Role of Macrophage Glycosaminoglycans in the Cellular Catabolism of Oxidized LDL by Macrophages

From the Lipid Research Laboratory, The Bruce Rappaport Faculty of Medicine, Technion, the Rappaport Family Institute for Research in the Medical Sciences, and Rambam Medical Center, Haifa, Israel (M.K., H.M., M.A.); and the Dorrance Hamilton Research Laboratories, Division of Endocrinology, Diabetes & Metabolic Diseases, Department of Medicine, Jefferson Medical College of Thomas Jefferson University, Philadelphia, Pa (K.J.W.).

Correspondence to Dr 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—Macrophage binding sites for oxidized LDL (Ox-LDL) include class A scavenger receptors (SR-As), the CD-36 molecule, and an additional but hitherto unidentified binding site. Because cell-surface glycosaminoglycans (GAGs) were previously shown to be involved in the cellular uptake of native LDL and lipoprotein(a), several strategies to assess the participation of heparan sulfate (HS) and chondroitin sulfate (CS) in macrophage catabolism of Ox-LDL were used. First, incubation of J-774 A.1 macrophage-like cells with either heparinase or chondroitinase, or with both enzymes together, reduced the binding, uptake, and degradation of ¹²⁵I–Ox-LDL by 20% to 45%, in comparison with control nontreated cells, while catabolism of ¹²⁵I-labeled acetylated LDL (Ac-LDL) and native LDL were unaffected. Second, the proteoglycan (PG) cellular content was increased by cell enrichment with exogenous GAGs or by using human monocyte-derived macrophages from two patients with Sanfilippo mucopolysaccharidosis, which are characterized by cellular HS accumulation. In these macrophages, cellular uptake of ¹²⁵I–Ox-LDL increased, while catabolism of ¹²⁵I–Ac-LDL and native LDL were unaffected. Experiments using conditioned media from control, heparinase-digested, or chondroitinase-digested macrophages indicated that neither secreted GAGs nor released digestion products played any role in Ox-LDL catabolism. To evaluate potential interactions between cell-surface GAGs and known receptors for Ox-LDL, we used excess unlabeled Ac-LDL to block SR-As or anti–CD-36 antibodies to block CD-36, and then examined the catabolism of ¹²⁵I–Ox-LDL by GAG-enriched or -depleted macrophages. Both excess unlabeled Ac-LDL and anti–CD-36 antibodies reduced ¹²⁵I–Ox-LDL catabolism, but only excess unlabeled Ac-LDL completely abolished the increase in ¹²⁵I–Ox-LDL catabolism on GAG enrichment of the cells, indicating a cooperation between exogenous GAGs and cell-surface SR-As in the catabolism of OX-LDL. Moreover, the addition of GAGases to macrophages that were preincubated with anti–CD-36 antibodies and excess Ac-LDL further reduced macrophage degradation of Ox-LDL in comparison with cells that were pretreated only with anti–CD-36 antibodies and Ac-LDL, indicating a more complex role for endogenous GAGs. Overall, these studies demonstrate a substantial contribution of macrophage-associated GAGs in the catabolism of Ox-LDL, which is mediated in part by a cooperation between GAGs and cell-surface SR-As.

Key Words: proteoglycans • oxidized LDL • macrophages • glycosaminoglycans • mucopolysaccharidosis

	Introduction

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Oxidatively modified LDL is likely to play an important contributory role in the development of atherosclerosis. Oxidized epitopes, including oxidized apoB, the major protein of Ox-LDL, have been demonstrated in human lesions.^{1 2} Ox-LDL contains many biologically active molecules, such as lysophosphatidylcholine^{3 4} and oxidized sterols,^{5 6} that substantially alter the metabolism of arterial wall cells. Furthermore, Ox-LDL is taken up by macrophages via pathways that are not regulated by cellular cholesterol content,^{7 8 9} and it has been suggested that Ox-LDL may contribute to the formation of cholesterol-rich "foam cells,"^{10 11 12} a hallmark of atherosclerosis.

The existence of several binding sites on the surface of macrophages for Ox-LDL was initially suggested by cross-competition studies that showed sites recognizing both Ox-LDL and Ac-LDL^{13 14 15} and sites recognizing Ox-LDL alone.¹⁶ The former have been identified as the SR-As.¹⁷ The latter may be the class B scavenger receptors, particularly CD-36, which certainly recognizes Ox-LDL,¹⁸ but may recognize unoxidized particles as well.¹⁹ Nevertheless, the sum of the binding of ¹²⁵I-labeled Ox-LDL to SR-As, assessed by competition with excess unlabeled Ac-LDL, plus the binding to CD-36, assessed by blockage with anti–CD-36 antibodies, does not account for the total binding of Ox-LDL to macrophages.²⁰ Recently, cell-surface PGs have attracted attention as binding sites for lipoproteins.^{21 22 23} The lipoproteins adhere to the highly polyanionic GAG side chains of the PGs in a process facilitated by "bridging" molecules, such as lipoprotein lipase (LPL),²³ or apoE.²⁴ Internalization of bound ligands may proceed directly as the PG itself is internalized^{23 25} or may involve the transfer of ligand to an LDL receptor family member that then brings it into the cell.^{21 26}

Based on prior literature, there could be three possible interactions between Ox-LDL and macrophage GAGs. The first is direct binding. Despite the large increase in overall surface negative charges on oxidation, Ox-LDL binds only slightly less avidly than native LDL to heparin, the most polyanionic GAG.²⁷ The second possible interaction is binding mediated by a bridging protein. Ox-LDL has been shown to bind far better than native LDL to GAG-rich endothelial cell matrix in the presence of LPL.²⁸ The third possibility is based on the binding of GAGs to collagen and collagen-like domains, which are positively charged.^{29 30 31} Thus, GAGs would be expected to interact with SR-As through their ligand-binding domain, which forms a triple helical structure that is highly homologous to collagen. GAGs could block ligand binding to this domain, a phenomenon that has been documented for heparin³² but has not been studied for less charged GAGs. HS, as well as CS, PGs are found in several macrophage-like cell lines, including the J-774 A.1 cell line,^{33 34} and PGs were also shown to be secreted from these cells.³³ Both HSPGs and CSPGs contain membrane-anchored core proteins, and in the P388D₁ macrophage-like cell line, 82% of their CSPG core protein is expressed on the cell surface, whereas only 20% of the HSPG core protein is expressed on the cell surface and the rest is located intracellularly.³⁴ We used three experimental approaches to address the possible role of these molecules in Ox-LDL catabolism: digestion of cell-surface GAGs with heparinase and/or chondroitinase; addition of exogenous HS or CS, which have been reported to associate with the macrophage cell surface;^{35 36} and the use of Sanfilippo macrophages, which have abnormally high amounts of cell-associated GAGs, owing to a defect in GAG breakdown.³⁷

