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Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:386-393

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:386.)
© 2001 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Retention of Oxidized LDL by Extracellular Matrix Proteoglycans Leads to Its Uptake by Macrophages

An Alternative Approach to Study Lipoproteins Cellular Uptake

Marielle Kaplan; Michael Aviram

From the Lipid Research Laboratory, Bruce Rappaport Faculty of Medicine, Technion, The Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel.

Correspondence to Michael Aviram, DSc, Lipid Research Laboratory, Rambam Medical Center, Haifa 31096, Israel. E-mail aviram{at}tx.technion.ac.il


*    Abstract
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 *Abstract
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Abstract—Interaction between arterial macrophages and oxidized LDL (Ox-LDL) leads to foam cell formation, a critical step during early atherogenesis. Until now, cellular uptake of lipoproteins was studied through incubation of the media-soluble lipoprotein with cultured macrophages. However, as lipoproteins in the arterial wall are bound to subendothelial matrix, we questioned whether the retention (binding) of Ox-LDL to a macrophage-derived extracellular matrix (ECM) could lead to enhanced uptake by macrophages. The uptake of ECM-bound Ox-LDL by activated macrophages (by phorbol myristate acetate) was lipoprotein dose dependent, time dependent and higher (by 1.5-fold) than the uptake of ECM-bound native LDL. Preincubation of the ECM with lipoprotein lipase before the addition of Ox-LDL was essential for the uptake of ECM-bound Ox-LDL by the macrophages. After radiolabeling of the ECM glycosaminoglycans (GAGs), we found that ECM-bound Ox-LDL is taken up by the macrophages together with the ECM-GAG. Finally, these results were further confirmed through the use of ECM obtained from mouse peritoneal macrophages (MPMs), derived from atherosclerotic, apoE-deficient mice. In 24-week-old mice with developed atherosclerosis, the GAG content of their MPM-derived ECM increased by 52%, the ability of their MPM-derived ECM to bind Ox-LDL increased by 57%, and macrophage uptake of Ox-LDL that was retained by the MPM-derived ECM increased by 86%. In conclusion, the present study demonstrated that ECM-bound Ox-LDL is taken up by activated macrophages. This may represent a physiopathological phenomenon that leads to cholesterol and oxysterol accumulation in arterial macrophages, the hallmark of early atherosclerosis.


Key Words: extracellular matrix • binding • oxidized LDL • macrophages • atherosclerosis


*    Introduction
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 up arrowAbstract
 *Introduction
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Early atherogenesis is characterized by lipoprotein-induced accumulation of cholesterol and oxysterols in arterial macrophages,1 2 3 as well as by subendothelial accumulation of atherogenic lipoproteins in extracellular matrix (ECM).4 5 6 ECM contains collagen and elastic fibers embedded in a viscoelastic gel that consists of proteoglycans (PGs), hyaluronan, and glycoproteins.7 Arterial ECM contributes to the trapping of LDL in the arterial wall, a phenomenon called "lipoprotein retention,"8 9 10 and ECM PGs were shown to be responsible for the entrapment of LDL and modified forms of LDL in the arterial wall.11 12 ECM can be produced in vitro by arterial cells, including endothelial cells,13 smooth muscle cells,14 and macrophages.15 Lipoprotein lipase (LPL) was shown to be required for the interaction of native LDL with ECM heparan sulfate PGs.16 17 There has been some controversy regarding the effect of the oxidation of LDL on its binding to PGs. Some studies showed that the oxidation of LDL particles decreases their ability to bind human aortic PGs,18 19 whereas in other studies, LDL oxidation was shown to further enhance its association with heparan sulfate PGs anchored to endothelial cell matrix.20 21 Recently, we have shown that macrophage-derived ECM exhibited an increased binding to oxidized LDL (Ox-LDL) compared with native LDL, and this binding was mediated by the ECM heparan sulfate as well as by chondroitin sulfate PGs.15 It is thus possible that LDL oxidation can occur under certain conditions before binding of the native lipoprotein.

