40 µg/mL). LDL was added to the culture medium, to a final concentration of 0.2 mg LDL protein per milliliter, and the medium was supplemented in defined experiments with CuCl2 (2.5 µmol/L) in the presence or absence of the drugs (final concentration, 1 µmol/L to 100 µmol/L). Nifedipine and atenolol were dissolved in ethanol and introduced into the incubation medium in a volume not exceeding 5% of the medium. Equivalent amounts of ethanol were added to control-cell cultures. Incubation was then carried out for 24 hours (unless otherwise indicated) at 37°C in a humidified atmosphere containing 5% CO2. Control dishes, in which either cells or LDL were omitted, were incubated under identical conditions. In some experiments, cells were first cultured for 24 hours in the presence of the drugs; cells were then washed (twice for 10 minutes) and their capacity to modify LDL was tested in Ham's F-10 medium in the absence of the drugs but in the presence of LDL (0.2 mg LDL protein per milliliter) over a period of 48 hours. At the end of the incubation period, EDTA (0.3 mmol/L) was added to chelate copper and to terminate metal-catalyzed oxidation. The media and cells were immediately separated by centrifugation at 500g for 10 minutes at 4°C. The content of TBARS and the electrophoretic mobility of LDL were measured in the cell-free culture supernatant.

Assessment of LDL Modification
The time course of the copper-induced oxidation of LDL (62 µg protein per milliliter) in PBS containing 1.6 µmol/L CuCl2 was monitored continuously for periods up to 8 hours by the formation of conjugated dienes, measured as the increase in absorbance at 234 nm.25 Kinetic studies of the formation of lipid peroxides during copper-induced oxidation were performed using the lipid peroxide assay of El-Saadani et al26 modified by Wallin et al.27 The principle of this assay is based on the oxidative capacity of lipid peroxides to convert iodide to iodine, which can be measured spectrophotometrically at 365 nm. Lipid peroxidation products of copper- and cell-oxidized LDL were estimated before dialysis as the fluorescent products obtained on reaction with thiobarbituric acid as described by Wallin and Camejo.28 Results are expressed as nanomoles equivalent MDA per milligram LDL protein. We have determined the electrophoretic mobility of LDL as a measure of the degree of protein modification. The total (net) electrical charge on both native and modified LDL was assessed by electrophoresis in agarose gel (Corning).29 The REM is the ratio of the mobility of modified LDL to that of the native LDL from which it was derived. To avoid interference due to the absorbance of nifedipine and atenolol at 234 nm and 365 nm, quantitation of dienes, lipoperoxides, or TBARS was performed in parallel in experiments in which LDL was omitted.

Culture and Cholesterol Loading of Human Monocyte-Derived Macrophages
The monocytes isolated as described above were cultured and grown in 35x10-mm plastic tissue-culture dishes (Primaria, Falcon, Becton Dickinson) with RPMI-1640 medium containing 10% heat-inactivated pooled human serum and 40 µg/mL gentamicin.24 At day 8 of culture, cells were washed three times with serum-free RPMI-1640 medium containing 40 µg/mL gentamicin. The cells were differentiated into adherent monocyte-derived macrophages (hereafter denoted as macrophages) and were free of lymphocytes. Indeed, all cells were CD68 positive but negative for CD3 antibody, as visualized by the indirect immunostaining method. Cholesterol-loaded macrophages were obtained by incubation of macrophages for 48 hours in RPMI-1640 medium supplemented with 40 µg/mL gentamicin, 1% Nutridoma HU, and 100 µg ac-LDL protein per milliliter or 100 µg ox-LDL protein per milliliter.24 30

Determination of Intracellular Cholesterol, Protein Content, and Cell Viability
After incubation, the culture medium was removed and the cells were washed with PBS and recovered in 0.2N NaOH. Cell protein contents were determined on aliquots of this solution by the BCA protein assay (Pierce). Cellular lipids were extracted with chloroform-methanol (2:1 vol/vol) according to a modification of the method of Folch et al.31 After drying of the lower chloroform phase, the lipids were dissolved in 95% (vol/vol) isopropanol. Total and free cellular cholesterol contents were determined using an enzymatic kit (Biomerieux). Esterified cellular cholesterol was estimated by subtraction of FC from TC; CE mass was calculated as the FC content multiplied by a factor of 1.67.32 The viability of cells after incubation for 48 hours with ac-LDL and of control cells was assessed by measuring the release of LDH into the extracellular medium (LDH kit, Boehringer Mannheim). The results indicated that there was no statistical difference in the level of cytotoxicity between cells incubated with atenolol, nifedipine, and ethanol (data not shown).

Determination of Free Radical–Scavenging Properties of Compounds In Vitro
DPPH, a stable free radical, is currently used for screening antioxidant molecules.33 DPPH has a deep purple color in ethalonic solution and an absorbance maximum at 517 nm but becomes pale yellow when trapped by an antioxidant. DPPH was dissolved to give a solution of 50 µmol/L; test compounds were dissolved in DMSO to a concentration of 4 mmol/L and then diluted to 2, 1, 0.5, 0.25, 0.12, 0.06, and 0.03 mmol/L. During the assay, 5 µL of test compound or DMSO (control) was dispensed into 96-well plates. To each well was added 200 µL of DPPH (50 µmol/L) in ethanol, or ethanol alone (blank), and the plate was incubated at room temperature for 30 minutes, after which the absorbance at 520 nm was measured.