	Methods

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Cells
The J-774 A.1 murine macrophage-like cell line was purchased from the American Type Culture Collection (Rockville, MD). J-774 A.1 cells were plated at 2.5x10⁵ cells per 16-mm dish in DMEM supplemented with 10% fetal calf serum, 100 U penicillin per milliliter, 100 µg streptomycin per milliliter, and 2 mmol/L glutamine (P/S/G). The cells were fed every 3 days and used for experiments within 7 days of plating. Mouse peritoneal macrophages were harvested from the peritoneal fluid of female BALB/c mice (15 to 25 g) 4 days after intraperitoneal injection into each mouse of 3 mL of thioglycolate (24 g/L) in saline. The cells (10 to 20x10⁶ per mouse) were washed and centrifuged three times with PBS at 1000g for 10 minutes, then resuspended to 10⁹ per liter in DMEM containing 10% horse serum (heat-inactivated at 56°C for 30 minutes) and P/S/G. The cell suspension was dispensed into plastic Petri dishes and incubated in a humidified 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. HMDMs were prepared from the blood of fasting normolipidemic subjects by density gradient centrifugation.³⁸ Twenty milliliters of blood anticoagulated with sodium heparin (final concentration 10 U/mL) was layered over 15-mL Ficoll-Paque. After centrifugation at 500g for 30 minutes at 23°C, the mixed mononuclear cell band was removed by aspiration and the cells were washed twice at 4°C in DMEM supplemented with P/S/G. The cells were plated at 3x10⁵ monocytes per 16-mm dish (Primaria Brand, Falcon Labware) in the same medium (0.5 mL) containing 20% autologous serum. After 2 hours of incubation at 37°C in 5% CO₂, 95% air, nonadherent cells were removed by three washes with serum-free medium. The cells were placed in fresh medium containing 20% autologous serum, fed twice weekly, and used for experiments after 7 days in culture.

Patients
We studied MDMs from two patients with Sanfilippo's syndrome and compared them with MDMs from control subjects. The patients are two sisters, 9 and 11 years old, who initially exhibited developmental delays and mild coarsening of the facies, but progressed to severe mental retardation, seizures, and valvular heart disease. Urinary separation of GAGs by thin-layer chromatography revealed increased excretion of HS. The heparan N-sulfatase activities in cultured skin fibroblasts from these patients were 2.0% and 2.3% of normal controls, respectively, indicating Sanfilippo's syndrome (type IIIA mucopolysaccharidosis).³⁷

Cell Subfractionation
J-774 A.1 macrophages were incubated without or with heparinase III or chondroitinase ABC (0.075 U/mL final concentration; Sigma Chemical Co) for 1 hour at 37°C. At the end of the incubation, cells (2x10⁶ per 35-mm dish) were washed with cold PBS (x3), harvested, and suspended in 2 mL of 250 mmol/L sucrose containing 5 mmol/L Tris-HCl buffer, pH 7.4. The cells were then sonicated for 20 seconds at 20 W (x2), homogenized in a polytetrafluoroethylene (Teflon)/glass homogenizer (15 strokes), and centrifuged at 500g for 10 minutes. The supernatant was then centrifuged at 10 000g for 45 minutes to precipitate the lysosome-rich fraction. The remaining supernatant was centrifuged at 100 000g for 60 minutes to precipitate the plasma membrane fraction.³⁹ The plasma membrane fraction was then resuspended in saline and analyzed for its GAG content, as described below.

Lipoproteins
LDL was prepared from human plasma (drawn into 1 mmol/L Na₂EDTA) from fasted normolipidemic volunteers. LDL (d=1.019 to 1.063 g/mL) was prepared by discontinuous density gradient ultracentrifugation as described previously.⁴⁰ The lipoprotein was washed at d=1.063 g/mL and dialyzed against 150 mmol/L NaCl, 1 mmol/L Na₂EDTA, pH 7.4. LDL was then sterilized by filtration and was used within 2 weeks. The protein content of the lipoproteins was determined with the Folin phenol reagent,⁴¹ and LDL was radioiodinated by using the iodine monochloride method.⁴² LDL was acetylated by repeated additions of acetic anhydride to 5 mg of protein per milliliter of LDL diluted 1:1 (vol/vol) with saturated ammonium acetate at 4°C.⁷ Acetic anhydride was added at a 40-fold molar excess with regard to total lysines in LDL, and the modification was confirmed by electrophoresis on cellulose acetate at pH 8.6 in barbital buffer.⁴³ Oxidized LDL was prepared by an overnight dialysis of LDL against PBS to remove EDTA, followed by incubation in 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 by analysis of MDA equivalents using the thiobarbituric acid–reactive substances assay,⁴⁴ and it ranged between 20 and 30 nmol MDA per milligram lipoprotein protein. Changes in apoB fragmentation and size were analyzed by SDS polyacrylamide gels.⁴⁵ The degree of Ox-LDL aggregation was determined by measuring the lipoprotein absorbance at 680 nm.⁴⁶

Macrophage Metabolism of Lipoproteins
Cellular Degradation of Lipoproteins
Cell-mediated degradation of Ox-LDL, Ac-LDL, and native LDL was measured after incubation of the cells (1x10⁶ per 16-mm dish) with increasing concentrations of the radioiodinated lipoproteins (25 to 100 µg of protein per milliliter) in serum-free DMEM containing 0.2% BSA. Cells were preincubated with heparinase III (0.075 U/mL), chondroitinase ABC (0.075 U/mL), neither enzyme, or both enzymes for 1 hour at 37°C, followed by the addition of the labeled lipoproteins. When present, the enzymes were maintained on the cells during the entire incubation period with labeled lipoproteins, to avoid quick regeneration of the GAG side chains.²³

