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Originally published In Press as doi:10.1074/jbc.M001659200 on April 3, 2000

J. Biol. Chem., Vol. 275, Issue 24, 18085-18092, June 16, 2000
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Altered Dermatan Sulfate Structure and Reduced Heparin Cofactor II-stimulating Activity of Biglycan and Decorin from Human Atherosclerotic Plaque*

Rebecca A. ShirkDagger §, Narayanan Parthasarathy, James D. San Antonio||, Frank C. Church**, and William D. WagnerDagger Dagger Dagger

From the Departments of Dagger  Pathology and  Cancer Biology, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27157-1040, the || Department of Medicine and Cardeza Foundation of Hematological Research, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and the ** Department of Pathology and Laboratory Medicine and the Center for Thrombosis and Hemostasis, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599-7035

Received for publication, February 25, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biglycan and decorin are small dermatan sulfate-containing proteoglycans in the extracellular matrix of the artery wall. The dermatan sulfate chains are known to stimulate thrombin inhibition by heparin cofactor II (HCII), a plasma proteinase inhibitor that has been detected within the artery wall. The purpose of this study was to analyze the HCII-stimulatory activity of biglycan and decorin isolated from normal human aorta and atherosclerotic lesions type II through VI and to correlate activity with dermatan sulfate chain composition and structure. Biglycan and decorin from plaque exhibited a 24-75% and 38-79% loss of activity, respectively, in thrombin-HCII inhibition assays relative to proteoglycan from normal aorta. A significant negative linear relationship was observed between lesion severity and HCII stimulatory activity (r = 0.79, biglycan; r = 0.63, decorin; p < 0.05). Biglycan, but not decorin, from atherosclerotic plaque contained significantly reduced amounts of iduronic acid and disulfated disaccharides Delta Di-2,4S and Delta Di-4,6S relative to proteoglycan from normal artery. Affinity coelectrophoresis analysis of a subset of samples demonstrated that increased interaction of proteoglycan with HCII in agarose gels paralleled increased activity in thrombin-HCII inhibition assays. In conclusion, both biglycan and decorin from atherosclerotic plaque possessed reduced activity with HCII, but only biglycan demonstrated a correlation between activity and specific glycosaminoglycan structural features. Loss of the ability of biglycan and decorin in atherosclerotic lesions to regulate thrombin activity through HCII may be critical in the progression of the disease.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Biglycan and decorin are small leucine-rich dermatan sulfate (DS)1-containing proteoglycans (PGs) found in the extracellular matrix of connective tissues such as skin, bone, and cartilage. Biglycan and decorin have also been detected in the artery wall (1). They are composed of distinct core proteins linked to one (decorin) or two (biglycan) DS chains (2) that consist of alternating hexuronic acid and N-acetylgalactosamine residues. The DS chains are heterogeneous in the extent of post-translational modifications such as the conversion of glucuronic acid to its epimer iduronic acid and sulfation at the C-4 and C-6 positions of N-acetylgalactosamine and the C-2 position of iduronic acid (3). The predominant disaccharide in mammalian DS is hexuronic acid-N-acetylgalactosamine-4-sulfate, but small amounts of "oversulfated" disaccharides containing two or three sulfates are also usually detectable. Numerous structural studies have been carried out on DS from mucosa, skin, and cartilage. The chain composition appears to be distinct for specific tissues and species (2, 4). However, relatively little is known about the structure of human arterial DSPG in health or disease. DS content is elevated in atherosclerotic plaque compared with normal aorta (5), and DSPGs produced by cultured aortic smooth muscle cells exhibit altered sulfation patterns after treatment with platelet-derived growth factor and transforming growth factor-beta (6), two cytokines implicated in atherosclerosis. Thus, changes in arterial DSPG may occur in the human atherosclerotic plaque but have not been reported.

DS chains (7, 8) and DSPG (9, 10) greatly increase the rate of thrombin inhibition by heparin cofactor II (HCII). Thrombin is an enzyme with procoagulant (11), chemoattractant (12, 13), and mitogenic activities (14, 15) that is generated at sites of vascular injury. Thrombin is thought to contribute to atherogenesis (16-18). HCII, a glycosaminoglycan (GAG)-binding plasma proteinase inhibitor and member of the serpin superfamily of proteins (19), inhibits thrombin by forming an inactive bimolecular complex with the enzyme. The inhibition reaction is accelerated by DS or DSPG (through the DS moiety) in a dose-dependent manner, up to several thousand-fold at optimal concentrations (7, 8). A specific DS hexasaccharide sequence composed of repeats of iduronic acid 2-sulfate right-arrow N-acetylgalactosamine 4-sulfate has been shown to bind to HCII (20). Given the selectivity with which DS activates HCII among all of the GAG-binding serpins and the presence of DSPG in the extracellular matrix of a wide variety of tissues, HCII has been proposed to be an inhibitor of "extravascular" thrombin activity (i.e. released or generated outside the circulation due to vascular damage) and to be activated physiologically by DSPG (7-10). Isolated biglycan and decorin from skin and cartilage have been shown to accelerate thrombin-HCII inhibition reactions (10). In addition, DSPGs synthesized by cultured fibroblasts (9) and arterial smooth muscle cells (21) accelerate the rate of thrombin inhibition by HCII. Recently, an immunohistochemical study indicated that HCII is distributed throughout the intima beneath the endothelium of normal human arteries (22). The presence of both DSPG and HCII within the arterial wall is consistent with a role for DSPG-stimulated HCII inhibition of intramural thrombin activity. If changes in arterial DSPG structure occur during the progression of atherosclerosis, the thrombin-inhibitory activity of HCII may be altered, thus affecting the proatherogenic activity of thrombin.

