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Angiotensin II Reduces Macrophage Cholesterol Efflux: A Role for the AT-1 Receptor but Not for the ABC1 Transporter
Biochemical and Biophysical Research Communications 290, 1529 –1534 (2002) doi:10.1006/bbrc.2002.6376, available online at http://www.idealibrary.com on
Angiotensin II Reduces Macrophage Cholesterol Efflux: A Role for the AT-1 Receptor but Not for the ABC1 Transporter Marielle Kaplan,* Michael Aviram,* Carlos Knopf,† and Shlomo Keidar* ,1 *Lipid Research Laboratory, Bruce Rappaport Faculty of Medicine, Technion, Rappaport Family Institute for Research in Medical Sciences and Rambam Medical Center, Haifa, Israel; and †Section of Metabolic Diseases, Department of Clinical Biochemistry, Rambam Medical Center, Haifa, Israel
Received January 2, 2002
Impaired cellular cholesterol efflux in cells of the arterial wall is suggested to be involved in the pathogenesis of atherosclerosis. Since angiotensin II (AngII) is implicated in the development of atherosclerosis, the aim of the present study was to determine whether Ang-II could affect macrophage cholesterol efflux. Incubation of increasing concentrations of Ang-II (10 ⴚ10– 10 ⴚ7 M) with mouse peritoneal macrophages that were prelabeled with [ 3H]cholesterol led to a significant decrease in HDL-induced macrophage cholesterol efflux, by up to 70% compared to control cells incubated without Ang-II. Ang-II specifically increased the plasma membrane unesterified cholesterol content, the substrate for HDL-induced cholesterol efflux. The inhibitory effect of Ang-II on macrophage cholesterol efflux was found to be mediated by the angiotensin II type 1 (AT-1) receptor, since addition of the AT-1 antagonist Losartan completely blocked the inhibitory effect of Ang-II on the macrophage cholesterol efflux. We thus conclude that Ang-II atherogenicity may be related, at least in part, to its inhibitory effect on macrophage cholesterol efflux, thus leading to cellular cholesterol accumulation, the hallmark of early atherogenesis. © 2002 Elsevier Science (USA)
Key Words: macrophages; cholesterol efflux; angiotensin II; Losartan.
Foam cell formation is the result of cholesterol accumulation in arterial macrophages and it is a prominent finding in atherosclerotic plaques (1–3). Cellular cholesterol accumulation depends on the balance between cholesterol influx resulting from the uptake of lipoproteins such as oxidized LDL (Ox-LDL), and cholesterol efflux (4 –7). Cholesterol efflux from cells by extracel1 To whom correspondence and reprint requests should be addressed at Rambam Medical Center, Haifa 31096 Israel. Fax: 9724-854-2721. E-mail: [email protected].
lular acceptors such as high-density lipoprotein (HDL) is the first step in reverse cholesterol transport. This process is responsible for the transport of cellular cholesterol from the periphery to the liver for excretion (8 –12). Thus, understanding of the cellular events that contribute to cholesterol efflux is vital for elucidating the mechanisms of cholesterol accumulation in foam cells during the development of atherosclerotic lesions. Cellular cholesterol, subject to efflux, must be in its unesterified form and should be present in the cell membrane. Efflux of unesterified cholesterol (UC) from plasma membranes into the extracellular spaces is therefore determined by the lipids composition of cell membranes and by the acceptor such as the HDL particle (13). Cholesterol ester (CE) droplets are formed by esterification of UC by the acyl-coenzyme cholesterol acyltransferase (ACAT) and give lipid-laden macrophages their foamy appearance. Cytosolic CE can be hydrolyzed by neutral cholesteryl ester hydrolase (NCEH) and the UC formed can then be either transferred to the cell membrane or reesterified by ACAT (7, 13). Recently, the ATP binding cassette transporter 1 (ABC1) has been shown to play an important role in lipid efflux (14). This transporter is formed of two cytoplasmic domains as well as two transmembranal domains. This allows it to act as a channel and substrates for ABC1 include cholesterol phospholipids vitamins and interleukins. It has been suggested that ABC1 allows transport of cholesterol from Golgi structures to the cellular plasma membrane. Angiotensin II (Ang-II), a vasoconstrictor produced by the renin–angiotensin system, is implicated in atherosclerosis (15, 16), and has been identified also in atherosclerotic lesion’s macrophages (17). Angiotensin receptor type 1 (AT-1) is distributed in almost all the tissues, and was shown to mediate most of Ang-II proatherogenic effects (18). Ang-II injection to the atherosclerotic apolipoprotein E deficient (E 0) mice signif-
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icantly increases the atherosclerotic lesion area (19), and this is associated with its stimulatory effects on macrophage-mediated oxidation of LDL (20), macrophage cholesterol biosynthesis (19), as well as Ox-LDL macrophage uptake (21), leading to macrophage cholesterol accumulation and foam cell formation. The goal of the present study was to analyze whether Ang-II could affect macrophage cholesterol accumulation, secondary to its effect on cellular cholesterol efflux and to elucidate possible mechanisms for such effects. METHODS Mouse peritoneal macrophage isolation and cell fractionation. Mouse peritoneal macrophages (MPM) were harvested from the peritoneal fluid of C57Bl mice 4 days after intraperitoneal injection into each mouse of 3 ml of thioglycolate (24 g/L) in saline. The cells (10 –20 ⫻ 10 6/mouse) were washed and centrifuged three times with phosphate-buffered saline (PBS) at 1000g for 10 min, then resuspended to 10 9/L in Dulbecco’s modified Eagle medium (DMEM) containing 10% horse serum (heat-inactivated at 56°C for 30 min) and 100 U penicillin/ml, 100 g streptomycin/ml, and 2 mM glutamine (P/S/G). The dishes were incubated in a humidified incubator (5% CO 2, 95% air) for 2 h, washed with DMEM to remove nonadherent cells, and the monolayer was incubated under similar conditions for 18 h. Membrane and cytosol fractions were prepared as described previously (22). Cells were suspended at 10 8 cells/ml in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl 2, 1.25 mM EGTA, 1 mM ATP, 10 mM Hepes, pH 7.4). containing 1 mM phenylmethanesulfonyl fluoride and 100 M leupeptin at 4°C, following sonication for 3 ⫻ 10 s, resulting in about 95% of cell breakage. Nuclei, granules, and unbroken cells were removed by centrifugation (2 min, 15,600g), and the postnuclear supernatant was made in 5 mM EDTA, 1 mM Na 3VO 4, and 5 mM NaF. The supernatant was centrifuged in a Beckman airfuge (30 min 134,000g) to obtain a cell membrane pellet and a cytosolic supernatant. Membranes were suspended at 10 9 cell equivalents/ml in 0.34 M sucrose/half strength relaxation buffer containing 1 mM dithiothreitol. Solubilized membrane and cytosolic fractions were stored at ⫺70°C. Lipoproteins preparation. High density lipoprotein (HDL) were prepared from human plasma (drawn into 1 mM Na 2 EDTA) from fasted normolipidemic volunteers. HDL (d ⫽ 1.064–1.21 g/ml) was prepared by discontinuous density gradient ultracentrifugation (23). The lipoproteins were then dialyzed against 150 mM NaCl, 1 mM Na 2 EDTA, pH 7.4. LDL was sterilized by filtration and was used within 2 weeks. The protein content of the lipoproteins was determined with the folin phenol reagent (24).