In the same way, cells were preincubated for 1 hour with either heparan monosulfate or CS A (50 µg/mL final concentration, Sigma Chemical Co) for 1 hour at 37°C. Cells were then washed and further incubated with labeled lipoproteins for 4 hours at 37°C. Following incubation of ¹²⁵I–Ox-LDL with the cell layer, the lipoprotein was released, if needed, by addition of 50 U of heparin on the washed cell layer.²⁸ Cell-mediated hydrolysis of LDL protein was assayed by determination of the trichloroacetic acid–soluble, chloroform-insoluble radioactivity in the incubation medium.⁴⁷ Degradation of LDL in the absence of cells was minimal and was always subtracted from the total LDL degradation. The cell layer was washed three times with PBS and extracted by a 1-hour incubation at room temperature in 0.1 mol/L NaOH for the measurement of cell-associated ¹²⁵I-labeled lipoproteins⁴⁸ and of total cellular protein mass.⁴¹

Incubation of Ox-LDL with the cells did not affect the lipid peroxidation level of the lipoprotein as measured by the thiobarbituric acid–reactive substance assay (27.5±1.9 nmol MDA per milligram LDL protein in the bulk Ox-LDL and 25.7±2.1 nmol MDA per milligram LDL protein in the same lipoprotein released from cellular PGs). Moreover, incubation of Ox-LDL with the GAG-depleted or -enriched cells did not affect the lipoprotein aggregation state (OD⁶⁸⁰ of 0.027 for the bulk Ox-LDL at 100 µg/mL, OD⁶⁸⁰ of 0.032 or 0.031 when incubating the same lipoprotein with either HS or CS at 50 µg/mL, and OD⁶⁸⁰ of 0.025 in the same lipoprotein released from cellular PGs).

To assess the involvement of known receptors for Ox-LDL, several incubations were performed in the presence of excess unlabeled Ac-LDL (200 µg/mL) to block SR-As⁴⁹ or excess anti–CD-36 antibodies at 5 µg/mL final concentration (the specific clone is SM–anti–CD-36, Immuno Quality Products) to block CD-36.⁵⁰

Cell-Surface Binding of Lipoproteins
Binding of lipoproteins to cells was studied by incubation of ¹²⁵I-labeled lipoproteins with cells for 4 hours at 4°C. Cells were preincubated in the presence or absence of 0.075 U/mL heparinase III or chondroitinase ABC for 1 hour at 37°C followed by the transfer of the cells on ice and the addition of labeled lipoproteins at 4°C for 4 hours. After extensive washing with PBS (x4), cells were solubilized by incubation in 0.1N NaOH for 18 hours at room temperature, and the bound radiolabeled LDL was then counted in a gamma counter.

Cellular Accumulation of Cholesterol Mass From Lipoproteins
Cellular cholesterol mass was determined after incubation of macrophages with 200 µg of Ox-LDL protein per milliliter for 18 hours at 37°C, in the absence or presence of heparinase or chondroitinase (0.075 U/mL). Cellular lipids were extracted with hexane/isopropanol (3:2, vol/vol) and then hydrolyzed with 0.1N NaOH. The free sterols were converted to their trimethylsilyl ethers and analyzed by capillary gas chromatography on a polar CP-Wax 57CB (PEG CARBOWAX) column using a flame ionization detector.⁵¹

Effects of Heparinase and Chondroitinase on Macrophage GAGs
Macrophage GAG content was analyzed using the DMMB spectrophotometric assay for sulfated GAGs.⁵² Briefly, 2.5 mL of ice-cold DMMB working solution (46 µmol/L DMMB, 40 mmol/L glycine, 40 mmol/L NaCl in 5% ethanol adjusted to pH 3.0) was added to 100 µL of cell sonicate or plasma membrane fraction on ice. The absorbency at 525 nm was then immediately measured. CS A was taken as a standard and included within each series of assays. The presence of uronic acid, released into culture media as a hydrolytic product of the enzymatic digestion of HS or CS, was analyzed either by continuous monitoring at 232 nm⁵³ or by using the 1-m-hydroxybiphenyl spectrophotometric assay for uronic acid.⁵⁴ Briefly, 1.2 mL of ice-cold borate solution (4.77g of Na₂B₄O₇ 10 H₂O per liter of concentrated H₂SO₄) was added to 1-mL aliquots of culture media. The mixture was then heated to 100°C for 5 minutes and then cooled immediately in an ice-water bath. Then, 20 µL of 0.15% 1-m-hydroxybiphenyl in 0.5% NaOH was added. The solution was vortexed, allowed to stand for 5 minutes, and the absorbency was then read at 520 nm.

Statistical Analyses
All results are given as mean±SD, n=3. For comparisons between a single experimental group and a control, the unpaired, two-tailed t test was used. For comparisons involving several groups simultaneously, analysis of variance (ANOVA) was initially used. When the ANOVA indicated differences among the groups, pairwise comparisons of each experimental group versus the control group were performed using the Dunnett q' statistic.⁵⁵

	Results

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Effect of Heparinase and Chondroitinase on Ox-LDL Uptake by Macrophages
To determine whether endogenous HS or CS play a role in the catabolism of Ox-LDL by macrophages, J-774 A.1 cells were preincubated for 1 hour at 37°C with heparinase III or chondroitinase ABC (0.075 U/mL), with both enzymes, or with neither enzyme (control), followed by addition of increasing concentrations of ¹²⁵I-labeled Ox-LDL, native LDL, or Ac-LDL for an additional 4 hours at 37°C, and cell-specific degradation (Fig 1A through 1C) and association (Fig 1D through 1F) of the labeled lipoproteins were assayed. Degradation of ¹²⁵I–Ox-LDL was substantially impaired by digestion of the cells with either heparinase or chondroitinase, or with both enzymes together (Fig 1A), depending on the concentration of the lipoprotein. At the lowest concentration tested (10 µg/mL), Ox-LDL degradation was not affected, whereas at the two highest concentrations, degradation was reduced by 24% and 36%, respectively, by each enzyme or by 44% by both enzymes together (P<.01; Fig 1A). In contrast, GAG digestion did not significantly affect cellular degradation of ¹²⁵I-LDL (Fig 1B) or ¹²⁵I–Ac-LDL (Fig 1C) at any lipoprotein concentration tested. Cell association exhibited a similar pattern of results: digestion of macrophage HS or CS, or of both HS and CS together, significantly (P<.01) reduced the cellular accumulation of ¹²⁵I–Ox-LDL radioactivity by 23%, 31%, and 43%, respectively (Fig 1D), but only at the two highest lipoprotein concentrations. GAG digestion under these conditions had no effect on cellular interactions with either ¹²⁵I-LDL (Fig 1E) or ¹²⁵I–Ac-LDL (Fig 1F). These results indicate that GAG digestion specifically impairs the catabolism of high concentrations of Ox-LDL, with little or no effect on low concentrations of Ox-LDL or on any concentration of native or acetylated LDL.

