This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Edwards, I. J. Articles by Wagner, W. D. Search for Related Content PubMed PubMed Citation Articles by Edwards, I. J. Articles by Wagner, W. D.

(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:400-409.)
© 1995 American Heart Association, Inc.

Articles

Differentiated Macrophages Synthesize a Heparan Sulfate Proteoglycan and an Oversulfated Chondroitin Sulfate Proteoglycan That Bind Lipoprotein Lipase

Iris J. Edwards; Hongzhi Xu; Joseph C. Obunike; Ira J. Goldberg; William D. Wagner

From the Department of Comparative Medicine, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, NC (I.J.E., H.X., W.D.W.), and the Department of Medicine of the College of Physicians and Surgeons of Columbia University, New York, NY (J.C.O, I.J.G.).

Correspondence to Iris J. Edwards, PhD, Department of Comparative Medicine, Bowman Gray School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1040.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract Lipoprotein lipase (LpL), which facilitates lipoprotein uptake by macrophages, associates with the cell surface by binding to proteoglycans (PGs). Studies were designed to identify and characterize specific PGs that serve as receptors for LpL and to examine effects of cell differentiation on LpL binding. PG synthesis was examined by radiolabeling THP-1 monocytes and macrophages (a cell line originally derived from a patient with acute monocytic leukemia) with [³⁵S]sodium sulfate and [³H]serine or [³H]glucosamine. Radiolabeled PGs isolated from the cell surface were purified by chromatography and identified as chondroitin-4–sulfate (CS) PG and heparan sulfate (HS) PG. A sixfold increase in CSPG and an 11-fold increase in HSPG accompanied cell differentiation. Whereas HS glycosaminoglycan chains from both monocytes and macrophages were 7.5 kD in size, CS chains increased in size from 17 kD to 36 kD with cell differentiation, and contained hexuronyl N-acetylgalactosamine-4,6-di-O sulfate disaccharides. LpL binding was sevenfold higher to differentiated cells, and affinity chromatography demonstrated that two cell surface PGs bound to LpL: HSPG and the oversulfated CSPG produced only by differentiated cells. We conclude that differentiation-associated changes in cell surface PG of human macrophages have functional consequences that could increase the atherogenic potential of the cells.

Key Words: macrophages • THP-1 cells • lipoprotein lipase • proteoglycans

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The process of differentiation of peripheral blood monocytes into tissue macrophages is characterized by a series of metabolic shifts to prepare the cells for a role in the immune system. With differentiation comes the ability to produce a wide variety of secretory products and the capacity for antigen presentation, phagocytosis, and microbicidal and tumoricidal activities. Changes in the expression of genes involved in lipid metabolism also accompany monocyte differentiation. Expression of the LDL receptor is replaced by scavenger receptor expression,¹ and apoE² and lipoprotein lipase (LpL)^{3 4} are produced by the differentiated cells. Similar properties have been described for macrophages of atherosclerotic lesions (reviewed in Reference 5⁵ ).

Several processes that regulate macrophage lipid metabolism involve cell surface proteoglycans (PGs). These molecules consist of a plasma membrane–associated core protein to which are linked pendant glycosaminoglycan (GAG) chains of repeating disaccharides. Some cell surface PGs, such as syndecan, contain both chondroitin sulfate (CS) and heparan sulfate (HS) GAG chains,^{6 7} whereas others containing only CS chains have been reported on fibroblasts,^{8 9} smooth muscle cells,¹⁰ and melanoma cells.¹¹ The functions of most of these PGs are presently unknown. The most widely studied cell surface PGs are HSPGs. HSPGs bind apoE,¹² basic fibroblast growth factor,¹³ and antithrombin III¹⁴ and interact with extracellular matrix proteins, including thrombospondin,¹⁵ collagen,¹⁶ and fibronectin.¹⁷ HSPGs also function as receptors for LpL¹⁸ and enhance LpL-mediated cellular uptake of LDL and VLDL.^{19 20 21 22}

Recently, LpL has been shown to enhance the binding of LDL to both CSPG and dermatan sulfate PG, the major extracellular matrix PGs of the artery wall.²³ The interaction of LDL with intercellular arterial PG has for many years been regarded as a principal mechanism for retention and increased macrophage uptake of plasma LDL (for review see Reference 24²⁴ ). The interaction of LDL with cell surface PG has received little attention, and the mechanisms of LpL-facilitated LDL uptake by cell surface PG are poorly understood. Recent evidence that the LDL receptor–related protein is involved in the process in fibroblasts^{19 20 25} but not in macrophages²⁵ indicates that the mechanism may differ between specific cell types. Little is known about the cell surface PG involved, which may also differ according to cell type.

In view of the potential importance of cell surface HSPG to lipid metabolism in monocytes/macrophages, the present studies were designed to characterize cell surface PG of human macrophages and to examine differentiation-associated changes in the PG with functional consequences. The studies were conducted with THP-1 cells, a human monocytic cell line originally derived from a patient with acute monocytic leukemia. These cells can be induced to differentiate by treatment with phorbol myristate acetate (PMA).²⁶ Characteristics of the differentiated cells include macrophage-like expression of membrane antigens, such as CD4, C3b, and FcR II, and the production of cytokines, including interleukins-1 and -1ß and tumor necrosis factor–. The differentiated cells are also phagocytic (for review see Reference 27²⁷ ). Several investigators have used THP-1 cells to model cellular mechanisms of lipid metabolism relative to atherosclerosis. The differentiated cells synthesize and secrete both apoE and LpL.²⁸ Moreover, differentiation leads to downregulation of the LDL receptor and upregulation of scavenger receptors for modified LDL,²⁹ events considered central to the accumulation of cholesteryl ester in monocyte-derived macrophages of the artery wall. The cells also have been shown to bind exogenous LpL in a PG-dependent process, leading to increased uptake and degradation of plasma LDL.¹⁹

The present studies indicate that LpL binds to a cell surface HSPG of both monocytes and macrophages, and also to an oversulfated cell surface CSPG produced only by differentiated cells. Moreover, differentiation of monocytes into macrophages leads to a marked increase in PG synthesis that, we hypothesize, could increase the atherogenic potential of these cells.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
THP-1 cells were purchased from the American Type Culture Collection. Tissue culture reagents were from JRH Biosciences. [³⁵S]Sodium sulfate (600 to 900 Ci/mmol), L-serine [3-³H] (10 to 20 Ci/mmol), and D-[6-³H]glucosamine hydrochloride (20 to 40 Ci/mmol) were purchased from Amersham Corp. Ultrapure guanidine hydrochloride (GdnHCl) and urea were from ICN; Triton X-100 was from Pierce Chemical; and benzamidine hydrochloride, ß-aminohexanoic acid, and ß-mercaptoethanol were from Eastman. Fetal calf serum, tryptamine hydrochloride, papain, PMSF, and pepstatin were from Sigma; CS was from Nutritional Biochemicals Corp; chondroitin ABC lyase (Proteus vulgaris), chondroitin AC II lyase (Arthrobacter aurescens), and unsaturated disaccharide standards containing Di-0S (unsulfated disaccharides), Di-4S, Di-6S (sulfated disaccharides), and Di-diS_E (disulfated disaccharides) were from Seikagaku America, Inc. Ready Safe liquid scintillation cocktail was from Beckman Instruments, and Sepharose CL-4B, Sepharose 6B, and DEAE-Sephacel were from Pharmacia. All other reagents were from Fisher; plastic ware was from Marsh Biomedical or Fisher.

Cell Culture and Labeling
Cells were maintained at 37°C in a 5% CO₂ atmosphere in a culture medium consisting of RPMI-1640 supplemented with 10% fetal bovine serum and 2x10^-5 mol/L ß-mercaptoethanol. Maintenance in the absence of differentiation-inducing stimuli allows the cells to proliferate in a nonadherent state, whereas treatment with the protein kinase C inducer PMA causes the cells to become adherent, growth arrested, and differentiated.²⁶ To prepare differentiated cells, 5x10⁶ cells were plated in 100-mm Petri dishes and cultured for 72 to 96 hours in 10 mL medium containing 200 ng/mL PMA. Control dishes of undifferentiated cells were maintained in the absence of PMA. For radiolabeling, the media of differentiated and undifferentiated cells were replaced with fresh media containing 30 µCi/mL [³⁵S]sodium sulfate and either 30 µCi/mL [³H]glucosamine or 30 µCi/mL [³H]serine. Triplicate cultures were used for all observations.

