American Journal ofPathology, Vol. 136, No. 3, March 1990 Copyright © American Association ofPathologists

Macrophage Secretory Products Selectively Stimulate Dermatan Sulfate Proteoglycan Production in Cultured Arterial Smooth Muscle Cells

Iris J. Edwards, William D. Wagner, and Rick T. Owens From the Department of comparative Medicine, Bowman Gray, School ofMedicinie, Wake Forest Unitersity, Witnstoni-Salem, North Carolinia

Arterial dermatan sulfate proteoglycan has been shown to increase with atherosclerosis progression, but factors responsible for this increase are unknown. To test the hypothesis that smooth muscle cellproteoglycan synthesis may be modified by macrophageproducts, pigeon arterial smooth muscle cells were exposed to the media of either cholesteryl ester-loaded pigeon peritoneal macrophages or a macrophage cell line P388D-,. Proteoglycans radiolabeled with [35S]sulfate and [31]serine were isolated from culture media and smooth muscle cells and purified following precipitation with 1hexadecylpyridinium chloride and chromatography. Increasing concentrations of macrophageconditioned media were associated with a dose-response increase in [35S]sulfate incorporation into secreted proteoglycans, but there was no change in cell-associated proteoglycans. Incorporation of [3H]serine into totalproteoglycan core proteins was not significantly different (5.2 X 105 dpm and 5.5 X 105 disintegrations per minute (dpm) in control and conditioned media-treated cultures, respectively), but selective effects were observed on individual proteoglycan types. Twofold increases in dermatan sulfate proteoglycan and limited degradation of chondroitin sulfate proteoglycan were apparent based on core proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoinhibition studies indicated that interleukin-1 was involved in the modulation of proteoglycan synthesis by macrophage-conditioned media. These data provide supportfor the role of macrophages in alteration of the matrix proteoglycans synthesized by smooth mus-

cle cells and provide a mechanism to account for the reported increased dermatan sulfate/chondroitin sulfate ratios in the developing atherosclerotic lesion. (Am JPathol 1990, 136:609-621)

Accumulations of sulfated proteoglycans (PG) as well as altered PG compositions accompany atherosclerosis development in several species.1-3 Wagner and Nohlgren4 demonstrated in White Carneau (WC) pigeons that as aortic cholesterol content increased, dermatan sulfate (DS) and chondroitin sulfate (CS) content increased. Based on an increasing ratio of DS:CS, greater accumulations of DS were noted as atherosclerosis progressed. Recently, using immunohistochemical techniques, Robbins et a15 examined the distribution of PG in normal and atherosclerotic aortas of WC pigeons and observed significant accumulations of DS-PG in naturally occurring and cholesterol diet-induced lesions. The source of the DS-PG localized in the intimal lesion is unknown. It has been reported that macrophages, an important cell type in the lesion, are capable of synthesizing PG.6 6 However, recent experiments have demonstrated the production of CS-PG and heparan sulfate (HS)-PG but no detectable DS-PG by cultured pigeon macrophages.9 DS-PG is produced by cultured pigeon smooth muscle cells, but like the cells in the normal artery, the major PG product is a CS-PG.10 In the changing environment of the developing atherosclerotic lesion, however, smooth muscle cell synthetic processes may be modified to account for an increased synthesis and the subsequent arterial accumulation of DS-PG. One source of factors capable of modification of smooth muscle cell metabolism is the lipid-filled macroSupported by grants HL-14164 and HL-25161 from the National Heart, Lung, and Blood Institute. Accepted for publication October 20, 1989. Address reprint requests to Dr. William D. Wagner, Department of Comparative Medicine, Bowman Gray School of Medicine, 300 S. Hawthorne Road, Winston-Salem, NC 27103.

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phages whose presence in the intima is an early event in atherogenesis. Macrophage products are known to affect the in vitro metabolism of a variety of cell types. Conditioned media and purified media fractions from cultured monocytes have been shown to modulate the proliferation of fibroblasts,' 1-13 endothelial cells, and artery smooth muscle cells.14"15 Secreted macrophage factors such as interleukin-1 (IL-1), platelet-derived growth factor (PDGF), and tumor necrosis factor (TNF) have been implicated in such activities. In addition, macrophage products have been shown to affect the synthesis of connective tissue components. In response to IL-1, an increase in glycosaminoglycan (GAG) and a decrease in collagen synthesis has been reported for synovial cells.16 IL-1 induction of structurally aberrant PG has been reported in chondrocytes.17 Prostaglandins have been shown to modulate PG synthesis in rat granulosa cells"8 and in rabbit aortic smooth muscle cells.19 Recently, transforming growth factor-,B (TGF-fl), a potent modulator of extracellular matrix components, including collagen,20 22fibronectin2022 and PG2324 in a vari ety of cell types, has been identified as a macrophage

product.25 The presence of such factors in the developing atherosclerotic lesion therefore may have important consequences regarding smooth muscle cell metabolism. This report is a study of the effects of secretory products of cholesteryl ester-loaded peritoneal macrophages and macrophage cell line (P388DI) on the production of PG types by aortic smooth muscle cells in culture. A specific enhancement of DS-PG, the predominant PG of the atherosclerotic lesion, is described.