View larger version (35K): [in this window] [in a new window]   Figure 1. Effect of heparinase or chondroitinase on macrophage uptake of Ox-LDL, native LDL, or Ac-LDL. J-774 A.1 macrophages were incubated with either heparinase or chondroitinase (0.075 U/mL), or both enzymes together, for 1 hour at 37°C, followed by the addition of 125I-labeled lipoprotein (10 to 50 µg of protein per milliliter) and a further incubation for 4 hours at 37°C. Cellular degradation of Ox-LDL (A), LDL (B), or Ac-LDL (C), as well as cell association of Ox-LDL (D), LDL (E), or Ac-LDL (F), were then analyzed. Results represent mean±SD (n=3).

We next studied the effect of GAG digestion on the accumulation of cholesterol mass from Ox-LDL by macrophages. J-774 A.1 cells were preincubated with either heparinase or chondroitinase for 1 hour at 37°C, followed by addition of unlabeled Ox-LDL (100 µg of protein per milliliter) and further incubation for 18 hours at 37°C. In undigested cells, Ox-LDL increased the total cellular cholesterol mass to 1.8 times the control value, whereas the cholesterol mass of heparinase- and chondroitinase-treated cells incubated with Ox-LDL increased to only 1.5 and 1.4 times the control, respectively (Table 1). Incubation of macrophages with GAGases in the absence of Ox-LDL did not affect cellular cholesterol content.

Next we analyzed two types of macrophages that are not transformed cell lines: MPMs and HMDMs. Heparinase digestion of J-774 A.1 cells, MPM, and HMDM significantly (P<.01) reduced the degradation of a high concentration of ¹²⁵I–Ox-LDL (25 µg/mL) by 26%, 43%, and 39%, respectively, in comparison with control nondigested cells (Fig 2). Chondroitinase treatment of the three macrophage preparations reduced ¹²⁵I–Ox-LDL degradation by 40%, 52%, and 41%, respectively (Fig 2). Treatment of these macrophages with GAGases had similar inhibitory effects on cell association of ¹²⁵I–Ox-LDL (Fig 1 and data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 2. Effect of heparinase or chondroitinase on cellular degradation of Ox-LDL by different types of macrophages. J-774 A.1 macrophages (A), MPMs (B), or HMDMs (C) were incubated with either heparinase or chondroitinase (0.075 U/mL) for 1 hour at 37°C. Then 125I-labeled Ox-LDL (25 µg of protein per milliliter) was added to the cells and further incubated for an additional 4 hours at 37°C. Results represent mean±SD (n=3). *P<.01 (vs control).

Furthermore, to determine the effect of different concentrations of GAGases on the cellular uptake of ¹²⁵I–Ox-LDL, J-774 A.1 cells were preincubated with several concentrations of either heparinase or chondroitinase for 1 hour at 37°C, followed by addition of ¹²⁵I-labeled Ox-LDL (25 µg of protein per milliliter) for 4 hours at 37°C prior to the determination of cell-mediated ¹²⁵I–Ox-LDL degradation. Cells treated with chondroitinase induced a significant reduction in Ox-LDL uptake already at low enzyme concentration. Ox-LDL degradation decreased from 1.28±0.54 µg of lipoprotein protein by nontreated cells to 0.89±0.06, 0.79±0.04, 0.75±0.05, and 0.72±0.06 µg of lipoprotein protein by cells treated with 0.025, 0.05, 0.075, or 0.1 U/mL chondroitinase, respectively. Heparinase treatment, on the contrary, affected the cellular uptake of Ox-LDL only from 0.075 U/mL of the enzyme concentration, with no effect at lower concentrations. Ox-LDL degradation decreased from 1.28±0.54 µg of lipoprotein protein by nontreated cells to 1.25±0.05, 1.22±0.06, 1.01±0.10, and 0.86±0.07 µg of lipoprotein protein by cells treated with 0.025, 0.05, 0.075, or 0.1 U/mL of heparinase, respectively.

Effect of Heparinase and Chondroitinase on Cellular GAG Content
To examine the effects of GAGases on the amount of cell-associated GAGs, J-774 A.1 macrophages were incubated for 1 hour at 37°C with increasing concentrations of heparinase or chondroitinase, followed by analyses of cellular GAG content and of the content of released uronic acid in the media. At the highest enzyme concentration studied (0.10 U/mL), chondroitinase and heparinase each caused a significant (P<.01) reduction in cellular GAG content (by 48% and 43%, respectively; Fig 3A) and in parallel increased medium content of uronic acid (Fig 3B), illustrating a dose-dependent digestion of cellular PGs. For the time-course studies, we chose an enzyme concentration that was shown to substantially digest macrophage GAGs (0.075 U/mL; see Fig 3A). After 40 minutes of incubation, both enzymes resulted in a similar significant (P<.01) reduction in cellular GAG content by 25% to 30% (Fig 3C) and in parallel, the uronic acid released to the medium was substantially increased (Fig 3D), illustrating a time-dependent effect of the enzymes on macrophage GAGs. At incubation periods longer than 40 minutes, the macrophage GAG content and medium uronic acid reached a plateau (Fig 3C and 3D). Next, to determine whether the enzymatic action on macrophages can be localized to the plasma membrane, cells were incubated with either heparinase or chondroitinase (0.075 U/mL) for 1 hour at 37°C followed by cell homogenization, subfractionation, and determination of the GAG content in the plasma membrane. The effects of heparinase or chondroitinase on plasma membrane PGs was shown by a significant (P<.01) 35% or 53% reduction in the plasma membrane GAGs content, respectively (55.7±4.3 and 40.6±5.1 µg of GAG per milligram protein in heparinase- and chondroitinase-treated cells in comparison with 85.7±7.9 µg GAG per milligram protein in control nontreated cells, n=3).

