Acute effects of oxidized low density lipoprotein on metabolic responses in macrophages

^a Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, University of Leiden, Leiden, The Netherlands
^b CNS Pharmacology, Solvay Duphar, Weesp, The Netherlands

	ABSTRACT

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The immediate effects of oxidized low density lipoprotein (OxLDL) on the metabolic activity of cultured macrophages (RAW 264.7) were studied using a microphysiometer. Administration of OxLDL acutely induced a concentration-dependent increase in metabolic activity, with an EC₅₀ of 16 ± 3 µg/ml OxLDL and a maximal effect of 35% ± 4% (mean ± SEM; n=5). A biphasic response was measured after administration of 75 or 100 µg/ml OxLDL consisting of an initial sharp increase, followed by the induction of a long-lasting hypoactivity of 80% of the control value. Incubation of cells with polyinosinic acid (polyI; 100 µg/ml) for 30 min prior to OxLDL administration could completely block the effect of 25 µg/ml OxLDL. In addition, polyI acted as a full antagonist on the decrease of the biphasic response of cells generated by 75 and 100 µg/ml OxLDL. Macrophages used in this study possessed a specific binding site for OxLDL, with a dissociation constant (K_D) of 9 ± 2 µg/ml and a maximal binding of 610 ± 32 ng ¹²⁵I-OxLDL/mg cell protein. Binding of ¹²⁵I-OxLDL to macrophages could be completely competed for by unlabeled OxLDL, by polyI for 58%, and by AcLDL for 46%. In conclusion, OxLDL can acutely activate the metabolic state of macrophages by a receptor-mediated process in a concentration-dependent fashion, which could be antagonized by polyI. Metabolic responses to OxLDL may underlie the changes observed in macrophages in the early atherosclerotic plaque.—de Vries, H. E., Ronken, E., Reinders, J.-H., Buchner, B., van Berkel, T. J. C., and Kuiper, J. Acute effects of oxidized low density lipoprotein on metabolic responses in macrophages. FASEB J. 12, 111–118 (1998)

Key Words: microphysiometer • oxLDL • scavenger receptor • atheroclerosis

	INTRODUCTION

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IN THE PROCESS OF ATHEROSCLEROSIS, monocytes infiltrate the vessel wall and reside in the subendothelium (1). After the uptake of oxidatively modified low density lipoprotein (OxLDL),² these cells can transform into foam cells. OxLDL plays a key role in the induction of the transformation of monocyte-derived macrophages into foam cells, and OxLDL may also act as a chemoattractant for monocytes (2).

Scavenger receptors, expressed at the surface of macrophages, mediate the uptake of OxLDL; so far, four different scavenger receptors have been identified. Two isoforms of a scavenger receptor with similar binding properties are characterized—type I and type II scavenger receptors (SRAI/SRAII)—that can bind a broad range of modified (lipo)proteins and polyanions (3, 4). A member of the class B scavenger receptors, the membrane glycoprotein CD36 (94–105 kDa), has also been described to specifically bind OxLDL (5,6). In addition, a 94–97 kDa macrophage protein was reported that specifically recognizes OxLDL, which is identified as macrosialin, the mouse homologue of human CD68 (7, 8).

Since the interaction of OxLDL with macrophages is of crucial importance for the initial stages of atherosclerosis, we studied the acute effects of OxLDL on macrophages by using a microphysiometer. The microphysiometer is highly sensitive to changes in extracellular pH. Generally, cell activation leads to acidification of the extracellular pH due to the excretion of acidic metabolites like lactate and CO₂. These changes are associated with activation of the cells via various signal transduction pathways (9). Using the microphysiometer, we were able to monitor the direct effects of OxLDL on the cellular metabolism of macrophages. These observations will contribute to the understanding of underlying mechanisms in macrophages in the onset of atherosclerosis.

	MATERIALS AND METHODS

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Materials
RAW 264.7 leukemia virus-transformed murine macrophages were obtained from American Type Culture Collection (Rockville, Md.). Dulbecco's modified Eagle's medium (DMEM), bovine calf serum (BCS), and trypsin were obtained from Gibco BRL Life Technologies (Breda, Netherlands). Transwell filters (polycarbonate, pore size 3.0 µm, 12 mm) were purchased from Costar (Cambridge, Mass.). ¹²⁵I in NaOH was purchased from Amersham Life Science (Buckinghamshire, U.K.). Bicarbonate-free RPMI1640 (running medium) and microphysiometer capsule cups were obtained from Molecular Devices Corporation (Sunnyvale, Calif.). The antibodies 2F8 and FA11 were a kind gift from Dr. S. Gordon (Sir William School of Pathology, Oxford, U.K.). All other chemicals were of analytical grade.