Materials and Methods Experimental Procedures Materials Tissue culture reagents were purchased from Flow Laboratories, Inc., Rockville, MD; plasticware from Corning Glass Works, Corning, NY; [3H]serine from ICN Biomedicals, Irvine, CA; [35S]sodium sulfate from Amersham Corp., Arlington Heights, IL; ENLIGHTNING from DuPontNew England Nuclear, Boston, MA; Liquiscint from National Diagnostics, Inc., Somerville, NJ; 1 -hexadecyl-pyridinium chloride (CPC), 6-aminohexanoic acid, and Coomassie blue R250 from Eastman Kodak, Rochester, NY; chondroitin ABC lyase (Proteus vulgaris), chondroitin AC lyase (Arthrobacter aurescens), and unsaturated disaccharide standards containing ADi-OS, ADi-4S, ADi-6S, and ADi-diSE from Seikagaku America, Inc., St. Petersburg, FL; chondroitin sulfate from Nutritional Biochemicals

Corp., Cleveland, OH; polyacrylamide gel electrophoresis reagents from Bio-Rad, Richmond, CA; phenylmethylsulfonyl fluoride (PMSF), pepstatin, and lipopolysaccharide (LPS) from Sigma Chemical Co., St. Louis, MO; rabbit antiPGE2 from Advanced Magnetics, Inc., Cambridge, MA; rabbit anti-porcine platelet TGF-# from R & D Systems, Minneapolis, MN; and rabbit anti-human IL-1 from Genzyme, Boston, MA. All other reagents used were purchased from Fisher Scientific.

Preparation of Conditioned Media from Pigeon Macrophages Six WC pigeons fed a cholesterol-containing diet (0.5% cholesterol by weight dissolved in 10% melted lard coated on pigeon pellets) for 6 months were used as a source of cholesteryl ester-loaded peritoneal macrophages. Macrophages were elicited by abdominal injection of 5 ml of sterile 3% thioglycolate broth or 5 ml of lipopolysaccharide (30 ,g/ml saline) 48 hours before necropsy. Two birds received no eliciting agent. At necropsy, the peritoneal cells were harvested by lavage with phosphate-buffered saline (PBS) (13.7 millimolar [mmol/l] NaCI, 2.7 molar [mol/l] KCI, 1.5 mmol/l KH2PO4, 8.1 mmol/l Na2HPO4, pH 7.4) containing 10 ,u/ml heparin by use of the sterile procedures described by St. Clair et al.26 Cells from birds with similar modes of elicitation were pooled, washed with PBS, and plated in two 1 00-mmol/l dishes in 10 ml of Eagle's minimal essential medium (EMEM) with Earle's base salts supplemented with 10% fetal bovine serum, 200 mmol/l L-glutamine, EMEM vitamins, 100 U/ml penicillin, and 100 ,g/ml streptomycin sulfate. The cultures were incubated at 37 C in 95% air, 5% C02, and media were replaced to remove nonadherent cells at 2 and 24 hours. After the 24-hour media change, the macrophages were incubated for 24 hours to obtain conditioned media. Control media consisted of EMEM supplemented as described and incubated for 48 hours at 37 C in the absence of cells. The conditioned media were collected and centrifuged at 400g for 10 minutes to remove cell debris, and aliquots were removed to prepare dilutions in EMEM as described for addition to smooth muscle cell cultures. Each dilution was adjusted to 30,uCi [35S]sodium sulfate/ml medium, 30 ,Ci [3H]serine/ml medium, and 200 mmol/l L-glutamine. The macrophages were harvested and analyzed for cell protein, cholesterol, and cholesteryl ester.26 Cholesteryl ester content of the cells was >40 ,ug/mg cell protein.