of them was directly silylated in order to measure free cholesterol, and the other was saponified to measure total cholesterol (26). The amount of esterified cholesterol was calculated as the difference between the total and free fractions. To measure total cholesterol, 2 ml 20% KOH in methanol (w/v) was added to the 2 ml ether extract, and saponified for 3 h at room temperature in a multivortex in pulse mode. Two milliliters of 25% citric acid in water were added for neutralize, and the organic phase was collected. The aqueous layer was washed twice with 2-ml portions of diethyl ether, and the three organic layers were combined and evaporated under nitrogen. The residue was redissolved in 1.5 ml hexane and Na 2SO 4 anhydrous (⫾50 mg) was added in order to remove traces of water. This extract was transferred to a 2-ml vial, and evaporated to dryness under nitrogen at 55°C. Na 2SO 4 was added to the other sample (for free cholesterol) and evaporated to dryness under nitrogen. Both samples were silylated with 40 l of BSTFA ⫹ TMCS, 99:1 (Supelco Inc., Supelco Park, Bellefonte, PA) and 40 l of dried pyridine at 60°C for 30 min. Sample analysis was performed by capillary GC–MS in selected ion monitoring (SIM) mode, in a Hewlett–Packard 6890/5972A instrument. A 30-m ⫻ 0.25-mm-i.d. 5% phenylmethyl siloxane column (0.25 m) (HP-5 MS, Cat No. 19091S-433) was used. One microliter in splitless mode was injected. Helium was used as the carrier gas at a flow rate of 1 ml/min and a linear velocity of 33 cm/s. The detector temperature was 180°C, and the injector temperature was 300°C. The oven temperature was programmed, starting at 200°C for 3 min, increased to 250°C at 25°C/min and then to 290°C at 10°C/min and maintained there for 4.5 min. The sensitivity of the mass detector was increased by means of manual tuning. An additional increment of the sensitivity was achieved using the exact mass (one decimal) of each ion, previously evaluated analyzing the spectra in scan mode. The amounts of cholesterol were calculated from a calibration curve, with a linearity of 1.00. mRNA expression of ABC1 (by RT-PCR analysis). Total RNA from macrophages was extracted with TRI-reagent (Molecular Research Center, Inc.). cDNA was generated from 1 g of total RNA using reverse transcriptase (RT) (Boehringer Mannheim, Germany) and oligo dT primers (Boehringer Mannheim, Germany). The RT reaction was carried out at 42°C for 50 min and at 99°C for 5 min. Products of the RT reaction were subjected to polymerase chain reaction (PCR) amplification. The forward primer used for ABC1 was 5⬘- ATGGTGGGAATGGGTCAGAAG-3⬘ and the reverse primer was 5⬘-CACGCAGCTCATTGTAGAAGG-3⬘ (27). The amplification conditions involved were denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C at 1.5 min. Similar conditions were used to amplify the housekeeping gene GAPDH using the forward primer 5⬘-CTG-CCA-TT-GCA-GTG-GCA-AAG-TGG-3⬘ and the reverse primer 5⬘-TTG-TCA-TGG-ATG-ACC-TTG-GCC-AGG-3⬘. Linearity of amplification was confirmed up to 32 cycles from 2 g of total RNA in the reverse transcription step. Specific PCR products obtained for ABC1 and GAPDH were separated on 6% polyacrylamide gel. Statistics. Student’s t test was performed for all statistical analyses. Results are given as means ⫾ SD.
Cholesterol efflux determination. Mouse peritoneal macrophages were incubated with 3H-labeled cholesterol for 18 h at 37°C followed by cell wash in ice-cold PBS (⫻3) and further incubation in the absence or presence of 100 g of HDL protein/ml for 3 h at 37°C. Cellular and medium [ 3H]-labels were quantitated and HDLmediated cholesterol efflux was calculated as the ratio of [ 3H]-label in the medium/[ 3H]-label in the medium ⫹ [ 3H]-label in cells (25).
RESULTS
Cholesterol mass determination [by gas chromatography–mass spectrometry (GC–MS)]. Macrophage cholesterol mass was determined after cell incubation in the absence or presence of Ang-II for 18 h at 37°C. Cellular lipids were extracted with hexane:isopropanol (3:2, v/v) and the solvents were evaporated. The dried extracts we were redissolved in 4 ml of diethyl ether and divided in two equal parts. Fifty microliters of ␣-cholestane (20 g/ml, Sigma Chemical Co., St. Louis, MO) was added to each one as internal standard. One
Macrophage cholesterol efflux was determined in mouse peritoneal macrophages following their radiolabeling with [ 3H]cholesterol. Incubation of [ 3H]cholesterol-radiolabeled mouse peritoneal macrophage with increasing concentrations of Ang-II (10 ⫺10–10 ⫺7 M) for 18 h at 37°C, led to a significant decrease, by up to 70%
Effect of Angiotensin II on Macrophage Cholesterol Efflux
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binding to its AT-1 receptor, we used the AT-1 specific receptor antagonist Losartan. Preincubation of the 3Hcholesterol radiolabeled macrophages with Losartan at increasing concentrations (10 ⫺7–10 ⫺5 M) prior to Ang-II addition (10 ⫺7 M), dose dependently reduced the inhibiting effect of Ang-II (Table 1). In the presence of Losartan (10 ⫺5 M), Ang-II completely failed to inhibit macrophage cholesterol efflux, suggesting that the Ang-II effect was mediated via its binding to the macrophage AT-1 receptor. Effect of Angiotensin II on Macrophage Cholesterol Distribution between Cytosol and the Plasmatic Membrane
FIG. 1. The effect of angiotensin II on macrophage cholesterol efflux. (A) Mouse peritoneal macrophages were radiolabeled with [ 3H]cholesterol for 18 h in the absence or presence of increasing concentrations of angiotensin II (10 ⫺10–10 ⫺7 M) prior to washes and addition of HDL. Then the HDL-mediated macrophage cholesterol efflux was determined as described under Methods. (B) Mouse peritoneal macrophages were radiolabeled with [ 3H]cholesterol for increasing periods of time (1–18 h), in the absence or presence of angiotensin II (10 ⫺7 M) prior to washes and addition of HDL. Then the HDL-mediated macrophage cholesterol efflux was determined as described under Methods.
