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© 1995 American Heart Association, Inc.
Articles |
HMG-CoA Reductase Inhibitors Reduce Acetyl LDL Endocytosis in Mouse Peritoneal Macrophages
From the Institute of Pharmacology and Pharmacognosy (F.B.), University of Parma, Parma, and the Institute of Pharmacological Sciences (N.S., G.B., R.F.), University of Milan, Italy.
Correspondence to Prof Franco Bernini, Institute of Pharmacology and Pharmacognosy, University of Parma, Via delle Scienze, 43100 Parma, Italy.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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Abstract We previously reported that mevalonate starvation elicited by hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase inhibitors reduced cholesterol accumulation promoted in murine macrophages by acetylated LDL (AcLDL). In the present study we investigated the cellular mechanism of this effect. Our results indicate that the HMG-CoA reductase inhibitors fluvastatin and simvastatin reduce, in a concentration-dependent manner, more then 50% of the 125I-AcLDL degradation by macrophages. This effect was not due to a decrease of lysosomal enzyme activity, and it was paralleled by the retention of AcLDL-associated cholesteryl ester in the incubation medium. The ability of fluvastatin to inhibit AcLDL degradation was completely overcome by mevalonate and its derivative geranylgeraniol. Evaluation at 4°C of 125I-AcLDL binding to plasma membrane suggested that the inhibitory effect of fluvastatin on lipoprotein catabolism was not due to a decreased expression of scavenger receptors. Fluorescent microscope analysis of cellular internalization of AcLDL labeled with the fluorochrome 3,3'-dioctadecyl indocarbocyanine demonstrated that fluvastatin inhibits lipoprotein endocytosis, an effect reversed by mevalonate. Studies performed with native 125I-LDL indicated that fluvastatin did not inhibit but rather increased the degradation of LDL taken up by the normal LDL receptor. These results exclude a generalized depression of the cellular endocytotic activity by the drug. The ability of fluvastatin to reduce AcLDL catabolism and cholesterol esterification was more pronounced in cholesterol-enriched macrophages compared with normal cells. In conclusion, the present results demonstrate that HMG-CoA reductase inhibitors may reduce the in vitro cholesterol accumulation in macrophages by inhibiting AcLDL endocytosis.
Key Words: acetyl LDL • HMG-CoA reductase inhibitors • mevalonate • macrophages • atherosclerosis
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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A major process in atheroma formation is the accumulation of cholesterol in macrophages present in the arterial wall. These cells have been demonstrated in vitro to accumulate an excess of cholesterol via scavenger receptors that mediate the internalization and degradation of modified lipoproteins such as AcLDL and oxidized LDL.1 2 The cholesteryl esters transported by these lipoproteins are hydrolyzed in the lysosomes, and free cholesterol is released to a pool(s) where it participates as a substrate for the esterification reaction catalyzed by the microsomal enzyme ACAT.3 Several compounds have been reported to reduce cholesterol esterification. Some act directly by inhibiting ACAT activity,4 others indirectly by reducing lipoprotein degradation and their cholesteryl ester hydrolysis in the lysosomes,5 6 slowing down intracellular cholesterol movement,7 8 and inhibiting scavenger receptor expression and cellular influx of modified lipoproteins.9 10 Depending on the mechanism involved, each agent may have different effects on cellular cholesterol content and localization and on the esterified-to–free form ratio. ACAT inhibitors may increase free cholesterol content of the plasma membrane, with a minor effect on total cellular cholesterol.11 Free cholesterol accumulation in lysosomes is observed with compounds active on intracellular cholesterol movement.7 8 Accumulation of cholesteryl esters in these organelles is achieved with agents inhibiting the activity of lysosomal enzymes.5 6 Finally, a decrease of total cellular cholesterol is observed with compounds that inhibit the expression of scavenger receptors.10
Previous studies demonstrated that HMG-CoA reductase inhibitors (vastatins) decreased cholesterol esterification and deposition in macrophages.12 13 This action did not occur in a cell-free homogenate12 and was observed only under conditions of simultaneous incubation of vastatins with AcLDL but not in cholesterol-preloaded cells,13 thus indicating that these drugs are not direct ACAT inhibitors. The inhibition of cholesterol esterification by vastatins was completely overcome by mevalonate and geranylgeraniol13 and took place in the presence of an excess of exogenous cholesterol.12 13 These results showed that vastatins did not effect esterification by preventing the intracellular formation of substrate for ACAT (ie, cholesterol) but rather by inhibiting the formation of nonsterol mevalonate product(s).