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of heparinase and chondroitinase on macrophage PG content. J-774 A.1 macrophages were incubated with either heparinase or chondroitinase at increasing concentrations (0.025 to 0.10 U/mL) for 1 hour at 37°C (A and B). Then cellular GAG content (A), as well as the medium content of uronic acid (B), were determined. J-774 A.1 macrophages were also incubated with a fixed concentration of heparinase or chondroitinase for increasing periods of time (C and D). At the end of the incubation, cellular GAG content (C) and the medium content of uronic acid (D) were determined. Results represent mean±SD (n=3).

Inhibitory Mechanisms of Heparinase and Chondroitinase on the Catabolism of Ox-LDL by Macrophages
Three possible mechanisms by which the GAGases could inhibit the catabolism of high concentrations of Ox-LDL by macrophages were examined: (1) alterations in the Ox-LDL itself; (2) generation of GAG fragments that might interfere with Ox-LDL catabolism; and (3) destruction of cell-associated, particularly cell-surface, GAGs.

Alterations of Ox-LDL by Heparinase or Chondroitinase
First, incubation of Ox-LDL for 4 hours at 37°C in the absence or presence of heparinase or chondroitinase (0.075 U/mL) did not affect the pattern of protein bands obtained for oxidized apoB, as analyzed by SDS-polyacrylamide gel electrophoresis (data not shown), suggesting that there was no artifactual proteolytic effect of the GAGases on Ox-LDL apoB-100. Second, ¹²⁵I–Ox-LDL (25 µg of protein per milliliter) that was incubated in the presence or absence of heparinase or chondroitinase (0.075 U/mL) for 4 hours at 37°C was then isolated by ultracentrifugation and subjected to cell-mediated degradation. In all cases, <5% of the total ¹²⁵I radioactivity was found in the infranatant. Essentially identical degradation was observed with ¹²⁵I–Ox-LDL pretreated in the absence of enzymes, or in the presence of heparinase or chondroitinase (2.19±0.18, 2.23±0.20, and 2.32±0.16 µg of lipoprotein protein was degraded per milligram cell protein, respectively), indicating that the GAGases produced no functional alteration in the ¹²⁵I–Ox-LDL. Finally, J-774 A.1 macrophages were preincubated with either heparinase or chondroitinase, or with both enzymes (0.075 U/mL) for 1 hour at 37°C, followed by addition of ¹²⁵I-labeled Ox-LDL, LDL, or Ac-LDL (25 µg of protein per milliliter), for 4 hours at 4°C to determine lipoprotein cellular binding. Thus, the enzymes are in contact with the lipoproteins only at 4°C and cannot hydrolyze the lipoproteins. Cell treatment with either heparinase or chondroitinase or with both enzymes together resulted in a significant (P<.01) inhibition of ¹²⁵I–Ox-LDL binding to the cells (by 15%, 38%, and 44%, respectively), whereas no significant effect was observed on the cellular binding of LDL or Ac-LDL (Table 2). The results of these three approaches exclude any structural or functional alteration of Ox-LDL that could be caused by the GAGases to impair its uptake by cells.

Involvement of GAG Fragments Generated by GAGase Action on Cellular PGs in Ox-LDL Cellular Metabolism
To directly determine whether GAGases impair Ox-LDL catabolism by macrophages through the generation or destruction of secreted GAGs in the media, macrophage-conditioned media were used. Incubation of J-774 A.1 cells for 10, 30, 60, 120, and 180 minutes resulted in 0, 5, 15, 35, and 37 µg of GAG accumulation per milliliter of medium, respectively. To determine whether these secreted GAGs participate in the cellular uptake of ¹²⁵I–Ox-LDL, we compared the effects of macrophage-conditioned medium collected after 10 minutes of cell incubation (no detectable GAGs) to that obtained after 120 minutes (37 µg of GAG per milliliter) on the binding capacity of ¹²⁵I–Ox-LDL by heparinase- (Fig 4A) or chondroitinase- (Fig 4B) treated J774 A.1 cells. In cells that were treated with heparinase or chondroitinase (in which both cellular GAGs, as well as medium GAGs, are susceptible to hydrolysis) macrophage degradation of ¹²⁵I–Ox-LDL was reduced by 32% and 37%, respectively (Fig 4A and 4B). The addition of macrophage-conditioned medium that contained either no GAGs or 37 µg of GAG per milliliter to the GAGase-treated cells (after cell wash to remove any excess of free enzyme) did not affect the binding of ¹²⁵I–Ox-LDL (Fig 4A and 4B), suggesting that the medium GAG content is not involved in the enhanced macrophage uptake of Ox- LDL. To examine the role in Ox-LDL catabolism of secreted PGs or short GAG fragments generated by the action of heparinase or chondroitinase, medium from incubation of J-774 A.1 macrophages with both heparinase and chondroitinase (0.075 U/mL) for 4 hours at 37°C was removed from the cell layer and the enzymes were completely inactivated by heating at 100°C for 10 minutes. These conditioned media were then added to a second set of macrophages, followed by the addition of ¹²⁵I–Ox-LDL (25 µg of protein per milliliter) for 4 hours prior to the determination of both cellular lipoprotein degradation at 37°C and cellular lipoprotein binding at 4°C. In macrophages that were incubated with conditioned medium pretreated with both enzymes, Ox-LDL cellular degradation and binding were 2.02±0.35 and 2.02±0.21 µg of lipoprotein protein per milligram cell protein, respectively, in comparison to a similar (2.22±0.25 and 2.23±0.18 µg of lipoprotein protein per milligram cell protein) cellular Ox-LDL degradation and binding, respectively, obtained by cells that were similarly incubated with control, nontreated medium. These results suggest that neither secreted PGs nor short GAG fragments generated by the action of heparinase and chondroitinase on cell surface PGs were involved in the reduced macrophage uptake of Ox-LDL.