Since cholesterol efflux is determined by cellular cholesterol composition and distribution, we determined macrophage cholesterol (unesterified and esterified) distribution in Ang-II-treated cells. For this purpose, macrophages were incubated in the absence or presence of increasing concentrations of Ang-II and the content of unesterified cholesterol (UC) and that of cholesteryl ester (CE) were analyzed in the macrophage plasmatic membrane and in the cytosol. Incubation of mouse peritoneal macrophages with Ang-II led to a dose-dependent increase, by up to 35%, in the plasmatic membrane UC whereas the CE content of the membrane was not significantly affected (Fig. 2A). In parallel, incubation of the macrophages with Ang-II resulted in a significant increase, by up to 4-fold at Ang-II 10 ⫺7 M, in the cytosolic cholesterol ester content (Fig. 2B), whereas, cytosolic UC levels were not affected (Fig. 2B).
TABLE 1
The Effect of the AT-1 Antagonist Losartan on the Angiotensin II-Induced Inhibition on Macrophage Cholesterol Efflux Macrophage treatment
in HDL-induced cellular cholesterol efflux, in comparison to control cells (Fig. 1A). We next analyzed whether this inhibitory effect of Ang-II on macrophage cholesterol efflux is dependent on the time of macrophages radiolabeling with [ 3H]cholesterol. For this purpose, mouse peritoneal macrophages were first radiolabeled for increasing period of times (1–24 h) with [ 3H]cholesterol followed by the addition of angiotensin II (10 ⫺7 M) for 18 h. As seen in Fig. 1B, Ang-II significantly inhibited macrophage cholesterol efflux (by up to 41%) already after 4 h of radiolabeling of cells with [ 3H]cholesterol. To determine whether the Ang-II effect on macrophage cholesterol efflux was mediated via
Control ⫹Angiotensin II (10 ⫺7 ⫹Losartan (10 ⫺7 M) ⫹Angiotensin II (10 ⫺7 ⫹Losartan (10 ⫺6 M) ⫹Angiotensin II (10 ⫺7 ⫹Losartan (10 ⫺5 M) ⫹Angiotensin II (10 ⫺7
Macrophage cholesterol efflux (%)
M)
19.4 ⫾ 2.0 8.1 ⫾ 2.1*
M)
10.9 ⫾ 2.2*
M)
13.5 ⫾ 0.9*
M)
20.0 ⫾ 1.1
Note. Mouse peritoneal macrophages were radiolabeled with [ 3H]cholesterol for 18 h in the absence or presence of increasing concentrations of Losartan (10 ⫺7–10 ⫺5 M) prior to addition of angiotensin II at 10 ⫺7 M. Then the HDL-mediated macrophage cholesterol efflux was determined as described under Methods. Results are presented as means ⫾ SD. * P ⬍ 0.05 versus control.
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FIG. 2. The effect of angiotensin II on macrophage cholesterol composition. Mouse peritoneal macrophages were incubated in the absence or presence of increasing concentrations of angiotensin II (10 ⫺8–10 ⫺7 M) for 18 h, then the cells were subfractionated and their unesterified cholesterol (UC) as well as esterified cholesterol (CE) were analyzed by GC–MS in the macrophage plasmatic membrane (A) and cytosol (B).
cellular cholesterol efflux is considered as an important antiatherogenic function of this lipoprotein (28). In the present study, angiotensin II was shown to significantly reduce HDL-induced cellular cholesterol efflux from macrophages. This was associated with Ang-II-induced increment in unesterified cholesterol (UC) content in the macrophage plasma membranes, and in cholesteryl ester (CE) accumulation in the macrophage cytosol. Indeed, foam cells in the atherosclerotic lesions are characterized by increased cellular UC content and by the presence of elevated levels of cytosolic droplets of CE. Therefore, Ang-II by affecting cellular cholesterol trafficking and cholesterol efflux could led to the formation of macrophage-derived foam cells. The effect of Ang-II on the macrophage cholesterol efflux was dependent on the time of cells radiolabeling, and 1 h of labeling was not sufficient to obtain the maximal Ang-II induced inhibiting effect. This could point out that the effect of angiotensin II is linked to the cholesterol transport in the cells, rather than to a downregulation of receptors involved in HDL-mediated cholesterol efflux, such as the SRB-I or the ABC-1 HDL receptors. Indeed, in the present study angiotensin II was not able to downregulate the mRNA expression of the ABC1 transporter, the main efflux transporter system present in macrophages (29). If angiotensin II would have downregulated macrophage receptors, which are involved in cholesterol efflux, its effect should not have been dependent on the time of radiolabeling of the cells, since Ang-II was present 18 h, whether the macrophages were preradiolabeled for 1 or for 24 h. However, if the angiotensin II effect could be linked to the intracellular cholesterol transport and the time of the macrophage prelabeling was shown to be associated with cellular cholesterol efflux.
Effect of Angiotensin II on mRNA Expression of ABC1 We next analyzed whether the inhibitory effect of angiotensin II on the macrophage cholesterol efflux was mediated by its ability to downregulate the ABC1 transporter. For this purpose macrophages (J-774 A.1 cell line) were incubated with increasing concentrations of Ang-II (0, 10 ⫺8, 10 ⫺7 M) for 18 h at 37°C. Then, mRNA was extracted from the cells and subjected to RT-PCR using specific primers for ABC1. The mRNA expression of ABC1 in macrophages was not affected by their incubation with angiotensin II (Fig. 3). DISCUSSION Cellular cholesterol efflux is one of the essential events in maintaining cellular cholesterol content since most peripheral cells are unable to catabolize cholesterol. Low levels of plasma HDL were shown to be a major cardiovascular risk factor, as HDL-mediated
FIG. 3. Effect of angiotensin II on mRNA expression in macrophages. Mouse peritoneal macrophages were incubated in the absence or presence of increasing concentrations of angiotensin II (10 ⫺8–10 ⫺7 M) for 18 h, and then mRNA was extracted and subjected to RT-PCR for determination of mRNA of ABC1.