PG Isolation
Secreted PGs
After 36 hours of radiolabeling, undifferentiated cells were separated from the culture media containing the secreted PG by centrifugation at 450g for 10 minutes. For the differentiated cells that adhered to the dishes, the culture media were removed and cleared of cell debris by centrifugation at 450g for 10 minutes to obtain secreted PG.

Cell Surface PG
Trypsin treatment was used to isolate cell surface PG. The cell pellets of undifferentiated cells and cell sheets of differentiated cells were washed with PBS before the addition of 0.05% trypsin and 0.02% EDTA in PBS. The cell pellets were resuspended in trypsin and incubated for 10 minutes at 37°C. The cell sheets were treated with trypsin for 5 minutes at 37°C, and then cells were scraped into the trypsin and further incubated for 5 minutes at 37°C. The cell suspensions were removed to an ice bath, and a medium rinse of the culture vessel was pooled with each suspension. Aliquots (10 µL) were removed for cell counting by hemocytometry, and the cells were separated from the trypsin fluids by centrifugation for 5 minutes at 450g. The trypsin fluids contained the ectodomains of cell surface PG.

Intracellular Fraction
The pelleted cells were washed once with PBS and treated with 4 mol/L GdnHCl, pH 5.8, containing protease inhibitors³⁰ for 18 hours at 4°C to extract intracellular PG.

All three fractions were dialyzed exhaustively against 0.03 mol/L Na₂SO₄ and 0.02 mol/L NaCl at 4°C to remove unincorporated radiolabel before further purification and characterization of PG.

PG Purification
DEAE Ion-Exchange Chromatography
Samples were dialyzed into a buffer consisting of 7 mol/L urea and 0.1% Triton X-100 in 0.05 mol/L Tris, pH 7.2, and applied to a 2-mL DEAE-Sephacel column. The column was washed with five bed volumes of the buffer with 0.15 mol/L NaCl added, followed by five bed volumes of the buffer without NaCl. A 40-mL continuous gradient of 0 to 1 mol/L NaCl in the buffer was used to elute the PG from the column. Fractions (0.5 mL) were collected and measured for radioactivity and conductivity.

Size-Exclusion Chromatography
Pools of ³⁵S-labeled material from DEAE chromatography were dialyzed into 4 mol/L GdnHCl in 0.05 mol/L sodium acetate, pH 5.8, and chromatographed on a 60x0.9-cm Sepharose CL-4B column eluted with the loading buffer at 4°C. Fractions (0.5 mL) were collected and analyzed for radioactivity.

High-Performance Liquid Chromatography
Aliquots of PGs were diluted in 0.25 mol/L Tris phosphate, pH 7.6, and chromatographed on an HPLC Beckman SEC 4000 column (300x7.5 mm) eluted with 0.25 mol/L Tris phosphate, pH 7.6, at a flow rate of 0.5 mL/min. Radioactivity was measured in 0.25-mL collected fractions by scintillation counting.

PG Characterization
PG Identification by Enzymatic Degradation of GAGs
Chondroitinase ABC and chondroitin AC II lyase sensitivity³¹ were used to identify CS GAG. Aliquots of cell surface PGs were incubated with 0.05 U chondroitin ABC lyase in 0.03 mol/L sodium acetate, 0.1 mol/L Tris, pH 8.0, containing protease inhibitors³¹ or with chondroitin AC II lyase (A aurescens) in the same buffer at pH 7.4 for 16 hours at 37°C. To identify HS GAGs, aliquots of PGs were subjected to nitrous acid degradation by incubation with an equal volume of 20% butyl nitrite: 1 mol/L HCl for 2 hours at 25°C with constant shaking, followed by neutralization with 1 mol/L NaOH.³² Products were chromatographed on a Beckman SEC 4000 column, and movement of ³⁵S radioactivity to the column total volume (V_t) was taken to represent degradation of the GAG chains.

GAG Size Estimation
To prepare GAG chains, 2-mL aliquots of purified cell surface PGs were treated with 200 µL 10N NaOH for 18 hours at 26°C with constant shaking and then neutralized with 10N HCl. To remove core proteins, the samples were adjusted to 7 mol/L urea and loaded on a 1-mL DEAE minicolumn that was washed with 3 bed volumes of 7 mol/L urea, 0.1% Triton X-100, and 0.2 mol/L NaCl in 0.05 mol/L Tris, pH 7.2. GAGs were eluted with a solution of 7 mol/L urea, 0.1% Triton X-100, and 1 mol/L NaCl in 0.05 mol/L Tris, pH 7.2. Urea was removed by dialysis against 10 mmol/L Tris and 0.1% Triton X-100, pH 7.0. CS GAGs were degraded by chondroitin ABC lyase, as described above, to prepare a sample of HS chains. HS GAGs were degraded by treatment with nitrous acid, as described above, to prepare a sample of CS chains. Purified HS or CS GAGs were chromatographed on a Sepharose 6B column using 0.2 mol/L NaCl as eluant. Apparent molecular weights were determined based on a comparison of elution position with a published standard curve.³³

Disaccharide Analysis
Aliquots of CS GAGs were treated with chondroitin ABC lyase as described above, and disaccharides were purified by the addition of three volumes of absolute ethanol for 18 hours at -20°C followed by centrifugation at 8000g for 10 minutes. The ethanol supernatants were air dried, resuspended in 20 µL HPLC solvent containing 1 µg each of Di-0S, Di-4S, Di-6S, and Di-diS_E disaccharide standards, and chromatographed on a 4.6x250-mm Whatman Partisil-10 PAC aminocyano-substituted normal-phase silica column according to the method of Selden et al.³⁴ The HPLC solvent used for elution was 70% acetonitrile/methanol (3:1, vol/vol) and 30% 0.5 mol/L ammonium acetate, pH 5.3.

PG-LpL Interactions
LpL was purified from fresh bovine milk as described by Saxena et al,³⁵ following the method of Socorro et al,³⁶ and stored at -70°C. LpL-Sepharose affinity columns were prepared as previously described,³⁷ using 100 µL gel per minicolumn. PG and GAG samples were dialyzed into 10 mmol/L Tris, pH 7.0, containing 0.1% Triton X-100 and loaded onto the LpL-Sepharose columns. Unbound material was reapplied to the column twice. After four washes with 500 µL 10 mmol/L Tris, PGs were eluted from the column with sequential applications of 3x 200-µL aliquots of 0.15 mol/L NaCl, 3x 200-µL aliquots of 0.4 mol/L NaCl, and 3x 200-µL aliquots of 1.5 mol/L NaCl. All NaCl solutions were in 10 mmol/L Tris, pH 7.0, containing 0.1% Triton X-100. The flow-through from each application was collected separately, and an aliquot was removed for measurement of radioactivity.

Cellular Binding of LpL
Purified LpL was iodinated with 1 mCi of ¹²⁵I, as described by Saxena et al,³⁵ with the use of lactoperoxidase and glucose oxidase enzymes. Binding of iodinated LpL to THP-1 monocytes and macrophages was measured as previously described²⁵ by incubating the cells for 2 hours at 4°C with increasing concentrations of ¹²⁵I-LpL.