View larger version (22K): [in this window] [in a new window]   Figure 4. Effect of macrophage-released GAG on Ox-LDL degradation. J-774 A.1 macrophages were preincubated in the absence (control) or presence of either heparinase (A) or chondroitinase (B) for 1 hour at 37°C followed by cell wash in fresh medium. Then 125I-labeled Ox-LDL (25 µg of protein per milliliter) was added to the cells and further incubated for an additional 4 hours in either fresh medium or macrophage-conditioned medium containing no GAG or high GAG levels (37 µg/mL). These conditioned media were obtained by cell incubation at 37°C for 10 minutes and 2 hours, respectively. At the end of the incubation, macrophage degradation of Ox-LDL results represent mean±SD (n=3).

Role of Cell-Associated GAGs in the Catabolism of Ox-LDL
To directly assess the role of cell-associated GAGs in the catabolism of Ox-LDL by macrophages, macrophage binding of ¹²⁵I–Ox-LDL was determined using an experimental design that eliminates GAGs and GAG fragments in the media. J-774 A.1 cells were preincubated for 1 hour at 37°C with heparinase (0.075 U/mL), chondroitinase (0.075 U/mL), both enzymes, or neither enzyme followed by chilling at 4°C and extensive washes at 4°C to remove GAGs and GAG fragments from the media. Fresh, chilled medium containing ¹²⁵I–Ox-LDL (25 µg/mL) was then added, for 4 hours at 4°C, and lipoprotein cellular binding was determined. Thus, in this design, the only differences between treated and untreated cells were the cellular content of GAGs. Predigestion with GAGases produced a substantial reduction in ¹²⁵I–Ox-LDL binding (from 1.96±0.11 µg of lipoprotein protein per milligram cell protein in nontreated cells to 1.76±0.06 or 1.64±0.06 µg of lipoprotein protein per milligram cell protein in heparinase- or chondroitinase-treated cells, respectively), implicating the involvement of cell-associated GAGs in the binding of ¹²⁵I–Ox-LDL to macrophages. To further investigate the role of cellular GAGs in the cellular catabolism of Ox-LDL, macrophages were enriched by the addition of exogenous GAG chains, specifically HS and CS, which have been reported to bind to the several types of cell surface.^{35 36} J774 A.1 cells were incubated in the absence or presence of 50 µg/mL heparan monosulfate or CS A (Sigma Chemical Co) for 1 hour at 37°C, followed by cell wash and analyses of cellular GAG content and macrophage degradation of ¹²⁵I–Ox-LDL (Fig 5). Incubation of J-774 A.1 macrophages with HS or CS significantly (P<.01) increased cellular GAG content by 29% or 41%, respectively, compared with control, nontreated cells (Fig 5A). Most of this increase in macrophage GAG content could be localized to the plasma membrane (data not shown). Degradation of ¹²⁵I–Ox-LDL by macrophages enriched in HS or CS was significantly (P<.01) increased by 15% and 27% over control cells (Fig 5B). No significant effect of GAG enrichment was observed on either cellular degradation of ¹²⁵I-LDL (Fig 5C) or of ¹²⁵I–Ac-LDL (Fig 5D).

View larger version (19K): [in this window] [in a new window]   Figure 5. Effect of macrophage enrichment with GAGs on the cellular uptake of Ox-LDL. J-774 A.1 macrophages were preincubated with HS or CS (50 µg/mL) for 1 hour at 37°C. Then cells were washed with PBS and their total GAG content was determined (A). Macrophage degradation rates of Ox-LDL (B), LDL (C), or Ac-LDL (D) were studied after 4 hours of incubation using 25 µg of 125I-labeled lipoprotein protein per milliliter. Results represent mean±SD (n=3). *P<.01 (vs control, nontreated cell).

To verify that the effects of GAG enrichment were in fact a result of the increased cellular content of GAGs, control, HS-enriched, and CS-enriched cells were digested with heparinase and chondroitinase (0.1 U/mL), prior to determination of ¹²⁵I–Ox-LDL cellular degradation (Table 3). Importantly, heparinase completely abolished the difference in ¹²⁵I–Ox-LDL catabolism between HS-enriched and control cells, whereas chondroitinase had only a slight effect on this difference. Similarly, heparinase had no effect on the difference in ¹²⁵I–Ox-LDL catabolism between CS-enriched and control cells, whereas chondroitinase completely abolished this difference (Table 3). Thus, the GAGases exert their effects on ¹²⁵I–Ox-LDL catabolism by acting specifically on cell-associated GAGs.

MDMs from patients with Sanfilippo's mucopolysaccharidosis, characterized by the accumulation of HSPG in their tissues, were isolated and their cellular uptake of ¹²⁵I–Ox-LDL, -LDL, and -Ac-LDL was analyzed and compared with that of control MDMs. Because these patients are characterized by a lysosomal defect, the parameter used to evaluate ¹²⁵I–Ox-LDL metabolism was the total cellular uptake, rather the lysosomal degradation. The cell association of ¹²⁵I–Ox-LDL to Sanfilippo MDMs was more than double the association to control HMDMs (5.91±0.79 µg of lipoprotein protein in Sanfilippo HMDMs compared with 2.52±0.24 µg of lipoprotein protein in control HMDMs), whereas no significant difference was found in the uptake of ¹²⁵I-labeled native LDL (0.94±0.06 µg of lipoprotein protein in Sanfilippo MDMs compared with 0.96±0.15 µg of lipoprotein protein in control HMDMs) or Ac-LDL (3.23±0.28 in Sanfilippo MDMs compared with 2.98±0.15 in control HMDMs). Finally, a clear correlation between GAG cellular content and Ox-LDL cellular uptake is apparent. When plotting macrophage GAG content versus the cellular uptake of Ox-LDL (as studied by enzymatic digestion of macrophage PGs, as well as by GAG-enriched macrophages), a positive correlation (r=.84, P<.01) was obtained (Fig 6).