The cellular mechanism by which vastatins limit cholesterol esterification has not been elucidated. Since the blockade of cholesteryl ester formation is concomitant with a net loss in cellular cholesterol content,13 it can be inferred that vastatins either induce cholesterol efflux or reduce cholesterol uptake from AcLDL.
In the present study we report that mevalonate starvation induced by vastatins does not influence cholesterol efflux but rather reduces cellular uptake of exogenous cholesterol by inhibiting AcLDL endocytosis.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Materials
DMEM, HEPES buffer solution (1 mol/L), penicillin-streptomycin (10 000 IU/mL to 10 000 UG/ml), and FCS were purchased from GIBCO Laboratories. EFAF-BSA was obtained from Sigma Chemical Co. Cell culture wells (35 mm, Multidish Nunclon) were obtained from NUNC.
Simvastatin, kindly provided by Merck Sharp & Dohme Research Laboratories, was added in the active form in 0.1 mol/L NaOH adjusted to pH 7.4 with HCl. Fluvastatin and S-58035, kindly provided by Sandoz Prodotti Farmaceutici, were added to monolayers in ethanol and DMSO, respectively, whose concentrations did not exceed 0.5% of culture medium; control dishes contained the same percentage of solvent. Farnesol and mevalonic acid lactone were also obtained from Sigma. (AII-trans)-geranylgeraniol was kindly provided by Prodotti Roche. [1-14C]oleic acid (56 mCi/mmol), [1a,2a(n)-3H]cholesteryl linoleate (40 Ci/mmol), and sodium [125I]iodide (17 mCi/mg) were from Amersham.
Cells
MPMs were obtained by peritoneal lavage from mice (BALB/c,
Charles River, Calco, Italy) 3 days after
intraperitoneal injection of thioglycollate. Cells
(2 to 3x106) were plated in 35-mm wells with DMEM
containing 10% FCS. After 3 hours, the dishes were washed to eliminate
unattached cells and maintained in DMEM plus 10% FCS for 24 hours
before use. Afterwards cell-plating experiments were performed at
37°C (except where indicated) in serum-free DMEM containing 0.2%
EFAF-BSA plus the indicated additions.
In experiments conducted at 4°C, bicarbonate was substituted with HEPES 10 mmol/L. Protein was measured according to method reported by Lowry et al.14
Lipoprotein Preparations
Human LDL (d=1.019 to 1.063 g/mL) was isolated from
plasma of healthy volunteers by sequential
ultracentrifugation (Beckman L5-50).15
For acetylation, LDL was dialyzed against 0.15 mol/L NaCl,
pH 7.4, diluted with an equal volume of saturated sodium acetate, and
treated with acetic anhydride, according to technique used by Basu et
al.16
For 125I-AcLDL and 125I-LDL preparations, lipoproteins were labeled with sodium [125I]iodide according to technique reported by Bilheimer et al,17 and desalted by gel filtration on Sephadex G-25 eluted with PBS. Specific activity was 100 to 200 cpm/ng protein. TCA nonprecipitable radioactivity was below 2% of total.
Cholesteryl linoleate–labeled AcLDL (specific activity, 20 disintegrations per minute/ng) was prepared by incubation of lipoproteins with 10 mCi/mg protein of [3H]cholesteryl linoleate in the presence of rabbit serum containing the cholesteryl ester transfer protein.18 Dil-AcLDL was kindly provided by Dr David Via (Baylor College of Medicine, Houston, Tx). All lipoproteins were sterile filtered.
Cellular Cholesterol Efflux
Cells were incubated for 24 hours with the tested compounds,
followed by 24 hours in the same fresh medium with
[3H]CE-AcLDL (25 µg/mL) added. Cells and medium were
subsequently analyzed as described below for radioactivity
content in free and esterified cholesterols.