Preparation of Conditioned Media from P388D, Cells Suspension cultures of mouse monocyte-macrophage cell line P388D, were a gift from Dr. S. Mizel (Bowman

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Gray School of Medicine). The cells were transferred to 75 cm2 flasks containing RPM1 -1640 medium supplemented with 1% fetal bovine serum and allowed to adhere for 24 hours at 37 C. The media were replaced with fresh RPM1 1640 containing 1% fetal bovine serum and 10 jig/ml lipopolysaccharide. Parallel cultures received no lipopolysaccharide. After a 48-hour incubation, the conditioned media were removed and centrifuged at 400g for 10 minutes, and the supernatant was dialyzed (3500 molecular weight cutoff) vs. PBS, pH 7.4, at 4 C with three buffer changes and a final change to EMEM. The dialyzed samples were adjusted to 10% fetal bovine serum and filter sterilized with Millex-HA 0.45 ,um filter units (Millipore Corp., Bedford, MA). Dilutions of the conditioned media were prepared in EMEM containing 10% fetal bovine serum, and each dilution was adjusted to 30 ACi [3S]sodium sulfate/ ml medium, 30 ,uCi [3H]serine/ml medium, and 200 mmol/ L-glutamine before use.

Immunoinhibition Studies Conditioned media from thioglycolate-elicited pigeon peritoneal macrophages were incubated with selected antibodies before addition to smooth muscle cell cultures. Rabbit anti-human IL-1, rabbit anti-porcine platelet TGF-f, and rabbit anti-PGE2 were diluted to a concentration of 1: 20 and 1:200 in the macrophage-conditioned media or in control media. All media were then incubated for 2 hours at 37 C. These concentrations are within the range of neutralizing activity indicated by the manufacturers for anti-IL1 and anti-TGF-f3. No information on the neutralizing activity of anti-PGE2 was available. The antibody-treated media were diluted 1:2 in labeling media and adjusted to 30 ACi [3S]sodium sulfate/ml medium, 30 ,Ci [3H]serine/ml medium, and 200 mmol/l L-glutamine before use.

cultures were prepared for determination of cell number by hemocytometry as previously described.10

Isolation of Proteoglycans Media Fraction After 48 hours of incubation, the labeling media were removed from the smooth muscle cell cultures, cleared of cell debris by centrifugation at 400g for 10 minutes, and dialyzed exhaustively against 0.02 mol NaCI, 0.03 mol Na2SO4 at 4 C to remove unincorporated radiolabel. Aliquots of the dialyzed samples were analyzed for radioactivity incorporated into total secreted protein. PG were then precipitated along with 100 ,tg of added carrier chondroitin sulfate for 48 hours at 25 C in 1% 1 -hexadecylpyridinium chloride (CPC). The PG-CPC complexes were pelleted at 800g for 2 hours, resuspended in 1 ml of 2 mol NaCI in water/absolute ethanol (100:14), and diluted to 3 ml with absolute ethanol to precipitate the PG free from the CPC. After 24 hours at 25 C, PG were separated by centrifugation at 800g for 1 hour, dissolved in 1 ml PBS, pH 7.4, and analyzed for radioactivity.

Cell-associated Fraction After removal of the culture media, the cell monolayers were rinsed twice with PBS, pH 7.4, before a 24-hour extraction at 4 C with 4 mol/l GdnHCI, pH 5.8, containing protease inhibitors28 and 0.1% Triton X-1 00. The extracts were dialyzed and purified as described for the media PG and analyzed for radioactivity incorporated into total protein and PG core protein.

Proteoglycan Characterization Size Exclusion Chromatography

Smooth Muscle Cell Culture and Radiolabeling Medial smooth muscle cell explant cultures were prepared from aortas of 1-day-old WC squabs as previously described27 and passaged in EMEM with Earle's base salts supplemented with 200 mmol/I L-glutamine, EMEM vitamins, 100 U/mI penicillin, 100 Ag/ml streptomycin sulfate, and 10% fetal bovine serum. Cells of the third to fifth passage were plated in 35-mm tissue culture dishes and allowed to grow to confluence. Media were then removed and replaced with the prepared macrophage-conditioned media or control media containing 30 ,Ci [35S]sodium sulfate/ml medium and 30 gCi [3H]serine/ml medium. Triplicate cultures were used for all observations. Additional

Aliquots of PG were diluted in 0.25 mol/l TRIS-phosphate buffer, pH 7.6, and chromatographed at a flow rate of 0.5 ml/minute on an HPLC Altex Spherogel TSK 5000 PW column (600 X 7.5 mm) by use of 0.25 mol/l TRISphosphate buffer, pH 7.6. Fractions (0.5 ml) were collected and analyzed for radioactivity.