Cell culture
Murine RAW 264.7 macrophages were cultured in DMEM (10 g/l DMEM, 25 mM HEPES, 3.7 g/l NaHCO₃, pH 7.4) containing 10% BCS, 2 mM L-glutamine, and 100 µg/ml penicillin-streptomycin in an incubator containing 5% CO₂ and 95% air in a humified atmosphere. Passaging of cells was performed by washing twice in phosphate-buffered saline (PBS) (150 mM NaCl, 10 mM NaPO₄, pH 7.4), followed by 10 min trypsinisation with 0.25% trypsin and 0.1% EDTA. Two days before acidification rate measurements, cells were plated on Transwell filters with a density of 2.5 x 10⁵ cells per filter.

Lipoprotein isolation, modification, and labeling
LDL was isolated from fresh human serum according to Redgrave et al. (10) and acetylated as described by Basu et al. (11). LDL was oxidatively modified by incubation of 200 µg/ml of LDL with 10 µM CuSO4 at 37°C for 20 h. LDL was iodinated prior to oxidative modification by the ICl method of McFarlane (12). OxLDL and AcLDL were extensively dialyzed against 1 mM PBS without EDTA 24 h before use in the microphysiometer. A Limulus amebocyte lysate assay was used to estimate endotoxin contents of various batches of lipoproteins. Levels ranged between 20 and 40 pg/ml. To determine the effect of similar concentrations of endotoxin, RAW macrophages were exposed to these concentrations (20 and 50 pg/ml) of endotoxin (lipopolysaccharide type Re595) for 6 min, and the metabolic activity of the cells was monitored with the microphysiometer. These concentrations of endotoxin exerted no significant effect on the acidification rates of the macrophages.

Receptor binding and competition studies
Total binding of ¹²⁵I-OxLDL was measured after incubating cells for 2 h at 4°C with various amounts of ¹²⁵I-OxLDL in concentrations ranging from 0.5 µg/ml to 50 µg/ml. Nonspecific binding was determined after incubating the cells with various amounts of ¹²⁵I-OxLDL in the presence of at least a 10-fold excess of unlabeled OxLDL with a minimum of 100 µg/ml. After 2 h, cells were washed three times with buffer A (50 mM Tris, 150 mM NaCl; pH 7.4) containing 0.2% bovine serum albumin (BSA) and 5 mM CaCl₂, followed by three washes with buffer A without albumin. Subsequently, the cells were solubilized in 0.1 N NaOH. Solubilized cells were counted for radioactivity and the protein content was measured. To determine the specific binding of ¹²⁵I-OxLDL to macrophages, nonspecific binding values were subtracted from the total binding values. Dissociation constant (K_D) and maximal binding (B_max) were determined from the specific binding curve by using a computerized nonlinear fitting program (Graphpad Prism; Graphpad Software).

Specifity of the binding of ¹²⁵I-OxLDL to macrophages was determined by incubating cells with 5 µg/ml ¹²⁵I-OxLDL at 4°C for 2 h in the presence of various concentrations of unlabeled competitors: OxLDL, AcLDL, lysophosphatidylcholine (lysoPC), or polyI. After 2 h, cells were washed three times with a buffer A containing 0.2% BSA and 5 mM CaCl₂, followed by three washes with buffer A without albumin. The cells were then solubilized in 0.1 N NaOH. Cell-associated radioactivity was determined as described above.

Microphysiometry
Cells were immobilized using cell capsule cups and placed into the sensor chamber of the microphysiometer. Culture medium was replaced by low-buffered (1 mM), serum-free/bicarbonate-free RPMI-1640 (running medium). The running medium flows at 100 µl/min through the chamber and disposable assembly. A voltage signal proportional to pH is measured and recorded every second. To determine the acidification rate, the medium flow is interrupted for 30 s, allowing the accumulation of extracellular acidification metabolites (lactate, H⁺,CO₂, bicarbonate) produced by the cells as a result of cellular metabolism. Measurements of the acidification rates were performed every 2 min. The chamber was held at 37°C. Basal acidification rates were monitored for at least 30 min.