View larger version (17K): [in this window] [in a new window]   Figure 6. Positive correlation between macrophage GAG content and Ox-LDL cellular uptake. J-774 A.1 macrophages were incubated for 1 hour at 37°C in the absence or presence of 0.075 U/mL heparinase or chondroitinase, or in the absence or presence of 50 µg/mL HS or CS. Then cellular GAG content, as well as cellular uptake of Ox-LDL, were determined (r=0.84, P<.01).

Interaction Between Cellular PGs and Ox-LDL
Since LPL was shown to be required for HSPG-mediated uptake of native LDL,¹⁵ we questioned whether LPL is also required for macrophage uptake of Ox-LDL. J-774 A.1 macrophages were preincubated with heparinase (0.075 U/mL) for 1 hour at 37°C, followed by the addition of ¹²⁵I-labeled Ox-LDL or LDL (25 µg of protein per milliliter), in the absence or presence of LPL (10 µg/mL) for a further 4 hours of incubation at 37°C. LPL increased macrophage degradation of Ox-LDL by only 15% in comparison with a 65% increment in cellular uptake of native LDL (Table 4). However, whereas exogenous LPL was obligatory for the stimulation of LDL degradation by the macrophage PGs, most of the effect of cellular PGs on the stimulation of Ox-LDL degradation did not require the participation of LPL, as heparinase substantially affect the cellular uptake of Ox-LDL but not that of native LDL, in the absence of LPL (Table 4).

To prove that a direct interaction between Ox-LDL and cellular PGs could occur, Ox-LDL (100 µg/mL) was incubated in the absence or presence of 30 µg/mL HS or CS in PBS with 0.6 µmol/L CaCl_2, reseparated by ultracentrifugation, and the amount of lipoprotein-associated GAGs was determined using the DMMB assay. The amount of lipoprotein-associated GAGs increased from 16 µg/mg lipoprotein in Ox-LDL to 41 and 84 µg/mg lipoprotein in Ox-LDL incubated with either HS or CS, respectively, illustrating a direct interaction of GAG with Ox-LDL.

Interactions of GAGs With Known Receptors in the Catabolism of ¹²⁵I–Ox-LDL
Since cell-associated GAGs could participate in the catabolism of ¹²⁵I–Ox-LDL by macrophages by cooperating with scavenger receptors or by acting as independent binding sites, we sought to determine the roles of SR-As and of CD-36 in the enhanced catabolism of ¹²⁵I–Ox-LDL by GAG-enriched or -depleted macrophages. In preliminary experiments, the addition of unlabeled Ac-LDL (200 µg/mL) to J-774 A.1 macrophages (to maximally block SR-As) inhibited ¹²⁵I–Ox-LDL degradation by 42%, and the addition of anti–CD-36 antibodies (5 µg/mL) to cells (to maximally block CD-36) inhibited cellular degradation of ¹²⁵I–Ox-LDL by 17%. Higher concentrations of Ac-LDL and anti–CD-36 had no additional inhibitory effects on ¹²⁵I–Ox-LDL degradation by J-774 A.1 macrophages (data not shown).

To determine whether the increase in ¹²⁵I–Ox-LDL cellular catabolism observed in macrophages enriched with HS or CS could involve participation of SR-As or CD-36, J-774 A.1 macrophages were incubated for 1 hour at 37°C in the absence or presence of HS or CS (50 µg/mL), followed by cell washes and a 1-hour incubation at 37°C with 200 µg of unlabeled Ac-LDL protein per milliliter or 5 µg of anti–CD-36 antibodies per milliliter and addition of ¹²⁵I–Ox-LDL (25 µg/mL) at 37°C for determination of the lipoprotein cellular degradation or uptake, or addition of ¹²⁵I–Ox-LDL (25 µg/mL) at 4°C for determination of the lipoprotein cellular binding (Table 5). In the absence of Ac-LDL or anti–CD-36 antibodies, HS- and CS-enriched macrophages exhibited increased degradation of ¹²⁵I–Ox-LDL compared with control, unenriched cells (Table 5), as noted above (Fig 6). The presence of excess unlabeled Ac-LDL completely abolished the increase in ¹²⁵I–Ox-LDL degradation by GAG-enriched macrophages over control, whereas anti–CD-36 had no effect on the difference in catabolism between enriched and control cells (Table 5), implicating an interaction between exogenous GAGs and SR-As in the catabolism of high concentrations of Ox-LDL by macrophages. To determine whether similar interactions occur between endogenous GAGs and SR-As, the ability of GAGases to affect ¹²⁵I–Ox-LDL catabolism in the presence or absence of agents that block SR-As or CD-36 was determined. The addition of heparinase or chondroitinase or of both enzymes to macrophages that were preincubated with anti–CD-36 antibodies together with Ac-LDL further reduced macrophage degradation of Ox-LDL (by 19%, 25%, or 39%, respectively) in comparison with cells that were pretreated only with anti–CD-36 antibodies and Ac-LDL (Fig 7), suggesting that macrophage PGs play a role in the cellular binding of Ox-LDL.

View larger version (19K): [in this window] [in a new window]   Figure 7. Unique contribution in addition to that of the cellular CD-36 and Ac-LDL receptor of the macrophage PGs to the cellular uptake of Ox-LDL. J-774 A.1 macrophages were incubated with Ac-LDL (200 µg/mL) or anti CD-36 antibodies (5 µg/mL) together for 1 hour at 37°C. Then heparinase or chondroitinase (0.075 U/mL) or both were added to the incubation medium and further incubated for an additional 1 hour at 37°C. 125I-labeled Ox-LDL (25 µg of protein per milliliter) was added to the cells for an additional 4 hours of incubation at 37°C. Cellular degradation of the lipoprotein was then determined. Results represent mean±SD (n=3); *P<.01 (vs +Ac-LDL +anti–CD-36).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates, for the first time, that macrophage surface GAGs are involved in the catabolism of Ox-LDL and in the accumulation of cholesterol mass by macrophages. The effect is specific to Ox-LDL, particularly when it is present at high concentrations, but there was no role for macrophage surface GAGs under our experimental conditions in the catabolism of native LDL or of Ac-LDL at any concentration. The effect is substantial: removal of cell-surface GAGs can inhibit Ox-LDL catabolism by nearly 50%, and augmentation of cell-associated GAGs can double the catabolic rate. The inhibitory effects of both heparinase and chondroitinase on Ox-LDL cellular uptake and degradation were dose dependent as well as additive, with a more potent effect of chondroitinase. Unlike the uptake of LDL via the macrophage PGs, which required the presence of LPL, LPL was not required for the enhanced uptake of Ox-LDL via the macrophage PGs. Most of the inhibitory effect of the GAGases was observed at a relatively high concentration of Ox-LDL (25 µg of lipoprotein protein per milliliter). Since LDL concentration within the intima and in atherosclerotic lesions was previously shown to be higher than its concentration in circulating plasma,^{56 57} we can assume that the local concentration of oxidized LDL in the atherosclerotic lesion will also be elevated. Moreover, since interaction of Ox-LDL with cellular PGs did not affect the lipoprotein aggregation state, the aggregation modification does not seem to be involved in the GAG-dependent cellular uptake of Ox-LDL.