Macrophage cholesterol accumulation and foam cell formation, the hallmark of early atherogenesis, can be obtained in vitro through cell interaction with Ox-LDL, which, unlike native LDL, can bind to scavenger receptors, leading to macrophage cholesterol accumulation.22 23 24 This is related to the fact that unlike the LDL receptor, the macrophage scavenger receptors are not regulated by the cellular cholesterol content, and hence, macrophage uptake of Ox-LDL can contribute to the cellular accumulation of cholesterol and oxysterols, an event that leads to accelerated atherosclerosis. Studies of macrophage lipoprotein uptake are usually performed through cell incubation with the lipoprotein in an appropriate medium.25 26 27 However, in the arterial wall, the lipoprotein may not be in a soluble form but rather attached to ECM. With the use of endothelial cell– and smooth muscle cell–derived ECM, a recent study showed that the uptake of matrix retained aggregated LDL by macrophages and that this uptake was not receptor mediated.28 However, the ability of ECM-bound Ox-LDL to be taken up by cells was not studied until now.

The ECM composition can be affected during atherogenesis by oxidative stress, involving the synthesis of lipoprotein-retentive molecules such as PGs or LPL.29 30 The apoE-deficient mice (E0) are used as a model to study atherogenesis because they develop severe hypercholesterolemia, oxidative stress, and accelerated atherosclerosis within a few months.31 32 The production of ECM by macrophages from these atherosclerotic mice and the possible role of such ECM in the promotion of macrophage uptake of ECM-bound Ox-LDL also were not previously studied. Thus, the goal of the present study was to determine whether Ox-LDL bound to ECM could be taken up by macrophages and, if so, via what mechanism. Furthermore, using the atherosclerotic E0 mouse model, we sought to determine the effect of the extent of atherosclerosis on the production of macrophage ECM, the ability of this ECM to bind Ox-LDL, and the effect on macrophage uptake of such an ECM-bound Ox-LDL.


*    Methods
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 *Methods
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Cell Culture and PG Radiolabeling
The J-77A-A.1 murine macrophage-like cell line was purchased from American Type Culture Collection. J-77A-A.1 cells were plated at 2.5x105 cells per 35-mm dish in DMEM supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine (P/S/G). In several experiments, after PBS washing, the macrophages were radiolabeled with 30 µCi/mL [35S]sodium sulfate for 24 hours at 37°C in DMEM supplemented with 10% FCS to label PGs from the ECM.33

Mouse peritoneal macrophages (MPMs) were harvested from the peritoneal fluid of apoE-deficient (E0) mice (weight 15 to 25 g) 4 days after intraperitoneal injection of 3 mL of 24 g/L thioglycolate in saline. The cells (10 to 20x106 per mouse) were washed with PBS, centrifuged at 1000g for 10 minutes (3 times), and then resuspended to 109/L in DMEM containing 10% inactivated horse serum and P/S/G. The cell suspension was dispensed into Petri dishes and incubated in a incubator (5% CO2, 95% air) for 2 hours. The dishes were washed once with DMEM to remove nonadherent cells, and the monolayer was then incubated under similar conditions for 18 hours.34

ECM Preparation
Macrophages (J-77A-A.1 or MPMs from E0 mice) were cultured in DMEM containing 10% FCS supplemented for 4 days at 37°C. Then, cells were removed with 0.5% Triton X-100 and 20 mmol/L NH4OH. Dishes coated with the ECM were then washed with PBS and kept at 4°C for use within 2 weeks.35 The macrophage ECM remained intact and was free of cellular elements. This treatment has been shown previously to completely remove only the cell layer, leaving behind the matrix secreted by the cells.

Glycosaminoglycan Determination
Macrophage glycosaminoglycan (GAG) content was analyzed with the 1,9-dimethylmethylene blue spectrophotometric assay for sulfated GAGs.36

Lipoproteins
Lipoprotein Preparation
LDL was prepared from plasma (drawn into 1 mmol/L Na2EDTA) from fasted normolipidemic volunteers. LDL (d=1.019 to 1.063 g/mL) were prepared through discontinuous density gradient ultracentrifugation as described previously.37 LDL was washed at d=1.063 g/mL and dialyzed against 150 mmol/L NaCl and 1 mmol/L Na2EDTA, pH 7.4. LDL was then sterilized through filtration and used within 2 weeks. The protein content of the lipoproteins was determined with the Folin phenol reagent according to the Lowry method.38

Ox-LDL was prepared through overnight dialysis of LDL or 125I-labeled LDL (1 mg lipoprotein protein/mL) against PBS to remove any residual EDTA, followed by incubation with 10 µmol/L CuSO4 for 18 hours at 37°C. Oxidation was terminated by refrigeration and the addition of 0.1 mmol/L Na2EDTA. The degree of LDL oxidation was determined through analysis of malondialdehyde (MDA) equivalents with the thiobarbituric acid reactive substances assay39 and ranged between 18 to 25 nmol MDA/mg protein compared with 0.5 to 1.0 nmol MDA/mg protein in native LDL.