Identification of PG by Enzymatic Degradation Aliquots of PG were diluted in 0.1 mol/I TRIS (pH 8.0), 0.03 mmol/l sodium acetate containing protease inhibitors (10 mmol/l) ethylenediaminetetraacetic acid [EDTA], 0.036 mmol/l pepstatin, 0.5 mmol/l PMSF, and 10 mmol/l N-ethylmaleimide (NEM), and digested with 0.05 units of chondroitin ABC lyase or chondroitin AC lyase for 3 hours

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-1 Media Cell associated Cell associated Media Media Cell associated Figure 1. The effect of conditioned media obtainedfrom cultured pigeon peritoneal macrophages on PG synthesis ofpigeon aortic smooth muiscle cells. Conditioned media prepared from elicited or resident peritoneal macrophages from cholesterol-fed pigeons durintg a 24-hour incubation were diluted in EMEM 10% FCS. Media were adjusted to contain 30 jCi [35S sulfate/ml and 30 tsCi [3H]serine/ml atnd added to 35-mm dish cultures of conjluenttsmooth muscle cells. After 48-hour incubation at 37 C, the media were removed, centrifuged 400g, 5 mittutes, dialyzed versus 0.02 mol/l NaCI, 0.03 mol/l, Na2SO4, and the radiolabeled PG were precipitated with CPC. The cell-associated PG were extracted 24 hours, 4 C with 4 mol/l GdnHCl in 0.05 mol/l sodium acetate, pH 4.5, 0. 1% Tritotn X-100. Extracts were dialyzed and precipitated with CPC. Smooth muscle cell PG synthesis was measured in the presence of conditioned media from A, resident; B, thioglycolate-elicited; C, LPS-elicited macrophages. Control media diluted 1:2E, macropha1:5 E, 1:10 El. Values are means ± SEM of triplicate cultures. Statistical summary: Within ge-cotnditionzed media diluted 1:2 group di.fferences-(P < 0. 05). Resident macrophages-1:2, 1:5> 1:10, control. TG-elicited macrophages-1:2> 1:5, 1:10> control. LPS-elicited macrophages- 1:2> 1:10> control; 1:5> conttrol. E,

at 37 C. Products were chromatographed on a TSK 5000 PW column, and movement of 35S radioactivity to the column Vt was taken to represent degradation of PG.

DEAE Ion Exchange Chromatography To obtain PG core proteins, aliquots of PG were dialyzed against 7 mol/l urea in 0.05 mol/l TRIS, pH 7.2, and loaded on a 0.5-ml DEAE-Sephacel mini-column. The column was eluted with a stepwise gradient of 1.5 ml 7 mol/ I urea in 0.05 mol/l TRIS; 1.5 ml 0.15 mol/l NaCI in 7 mol/l urea, 0.05 mol/l TRIS; and 2.0 ml 1 mol/l NaCI in 7 mol/l urea, 0.05 mol/l TRIS. The 1 mol/l NaCI fraction containing 70% to 73% of the [3H]serine radioactivity was dialyzed against 0.05 mol/l sodium acetate and precipitated with 4 volumes absolute ethanol at -20 C for 48 hours. Core Protein Analysis

PG core proteins were prepared following chondroitin ABC lyase hydrolysis of either CPC or CPC-DEAE purified PG. The samples were adjusted to 0.05 mol/l TRIS, pH 8.0, 1% sodium dodecyl sulfate, 1% p3-mercaptoethanol, and boiled for 2 minutes. Samples were electrophoresed on 7.5% polyacrylamide gels by the method of Laemmli' with known proteins used as molecular weight markers. The gels were stained for 1 hour with 0.05% Coomassie blue in 50% methanol, 10% acetic acid, destained in 50%

methanol, 10% acetic acid, for 1 hour, treated with ENLIGHTNING for 30 minutes, and dried for 2 hours on a BioRad Model 224 gel dryer. Autoradiograms were prepared by exposing the dried gels to Kodak X-Omat AR x-ray films for 2, 5, 7, or 10 days at -60 C. Densitometric scans were obtained from the autoradiograms with a Gelman ACD-1 8 densitometer.

Disaccharide Analysis Unsaturated disaccharides were prepared following chondroitin ABC lyase digestion of CPC-purified PG. The disaccharides were purified by the addition of nine volumes of absolute ethanol for 2 hours at -20 C. Following centrifugation at 800g for 5 minutes, the ethanol supernatants containing the disaccharides were air dried and solubilized in 20 ,ul HPLC elution buffer containing 1 jig each of mixed disaccharide standards. A Whatman Partisil-10 PAC (4.6 x 250 mm) HPLC column was used to separate the disaccharides with 70% acetonitrile/methanol (3:1 volume/volume) and 30% 0.5 mol/l ammonium acetate, pH 5.3, as the mobile phase. Fractions (0.5 ml) were collected and analyzed for radioactivity.