The purpose of the present study was to investigate the structure and HCII-stimulatory activity of human aortic biglycan and decorin and to determine possible changes that occur in atherosclerosis. Biglycan and decorin were isolated from normal aorta and atherosclerotic lesions of varying severity. The DSPGs were compared for activity in thrombin-HCII assays and for GAG composition. The results indicate that biglycan and decorin from atherosclerotic lesions exhibit both altered structure and reduced activity compared with DSPG from normal aorta.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Human arterial biglycan and decorin were isolated as described below. Porcine skin DS was obtained from Calbiochem and nitrous acid-treated as described previously (23) to remove contaminating heparin. HCII (24) and thrombin (25) were purified as described. The thrombin chromogenic substrate, tosyl-Gly-Pro-Arg-p-nitroanilide (Chromozym-TH), was purchased from Roche Molecular Biochemicals, and Polybrene was from Sigma. Chondroitinase ABC (EC 4.2.2.4) and chondroitinase ACII (EC 4.2.2.5) were obtained from Seikagaku America, Inc. Low melting point agarose (SeaPlaque) and GelBond were from FMC Bioproducts. For affinity coelectrophoresis (ACE) experiments, heparin from porcine intestinal mucosa (Sigma) was substituted with tyramine at the reducing end, radiolabeled with 125I to a specific activity of ~30,000 cpm/ng using the IODO-GEN method (Pierce), and size-fractionated by Sephadex G-100 chromatography as described (26). Porcine skin DS was radioiodinated using the IODO-GEN method without prior substitution with tyramine, and the HCII-binding fraction was recovered by adsorption to HCII-Sepharose in 50 mM Tris-HCl (pH 7.4), 50 mM NaCl (20).

Isolation of Human Arterial Biglycan and Decorin-- Human aortas from four individuals were obtained at autopsy less than 8 h postmortem through the autopsy service of Wake Forest University Medical Center (Winston-Salem, NC). The four autopsy cases were as follows: A421, a 31-year-old female with nephritis determined as the cause of death; A127, a 59-year-old male with heart failure; A243, a 27-year-old female with sepsis; and A507, a 52-year-old female with emphysema. Intima preparations were made by stripping vessels under × 10 magnification. Atherosclerotic lesions, identified grossly according to the American Heart Association classification scheme (27), and adjacent normal aorta were excised, minced, and then extracted with buffer (15 ml/g of wet tissue) containing 4 M GdnHCl in 0.05 M sodium acetate (pH 4.5) with 100 mM 6-aminohexanoic acid, 5 mM benzamidine, 3 mM o-phenanthroline, and 5 mM tryptamine HCl. Extracts were filtered on Whatman no. 1 paper, concentrated in an Amicon stirred-cell concentrator with YM30 membrane, and dialyzed into buffer containing 7 M urea, 0.05 M sodium acetate (pH 7.2), 0.15 M NaCl. Dialyzed samples were applied to DEAE Sephacel columns (15-25-ml bed volume) equilibrated and washed with 7 M urea in 0.05 M Tris-HCl (pH 7.2), 0.15 M NaCl. The column was eluted stepwise with equilibrating buffer containing 0.35 M NaCl to elute heparan sulfate PG and then with 1 M NaCl to elute the DSPG and chondroitin sulfate-containing PG pool. The 1 M NaCl fraction was concentrated; dialyzed against 4 M GdnHCl, 0.05 M sodium acetate (pH 5.8); and chromatographed on a Sepharose CL-4B column (1.5 × 78 cm). Collected fractions were analyzed for PG with the dimethylmethylene blue dye-binding assay (28). The CL-4B peak eluting at a Kav of ~0.6 contained the biglycan and decorin, which were subsequently separated by octyl-Sepharose chromatography as described previously (2). First an aliquot of DSPG was radiolabeled with 14C and mixed with unlabeled DSPG to monitor the separation of biglycan and decorin. 14C labeling of PG was done by reductive alkylation with 14C-labeled formaldehyde by the procedure of Jentoft and Dearborn (29) as adopted by Parthasarathy and Tanzer (30). For each sample, DSPG plus 106 dpm of 14C-labeled PG was loaded onto a 1-ml octyl-Sepharose column equilibrated with 4 M GdnHCl, 0.15 M sodium acetate (pH 6.3). Decorin was obtained in the column flow-through (unbound fraction), and biglycan was eluted with 1% CHAPS in GdnHCl/acetate buffer. Fig. 1 illustrates the effective separation of labeled decorin and biglycan in normal aorta and type IV lesion from autopsy case A421. For all samples used in this study, the octyl-Sepharose elution profiles were similar to Fig. 1. Purity of decorin and biglycan was evaluated based on the absence of extraneous protein on Coomassie Blue-stained 4-12% SDS-polyacrylamide gels. The identity of decorin and biglycan was assessed by SDS-polyacrylamide gel electrophoresis of intact PG and core proteins prepared with chondroitinase ABC treatment and by recognition by core-specific antibodies on Western blots. For normal and atherosclerotic lesions, molecular weights were similar to reported values for intact decorin and biglycan and enzyme-generated core proteins (2). In addition, in view of the limited information available on aortic biglycan, one 20-µg sample representing 5 µg of protein pooled from each of the four biglycan samples from normal aorta was examined by N-terminal sequence analysis. This sample was digested with chondroitinase ABC, subjected to SDS-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membrane. One band of 47 kDa was generated following digestion. The band was divided into top, middle, and bottom. Each part was excised from the polyvinylidene difluoride membrane and sequenced. The amino acid sequence obtained for all three samples (11, 16, and 16 residues, respectively) was identical to the reported sequence of human bone biglycan (31).