Lipoprotein Labeling
LDL was radioiodinated with 125I according to the iodine monochloride method as modified for lipoproteins.40 LDL was also radiolabeled with 1 mCi/mL 3H-cholesteryl linoleate labeled in its cholesterol moiety.41 The final LDL preparation contained 98% of the radioactivity in cholesteryl ester (CE) and had a specific radioactivity of 315 cpm/µg CE.

Preparation of Ox-LDL-GAG Complex
125I-Ox-LDL (2 mg LDL protein/mL) was incubated with LPL (10 µg/mL) and with chondroitin sulfate (50 µg/mL) in PBS for 4 hours at 37°C. Then, the mixture was subjected to a density gradient ultracentrifugation for 24 hours at 4°C, and the GAG-LPL-125I-Ox-LDL complex was reseparated42 at the LDL density range (d=1.019 to 1.063 g/mL). The complex was washed at d=1.063 g/mL and dialyzed against 150 mmol/L NaCl and 1 mmol/L Na2EDTA, pH 7.4. GAGs were analyzed with the 1,9-dimethylmethylene blue spectrophotometric assay in 125I-Ox-LDL alone or in the GAG-LPL-125I-Ox-LDL complex. The GAG-LPL-125I-Ox-LDL complex (10 µg lipoprotein protein/mL) was then incubated with macrophages for 4 hours at 37°C, and its cellular degradation was determined and compared with that of 125I-Ox-LDL.

LPL Purification
Bovine milk LPL was purified from fresh nonpasteurized milk with Affi-Gel heparin chromatography as described previously.43

Lipoprotein Cellular Metabolism
Degradation and Cell Association of ECM-Retained Lipoproteins by Macrophages
Dishes coated with the macrophage-derived ECM were preincubated with 8 µg/mL LPL for 1 hour at 4°C. After extensive washes, varying concentrations of radioiodinated lipoproteins (10 to 100 µg protein/mL) were added for 4 hours at 37°C. Finally, after additional washes, fresh macrophages were seeded onto the ECM layer in the absence or presence of cellular activators (phorbol myristate acetate [PMA] or lipopolysaccharide [LPS]) for 18 hours at 37°C. Cell-mediated hydrolysis of LDL protein was assayed through determination of trichloroacetic acid–soluble, chloroform-insoluble radioactivity in the incubation medium.44 Degradation of lipoproteins that were added to control empty plates was minimal and was always subtracted from the degradation of the ECM-bound lipoproteins. The content of the cell-associated radiolabeled lipoproteins was determined after removal of the cells with Triton X-100 and NH4OH, followed by dissolution of the cell layer with NaOH and quantification of the 125I radioactivity in a {gamma}-counter.

Intracellular Cholesterol Accumulation After Macrophage Uptake of ECM-Retained Ox-LDL
Cellular uptake of ECM-bound Ox-LDL was also analyzed through the incubation of 3H-CE-Ox-LDL with LPL-anchored macrophage-derived ECM layer. Then, the unbound lipoproteins were washed, and fresh macrophage cells were added for 18 hours at 37°C. The macrophage cell layer was then detached with NH4OH and Triton X-100 and further dissolved in NaOH, and intracellular cholesterol was determined through analyses of intracellular 3H-radioactivity in a {gamma}-counter. In some of the plates, the levels of unesterified cholesterol (UC) and cholesteryl ester (CE) were then determined after lipid separation with thin-layer chromatography.41


*    Results
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 *Results
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Macrophage Uptake of ECM-Retained Ox-LDL
Cellular degradation of ECM-bound Ox-LDL and ECM-bound native LDL by J-774.A1 macrophages was studied. As seen in Figure 1ADown, the addition of increasing concentrations (10 to 100 µg protein/mL) of either Ox-LDL or native LDL to the ECM layer that was preincubated with LPL led to a gradual increase in the cellular uptake of the ECM-bound lipoproteins by macrophages that were activated with PMA. After 24 hours of incubation with 100 µg lipoprotein protein/mL, ECM-bound Ox-LDL uptake by macrophages was 1.5-fold higher than the cellular uptake of ECM-bound native LDL. Similarly, the incubation of either Ox-LDL or native LDL for increasing periods (4 to 24 hours) with LPL-anchored ECM layer led to a gradual increase in the cellular uptake of ECM-bound lipoproteins, up to 18 hours of incubation, with a 1.5-fold elevated degradation rate for Ox-LDL in comparison with native LDL at this time point (Figure 1BDown). In addition, the binding capabilities of increasing concentrations (5 to 50 µg lipoprotein protein/mL) of 125I-Ox-LDL or 125I-native LDL to the matrix layer were determined. As seen in Table 1Down, the binding of Ox-LDL to the ECM was dose dependent and increased 3-fold in comparison with native LDL.