Radioactivity Measurements
³⁵S and ³H radioactivity measurements were made in a Packard 1500 Tri-Carb scintillation counter with Ready Safe as the scintillation cocktail.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Increased PG Production in Differentiated Cells
To examine alterations in PG production with cell differentiation, THP-1 cells, either untreated or pretreated for 72 hours with PMA, were radiolabeled for 36 hours with [³⁵S]sodium sulfate. To determine relative distribution of newly synthesized PG, high–molecular weight, ³⁵S-labeled macromolecules were isolated from the culture media, cell surface, and intracellular compartments (Fig 1). Secreted PGs constituted the major fraction of ³⁵S-labeled molecules in both undifferentiated and differentiated cells. The relative distributions for the media, cell surface, and intracellular compartments were 85:6:8 and 81:13:6 for monocytes and macrophages, respectively. Incorporation of ³⁵S was significantly greater in the media (P<.0001) and cell surface (P<.001) fractions for differentiated cells than for undifferentiated cells. Size-separation chromatography demonstrated a reduced molecular size following base treatment of media and cell surface fractions, indicating that these fractions consisted of PGs. Intracellular fractions were unaffected by base treatment and therefore consisted only of GAG chains (data not shown). Because trypsin was used to isolate the cell surface PGs, these PGs had to have been the ectodomains of membrane-associated PGs rather than intact PGs. In addition, because there were no detectable PGs associated with the cells after trypsin treatment, there appeared to have been little expression of trypsin-resistant PGs on the surface of these cells. When Triton X-100 was used to extract total cell PG, levels of radiolabeled PGs similar to the sum of trypsin-released and intracellular PG were measured in both monocytes and macrophages. This indicated that the macrophages, although adherent, were laying down very little PG-containing extracellular matrix.

View larger version (13K): [in this window] [in a new window]   Figure 1. Bar graph showing distribution of 35S-labeled proteoglycans to different cellular pools in monocytes and macrophages. THP-1 monocytes (shaded bars) and macrophages (open bars) were labeled with 50 µCi/mL [35S]sodium sulfate for 36 hours. Radiolabeled proteoglycans were measured in media, cell surface, and intracellular fractions after exhaustive dialysis. Values are mean±SEM of triplicate samples.

Production of CSPG and HSPG by THP-1 Cells
To identify specific PGs, aliquots of each fraction were treated with GAG-degrading enzymes or chemicals and chromatographed on an HPLC size-exclusion column. Degradation of GAG chains was measured by movement of radiolabel to the column V_t. Fig 2 shows the HPLC profiles of cell surface PGs. The untreated PGs for both monocytes and macrophages had a broad distribution, with a slight separation of two peaks apparent in the sample from differentiated cells. Treatment with nitrous acid, which specifically cleaves the N-sulfated residues of HSPG, resulted in degradation of 23% of radiolabel from monocytes and 36% from macrophages. Seventy-seven percent and 64% of ³⁵S-labeled PGs from monocytes and macrophages, respectively, were degraded with chondroitinase ABC, indicating the presence of CSPG. Treatment with chondroitin AC II lyase, which cleaves only glucuronosyl-galactosamine linkages and not the iduronosyl-galactosamine linkages present in dermatan sulfate, resulted in profiles similar to those produced by chondroitinase ABC, indicating no detectable dermatan sulfate PG produced by THP-1 cells (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 2. Size-exclusion chromatograph of 35S-labeled cell surface proteoglycans (PGs) treated chemically or enzymatically to identify PG type. PGs, isolated from the cell surface of THP-1 monocytes (A) or macrophages (B) by trypsin treatment, were separated on a high-performance liquid chromatography SEC 4000 column eluted with 0.25 mol/L Tris phosphate, pH 7.6, at 0.5 mL/min after no treatment (solid line) or treatment with chondroitinase ABC for 16 hours at 37°C (dashed line) or nitrous acid for 2 hours at 26°C (broken line). Vo indicates void volume; Vt, total volume.

On the basis of the enzyme and chemical analyses, it was possible to calculate the radioactivity associated with CSPG and HSPG (Fig 3). With cell differentiation, there was an increase in both CSPG and HSPG in all compartments. CS increased threefold in media and twofold in intracellular compartments, whereas HS increased fivefold in both. The largest differences were observed in the cell surface PG, of which HSPG increased 11-fold and CSPG increased sixfold.

View larger version (15K): [in this window] [in a new window]   Figure 3. Bar graphs showing comparison of chondroitin sulfate proteoglycans (CSPG, hatched bars) and heparan sulfate proteoglycans (HSPG, solid bars) in different cellular pools in monocytes and macrophages. Media, cell surface, and intracellular pools were obtained as described in Fig 1. Radioactivity associated with CSPG and HSPG in each fraction was measured based on susceptibility to chondroitinase ABC or nitrous acid, as described in Fig 2. -PMA indicates no treatment with phorbol myristate acetate (cells not induced to differentiate); +PMA, treatment with phorbol myristate acetate (cells induced to differentiate).

Increased Numbers of PG Molecules Produced by Differentiated Cells
The increased ³⁵S incorporation into PG produced by macrophages could indicate (1) increased numbers of PGs, (2) greater sulfation of GAG chains, (3) increased numbers of GAGs per PG molecule, or (4) longer GAG chains in differentiated cells. To test whether the amount of sulfation per core protein was increased, cells were radiolabeled for 36 hours with both [³⁵S]sodium sulfate and [³H]serine. PGs isolated from the cell surface were purified by DEAE-Sephacel ion-exchange chromatography using a linear gradient of 0 to 1 mol/L NaCl for elution (Fig 4). The ³⁵S-labeled PGs from both undifferentiated and differentiated cells eluted with 0.5 mol/L NaCl, and were associated with 17% and 22% of the total bound ³H label from monocytes and macrophages, respectively. The separations failed to resolve distinct ³H peaks coeluting with the ³⁵S peaks, indicating that additional purification steps were necessary.

View larger version (17K): [in this window] [in a new window]   Figure 4. Elution profiles of cell surface proteoglycans from monocytes (A) and macrophages (B) on DEAE-Sephacel. Cells were radiolabeled for 36 hours with 30 µCi/mL [35S]sodium sulfate and 30 µCi/mL [3H]serine. Proteoglycans were isolated from the cells by trypsin treatment, dialyzed into a solution composed of 7 mol/L urea and 0.1% Triton X-100 in 0.05 mol/L Tris, pH 7.2, and chromatographed on a DEAE-Sephacel column equilibrated with the same buffer. Proteoglycans were eluted with a 40 mL linear gradient of 0 to 1 mol/L NaCl in urea/Tris buffer. Fractions (0.5 mL) were collected and analyzed for radioactivity. 35S, solid line; 3H, dotted line; NaCl, dashed line.

To further remove non-PG ³H-labeled proteins, peak ³⁵S fractions from DEAE-Sephacel were pooled and chromatographed under dissociative conditions on Sepharose CL-4B (not shown). This resulted in the removal of 61% and 70% of the ³H-labeled material from monocytes and macrophages, respectively, and provided a distinct peak of ³⁵S/³H-labeled PG from each cell type. The radioactivity associated with the purified PG is shown in Table 1. Incorporation of both ³H and ³⁵S was higher in macrophages (ninefold and 17-fold, respectively), indicating increased production of both PG core protein and GAG, ie, increased numbers of PG molecules. However, the ratio of ³⁵S to ³H was doubled in PG from macrophages, suggesting that the relationship of PG core protein to GAG was altered in differentiated cells. This interpretation of the data assumes that labeling of the core proteins results in similar specific activities and that similar types of PG are being produced. Although there is no evidence from other cells to suggest that distinct new types of PG are associated with differentiation, this possibility cannot be ruled out without further studies.

View this table: [in this window] [in a new window]   Table 1. Incorporation of [35S]Sodium Sulfate and [3H]Serine Into Cell Surface Proteoglycans

Longer GAG Chains of Cell Surface CSPG of Macrophages
To assess whether the increased ³⁵S in macrophage PG was due to longer GAGs or increased sulfation, [³⁵S]sodium sulfate and [³H]glucosamine were used to radiolabel GAG sulfate and sugar residues. PGs isolated from the cell surface were base treated to release GAG chains from the core proteins, and GAGs were separated by DEAE-Sephacel ion-exchange chromatography. GAGs were further purified by chromatography on Sepharose 6B columns. Table 2 shows the radioactivity associated with total GAG. Incorporation of ³⁵S was 30-fold higher and ³H was 23-fold higher in differentiated cells, the higher ratio of ³⁵S/³H indicating a possible increase in sulfation of macrophage GAG.