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  		Fig. 1.   Elution profile of decorin and biglycan from octyl-Sepharose. DSPG isolated from A421 normal aorta (A) and A421 type IV lesion (B) that eluted from Sepharose CL-4B were chromatographed on octyl-Sepharose. Decorin (peak 1) was eluted with 4.0 M GdnHCl, 0.15 M sodium acetate (pH 6.3). Biglycan (peak 2) was eluted with the same buffer containing 1% CHAPS.

For structural analysis of the GAG chains, biglycan and decorin core proteins were removed by digestion with papain as described previously (32). For ACE experiments, biglycan and decorin core proteins were labeled with 125I by the chloramine T method (33).

Thrombin Inhibition Assays-- Inhibition assays were performed as described previously (21) at ambient temperature. Reaction mixtures contained 50 nM HCII, 5 nM thrombin, 2 mg/ml bovine serum albumin, and PG or GAG at the indicated concentration of hexuronic acid in buffer containing 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mg/ml polyethylene glycol, and 0.02% NaN3. Fifty-µl inhibition reactions were begun with the addition of thrombin. After the appropriate incubation period, the reaction was quenched, and residual thrombin activity was measured by the addition of 135 µl of 0.2 mM Chromozym TH containing 2 mg/ml Polybrene. Color development was quenched with the addition of 50 µl of 8.7 M glacial acetic acid, and the absorbance was read at 405 nm. Second order inhibition rate constants were calculated as k2 = -ln(a)/t[I] where a is the fractional thrombin activity remaining relative to a thrombin control containing the same components minus HCII, t is incubation time, and I is the initial inhibitor concentration. Each assay consisted of triplicate determinations, and except where indicated, the mean ± S.E. of three assays is reported.

Determination of GAG Molecular Weight-- DS apparent molecular weights were determined by Sepharose 6B chromatography essentially as described (34). DS isolated by papain digestion was pooled using equivalent amounts of hexuronic acid from either normal artery samples or type V lesion samples. They were subject to beta -elimination followed by reduction with [3H]NaBH4 (35) and chromatographed on a 1.5 × 90-cm column of Sepharose 6B (Amersham Pharmacia Biotech) that was eluted with 0.2 M NaCl at a flow rate of 11.5 ml/h. Apparent molecular weights were determined by comparison of elution position with published calibration curves (34) that were confirmed with three [3H]GAG standards of known molecular weight obtained from the National Institutes of Health.

Determination of Iduronic Acid Content-- Iduronic acid content was determined by measuring the amount of unsaturated disaccharides produced from 10 µg of hexuronic acid of DS chains by digestion with either chondroitinase ABC (for total hexuronic acid-containing disaccharides, denoted UA below; Delta UA represents 4-deoxy-alpha -L threo-hex-4-enopyranosyluronic acid) or chondroitinase ACII (for glucuronic acid-containing disaccharides, denoted GluA) (36). Iduronic acid content was calculated as follows.
% <UP>IdoA</UP>=[(<UP>total &Dgr;UA</UP>−<UP>GluA</UP>)<UP>/total &Dgr;UA</UP>]×100% (Eq. 1)

Disaccharide Analysis-- DS (2 µg of hexuronic acid) was digested with chondroitinase ABC to produce unsaturated disaccharides. One-tenth of the sample (~200 ng) was chromatographed in buffer containing 70% acetonitrile/methanol (3:1, v/v) and 30% 0.5 M ammonium acetate, pH 5.3, on a 250 × 4.6-mm Partisil-10 PAC column (Whatman). Peaks detected at 232 nm were identified by comparison with retention times for unsulfated, monosulfated, and disulfated disaccharide standards obtained from Seikagaku America, Inc. Percentage composition was calculated from the sum of peak areas. The mean value of three or more runs is reported.