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  		Figure 1. Lipoprotein dose and incubation time studies of the cellular uptake of ECM-bound Ox-LDL and native LDL by J-774-A.1 macrophages. A, Macrophage-derived ECM layer was preincubated with 8 µg/mL LPL for 1 hour at 4°C, followed by washes and the addition of increasing concentrations (10 to 100 µg protein/mL) of 125I-Ox-LDL or 125I-LDL for 4 hours at 37°C. Fresh J-774-A.1 macrophages were seeded onto the ECM layer for 18 hours at 37°C, together with 100 nmol/L PMA. Cellular degradation rates of Ox-LDL and LDL were then determined. B, J-774 A.1 macrophage-derived ECM layer was preincubated with 8 µg/mL LPL for 1 hour at 4°C, followed by washes and incubation with 25 µg protein/mL of either 125I-Ox-LDL or 125I-LDL for 4 hours at 37°C. Fresh J-774-A.1 macrophages were seeded onto the ECM layer for increasing periods (4 to 24 hours) together with 100 nmol/L PMA at 37°C. Then, macrophage degradation rates of Ox-LDL and native LDL were determined. Results are given as mean±SD (n=3).


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  		Table 1. Binding of Ox-LDL and Native LDL to the Macrophage-Derived ECM

We next studied whether complexes such as those formed by the binding of Ox-LDL to the matrix (ie, GAG-LPL-Ox-LDL) can be taken up by macrophages. Levels of GAGs in the GAG-LPL-125I-Ox-LDL complex increased by 6.8-fold compared with levels of GAGs in the lipoprotein alone (33.9±1.5 µg GAG/mg LDL protein in GAG-LPL-125I-Ox-LDL complex and 5.0±0.6 µg GAG/mg LDL protein in 125I-Ox-LDL). The GAG-LPL-125I-Ox-LDL complex (5 to 50 µg lipoprotein protein/mL) was then incubated with macrophages, and its cellular uptake was determined and compared with that of 125I-Ox-LDL. As shown in Table 2Down, the GAG-LPL-125I-Ox-LDL complex was taken up by macrophages in a lipoprotein dose-dependent manner, and at 50 µg/mL GAG-LPL-125I-Ox-LDL, its uptake was slightly higher (by up to 25%) than that of 125I-Ox-LDL.


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  		Table 2. Macrophage Uptake of GAG-LPL-Ox-LDL Complex

The requirement for PMA (compared with LPS) and LPL for ECM-bound Ox-LDL uptake by macrophages was next studied. PMA, but not LPS, in the presence of LPL, led to significant cellular degradation of ECM-bound Ox-LDL (Figure 2ADown). LPL (8 µg/mL) led to 20-fold increased ECM-bound Ox-LDL degradation by macrophages that were activated with PMA but not with LPS (Figure 2ADown).



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  		Figure 2. The role of macrophage activation in the uptake of ECM-bound Ox-LDL by macrophages, showing the requirement for LPL. A, J-774-A.1 macrophage-derived-ECM layer was preincubated without or with 8 µg/mL LPL for 1 hour at 4°C, followed by washes and incubation with 25 µg protein/mL of 125I-Ox-LDL for 4 hours at 37°C. Fresh J-774-A.1 macrophages were seeded onto the ECM layer in the absence (Control) or presence of either 200 µg/mL LPS or with 100 nmol/L PMA. After 18 hours of incubation, macrophage degradation of Ox-LDL was determined. Results are given as mean±SD (n=3, *P<0.01). B, J-774-A.1 macrophage-derived ECM layer was preincubated with 8 µg/mL LPL for 1 hour at 4°C, followed by washes and incubation with 25 µg protein/mL of 125I-Ox-LDL for 4 hours at 37°C. Fresh J-774-A.1 macrophages were seeded onto the ECM layer in the absence (-) or presence (+) of either 100 nmol/L PMA or 100 nmol/L 4{alpha}-phorbol-12,13-didecanoate. After 18 hours of incubation, macrophage degradation of Ox-LDL was determined. Results are given as mean±SD (n=3, *P<0.01).