Statistical Analysis
Statistical analysis was performed by Student's t test.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effect of Nifedipine and Atenolol, Alone or in Combination, on Copper-Induced Oxidation of LDL
The effect of nifedipine, atenolol, or both on the copper-ion–mediated oxidation of LDL was first studied by incubating LDL in PBS with copper ions at a final concentration of 1.6 µmol/L. BHT was introduced as a reference antioxidant compound in control assays. The early peroxidative changes occurring during oxidation were determined by monitoring the formation of conjugated dienes at 234 nm (Fig 1Down). Treatment of LDL isolated from a normolipidemic subject with 50 µmol/L or 100 µmol/L atenolol did not significantly affect the lag time (respectively 94 minutes and 98 minutes versus 99 minutes for control) for diene formation. In contrast, nifedipine at final concentrations of 50 µmol/L and 100 µmol/L retarded the onset of LDL oxidation (ie, increased the lag time) in a dose-dependent manner (respectively 126 minutes and 161 minutes versus 99 minutes for control) (Fig lDown). The association of atenolol with nifedipine (50 µmol/L or 100 µmol/L) did not further modify the observed effect of nifedipine on the prolongation of the lag phase (respectively 122 minutes and 159 minutes versus 99 minutes for control). The lag time for diene production in the presence of the antioxidant BHT (100 µmol/L) was >400 minutes under our experimental conditions. Determination of the lag time for diene production in the presence or absence of the two drugs revealed that the dose of nifedipine prolonged the lag phase at concentrations >=50 µmol/L in a dose-dependent manner. Indeed, in the presence of 50 µmol/L and 100 µmol/L nifedipine, the mean lag time was prolonged by 50% to 118%, respectively (P<=.0001 versus control; n=7). Addition of atenolol did not significantly modify lag time values obtained with nifedipine.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Continuous monitoring of the formation of conjugated dienes during the copper-mediated oxidation of LDL in the presence of nifedipine and/or atenolol. Preparations of LDL (n=7; 62 µg of LDL protein per milliliter in PBS) were supplemented with 1.6 µmol/L CuSO4; the absorption at 234 nm was recorded at intervals of 4 minutes at 37°C. The absorption data were stored on a personal computer and the diene curve was determined by subtracting the initial absorbance from all data using the Microsoft Excel spread sheet program. The values for lag time were subsequently determined graphically, as described by Esterbauer et al.25 Data from one LDL preparation representative of seven are shown. Nifedipine and/or atenolol were added at concentrations of 100 µmol/L and 50 µmol/L.

The production of thiobarbituric acid–reactive aldehydes derived from lipid oxidation was equally delayed by nifedipine (Fig 2Down). Furthermore, the formation of aldehyde-like compounds was inhibited in a dose-dependent manner in the presence of nifedipine itself or in combination with atenolol (Fig 2Down). The period required for half maximum thiobarbituric acid–reactive aldehyde formation was almost 3 hours for both control LDL and atenolol-treated LDL. These values increased to 5 hours and 12 hours in the presence of increasing concentrations of nifedipine (50 µmol/L and 100 µmol/L, respectively); the addition of atenolol was without effect.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. Effect of nifedipine and/or atenolol treatment on the time course of TBARS formation during the copper-mediated oxidation of LDL. The formation of TBARS in the presence of nifedipine and/or atenolol was monitored in three different LDL preparations. Results are presented for a representative LDL preparation (62 µg protein per milliliter) in the presence of 1.6 µmol/L copper ions. Nifedipine and atenolol were added at final concentrations of 50 µmol/L and 100 µmol/L, respectively. Values are the means of triplicate determinations. BHT was used as a control antioxidant at a final concentration of 100 µmol/L.

The effect of nifedipine on the oxidative modification of apolipoprotein B100 in LDL was equally demonstrated by analysis of the REM of LDL in the presence of nifedipine associated or not with atenolol (Fig 3Down). A significant reduction in REM of almost 20% (P<=.001 vs control) was observed at 16 hours of incubation with nifedipine (100 µmol/L). The REM of LDL particles was not affected by incubation with atenolol (100 µmol/L). Addition of atenolol to nifedipine did not result in potentialization or inhibition of the observed effect in the presence of nifedipine alone.



View larger version (21K):
[in this window]
[in a new window]
  		Figure 3. Time course of the effect of nifedipine and/or atenolol on the net electronegative charge of LDL during copper-mediated oxidation. LDL was treated under the conditions shown in Fig 2Up. The net electrical charge on LDL is presented as the ratio of the electrophoretic mobility of ox-LDL versus nonoxidized LDL. Values are the mean±SD of duplicate determinations on three separate LDL preparations. *P<.001 vs control.