ACE-- GAG- and PG-protein interactions were analyzed by ACE as described (26, 37). ACE gels were made of 1% SeaKem low melting point agarose in 50 mM MOPSO (pH 7.0), 125 mM sodium acetate, 0.5% CHAPS buffer. They contained nine parallel rectangular wells (4 × 15 mm) filled with nine different concentrations of HCII embedded in agarose and a single slot (65 × 1 mm) positioned 3 mm above the top of and perpendicular to the wells. The gels were submerged in MOPSO/sodium acetate buffer in a submarine electrophoresis chamber, and ~10,000 cpm (<1 ng) of radiolabeled PG or GAG in electrophoresis buffer containing 5% sucrose and tracking dyes was loaded into the slot above the well. The PG or GAG was electrophoresed through the HCII-containing wells at 20 °C for 1 h at 76 V. The gels were dried, and PG or GAG mobility was measured with a PhosphorImager (Molecular Dynamics). Each protein lane was scanned from top to bottom to measure relative radioactivity content per 88-µm pixel, and a distribution curve for each lane was determined. PG or GAG mobility in each lane was defined as the pixel position that divided the distribution curve into halves. The PG or GAG retardation coefficient (R) was calculated for each lane as the mobility shift in the protein-containing lane divided by the mobility in a protein-free zone (r = (M0 - M)/M0, where M0 is the mobility of free PG or GAG and M is the mobility through protein). Curve fitting of binding isotherms and calculation of apparent Kd were performed as described (26).

ACE analysis of biglycan and decorin required the following modifications. The radiolabeled DSPG samples were found to contain a co-purifying minor contaminant that may have potentially interfered with analysis of PG migration on ACE gels. The contaminant migrated as a discrete slow-moving front on ACE gels, was resistant to chondroitinase ABC digestion and noninteractive with 5 µM HCII or 2 µM collagen type I in ACE gels, and was thus determined not to be a DSPG. To remove the contaminant, radiolabeled decorin and biglycan were subjected to preparative ACE in block gels containing 1 µM collagen type I, as described (26). The DSPGs were retained at the top of the gels through binding to collagen and were thus resolved from the faster migrating unbound contaminant. After electrophoresis, the portion of the ACE gel containing the contaminant was cut away and discarded, and the DSPG-containing gel segments were then melted, pooled, and made 6 M urea for loading into analytical ACE gels (urea prevents the molten agarose from gelling, does not migrate in an electrophoretic field, and prevents renaturation of the collagen). Analytical gels were poured that each contained only three agarose lanes, one with 5 µM HCII and two lacking protein. Following electrophoresis of biglycan and decorin through these gels, DSPG retardation coefficients (R) at 5 µM HCII were calculated as described above.

Statistical Analysis-- Results of the experimental studies are reported as mean ± S.E. unless otherwise noted. Differences between means were assessed either by Student's t test or by one-way analysis of variance followed by Tukey's test for mean separation. Probability values of <0.05 were considered significant. No data were transformed prior to analyses.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Properties of Proteoglycans in Human Aorta-- Human arterial biglycan and decorin were isolated from the aorta intima tissue of four different individuals obtained at autopsy. Total proteoglycan isolated by GdnHCl extraction was similar to our previous reports (5, 38) and the reports of others (39) for human aorta. For this study, mean hexuronic acid concentrations ranged from 379 to 502 µg/g of wet tissue (Table I). Depending upon lesion type, approximately 63-83% of the PG was accounted for by a mixture of chondroitin sulfate PG (versican) and DSPG (decorin and biglycan) eluting from DEAE-Sephacel with 1.0 M NaCl (Table I). Heparan sulfate PG accounted for approximately 12-22% of the total PG based on hexuronic acid analysis of the 0.35 M NaCl eluate. DSPG were separated from versican by chromatography on Sepharose CL-4B. Following hexuronic acid analysis of the DSPG, normal aorta and lesion types II, IV, and V, respectively, comprised 27 ± 6, 37 ± 3, 31 ± 2, and 43 ± 5% (mean ± S.E.) total PG (Table I). Increased amounts of biglycan were observed in type IV lesions, where the biglycan/decorin ratio was significantly greater (p < 0.05) compared with normal aorta.

                              
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Table I
Proteoglycans prepared from human aortic tissue
All values are means ± S.E. µg/g, µg of hexuronic acid/g of wet tissue.

Human Arterial Biglycan and Decorin from Normal Aorta Accelerate Thrombin Inhibition by HCII-- The DSPG samples were analyzed for activity in inhibition assays to determine the ability to accelerate the rate of thrombin inhibition by HCII. At 1 µg of hexuronic acid/ml, human DSPG from normal aorta increased the rate of thrombin inhibition several hundred-fold over the inhibition rate in the absence of PG (k2 = 4.1 × 104 M-1 min-1) (Fig. 2A). Some variation between individuals was observed, but normal arterial biglycan consistently exhibited between 2- and 4-fold greater activity than decorin from the same tissue source. Human arterial DSPG had less activity than porcine skin DS (see legend to Fig. 2A). The effect of biglycan and decorin concentration on thrombin inhibition rates was determined for selected samples. Over the range of concentrations tested, a dose-dependent effect was observed for normal artery biglycan and decorin from autopsy A243 (Fig. 2B) and for porcine skin DS (data not shown). At each concentration, the consistent decreasing order of activity was as follows: skin DS > arterial biglycan > arterial decorin (Fig. 2B).