Voltage measurement of activity is a least-squares fit to linear regression of the declining slope of the line for signal vs. time during flow-off cycles, and is repeated for every measurement of the acidification rate. Cells were exposed for 6 min to various concentrations of modified lipoproteins, polyanions, and lysophosphatidylcholine. Data were recorded automatically on a MacIntosh IIc interfaced with the microphysiometer; 1 µV/s is equal to 1.10^-3 pH units/min. Acidification rates were expressed as percent change of the baseline activity before the administration of various stimuli (9).

The cytotoxicity of RAW macrophages after incubation with high concentrations of OxLDL (75 and 100 µg/ml) was measured by an MTT assay. No cytotoxicity of the cells could be detected after 6 min of exposure of the cells to 75 and 100 µg/ml OxLDL. Cytotoxicity could be observed only after an incubation of the cells for more than 5 h with 75 and 100 µg/ml OxLDL.

	RESULTS

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Binding of OxLDL to RAW macrophages
The binding of radiolabeled OxLDL to RAW macrophages was determined, and a specific binding of ¹²⁵I-OxLDL with a dissociation constant (K_D) of 9 ± 2.1 µg/ml ¹²⁵I-OxLDL and a maximal specific binding of 597 ± 32 ng bound ¹²⁵I-OxLDL/mg cell protein was determined ( Fig. 1A). The specificity of the binding of ¹²⁵I-OxLDL to cultured RAW macrophages was determined by competition experiments ( Fig. 1B). Binding of ¹²⁵I-OxLDL to macrophages was blocked by unlabeled OxLDL for 90%, by AcLDL for 46%, by polyI for 58%, and not by lysoPC ( Fig. 1B). The antibody 2F8, directed against the scavenger receptor SRIA/IIA, was able to displace binding of ¹²⁵I-OxLDL for 25% at a concentration of 5 µg/ml 2F8 and for 40% at a concentration of 25 µg/ml 2F8, respectively.

View larger version (47K): [in this window] [in a new window]   Figure 1. A) Binding of 125I-OxLDL to RAW macrophages. Cells were incubated for 2 h at 4°C with increasing concentrations of 125I-OxLDL in the absence () or presence () of an excess of unlabeled OxLDL. Specific binding () was determined as described in Materials and Methods. Data are expressed as nanograms of bound 125I-OxLDL/mg of cell protein, and represent the mean ± SEM of three individual measurements. B) Specific binding of 125I-OxLDL by RAW macrophages. Cells were incubated for 2 h at 4°C in the presence of radiolabeled OxLDL (5 µg/ml) and increasing concentrations of the competitors OxLDL (), AcLDL (), polyI (), and lysoPC (). Data represent the mean ± SEM of four individual experiments. Binding of 125I-OxLDL in the absence of competitors was regarded as 100% (100%=202 ± 44 ng 125I-OxLDL bound/mg cell protein).

Microphysiometry
Effects of OxLDL on metabolic rates of macrophages
We determined the acidification rate of macrophages in response to various concentrations of OxLDL by using a cytosensor microphysiometer ( Fig. 2A). Metabolic activity of macrophages increased in the presence of OxLDL in a concentration-dependent manner, and peak activities were reached 10–22 min after administration of OxLDL. Exposure of RAW macrophages to 5 µg/ml OxLDL resulted in an acute increase of 8% in the acidification rate, whereas in the presence of 50 µg/ml OxLDL, acidification rates were enhanced by 41%. Native LDL (100 µg/ml) exerted no significant effect on acidification rates.

View larger version (71K): [in this window] [in a new window]   Figure 2. A) Concentration–time profile of the effect of OxLDL on the acidification rate of RAW macrophages. Acidification rate of cultured RAW macrophages was measured every 2 min after administration of 3.25 µg/ml (), 5 µg/ml (), 10 () µg/ml, 25 µg/ml (), 37.5 µg/ml (), and 50 µg/ml () OxLDL for 6 min, using a cytosensor. Basal metabolic activity levels (160±13 µV/s; n=8) of the cells are regarded as 100%. Data are expressed as the mean ± SEM of five different experiments. B) Concentration–response profile of OxLDL on the acidification rate of RAW macrophages. Cells were exposed to various concentrations of OxLDL, and the area under the concentration–time profiles was calculated. An EC50 value of 16 ± 3 µg/ml for OxLDL was calculated with a maximal effect of 35 ± 4% (mean ± SEM; n=5). C) Concentration–time profile of the effect of high concentrations of OxLDL on the acidification rate of RAW macrophages. Response of RAW macrophages to 75 µg/ml () and 100 µg/ml () of OxLDL was measured. Basal levels of metabolic activity (160±13 µV/s, n=8) of the cells are regarded as 100%. Data are expressed as the mean ± SEM of five different experiments.