Because PMA activates protein kinase C (PKC), the possible involvement of PKC activation in the cellular uptake of ECM-bound Ox-LDL was studied. The LPL-anchored ECM layer was preincubated with 125I-Ox-LDL, followed by supplementation with fresh J-774-A.1 macrophages added with PMA or with the PMA analog 4{alpha}-phorbol-12,13-didecanoate. Like PMA, this analog binds to the hydrophobic chains on the plasma membrane of the cell, but unlike PMA, it does not activate PKC. Unlike PMA, the PMA analog did not promote the cellular uptake of ECM-bound Ox-LDL (Figure 2BUp).

We next analyzed the effects of LPS and PMA on the uptake of ECM-bound native LDL by macrophages in the absence or presence of LPL. As shown for ECM-bound Ox-LDL, PMA (100 nmol/L), but not LPS (200 µg/mL), in the presence of LPL led to significant cellular degradation of ECM-bound native LDL (93±5 and 18±4 nmol of ECM-bound native LDL degraded/mg cell protein in PMA- and LPS-activated cells compared with 15±6 nmol of ECM-bound native LDL degraded/mg cell protein in control cells). LPL (8 µg/mL) increased by 7-fold ECM-bound native LDL degradation by macrophages that were activated with PMA, but not with LPS (93±5 and 18±4 nmol of ECM-bound native LDL degraded/mg cell protein in PMA- and LPS-activated cells in the presence of LPL compared with 14±4 and 16±4 nmol of ECM-bound native LDL degraded/mg cell protein in PMA- and LPS-activated cells without LPL, respectively).

The cellular uptake of ECM-bound Ox-LDL by macrophages resulted from an interaction between the lipoprotein and specific macrophage receptors for Ox-LDL. Indeed, the addition of excess concentrations of nonlabeled Ox-LDL (100-fold higher than that of the labeled 125I-Ox-LDL) almost totally abolished, by up to 96%, the ability of the macrophages to take up ECM-bound 125I-Ox-LDL, whereas similar excess concentrations of nonlabeled native LDL did not significantly affect the cellular uptake of ECM-bound 125I-Ox-LDL (Figure 3ADown). To verify that the ECM-bound Ox-LDL degradation data represent an accurate measurement of cellular uptake of the lipoprotein, cellular uptake of 125I-Ox-LDL as well as uptake of lipoprotein labeled in its CE rather than in its protein moiety were analyzed (Figure 3BDown). Cellular association of ECM-bound 125I-Ox-LDL, as well as that of 3H-CE-Ox-LDL, demonstrated a similar gradual increase in the uptake of the lipoprotein protein and CE, respectively (Figure 3BDown).



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  		Figure 3. Specificity of ECM-bound Ox-LDL uptake by macrophages. A, J-774-A.1 macrophage-derived ECM layer was preincubated with 8 µg/mL LPL for 1 hour at 4°C, followed by washes and incubation with 5 to 50 µg protein/mL of 125I-Ox-LDL for 4 hours at 37°C. Fresh J-774-A.1 macrophages were seeded onto the ECM layer together with 100 nmol/L PMA in the absence (Control) or presence of excess (50-fold concentration) of nonlabeled Ox-LDL or of native LDL. Macrophage degradation of Ox-LDL was then determined. B, Macrophage uptake of ECM-bound Ox-LDL measured as 125I-Ox-LDL or as 3H-CE-Ox-LDL cell association. 125I-Ox-LDL or 3H-CE-Ox-LDL (10 to 50 µg protein/mL) was bound to J-774-A.1 macrophage-derived ECM in the presence of 8 µg/mL LPL, followed by incubation with J-774-A.1 macrophages in the presence of 100 nmol/L PMA at 37°C. After 18 hours of incubation, the macrophage cell layer was removed with NH4OH and Triton X-100 and analyzed for its Ox-LDL protein (125I) and cholesterol ester (3H) cell association. Results are given as mean±SD (n=3).

We next analyzed the effect of the extent of LDL oxidation on the uptake of ECM-bound Ox-LDL. LDL was oxidized with increasing concentrations of CuSO4 (1 to 20 µmol/L), and LDL oxidation was terminated by the addition of Na2EDTA and BHT (25 µmol/L). The resulting preparations of Ox-LDL (with increased oxidation extents) were then analyzed for their ability to bind to the macrophage-derived ECM, as well as for further uptake by macrophages after binding to the matrix (Table 3Down). The capacity of Ox-LDL to bind to the matrix increased by up to 2.5-fold as a function of LDL oxidation up to LDL oxidation of 22 nmol MDA equivalents/mg lipoprotein protein, followed by a gradual decrease in the binding of heavily oxidized Ox-LDL to the matrix (Table 3Down). In correlation with this observation, the uptake of ECM-bound Ox-LDL by macrophages similarly increased up to 22% (with LDL oxidation of to 20 nmol MDA equivalents/mg lipoprotein protein), followed by a sharp decrease in the uptake of ECM-bound heavily oxidized LDL (Table 3Down).