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  		Fig. 2.   Human arterial biglycan and decorin from normal aorta accelerate thrombin-HCII inhibition reactions. A, the activity of normal human artery biglycan DSPG (black bars) and decorin DSPG (hatched bars) from four separate autopsies (denoted with the letter A followed by a number) was analyzed in thrombin-HCII inhibition reactions. Reactions contained 50 nM HCII, 5 nM thrombin, and 1 µg of hexuronic acid/ml of DSPG. For comparison, the rate constant (k2) obtained with 1 µg/ml porcine skin DS was 9.2 ± 0.3 × 107 M-1 min-1, and the k2 in the absence of added DSPG or DS was 4.1 ± 0.4 × 104 M-1 min-1. The mean ± S.E. of three independent assays is reported. B, the effect of DSPG concentration on the thrombin-HCII inhibition rate was determined using normal arterial biglycan (open circle ) and decorin (black-square) from autopsy A243. The mean of two independent assays is reported.

Arterial Biglycan and Decorin from Atherosclerotic Lesions Have Reduced Activity in Thrombin-HCII Inhibition Reactions-- The activity of biglycan and decorin isolated from atherosclerotic lesion types II through VI was next compared with normal artery DSPG. Lesion biglycan exhibited a 24-75% loss of activity, and lesion decorin exhibited a 38-79% loss of activity in thrombin-HCII inhibition assays relative to normal artery DSPG from the same autopsy case (Fig. 3). In no case was the activity of lesion decorin or biglycan equal to or greater than the corresponding normal artery DSPG. For both biglycan and decorin, a significant negative linear relationship of inhibition rate and atherosclerosis progression was observed. The correlation coefficient for biglycan was 0.79 (p < 0.001), and the value for decorin was 0.63 (p < 0.05).


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  		Fig. 3.   Comparison of activity of human arterial DSPG from normal aorta and atherosclerotic lesion. The activity of biglycan (A) and decorin (B) isolated from normal aorta (NA) and atherosclerotic lesion types II, IV, V, and VI of four autopsy cases (, A421; , A127; black-triangle, A243; down-triangle, A507) was analyzed in thrombin-HCII inhibition assays at a fixed concentration of 1 µg hexuronic acid/ml. Inhibition rates (mean ± S.E., n = 3) for plaque DSPG are reported relative to the inhibition rate for normal artery DSPG from the corresponding autopsy case (defined as 1.0).

Biglycan and Decorin from Atherosclerotic Plaque Exhibit Altered Structural Features-- The structure of biglycan and decorin GAG chains was analyzed in an attempt to explain the functional differences observed. We first determined by using gel filtration chromatography whether there were differences in the GAG molecular size between normal artery versus plaque DSPG. However, the lack of sufficient material for several samples precluded molecular weight determinations for all 30 samples. Instead, DS chains were analyzed from normal artery and type V atherosclerotic lesion. For this purpose, equal amounts of DS chains were pooled from two autopsy samples: A421 and A507 for normal artery biglycan and A421 and A127 for type V lesion biglycan, type V lesion decorin, and normal artery decorin. The apparent molecular mass of the pooled normal artery biglycan and type V lesion biglycan was 30,521 and 41,058 daltons, respectively. The molecular mass of pooled normal artery decorin and type V lesion decorin was 28,566 and 33,797 daltons, respectively.

Iduronic acid-rich DS sequences have been associated with the stimulation of HCII activity. Iduronic acid content was measured by susceptibility to chondroitinase ABC and resistance to chondroitinase ACII digestion. The mean iduronic acid content for normal arterial biglycan and decorin comprised 67 and 22%, respectively, of total hexuronic acid and differed significantly (p < 0.01) (Table II). In addition, biglycan from atherosclerotic lesion consistently contained reduced (p < 0.002) amounts of iduronic acid relative to normal artery (Table II). The mean iduronic acid content for biglycan from all lesion types was 32%. No significant reductions in iduronic acid content were observed for decorin from atherosclerotic plaques.

                              
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Table II
Disaccharide composition (percentage of total)
Iduronic acid content (IdoA, percentage of total hexuronic acid) and unsaturated disaccharide (Delta Di-) composition were determined as described under "Experimental Procedures." Significant differences for biglycan are as follows: IdoA, normal > all lesion types, p < 0.002; IdoA, normal > lesion II, p < 0.003; IdoA, normal > lesion IV, p < 0.03; IdoA, normal > lesion V, p < 0.02; 2,4-diS, normal > all lesion types, p < 0.05; 4,6-diS, normal > all lesion types, p < 0.04. ND, not determined.