Radioactivity Measurements All radioactivity measurements were made in a Packard 1500 Tri-Carb (Packard Instruments Inc., Downer's

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a)

Figure 2. The effect of conditioned media from P388D, cells on PG synthesis by pi-

geon aortic smooth muscle cells. Conditioned media from unstimulated and 48hour LPS-stimulated P388D, cells were dialyzed versus PBS, adjusted to 10% FCS anid filter sterilized. Media were diluted in EMEM 10% fetal bovine serum to contain 30 MCi [35S]sulfate/ml and 30 jCi [3HJserine/ml) and added to 35-mm dish cultures of confluent smooth muscle cells. After 24-hour incubation at 37 C, media were removed, centrifuged 400g 5 minutes, dialyzed versus 0. 02 mol/l NaCI, 0. 03 mol/l Na2SO4 and radiolabeled PG were precipitated with CPC. Cell-associated PG were extracted 24 hours, 4 C with 4 mol/l GdnHCI in 0.05 mol/l sodium acetate, pH 4.5, 0. 1% Triton X-100. The extracts were dialyzed and precipitated with CPC as describedfor the media PG. A: media PG; B: cell-associated PG. Media diluted 1:2 i, 1:5 2, 1:10 El, 1:50 . Values are the means ± SEM oftriplicate cultures. Statistical summary: a) Within group differences-(P < 0.05). Control-1:2 > 1:10. Unstimulated macrophages-1:2 > 1:5, 1:10. LPS stimulated macrophages-1:2 > 1:5 > 1:10 > 1:50. b) Between group differences-(P < 0.05). For 1:10 dilutioni-LPS > control. For 1:5 dilutionLPS > unstimulated. For 1:2 dilutionLPS > unstimulated > control. There was a significant interaction between dosage of conditioned media and group.

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Statistical Analyses The significance of difference between means was determined by analysis of variance and Duncan's multiple range test.

Results Effect of Macrophage-conditioned Media on [35S] Incorporation into PG by Pigeon Aortic Smooth Muscle Cells Synthesis of PG by WC pigeon artery smooth muscle cells in the presence of WC peritoneal macrophage-con-

Control

Unstimulated macrophages

LPS stimulated macrophages

Media Source ditioned media was assessed by incorporation of [3S]sulfate into nondialyzable CPC-precipitable macromolecules. Three different methods of macrophage harvest were used to obtain macrophages for conditioned media preparation (Figure 1). With increasing concentrations of macrophage-conditioned media, increased radiolabel was incorporated into the PG secreted into the culture media by the smooth muscle cells. The stimulation was obtained with media conditioned by both resident and elicited macrophages. Both methods of elicitation of macrophages gave comparable effects at a given dose of conditioned media. The cultures of elicited macrophages from which the conditioned media were obtained were higher in cell protein (470 ug/dish in thioglycolate-elicited and 700 ,ug/dish in lipopolysaccharide-elicited) than the cultures of resident macrophages (211 ,ug/dish), in which

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Figure 3. Immunoinhibition of the stimulatory effiect of macrophage-conditioned media on smooth muscle cell PG synthesis. Conditioned media were prepared from thioglycolate-elicited peritoneal macrophages from cholesterol-fed pigeons during a 24-hour incubation. The indicated antibodies were diluted to a concentration of 1:20 or 1:200 in the macrophage-conditioned media or in control media and incubatedfor 2 hours at 37 C The antibody-treated media were diluted 1:2 in EMEM 10% fetal bovine serum containing[35Sl sulfate (30,gCi/ml media,final concentration) and added to 24-well clusterdish culturesofconJluentsmooth muscle cells. After 30-hour incubation at 37 C, the media were removed, centrifuged 4O0g 5 minutes, dialyzed versus 0.02 mol/l NaCI, 0.03 mol/l Na2SO4, and the radiolabeled PG were precipitated with CPC. * No antibody, E 1:20 antibody dilution, O 1:200 antibody dilutioni. Values are the means ± SEM of triplicate cultures. Statistical summary: Conditioned media within group differences-(P < 0. 05). PGE2-1:20 < 1:200; IL- 1-1:20 < 1:200. Between group differences-(P < 0. 05). For a giien antibody dilutionl, all means for conditioned media treatment were greater than meatnsfor conitrol media with the exception of IL-1 at a 1:20 dilution. For this experiment, macrophages and smooth muscle cells were deriiedfrom differenit animals than those described in Figure 1.

