INTRODUCTION

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Interactions of the sulfated glycosaminoglycan (GAG)¹ heparan sulfate (HS) with various proteins affect the biological activity, tissue localization, and turnover of the protein ligands (1-3). Such interactions, generally electrostatic in nature, regularly involve specific oligosaccharide domains generated by an elaborate biosynthetic machinery in the Golgi apparatus. HS formation starts by assembly of an initial (GlcA-GlcNAc)_n polymer. Parts of the nascent polymer are subsequently modified by N-deacetylation/N-sulfation of GlcNAc residues, and further modifications, including C-5 epimerization of GlcA residues into IdceA residues as well as O-sulfation at various positions, occur mainly in the vicinity of the previously incorporated N-sulfate groups (3, 4). The O-sulfate groups are predominantly found at the C-2 position of IdceA residues and the C-6 position of GlcN residues. The protein-binding HS domains typically reside within the N-sulfated regions, their functional specificity being determined by the pattern of modification, particularly the positioning of sulfate groups. Although information regarding the structures of the recognition sites for individual proteins (5-11) and the general features of HS biosynthesis (3) is accumulating, the biological control of HS structure and function remains poorly understood. Nevertheless, HS species from various cells and tissues clearly differ in their structure and in some studies such differences have been correlated to differential protein-binding properties (12-15). These and other findings (discussed in Refs. 1 and 3) suggest that the biosynthesis of HS is subject to regulation during development or aging. Control of the appropriate expression of functional HS domains in given organs or at particular developmental stages would appear essential, whereas, conversely, perturbed regulation could contribute to various pathologies. In the present study we have explored the aging aortic wall as a model to gain insight into the control of HS structure and function in humans. Aging is a strong predisposing factor to atherosclerosis, characterized by endothelial damage, lipid accumulation, and cell proliferation in arterial wall plaques (16). HS has been attributed multiple roles in these processes by interacting with lipoprotein lipase (17) and with growth factors such as basic fibroblast growth factor (FGF-2) and platelet-derived growth factors (PDGFs) (18-21). HS binds to and enhances the mitogenic activity of FGF-2 (22) and regulates the tissue localization of PDGFs (20). PDGFs are homo- or heterodimers of two closely related, A and B, polypeptide chains. The PDGF-A chain exists as two variants due to alternative mRNA splicing. The longer variant (PDGF-A_L) contains a C-terminal polybasic sequence that serves as a cell retention signal and is associated with the localization of PDGF to the surface of the producer cell or to the extracellular matrix (21), presumably due to interactions with HS (20). A similar but not identical retention signal is found at the C terminus of the PDGF-B propeptide chain (PDGF-B_L) (21). This propeptide may undergo various N- and C-terminal proteolytic processing events that give rise to multiple forms of cell-associated PDGF-B species (23).

	MATERIALS AND METHODS

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Isolation and Radiolabeling of Heparan Sulfate-- Tissue samples from human abdominal aorta were obtained at autopsy and stored at 70 °C until processed for HS isolation. The surrounding connective tissue was removed, and the sample, encompassing the entire thickness of the vessel wall, was cut into fine pieces with a scalpel. The samples were defatted essentially as described (15) with the exception that ethyl ether was replaced by ethanol. Defatted samples (dry weight, 0.15-2.0 g) were subjected to protease digestion with papain (Sigma; 5 mg/g of defatted tissue) in 25 ml of 0.05 M Tris, pH 5.5, 0.01 M EDTA, 2 M NaCl, 0.01 M cysteine/HCl at 60 °C for 18 h and centrifuged (10 min at 2000 × g), and the supernatants were applied to columns of DEAE-Sephacel (1.5 × 5 cm; Amersham Pharmacia Biotech) equilibrated with 0.05 M Tris, pH 7.2. The column was washed with 0.05 M sodium acetate, pH 4.0, followed by elution of the DEAE-bound material (containing sulfated GAGs) by a linear gradient of LiCl (0.15-2.0 M) in the acetate buffer (15). 2-ml fractions were collected and analyzed for uronic acid content by the carbazole reaction (24). Fractions containing GAGs were pooled, dialyzed against water, lyophilized, and digested with 1 unit of chondroitinase ABC (Seikagaku Corporation) and 125 units of endonuclease (Benzonase, Benzon Pharma A/S) in 0.05 M Tris-HCl, pH 8.0, 1 mM MgCl₂, 0.05 M sodium acetate at 37 °C overnight (25). The digest was heated for 2 min and applied to a DEAE-Sephacel column (1.5 × 5 cm) equilibrated with 0.2 M NH₄HCO₃ and eluted by a linear gradient of NH₄HCO₃ (0.2-2.0 M). Fractions containing HS were identified by the carbazole reaction, pooled, and freeze-dried, and the material was stored at 20 °C until further use. Treatment of the purified HS preparations with HNO₂ at pH 1.5 resulted in quantitative degradation of the material into lower molecular weight species as demonstrated by chromatography of intact and HNO₂-treated samples on a column of Superose 12 (data not shown), indicating that the purification procedure yielded pure HS.