Disaccharide composition was determined by reverse phase high performance liquid chromatography analysis on a Partisil PAC 10 column after GAG digestion with chondroitinase ABC. Monosulfated disaccharides constituted a major portion (78-97%) of total disaccharides, with Delta Di-4S being the predominant disaccharide for both biglycan and decorin (Table II). However, the ratio of Delta Di-6S to Delta Di-4S for decorin was approximately twice as high as that of biglycan. The disulfated disaccharides Delta Di-2,4diS and Delta Di-4,6diS together constituted a small percentage of the total, ranging from 0 to 8%. The percentage of disulfated disaccharides was reduced relative to normal artery biglycan in 8 of 11 plaque biglycan samples for Delta Di-2,4diS and in 9 of 11 samples for Delta Di-4,6diS (Table II). The mean Delta Di-2,4diS content for normal artery biglycan was 2.7% of the total disaccharides and decreased to 1.3, 0.7, 0.2, and 0% for lesion type II, type IV, type V, and type VI biglycan, respectively. The mean ± S.E. for Delta Di-2,4diS content in biglycan for all lesion types (0.78 ± 0.25%) was significantly reduced (p < 0.05) compared with normal aorta. Likewise, Delta Di-4,6diS content for biglycan from all lesion types (0.63 ± 0.32%, mean ± S.E.) was significantly lower compared with normal aorta (2.2 ± 0.72%). There was no significant change in disulfated disaccharide content for plaque decorin.

Relationship between DSPG Activity and Composition-- Normal artery biglycan and normal artery decorin demonstrated a positive correlation between activity in thrombin-HCII inhibition assays (i.e. inhibition rates) and iduronic acid content (Table III). Normal artery and plaque biglycan samples also exhibited a significant positive correlation between iduronic acid content and activity in thrombin-HCII assays (Table III). For biglycan, there was a positive correlation between activity in thrombin-HCII assays and Delta Di-2,4diS content or Delta Di-2,4diS plus Delta Di-4,6diS content, but there was no significant correlation for biglycan activity versus Delta Di-4,6diS content alone (Table III). Decorin did not show a significant correlation between activity and Delta Di-2,4diS content, Delta Di-4,6diS content, or Delta Di-2,4diS plus Delta Di-4,6diS content.

                              
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Table III
Relationship of DSPG activity and GAG composition
Linear regression analysis of thrombin-HCII inhibition rate (k2, measured at 1 µg of hexuronic acid/ml of DSPG) plotted versus percentage composition of specified disaccharide components. n is the sample size, and r is the correlation coefficient.

Differences in DSPG Activity in Thrombin-HCII Assays Correlate with Differences in Binding to DSPG-- The accelerating effect of DS and DSPG on thrombin inhibition by HCII is thought to be due to binding of GAG to HCII. Because the observed differences in DS chain composition did not appear to account for all of the differences in DSPG activity, we next determined if variation in DSPG activity reflects differences in binding affinity for HCII. The technique of ACE was applied because it detects even weak binding events characteristic of protein interactions with PG or GAG. Binding is detected under nondenaturing conditions and at physiological ionic strength as the retardation of PG electrophoretic migration through protein-containing lanes of a 1% agarose gel. The reduction in PG mobility (i.e. retardation coefficient, R) is proportional to fractional saturation of the PG or GAG by protein. The equilibrium binding constant (Kd) can be determined from the relationship between R and protein concentration, as reported previously for the interaction of heparin with antithrombin and selected matrix proteins (37). To first validate the ACE method for the study of HCII-GAG interactions, the binding of HCII to size-fractionated heparin was examined. The Kd of 212 nM (Fig. 4) was measured for medium molecular weight 125I-tyramine-heparin (excluding the ~12% of molecules lowest in Mr as well as the ~12% highest in Mr and chosen as representative of the "average" heparin molecule), which compares favorably with a previously reported Kd of 230 nM for unfractionated heparin and HCII (8). Four aortic DSPG samples with different HCII activities and porcine skin DS were next analyzed by ACE. Saturating concentrations of HCII could not be achieved due to weak binding (Kd > 5 µM) and limited supply of protein, and therefore complete binding isotherms and Kd values were not obtained. Instead, retardation coefficients were measured at a fixed concentration of 5 µM HCII to determine differences between 125I-DSPG samples in binding to HCII. Fig. 5 illustrates that while 5 µM HCII induces less than 25% retardation in DSPG or DS migration, the ranking of DSPG/DS samples by apparent binding to HCII (represented by R') or by HCII cofactor activity (k2) is identical. A second experiment showed the same pattern with the exception of a single outlier. These results demonstrate that the increased activity of artery-derived DSPG in thrombin-HCII inhibition assays is associated with increased apparent affinity for HCII.


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  		Fig. 4.   ACE analysis of HCII-heparin interaction. Calculation of apparent affinity of HCII for size-fractionated medium molecular weight 125I-Tyr-heparin. The heparin retardation coefficient (R) for each protein-containing lane was determined as described under "Experimental Procedures" and is plotted versus HCII concentration. The smooth curve represents nonlinear least-squares fit to the equation, r = Rinfinity /(1 + Kd/[protein]2), where Rinfinity  = R at saturating concentrations of protein. Rinfinity  = 0.73 for HCII, due to high mobility of HCII toward the cathode in the electrophoretic field. A representative experiment is shown.