Effect of Cell-Induced Oxidation of LDL During Treatment With Nifedipine and Atenolol, Alone or in Combination
The potential actions of nifedipine and atenolol on the oxidative modification of LDL by (1) cells of the promonocytic cell line U937, treated or not by PMA (50 ng/mL), and (2) primary cultures of human monocytes and human macrophages were first evaluated. This study was performed in the presence of the two agonists in the culture medium to detect a potentially direct effect of these drugs on cell-mediated modification of LDL (Tables 1Down and 2Down). In a second step, we evaluated the question as to whether the effect of the ß-blockers and calcium antagonist could be preserved after preincubation of the drugs with cells during 24 hours; their subsequent capacity to modify LDL was tested over a period of 48 hours' incubation in the absence of the agonists and the presence of LDL (Tables 1Down and 2Down). The results (Table 1Down) clearly demonstrate that nifedipine alone or in association with atenolol is capable of inhibiting cell-mediated modification of LDL. This effect was dose dependent; for concentrations of nifedipine of 1 µmol/L, 10 µmol/L, and 100 µmol/L in the presence or absence of atenolol at the same concentration, we observed significant inhibition of TBARS formation in cell-culture supernatants (10%, 58%, and 82%, respectively, versus control; P<.001).


View this table:
[in this window]
[in a new window]
  		Table 1. Effect of Nifedipine and/or Atenolol on Human Monocyte-Mediated Modification of LDL as Determined by TBARS Content


View this table:
[in this window]
[in a new window]
  		Table 2. Effect of Nifedipine and/or Atenolol on the Capacity of Different Cellular Types to Induce Oxidative Modification of LDL

The results of studies involving pretreatment of human monocytes for 24 hours with these drugs followed by evaluation of the capacity of cells to modify LDL are shown in Table 2Up. An antiperoxidative effect of nifedipine was detected that was smaller than that observed when the drugs were coincubated in the culture medium with cells and LDL. Indeed, this minor antioxidant effect was statistically significant (P<=.05) at a nifedipine concentration of 10-4 mol/L.

Our results were confirmed in different cellular systems (Table 2Up). In human monocyte-derived macrophages incubated with nifedipine, MDA-like product formation was inhibited (100 µmol/L; P<.001), while the REM of LDL was decreased. Coincubation of nifedipine with atenolol did not modify the results observed in the presence of nifedipine alone (inhibition of TBARS formation was almost 75% for 10-4 mol/L nifedipine). Similarly, this effect was observed for both PMA-treated U937 and nonactivated U937 cells. The degree of inhibition was 50% and 65%, respectively, for U937-monocytes and U937-macrophages in the presence of 10-4 mol/L nifedipine; these results were strongly correlated with the modification of REM (r=.84; P<.0001). Indeed, the electrophoretic mobility of LDL was decreased by 30% compared with LDL treated in the absence of nifedipine in all cellular systems.

Finally, the capacity of different cellular types to modify LDL after treatment with these pharmacological drugs is shown in Table 2Up. We again observed that cells pretreated with a high concentration of nifedipine (100 µmol/L) maintained their capacity to modify LDL. The addition of atenolol (100 µmol/L) did not modify the above results.

Effect of Nifedipine, Atenolol, or Both on the Cellular Cholesterol Content of Macrophages Incubated in the Presence of Native or Modified LDL
After 48 hours of in vitro incubation of native LDL with human monocyte-derived macrophages in the absence of the drugs, total intracellular cholesterol content represented 22.5±2.9 µg/mg cell protein, consisting of 21.9±2.5 µg/mg cell protein for FC and 1.07±0.7 µg/mg protein for CE (Fig 4ADown). The addition of nifedipine, atenolol, or both drugs to the culture medium did not alter the cellular contents of TC, FC, and CE, which resembled closely those in control cells treated with ethanol (0.5%).



View larger version (27K):
[in this window]
[in a new window]
  		Figure 4. Effect of nifedipine and/or atenolol on the cellular content of TC, CE, and FC in human monocyte-derived macrophages incubated in the presence of native LDL, ox-LDL, or ac-LDL. Cultured human monocyte-macrophages were incubated for 48 hours in the presence of 100 µg native LDL protein per milliliter (A), 100 µg ox-LDL protein per milliliter (B), or 100 µg ac-LDL protein per milliliter (C). Ethanol (0.5% final concentration in control dishes) and drugs (atenolol and/or nifedipine at a final concentration of 1, 10, and 100 µmol/L) were added at the beginning of the incubation period. The cellular content of TC and FC was measured by enzymatic procedures as described in the "Methods" section. The cellular mass of esterified cholesterol was estimated as (TC-FC mass)x1.67; 1.67 is the factor representing the ratio of the average molecular weight of CE to FC. Results are given as the mean±SD of three different cell preparations, each assayed in quadruplicate.

