Esterified Cholesterol Accumulation Induced by Aggregated LDL Uptake in Human Vascular Smooth Muscle Cells Is Reduced by HMG-CoA Reductase Inhibitors

From the Cardiovascular Research Center, CSIC-HSCSP-UAB (V.L.-C., J.M.-G., L.B.); and the Institut de Recerca de l' Hospital de la Santa Creu i Sant Pau (J.M.-G.), Barcelona, Spain.

Correspondence to Prof Lina Badimon, Centro de Investigacion y Desarrollo (CSIC), C/Jordi Girona 18–26, 08034 Barcelona, Spain. E-mail lbmucv{at}cid.csic.es

	Abstract

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Abstract—Vascular smooth muscle cell (VSMC) proliferation is a key event in the development of atherosclerotic lesions. VSMCs synthesize extracellular matrix, where low density lipoproteins (LDLs) are trapped and become aggregated (agLDL). The objective of this study was to investigate the cholesterol uptake and accumulation triggered by agLDL in comparison with native LDL (nLDL) on unstimulated and platelet-derived growth factor–stimulated human aortic VSMCs and the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on these processes. Esterified cholesterol (EC) accumulation induced by agLDL in VSMCs was correlated with the degree of aggregation and concentration. The EC content of VSMCs treated with 100 µg/mL of agLDL (80% aggregated) increased 70-fold over that in VSMCs incubated with the same concentration of nLDL. Whereas nLDL-derived EC was increased approximately twofold in platelet-derived growth factor–stimulated VSMCs, there was no effect of platelet-derived growth factor (10^-9 mol/L) on the uptake of agLDL. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (5 µmol/L) reduced EC accumulation derived from agLDL uptake by 58% and 35% in platelet-derived growth factor—stimulated and unstimulated VSMCs, respectively. This inhibition was overcome by geranylgeraniol (10 µmol/L) and partially by farnesol (10 µmol/L). Fluorescence microscopy of the cellular internalization of agLDL labeled with the fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine demonstrated that simvastatin reduces EC accumulation derived from agLDL by inhibiting its endocytosis and that the effect is completely reversed by geranygeraniol. These results indicate that agLDLs are rapidly internalized by human VSMCs and that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors modulate EC accumulation. These data suggest a possible mechanism by which statins could contribute to the passivation and stabilization of actively growing atherosclerotic lesions.

Key Words: LDL aggregation • vascular smooth muscle cells • HMG-CoA reductase inhibitors

	Introduction

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Atherosclerosis is a complex process that involves multiple factors, which interact to promote lesion development. Hence, there are a number of possible targets for intervention aimed at regression, stabilization, or deceleration of the formation and progression of the lesions.

Two key events in the atherogenic cascade are the deposition of lipids, mainly cholesterol esters, and the migration and proliferation of VSMCs.^{1 2} Other evidence indicates that lipids deposited in atherosclerotic lesions are derived from modified LDLs.³ VSMCs and macrophage-derived foam cells are the main cellular constituents of human atherosclerotic lesions. Macrophages accumulate lipoprotein cholesterol through scavenger receptors that recognize chemically modified LDL, such as acetylated LDL and oxidized LDL.^{4 5} In addition, the macrophage uptake of LDL associated with arterial PGs^{6 7} or agLDL^{8 9} seems to be receptor independent. Cholesterol escapes feedback regulation and consequently is extensively accumulated in an esterified form in the cells. VSMCs represent, on average, 50% of the cellular component in an advanced atherosclerotic plaque and can reach 90% to 95% in early lesions.^{10 11} In addition, VSMCs contribute to the lesion by synthesizing extracellular matrix; these cells can also accumulate esterified cholesterol, characteristic of foam cell formation. Proliferative VSMCs have a high capacity to synthesize sulfated PGs, and it is well established that PGs in the arterial wall are involved in the focal deposition of cholesterol-rich particles in the early phases of atherogenesis.^{12 13} In fact, increased uptake of lipoprotein-PG complexes and agLDL by VSMCs has been demonstrated.^{14 15 16} In addition, the presence of scavenger receptors in intimal VSMCs has been reported.^{17 18}

A major rate-limiting step in the cholesterol biosynthesis pathway is at the level of HMG-CoA reductase [mevalonate: NADP+oxidoreductase (CoA-acylating); EC 1.1.1.34]. HMG-CoA reductase is an inducible enzyme that catalyzes the formation of mevalonate, the first metabolite committed to the synthesis of sterols.¹⁹ In the last decade, different competitive inhibitors of HMG-CoA reductase (statins) have been introduced into human therapy as systemic lipid-lowering agents. These drugs strongly inhibit cholesterol synthesis in the liver and intestine, and their beneficial effect has been established in large clinical trials and regression studies.^{20 21 22} Stabilization of coronary lesions has been proposed as the most likely explanation for the improvement in clinical events and survival. Previous studies have demonstrated the capacity of statins to inhibit VSMC growth.^{23 24} However, mevalonate derivatives are essential not only for cell cycle progression but also for endocytotic processes.^{25 26} Thus, inhibition of the mevalonate pathway by HMG-CoA reductase inhibitors could affect not only VSMC proliferation but also lipoprotein internalization.