View this table: [in this window] [in a new window]   Table 2. Incorporation of [35S]Sodium Sulfate and [3H]Glucosamine Into Glycosaminoglycans

The most likely reason for the increased chain labeling in macrophage PG was longer GAG chains. GAG length was measured by elution position on a Sepharose 6B column compared with published calibration curves.³³ When nitrous acid treatment was used to degrade HS chains, degradation products appeared at the column V_t, and the remaining CS GAG had an average molecular size of 17 kD for CS from monocytes and 36 kD for CS from macrophages (Fig 5A). By contrast, when chondroitinase ABC was used to degrade CS chains, the remaining HS GAGs from both undifferentiated and differentiated cells had an average molecular size of 7.5 kD (Fig 5B). Therefore, only the CS chains increased in size with cell differentiation.

View larger version (13K): [in this window] [in a new window]   Figure 5. Elution profiles of 35S-labeled glycosaminoglycans (GAGs) chromatographed on Sepharose 6B. GAGs were prepared by base treatment of cell surface proteoglycans and purified by chromatography on DEAE-Sephacel. Purified GAGs were treated with either nitrous acid (A) or chondroitinase ABC (B) and chromatographed on Sepharose 6B using 0.2 mol/L NaCl as eluant. Fractions (0.5 mL) were collected and analyzed for radioactivity. CS indicates chondroitin sulfate; HS, heparan sulfate; Vo, void volume; and Vt, total volume. GAG from monocytes, dashed line; GAG from macrophages, solid line.

Disulfated Disaccharides in GAGs of Macrophage Cell Surface CSPG
CS GAG chains can be sulfated at C4 or C6 of the hexosamine. To examine a possible alteration in sulfation of CS GAG with cell differentiation, chains from Sepharose 6B were treated with chondroitinase ABC, and the disaccharides were identified by HPLC (Table 3). As determined with a detection limit of approximately 100 dpm, the digestion products of CS from monocytes contained only Di-4S. CS from macrophages, however, contained a second component that eluted with the Di-diS_E standard, indicating that some of the chains contained hexuronyl N-acetylgalactosaminyl-4,6-di-O sulfate disaccharides. No unsulfated disaccharides were detected in GAGs from either cell type.

View this table: [in this window] [in a new window]   Table 3. Disaccharides of Cell Surface Chondroitin Sulfate

Greater Amounts of LpL Bound by Macrophages
The next set of experiments was designed to determine whether the differentiation-associated changes in PG biochemistry lead to functional changes in the PG molecules. Because LpL is known to bind to cell surface PG, the first study compared the binding of ¹²⁵I-LpL to undifferentiated and differentiated cells. As shown in Fig 6, during a 4-hour incubation at 4°C, binding of LpL was increased in the differentiated macrophages.

View larger version (13K): [in this window] [in a new window]   Figure 6. Line graph showing lipoprotein lipase (LpL) binding to THP-1 monocytes and macrophages. Monocytes and macrophages were incubated for 2 hours at 4°C with increasing concentrations of 125I-LpL. Values represent mean±SEM of cell-bound radioactivity released with 100 U/mL heparin. Monocytes, dashed line; macrophages, solid line.

LpL Bound by CSPG and HSPG From Macrophages
To further examine the differentiation-associated changes in cell surface PG on interaction with LpL, purified PGs were added to an LpL affinity column. A stepwise NaCl gradient of 0.15 mol/L and 0.4 mol/L was used to identify PG-LpL interactions that would dissociate (0.15 mol/L NaCl) or remain stable (0.4 mol/L NaCl) under physiological conditions (Fig 7). Thirty-four percent of the PG from monocytes demonstrated stable binding. This high-affinity fraction was higher in macrophages, comprising 58% of the PG in these cells. The high proportion of LpL-binding PG in differentiated cells was surprising because studies (Fig 2) had determined that HSPG (the major reported LpL-binding PG) accounted for only 30% of cell surface PG in these cells.

View larger version (14K): [in this window] [in a new window]   Figure 7. Chromatograph showing lipoprotein lipase (LpL) affinity chromatography of 35S-labeled cell surface proteoglycans (PGs) from monocytes (- - - -) and macrophages (——). PGs, purified by DEAE-Sephacel and CL-4B–Sepharose chromatography, were dialyzed into 10 mmol/L Tris, pH 7.0, containing 0.1% Triton X-100, loaded on an LpL-Sepharose affinity column, and eluted using a stepwise NaCl gradient. Fractions (0.2 mL) were collected and analyzed for radioactivity.

To determine whether cell surface CSPG was also interacting with the lipase, the identity of the PG in the 0.15 mol/L and 0.4 mol/L NaCl eluates was examined. Aliquots of each peak from the LpL column were treated with either chondroitinase ABC or nitrous acid and chromatographed by HPLC to assess degradation of the GAG chains. On the basis of susceptibility to chondroitinase ABC, the weakly interacting PG (0.15 mol/L NaCl) was shown to be primarily CSPG (Fig 8A). On the basis of susceptibility to nitrous acid (Fig 8B) and chondroitinase ABC (data not shown), the stably bound PG requiring >0.4 mol/L NaCl for removal from the lipase consisted of both CSPG and HSPG.

View larger version (12K): [in this window] [in a new window]   Figure 8. Size-exclusion chromatograph of 35S-labeled macrophage proteoglycans (PGs) eluted from a lipoprotein lipase–Sepharose affinity column (see Fig 7) and treated enzymatically or chemically to identify PG type. The 0.15 mol/L peak (A) was treated with chondroitinase ABC and the 0.4 mol/L peak (B) was treated with nitrous acid before chromatography on a high-performance SEC 4000 column. Vo indicates void volume; Vt, total volume; untreated PG, solid line; PG treated with chondroitinase ABC or nitrous acid, dashed line.

To confirm these data, aliquots of the 0.4 mol/L NaCl material were treated with either chondroitinase ABC or nitrous acid and reloaded on the lipase column. As shown in Table 4, when an aliquot of the peak was reapplied to the column, 97% bound a second time. Chondroitinase ABC treatment to hydrolyze CS chains resulted in only 23% of the PG being able to bind to the column, indicating that the peak consisted of 77% CSPG. To confirm this, nitrous acid treatment was used to degrade HS chains; after this treatment, 80% of the PGs were still able to bind to the lipase.

View this table: [in this window] [in a new window]   Table 4. Identification of Lipoprotein Lipase–Bound Proteoglycans

LpL Binding by GAG Chains of the PGs
The previous experiments, though indicating a PG-LpL interaction by means of the GAG chains, did not rule out possible core protein–LpL interactions. To investigate such interactions, cell surface PGs were base treated, separated on DEAE to isolate protein-free chains, and applied to LpL-Sepharose. Eighty-eight percent of GAG, compared with 81% of control PG, demonstrated stable binding (>0.4 mol/L NaCl) to the LpL, indicating that the core protein is not required for PG-LpL interaction.

Double-radiolabeled PGs purified through DEAE and Sepharose CL-4B were also used to determine whether the LpL-binding CS and HS chains were attached to the same core protein. When these PGs were treated with chondroitinase ABC before LpL affinity chromatography, 84% of the ³H label was removed in the unbound fraction. This indicates that the CS and HS chains are on separate core proteins rather than being part of a hybrid molecule, because the radiolabeled protein of a hybrid molecule would be expected to retain association with the LpL by means of the HS chains even after removal of the CS chains.