Free-cholesterol radioactivity in the medium indicated the
amount of cholesterol efflux from cells.
Lipoprotein Degradation and Binding
Cells were incubated for 24 hours with the tested compounds.
After substitution of the medium with an identical one, incubation was
continued for an additional 24 hours. During this second incubation
125I-labeled lipoproteins were added at the times and
concentrations indicated in the figures and tables. Then medium was
removed and lipoprotein degradation was measured as the accumulation of
noniodide TCA–soluble 125I in excess of that occurring in
the absence of cells19 (less than 0.2% of total
degradation of control). Lipoprotein binding was determined after
incubation of cells at 4°C for 4 hours with 125I-AcLDL
followed by extensive washing of the monolayers with albumin
buffer and their digestion with 0.1 mol/L NaOH.20 The
nonspecific lipoprotein binding was evaluated in the presence of a
50-fold excess of unlabeled lipoprotein.21 Nonspecific
values were less than 15% of total binding.
Cholesterol Esterification Assay
Cells were incubated for 24 hours with the drug, followed by 24
hours in the same fresh medium added with 5 µg/mL of AcLDL.
Cholesterol esterification was measured after addition of
[1-14C]oleic acid (0.68 mCi sample) complexed with
BSA during the last 2 hours of incubation and subsequent
determination of radioactivity associated with cellular
cholesteryl esters.22
Cholesterol Enrichment of Cells
Where indicated, cells were enriched with
cholesterol by 24-hour incubation with 10 to 25 µg/mL of
AcLDL before drug addition and experimental determinations.
Lipoprotein–Cholesteryl Ester Hydrolysis
After the 24-hour drug treatment, cells were incubated in the
same fresh medium with [3H]CE-AcLDL (25 µg/mL) added
and the ACAT inhibitor S-58035.23 Cells and
media were then assayed for radioactivity content of the free and
esterified cholesterol fractions. The percentage of
radioactivity associated with free cholesterol (cell plus
medium) indicated the amount of AcLDL-derived cholesteryl ester
hydrolyzed by cells.
Determination of Free and Esterified
Cholesterol
After the indicated incubations cells were washed with PBS and
extracted with hexane/isopropanol (3:2, vol/vol). Media were
extracted with chloroform/methanol (2:1 vol/vol).
After solvent removal, free and esterified cholesterol was partitioned by thin-layer chromatography (isooctane/diethyl ether/acetic acid, 75:25:2 by volume).21 Cholesterol mass or radioactivity of the spots was determined by an enzymatic method (Boeringher Mannheim)13 24 or by liquid scintillation counting (Lipoluma Lumac, Landgraf), respectively.
Fluorescence Microscopy
The cells to be used for fluorescence microscopy were
seeded (106 cells/sample) on 18x24-mm coverslips.
After treatment with the tested compounds as described above, cells
were added with Dil-AcLDL (5 µg/mL) and incubated for an additional 5
hours. Monolayers were subsequently washed and fixed with 3%
formaldehyde. Fluorescent photomicrographs were made on a Zeiss
Axiovert 100 with an excitation filter for rhodamine using Kodak TMY
film (ASA 400). Since fluvastatin was more active in
cholesterol-loaded cells (see Fig 4
) the reported results
were obtained in this experimental condition. Consistent
results were observed in unloaded cells (data not shown).
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)
and cholesterol-loaded (
) MPMs. Macrophages
were cholesterol enriched as described in "Methods."
Cells were further incubated for 24 hours in a lipoprotein-free medium
containing 0.2% EFAF-BSA and drug at the indicated concentrations.