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  		Fig. 5.   Relationship between DSPG activity and interaction with HCII in ACE gels. Porcine skin DS (), human normal aorta biglycan (black-square) and normal aorta decorin (black-triangle), and type II lesion biglycan (black-down-triangle ) and type II lesion decorin (black-diamond ) from autopsy A421 were subjected to ACE analysis. The thrombin-HCII inhibition rate (k2) measured with 1 µg of hexuronic acid/ml of DSPG is plotted versus the normalized retardation coefficient (R') of 125I-DSPG or DS in ACE gel lanes containing a 5 µM HCII. R' = R/Rinfinity , where Rinfinity is as defined in the legend to Fig. 3. Results shown are from a single experiment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Arterial DSPG has been implicated as having various roles in the artery wall, including the regulation of collagen fibrillogenesis (40), the binding of cytokines (40) and lipoproteins (41), and the stimulation of HCII activity (9, 10). All of these activities have been attributed at least in part to the DS moiety, although the fine structure of the DS may vary between different cells or tissues. Despite these reports indicating structure-function interrelationships of DS, no information is available on human aortic DSPG structure. The goal of the present study was to assess and compare the structure and HCII stimulatory activity of biglycan and decorin from normal aorta and to determine whether functional and structural alterations occur in these PGs during atherosclerosis. The main results of the study indicate that there is a loss in the ability of DS to inhibit artery wall thrombin through the activity of HCII as atherosclerosis progresses.

Both biglycan and decorin from normal artery were found to accelerate the rate of thrombin inhibition by HCII in a dose-dependent manner, with biglycan stimulating a larger rate increase than equal hexuronic acid amounts of decorin. Interestingly, both biglycan and decorin isolated from atherosclerotic plaque exhibited reduced activity in thrombin-HCII inhibition assays compared with the normal artery-derived PG. Compositional analysis indicated that iduronic acid content was significantly greater in biglycan than decorin from normal artery and that iduronic acid content was significantly reduced in plaque biglycan versus normal artery biglycan. The predominant disaccharide for both biglycan and decorin was Delta Di-4S, but decorin contained on average a greater percentage of Delta Di-6S than biglycan. The majority of biglycan samples from atherosclerotic plaque contained reduced amounts of the disulfated disaccharides. On average, lesion types had significant reductions in both Delta Di-2,4diS and Delta Di-4,6diS compared with normal aorta. There was no such pattern of reduced iduronic acid or disulfated disaccharide content for plaque decorin.

With one notable exception, previous studies of DS structure (4, 42-45) used material isolated without knowing whether they were derived from decorin or biglycan. These studies, which were performed on total DS derived from a variety of mostly nonhuman tissues such as skin, intestinal mucosa, liver, spleen, and aorta, demonstrated that DS was a copolymer containing both glucuronic acid- and iduronic acid-containing disaccharides that are sulfated mainly on the C-4 position of N-acetylgalactosamine. Differences in DS disaccharide composition have been observed between preparations from different tissue sources or from the same tissue but different species (2, 4). Our data are consistent with the range of values previously reported. An iduronic acid content of 60% in normal artery biglycan approaches the ~80% iduronic acid found in bovine skin DS (2) and porcine skin DS (42), while the iduronic acid content of ~30% in normal artery decorin is closer to the reported levels for human skin DS (43), equine aorta DS (45), and bovine cartilage DS (2). Human arterial biglycan and decorin have a Delta Di-4S content comparable with horse aorta DS (~67%) (45) but appreciably less than the Delta Di-4S of ~85% reported for porcine skin DS and bovine mucosa DS (44).

In only one other study have DS chains from biglycan and decorin been examined separately. Choi and Rosenberg (2) isolated intact biglycan and decorin from bovine cartilage and skin and found striking tissue specificity of iduronic acid content. Biglycan and decorin from bovine skin contained ~80% iduronic acid, whereas both PG in cartilage contained only ~35% iduronic acid (2), demonstrating that DSPG containing the same core protein can have very different DS chains. Our findings suggest, however, that two different core proteins from the same tissue can have distinct DS compositions. While both biglycan and decorin are synthesized by smooth muscle cells in the artery wall, their distribution between interstitial and pericellular matrices differ (46). Apparently, the cellular GAG modification machinery is sensitive to a variety of regulatory influences. The changes observed in GAG composition of biglycan from normal artery versus atherosclerotic plaque suggest that the presence of disease can also modify smooth muscle cell metabolism and result in altered structure of DS chains.

The identification of structural features required for the stimulation of HCII by DS has been the subject of numerous studies. Highly charged DS preparations enriched in iduronic acid and the disulfated disaccharides have been shown to have the greatest activity. A high affinity hexasaccharide composed of the repeating disaccharide iduronic acid 2-sulfate right-arrow N-acetylgalactosamine 4-sulfate has been identified in porcine DS (20), and Delta Di-4,6diS has been found to contribute to the activity expressed by Delta Di-2,4S-enriched sequences (44, 47). Iduronic acid seems to be important in that it can be sulfated at the C-2 position, while its epimer glucuronic acid is rarely sulfated. Because of the conformational flexibility of iduronic acid in the polymer, the resulting oligosaccharide could provide highly charged clusters of sulfate groups with high specificity for HCII, as has been described for other GAG-protein interactions (48). The positive correlation between the disulfated disaccharides and iduronic acid content of human artery biglycan and the activity in thrombin-HCII inhibition assays supports the importance of a specific sulfation pattern for the interaction with HCII. In contrast, human arterial decorin did not demonstrate the same correlation. However, the organization of disulfated disaccharides within DS appears to be crucial, and the current study does not attempt to determine the sequence of the DS chains. Oligosaccharide blocks consisting of three or more iduronic acid-containing disulfated disaccharides are required for binding to HCII (20). The reduced activity of plaque decorin compared with normal artery decorin may result from a lack of block structure due to random distribution of the disulfated disaccharides.