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  		Table 3. Effect of the Extent of LDL Oxidation in Its Binding to Macrophage-Derived ECM and on Its Cellular Uptake After Its Retention in the Matrix

Macrophage Intracellular Pathway of ECM-Retained Ox-LDL
Both native LDL and Ox-LDL bind to ECM GAGs in the presence of LPL, which acts as a bridging element.15 16 17 20 We next questioned whether Ox-LDL is taken up by the cells together with the GAGs. For this purpose, ECM layer was prepared from 35S-labeled macrophages and then incubated with 125I-Ox-LDL. With increasing concentrations of Ox-LDL, the ECM-bound Ox-LDL was taken up by macrophages in a dose-dependent manner (0, 315, and 534 nmol ECM-bound Ox-LDL degraded/mg cell protein after the incubation of 0, 10, and 25 µg Ox-LDL/mL, respectively). Similarly, the macrophage content of ECM-labeled GAGs was similarly gradually increased (150, 2984, and 5296 cpm 35S-GAG/mL after the incubation of 0, 10, and 25 µg Ox-LDL/mL, respectively). These results suggest that ECM-bound Ox-LDL is taken up by macrophages together with the ECM-labeled GAG, which is bound to the lipoprotein.

Studies on lipoprotein uptake by cells involve cell incubation with the lipoprotein added into the culture medium; the lipoprotein is taken up by the cells via receptor-mediated endocytosis. The lipoprotein then reaches the lysosome, where its CE moiety is hydrolyzed to UC. Analysis of the distribution of CE and UC in macrophages after their incubation with ECM-bound 3H-labeled CE-Ox-LDL (25 µg/mL) for 18 hours at 37°C revealed a similar pattern as that obtained with 3H-labeled CE-Ox-LDL added to the culture medium (data not shown).

To determine whether the uptake of ECM-bound Ox-LDL by macrophages also involves transfer of the lipoprotein to the lysosome compartment, we used chloroquine, an inhibitor of lysosomal proteolytic enzymes. The addition of chloroquine (10 mmol/L) with the PMA-activated cells to ECM-bound Ox-LDL totally abolished (by up to 98%) the cellular uptake of ECM-bound Ox-LDL. These data suggest that the macrophage lysosomal compartment is involved in the cellular uptake of ECM-bound Ox-LDL, as is the case for Ox-LDL in culture medium.

ECM Derived From Atherosclerotic apoE-Deficient Mice
MPMs harvested from the atherosclerotic apoE-deficient (E0) mice during their aging (10 to 24 weeks) were used to produce ECM. ECM derived from these E0 MPMs was analyzed for GAG content and its ability to bind Ox-LDL. In addition, we analyzed the ability of Ox-LDL bound to ECM from E0 mice at different ages (10 to 24 weeks old) to be taken up by J-774-A.1 macrophages. The GAG content in MPM-derived ECM was increased by up to 52% with aging of the mice (718±56, 901±68, and 1088±76 µg/mg ECM protein in ECM from 10-, 16-, and 24-week-old E0 mice). Binding of Ox-LDL to ECM derived from MPMs that were harvested from the old mice (24 weeks old) was increased by 57% compared with the binding of Ox-LDL to ECM derived from MPMs isolated from young, 10-week-old mice (154±22, 220±24, and 242±25 ng Ox-LDL bound/mL to ECM from 10-, 16-, and 24-week-old E0 mice). Finally, macrophage degradation of Ox-LDL that was bound to ECM from MPMs that were harvested from 24-week-old mice increased by 86% compared with ECM derived from MPMs isolated from the young mice (77±10, 92±12, and 143±10 ng Ox-LDL degraded/mg cell protein retained to ECM from 10- 16-, and 24-week-old E0 mice).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The present study proposed an alternative approach to analysis of the macrophage uptake of Ox-LDL. We presented strong evidence that Ox-LDL bound to ECM can be taken up by activated macrophages. The uptake of ECM-bound Ox-LDL was higher than that of ECM-bound LDL and was found to be dose and time dependent, as well as specific for Ox-LDL. In addition, the presence of LPL as a bridging molecule between the ECM and Ox-LDL was required for this cellular uptake. Finally, ECM produced by MPMs harvested from old E0 mice compared with young, less atherosclerotic mice was shown to contain more GAGs, to bind more Ox-LDL, and to lead to an increased uptake of ECM-bound Ox-LDL.