Based on these findings, the objectives of this work were to study whether VSMCs isolated from the human arterial wall were able to accumulate EC derived from agLDL and to demonstrate the effect of HMG-CoA reductase inhibitors on lipoprotein internalization. Since PDGF has been implicated in atherogenesis by effects such as increases in both cellular proliferation and cholesterol availability, the studies were performed in parallel in unstimulated and PDGF-stimulated VSMCs. Our results indicate that VSMCs significantly accumulate EC derived from agLDL and that HMG-CoA reductase inhibitors regulate its accumulation in VSMCs.

	Methods

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Materials
Cell culture medium and reagents were from GIBCO Laboratories. [2-¹⁴C]acetic acid sodium salt (56 mCi/mmol) and human recombinant PDGF-BB were purchased from Amersham. R(-), S(+)mevalonic acid, farnesol, geranylgeraniol, and monoclonal antibodies of anti-human fibroblast surface protein were obtained from Sigma Chemical Co. DiI was purchased from Molecular Probes, Inc. Mouse monoclonal antibodies specific for human - SM actin, human von Willebrand factor, and fluorescein-conjugated goat anti-mouse IgG were purchased from Dako.

Simvastatin (MK-733, sinvinolin) was kindly provided by Merck Sharp & Dohme Laboratories. The inactive lactone forms were converted to the active forms by dissolving 20 mg of the lactone in 0.5 mL of 100% ethanol, adding 750 µL of 0.1 mol/L NaOH, heating at 50°C for 2 hours, neutralizing the solution with 0.1 mol/L HCl to pH 7.2, and adjusting the pH with PBS to a final concentration of 5 mmol/L. Stock solutions were sterilized by filtration and aliquots were stored at -20°C. Pravastatin sodium salt (CS-514, mevalotin) was kindly provided by Bristol-Myers Squibb Laboratories. Pravastatin was dissolved in PBS to reach a final concentration of 500 mmol/L. Stock solutions were sterilized by filtration and aliquots were stored at -20°C.

VSMC Culture
Primary cultures of human VSMCs were obtained from segments of macroscopically healthy aortas (as deduced from the absence of fibrofatty tissue or visible plaques) obtained at heart transplant operations by a modification of the explant technique as we described previously.²⁷ Transplanted patients were men between 40 and 60 years old. Explants were incubated at 37°C in a humidified atmosphere of 5% CO₂. Cells grown out of explants were suspended in a solution of trypsin/EDTA and subcultured. They grew in monolayers in medium 199 supplemented with 5% fetal calf serum and 5% human serum, 2 mmol/L L-glutamine, 100 U/mL penicillin G, and 100 µg/mL streptomycin. VSMCs were grown under the previous conditions until near confluence.

VSMCs were identified by their growth behavior, morphology, and immunofluorescence. Mouse monoclonal antibodies specific for human -SM actin (clone 1A4), human von Willebrand factor (clone F8/86), and human fibroblast surface protein (clone 1B10) were used. Cells were seeded on coverslips, grown to confluence, and then fixed with methanol for 5 minutes. A solution of BSA at 1% was used as a blocking agent. Monoclonal antibodies were added after they were diluted in 1% BSA and 0.1% Triton X-100. Finally, a fluorescein-conjugated goat anti-mouse IgG was used as a secondary antibody. VSMCs were used between passages 2 and 6. Cell viability was determined by trypan blue exclusion. VSMCs were stained and counted using a hemocytometer.

LDL Preparation and DiI Labeling
Human LDLs (d_1.019 to d_1.063 g/mL) were obtained from pooled sera of normocholesterolemic volunteers and isolated by sequential ultracentrifugation.²⁸ LDLs were dialyzed three times against 200 volumes of 150 mmol/L NaCl, 1 mmol/L EDTA, and 20 mmol/L Tris-HCl, pH 7.4, overnight and once against 150 mmol/L NaCl. LDL protein concentration was determined by the bicinchoninic acid method²⁹ and cholesterol concentration by a commercial kit (Boehringer). The average total cholesterol content of human LDL was 2 mg/mg LDL protein. The LDLs used in the experiments were <48 hours old. The purity of LDL was assessed by agarose gel electrophoresis (Paragon system, Beckmann). TBARS were measured as an indirect evaluation of lipid peroxidation. TBARS levels were <1.2 mmol malonaldehyde per milligram of protein LDL.

LDLs were labeled with DiI by a modification of the method described by Beisiegel et al³⁰ by incubating LDL (1 mg/mL) in PBS–0.5% BSA with 100 µL of DiI in DMSO (3 mg/mL) for 8 hours at 37°C. The density of the LDL solution was adjusted to 1.063, and LDLs were reisolated by ultracentrifugation, dialyzed, and filtered through a 0.22-µm filter. No alterations of electrophoretic mobility were detected against unlabeled LDL.

agLDLs (unlabeled or DiI labeled) were prepared by vortexing in PBS at room temperature. The formation of LDL aggregates by vortexing was monitored by measuring the turbidity (absorbance at 680 nm) as previously described.^{8 9} The percentage of LDL in aggregated form was calculated by measuring the fraction of protein recovered in the pellet obtained after centrifugation at 10 000g for 10 minutes.^{8 9 31} The different fractions were analyzed by agarose electrophoresis (Paragon system, Beckmann). No significant alterations of TBARS levels against nLDL were detected after LDL aggregation.