Disulfated Disaccharides in CS GAG That Bind LpL
To determine whether a unique disaccharide structure in CS chains could account for enhanced binding to LpL, CS GAGs were eluted from LpL by either 0.15 mol/L or 0.4 mol/L NaCl, and disaccharides were prepared by chondroitinase ABC digestion. As shown in Fig 9, Di-4S, Di-6S, and Di-diS_E (4- and 6-sulfated) were separated by HPLC. Very little radiolabeled disaccharide eluted with the 6-sulfated standard. Di-diS_E disaccharide constituted approximately 18% of the total disaccharides of the 0.4 mol/L NaCl fraction but <3% of the 0.15 mol/L fraction. Thus, the disulfated disaccharides were found predominantly in the CS which demonstrated stable interaction with LpL.

View larger version (16K): [in this window] [in a new window]   Figure 9. Chromatograph showing separation of disaccharides of [3H]glucosamine-radiolabeled chondroitin-4–sulfate glycosaminoglycans (GAGs) with low and high affinity for lipoprotein lipase (LpL). Proteoglycans were chromatographed on an LpL-Sepharose affinity column, as described in Fig 7, and peak material was treated with chondroitinase ABC. Disaccharides were purified with ethanol and chromatographed on a 250x4.5-mm Whatman Partisil-10 PAC aminocyano-substituted normal-phase silica column eluted with 70% acetonitrile/methanol (3:1 vol/vol) and 30% ammonium acetate/acetic acid (0.5 mol/L), pH 5.3. Mixed standards added to each sample were detected by absorbance at 254 nm.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Many macrophage functions are associated with differentiated cells. The progression of blood monocytes to tissue macrophages requires changes in cell surface molecules, both to facilitate the interaction of the cells with their new environment and to reflect new cell functions. The results of the present studies provide important evidence to link differentiation-associated changes in cell surface PG to altered macrophage lipid metabolism.

Several important observations were made in these studies: (1) Both CSPG and HSPG were identified as cell surface PG of THP-1 macrophages; (2) when the cells were induced to differentiate, there was a sixfold increase in cell surface CSPG and an 11-fold increase in cell surface HSPG; (3) structural changes in cell surface CSPG associated with cell differentiation included longer GAG chains and the production of CS chains containing hexuronyl N-acetylgalactosaminyl-4,6-di-O sulfate disaccharide units; and (4) both HSPG and the oversulfated CSPG of differentiated cells were capable of binding LpL, suggesting that both may be involved in modification of macrophage lipid metabolism.

The identification of HSPG as a cell surface PG was not surprising, because HSPG is ubiquitous on most cell types. Both pigeon^{38 39} and guinea pig⁴⁰ peritoneal macrophages produce a cell surface HSPG, as do several murine macrophage cell lines.⁴¹ However, in some studies HSPG was not detected on the cell surface of monocyte/macrophage cell lines. Murine cell lines M1⁴² and P388D1⁴³ as well as the human monocytic U937 cells⁴⁴ were shown to produce exclusively CSPG. Recently, CSPG was reported to be the only cell-associated PG of human peritoneal macrophages.⁴⁵

Cell surface HSPGs may act as receptors for several ligands, including LpL,¹⁸ apoE,¹² and basic fibroblast growth factor,¹³ as well as the herpes simplex virus⁴⁶ and the human immunodeficiency virus.⁴⁷ The proposed model for involvement of PG binding with cell uptake of these ligands involves interaction with cell surface HSPG as an essential first step for binding the ligand to its high-affinity receptor. Little is known about these processes in macrophages, but the present studies have identified a cell surface HSPG that may be similar in function to the HSPG of other cell types.

Although the amount of cell surface HSPG was increased in cell differentiation, we were unable to detect any differentiation-associated structural changes such as increased GAG chain size. Observation of more discrete changes involving saccharide composition will require further studies. Differentiation-associated structural changes in cell surface CSPG were apparent in these studies. CS GAG chain size increased from an average of 17 kD in monocytes to 36 kD in macrophages. This is in contrast to the secreted CSPG in the human monoblastic cell line U937, which was substituted with 30-kD chains before differentiation and 17 kD after differentiation.⁴⁴ Because THP-1 cells become adherent when induced to differentiate, the increased GAG length of cell surface CSPG may be related to the process of adherence rather than to differentiation per se, or it may represent a link between the two processes. However, no differentiation-associated changes in GAG size were observed in the cell-associated CSPG of the murine leukemic cell line, M1, which also become adherent when they differentiate.⁴² As in the THP-1 cells of the present study, induction of differentiation in the human myeloid leukemic cell line HL-60 is associated with an increase in GAG chain length.⁴⁸

The production of oversulfated CS chains is not unique to THP-1 cells. Kolset and coworkers^{49 50} described such chains in the secreted PGs of both human peritoneal macrophages and monocyte-derived macrophages. N-acetylgalactosamine-4,6-di-O sulfate units have also been identified in CSPG from mouse bone marrow,⁵¹ rat serosal mast cells,⁵² human lung mast cells,^{53 54} and human basophilic leukocytes from patients with leukemia.⁵⁵ In the latter cells, however, the oversulfated CSPG was found within the secretory granules. A proposed role for secretory granule PG involves their concentration and the stabilization of secretory granule enzymes, although there is little experimental evidence to support such a role (reviewed in Reference 56⁵⁶ ).

The role and biological significance of the oversulfated CSPG on the cell surface are unknown, but its interaction with LpL suggests a potential for alteration of cell lipid metabolism. Previous studies have not indicated involvement of a cell surface CSPG in binding LpL. Pretreatment with chondroitinase ABC failed to reduce LpL binding to endothelial cells¹⁹ or THP-1 cells.²⁵ However, in those studies, only 80% of LpL binding was prevented by heparinase treatment. It is possible that cell surface CSPG may be located in cell crevices that protect it from enzymatic removal. Because our studies indicate that disulfation of the CS hexosamine imparts its LpL-binding property to the molecule, LpL binding to CSPG may occur only in macrophages and not in endothelial cells that do not produce an oversulfated CS.

Several lines of evidence suggest that a number of the biological manifestations of atherosclerosis are due to changes in arterial PG. Rapidly proliferating arterial smooth muscle cells from atherosclerosis-susceptible pigeons are associated with decreased HSPG¹⁰ and HS chains of altered disaccharide structure (Wagner WD, Edwards IJ, Flory DM, manuscript in preparation). CSPG is a major component of atherosclerotic lesions, and a correlation between apoB and artery CS content has been demonstrated in lesion tissue.⁵⁷ The highly sulfated CSPG identified in the present studies may be especially atherogenic. LpL is also a component of the atherosclerotic lesion, and Zilversmit⁵⁸ has proposed that LpL may promote atherogenesis by producing remnant lipoproteins for macrophage uptake. Several experimental studies have supported the concept of an atherogenic role for LpL by demonstrating enhanced cell uptake of lipoproteins in the presence of LpL.^{18 19 20 21 22} This study provides further evidence that changes in PGs may have several and varied effects on events within the atherosclerotic lesion. We hypothesize that such PG biochemical changes might, in fact, be central to the pathogenesis of atherosclerosis.

	Acknowledgments

 
This work was supported by grants HL-25161, HL-45848, and HL-45095 from the National Heart, Lung, and Blood Institute and grant NC91-G-7 from the North Carolina Heart Association. The authors thank Mary Jo Wright for maintenance of cell cultures, Lonnie Ellis and Judy Anderson for secretarial assistance, and Karen Klein for editorial assistance.

Received September 19, 1994; accepted December 28, 1994.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Shimano H, Yamada N, Ishibashi S, Harada K, Matsumoto A, Mori N, Inaba T, Motoyoshi K, Itakura H, Takaku F. Human monocyte colony-stimulating factor enhances the clearance of lipoproteins containing apolipoprotein B-100 via both low density lipoprotein receptor-dependent and -independent pathways in rabbits. J Biol Chem. 1990;265:12869-12875. [Abstract/Free Full Text]

2. Wang-Iverson P, Gibson JC, Brown WV. Plasma apolipoprotein E secretion by human monocyte derived macrophages. Biochim Biophys Acta. 1985;834:256-262. [Medline] [Order article via Infotrieve]

3. Chait A, Iverius PH, Brunzell JD. Lipoprotein lipase secretion by human monocyte-derived macrophages. J Clin Invest. 1982;69:490-493.