somewhat lower effects at comparable doses were observed. Of the newly synthesized PG produced by smooth muscle cells, approximately 8% remained associated with the cells. No effect of macrophage-conditioned media was seen on this PG fraction (Figure 1). No differences in cell numbers were observed, indicating that the conditioned media were not affecting smooth muscle cell proliferation (data not shown). The stimulatory effect on PG synthesis was specific for macrophage-conditioned media. In another experimenrt in which a 1:2 dilution of conditioned media from smooth muscle cells was used, PG synthesis was the same (1.61 ± 0.12 dpm 35S per cell, x ± SEM) as control (1.67 ± 0.12 dpm 35S per cell), whereas conditioned media from pigeon peritoneal macrophages was 7.52 ± 0.9 dpm IS per cell. In an additional study, the conditioned media from a continuous macrophage cell line, P388D1, was examined. This cell line is known to produce IL-1 in response to LPS stimulation.x Figure 2 illustrates the effect of P388D1-

conditioned media on [3S]sulfate incorporation by WC smooth muscle cells into secreted (Figure 2A) and cellassociated (Figure 2B) nondialyzable CPC-precipitable macromolecules. Stimulatory effects similar to those of the pigeon macrophages were noted: a dose-dependent increase in [35S]sulfate incorporation into secreted PG, but no stimulation of incorporation into cell-associated PG. Stimulation of the P388D1 macrophages by lipopolysaccharide treatment resulted in significant enhancement of the stimulatory effect on the smooth muscle cell-secreted PG (Figure 2A). This occurred at a level as low as a 1 :10 dilution.

Identification of the Stimulatory Factor by Immunoinhibition In an attempt to identify the macrophage product responsible for the PG stimulatory activity, the conditioned media from pigeon peritoneal macrophages were preincubated

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Table 1. Effect ofMacrophage-conditioned Media on the Synthesis of Secreted Protein by Aortic Smooth Muscle Cells Mode of macrophage recruitment None Thioglycolate LPS

Control

1:10

Conditioned media dilution 1:5

1:2

50.50 ± 1.5

47.8 ± 2.8 49.7 ± 1.4 49.5 ± 1.4

50.2 ± 2.3 48.3 ± 3.2 50.6 ± 1.3

44.3 ± 0.7 43.0 ± 1.4 44.4 ± 1.3

Numbers are dpm [3H]serine X 10-5 per 105 cells and represent mean ± SEM of triplicate cultures. Conditioned media were prepared from elicited or nonelicited peritoneal macrophages of cholesterol-fed pigeons during a 24-hour incubation period in culture. Media diluted in EMEM 10% fetal bovine serum were adjusted to 30 ACi [3S]sulfate/ml and 30 uCi [3H]serine/ml and added to 35-mm dish cultures of confluent smooth muscle cells. After a 48-hour incubation period at 37 C, the media were removed, centrifuged 400g for 5 minutes, and dialyzed versus 0.02 mol/l NaCI, 0.03 mol/l Na2SO4. Radioactivity was measured as [3H]serine incorporated into nondialyzable (molecular weight > 3500) material. The control included nonconditioned media at a dilution of 1:2. Statistical summary: Within group differences-(P < 0.05). LPS stimulated macrophages-1:2 < 1:5, 1:10. LPS stimulated macrophages-1:2 < control 1:2.

with antibodies to factors known to modulate PG synthesis in other cell types. As indicated in Figure 3, a 1:20 dilution of rabbit anti-IL-1 totally abrogated the stimulatory effect of the conditioned media. Anti-TGF-f and antiPGE2 had no significant inhibitory activity. However, because the activity of the antibodies was not verified by a positive control, the involvement of these factors cannot conclusively be ruled out. Between-group analysis of the control media alone indicated that the 1:20 dilution of anti-IL-1 had significant inhibitory activity on PG synthesis in control media.

served for the production of secreted PG core proteins (Table 2) or cell-associated core proteins (data not shown). Treatment of the smooth muscle cells with macrophage-conditioned media therefore resulted in an increased [35S]sulfate but unchanged [3H]serine incorporation into secreted PG and thus increased ratios of 3S/3H (Table 3). When high concentrations of conditioned media were used, increased ratios were observed. This finding suggested either an oversulfation of PG, a shift in PG population, or the release of protein-free GAG.