The incubation of ox-LDL (Fig 4BUp) or ac-LDL (Fig 4CUp) for 48 hours with monocyte-derived macrophages stimulated foam cell formation; the resulting intracellular accumulation of TC represented 32.0±8.4 µg/mg cell protein and 36.9±11.8 µg/mg cell protein, respectively, and 5.14±4.7 µg/mg cell protein and 11.5±8.9 µg/mg cell protein, respectively, for CE (Fig 4AUp). The addition of nifedipine, atenolol, or a combination of the two drugs at all concentrations tested did not induce a statistically significant effect on TC, FC, or CE accumulation during foam cell formation at concentrations of 1, 10, or 100 µmol/L.

In short-term experiments (6 hours' incubation), in which macrophage foam cell formation in J774 cells (a mouse-derived cell line) was induced by incubation with ox-LDL or ac-LDL, we observed a rapid accumulation of TC (31.3±5.4 µg/mg and 27.8±5.1 µg/mg cell protein, respectively; Table 3Down); this phenomenon was associated with a significant increase in intracellular CE content (21.7±8.2 µg/mg and 15.2±5.5 µg/mg cell protein, respectively, versus 10.7±4.1 µg/mg in control cells). In macrophages incubated with ac-LDL, treatment with atenolol resulted in an increase in TC content that attained significance (36.8±4.1 µg/mg versus 31.3±5.4 µg/mg cell protein; P<.05). CE content in nifedipine-treated cells was significantly decreased (13.6±3.7 µg/mg versus 21.7±8.2 µg/mg cell protein in control cells). The association of nifedipine and atenolol induced a cellular content of TC and CE that was intermediate between that of cells treated with nifedipine or atenolol alone. None of the above effects was observed in J774 cells incubated in the presence of ox-LDL or drugs at final concentrations of 1 or 10 µmol/L (data not shown).


View this table:
[in this window]
[in a new window]
  		Table 3. Effect of Nifedipine and/or Atenolol on Intracellular Cholesterol Content in J774 Macrophages Incubated With Ac-LDL or Ox-LDL

In the next experiment, we evaluated the effect of these drugs on the capacity of HDL3 to induce cholesterol efflux in preloaded human macrophages, using a 72-hour incubation with 100 µg/mL ac-LDL followed by extensive washing and 24 hours' incubation with 400 µg/mL HDL3 (Table 4Down). In this experiment, the incubation of cells with HDL3 in the absence of drugs resulted in a significant decrease in TC and CE content (78.1% and 69.6%, respectively). The addition of drugs in combination or alone resulted also in a decrease in TC and CE; however, these decrements were not statistically significant when comparison was made with control cells incubated in the presence or absence of HDL3.


View this table:
[in this window]
[in a new window]
  		Table 4. Effect of Nifedipine and/or Atenolol on Cellular Cholesterol Content on Incubation With HDL3


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The potential proatherogenic or antiatherogenic actions of ß-blockers and calcium antagonists appear to be highly variable between respective members of each of these groups of compounds; indeed, such drugs may exert antagonistic effects. For the first time, therefore, we evaluated the impact of a combination of nifedipine and atenolol on cell- and copper-mediated oxidation of LDL and on the accumulation and efflux of cholesterol in human macrophages and murine J774 cells. Our studies demonstrate first that the antioxidant action of nifedipine was maintained in the presence of atenolol, second that the combination was ineffective in inhibiting foam cell formation induced by ac-LDL or ox-LDL in human monocyte-macrophages, and third that the association was without significant effect on cholesterol efflux from preloaded human macrophages.

Analysis of several different aspects of LDL oxidation has allowed us to confirm that the calcium blocker nifedipine is capable of inhibiting the monocyte-macrophage or copper-mediated oxidation of LDL, consistent with earlier findings14 15 16 in which the protective effect of nifedipine on endothelial- and monocyte-mediated oxidation of LDL was demonstrated. Furthermore, Mak and Weglicki34 provided evidence for the antioxidant activity of nifedipine in a biological system in which LDL and copper ions were replaced by sarcolemmal membranes and iron ions. In addition, the recent studies of Lupo et al15 documented the antioxidant capacity of a large family of calcium antagonists, including nifedipine, in a copper-mediated oxidation system. These observations suggest that this calcium antagonist can directly exert an antioxidant activity that may be qualified as endogenous. Breugnot et al14 could not, however, confirm the scavenging properties of nifedipine when employing DPPH. By contrast, we have performed free radical–scavenging experiments using the DPPH method33 that were positive for nifedipine and may thus explain its properties. Indeed, the number of molecules of nifedipine necessary to trap one molecule of free radical derived from DPPH was 87.8±9.8 (n=3), while it was 0.77±0.16 (n=3) for {alpha}-tocopherol (molar ratio of radical-trapping activity, nifedipine:{alpha}-tocopherol, 1:114; no radical-trapping activity was detected, however, for atenolol). The difference between our data and the earlier study of Breugnot et al14 appears to result from differences in the experimental procedure; our test compounds were dissolved in DMSO rather than ethanol, and absorbance was measured after 30 minutes instead of 20 minutes. Our investigations equally confirm the antioxidant capacity of nifedipine in cell-mediated oxidation. Furthermore, the fact that cells pretreated with nifedipine displayed a reduction in their prooxidant activity suggests that nifedipine may inhibit cell-dependent oxidation mediated, for example, by prooxidant enzymes such as lipoxygenases and myeloperoxidases or by molecular free radical species (O2-, H2O2), which are known to mediate the modification of LDL. This latter aspect was documented by Li et al16 and may result from a separate cellular effect of nifedipine rather than solely from its endogenous antioxidant activity. An alternative hypothesis would involve the hydrophobic association of nifedipine with cell membranes, which would prevent complete elimination of nifedipine during the successive washing of cells and might thus account for a residual antioxidant activity associated with cells after drug pretreatment.