4. Khoo JC, Mahoney EM, Witztum JL. Secretion of lipoprotein lipase by macrophages in culture. J Biol Chem. 1981;256:7105-7108. [Abstract/Free Full Text]

5. Libby P, Clinton SK. The role of macrophages in atherogenesis. Curr Opin Lipidol. 1993;4:355-363.

6. Rapraeger A, Jalkanen M, Endo E, Koda J, Bernfield M. The cell surface proteoglycan from mouse mammary epithelial cells bears chondroitin sulfate and heparan sulfate glycosaminoglycans. J Biol Chem. 1985;260:11046-11052. [Abstract/Free Full Text]

7. David G, Van Den Berghe H. Cell-surface heparan sulfate and heparan-sulfate/chondroitin-sulfate hybrid proteoglycans of mouse mammary epithelial cells. Eur J Biochem. 1989;178:609-617. [Medline] [Order article via Infotrieve]

8. Hedman K, Christner J, Julkunen I, Vaheri A. Chondroitin sulfate at the plasma membranes of cultured fibroblasts. J Cell Biol. 1993;97:1288-1293. [Abstract/Free Full Text]

9. David G, Lories V, Heremans A, Van Der Schueren B, Cassiman JJ, Van Den Berghe H. Membrane-associated chondroitin sulfate proteoglycans of human lung fibroblasts. J Cell Biol. 1989;108:1165-1175. [Abstract/Free Full Text]

10. Edwards IJ, Wagner WD. Cell surface heparan sulfate proteoglycan and chondroitin sulfate proteoglycan of arterial smooth muscle cells. Am J Pathol. 1992;140:193-205. [Abstract]

11. Bumol TF, Walker LE, Reisfeld RA. Biosynthetic studies of proteoglycans in human melanoma cells with a monoclonal antibody to a core glycoprotein of chondroitin sulfate proteoglycans. J Biol Chem. 1984;259:12733-12741. [Abstract/Free Full Text]

12. Zhong-Sheng J, Brecht WJ, Miranda RD, Hussain MM, Innerarity TL, Mahley RW. Role of heparan sulfate proteoglycans in the binding and uptake of apolipoprotein E-enriched remnant lipoproteins by cultured cells. J Biol Chem. 1993;268:10160-10167. [Abstract/Free Full Text]

13. Moscatelli D. High and low affinity binding sites for basic fibroblast growth factor on cultured cells: absence of a role for low affinity binding in the stimulation of plasminogen activator production by bovine capillary endothelial cells. J Cell Physiol. 1987;131:123-130. [Medline] [Order article via Infotrieve]

14. Marcum JA, Rosenberg RD. Anticoagulantly active heparan sulfate proteoglycan and the vascular endothelium. Semin Thromb Hemost. 1987;13:464-467. [Medline] [Order article via Infotrieve]

15. Sun X, Mosher DF, Rapraeger A. Heparan sulfate-mediated binding of epithelial cell surface proteoglycans to thrombospondin. J Biol Chem. 1989;264:2885-2889. [Abstract/Free Full Text]

16. Koda JE, Bernfield M. Heparan sulfate proteoglycans from mouse mammary epithelial cells: basal extracellular proteoglycan binds specifically to native type I collagen fibrils. J Biol Chem. 1984;259:11763-11770. [Abstract/Free Full Text]

17. Saunders S, Bernfield M. Cell surface proteoglycan binds mouse mammary epithelial cells to fibronectin and behaves as a receptor for interstitial matrix. J Cell Biol. 1988;106:423-430. [Abstract/Free Full Text]

18. Saxena U, Klein MG, Goldberg IJ. Identification and characterization of the endothelial cell surface lipoprotein lipase receptor. J Biol Chem. 1991;266:17516-17521. [Abstract/Free Full Text]

19. Rumsey SC, Obunike JC, Arad Y, Deckelbaum RJ, Goldberg IJ. Lipoprotein lipase-mediated uptake and degradation of low density lipoproteins by fibroblasts and macrophages. J Clin Invest. 1992;90:1504-1512.

20. Williams KJ, Fless GM, Petrie KA, Snyder ML, Brocia RW, Swenson TL. Mechanisms by which lipoprotein lipase alters cellular metabolism of lipoprotein(a), low density lipoprotein, and nascent lipoproteins. J Biol Chem. 1992;267:13284-13292.[Abstract/Free Full Text]

21. Mulder M, Lombardi P, Jansen H, van Berkel TJC, Frants RR, Havekes LM. Heparan sulphate proteoglycans are involved in the lipoprotein lipase-mediated enhancement of the cellular binding of very low density and low density lipoproteins. Biochem Biophys Res Commun. 1992;185:582-587. [Medline] [Order article via Infotrieve]

22. Eisenberg S, Sehayek E, Olivecrona T, Vlodavsky I. Lipoprotein lipase enhances binding of lipoproteins to heparan sulfate on cell surfaces and extracellular matrix. J Clin Invest. 1992;90:2013-2021.

23. Edwards IJ, Goldberg IJ, Parks JS, Xu H, Wagner WD. Lipoprotein lipase enhances the interaction of low density lipoproteins with artery-derived extracellular matrix proteoglycans. J Lipid Res. 1993;34:1155-1163. [Abstract]

24. Camejo G, Camejo EH, Olsson U, Bondjers G. Proteoglycans and lipoproteins in atherosclerosis. Curr Opin Lipidol. 1993;4:385-391.

25. Obunike JC, Edwards IJ, Rumsey SC, Strickland DK, Curtiss LK, Wagner WD, Deckelbaum RJ, Goldberg IJ. Cellular differences in lipoprotein lipase-mediated uptake of low density lipoproteins. J Biol Chem. 1994;269:13129-13157. [Abstract/Free Full Text]

26. Tsuchiya S, Kobayashi Y, Goto Y, Okumura H, Nakae S, Konno T, Tada K. Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. Cancer Res. 1982;42:1530-1536. [Abstract/Free Full Text]

27. Auwerx J. The human leukemia cell line, THP-1: a multifaceted model for the study of monocyte-macrophage differentiation. Experientia. 1991;47:22-31. [Medline] [Order article via Infotrieve]

28. Tajima S, Hayashi R, Tsuchiya S, Miyake Y, Yamamoto A. Cells of human monocytic leukemia cell line (THP-1) synthesize and secrete apolipoprotein E and lipoprotein lipase. Biochem Biophys Res Commun. 1985;126:526-531. [Medline] [Order article via Infotrieve]

29. Hara H, Tanishita H, Yokoyama S, Tajima S, Yamamoto A. Induction of acetylated low density lipoprotein receptor and suppression of low density lipoprotein receptor on the cells of a human monocytic leukemia cell line. Biochem Biophys Res Commun. 1987;146:802-808. [Medline] [Order article via Infotrieve]

30. Salisbury BGJ, Wagner WD. Isolation and preliminary characterization of proteoglycans dissociatively extracted from human aorta. J Biol Chem. 1981;256:8050-8057. [Abstract/Free Full Text]

31. Oike Y, Kimata K, Shinomura T, Nakazawa K, Suzuki S. Structural analysis of chick-embroyo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-ß-galactosidase (keratanase). Biochem J. 1980;191:193-207. [Medline] [Order article via Infotrieve]

32. Shively JE, Conrad HE. Formation of anhydrosugars in the chemical depolymerization of heparin. Biochemistry. 1976;15:3932-3942. [Medline] [Order article via Infotrieve]

33. Wasteson A. A method for the determination of the molecular weight and molecular-weight distribution of chondroitin sulphate. J Chromatogr. 1971;59:87-97. [Medline] [Order article via Infotrieve]

34. Selden DS, Seno N, Austen KF, Stevens RL. Analysis of polysulfated chondroitin disaccharides by high-performance liquid chromatography. Anal Biochem. 1984;141:291-300. [Medline] [Order article via Infotrieve]