LDL Binding and Internalization by VSMCs
VSMCs were seeded in glass chamber slides at 100 cells/mm² and incubated with medium 199 containing 0.2% FCS for 24 hours prior to the experiment. Cells were prechilled to 4°C and washed with cold medium 199 containing 1% BSA. Binding experiments were performed according to a previously described method³² with minor modifications. VSMCs were incubated with 50 µg/mL of DiI-labeled LDL (n or ag) at 4°C for 30 minutes. After binding, the medium was removed, and previously warmed, fresh medium 199–BSA was added to cells, which were then incubated at 37°C for 4 hours in the absence or presence of the different compounds tested. Cells were then washed in medium 199–BSA containing 100 U heparin/mL for 15 minutes at 4°C with constant shaking. The cells were then fixed at room temperature for 10 minutes in PBS containing 3% paraformaldehyde and 2% sucrose before staining with Hoechst 33258 colorant (1:1000) for 10 minutes and washed twice with PBS. Finally, fluorescence photomicrographs were taken in an Olympus Vanox AHBT3 microscope with an excitation filter for rhodamine with Kodak Ektachrome (ASA 400) daylight film. Confocal laser scanning microscopy was performed on an invert laser scan microscope (Leica TCS NT). The excitation wavelengths used were 568 nm for the LP 590 filter with an absorption window >590 nm. The software program used was TCSNT, version 1.3.237.

Synthesis of Sterols
Synthesis of cholesterol was determined by measuring the incorporation of radioactive acetate into cellular sterols. VSMCs were seeded into six-well plates at 100 cells/mm², maintained for 48 hours, arrested for 24 hours with 0.2% FCS, and maintained for an additional 24 hours with or without PDGF-BB (10^-9 mol/L). The cells were then incubated overnight with [¹⁴C]acetate (5 µCi/mL) in the absence or presence of 100 µg/mL LDL (n or ag) and in the absence or presence of simvastatin 3.5 µmol/L. Cells were then washed with PBS and harvested into 1 mL of 0.15 mol/L NaOH. Lipid extraction was done according to the method of Bligh and Dyer³³ with minor modifications.³⁴ One aliquot of the cell suspension was extracted with methanol/dichloromethane (2:1, vol/vol). After solvent removal under an N₂ stream, the lipid extract was redissolved in dichloromethane and one aliquot was partitioned by TLC, which was performed on silica G-24 plates. Three different concentrations of standards (a mixture of cholesterol and cholesterol palmitate) were applied to each plate. The chromatographic developing solution was heptane/diethyl ether/acetic acid (74:21:4, vol/vol/vol). The plate was completely dried and spots were stained according to Huber et al.³⁵ The incorporation of labeled acetate into cellular cholesterol was determined by scintillation counting of the scraped cholesterol spot by using OptiScint Hisafe (LKB).

Determination of FC and EC Contents
VSMCs were seeded into six-well plates at 100 cells/mm², maintained for 48 hours, arrested for 24 hours with 0.2% FCS, and maintained for an additional 24 hours with or without PDGF-BB (10^-9 mol/L). Cells were then incubated overnight with nLDL or agLDL in the absence or presence of the different compounds tested. At the end of this period, cells were exhaustively washed, twice with PBS, twice with PBS–1% BSA, and twice with PBS–1% BSA–heparin 100 U/mL before they were harvested into 1 mL of 0.15 mol/L NaOH. Lipid extraction and TLC were performed as explained above. The spots corresponding to FC and EC were quantified by densitometry against the standard curve of cholesterol and cholesterol palmitate, respectively, by using a computing densitometer (Molecular Dynamics).

Measurement of Cellular DNA
DNA determination was done according to the method of Switzer and Summer³⁶ with small modifications.³⁴ One aliquot of the alkaline cell homogenate was treated with 50% trichloroacetic acid and maintained at 4°C for 30 minutes. After centrifugation (3000g, 4°C, 10 minutes), the pellet was treated with 0.01 mol/L potassium acetate, and DNA was extracted by centrifugation (3000g, 10 minutes). DNA was measured fluorometrically after its reaction with diaminobenzoic acid and quantified against a standard curve of DNA by using a luminescence spectrometer (Perkin-Elmer LS 50B).

The results for cellular cholesterol content were normalized by DNA content. For human aortic VSMCs we obtained a DNA content of 6.5 µg DNA/10⁶ cells. In different experiments, a constant relation between cellular DNA and protein content was found (1:100).

Data Analysis
Data were expressed as mean±SEM. Results were analyzed by ANOVA. A Statview (Abacus Concepts) statistical package for the Macintosh computer system was used for all analyses. Multiple groups were compared by one-factor ANOVA. Statistical significance was considered when P<.05.