As previously demonstrated, atenolol, a hydrophilic ß-blocker, did not exhibit antioxidant properties when oxidation was catalyzed by cells, copper, or iron ions.10 11 On the contrary, Mak and Weglicki35 revealed a slight inhibitory effect of atenolol in a peroxidative system that included sarcolemmal membranes in the presence of iron ions. In our present study, we were able to confirm the results of Yue et al11 and Jenkins et al,10 as we did not detect significant inhibition of lipid peroxidation in LDL by atenolol in the presence of copper ions or cells (U937, human monocyte, and human macrophages), even at elevated concentrations. Furthermore, we demonstrated that association of this ß-blocker with nifedipine did not modify the antioxidant properties of nifedipine itself. We conclude, therefore, that the cardioprotective effects of atenolol are unlikely to be mediated by modulation of the oxidative behavior of LDL.

The second aim of our study was to investigate the capacity of nifedipine and atenolol to modulate cholesterol metabolism, first during foam cell formation and second during HDL3-induced cholesterol efflux from macrophage foam cells.

In long-term experiments (2 days) with human macrophages, our data did not reveal any specific effect of atenolol, nifedipine, or the combination of the two drugs on cellular cholesterol accumulation. Previous studies on the effect of nifedipine and atenolol have focused on foam cell formation induced by ac-LDL, but it is noteworthy that such chemically modified lipoproteins do not constitute a physiological model for biologically modified LDL.12 13 36 37 Indeed, ox-LDL may represent a more appropriate ligand.38 Our experimental data, obtained with ox-LDL, failed to reveal any significant activity of nifedipine and/or atenolol on macrophage cholesterol metabolism.

In a mouse macrophage model (J774 cells), in which short-term experiments (6 hours) were performed in the presence of ac-LDL, we observed two significant drug-induced effects: first, atenolol induced an increase (+17%) in total cellular cholesterol and second, nifedipine induced a decrease in cellular CE (-37.4%). The combination of the two drugs neutralized these antagonistic effects. The use of ox-LDL in our murine system did not confirm the results obtained in the presence of ac-LDL. Indeed, substantial evidence indicates that ac-LDL and ox-LDL are not catabolized by the same pathways during foam cell formation, as demonstrated earlier.39 40 41 42 Furthermore, the results obtained in the presence of ox-LDL and which may have relevance to in vivo mechanisms argue for the absence of a significant effect of this drug combination in the long-term formation of foam cells.

Macrophages do not possess voltage-dependent calcium channels; however, the investigations of Daugherty et al36 and Dushkin and Schwartz13 demonstrated that a reduced rate of cholesterol esterification occurs in the presence of nifedipine, in addition to promotion of cholesterol efflux.12 13 These results were obtained in macrophages of murine origin, in which the effect of nifedipine was mediated by inhibition of cholesterol esterification by acyl-CoA:cholesterol acyltransferase43 and by enhancement of cholesterol hydrolysis mediated by the CE hydrolase present in mouse macrophages.44 Our failure to detect any modification in the intracellular pools of cholesterol in a human monocyte-derived macrophage model could be partially explained by the absence of detectable neutral esterase activities in mature human macrophages (M.C. Schotz, unpublished observations, 1995). Under these conditions, the effect of nifedipine on lipid metabolism in arterial intimal cells may be more closely related to inhibition of foam cell formation of smooth muscle cell origin, as demonstrated by Etingin and Hajjar.37 45 Furthermore, our experimental data suggest that the antiatherogenic effect of nifedipine may be more related to an antioxidant effect than to its effect on CE metabolism. As argued by others, the antiatherogenic effect of nifedipine may result from potential effects on smooth muscle cell proliferation and migration, on the preservation of endothelium function, on antithrombotic and antiplatelet activity, or on reduction of arterial pressure.4 46 Indeed, previous studies have demonstrated the beneficial effect of nifedipine on the development of atherogenesis in the cholesterol-fed rabbit,47 48 although other investigators found no effect.49 50 Such apparently conflicting data have also been observed in clinical trials, and indeed the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT) demonstrated that nifedipine treatment had no effect on overall progression and regression of existing lesions; in contrast, the number of new coronary lesions was reduced.18 It is important to note that the discrepancy between some of these data may be explained first by the dissimilarity of certain features of the atherosclerotic process in animal and human models and second by the doses of calcium antagonist used. Indeed, the studies on cholesterol-fed rabbits which involved low doses of nifedipine49 50 compared with those in which high doses of nifedipine were employed (at least tenfold higher than the dose given so far clinically)47 48 revealed no effect on cholesterol metabolism. Similarly, the effect of nifedipine in J774 mouse macrophages was specifically observed at high doses (100 µmol/L). Such doses, used to obtain statistically significant effects, raise the question as to the clinical relevance of these findings.