From the concentration–time profiles in Fig. 2A, a concentration–response curve was calculated ( Fig. 2B). The response of the cells to increasing concentrations of OxLDL was calculated as the area under the acidification curve (AUC) of the concentration–time profiles shown in Fig. 2A in the time period beginning directly after exposure (t=0) until 36 min after administration. From this curve, a half-maximal activation concentration of OxLDL (EC₅₀) of 16 ± 3 µg/ml of OxLDL was calculated; from the maximal AUC, the maximal effect (E_max) of OxLDL was calculated to be 35 ± 4% (mean ± SEM; n=5).

In contrast to the stimulatory response of RAW cells to increasing amounts of OxLDL up to 50 µg/ml, incubation of the cells with higher concentrations of OxLDL resulted in a biphasic profile in the acidification rates ( Fig. 2C). Exposure of the cells to 75 or 100 µg/ml of OxLDL resulted in an initial rise in metabolic activity to 25% of basal value, followed by a long-lasting decrease in activity to 80% of basal metabolism values. This decrease lasted more than 30 min before acidification rates returned to basal levels ( Fig. 2C).

Effect of scavenger receptor ligands on activation by OxLDL
To determine the effect of other ligands for scavenger receptors on the acidification rate of macrophages, various concentrations (up to 100 µg/ml) of AcLDL, polyC, or polyI were administered and the cellular responses monitored, but no significant changes of the acidification rate could be observed. The monoclonal antibody directed against SRIA/IIA, and the 2F8 antibody (5 µg/ml and 25 µg/ml) and polyclonal antibody FA11 (500 µg/ml) directed against murine macrosialin, exerted no effect on the metabolic activity of the macrophages.

The potential inhibitory effects of these ligands for scavenger receptors on the stimulation generated by OxLDL were examined. Cells were incubated with AcLDL (125 µg/ml) or polyI (100 µg/ml) for 30 min. OxLDL (25 µg/ml) generated a 25% increase in metabolic activity in the absence or presence of AcLDL (100 µg/ml). However, in the presence of polyI, the effect of OxLDL on the acidification rate of the macrophages was completely antagonized ( Table 1). Furthermore, incubation of the cells with 2F8 (25 µg/ml) or FA11 (500 µg/ml) for 30 min did not inhibit the effect of OxLDL (25 µg/ml) ( Table 1).

To further examine the antagonizing effects of polyI, macrophages were incubated with polyI (100 µg/ml) for 30 min. Stimulation of the cells with 25 µg/ml and 50 µg/ml of OxLDL activated cellular metabolism by 25 and 41%, respectively ( Fig. 3). PolyI was able to completely block the effect of 25 µg/ml OxLDL, whereas in the presence of polyI, the effect of 50 µg/ml OxLDL was diminished to a 13% increase in basal level ( Fig. 3A).

View larger version (55K): [in this window] [in a new window]   Figure 3. Effects of polyinosinic acid on OxLDL-induced activation of the acidification rate. A) RAW cells were incubated with 100 µg/ml polyI for 30 min; 25 µg/ml () and 50 µg/ml of OxLDL () were subsequently administered. OxLDL was also administered to control cells at concentrations of 25 µg/ml () and 50 µg/ml (). B) RAW cells were incubated with 100 µg/ml polyI for 30 min; subsequently, 75 µg/ml () and 100 µg/ml OxLDL () were administered. OxLDL was also administered to control cells at the concentrations of 75 µg/ml () and 100 µg/ml (). Basal levels of cellular metabolic activity (160±13 µV/s; n=8) are regarded as 100%. Data are expressed as the mean ± SEM of five different experiments.

Figure 3B shows the effect of polyI on the cellular responses to higher concentrations of OxLDL. OxLDL initially induced a sharp peak of 24 and 29% at 75 µg/ml and 100 µg/ml OxLDL, respectively. In the presence of polyI (100 µg/ml), the effect of 75 and 100 µg/ml OxLDL was inhibited for 50 and 58%. The decrease in metabolic activity observed after stimulation with 75 or 100 µg/ml OxLDL could be completely antagonized by polyI (100 µg/ml); activity remained at basal levels ( Fig. 3B).