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The effect of angiotensin II on the macrophage cholesterol efflux was mediated by the specific angiotensin II receptor type 1 (AT-1) as shown by using the AT-1 antagonist receptor, Losartan. Some atherogenic effects of angiotensin II are mediated by the G-protein coupled receptors AT-1 and AT-2, and the AT-1 receptor mediates virtually all the known physiological actions of angiotensin II, including its pro-atherogenic effects (18, 19). Furthermore, AT-1 receptor has been identified in the cells of the arterial wall, including macrophages (18). Angiotensin-II was shown to stimulate macrophage foam cell formation and angiotensin converting enzyme inhibitors reduce atherosclerosis in animal models (15, 16). Ang-II can induce cholesterol accumulation in macrophages by several mechanisms including induction of cellular oxidative stress. Ang-II activates NAD(P)H-dependent oxidases, leading to increased lipid peroxidation in arterial macrophages and also increased macrophage-mediated oxidation of LDL (19 – 21). The ability of Ang-II to induce oxidative stress in macrophages could also be linked to its ability to inhibit cellular cholesterol efflux. Although, little is known about the regulation of macrophage cholesterol efflux, cellular oxidative stress and specifically oxysterols have been linked to reduced cellular cholesterol efflux (30 –33). 7-Ketocholesterol, which is a prominent product of cholesterol oxidation, accumulates in macrophages as a result of enhanced cellular uptake of Ox-LDL (34), and macrophage foam cells enriched in 7-ketocholesterol, exhibits reduced cellular cholesterol efflux. Furthermore, depletion of n-3 fatty acids, which occurs during oxidation, reduces the efficiency of cellular cholesterol efflux (32). It is also possible that products of fatty acid oxidation may have direct effects on the diffusional transfer or on receptor-mediated removal of cholesterol through changes in membrane fluidity. We conclude that Ang-II atherogenicity could be related to its prooxidative properties (20). Ang-II-induced oxidative stress not only increased macrophage cholesterol influx (uptake of LDL and oxidized LDL, cholesterol biosynthesis; 19, 21), but also could inhibit cellular cholesterol efflux and reverse cholesterol transport. REFERENCES 1. Ross, R. (1986) The pathogenesis of atherogenesis—An update. N. Engl. J. Med. 314, 498 –500. 2. Lusis, A. J. (2000) Atherosclerosis. Nature 407, 233–241. 3. Aviram, M. (1993) Modified forms of low density lipoprotein and atherosclerosis. Atherosclerosis 98, 1–9. 4. Aviram, M., and Fuhrman, B. (1998) LDL oxidation by arterial wall macrophages depends on the oxidative status in the lipoprotein and in the cells: Role of prooxidants vs antioxidants. Mol. Cell. Biochem. 188, 149 –159. 5. Steinberg, D. (1997) Low density lipoprotein oxidation and its pathobiological significance. J. Biol. Chem. 272, 20963–20966.
6. Aviram, M. (1996) Interaction of oxidized low density lipoprotein with macrophages in atherosclerosis, and the antiatherogenicity of antioxidants. Eur. J. Clin. Chem. Clin. Biochem. 34, 599 – 608. 7. Fielding, C. J., and Fielding, P. E. (2001) Cellular cholesterol efflux. Biochim. Biophys. Acta 1533, 175–189. 8. Navab, M., Van Lenten, B. J., Reddy, S. T., and Fogelman, A. M. (2001) High-density lipoprotein and the dynamics of atherosclerotic lesions. Circulation 104, 2386 –2387. 9. Shah, P. K., Kaul, S., Nilsson, J., and Cercek, B. (2001) Exploiting the vascular protective effects of high-density lipoprotein and its apolipoproteins: An idea whose time for testing is coming, Part I. Circulation 104, 2376 –2383. 10. Shah, P. K., Kaul, S., Nilsson, J., and Cercek, B. (2001) Exploiting the vascular protective effects of high-density lipoprotein and its apolipoproteins: An idea whose time for testing is coming, Parts II and III. Circulation 104. 11. Stein, Y., and Stein, O. (1999) Atheroprotective mechanisms of HDL. Atherosclerosis 144, 285–301. 12. Rong, J. X., Li, J., Reis, E. D., Choudhury, R. P., Dansky, H. M., Elmalem, V. I., Fallon, J. T., Breslow, J. L., and Fisher, E. A. (2001) Elevating high-density lipoprotein cholesterol in apolipoprotein e-deficient mice remodels advanced atherosclerotic lesions by decreasing macrophage and increasing smooth muscle cell content. Circulation 104, 2447–2452. 13. Eckardstein, A., Nofer, J. R., and Assman, G. (2001) High density lipoproteins and arteriosclerosis: Role for cholesterol efflux and reverse cholesterol transport. Arterioscler. Thromb. Vasc. Biol. 21, 13–27. 14. Schmitz, G., and Langmann, T. (2001) Structure, function and regulation of the ABC1 gene product. Curr. Opin. Lipidol. 12, 129 –140. 15. Keidar, S. (1998) Angiotensin, LDL peroxidation and atherosclerosis. Life Sci. 63, 1–11. 16. Daugherty, A., Manning, M. W., and Cassis, L. A. (2000) Angiotensin II promotes atherosclerotic lesions and aneurysms in apolipoprotein E-deficient mice. J. Clin. Invest. 105, 1605–1612. 17. Potter, D. D., Sobey, C. G., Tompkins, P. K., et al. (1998) Evidence that macrophages in atherosclerotic lesions contain AngII. Circulation 98, 800 – 807. 18. Griendling, K. K., Lassegue, B., and Alexander, R. W. (1996) Angiotensin receptors and their therapeutics implications. Annu. Rev. Pharmacol. Toxicol. 36, 281–306. 19. Keidar, S., Attias, J., Heinrich, R., Coleman, R., and Aviram, M. (1999) Ang-II atherogenicity in E 0 mice is associated with increased cellular cholesterol biosynthesis. Atherosclerosis 46, 249 –257. 20. Keidar, S., Kaplan, M., Hoffman, A., and Aviram, M. (1995) Ang-II stimulates macrophage-mediated oxidation of LDL. Atherosclerosis 115, 201–215. 21. Keidar, S., Heinrich, R., Kaplan, M., Hayek, T., and Aviram, M. (2001) Angiotensin II administration to atherosclerotic mice increases macrophage uptake of oxidized LDL: A possible role for interleukin-6. Arterioscler. Thromb. Vasc. Biol. 21, 1464 –1469. 22. Rosenblat, M., Belinky, P., Vaya, J., Levy, R., Hayek, T., Coleman, R., Merchav, S., and Aviram, M. (1999) Macrophage enrichment with the isoflavan glabridin inhibits NADPH oxidase-induced cell-mediated oxidation of low density lipoprotein. A possible role for protein kinase C. J. Biol. Chem. 274, 13790 –13799. 23. Aviram, M. (1983) Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipoproteinemic patients. Biochem. Med. 10, 111–118. 24. Lowry, O. H., Rosenbrough, N. J., Farr, J. L., and Randell, R. I. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275.