	Results

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Preparation and Properties of agLDL
The susceptibility of human LDL to undergo aggregation was assayed in a time- and concentration-dependent fashion. Both the turbidity (Fig 1A) and the percentage of protein (Fig 1B) recovered in the precipitate (aggregated form of LDL) increased with vortexing time. The percentage of LDL in aggregated form was also dependent on LDL protein concentration. The higher percentages of aggregation were obtained at the lowest protein concentrations (0.5 and 1 mg/mL). To best characterize the effect of agLDL uptake on EC accumulation, cells were incubated with identical concentrations (100 µg/mL) of total, pellet (precipitable agLDL), or supernatant (unprecipitable agLDL) LDL obtained from an LDL solution (1 mg/mL) vortexed for 4 minutes and centrifuged under the conditions described in "Methods." As shown in Fig 2, total, unprecipitable, and precipitable fractions of agLDL but not nLDL produced a high EC accumulation in VSMCs. The effect of the unprecipitable fraction was 1.5-fold higher than that of the total or precipitable fraction. In this work, experiments focused on studying the effect of agLDL uptake on EC accumulation were performed with total agLDL obtained from a 1 mg/mL protein solution vortexed for 4 minutes. In fact, agLDL are formed after only a few seconds of vortexing, but LDL aggregation is complete only after 4 minutes.³⁷ Since nLDL binds with high affinity to the plasma membrane, in fluorescence microscope experiments the precipitable fraction was tested to clearly distinguish the nLDL from the agLDL uptake pattern.

View larger version (17K): [in this window] [in a new window]   Figure 1. Formation of LDL aggregates by vortexing. Preparations of LDL at three different concentrations (0.5 [], 1 [], and 2 [] mg/mL) were vortexed for increasing times. The turbidity of the LDL solution was determined by measuring the absorbance at 680 nm (A). After vortexing, preparations were centrifuged at 10 000g, and protein content recovered in the unprecipitable and precipitable fractions was determined. Percentage of agLDL (precipitable form) was calculated by dividing the content of protein recovered in the pellet by total protein (B). Results are mean±SEM of three determinations.

View larger version (30K): [in this window] [in a new window]   Figure 2. Effect of nonprecipitable and precipitable fractions of agLDL on cholesterol content of VSMCs. LDL solution (1 mg protein per mL) was vortexed for 4 minutes and centrifuged as described in "Methods." Cells were incubated with LDL (100 µg protein per mL) from the nonprecipitable fraction (supernatant), precipitable fraction (pellet), total agLDL, and nLDL. A, TLC showing the bands corresponding to FC and EC of cells incubated with different preparations of LDL. B, Bar graph showing quantification of EC (filled bars) and FC (open bars). Results are expressed as µg cholesterol per µg DNA and are shown as mean±SEM of three independent samples.

Effect of agLDL on VSMC Cholesterol Content: Comparison Between Unstimulated and PDGF-Stimulated VSMCs
To study the ability of nLDL and agLDL to induce accumulation of cholesterol in human VSMCs, cells were incubated in parallel with increasing concentrations of these lipoproteins. Since the EC content in control VSMCs (in the absence of LDL) was negligible and LDL (n or ag) reduced endogenous cholesterol synthesis to undetectable levels (data not shown), the increase in EC content observed in VSMCs reflects the cholesterol that enters through LDL. The FC content of VSMCs remained unaltered by the presence of nLDL or agLDL at any concentration. On the contrary, whereas the EC content of VSMCs incubated with nLDL increased only slightly (from undetectable levels to 0.15±0.02 µg EC/µg DNA), the EC content increased proportionally with the concentration of agLDL (from undetectable levels to 14.5±0.5 µg EC/µg DNA at 200 µg/mL) (Fig 3). To investigate the relation between the degree of LDL aggregation and EC accumulation, unstimulated and PDGF-stimulated VSMCs were incubated with LDLs that had a progressively higher content of aggregates, measured as described in "Methods." These LDL preparations increased the EC content of VSMCs in proportion to their degree of aggregation. As shown in Fig 4, the EC content of VSMCs incubated with 100 µg/mL of agLDL (25% of agLDL) increased about 20-fold compared with VSMCs incubated with the same concentration of nLDL. As deduced from Fig 3, VSMCs incubated with 100 µg/mL of agLDL (80% of agLDL) increased the EC content by 70-fold. Stimulation of VSMCs with PDGF-BB induced a slight increase in EC content when the percentage of agLDL was <5%. This result could indicate that the EC that derives from agLDL uptake, in contrast to EC that derives from nLDL uptake, was not influenced by PDGF. PDGF increased endogenous cholesterol synthesis only in the absence of lipoproteins (approximately twofold).

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of increasing concentrations of agLDL and nLDL on EC content of VSMCs. VSMCs were incubated with increasing concentrations (50, 100, and 200 µg/mL) of agLDL (circles) and nLDL (squares) for 24 hours. After this period, VSMCs were exhaustively washed and harvested for measurement of EC (filled symbols) and FC (open symbols). Results are expressed as µg cholesterol per µg DNA and are shown as mean±SEM of three independent samples.

View larger version (43K): [in this window] [in a new window]   Figure 4. Effect of degree of LDL aggregation on EC content of VSMCs. VSMCs were incubated with LDL (100 µg protein per mL), with increasing percentage of aggregates, for 24 hours in the absence or presence of PDGF-BB (10-9 mol/L). After this period, VSMCs were exhaustively washed and harvested for measurement of EC and FC. A, TLC showing the effect of increasing percentage of agLDL on FC and EC in unstimulated VSMCs. B, Line graph showing quantification of EC bands in unstimulated () and PDGF-stimulated () VSMCs. Results are expressed as µg cholesterol per µg DNA and are shown as mean±SEM of three independent samples.