Assay of Heparan Sulfate-Protein Interaction-- The binding of [³H]HS preparations to recombinant PDGF-AA_L (27), PDGF-BB_L (33 pmol/incubation) (prepared as described for PDGF-AA_L (27)),² and FGF-2 (29 pmol/incubation) (Pepro Tech EC) was studied by a nitrocelloluse filter trapping assay as described previously (8, 11).

PDGF Affinity Chromatography-- 5 mg of recombinant PDGF-AA_L was mixed with an equimolar amount of heparin and immobilized to 3 ml of CH-Sepharose CL4B (Amersham Pharmacia Biotech) according to the manufacturer's instructions (8). The column was equilibrated with Tris-buffered saline (50 mM Tris-HCl pH 7.4, 150 mM NaCl) prior to the application of [³H]HS. Subsequently, the column was washed with 10 ml of Tris-buffered saline, and the bound material was eluted using a linear NaCl gradient (0.15-2.0 M in 50 mM Tris-HCl, pH 7.4). Fractions of 1 ml were collected and analyzed for radioactivity in a liquid scintillation counter.

Structural Analysis of Heparan Sulfate-- Samples of HS (~30 µg) were subjected to cleavage with nitrous acid at pH 1.5 (28). The cleavage products were end-labeled by reduction with 250 µCi of NaB³H₄ (Amersham Pharmacia Biotech) overnight as described previously (4) and desalted on a Sephadex G-15 column (1 × 190 cm, Amersham Pharmacia Biotech) in 0.2 M NH₄HCO₃. Fractions containing disaccharides were analyzed by anion exchange HPLC on a Partisil-10 SAX column (4.6 × 250 mm, Whatman Inc.) (29).

(Eq. 1)

	RESULTS

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We first examined the binding of radiolabeled human aorta HS from a young and an old individual to FGF-2 and to dimers of the long isoforms of PDGF A or B chains (designated PDGF-AA_L and PDGF-BB_L below) (30). A filter trapping procedure was employed, involving interaction of labeled HS and the protein ligand in solution followed by rapid passage of the mixture through a nitrocellulose filter. Protein and protein-bound HS chains (but not free HS chains) were retained on the filter (8). We found that the binding capacity for PDGF-AA_L and PDGF-BB_L of HS from a 76-year-old individual was 4-5 times higher than that of HS from a 21-year-old individual (Fig. 1A), whereas the binding to FGF-2 differed only marginally between the two HS samples. To assess the inter-individual variation in these interactions, we next compared the binding of six HS preparations, from three young and three old subjects, to PDGF and FGF-2. The results of this experiment (Fig. 1B) indicated a virtually invariable level of binding of HS to a given protein ligand within each of the two age groups but marked differences in binding characteristics between these groups. The effect of age thus was selectively expressed for different proteins, in accord with the data shown in Fig. 1A. These findings clearly point to the occurrence of age-dependent differences in HS structure that affect the binding of HS to PDGF and FGF-2 in distinct ways. The effect of this structural transition with regard to PDGF binding was further examined by affinity chromatography of HS from young and old subjects on a column of immobilized PDGF-AA_L (Fig. 1C). Both types of HS contained material that remained bound to the growth factor at physiological pH and ionic strength, as well as a fraction of unbound material. The PDGF-binding chains amounted to 50.0 ± 2.4% versus 81.8 ± 1.5% (mean ± S.E. from three and two samples, respectively) of the total HS from young and old subjects, respectively. These results thus confirm not only the correlation between individual age and PDGF binding ability of aortic HS but also the striking inter-individual similarity within each age group.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Effect of age on binding of HS from human aorta to growth factors. A, PDGF-AAL, PDGF-BBL, and FGF-2 were tested for binding to [3H]HS isolated from a young (21 years) and an old (76 years) individual. Increasing amounts of [3H]HS (up to 19 pmol) were incubated with recombinant PDGF-AAL, PDGF-BBL (33 pmol/incubation), and FGF-2 (29 pmol/incubation) as described under "Materials and Methods." The data shown represent means of duplicate incubations. B, samples of [3H]HS (7.5 pmol/incubation) from three young (20, 21, and 22 years) and three old (70, 76, and 81 years) subjects were tested for binding to PDGF-AAL, PDGF-BBL, and FGF-2 (same amounts as in A). The binding of HS from old subjects is set as 100%. The means ± S.D. are shown. C, samples of [3H]HS from three subjects aged 20 (solid line), 21(dashed line), and 22 (dotted line) years (Young subjects) and two subjects aged 76 (dashed line) and 80 years (solid line) (Old subjects) were subjected to chromatography on a column of PDGF-AAL- Sepharose as described under "Materials and Methods." The arrow indicates the start of the gradient.