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25. Keidar, S., Aviram, M., Maor, I., Oiknine, J., and Brook, J. G. (1994) Pravastatin inhibits cellular cholesterol synthesis and increases low density lipoprotein receptor activity in macrophages: In vitro and in vivo studies. Br. J. Clin. Pharmacol. 38, 513–519. 26. Aviram, M., and Vaya, J. (2001) Markers for low-density lipoprotein oxidation. Methods Enzymol. 335, 244 –256. 27. Tsujita, M., Tomimoto, S., Okumura-Noji, K., Okazaki, M., and Yokoyama, S. (2000) Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice. Biochim. Biophys. Acta 1485, 199 –213. 28. Aviram, M., Bierman, E. L., and Oram, J. F. (1989) High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages. J. Lipid Res. 30, 65–76. 29. Lawn, R. M., Wade, D. P., Couse, T. L., and Wicox, J. N. (2001) Localization of human ATP-binding cassette transporter 1
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(ABC1) in normal and atherosclerotic tissues. Arterioscler. Thromb. Vasc. Biol. 21, 378 –385. Gesquiere, L., Loreau, N., Minnich, A., Davignon, J., and Blache, D. (1999) Oxidative stress leads to cholesterol accumulation in vascular muscle cells. Free Radical Biol. Med. 27, 134 –145. Blache, D., Gesquiere, L., Loreau, N., and Durand, P. (1999) Oxidant stress: The role of nutrients in cell–lipoprotein interactions. Proc. Nutr. Soc. 58, 559 –563. Gelissen, I. C., Rye, K. A., Brown, A. J., Dean, R. T., and Jessup, W. (1999) Oxysterol efflux from macrophage foam cells: The essential role of acceptor phospholipid. J. Lipid Res. 40, 1636 –1646. Maor, I., and Aviram, M. (1994) Oxidized low density lipoprotein leads to macrophage accumulation of unesterified cholesterol as a result of lysosomal trapping of the lipoprotein hydrolyzed cholesteryl ester. J. Lipid Res. 35, 803– 819. Lyons, M. A., and Brown, A. J. (1999) 7-Ketocholesterol. Int. J. Biochem. Cell. Biol. 31, 369 –375.
Angiotensin II Reduces Macrophage Cholesterol Efflux: A Role for the AT-1 Receptor but Not for the ABC1 Transporter Marielle Kaplan,* Michael Aviram,* Carlos Knopf,† and Shlomo Keidar* ,1 *Lipid Research Laboratory, Bruce Rappaport Faculty of Medicine, Technion, Rappaport Family Institute for Research in Medical Sciences and Rambam Medical Center, Haifa, Israel; and †Section of Metabolic Diseases, Department of Clinical Biochemistry, Rambam Medical Center, Haifa, Israel
Received January 2, 2002
Impaired cellular cholesterol efflux in cells of the arterial wall is suggested to be involved in the pathogenesis of atherosclerosis. Since angiotensin II (AngII) is implicated in the development of atherosclerosis, the aim of the present study was to determine whether Ang-II could affect macrophage cholesterol efflux. Incubation of increasing concentrations of Ang-II (10 ⴚ10– 10 ⴚ7 M) with mouse peritoneal macrophages that were prelabeled with [ 3H]cholesterol led to a significant decrease in HDL-induced macrophage cholesterol efflux, by up to 70% compared to control cells incubated without Ang-II. Ang-II specifically increased the plasma membrane unesterified cholesterol content, the substrate for HDL-induced cholesterol efflux. The inhibitory effect of Ang-II on macrophage cholesterol efflux was found to be mediated by the angiotensin II type 1 (AT-1) receptor, since addition of the AT-1 antagonist Losartan completely blocked the inhibitory effect of Ang-II on the macrophage cholesterol efflux. We thus conclude that Ang-II atherogenicity may be related, at least in part, to its inhibitory effect on macrophage cholesterol efflux, thus leading to cellular cholesterol accumulation, the hallmark of early atherogenesis. © 2002 Elsevier Science (USA)
Key Words: macrophages; cholesterol efflux; angiotensin II; Losartan.
Foam cell formation is the result of cholesterol accumulation in arterial macrophages and it is a prominent finding in atherosclerotic plaques (1–3). Cellular cholesterol accumulation depends on the balance between cholesterol influx resulting from the uptake of lipoproteins such as oxidized LDL (Ox-LDL), and cholesterol efflux (4 –7). Cholesterol efflux from cells by extracel1 To whom correspondence and reprint requests should be addressed at Rambam Medical Center, Haifa 31096 Israel. Fax: 9724-854-2721. E-mail: [email protected].