Most of our present data on the effect of atenolol on cellular cholesterol metabolism are consistent with those of Orekhov et al.8 9 Thus, we observed no significant effect of atenolol on cholesterol metabolism in human monocyte-derived macrophages. Orekhov et al8 demonstrated that among all the ß-blockers tested in human cells isolated from atherosclerotic lesions, only atenolol displayed no significant effect on intracellular cholesterol levels. Second, a more recent report showed an increase of total cellular cholesterol content in experiments conducted in mouse peritoneal macrophages under ex vivo and in vivo conditions9 ; in agreement with these results, we observed a minor increase (+17%) in TC in the mouse macrophage cell line J774 treated with high concentrations of atenolol (100 µmol/L) in the presence of ac-LDL. These differences between human and mouse cells may be explained by dissimilarities in cholesterol metabolism. In addition, and as observed during nifedipine treatment, the results obtained during ac-LDL stimulation of foam cell formation were not reproduced on ox-LDL treatment. Third, no effect was detected at low doses of atenolol. In the absence of extensive clinical trials with atenolol,51 and independently of the fact that atenolol tends to induce minor increases in the plasma triglyceride levels,7 these observations argue for a neutral effect of atenolol on in vivo cholesterol metabolism in human macrophages.


*    Selected Abbreviations and Acronyms
 
ac-LDL = acetylated LDL
CE = cholesteryl ester
DPPH = 1,1-diphenyl-2-picrylhydrazyl
FC = free cholesterol
MDA = malondialdehyde
ox-LDL = oxidatively modified LDL
PMA = phorbol myristate acetate
REM = relative electrophoretic mobility
TBARS = thiobarbituric acid–reactive substances
TC = total cholesterol


*    Acknowledgments
 
These studies were supported by INSERM and by Zeneca-Pharma. We are indebted to C. Debets-Albertini (Centre Départemental de Transfusion Sanguine, Créteil, France) for the generous gift of thrombopheresis residues.