Internalization Pattern of nLDL and agLDL by VSMCs
Fluorescence microscopy experiments were carried out to visualize the pattern of internalization of both agLDL and nLDL by VSMCs. To obtain a fraction of 100% agLDL, the LDL solution was centrifuged at 10 000g for 10 minutes after vortexing for 4 minutes, and the pellet was recovered. VSMCs were incubated at 4°C for 30 minutes with either nLDL or 100% agLDL. After removal of unbound DiI-LDL by extensive washing, the DiI-LDL internalized during the 4-hour incubation was observed under fluorescence microscopy. Endocytosed nLDLs were found in bright vesicles that were homogeneously distributed in the perinuclear space leading to an unstained cytoplasm surrounding the fluorescent vesicles. In contrast, agLDLs were found in bigger and more diffuse vesicles (Fig 5) distributed throughout the cytoplasm.

View larger version (68K): [in this window] [in a new window]   Figure 5. Uptake of nLDL and agLDL labeled with DiI in VSMCs. Human VSMCs were incubated with DiI-nLDL and DiI-agLDL (100% aggregated; 50 µg protein per mL) for 30 minutes at 4°C. They were then washed and incubated at 37°C for 4 hours. Cells were washed, fixed, and photographed (magnification x2500).

Confocal laser scanning microscopy was performed to demonstrate that agLDL was not associated with the plasma membrane but was inside the cytoplasm. The majority of labeled LDL was located in the internal optical sections of the cell, showing that agLDL had been clearly internalized by VSMCs (Fig 6).

View larger version (111K): [in this window] [in a new window]   Figure 6. Confocal microscopy of VSMCs incubated with LDL. Human VSMCs incubated with DiI-agLDL (100% aggregated, 50 µg/mL) were analyzed by confocal microscopy on an invert laser scan microscope. Photo shows several of 16 consecutive images obtained by optical sectioning of one cell: A, section 4; B, section 5; C, section 10; D, section 11; E, section 14; and F, section 15.

Effect of HMG-CoA Reductase Inhibition on Cholesterol Accumulation Induced by agLDL Uptake
To investigate the effect of HMG-CoA reductase inhibitors on cholesterol accumulation induced by agLDL uptake, unstimulated and PDGF-stimulated VSMCs were incubated in parallel with 100 µg/mL of agLDL (>80% aggregated) and simultaneously with increasing concentrations of simvastatin (2.5, 5, and 10 µmol/L) or pravastatin (250, 500, and 1000 µmol/L) for 24 hours. The Table shows that simvastatin decreased EC accumulation derived from agLDL in unstimulated VSMCs in a dose-dependent manner. A significant inhibitory effect was observed at the lowest simvastatin concentration (2.5 µmol/L). The inhibitory effect of simvastatin was higher in PDGF-stimulated VSMCs at any concentration; the maximal inhibitory effect (58%) was observed in PDGF-stimulated VSMCs treated with 5 µmol/L simvastatin. Similar results were obtained with pravastatin, but the concentration required to observe 50% inhibition was much higher (1 mmol/L) owing to its higher hydrophilicity (data not shown). To investigate whether the decrease in cholesterol accumulation was due to a lack of agLDL endocytosis, we studied the effect of simvastatin on DiI-agLDL uptake (100% aggregated). As shown in Fig 7, 4 hours of simvastatin treatment (10 µmol/L) almost completely inhibited endocytosis of the DiI-agLDL previously bound to the plasma membrane during 30 minutes of preincubation at 4°C.

View larger version (128K): [in this window] [in a new window]   Figure 7. Internalization of DiI-agLDL in simvastatin-treated VSMCs. VSMCs were incubated with DiI-agLDL (100% aggregated, 50 µg/mL) for 30 minutes at 4°C. They were washed and then incubated at 37°C for 4 hours with normal medium (A), medium with simvastatin (10 µmol/L) (B), medium with simvastatin (10 µmol/L) plus farnesol (10 µmol/L) (C), and medium with simvastatin (10 µmol/L) plus geranylgeraniol (10 µmol/L) (D). VSMCs were washed, fixed, and photographed (magnification x500).

Reversal of Simvastatin Inhibitory Effect by Isoprenyl Groups
To characterize the main prenyl groups implicated in the effect of simvastatin on agLDL uptake by VSMCs, we added either farnesol or geranylgeraniol during the simvastatin treatment. As shown in Fig 7, whereas farnesol (10 µmol/L) only slightly prevented the effect of simvastatin (10 µmol/L) on DiI-agLDL endocytosis, geranylgeraniol (10 µmol/L) completely prevented this effect. As shown in Fig 8, quantification of the spots obtained by TLC indicates that geranylgeraniol prevented the inhibition of simvastatin on EC accumulation, whereas farnesol only displayed a slight effect. Increasing the dose of farnesol did not improve its effect.

View larger version (33K): [in this window] [in a new window]   Figure 8. Reversal by farnesol (Far) and geranylgeraniol (Ger) of simvastatin (Sim) effects on EC accumulation derived from agLDL. VSMCs were incubated with agLDL (100 µg protein per mL, 100% aggregated) in the absence or presence of simvastatin and in the absence or presence of farnesol or geranylgeraniol for 24 hours. At the end of this period, cells were harvested and lipid extract was analyzed by TLC. A, TLC showing the effect of increasing concentrations of simvastatin (2.5, 5, and 10 µmol/L); simvastatin (10 µmol/L) plus farnesol (10 µmol/L); and simvastatin (10 µmol/L) plus geranylgeraniol (10 µmol/L). B, Bar graph showing quantification of EC (filled bars) and FC (open bars) bands of simvastatin (10 µmol/L) -treated VSMCs and simvastatin plus farnesol or geranylgeraniol–treated VSMCs. Results are expressed as µg cholesterol per µg DNA and are shown as mean±SEM of three samples.