Cells were subsequently incubated for 24 hours in the same fresh medium
added with 125I-AcLDL (5 µg protein/mL). Lipoprotein
degradation was then determined as described in "Methods." Data
are the mean of duplicate samples, which did not differ by more than
10%. Control value for normal cells, 13 200 ng 125I-AcLDL
degraded per milligram protein; cholesterol loaded cells,
14 250 ng 125I-AcLDL degraded per milligram protein. |
Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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To test whether the activity of HMG-CoA reductase inhibitors on cellular cholesterol metabolism could involve a stimulatory effect on cholesterol efflux, MPMs were incubated with [3H]CE-AcLDL. Incubation of cells with 25 µg/mL [3H]CE-AcLDL induced the accumulation of 20 µg/mg cell proteins of AcLDL-derived cholesterol, two thirds in the esterified form. Treatment of cells with fluvastatin reduced the amount of radioactive cholesterol esters by 60%, with minor changes of free [3H]cholesterol content (Table 1
). Analysis of radioactive
cholesterol present in the incubation medium revealed
that the decrease of [3H]cholesteryl esters in cells was
paralleled by an increase of extracellular radioactivity
associated with the esterified fraction of cholesterol,
whereas the content of labeled free cholesterol was
unaffected by the drug (Table 1). All these effects were abolished by
mevalonate (data not shown).
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We next evaluated whether HMG-CoA reductase inhibitors
could influence cellular catabolism of AcLDL. Fluvastatin and
simvastatin were tested for their actions on specific
degradation of 125I-AcLDL (50 µg/mL) by MPMs. Data
reported in Fig 1 demonstrate that both drugs inhibited
in a dose-dependent manner up to 50% 125I-AcLDL
degradation by macrophages. The inhibitory effects
of both drugs were overcome by the addition of 100 µmol/L mevalonate.
Mevalonate alone did not affect this parameter (Fig 1).
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In addition to mevalonate, fluvastatin inhibitory activity
on lipoprotein degradation was fully prevented by geranylgeraniol and
only slightly by farnesol (Table 2). The activity of
fluvastatin was also investigated on the catabolism by
macrophages of native LDL. The effects of the drug on
125I-LDL or 125I-AcLDL cell degradation were
compared in identical experimental conditions. Fluvastatin,
despite its ability to inhibit 125I-AcLDL degradation, did
not reduce degradation of 125I-LDL. On the contrary, the
drug showed a slight but significant stimulatory effect on
125I-LDL degradation (Table 3). To evaluate
the effect of fluvastatin on AcLDL specific binding to plasma membrane,
cells were treated for 44 hours with the drug, followed by a 4-hour
incubation with 125I-AcLDL (40 µg/mL) at 4°C. The
results indicate a lack of effect of fluvastatin in these experimental
conditions (539±21 and 545±12 ng/mg cell protein of specific binding
in control and treated cells, respectively).
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We next investigated whether the reduced lipoprotein degradation could
involve a nonspecific cloroquine-like inhibitory action of
fluvastatin on lysosomal enzyme activity. To answer this question we
performed experiments incubating the cells with
[3H]CE-AcLDL (25 µg/mL) in the presence of the specific
ACAT inhibitor S-58035. The blockade of intracellular
re-esterification of cholesterol allowed us to assess the
ability of lysosomes to hydrolyze cholesteryl esters
transported by AcLDL. In the presence of S-58035 
85% of the
radioactive cholesteryl ester released to cells by
[3H]CE-AcLDL was hydrolyzed (Table 4). The
addition of fluvastatin only slightly influenced this result and did
not induce any increase of radioactivity in the esterified fraction,
suggesting a minor effect on the cellular hydrolysis of esterified
cholesterol. By evaluating cellular cholesterol
mass we observed that the ability of fluvastatin to reduce total
cellular cholesterol was unaffected by S-58035 (Fig 2).
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We further investigated the effect of HMG-CoA reductase inhibition on
AcLDL catabolism by studying the effect of fluvastatin on lipoprotein
endocytosis. For this purpose we evaluated with fluorescence
microscopy the action of the drug on the internalization by MPMs of
Dil-AcLDL. As expected25 incubation of MPMs with Dil-AcLDL
resulted in intense perinuclear fluorescence (Fig 3A). The fluorescence was reduced in cells
exposed to fluvastatin compared with control cells (Fig 3B). The
addition of mevalonate alone did not influence Dil-AcLDL uptake (data
not shown), but in fluvastatin-treated cells it restored a pattern of
fluorescence similar to that of control cells (Fig 3C).