Effect of Macrophage-conditioned Media on Smooth Muscle Cell Protein Synthesis

Characterization of Stimulated PG

To determine whether conditioned media produced a generalized stimulatory effect on the metabolism of smooth cells, [3H]serine incorporation into nondialyzable material was used to assess an effect on total protein synthesis. No increase in response to the conditioned media treatment was seen in newly synthesized total protein secreted by the cells (Table 1), but rather a trend toward lower protein synthesis was observed with the 1:2 dilutions. In addition, no differences in [3H]serine incorporation into cell-associated proteins could be determined (data not shown). Surprisingly, no differences were ob-

were

For examination of PG size, aliquots of PG preparations chromatographed on an HPLC TSK 5000 column (Beckman Instruments, Fullerton, CA). Radiolabeled PG were separated in the control (Figure 4A) as peak 1 material eluting at a Ka. of 0.04 and peak 2 material eluting at a Ka, of 0.43. Peak 2 contained 59% of the [3S]sulfate and 54% of the [3H]serine radioactivity. In the cells treated with conditioned media, two peaks of radioactive PG were observed (Figure 4B). Compared with controls, no differences in elution positions of these PG were apparent. No significant amount of radioactive material indicative of free GAG chains was observed (see position of arrow, Figure

Table 2. Effect ofMacrophage-conditioned Media on the Synthesis of Secreted Proteoglycans by Aortic Smooth Muscle Cells Conditioned media dilution 1:2 Mode of macrophage recruitment Control 1:5 1:10 3.2 ± 0.05 None 3.4 ± 0.24 3.2 ± 0.27 3.5 ± 0.34 3.4 ± 0.11 3.2 ± 0.11 2.5 ± 0.30 Thioglycollate 3.3 ± 0.35 3.4 ± 0.35 LPS 3.5 ± 0.07 Numbers are dpm [3H]serine x 10-5 per 1 05 cells and represent mean ± SEM of triplicate cultures. Conditioned media were prepared from elicited or

nonelicited peritoneal macrophages of cholesterol-fed pigeons during a 24-hour incubation perod in culture. Media diluted in EMEM 10% fetal bovine serum were adjusted to 30 usCi [3S]sulfate/ml and 30 uCi [3H]serine/ml and added to 35-mm dish cultures of confluent smooth muscle cells. After a 24-hour incubation period at 37 C, the media were removed, centrifuged 400g for 5 minutes, dialyzed versus 0.02 mol/l NaCI, 0.03 mol/l Na2SO4, and the radiolabeled PG were precipitated with CPC. Radioactivity was measured as [31-1]serine incorporated into CPC precipitable material. The control includes nonconditioned media at a dilution of 1:2.

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Table 3. Effect ofMacrophage-conditioned Media on the Ratio ofi[35S]Sulfate to [3I]Serine in PG of CulturedAortic Smooth Muscle Cells Dilution of conditioned media Macrophage source 1:5 1:2 1:50 1:10 ND 25 58 Pigeon nonelicited 0 17 25 75 Pigeon TG-elicited ND 25 50 Pigeon LPS-elicited ND 17 P388D1 ND ND 6 unstimulated 0 21 37 P388D1 stimulated 0 16 Values are percentage increase over control in [35S]sulfate:[3H]serine. Counts are taken from Figures 1 and 2 and Table 2. ND, not determined.

lyase, which cleaves only glucuronosylgalactosamine linkages and not the iduronosylgalactosamine linkages found characteristically in dermatan sulfate, failed to hydrolyze 43% of the PG. These data indicate the presence of a DS-PG as a significant component of the PG mixture. Nitrous acid treatment indicated that about 6% of the [35S]sulfate (based upon movement of 35S label from V0 to Vj) in both control and conditioned media-treated samples was associated with HS-PG.

PG Core Protein Analysis

4A, B). As a percentage of total PG, peak 2 material increased 14% ([35S]sulfate) to 17% ([3H]serine) in conditioned media-treated cells. These data suggest the stimulation of intact PG rather than increased release of free GAG chains. Radioactive PG were identified by susceptibility to specific GAG-degrading enzymes (Figure 5). Using the total radiolabeled PG, >90% of the 3S was hydrolyzed with chondroitin ABC lyase, indicating that it contained chondroitin sulfate or dermatan sulfate. Chondroitin AC

To further characterize the PG, samples were purified by precipitation with CPC. [3H]serine-labeled PG and PG core proteins generated by chondroitin ABC lyase treatment were examined following electrophoresis on SDS polyacrylamide gel and autoradiography. An increase in a 200- to 250-kd band was observed in the PG from the cells treated with conditioned media (Figure 6, lane 3 vs. lane 1 control). The chondroitin ABC lyase-generated core proteins from these samples are shown in lane 2 (control) and lane 4 (conditioned media treated). While the

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Figure 4. Elution profiles of[3Mserine and [35S]sulfate radiolabeledPGproduced bypigeon aortic smooth muscle cells in the absence (A) or presence (B) of P388DI-conditioned media. PG precipitatedfrom media with CPCP were solubilized in 0.25 mol/l Tris-phosphate, pH 7.6, and aliquots were chromatographed on a HPLC TSK PW 5000 column using the same bufferfor the mobile pbase. Fractions (0.25 ml) were collected and analyzedfor radioactivity. Arrows indicate the elution position of chondroitin sulfate GAG chains (Mr 55, 000).