lular acceptors such as high-density lipoprotein (HDL) is the first step in reverse cholesterol transport. This process is responsible for the transport of cellular cholesterol from the periphery to the liver for excretion (8 –12). Thus, understanding of the cellular events that contribute to cholesterol efflux is vital for elucidating the mechanisms of cholesterol accumulation in foam cells during the development of atherosclerotic lesions. Cellular cholesterol, subject to efflux, must be in its unesterified form and should be present in the cell membrane. Efflux of unesterified cholesterol (UC) from plasma membranes into the extracellular spaces is therefore determined by the lipids composition of cell membranes and by the acceptor such as the HDL particle (13). Cholesterol ester (CE) droplets are formed by esterification of UC by the acyl-coenzyme cholesterol acyltransferase (ACAT) and give lipid-laden macrophages their foamy appearance. Cytosolic CE can be hydrolyzed by neutral cholesteryl ester hydrolase (NCEH) and the UC formed can then be either transferred to the cell membrane or reesterified by ACAT (7, 13). Recently, the ATP binding cassette transporter 1 (ABC1) has been shown to play an important role in lipid efflux (14). This transporter is formed of two cytoplasmic domains as well as two transmembranal domains. This allows it to act as a channel and substrates for ABC1 include cholesterol phospholipids vitamins and interleukins. It has been suggested that ABC1 allows transport of cholesterol from Golgi structures to the cellular plasma membrane. Angiotensin II (Ang-II), a vasoconstrictor produced by the renin–angiotensin system, is implicated in atherosclerosis (15, 16), and has been identified also in atherosclerotic lesion’s macrophages (17). Angiotensin receptor type 1 (AT-1) is distributed in almost all the tissues, and was shown to mediate most of Ang-II proatherogenic effects (18). Ang-II injection to the atherosclerotic apolipoprotein E deficient (E 0) mice signif-
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icantly increases the atherosclerotic lesion area (19), and this is associated with its stimulatory effects on macrophage-mediated oxidation of LDL (20), macrophage cholesterol biosynthesis (19), as well as Ox-LDL macrophage uptake (21), leading to macrophage cholesterol accumulation and foam cell formation. The goal of the present study was to analyze whether Ang-II could affect macrophage cholesterol accumulation, secondary to its effect on cellular cholesterol efflux and to elucidate possible mechanisms for such effects. METHODS Mouse peritoneal macrophage isolation and cell fractionation. Mouse peritoneal macrophages (MPM) were harvested from the peritoneal fluid of C57Bl mice 4 days after intraperitoneal injection into each mouse of 3 ml of thioglycolate (24 g/L) in saline. The cells (10 –20 ⫻ 10 6/mouse) were washed and centrifuged three times with phosphate-buffered saline (PBS) at 1000g for 10 min, then resuspended to 10 9/L in Dulbecco’s modified Eagle medium (DMEM) containing 10% horse serum (heat-inactivated at 56°C for 30 min) and 100 U penicillin/ml, 100 g streptomycin/ml, and 2 mM glutamine (P/S/G). The dishes were incubated in a humidified incubator (5% CO 2, 95% air) for 2 h, washed with DMEM to remove nonadherent cells, and the monolayer was incubated under similar conditions for 18 h. Membrane and cytosol fractions were prepared as described previously (22). Cells were suspended at 10 8 cells/ml in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl 2, 1.25 mM EGTA, 1 mM ATP, 10 mM Hepes, pH 7.4). containing 1 mM phenylmethanesulfonyl fluoride and 100 M leupeptin at 4°C, following sonication for 3 ⫻ 10 s, resulting in about 95% of cell breakage. Nuclei, granules, and unbroken cells were removed by centrifugation (2 min, 15,600g), and the postnuclear supernatant was made in 5 mM EDTA, 1 mM Na 3VO 4, and 5 mM NaF. The supernatant was centrifuged in a Beckman airfuge (30 min 134,000g) to obtain a cell membrane pellet and a cytosolic supernatant. Membranes were suspended at 10 9 cell equivalents/ml in 0.34 M sucrose/half strength relaxation buffer containing 1 mM dithiothreitol. Solubilized membrane and cytosolic fractions were stored at ⫺70°C. Lipoproteins preparation. High density lipoprotein (HDL) were prepared from human plasma (drawn into 1 mM Na 2 EDTA) from fasted normolipidemic volunteers. HDL (d ⫽ 1.064–1.21 g/ml) was prepared by discontinuous density gradient ultracentrifugation (23). The lipoproteins were then dialyzed against 150 mM NaCl, 1 mM Na 2 EDTA, pH 7.4. LDL was sterilized by filtration and was used within 2 weeks. The protein content of the lipoproteins was determined with the folin phenol reagent (24).
of them was directly silylated in order to measure free cholesterol, and the other was saponified to measure total cholesterol (26). The amount of esterified cholesterol was calculated as the difference between the total and free fractions. To measure total cholesterol, 2 ml 20% KOH in methanol (w/v) was added to the 2 ml ether extract, and saponified for 3 h at room temperature in a multivortex in pulse mode. Two milliliters of 25% citric acid in water were added for neutralize, and the organic phase was collected. The aqueous layer was washed twice with 2-ml portions of diethyl ether, and the three organic layers were combined and evaporated under nitrogen. The residue was redissolved in 1.5 ml hexane and Na 2SO 4 anhydrous (⫾50 mg) was added in order to remove traces of water. This extract was transferred to a 2-ml vial, and evaporated to dryness under nitrogen at 55°C. Na 2SO 4 was added to the other sample (for free cholesterol) and evaporated to dryness under nitrogen. Both samples were silylated with 40 l of BSTFA ⫹ TMCS, 99:1 (Supelco Inc., Supelco Park, Bellefonte, PA) and 40 l of dried pyridine at 60°C for 30 min. Sample analysis was performed by capillary GC–MS in selected ion monitoring (SIM) mode, in a Hewlett–Packard 6890/5972A instrument. A 30-m ⫻ 0.25-mm-i.d. 5% phenylmethyl siloxane column (0.25 m) (HP-5 MS, Cat No. 19091S-433) was used. One microliter in splitless mode was injected. Helium was used as the carrier gas at a flow rate of 1 ml/min and a linear velocity of 33 cm/s. The detector temperature was 180°C, and the injector temperature was 300°C. The oven temperature was programmed, starting at 200°C for 3 min, increased to 250°C at 25°C/min and then to 290°C at 10°C/min and maintained there for 4.5 min. The sensitivity of the mass detector was increased by means of manual tuning. An additional increment of the sensitivity was achieved using the exact mass (one decimal) of each ion, previously evaluated analyzing the spectra in scan mode. The amounts of cholesterol were calculated from a calibration curve, with a linearity of 1.00. mRNA expression of ABC1 (by RT-PCR analysis). Total RNA from macrophages was extracted with TRI-reagent (Molecular Research Center, Inc.). cDNA was generated from 1 g of total RNA using reverse transcriptase (RT) (Boehringer Mannheim, Germany) and oligo dT primers (Boehringer Mannheim, Germany). The RT reaction was carried out at 42°C for 50 min and at 99°C for 5 min. Products of the RT reaction were subjected to polymerase chain reaction (PCR) amplification. The forward primer used for ABC1 was 5⬘- ATGGTGGGAATGGGTCAGAAG-3⬘ and the reverse primer was 5⬘-CACGCAGCTCATTGTAGAAGG-3⬘ (27). The amplification conditions involved were denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C at 1.5 min. Similar conditions were used to amplify the housekeeping gene GAPDH using the forward primer 5⬘-CTG-CCA-TT-GCA-GTG-GCA-AAG-TGG-3⬘ and the reverse primer 5⬘-TTG-TCA-TGG-ATG-ACC-TTG-GCC-AGG-3⬘. Linearity of amplification was confirmed up to 32 cycles from 2 g of total RNA in the reverse transcription step. Specific PCR products obtained for ABC1 and GAPDH were separated on 6% polyacrylamide gel. Statistics. Student’s t test was performed for all statistical analyses. Results are given as means ⫾ SD.
Cholesterol efflux determination. Mouse peritoneal macrophages were incubated with 3H-labeled cholesterol for 18 h at 37°C followed by cell wash in ice-cold PBS (⫻3) and further incubation in the absence or presence of 100 g of HDL protein/ml for 3 h at 37°C. Cellular and medium [ 3H]-labels were quantitated and HDLmediated cholesterol efflux was calculated as the ratio of [ 3H]-label in the medium/[ 3H]-label in the medium ⫹ [ 3H]-label in cells (25).