	Discussion

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Aggregates of LDL formed during vortexing in vitro have been demonstrated to occur in vivo, and the physical changes in lipoprotein structure are similar to those described in atherosclerosis lesions.^{37 38 39} On the other hand, chemically modified LDLs, such as desialylated or glycosylated LDL, which produce EC accumulation in VSMCs, share a high degree of aggregation.^{15 16} Thus, we studied whether LDL aggregation by itself would lead to EC accumulation, the hallmark of foam cell formation, in VSMCs. Our results clearly show that agLDLs produce EC accumulation in human aortic VSMCs, depending on both the degree of aggregation and the concentration. In addition, not only large (precipitable by centrifugation) but also smaller (unprecipitable) aggregates caused significant cholesterol accumulation in these cells. The rapid internalization of agLDL by VSMCs could be relevant in vivo, owing to the high capacity of these cells to retain LDL and to modify these lipoproteins by "anchored" enzymes (lipoprotein lipases) that facilitate their aggregation.^{12 38 39} PDGF stimulates LDL receptor expression in different cell types, including VSMCs.⁴⁰ However, EC accumulation from agLDL was not affected by PDGF, suggesting an nLDL receptor–independent uptake of these modified lipoproteins. In addition, fluorescence microscopy provided evidence that agLDLs were internalized in diffuse and large vesicles, which were clearly different from the smaller, well-defined vesicles involved in nLDL uptake. This result suggests a different pathway for the internalization of LDL aggregates. It has recently been demonstrated that the internalization of LDL modified by lipoprotein lipase is independent of the LDL receptor.³²

Simvastatin inhibits HMG-CoA reductase activity and blocks the synthesis of cholesterol and other mevalonate derivatives. To investigate the effect of HMG-CoA reductase inhibition on VSMC–foam cell formation, the intracellular lipid accumulation induced by agLDL was studied in simvastatin- and pravastatin-treated VSMCs. HMG-CoA reductase inhibitors significantly reduced EC accumulation; in contrast, FC levels were unaltered. It seems that in VSMCs, similar to other cell types, the FC content is strictly controlled.³⁴ Simvastatin's effect was higher in PDGF-stimulated VSMCs than in unstimulated VSMCs. This differential effect could be explained by pathways "competing" for a limited pool of mevalonate. The affinity of prenyltransferase by polyprenylpyrophosphate is higher than that of squalene synthase.⁴¹ However, in cells stimulated by growth factors, cholesterol synthesis increases several-fold⁴⁰ and under these conditions, the synthesis of isoprenylated products, which are most likely related to agLDL uptake, could be more affected by HMG-CoA reductase inhibition.

The results obtained with DiI-agLDL performed to demonstrate agLDL internalization suggest that simvastatin inhibits agLDL-derived EC accumulation by preventing its endocytosis. The discovery that many G proteins are modified by isoprenoid lipids has added new light to our understanding of this process. It has been estimated that about 50 polypeptides in cultured mammalian cells are posttranslationally modified by isoprenoids.⁴² Some of these are GTP-binding proteins that regulate a wide variety of cellular processes, including endocytosis. Two different isoprenyl groups, farnesyl and geranylgeranyl, have been found to modify these proteins covalently. In our study, geranylgeraniol prevented simvastatin inhibition of agLDL uptake in the absence of other prenyl intermediates, suggesting that proteins modified by this isoprene have a key role in regulating VSMC EC accumulation. In fact, the prenylated proteins involved in the regulation of vesicle targeting and fusion during intracellular trafficking appear to be modified exclusively with the geranylgeranyl isoprenoid.^{43 44 45}

Our results indicate that VSMCs rapidly internalize agLDLs that accumulate EC and that HMG-CoA reductase inhibitors block the entry of agLDL and hence lipid accumulation. VSMCs are a major cellular component in developing atherosclerotic plaques, and lipid accumulation is one of the main processes that leads to the progression of small lesions to advanced plaque. The inhibitory effect of statins on EC accumulation could contribute to the stabilization of lipid-rich, growing atherosclerotic plaques. By interfering in the process of foam cell formation and reducing the focal inflammatory and thrombotic risk associated with the small lipid plaques,² this vascular effect of statins would be a mechanism to partially explain the beneficial effects observed in several randomized trials.^{20 21 22}

	Selected Abbreviations and Acronyms

 

ag = aggregated DiI = 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine EC = esterified cholesterol FC = free cholesterol HMG-CoA = 3-hydroxy-3-methylglutaryl coenzyme A n = native PDGF = platelet-derived growth factor PG = proteoglycan TLC = thin-layer chromatography VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was partly funded by PNSAF 94/712, FIS 95/0917, BMS/CDTI 96–0035, and Fundacion Investigacion Cardiovascular Catalana-Occidente (all grants awarded to L.B.). We thank the heart transplant team of the Division of Cardiology and Cardiac Surgery and the blood bank of Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. We thank Maria Berrozpe, Francisco Vidal, and Anna Bosch for their help in microscopy and image analysis.