Unlike most studies on lipoprotein-cell interactions, in which the lipoprotein is added to the cells in the incubation medium,25 26 27 in the present study, we analyzed the possibility that ECM-bound Ox-LDL could be taken up by macrophages (ie, Ox-LDL is transferred from the ECM compartment directly to the cellular compartment). Cellular uptake of this ECM-bound Ox-LDL by macrophages could then lead to cholesterol accumulation and foam cell formation, the hallmarks of early atherogenesis.

It is usually believed that oxidation can occur only after the binding of native LDL within the ECM.19 29 45 46 Once LDL is trapped in the arterial wall and isolated from the antioxidant-rich elements of the plasma, LDL would be more susceptible to oxidation,47 48 and oxidation of LDL would lead to its separation from the ECM and its further uptake by macrophages. In the present study, we proposed an additional pathway via which oxidation of LDL can possibly take place in the arterial wall even with no preceding binding of native LDL to ECM. The binding of Ox-LDL to the macrophage-derived ECM,15 as well as to endothelial cell–derived ECM,20 21 has been previously shown to be higher than that of native LDL. In the present study, we have shown that cellular uptake of ECM-bound Ox-LDL is higher than that of ECM-bound native LDL, and this phenomenon is probably related to increased binding of Ox-LDL to ECM compared with that of ECM-bound native LDL. Thus, oxidation of LDL can also occur before its binding, and it may not be obligatory that LDL oxidation take place only after its binding to ECM. Both the binding of Ox-LDL to the matrix and the binding of native LDL could contribute to foam cell formation.

There has been some controversy regarding the effect of LDL oxidation on its binding to ECM. Some studies showed that the oxidation of LDL decreases its ability to bind human aortic PGs,18 19 whereas in other studies, LDL oxidation was shown to further enhance its association with PGs anchored to endothelial cell matrix.20 21 49 The present study illustrates the increased capacity of mildly oxidized LDL to interact with ECM compared with native LDL. However, on increasing the extent of LDL oxidation, the binding of heavily oxidized LDL to ECM and the cellular uptake of ECM-bound heavily oxidized LDL were significantly reduced. Although the explanation for these discrepancies could be the presence of LPL as a bridging element, Ox-LDL has also been shown to bind PGs even in the absence of LPL.49 50

LPL certainly plays a central role in the binding of lipoproteins. We have previously shown15 that in the absence of LPL, both Ox-LDL and native LDL were able to bind to the ECM layer only to a limited extent. Preincubation of ECM layer with LPL, however, led to a substantial increase in the binding of both Ox-LDL and native LDL to the matrix. Macrophages isolated from human atherosclerotic aorta were shown to express mRNA of LPL and to secrete LPL.51 Moreover, LPL used at the same concentration as in this study was shown to mediate lipoprotein binding.17 20 In addition, a recent study showed that LPL was required for Ox-LDL binding to PGs (versican and biglycan) from smooth muscle cell–derived ECM.52 We have shown in the present study that macrophage uptake of ECM-bound Ox-LDL requires preincubation of ECM and the lipoprotein with LPL. The need of LPL for the cellular uptake of ECM-bound Ox-LDL (or native LDL) could be explained by the fact that the amount of lipoprotein bound to ECM was very limited in the absence of LPL. The structure of LPL could also be linked to the differences in binding and macrophage uptake between Ox-LDL and native LDL. It was recently shown that native LDL binds to monomeric LPL, whereas Ox-LDL binds to dimeric LPL.53 Although LPL is synthesized and active as a dimeric form, its nonomeric form is also present in vivo.53 Therefore, the ratio between dimeric and monomeric LPL in the arterial wall can affect the relative binding of Ox-LDL and native LDL.