The inhibitory effect of heparinase and chondroitinase on cellular uptake of Ox-LDL was not limited to J-774 A.1 cultured macrophages but was also observed in other types of macrophages, such as MPMs and HMDMs, suggesting that the PG pathway for the uptake of Ox-LDL is common to macrophages in general. Differences in the uptake of Ox-LDL by different types of macrophages may be explained by differences in the type of PG distribution. Indeed, in MPMs it was shown that HS and CS accumulated in a similar proportion,³⁴ as opposed to the higher content of HS versus CS in J-774 A.1 macrophages.⁵⁸ Moreover, MPMs are growth refractory, whereas the J-774 A.1 cell line can synthesize more new PGs during its incubation period, and this may also contribute to the differences in the interactions of these macrophages with Ox-LDL.

Most, if not all, of the effect of GAGs was on that portion of Ox-LDL catabolism that is mediated through SR-As (Table 5). Several interactions of Ox-LDL and of SR-As with GAGs are likely to occur. First, Ox-LDL can bind highly polyanionic GAGs nearly as well as LDL does, presumably through positively charged domains that remain on apoB after oxidation.²⁷ Because of the relatively low affinity, GAG binding would be most apparent at high concentrations of Ox-LDL, which is where the catabolic effects of GAGs were most evident (see Fig 1). Furthermore, Ox-LDL has unique properties that may allow its catabolism to be specifically enhanced by this binding: Ac-LDL binds GAGs much more poorly,²⁷ and the binding of LDL to GAGs shields the positively charged domains required for interaction with the LDL receptor.⁵⁹ In contrast, binding of Ox-LDL to GAGs (1) could shield positively charged domains on Ox-LDL that otherwise might repel the positively charged binding domain of SR-As; (2) may concentrate Ox-LDL on the cell surface for presentation to SR-As; and (3) might alter the conformation of Ox-LDL to enhance receptor binding.⁶⁰ Each of these potential effects depends largely on simple electrostatic interactions, which may explain the lack of specificity regarding the type (Fig 1 and Table 2 and elsewhere) or cellular origin (Fig 6 and Table 3) of the GAGs.⁶¹ Since the source of HS and CS used in the enrichment experiments was not macrophages, the saccharide sequence that is required for ¹²⁵I–Ox-LDL binding does not seem to be unique.

Binding of highly sulfated GAGs to scavenger receptors has been reported, presumably involving an interaction with the positively charged collagen-like domain of SR-As,^{29 30} but binding of less sulfated forms, such as HS and CS, has not been examined. Binding of highly sulfated GAGs has been reported to block Ox-LDL catabolism,⁶² but less sulfated forms could produce other effects, such as clustering of several SR-As that bind along the same GAG chain or alteration of the conformation of the ligand-binding domain of SR-As.^{63 64} Most of these interactions would be enhanced by close proximity of GAGs to SR-As, either by direct binding of GAGs to SR-As or by the known collagen-binding domains of several PG core proteins interacting with SR-As.

For several reasons, GAGs are likely to have an important role in macrophage catabolism of modified LDL in vivo. GAGs are abundant at all stages of lesion development, are associated with retained LDL and lipoprotein(a), and are regulated by atherogenic stimuli, such as platelet-derived growth factor, transforming growth factor-ß,^{65 66} shear stress,⁶⁷ and cholesterol enrichment.^{68 69} Most GAGs of the arterial wall are synthesized by smooth muscle and endothelial cells, but our results indicate that GAGs derived from nonmacrophage sources readily participate in macrophage-mediated catabolism of Ox-LDL. Differences in macrophage GAG content, either of endogenous or exogenous origin, may account for some of the differences between macrophages in lipid accumulation. Also, molecules that alter lipoprotein interactions with GAGs, such as LPL, are present in lesions in vivo and may further alter scavenger receptor–mediated catabolism of modified lipoproteins.

Overall, our results indicate a novel, regulated influence on Ox-LDL catabolism by macrophage PGs that affects cellular accumulation of cholesterol and may play a role in lesion development in vivo.

Presumably, this binding is the result of persistent positively charged domains within Ox-apoB. Other possible effects of GAG binding to this domain include altering its conformation without necessarily blocking all ligand binding, causing SR-As to cluster if several receptors bind along the same long GAG chain, or facilitating a transfer of ligands from GAGs to SR-As by maintaining these molecules in close proximity. We therefore sought to determine whether GAGs could play a role, either contributory or interfering, in the catabolism of Ox-LDL by macrophages.

	Selected Abbreviations and Acronyms

 

Ac-LDL = acetylated LDL apo = apolipoprotein CS = chondroitin sulfate DMEM = Dulbecco's modified Eagle's medium DMMB = 1,9-dimethylmethylene blue GAG = glycosaminoglycans HMDM = human MDM HS = heparan sulfate LPL = lipoprotein lipase MDA = malondialdehyde MDM = monocyte-derived macrophage MPM = mouse peritoneal macrophage OD = optical density Ox-LDL = oxidatively modified LDL PG = proteoglycan SR-As = class A (collagen-like) scavenger receptors

	Acknowledgments

 
This study was supported by a grant from the Israel Science Foundation and by a grant from the Rappaport Family Institute for Research in Medical Sciences, Haifa, Israel. K.J. Williams is an Established Investigator of the American Heart Association.

Received July 9, 1997; accepted November 11, 1997.

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