Received August 12, 1997; accepted December 3, 1997.

	References

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1. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes (part I). N Engl J Med. 1992;326:242–250.[Medline] [Order article via Infotrieve]

2. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and acute coronary syndromes (part II). N Engl J Med. 1992;326:310–318.[Medline] [Order article via Infotrieve]

3. Steinberg D, Parthasarathy S, Carew T, Khoo J, Witztum J. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915–924.[Medline] [Order article via Infotrieve]

4. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983;52:223–261.[Medline] [Order article via Infotrieve]

5. Goldstein JL, Ho YK, Basu SK, Brown MS. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein producing massive cholesterol deposition. Proc Natl Acad Sci U S A. 1979;76:333–337.[Abstract/Free Full Text]

6. Camejo G, Hurt-Camejo E, Rosengren B, Wiklund O, L-Pez F, Bondjers G. Modification of copper-catalyzed oxidation of low density lipoprotein by proteoglycans and glycosaminoglycans. J Lipid Res. 1991;32:1983–1991.[Abstract]

7. Vijayagopal P, Srinivasan SR, Radhakrishnamurthy B, Berenson GS. Factors regulating the metabolism of low density lipoprotein-proteoglycan complex in macrophages. Biochim Biophys Acta. 1990;1042:204–209.[Medline] [Order article via Infotrieve]

8. Khoo JC, Miller E, McLoughlin P, Steinberg D. Enhanced macrophage uptake of low density lipoprotein after self-aggregation. Arteriosclerosis. 1988;8:348–358.[Abstract/Free Full Text]

9. Khoo JC; Miller E, Pio F, Steinberg D, Witztum JM. Monoclonal antibodies against LDL further enhance macrophage uptake of LDL aggregates. Arterioscler Thromb. 1992;12:1258–1266.[Abstract/Free Full Text]

10. Katsuda S, Boyd HC, Fligner C, Ross R, Gown AM. Human atherosclerosis, III: immunocytochemical analysis of the cell composition of lesions of young adults. Am J Pathol. 1992;140:907–914.[Abstract]

11. Wissler RW, Vesselinovitch D, Komatsu A. The contribution of studies of atherosclerotic lesions in young people to future research. Ann N Y Acad Sci. 1990;598:418–434.[Medline] [Order article via Infotrieve]

12. Tabas I, Yueqing L, Brocia RW, Wen Xu S, Swenson TL, Williams KJ. Lipoprotein lipase and sphingomyelinase synergistically enhance the association of atherogenic lipoproteins with smooth muscle cells and extracellular matrix. J Biol Chem. 1993;268:20419–20432.[Abstract/Free Full Text]

13. Camejo G, Gunnar F, Rosengren B, Hurt-Camejo E, Bondjers G. Binding of low density lipoproteins by proteoglycans synthesized by proliferating and quiescent human arterial smooth muscle cells. J Biol Chem. 1993;268:14131–14137.[Abstract/Free Full Text]

14. Nermine A, Ismail E, Zafar Alavi M, Moore S. Lipoprotein-proteoglycan complexes from injured rabbit aortas accelerate lipoprotein uptake by arterial smooth muscle cells. Atherosclerosis. 1993;105:79–87.

15. Tertov VV, Orekhov AN, Sobenin ZA, Gabbasov, Popov EG, Yaroslavov AA, Smirnov VN. Three types of naturally occurring modified lipoproteins induce intracellular lipid accumulation due to lipoprotein aggregation. Circ Res. 1992;71:218–228.[Abstract/Free Full Text]

16. Tertov VV, Sobenin IA, Gabbasov ZA, Popov EG, Orekhov AN. Lipoprotein aggregation as an essential condition of intracellular lipid accumulation caused by modified low density lipoproteins. Biochem Biophys Res Commun. 1989;163:489–494.[Medline] [Order article via Infotrieve]

17. Inaba T, Gotoda T, Shimano H, Shimada M, Harada K, Kozaki K, Watanabe Y, Hoh E, Motoyoshi K, Yazaki Y, Yamada N. Platelet-derived growth factor induces c-fms and scavenger receptor genes in vascular smooth muscle cells. J Biol Chem. 1992;267:13107–13112.[Abstract/Free Full Text]

18. Li H, Freeman MW, Libby P. Regulation of smooth muscle cell scavenger receptor expression in vivo by atherogenic diets and in vitro by cytokines. J Clin Invest. 1995;95:122–133.

19. Goldstein JL, Brown MS. Regulation of mevalonate pathway. Nature. 1990;343:425–430.[Medline] [Order article via Infotrieve]

20. Scandinavian Simvastatin Survival Study Group. Randomized 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]

21. MAAS Investigators. Effect of simvastatin on coronary atheroma: the multicentre anti-atheroma study (MAAS). Lancet. 1994;344:633–638.[Medline] [Order article via Infotrieve]

22. Shepherd J, Cobbe SM, Ford I, Isles CG, Lorimer AR, MacFarlane PW, McKillop JH, Packard CJ. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia: West of Scotland Coronary Prevention Study Group. N Engl J Med. 1995;33:1301–1307.