35. Saxena U, Witte LD, Goldberg IJ. Release of endothelial cell lipoprotein lipase by plasma lipoproteins and free fatty acids. J Biol Chem. 1989;264:4349-4355. [Abstract/Free Full Text]

36. Socorro L, Green CC, Jackson RL. Preparation of a homogenous and stable form of bovine milk lipoprotein lipase. Prep Biochem. 1985;15:133-143. [Medline] [Order article via Infotrieve]

37. Saxena U, Goldberg IJ. Interaction of lipoprotein lipase with glycosaminoglycans and apolipoprotein C-II: effects of free-fatty-acids. Biochim Biophys Acta. 1990;1043:161-168. [Medline] [Order article via Infotrieve]

38. Owens RT, Wagner WD. Chondroitin sulfate proteoglycan and heparan sulfate proteoglycan production by cultured pigeon peritoneal macrophages. J Leukoc Biol. 1992;51:626-633. [Abstract]

39. Owens RT, Wagner WD. Metabolism and turnover of cell surface-associated heparan sulfate proteoglycan and chondroitin sulfate proteoglycan in normal and cholesterol-enriched macrophages. Arterioscler Thromb. 1991;11:1752-1758. [Abstract/Free Full Text]

40. Takasu Y, Hasumi F, Mori Y. Biosynthesis of glycosaminoglycans in peritoneal macrophages from the guinea pig. Biochim Biophys Acta. 1982;716:316-323. [Medline] [Order article via Infotrieve]

41. Yeaman C, Rapraeger AC. Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4-like heparan sulfate proteoglycan and a distinct chondroitin sulfate form. J Cell Physiol. 1993;157:416-425.

42. McQuillan DJ, Yanagishita M, Hascall VC, Bickel M. Proteoglycan biosynthesis in murine monocytic leukemic (M1) cells before and after differentiation. J Biol Chem. 1989;264:13245-13251. [Abstract/Free Full Text]

43. Christner JE. Biosynthesis of chondroitin sulfate proteoglycan by P388D₁ macrophage-like cell line. Arteriosclerosis. 1988;8:535-543. [Abstract/Free Full Text]

44. Kolset SO, Ivhed I, Øvervatn A, Nilsson K. Differentiation-associated changes in the expression of chondroitin sulfate proteoglycan in induced U-937 cells. Cancer Res. 1988;48:6103-6108. [Abstract/Free Full Text]

45. Uhlin-Hansen L, Wik T, Kjellen L, Berg E, Forsdahl F, Kolset SO. Proteoglycan metabolism in normal and inflammatory human macrophages. Blood. 1993;82:2880-2889. [Abstract/Free Full Text]

46. Shieh M-T, WuDunn D, Montgomery RI, Esko JD, Spear PG. Cell surface receptors for herpes simplex virus are heparan sulfate proteoglycans. J Cell Biol. 1992;116:1273-1281. [Abstract/Free Full Text]

47. Patel M, Yanagishita M, Roderiquez G, Bou-Mabib DC, Oravecz T, Hascall VC, Norcross MA. Cell-surface heparan sulfate proteoglycan mediates HIV-1 infection of T-cell lines. AIDS Res Hum Retroviruses. 1993;9:167-174. [Medline] [Order article via Infotrieve]

48. Hasumi F, Tanji N, Mori Y. Changes in glycosaminoglycan synthesis during the differentiation of HL-60 cells induced by 12-O-tetra-decanoylphorbol-13-acetate. J Pharmacobio Dyn. 1987;10:528-536. [Medline] [Order article via Infotrieve]

49. Kolset SO, Kjellen L, Seljelid R, Lindahl U. Changes in glycosaminoglycan biosynthesis during differentiation in vitro of human monocytes. Biochem J. 1983;210:661-667. [Medline] [Order article via Infotrieve]

50. Kolset SO. Oversulfated chondroitin sulfate proteoglycan in cultured human peritoneal macrophages. Biochem Biophys Res Commun. 1986;139:377-382. [Medline] [Order article via Infotrieve]

51. Razin E, Stevens RL, Akiyama F, Schmid K, Austen KF. Culture from mouse bone marrow of a subclass of mast cells possessing a distinct chondroitin sulfate proteoglycan with glycosaminoglycans rich in N-acetylgalactosamine-4,6-disulfate. J Biol Chem. 1982;257:7229-7236. [Abstract/Free Full Text]

52. Katz HR, Austen KF, Caterson B, Stevens RL. Secretory granules of heparin-containing rat serosal mast cells also possess highly sulfated chondroitin sulfate proteoglycans. J Biol Chem. 1986;261:13393-13396. [Abstract/Free Full Text]

53. Thompson HL, Schulman ES, Metcalfe DD. Identification of chondroitin sulfate E in human lung mast cells. J Immunol. 1988;140:2708-2713. [Abstract]

54. Stevens RL, Fox CC, Lichtenstein LM, Austen KF. Identification of chondroitin sulfate E proteoglycans and heparin proteoglycans in the secretory granules of human lung mast cells. Proc Natl Acad Sci U S A. 1988;85:2284-2287. [Abstract/Free Full Text]

55. Rothenberg ME, Caulfield JP, Austen KF, Hein A, Edmiston K, Newburger PE, Stevens RL. Biochemical and morphological characterization of basophilic leukocytes from two patients with myelogenous leukemia. J Immunol. 1987;138:2616-2625. [Abstract]

56. Kolset SO, Gallagher JT. Proteoglycans in haemopoietic cells. Biochim Biophys Acta. 1990;1032:191-211. [Medline] [Order article via Infotrieve]

57. Hoff HF, Wagner WD. Plasma low density lipoprotein accumulation in aortas of hypercholesterolemic swine correlates with modifications in aortic glycosaminoglycan composition. Atherosclerosis. 1986;61:231-236. [Medline] [Order article via Infotrieve]

58. Zilversmit DB. A proposal linking atherogenesis to the interaction of endothelial lipoprotein lipase with triglyceride-rich lipoproteins. Circ Res. 1973;33:633-638.[Free Full Text]

This article has been cited by other articles:

				N. Malla, E. Berg, L. Uhlin-Hansen, and J.-O. Winberg Interaction of Pro-matrix Metalloproteinase-9/Proteoglycan Heteromer with Gelatin and Collagen J. Biol. Chem., May 16, 2008; 283(20): 13652 - 13665. [Abstract] [Full Text] [PDF]

				C. N. Birts, C. H. Barton, and D. C. Wilton A Catalytically Independent Physiological Function for Human Acute Phase Protein Group IIA Phospholipase A2: CELLULAR UPTAKE FACILITATES CELL DEBRIS REMOVAL J. Biol. Chem., February 22, 2008; 283(8): 5034 - 5045. [Abstract] [Full Text] [PDF]

				J. G. Beeson, K. T. Andrews, M. Boyle, M. F. Duffy, E. K. Choong, T. J. Byrne, J. M. Chesson, A. M. Lawson, and W. Chai Structural Basis for Binding of Plasmodium falciparum Erythrocyte Membrane Protein 1 to Chondroitin Sulfate and Placental Tissue and the Influence of Protein Polymorphisms on Binding Specificity J. Biol. Chem., August 3, 2007; 282(31): 22426 - 22436. [Abstract] [Full Text] [PDF]

				K. Hashimura, K. Sudhir, J. Nigro, S. Ling, M. R. I. Williams, P. A. Komesaroff, and P. J. Little Androgens Stimulate Human Vascular Smooth Muscle Cell Proteoglycan Biosynthesis and Increase Lipoprotein Binding Endocrinology, April 1, 2005; 146(4): 2085 - 2090. [Abstract] [Full Text] [PDF]

				M. F. Khalil, W. D. Wagner, and I. J. Goldberg Molecular Interactions Leading to Lipoprotein Retention and the Initiation of Atherosclerosis Arterioscler. Thromb. Vasc. Biol., December 1, 2004; 24(12): 2211 - 2218. [Abstract] [Full Text] [PDF]