Effects of lysophosphatidylcholine on acidification rates
LysoPC is a major component of atherogenic lipoproteins and may mediate the atherogenic activities of OxLDL. LysoPC induced a concentration-dependent increase in the extracellular pH of macrophages ( Fig. 4A). Administration of 10 µM lysoPC resulted in a 10% increase of acidification rates, whereas 100 µM of lysoPC generated a rise of 32% ( Fig. 4A).

View larger version (45K): [in this window] [in a new window]   Figure 4. A) Concentration–time profile of the effect of lysoPC on the acidification rate of RAW macrophages. The acidification rate of cultured RAW macrophages exposed to 10 µM (), 25 µM (), 50 µM (), and 100 µM () was measured. Basal levels of metabolic activity (160±13 µV/s; n=8) of the cells are regarded as 100%. Data are the mean of five experiments. B) Concentration–response curve of lysoPC on the acidification rate of RAW macrophages. Rate of acidification was calculated as the area under the curve of the acidification time profiles of cells as shown in Fig. 4A. An EC50 value of 43 ± 3 µM was calculated with a maximal effect of 32% ± 3% (mean ± SEM; n=5).

From the data obtained in Fig. 4A, a concentration–response curve was calculated. The cellular response to increasing concentrations of lysoPC was calculated as the AUC of the concentration–time profiles of Fig. 4A during the time period directly after exposure until 36 min later ( Fig. 4B). From this curve, a half-maximal concentration (EC₅₀) of 43 ± 3 µM of lysoPC and a maximal effect of 37 ± 3% (mean ± SEM; n=4) were calculated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The interaction of oxidatively modified lipoproteins with macrophages is suggested to be an initial step in atherosclerosis (13). In this study, the acute effects of modified lipoproteins on cellular metabolism of the murine macrophage cell line RAW 264.7 are described when using a microphysiometer.

The presence of a high-affinity binding site for OxLDL on macrophages was determined, and revealed an apparent binding constant of 9 µg/ml and a maximal binding of 597 ng OxLDL/mg of cell protein. These values are in the same range as those obtained for other monocytes or macrophages from other sources (8, 14). The interaction of OxLDL with macrophages may be mediated by four different scavenger receptors. The classical scavenger receptors type IA/IIA bind OxLDL as well as AcLDL with similar affinity (3). CD36 and macrosialin are also identified as proteins that specifically bind OxLDL (5, 7, 15). In this study, the binding of ¹²⁵I-OxLDL to macrophages could be partially displaced by AcLDL, which indicates that a specific binding site for OxLDL is present on RAW macrophages. The presence of a specific binding site for OxLDL on macrophages has been reported previously (5, 7). In our study, the specific binding of OxLDL to macrophages appears to be mediated by two distinct binding sites: one site that interacts with polyI as well as with OxLDL, and an additional site that only binds OxLDL. These sites may correspond to macrosialin and CD36, respectively (6, 7).

In addition to its role in foam cell formation, OxLDL is an important atherogenic compound that can exert various biological effects such as the induction of adhesion molecules and the release of proinflammatory cytokines (2, 16). Besides the long-term effects of OxLDL, short-term effects of OxLDL on cells are also reported. Interaction of OxLDL with various signal transduction pathways is described, such as activation of protein kinase C or enhanced levels of cAMP due to stimulation of Gs complexes (17, 18). Using the microphysiometer, we show that exposure of macrophages to OxLDL induces acute effects on cellular metabolism.

The metabolic rate of macrophages is rapidly activated after exposure to OxLDL in a concentration-dependent manner, with an EC₅₀ value of 16 µg/ml and a maximal effect of 35%. EC₅₀ values obtained for OxLDL effects on the cellular acidification rate are in the same range as the apparent binding constant for ¹²⁵I-OxLDL binding to macrophages.