Activation of the macrophages with PMA was required for the cellular uptake of ECM-bound Ox-LDL. PMA is a potent cellular activator and an inducer of differentiation.54 55 56 PMA-induced macrophage uptake of ECM-bound Ox-LDL could be mediated by increasing the synthesis of high-affinity receptors for Ox-LDL.57 Macrophage activation through the stimulation of PKC can also induce the secretion of hydrolytic enzymes and free radicals.58 59 These secreted substances can then lead to the release of the Ox-LDL from the matrix, followed by its further cellular uptake. Indeed, PMA activation of NADPH oxidase through PKC leads to the production of superoxide radicals, which activate matrix metalloproteinases and thus leads to matrix remodeling.60 However, LPS, an additional cellular activator, failed to induce cellular uptake of ECM-bound Ox-LDL. Although PMA and LPS are both cellular activators, "cellular activation" is a very broad term, and previous studies have shown substantial differences in their effects on macrophages. In human monocytes, PMA increased the scavenger receptor CD36 mRNA, whereas LPS downregulated it.57 PMA induced substantial superoxide generation from alveolar macrophages, whereas LPS was only a weak stimulus for the generation of superoxide anion.61 PMA increased mouse macrophage elastase mRNA by 2.5-fold, whereas LPS did not induce any changes.62 These differences could result from differences in signal transduction. Although both PMA and LPS have been shown to induce PKC in MPMs, PMA was shown to induce translocation of several PKC subtypes (PKCß, {delta}, and {epsilon}), whereas LPS induced only PKCß translocation but failed to induce translocation of PKC{delta} and {epsilon}.63 This could explain why PMA, and not LPS, allowed the uptake of ECM-bound Ox-LDL by macrophages and thus may mimic pathophysiological events occurring during early atherogenesis.

The uptake of the ECM-bound Ox-LDL was accompanied by the uptake of ECM-GAGs from the ECM. The ECM-bound Ox-LDL thus seems to be released from ECM together with its bound GAG. Because lipoprotein binding was previously shown to be mediated by binding to PGs,5 8 9 it seems that PG GAGs are detached from the core protein and then are taken up by the cells together with their bound Ox-LDL. In arterial lesions of atherosclerotic animals, the presence of PG-lipoprotein complexes were demonstrated.64 65 These complexes were previously shown to be taken up at enhanced rate by macrophages and to cause cellular cholesterol accumulation.12 46

In the present study, we used an ECM secreted by macrophages. The ECM present in the atheroma is likely to be a mixture of products secreted from all 3 types of cells that are present in the arterial wall. Using an in vitro model, we previously demonstrated that macrophages (like smooth muscle and endothelial cells) can also secrete an ECM layer.15 Because macrophages are present at the site of the atheroma at an early stage of atherogenesis (before smooth muscle cell proliferation), it may be relevant to analyze the capability of macrophage-derived ECM to participate in lipoprotein binding and hence to participate in the uptake of ECM-bound lipoproteins.

The E0 mice develop accelerated atherosclerosis within 4 to 6 months, and their lesions have a similar morphology as those of humans.31 32 Multiple factors have been associated with atherogenesis in E0 mice, including severe hypercholesterolemia and increased oxidative stress.31 66 67 The present study thus provides new information to explain the atherogen-icity of MPMs harvested from E0 mice (ie, extensive production of ECM, which leads to Ox-LDL binding, followed by its delivery to arterial macrophages). We have shown in the present study a correlation between the aging of mice (which is associated with increased atherogenesis) and proatherogenic parameters of the MPM-derived ECM. Because lipoprotein binding is mediated via binding to PGs, MPM-derived ECM may be qualified as atherogenic, as shown by its increased GAG content during the aging of mice and the development of atherosclerosis. The increasing atherogenicity of the MPM-derived ECM can also be evaluated by its increased ability to bind Ox-LDL and, as a result, enhanced uptake of Ox-LDL bound in MPM-derived ECM. MPMs are widely used as a tool for understanding macrophage cholesterol and lipoprotein metabolism in vitro. Even though peritoneal macrophages are not arterial macrophages and obviously do not participate in the formation of atheroma, this model is widely used and accepted by the scientific community for studies of macrophage cholesterol metabolism and foam cell formation68 69 because these cells mimic macrophages that are present in atheroma areas.

In conclusion, the present study presented evidence for the first time that macrophages can take up ECM-bound Ox-LDL and that this uptake of Ox-LDL together with ECM GAGs is specific and requires preactivation of the macrophages. We thus suggest that binding of the lipoprotein to ECM PGs can also be achieved after LDL oxidation. ECM-bound Ox-LDL and activated macrophages are both present in the atherosclerotic lesion, and thus their interaction can lead to cellular accumulation of atherogenic sterols and accelerated atherogenicity.

Received January 31, 2000; accepted August 21, 2000.


*    References
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