DS accelerates the HCII-thrombin inhibition reaction in a dose-dependent manner (7, 8). Therefore, a rigorous comparison of the activity of GAG or PG in thrombin-HCII inhibition reactions requires that the assays be performed with equimolar concentrations of DSPG/DS. In the present study, equal amounts of hexuronic acid were used for activity comparisons instead, since limited sample size prohibited molecular weight measurements on all samples. Yet GAG molecular weight measurements made on mixtures of two normal artery or plaque biglycan and decorin samples suggest that DS chain length may increase as atherosclerosis progresses. To the best of our knowledge, there are no reports of the specific effect of chain length on HCII activity beyond the minimum of 14-18 saccharide units (~4000 daltons) required for full activity (49, 50). Therefore, the reduced activity of plaque versus normal artery DSPG may in part be due to a lower molar concentration of plaque DSPG used in the assay. However, the magnitude of the loss of plaque DSPG activity exceeds the expected effect of the differences in molar concentration. The concern over the potential influence of different chain lengths is further weakened by (i) the correlation between structural components and activity (discussed above) and (ii) the observation that 1 µg of hexuronic acid/ml of normal artery biglycan (with two DS chains per molecule) has approximately twice the activity of 1 µg of hexuronic acid/ml of decorin (which contains one DS chain per molecule and was therefore assayed at effectively twice the molar concentration of biglycan). Therefore, while chain size may contribute to the observed differences in activity, structural features appear to play the main role.

Both the differences observed between biglycan and decorin in the correlation of GAG composition and HCII stimulatory activity and the potential influence of differences in DS chain length prompted us to use a second method to assess the interaction of HCII with DSPG. ACE measures the direct binding of GAG to protein in a GAG concentration-independent manner. A single previous report measured a Kd of ~1.5 µM for the HCII-DS interaction using kinetic methods (8). The current findings confirm that the affinity of DS/DSPG for HCII is weak, but for the samples analyzed by ACE there is a positive relationship between the activity measurements and interactions with HCII in an ACE gel. Although the interaction is not strong and may not represent a classical binding phenomenon, DS at optimal concentrations stimulates a several thousand-fold rate increase in a reaction that depends on an HCII-DS interaction but not a thrombin-DS interaction (51, 52).

In recent publications, atherosclerotic plaques prone to thrombosis have been identified as "vulnerable" atherosclerotic lesions (53, 54) or type IV lesions (55). The presence of this lesion type is associated with unstable angina, ischemic stroke, myocardial infarction, and, potentially, sudden death (53, 54). While therapeutic modulators to slow or delay the thrombotic event are essential for eventually preventing extensive thrombotic complications, the results of this study suggest that an earlier intervention prior to the final stages of atherosclerosis is possible. The understanding of why DS produced by smooth muscle cells of developing atherosclerotic lesions does not maintain the structural properties necessary for inhibition of arterial wall thrombin by HCII may in turn permit the development of therapies to reduce the rate of progression of atherosclerosis and thus reduce the number of vulnerable atherosclerotic lesions.

    ACKNOWLEDGEMENTS

We acknowledge N. P. Wang and J. D. Bottoms for assistance and technical expertise in proteoglycan purification and disaccharide analysis, respectively.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL-017115 (to R. A. S.) and Grants HL-25161 (to W. D. W), HL-53590 (to J. D. S.), and HL-32656 (to F. C. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Women's Health Research Institute, Wyeth- Ayerst Research, Radnor, PA 19087.

Dagger Dagger To whom correspondence should be addressed: Dept. of Pathology, Bowman Gray School of Medicine of Wake Forest University, Medical Center Blvd., Winston-Salem, NC 27157-1040. Tel.: 336-716-4568; Fax: 336-716-6279; E-mail: wwagner@bgsm.edu.

Published, JBC Papers in Press, April 3, 2000, DOI 10.1074/jbc.M001659200

    ABBREVIATIONS

The abbreviations used are: DS, dermatan sulfate; PG, proteoglycan; DSPG, dermatan sulfate proteoglycan; HCII, heparin cofactor II; GAG, glycosaminoglycan; ACE, affinity coelectrophoresis; IdoA, iduronic acid; Delta Di-0S, 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha -L-threo-hex-4-enepyranosyluronic acid)-D-galactose; Delta Di-4S, 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha -L-threo-hex-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose; Delta Di-6S, 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha -L-threo-hex-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose; Delta Di-2, 4diS, 2-acetamido-2-deoxy-3-O-(4-deoxy-2-O-sulpho-alpha -L-threo-hex-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose; Delta Di-4, 6diS, 2-acetamido-2-deoxy-3-O-(4-deoxy-alpha -L-threo-hex-4-enepyranosyluronic acid)-4,6-di-O-sulpho-D-galactose; GdnHCl, guanidine hydrochloride; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPSO, sodium 2-(N-morpholino)-hydroxypropane sulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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