RESULTS
Cholesterol mass determination [by gas chromatography–mass spectrometry (GC–MS)]. Macrophage cholesterol mass was determined after cell incubation in the absence or presence of Ang-II for 18 h at 37°C. Cellular lipids were extracted with hexane:isopropanol (3:2, v/v) and the solvents were evaporated. The dried extracts we were redissolved in 4 ml of diethyl ether and divided in two equal parts. Fifty microliters of ␣-cholestane (20 g/ml, Sigma Chemical Co., St. Louis, MO) was added to each one as internal standard. One
Macrophage cholesterol efflux was determined in mouse peritoneal macrophages following their radiolabeling with [ 3H]cholesterol. Incubation of [ 3H]cholesterol-radiolabeled mouse peritoneal macrophage with increasing concentrations of Ang-II (10 ⫺10–10 ⫺7 M) for 18 h at 37°C, led to a significant decrease, by up to 70%
Effect of Angiotensin II on Macrophage Cholesterol Efflux
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binding to its AT-1 receptor, we used the AT-1 specific receptor antagonist Losartan. Preincubation of the 3Hcholesterol radiolabeled macrophages with Losartan at increasing concentrations (10 ⫺7–10 ⫺5 M) prior to Ang-II addition (10 ⫺7 M), dose dependently reduced the inhibiting effect of Ang-II (Table 1). In the presence of Losartan (10 ⫺5 M), Ang-II completely failed to inhibit macrophage cholesterol efflux, suggesting that the Ang-II effect was mediated via its binding to the macrophage AT-1 receptor. Effect of Angiotensin II on Macrophage Cholesterol Distribution between Cytosol and the Plasmatic Membrane
FIG. 1. The effect of angiotensin II on macrophage cholesterol efflux. (A) Mouse peritoneal macrophages were radiolabeled with [ 3H]cholesterol for 18 h in the absence or presence of increasing concentrations of angiotensin II (10 ⫺10–10 ⫺7 M) prior to washes and addition of HDL. Then the HDL-mediated macrophage cholesterol efflux was determined as described under Methods. (B) Mouse peritoneal macrophages were radiolabeled with [ 3H]cholesterol for increasing periods of time (1–18 h), in the absence or presence of angiotensin II (10 ⫺7 M) prior to washes and addition of HDL. Then the HDL-mediated macrophage cholesterol efflux was determined as described under Methods.
Since cholesterol efflux is determined by cellular cholesterol composition and distribution, we determined macrophage cholesterol (unesterified and esterified) distribution in Ang-II-treated cells. For this purpose, macrophages were incubated in the absence or presence of increasing concentrations of Ang-II and the content of unesterified cholesterol (UC) and that of cholesteryl ester (CE) were analyzed in the macrophage plasmatic membrane and in the cytosol. Incubation of mouse peritoneal macrophages with Ang-II led to a dose-dependent increase, by up to 35%, in the plasmatic membrane UC whereas the CE content of the membrane was not significantly affected (Fig. 2A). In parallel, incubation of the macrophages with Ang-II resulted in a significant increase, by up to 4-fold at Ang-II 10 ⫺7 M, in the cytosolic cholesterol ester content (Fig. 2B), whereas, cytosolic UC levels were not affected (Fig. 2B).
TABLE 1
The Effect of the AT-1 Antagonist Losartan on the Angiotensin II-Induced Inhibition on Macrophage Cholesterol Efflux Macrophage treatment
in HDL-induced cellular cholesterol efflux, in comparison to control cells (Fig. 1A). We next analyzed whether this inhibitory effect of Ang-II on macrophage cholesterol efflux is dependent on the time of macrophages radiolabeling with [ 3H]cholesterol. For this purpose, mouse peritoneal macrophages were first radiolabeled for increasing period of times (1–24 h) with [ 3H]cholesterol followed by the addition of angiotensin II (10 ⫺7 M) for 18 h. As seen in Fig. 1B, Ang-II significantly inhibited macrophage cholesterol efflux (by up to 41%) already after 4 h of radiolabeling of cells with [ 3H]cholesterol. To determine whether the Ang-II effect on macrophage cholesterol efflux was mediated via
Control ⫹Angiotensin II (10 ⫺7 ⫹Losartan (10 ⫺7 M) ⫹Angiotensin II (10 ⫺7 ⫹Losartan (10 ⫺6 M) ⫹Angiotensin II (10 ⫺7 ⫹Losartan (10 ⫺5 M) ⫹Angiotensin II (10 ⫺7
Macrophage cholesterol efflux (%)
M)
19.4 ⫾ 2.0 8.1 ⫾ 2.1*
M)
10.9 ⫾ 2.2*
M)
13.5 ⫾ 0.9*
M)
20.0 ⫾ 1.1
Note. Mouse peritoneal macrophages were radiolabeled with [ 3H]cholesterol for 18 h in the absence or presence of increasing concentrations of Losartan (10 ⫺7–10 ⫺5 M) prior to addition of angiotensin II at 10 ⫺7 M. Then the HDL-mediated macrophage cholesterol efflux was determined as described under Methods. Results are presented as means ⫾ SD. * P ⬍ 0.05 versus control.
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FIG. 2. The effect of angiotensin II on macrophage cholesterol composition. Mouse peritoneal macrophages were incubated in the absence or presence of increasing concentrations of angiotensin II (10 ⫺8–10 ⫺7 M) for 18 h, then the cells were subfractionated and their unesterified cholesterol (UC) as well as esterified cholesterol (CE) were analyzed by GC–MS in the macrophage plasmatic membrane (A) and cytosol (B).
cellular cholesterol efflux is considered as an important antiatherogenic function of this lipoprotein (28). In the present study, angiotensin II was shown to significantly reduce HDL-induced cellular cholesterol efflux from macrophages. This was associated with Ang-II-induced increment in unesterified cholesterol (UC) content in the macrophage plasma membranes, and in cholesteryl ester (CE) accumulation in the macrophage cytosol. Indeed, foam cells in the atherosclerotic lesions are characterized by increased cellular UC content and by the presence of elevated levels of cytosolic droplets of CE. Therefore, Ang-II by affecting cellular cholesterol trafficking and cholesterol efflux could led to the formation of macrophage-derived foam cells. The effect of Ang-II on the macrophage cholesterol efflux was dependent on the time of cells radiolabeling, and 1 h of labeling was not sufficient to obtain the maximal Ang-II induced inhibiting effect. This could point out that the effect of angiotensin II is linked to the cholesterol transport in the cells, rather than to a downregulation of receptors involved in HDL-mediated cholesterol efflux, such as the SRB-I or the ABC-1 HDL receptors. Indeed, in the present study angiotensin II was not able to downregulate the mRNA expression of the ABC1 transporter, the main efflux transporter system present in macrophages (29). If angiotensin II would have downregulated macrophage receptors, which are involved in cholesterol efflux, its effect should not have been dependent on the time of radiolabeling of the cells, since Ang-II was present 18 h, whether the macrophages were preradiolabeled for 1 or for 24 h. However, if the angiotensin II effect could be linked to the intracellular cholesterol transport and the time of the macrophage prelabeling was shown to be associated with cellular cholesterol efflux.