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Since the action of fluvastatin on AcLDL catabolism appears to be
linked to the inhibition of HMG-CoA reductase, and since the activity
of this enzyme is partially suppressed in
cholesterol-loaded cells, we explored the effect of
fluvastatin in cholesterol-enriched macrophages
compared with unloaded cells. Data reported in Fig 4
show that fluvastatin was able to inhibit AcLDL degradation with an
IC50 of about 2 µmol/L and 0.08 µmol/L in normal and
cholesterol-loaded cells, respectively. The efficacy of
fluvastatin was greater in loaded cells than in normal cells (48%
versus 32% inhibition). In the same experimental conditions a similar
pattern of activity was observed when the drug was tested on
cholesterol esterification (Fig 5).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Data presented in this study show that HMG-CoA reductase inhibitors reduce in vitro cholesterol accumulation elicited by AcLDL in MPMs by inhibiting the endocytosis of these lipoproteins by the cells. The experiments performed with [3H]CE-AcLDL indicate that fluvastatin does not increase the concentration of free cholesterol in the incubation medium; since no extracellular esterification activity was detected under our experimental conditions (data not shown), an effect of the drug on cholesterol efflux from cells can be excluded. The decrease of labeled cholesterol esters in cells elicited by fluvastatin is concomitant with an increase of radioactive cholesteryl ester in the incubation medium, suggesting its reduced influx into the cells. Altogether these results indicate that fluvastatin inhibition of cellular accumulation of esterified cholesterol elicited by AcLDL may involve a decreased influx of these lipoproteins into cells, with the consequent reduction of AcLDL-derived cholesterol release to ACAT. This hypothesis is consistent with our previous finding13 that vastatins inhibited cholesterol esterification when added to macrophages before AcLDL but not in cholesterol-preloaded cells, ie, when AcLDL-derived cholesterol has already been released to the substrate pool of ACAT. Confirmatory data of a decreased cellular influx of AcLDL are provided by the observation that fluvastatin inhibits the degradation of 125I-AcLDL by MPMs. This inhibitory effect was dose dependent in the same range of concentrations effective on cholesterol esterification and deposition. This effect was shared by simvastatin, suggesting that this property is related to the class of these drugs.
Experiments performed in the presence of the ACAT inhibitor S-58035 showed that fluvastatin does not induce the deposition of esterified cholesterol, indicating that the drug has a negligible effect on lysosomal esterase activity. Moreover, the ability of fluvastatin to reduce cellular cholesterol content in MPMs incubated with AcLDL was unaffected by the presence of the ACAT inhibitor. This observation indicates that the reduction of cellular cholesterol deposition elicited by vastatins is not related to ACAT activity and is consistent with an inhibitory effect on cholesterol delivery to cells. The results obtained with Dil-AcLDL, a means to visualize AcLDL internalization,25 offer direct evidence that fluvastatin inhibits AcLDL catabolism by preventing its endocytosis.
The inhibition by vastatins of AcLDL-induced cholesterol
esterification in human macrophages was previously reported by
Kempen et al.12 These authors reported a slight but not
statistically significant effect of these drugs on AcLDL degradation,
and concluded that the inhibitory effect of vastatins on
cholesterol esterification could not be attributed to a
decrease in receptor-mediated uptake or degradation of AcLDL. The
reasons for this discrepancy are not clear. A species difference is
possible; however, the results reported by Kempen et al, indicating a
net reduction of total cellular cholesterol content in
human macrophages (see Table 1 in Reference 1212 ), are
consistent with our observations. In any case, our results do
not exclude additional mechanisms (such as an interference with the
intracellular cholesterol trafficking)12 that
may contribute to the ability of vastatins to reduce
cholesterol esterification. This possibility is supported
by the observation that in our experiments the inhibition of
cholesterol esterification by fluvastatin was greater than
that elicited on AcLDL degradation (compare Figs 4 and 5).