23. Corsini A, Mazzoti M, Raiteri M, Soma MR, Gabbiani G, Fumagalli R, Paoletti R. Relationship between mevalonate pathway and arterial myocyte proliferation: in vitro studies with inhibitors of HMG-CoA reductase. Atherosclerosis. 1993;101:117–125.[Medline] [Order article via Infotrieve]

24. Martínez-González J, Badimon L. Human and porcine smooth muscle cells share similar proliferation dependence on the mevalonate pathway: implication for in vivo interventions in the porcine model. Eur J Clin Invest. 1996;26:1023–1032.[Medline] [Order article via Infotrieve]

25. Martínez-González J, Viñals M, Vidal F, Llorente-Cortés V, Badimon L. Mevalonate deprivation impairs IGF-I/insulin signaling in human vascular smooth muscle cells. Atherosclerosis.. 1997;135:213–223.[Medline] [Order article via Infotrieve]

26. Bucci C, Parton RG, Mather IH, Stunnenberg H, Simons K, Hoflack B, Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992;70:715–728.[Medline] [Order article via Infotrieve]

27. Castellano F, Wilson A, Maltese WA. Intracellular transport and maturation of nascent low density lipoprotein receptor is blocked by mutation in the ras-related GTP-binding protein, RAB1B. J Recept Signal Transduct Res. 1995;15:847–862.[Medline] [Order article via Infotrieve]

28. Havel R, Eder H, Bragdon J. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest. 1955;34:1345–1354.

29. Smith PK, Krohn RI, Ermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klent DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985;150:76–85.[Medline] [Order article via Infotrieve]

30. Beisiegel U, Weber W, Ihrke G, Herz J, Stanley KK. The LDL receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature. 1989;341:162–164.[Medline] [Order article via Infotrieve]

31. Lougheed M, Steinbrecher UP. Mechanism of uptake of copper oxidized low density lipoprotein in macrophages is dependent on its extent of oxidation. J Biol Chem. 1996;271:11798–11805.[Abstract/Free Full Text]

32. Fernández-Borja M, Bellido D, Vilella E, Olivecrona G, Vilar- S. Lipoprotein lipase-mediated uptake of lipoprotein in human fibroblasts: evidence for an LDL receptor-independent internalization pathway. J Lipid Res. 1996;37:464–481.[Abstract]

33. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–916.

34. Asmis R, Llorente-Cortés V, Gey F. Prevention of cholesteryl ester accumulation in P388D₁ macrophage-like cells by increased cellular vitamin E depends on species of extracellular cholesterol. Eur J Biochem. 1995;233:171–178.[Medline] [Order article via Infotrieve]

35. Huber LA, Xu Q B, Jurgens G, Bock G, Buhler E, Gey KF, Schonitzer D, Tralill K, Wick G. Correlation of lymphocyte lipid composition membrane viscosity and mitogen response in the aged. Eur J Immunol. 1991;21:2761–2765.[Medline] [Order article via Infotrieve]

36. Switzer BR, Summer GK. A modified fluorometric method for DNA. Clin Chem Acta. 1971;32:203–206.[Medline] [Order article via Infotrieve]

37. Guyton JR, Klemp KF, Mims MP. Altered ultra-structural morphology of self-aggregated low density lipoproteins; coalescence of lipid domain forming droplets and vesicles. J Lipid Res. 1991;32:953–962.[Abstract]

38. Hoff HF, O'Neil J. Lesion-derived LDL and oxidized LDL share a lability for aggregation, leading to enhanced macrophage degradation. Arterioscler Thromb. 1991;11:1209–1222.[Abstract/Free Full Text]

39. Aviram M, Maor I, Keidar S, Hayek T, Oiknine J, Bar EI Y, Adler Z, Kertzman V, Milo S. Lesioned low density lipoprotein in atherosclerotic apolipoprotein E-deficient transgenic mice and in humans is oxidized and aggregated. Biochem Biophys Res Commun. 1995;216:501–513.[Medline] [Order article via Infotrieve]

40. Roth M, Emmos LR, Perruchoud A, Block LH. Expression of the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl coenzyme A reductase genes are stimulated by recombinant platelet-derived growth factor isomers. Proc Natl Acad Sci U S A. 1991;88:1888–1892.[Abstract/Free Full Text]

41. Sinensky M, Beck L, Leonard S, Evans R. Differential inhibitory effects of lovastatin on protein isoprenylation and sterol synthesis. J Biol Chem. 1990;265:19937–19941.[Abstract/Free Full Text]

42. Leonard S, Beck L, Sinensky M. Inhibition of isoprenoid biosynthesis and the post-translational modification of pro-p21^ras. J Biol Chem. 1990;265:5157–5160.[Abstract/Free Full Text]

43. Casey PJ, Mooman JF, Zhang FL, Higgins JB, Thissen JA. Prenylation, and G protein signalling. Recent Prog Horm Res. 1994;49:215–239.

44. Kempen HJM, Vermeer M, de Wit E, Havekes LM. Vastatins inhibit cholesterol ester accumulation in human monocyte-derived macrophages. Arterioscler Thromb. 1991;11:146–153.[Abstract/Free Full Text]

45. Bernini F, Scurati N, Bonfadine G, Fumagalli R. HMG-CoA reductase inhibitors reduce acetyl LDL endocytosis in mouse peritoneal macrophages. Arterioscler Thromb Vasc Biol. 1995;15:1352–1358.[Abstract/Free Full Text]

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