The effect of OxLDL on cellular metabolism could be antagonized by the polyanion polyI. PolyI (100 µg/ml) completely abolished the stimulatory effect of 25 µg/ml OxLDL. These results suggest that interaction of OxLDL with macrophages leads to an activation via a receptor binding pathway that can be blocked by polyI. The scavenger receptors SRIA/IIA apparently are not involved in the activation of cellular acidification rates in reaction to OxLDL, since incubation of macrophages with ligands that specifically interact with SRIA/IIA, such as AcLDL or the monoclonal 2F8, revealed no significant effect on cellular metabolism, thus indicating that SRIA/IIA does not play a role in activation of cellular metabolism by OxLDL. The involvement of the OxLDL binding protein CD36 in the acidification activation rates of macrophages by OxLDL is not yet clear. CD36 is a member of the class B scavenger receptors and is reported to specifically bind a large variety of ligands such as OxLDL, thrombospondin, and anionic phospholipids, but does not interact with polyanions like polyI (6). That polyI acts as an full antagonist in the activation of the cellular metabolism suggests that CD36 is not involved in OxLDL-mediated activation of cellular metabolism. The final candidate accounting for the stimulation of cellular metabolism by OxLDL may be macrosialin. Macrosialin is a member of the lysosomal-associated membrane protein family and is characterized as a specific OxLDL binding protein (7), which also interacts with polyI (8).

High concentrations of OxLDL (75 and 100 µg/ml) induced a biphasic response in the acidification rate of macrophages. Incubation of macrophages with high concentrations of OxLDL induced a sharp increase in the acidification rate, which was followed by a long-lasting decrease to 80% of the control level. Thirty minutes after administration, however, cellular metabolism recovered to basal values. The decrease in metabolic activity may be the result of the interaction of compounds derived from the oxidized LDL with the macrophages generating an initial cytotoxic effect, from which the cells apparently recover. Alternatively, the decrease observed in cellular acidification rates after incubation with high concentrations of OxLDL may also be mediated by a distinct binding site. The decrease obtained after stimulation of the cells with 75 and 100 µg/ml OxLDL was antagonized completely in the presence of 100 µg/ml polyI, whereas in receptor binding competition experiments, binding of 100 µg/ml radiolabeled OxLDL could be competed for by polyI (100 µg/ml) only for 40% (results not shown). The decrease can be fully blocked by polyI, indicating that the specific binding site for OxLDL and polyI affects the cellular metabolism of macrophages, suggesting a potential role for macrosialin.

The atherogenic component of OxLDL, lysoPC, also generated a concentration-dependent increase in the metabolic state of the cells with an EC₅₀ of 43 µM. LysoPC acts as an important mediator of atherogenic effects of OxLDL (18–20). Like OxLDL, lysoPC may exert its effects through activation of various second messenger pathways, such as G-protein-dependent activation of adenylyl cyclase, activation of protein kinase C, or enhanced calcium influxes (18, 19, 21). In our study, the binding of OxLDL to macrophages could not be displaced by lysoPC, which indicates that lysoPC interacts with cells via another mechanism. Studies suggest that the phospholipid may intercalate into the membrane lipid bilayer and directly activate second messenger systems (19). LysoPC constitutes 40% of the total phosholipid content in totally oxidized LDL, with maximum concentrations of up to 0.3 µmol/mg apoprotein B in OxLDL (22–24). In this study, lysoPC was able to affect the cellular metabolism of macrophages at a slightly higher concentration than lysoPC concentrations in maximally oxidized LDL. These observations suggest that the effect of OxLDL on macrophage cellular metabolism may be mediated by lysoPC present in OxLDL.

In conclusion, OxLDL acutely induces a stimulation of extracellular acidification of macrophages in a concentration-dependent manner and can be antagonized by the polyanion polyI. Observations described in this study illuminate the role of modified lipoproteins in the early stages of atherosclerosis, and may lead to development of new therapeutic strategies for preventing atherosclerosis.

	ACKNOWLEDGMENTS

 
This work was supported by grant no. 94.124 of the Dutch Heart Foundation.

	FOOTNOTES

 
¹ Correspondence: Leiden/Amsterdam Center for Drug Research, University of Leiden, P.O. Box 9503, 2300 RA Leiden, The Netherlands. E-mail: e.vries{at}lacdr. LeidenUniv.nl

² Abbreviations: AUC, area under acidification curve; OxLDL, oxidized low density lipoprotein; BCS, bovine calf serum; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; lysoPC, lysophosphatidylcholine; K_D, dissociation constant; B_mas, maximal binding; PBS, phosphate-buffered saline.

Received for publication June 12, 1997. Accepted for publication October 13, 1997.

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