The mechanism involved in fluvastatin action on AcLDL endocytosis needs clarification. The effect is not related to a decreased expression of the scavenger receptors, since no decrease of cellular binding at 4°C could be detected. Although we cannot exclude an inhibitory effect of vastatins on the endocytosis of other ligands, the effect on AcLDL seems not to be due to a nonspecific depression of cellular endocytotic functions, since in the condition in which AcLDL degradation is reduced, a slight but significant increase in native LDL degradation is observed. A slight but consistent increase of receptor-mediated catabolism of LDL in cultured macrophages by HMG-CoA reductase inhibitors has been recently reported.26 Our results show that the inhibition by fluvastatin of AcLDL catabolism is reversed by mevalonate and its isoprenoid derivative geranylgeraniol. This result suggests the involvement of nonsterol products of the mevalonate pathway in AcLDL endocytosis. For example, geranylgeranylated proteins such as rab4 and rab5 may play a regulatory role in receptor-mediated endocytosis and recycling.27 28 Interestingly, we also observed that fluvastatin is more effective in cholesterol-loaded cells. These results may be explained when one considers that in cholesterol-rich cells, HMG-CoA reductase activity is already largely reduced compared with the activity in unloaded cells.29
The fluvastatin IC50s of 2 and 0.1 µmol/L,
evaluated in normal and loaded cells respectively, are in the range of
the reported therapeutic concentrations of the drug.30 The
accumulation of cholesterol in macrophages may
result from an imbalance between cholesterol influx and
cholesterol efflux from cells.3 Vastatin
inhibition of AcLDL endocytosis might therefore affect foam cell
formation. In vivo evidence indicates that HMG-CoA reductase
inhibitors may have beneficial effects on
atheroma generation not only by reducing plama
cholesterol levels but also by directly acting on the
arterial wall.31 32
In conclusion the present study demonstrates that HMG-CoA reductase inhibitors may reduce cholesterol accumulation in macrophages by inhibiting lipoprotein endocytosis through the scavenger receptors. This effect is related to the depletion of nonsterol products of the mevalonate pathway. The present results may help in the understanding of the mechanism of cholesterol accumulation in the arterial wall. The observation that fluvastatin is more active on cholesterol-loaded macrophages (ie, foam cells) suggests the possibility that the mevalonate pathway in the arterial lesions may represent a selective target for pharmacological intervention.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The research was partially supported by MPI and CNR (Italian Government). We are indebted to Dr David Via for providing Dil-AcLDL. The secretarial help of Laura Mozzarelli is also acknowledged.
Received May 31, 1994; accepted June 9, 1995.
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 N. Hrboticky, G. Draude, G. Hapfelmeier, R. Lorenz, and P. C. Weber Lovastatin Decreases the Receptor-Mediated Degradation of Acetylated and Oxidized LDLs in Human Blood Monocytes During the Early Stage of Differentiation Into Macrophages Arterioscler Thromb Vasc Biol, May 1, 1999; 19(5): 1267 - 1275. [Abstract] [Full Text] [PDF]
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S. Bellosta, D. Via, M. Canavesi, P. Pfister, R. Fumagalli, R. Paoletti, and F. Bernini HMG-CoA Reductase Inhibitors Reduce MMP-9 Secretion by Macrophages Arterioscler Thromb Vasc Biol, November 1, 1998; 18(11): 1671 - 1678. [Abstract] [Full Text] [PDF]
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A. Cignarella, B. Brennhausen, A. von Eckardstein, G. Assmann, and P. Cullen Differential Effects of Lovastatin on the Trafficking of Endogenous and Lipoprotein-Derived Cholesterol in Human Monocyte–Derived Macrophages Arterioscler Thromb Vasc Biol, August 1, 1998; 18(8): 1322 - 1329. [Abstract] [Full Text] [PDF]
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V. Llorente-Cortes, J. Martinez-Gonzalez, and L. Badimon Esterified Cholesterol Accumulation Induced by Aggregated LDL Uptake in Human Vascular Smooth Muscle Cells Is Reduced by HMG-CoA Reductase Inhibitors Arterioscler Thromb Vasc Biol, May 1, 1998; 18(5): 738 - 746. [Abstract] [Full Text] [PDF]
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