Effect of Angiotensin II on mRNA Expression of ABC1 We next analyzed whether the inhibitory effect of angiotensin II on the macrophage cholesterol efflux was mediated by its ability to downregulate the ABC1 transporter. For this purpose macrophages (J-774 A.1 cell line) were incubated with increasing concentrations of Ang-II (0, 10 ⫺8, 10 ⫺7 M) for 18 h at 37°C. Then, mRNA was extracted from the cells and subjected to RT-PCR using specific primers for ABC1. The mRNA expression of ABC1 in macrophages was not affected by their incubation with angiotensin II (Fig. 3). DISCUSSION Cellular cholesterol efflux is one of the essential events in maintaining cellular cholesterol content since most peripheral cells are unable to catabolize cholesterol. Low levels of plasma HDL were shown to be a major cardiovascular risk factor, as HDL-mediated
FIG. 3. Effect of angiotensin II on mRNA expression in macrophages. Mouse peritoneal macrophages were incubated in the absence or presence of increasing concentrations of angiotensin II (10 ⫺8–10 ⫺7 M) for 18 h, and then mRNA was extracted and subjected to RT-PCR for determination of mRNA of ABC1.
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The effect of angiotensin II on the macrophage cholesterol efflux was mediated by the specific angiotensin II receptor type 1 (AT-1) as shown by using the AT-1 antagonist receptor, Losartan. Some atherogenic effects of angiotensin II are mediated by the G-protein coupled receptors AT-1 and AT-2, and the AT-1 receptor mediates virtually all the known physiological actions of angiotensin II, including its pro-atherogenic effects (18, 19). Furthermore, AT-1 receptor has been identified in the cells of the arterial wall, including macrophages (18). Angiotensin-II was shown to stimulate macrophage foam cell formation and angiotensin converting enzyme inhibitors reduce atherosclerosis in animal models (15, 16). Ang-II can induce cholesterol accumulation in macrophages by several mechanisms including induction of cellular oxidative stress. Ang-II activates NAD(P)H-dependent oxidases, leading to increased lipid peroxidation in arterial macrophages and also increased macrophage-mediated oxidation of LDL (19 – 21). The ability of Ang-II to induce oxidative stress in macrophages could also be linked to its ability to inhibit cellular cholesterol efflux. Although, little is known about the regulation of macrophage cholesterol efflux, cellular oxidative stress and specifically oxysterols have been linked to reduced cellular cholesterol efflux (30 –33). 7-Ketocholesterol, which is a prominent product of cholesterol oxidation, accumulates in macrophages as a result of enhanced cellular uptake of Ox-LDL (34), and macrophage foam cells enriched in 7-ketocholesterol, exhibits reduced cellular cholesterol efflux. Furthermore, depletion of n-3 fatty acids, which occurs during oxidation, reduces the efficiency of cellular cholesterol efflux (32). It is also possible that products of fatty acid oxidation may have direct effects on the diffusional transfer or on receptor-mediated removal of cholesterol through changes in membrane fluidity. We conclude that Ang-II atherogenicity could be related to its prooxidative properties (20). Ang-II-induced oxidative stress not only increased macrophage cholesterol influx (uptake of LDL and oxidized LDL, cholesterol biosynthesis; 19, 21), but also could inhibit cellular cholesterol efflux and reverse cholesterol transport. REFERENCES 1. Ross, R. (1986) The pathogenesis of atherogenesis—An update. N. Engl. J. Med. 314, 498 –500. 2. Lusis, A. J. (2000) Atherosclerosis. Nature 407, 233–241. 3. Aviram, M. (1993) Modified forms of low density lipoprotein and atherosclerosis. Atherosclerosis 98, 1–9. 4. Aviram, M., and Fuhrman, B. (1998) LDL oxidation by arterial wall macrophages depends on the oxidative status in the lipoprotein and in the cells: Role of prooxidants vs antioxidants. Mol. Cell. Biochem. 188, 149 –159. 5. Steinberg, D. (1997) Low density lipoprotein oxidation and its pathobiological significance. J. Biol. Chem. 272, 20963–20966.
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25. Keidar, S., Aviram, M., Maor, I., Oiknine, J., and Brook, J. G. (1994) Pravastatin inhibits cellular cholesterol synthesis and increases low density lipoprotein receptor activity in macrophages: In vitro and in vivo studies. Br. J. Clin. Pharmacol. 38, 513–519. 26. Aviram, M., and Vaya, J. (2001) Markers for low-density lipoprotein oxidation. Methods Enzymol. 335, 244 –256. 27. Tsujita, M., Tomimoto, S., Okumura-Noji, K., Okazaki, M., and Yokoyama, S. (2000) Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice. Biochim. Biophys. Acta 1485, 199 –213. 28. Aviram, M., Bierman, E. L., and Oram, J. F. (1989) High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages. J. Lipid Res. 30, 65–76. 29. Lawn, R. M., Wade, D. P., Couse, T. L., and Wicox, J. N. (2001) Localization of human ATP-binding cassette transporter 1
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(ABC1) in normal and atherosclerotic tissues. Arterioscler. Thromb. Vasc. Biol. 21, 378 –385. Gesquiere, L., Loreau, N., Minnich, A., Davignon, J., and Blache, D. (1999) Oxidative stress leads to cholesterol accumulation in vascular muscle cells. Free Radical Biol. Med. 27, 134 –145. Blache, D., Gesquiere, L., Loreau, N., and Durand, P. (1999) Oxidant stress: The role of nutrients in cell–lipoprotein interactions. Proc. Nutr. Soc. 58, 559 –563. Gelissen, I. C., Rye, K. A., Brown, A. J., Dean, R. T., and Jessup, W. (1999) Oxysterol efflux from macrophage foam cells: The essential role of acceptor phospholipid. J. Lipid Res. 40, 1636 –1646. Maor, I., and Aviram, M. (1994) Oxidized low density lipoprotein leads to macrophage accumulation of unesterified cholesterol as a result of lysosomal trapping of the lipoprotein hydrolyzed cholesteryl ester. J. Lipid Res. 35, 803– 819. Lyons, M. A., and Brown, A. J. (1999) 7-Ketocholesterol. Int. J. Biochem. Cell. Biol. 31, 369 –375.