Received May 7, 1996; accepted August 7, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Blankenhorn DH. Blood lipids and human coronary artery atherosclerosis regression: the angiographic evidence. Curr Opin Lipidol. 1991;2:234-239.
  2. Scandinavian Simvastatin Survival Study. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet. 1994;344:1383-1389.[Medline] [Order article via Infotrieve]
  3. Cleland FGF, Krikler DM. Modification of atherosclerosis by agents that do not lower cholesterol. Br Heart J. 1993;69(suppl):S54-S62.
  4. Schmitz G, Hankowitz J, Kovacs EM. Cellular processes in atherogenesis: potential target of calcium channel blockers. Atherosclerosis. 1991;88:109-132.[Medline] [Order article via Infotrieve]
  5. Cruickshank JM, Smith JC. The beta-receptor, atheroma and cardiovascular damage. Pharmacol Ther. 1989;42:385-404.[Medline] [Order article via Infotrieve]
  6. Shimamoto T, Sunaga T. The concentration and blebbing of endothelial cells accompanied by acute infiltration of plasma substances into the vessel wall and their prevention. Excerpta Med Int Congr Ser. 1972;269:3-31.
  7. Leren P. Comparison of effects on lipid metabolism of antihypertensive drugs with alpha- and beta-adrenergic antagonist properties. Am J Med. 1987;82:31-35.
  8. Orekhov AN, Ruda MY, Baldenkov BN, Tertov VV, Khashimov KA, Ryong LH, Lyakishev AA, Koslov SG, Tkachuk VA, Smirnov VN. Atherogenic effects of beta blockers on cells cultured from normal and atherosclerotic aorta. Am J Cardiol. 1988;61:1116-1117.[Medline] [Order article via Infotrieve]
  9. Orekhov AN, Andrianova I, Rekhter MD, Tertov VV, Andreeva ER, Ragimov SE, Mironov AA. Beta blockers: propanolol, metaprolol, atenolol, pindolol, alprenolol and timolol, manifest atherogenicity on in vitro, ex vivo and in vivo models. Atherosclerosis. 1992;95:77-85.[Medline] [Order article via Infotrieve]
  10. Jenkins RR, Del-Signore CM, Sauer P, Skelly C. The effect of beta blocking drugs on lipid peroxidation in rat heart in vitro. Lipids. 1992;27:539-542.[Medline] [Order article via Infotrieve]
  11. Yue TL, McKenna PJ, Gu JL, Cheng HY, Ruffolo RR, Feuerstein GZ. Carvedilol, a new antihypertensive agent, prevents lipid peroxidation and oxidative injury to endothelial cells. Hypertension. 1993;22:922-928.[Abstract/Free Full Text]
  12. Schmitz G, Robeneck H, Beuck M, Krause R, Schurek A, Niemann R. Calcium antagonists and ACAT inhibitors promote cholesterol efflux from macrophages by different mechanisms. Arteriosclerosis. 1988;8:46-56.[Abstract/Free Full Text]
  13. Dushkin MI, Schwartz YS. Effect of verapamil and nifedipine on cholesteryl ester metabolism and low-density lipoprotein oxidation in macrophages. Biochem Pharmacol. 1995;49:389-397.[Medline] [Order article via Infotrieve]
  14. Breugnot C, Maziere C, Auclair M, Mora L, Ronveaux MF, Salmon S, Santus R, Morliere P, Lenaers A, Maziere JC. Calcium antagonists prevent monocyte and endothelial cell–induced modification of low density lipoproteins. Free Radic Res Commun. 1991;15:91-100.[Medline] [Order article via Infotrieve]
  15. Lupo E, Locher R, Weisser B, Vetter W. In vitro antioxidant activity of calcium antagonists against LDL oxidation compared with alpha-tocopherol. Biochem Biophys Res Commun. 1994;203:1803-1808.[Medline] [Order article via Infotrieve]
  16. Li Q, Tallant A, Cathcart MK. Dual calcium requirement for optimal lipid peroxidation of low density lipoprotein by activated human monocytes. J Clin Invest. 1993;91:1499-1506.
  17. Weidmann P, Uehlinger DE, Gerber A. Antihypertensive treatment and serum lipoproteins. J Hypertens. 1985;3:297-306.[Medline] [Order article via Infotrieve]
  18. Lichtlen PR, Hugenholtz PG, Rafflenbeul W, Hecker H, Jost S, Deckers JW. Retardation of angiographic progression of coronary artery disease by nifedipine: results of the International Nifedipine Trial on Antiatherosclerotic Therapy (INTACT). Lancet. 1990;335:1009-1013.
  19. Chapman MJ, Laplaud PM, Luc G, Forgez P, Bruckert E, Goulinet S, Lagrange D. Further resolution of the low-density lipoprotein spectrum in normal human plasma: physicochemical characteristics of discrete subspecies separated by density gradient ultracentrifugation. J Lipid Res. 1988;29:442-458.[Abstract]
  20. Lesnik P, Vonica A, Guerin M, Moreau M, Chapman MJ. Anticoagulant activity of tissue factor pathway inhibitor in human plasma is preferentially associated with dense subspecies of LDL and HDL and with Lp(a). Arterioscler Thromb. 1993;13:1066-1075.[Abstract/Free Full Text]
  21. Nigon F, Lesnik P, Rouis M, Chapman MJ. Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor. J Lipid Res. 1991;32:1741-1754.[Abstract]
  22. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275.[Free Full Text]
  23. Basu SK, Goldstein JL, Anderson RGW, Brown MS. Degradation of cationized low-density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc Natl Acad Sci U S A. 1976;73:3178-3182.[Abstract/Free Full Text]
  24. Rouis M, Nigon F, Lafuma C, Hornebeck W, Chapman MJ. Expression of elastase activity by human monocyte-macrophages is modulated by cellular cholesterol content, inflammatory mediators, and phorbol myristate acetate. Arteriosclerosis. 1990;10:246-255.[Abstract/Free Full Text]
  25. Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low-density lipoprotein. Free Radic Res Commun. 1989;6:67-75.[Medline] [Order article via Infotrieve]
  26. El-Saadani MH, Esterbauer H, El-Sayed M, Goher M, Nassar AY, Jurgens G. A spectrophotometric assay for lipid peroxydes in the serum lipoproteins using commercially available reagents. J Lipid Res. 1989;30:627-630.[Abstract]
  27. Wallin B, Rosengren B, Shertzer HJ, Camejo G. Lipoprotein oxidation and measurement of thiobarbituric acid reacting substances formation in a single microtiter plate: its use for evaluation of antioxidants. Anal Biochem. 1992;208:10-15.