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media-treated (Figure 7B) cultures, the GAG chains of the PG contained disaccharides that migrated with the ADi4S and ADi-6S standards. No oversulfated disaccharides were detected. Although nonsulfated disaccharides were not measured in this experiment, we have, previously shown that more than 90% of the disaccharides of PG produced by WC smooth muscle cells are sulfated.10 Therefore, the observed differences in sulfate incorporation cannot be due to sulfation of previously nonsulfated disaccharides.

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Discussion

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Vt Fraction Figure 5. Elution profiles of radiolabeled PG treated enzymaticallj' to identif/j PG tjpe. PG were precipitatedfrom the culture media ofpigeon smooth muscle cells treated with the indicated enzymes and aliquots were chromatographed on a PW5000 column using 0.25 mol/l Tris-phosphate, pH 7.6, as the mobile phase. Fractions (0.25 ml) were collected and analyzedfor - Chondrotin AC II lyaseintact PG, radioactivity treated PG, Chotndroitin ABC lyase-treated PG. .

intact CS-PG core proteins were too large to enter this gel, 45-kd protein, previously demonstrated to be the core protein of DS-PG,31'32 was seen after the enzyme treatment. Preparations purified on DEAE-Sephacel before PAGE gave a similar banding pattern, indicating that minimal contaminating proteins containing [3H]serine were present. Following autoradiography, densitometric scans of the autoradiograms were prepared and the [3H]serine in each peak was calculated from the total radioactivity applied (Table 4). From data using either core or intact PG, DS-PG increased 1.8- to 2.3-fold in conditioned media-treated cultures. For CS-PG, the presence of increased radioactivity as small-molecular-weight cores suggested limited cleavage of this PG. a

Sulfation of GAG Chains To determine whether treatment with macrophage-conditioned media caused an alteration in sulfation of the smooth muscle cell PG, the chain disaccharides generated by chondroitin ABC lyase hydrolysis were examined (Figure 7). In both control (Figure 7A) and conditioned

Investigations of the interaction of macrophages and smooth muscle cells, the two major cell types of the atherosclerotic plaque, are crucial to the understanding of lesion progression. Morphologic studies of early events of atherogenesis in cholesterol-fed animals indicate that in response to a cholesterol diet, blood monocytes adhere to the arterial intima, migrate through the endothelium, and invade the subendothelial space. Focal accumulations of lipid-laden macrophages are followed by the migration of smooth muscle cells into the intima and subsequent fibrotic complications of the lesion.37 This sequence of events has led to a hypothesis of macrophage secretory products affecting smooth muscle cell chemotaxis and proliferation. In addition, macrophage products that signal a repair process by mesenchymal cells in a normal healing situation may stimulate the smooth muscle cells to produce substances that lead to pathologic fibrogenesis in the atherosclerotic lesion. The purpose of the present study was to examine the effect of macrophages on artery smooth muscle cell PG synthesis. The results indicate that conditioned media from both pigeon peritoneal macrophages and a mouse monocyte-macrophage cell line, P388D1, caused altered PG synthesis by WC smooth muscle cells. There was a specific stimulation of the DS-PG released into the media. No apparent stimulatory effect was detected on the CSPG or HS-PG secreted by the cells or on the cell-associated PG, previously shown to consist solely of CS-PG and HS-PG.38 The effect observed was specific for macrophages, because no stimulatory effect on PG production was observed when smooth muscle cell cultures were treated with conditioned media from smooth muscle cells. Although stimulation of DS-PG was a consistent effect of macrophage-conditioned media, the magnitude of response varied with cells obtained from different animals (Figures 1 and 3). This observation may suggest regulatory differences between macrophages or response differences between smooth muscle cells. The increase in both [35S]sulfate and [3H]serine incorporation into DS-PG indicated stimulation of intact PG

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Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells.

Arterial dermatan sulfate proteoglycan has been shown to increase with atherosclerosis progression, but factors responsible for this increase are unkn...
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