				M. A. Gebska, I. Titley, H. F. Paterson, R. M. Morilla, D. C. Davies, A. M. Gruszka-Westwood, V. V. Kakkar, S. Eccles, and M. F. Scully High-affinity binding sites for heparin generated on leukocytes during apoptosis arise from nuclear structures segregated during cell death Blood, March 15, 2002; 99(6): 2221 - 2227. [Abstract] [Full Text] [PDF]

				M. O. Pentikainen, R. Oksjoki, K. Oorni, and P. T. Kovanen Lipoprotein Lipase in the Arterial Wall: Linking LDL to the Arterial Extracellular Matrix and Much More Arterioscler. Thromb. Vasc. Biol., February 1, 2002; 22(2): 211 - 217. [Abstract] [Full Text] [PDF]

				S. Ohtake, Y. Ito, M. Fukuta, and O. Habuchi Human N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase cDNA Is Related to Human B Cell Recombination Activating Gene-associated Gene J. Biol. Chem., November 16, 2001; 276(47): 43894 - 43900. [Abstract] [Full Text] [PDF]

				M. Kaplan and M. Aviram Retention of Oxidized LDL by Extracellular Matrix Proteoglycans Leads to Its Uptake by Macrophages : An Alternative Approach to Study Lipoproteins Cellular Uptake Arterioscler. Thromb. Vasc. Biol., March 1, 2001; 21(3): 386 - 393. [Abstract] [Full Text] [PDF]

				J.-C. Mamputu, L. Levesque, and G. Renier Proliferative Effect of Lipoprotein Lipase on Human Vascular Smooth Muscle Cells Arterioscler. Thromb. Vasc. Biol., October 1, 2000; 20(10): 2212 - 2219. [Abstract] [Full Text] [PDF]

				J. C. Obunike, S. Pillarisetti, L. Paka, Y. Kako, M. J. Butteri, Y.-Y. Ho, W. D. Wagner, N. Yamada, T. Mazzone, R. J. Deckelbaum, et al. The Heparin-Binding Proteins Apolipoprotein E and Lipoprotein Lipase Enhance Cellular Proteoglycan Production Arterioscler. Thromb. Vasc. Biol., January 1, 2000; 20(1): 111 - 118. [Abstract] [Full Text] [PDF]

				S. Clasper, S. Vekemans, M. Fiore, M. Plebanski, P. Wordsworth, G. David, and D. G. Jackson Inducible Expression of the Cell Surface Heparan Sulfate Proteoglycan Syndecan-2 (Fibroglycan) on Human Activated Macrophages Can Regulate Fibroblast Growth Factor Action J. Biol. Chem., August 20, 1999; 274(34): 24113 - 24123. [Abstract] [Full Text] [PDF]

				J.-C. Mamputu and G. Renier Differentiation of Human Monocytes to Monocyte-Derived Macrophages Is Associated With Increased Lipoprotein Lipase–Induced Tumor Necrosis Factor-{alpha} Expression and Production : A Process Involving Cell Surface Proteoglycans and Protein Kinase C Arterioscler. Thromb. Vasc. Biol., June 1, 1999; 19(6): 1405 - 1411. [Abstract] [Full Text] [PDF]

				U. Lundstam, E. Hurt-Camejo, G. Olsson, P. Sartipy, G. Camejo, and O. Wiklund Proteoglycans Contribution to Association of Lp(a) and LDL With Smooth Muscle Cell Extracellular Matrix Arterioscler. Thromb. Vasc. Biol., May 1, 1999; 19(5): 1162 - 1167. [Abstract] [Full Text] [PDF]

				F. de Beer, W. L. Hendriks, L. C. van Vark, S. W.A. Kamerling, K. W. van Dijk, M. H. Hofker, A. H.M. Smelt, and L. M. Havekes Binding of ß-VLDL to Heparan Sulfate Proteoglycans Requires Lipoprotein Lipase, Whereas ApoE Only Modulates Binding Affinity Arterioscler. Thromb. Vasc. Biol., March 1, 1999; 19(3): 633 - 637. [Abstract] [Full Text] [PDF]

				L. Paka, Y. Kako, J. C. Obunike, and S. Pillarisetti Apolipoprotein E Containing High Density Lipoprotein Stimulates Endothelial Production of Heparan Sulfate Rich in Biologically Active Heparin-like Domains. A POTENTIAL MECHANISM FOR THE ANTI-ATHEROGENIC ACTIONS OF VASCULAR APOLIPOPROTEIN E J. Biol. Chem., February 19, 1999; 274(8): 4816 - 4823. [Abstract] [Full Text] [PDF]

				M. Y. Chang, K. L. Olin, C. Tsoi, T. N. Wight, and A. Chait Human Monocyte-derived Macrophages Secrete Two Forms of Proteoglycan-Macrophage Colony-stimulating Factor That Differ in Their Ability to Bind Low Density Lipoproteins J. Biol. Chem., June 26, 1998; 273(26): 15985 - 15992. [Abstract] [Full Text] [PDF]

				M. Kaplan, K. J. Williams, H. Mandel, and M. Aviram Role of Macrophage Glycosaminoglycans in the Cellular Catabolism of Oxidized LDL by Macrophages Arterioscler. Thromb. Vasc. Biol., April 1, 1998; 18(4): 542 - 553. [Abstract] [Full Text] [PDF]

				C.-Y. Lin, M. Lucas, and T. Mazzone Endogenous apoE expression modulates HDL3 binding to macrophages J. Lipid Res., February 1, 1998; 39(2): 293 - 301. [Abstract] [Full Text]

				J. Li, L. F. Brown, R. J. Laham, R. Volk, and M. Simons Macrophage-Dependent Regulation of Syndecan Gene Expression Circ. Res., November 19, 1997; 81(5): 785 - 796. [Abstract] [Full Text]

				A. Kinoshita, S. Yamada, S. M. Haslam, H. R. Morris, A. Dell, and K. Sugahara Novel Tetrasaccharides Isolated from Squid Cartilage Chondroitin Sulfate E Contain Unusual Sulfated Disaccharide Units GlcA(3-O-sulfate)beta 1-3GalNAc(6-O-sulfate) or GlcA(3-O-sulfate)beta 1-3GalNAc(4,6-O-disulfate) J. Biol. Chem., August 8, 1997; 272(32): 19656 - 19665. [Abstract] [Full Text] [PDF]

				E. Hurt-Camejo, U. Olsson, O. Wiklund, G. Bondjers, and G. Camejo Cellular Consequences of the Association of ApoB Lipoproteins With Proteoglycans: Potential Contribution to Atherogenesis Arterioscler. Thromb. Vasc. Biol., June 1, 1997; 17(6): 1011 - 1017. [Abstract] [Full Text]

				M. Lucas and T. Mazzone Cell Surface Proteoglycans Modulate Net Synthesis and Secretion of Macrophage Apolipoprotein E J. Biol. Chem., June 7, 1996; 271(23): 13454 - 13460. [Abstract] [Full Text] [PDF]

				M. Ueno, S. Yamada, M. Zako, M. Bernfield, and K. Sugahara Structural Characterization of Heparan Sulfate and Chondroitin Sulfate of Syndecan-1 Purified from Normal Murine Mammary Gland Epithelial Cells. COMMON PHOSPHORYLATION OF XYLOSE AND DIFFERENTIAL SULFATION OF GALACTOSE IN THE PROTEIN LINKAGE REGION TETRASACCHARIDE SEQUENCE J. Biol. Chem., July 27, 2001; 276(31): 29134 - 29140. [Abstract] [Full Text] [PDF]

				Y. Ito and O. Habuchi Purification and Characterization of N-Acetylgalactosamine 4-Sulfate 6-O-Sulfotransferase from the Squid Cartilage J. Biol. Chem., October 27, 2000; 275(44): 34728 - 34736. [Abstract] [Full Text] [PDF]

This Article Abstract Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Edwards, I. J. Articles by Wagner, W. D. Search for Related Content PubMed PubMed Citation Articles by Edwards, I. J. Articles by Wagner, W. D.