  28. Wallin B, Camejo G. Lipoprotein oxidation and measurement of hydroperoxide formation in a single microtitre plate. Scand J Clin Lab Invest. 1994;54:341-346.[Medline] [Order article via Infotrieve]
  29. Noble RP. Electrophoretic separation of plasma lipoproteins in agarose-gel. J Lipid Res. 1968;9:693-700.[Abstract]
  30. Lesnik P, Rouis M, Skarlatos S, Kruth HS, Chapman MJ. Uptake of exogenous free cholesterol induces upregulation of tissue factor expression in human monocyte-derived macrophages. Proc Natl Acad Sci U S A. 1992;89:10370-10374.[Abstract/Free Full Text]
  31. Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem. 1957;226:497-501.[Free Full Text]
  32. Dejager S, Bruckert E, Chapman MJ. Dense low-density lipoprotein subspecies with diminished oxidative resistance predominate in combined hyperlipidemia. J Lipid Res. 1993;34:295-308.[Abstract]
  33. Blois MS. Antioxidant determination by the use of a stable free radical. Nature. 1958;181:1199-1200.
  34. Mak IT, Weglicki WB. Comparative antioxidant activities of propanolol, nifedipine, verapamil and diltiazem against sarcolemmal membrane lipid peroxidation. Circ Res. 1990;66:1449-1452.[Abstract/Free Full Text]
  35. Mak IT, Weglicki WB. Protection by beta-blocking agents against free radical–mediated sarcolemmal lipid peroxidation. Circ Res. 1988;63:262-266.[Abstract/Free Full Text]
  36. Daugherty A, Rateri DL, Schonfeld G, Sobel BE. Inhibition of cholesteryl ester deposition in macrophages by calcium entry blockers: an effect dissociable from calcium entry blockade. Br J Pharmacol. 1987;91:113-118.[Medline] [Order article via Infotrieve]
  37. Etingin OR, Hajjar DP. Nifedipine increases cholesteryl ester hydrolytic activity in lipid laden rabbit arterial smooth muscle cells. J Clin Invest. 1985;75:1554-1558.
  38. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;344:793-795.[Medline] [Order article via Infotrieve]
  39. Hoppe GO, Neil J, Hoff HF. Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages. J Clin Invest. 1994;94:1506-1512.
  40. Hoff HF, Zyromski N, Armstrong D, O'Neil J. Aggregation as well as chemical modification of LDL during oxidation is responsible for poor processing in macrophages. J Lipid Res. 1993;34:1919-1929.[Abstract]
  41. Arai H, Kita D, Yokode M, Narumiya S, Kawai C. Multiple receptors for modified LDL in mouse peritoneal macrophages: different uptake mechanisms for acetylated LDL and oxidized LDL. Biochem Biophys Res Commun. 1989;159:1375-1382.[Medline] [Order article via Infotrieve]
  42. Van Berkel TJC, De Rijke YB, Kruijt JK. Different fate in vivo of oxidatively modified LDL and acetylated LDL in rats: recognition by various scavenger receptors on Kupffer and endothelial liver cells. J Biol Chem. 1991;266:2282-2289.[Abstract/Free Full Text]
  43. Brown MS, Ho YK, Goldstein JL. The cholesteryl ester cycle in macrophage foam cells: continual hydrolysis and re-esterification of cytoplasmic cholesteryl esters. J Biol Chem. 1980;255:9344-9352.[Free Full Text]
  44. Khoo JC, Mahoney EM, Witztum JL. Secretion of lipoprotein lipase by macrophages in culture. J Biol Chem. 1981;256:7105-7108.[Abstract/Free Full Text]
  45. Etingin OR, Hajjar DP. Calcium channel blockers enhance cholesteryl ester hydrolysis and decrease total cholesterol accumulation in human aortic tissue. Circ Res. 1990;66:185-190.[Abstract/Free Full Text]
  46. Waters D, Lesperance J. Calcium channel blockers and coronary atherosclerosis: from the rabbit to the real world. Am Heart J. 1994;128:1309-1316.[Medline] [Order article via Infotrieve]
  47. Willis AL, Nagel B, Churchill V, Whyte MA, Smith DL, Mahmud I, Puppione DL. Antiatherosclerotic effects of nicardipine and nifedipine in cholesterol-fed rabbits. Arteriosclerosis. 1985;5:250-255.[Abstract/Free Full Text]
  48. Henry PD, Bentley KI. Suppression of atherogenesis in cholesterol-fed rabbit treated with nifedipine. J Clin Invest. 1981;68:1366-1369.
  49. Stender S, Stender I, Nordestgaard B, Kjeldsen K. No effect of nifedipine on atherogenesis in cholesterol-fed rabbit. Arteriosclerosis. 1984;4:389-394.[Abstract/Free Full Text]
  50. Van Niekert JLM, Hendricks T, DeBoer HHM, Van't Larr A. Does nifedipine suppress atherogenesis in WHHL rabbits? Atherosclerosis. 1984;53:91-98.[Medline] [Order article via Infotrieve]
  51. Kaplan JR, Manuck SB. Antiatherogenic effects of beta-adrenergic blocking agents: theoretical, experimental, and epidemiologic considerations. Am Heart J. 1994;128:1316-1328.[Medline] [Order article via Infotrieve]



This article has been cited by other articles:


Home page
 CirculationHome page
J. E. Nettleship, T. H. Jones, K. S. Channer, and R. D. Jones
Physiological Testosterone Replacement Therapy Attenuates Fatty Streak Formation and Improves High-Density Lipoprotein Cholesterol in the Tfm Mouse: An Effect That Is Independent of the Classic Androgen Receptor
Circulation, November 20, 2007; 116(21): 2427 - 2434.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
M. C. Verhaar, M. L.H. Honing, T. van Dam, M. Zwart, H. A. Koomans, J. J.P. Kastelein, and T. J. Rabelink
Nifedipine improves endothelial function in hypercholesterolemia, independently of an effect on blood pressure or plasma lipids
Cardiovasc Res, June 1, 1999; 42(3): 752 - 760.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lesnik, P.
Right arrow Articles by Chapman, M. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lesnik, P.
Right arrow Articles by Chapman, M. J.

ATVB Home | Subscriptions | Archives | Feedback | Authors | Help | AHA Journals Home | Search
Copyright © 1997 American Heart Association, Inc. All rights reserved. Unauthorized use prohibited.