Binding of HS to PDGF-AA_L and FGF-2 involves structurally distinct oligosaccharide domains. The former domain is comprised by N-sulfated ~8-mer sequences with at least one trisulfated IdceA(2-OSO₃)-GlcNSO₃(6-OSO₃) disaccharide unit (8), whereas the minimal FGF-2-binding site contains an essential IdceA(2-OSO₃) residue but no 6-O-sulfate groups (5, 7, 31). Given these data, we wanted to examine whether the differential protein binding of aorta HS from young and old subjects would correlate with the O-sulfate substitution pattern of the N-sulfated regions of HS. We therefore determined the disaccharide composition of these regions in a total of 15 HS preparations from subjects aged 20-84 years (including the six preparations used in the binding experiments). The results of this analysis (Table I) demonstrated an age-dependent increase in the proportion of the IdceA(2-OSO₃)-GlcNSO₃(6-OSO₃) unit (Fig. 2). Calculation of the overall extent of 2-O- and 6-O-sulfation indicated that this increase was due to an approximate doubling of the level of 6-O-sulfate substitution of GlcNSO₃ residues in the old subjects (Fig. 2), whereas the IdceA 2-O-sulfation remained high and essentially unchanged (Fig. 2). The 6-O-sulfation of GlcNSO₃ residues showed an almost linear increase between the age of 20 and 40 years; in the still older subjects the levels of 6-O-sulfation were somewhat scattered but consistently higher than in the young individuals (Fig. 2).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Age-dependent changes in O-sulfate substitution of human aorta HS. The composition of O-sulfated disaccharide units within the contiguous N-sulfated HS domains was determined in 15 HS preparations (Table I) by anion exchange HPLC as shown in Fig. 2. The panels show the proportion of the trisulfated disaccharide units (IdceA(2-OSO3)-GlcNSO3(6-OSO3), as present in the corresponding intact polysaccharides), and the contribution of GlcN 6-O- and HexA 2-O-sulfate groups to total O-sulfation.

We further determined the degree of N-sulfate substitution of GlcN residues from the pattern of HS depolymerization following cleavage with HNO₂ (at pH 1.5; see "Materials and Methods"). Five of the HS samples (subject aged 20, 21, 74, 76, and 80 years) were analyzed (Fig. 3) and found to contain essentially similar proportions of GlcNSO₃ units (means ± S.D., 39 ± 2% of total GlcN units).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Analysis of the extent of N-sulfation of GlcN residues. Samples of aortic HS from a young (20 years) and an old (76 years) subject were subjected to cleavage with HNO2 at pH 1.5. The cleavage products were radiolabeled by reduction with NaB3H4 and separated by chromatography on Bio-Gel P-10 as described under "Materials and Methods."

Finally, we assessed the degree of sulfation of the sequences comprised of alternating N-sulfated and N-acetylated disaccharide units. These domains typically harbor GlcN 6-O-sulfate groups but few or no IdceA 2-O-sulfate groups (4) and are recovered as tetrasaccharides after cleavage of HS with HNO₂ at pH 1.5. Analysis of tetrasaccharides from four HS samples (subject aged 20, 30, 50, and 80 years) indicated essentially similar proportions of nonsulfated, monosulfated, and disulfated species (76 ± 3, 20 ± 3, and 4 ± 1% of all tetrasaccharides, respectively, expressed as means ± S.D.). The increase in 6-O-sulfate substitution of GlcN units in the old individuals thus is essentially confined to the contiguous N-sulfated domains of HS.

The major alteration in human aorta HS in association with aging is increased 6-O-sulfation of GlcNSO₃ residues. The 6-O-sulfate groups were largely incorporated into GlcNSO₃ units linked at C-4 to IdceA(2-OSO₃) residues, leading to increased formation of the trisulfated IdceA(2-OSO₃)-GlcNSO₃(6-OSO₃) disaccharide units. These results are in good agreement with the protein binding properties of the polysaccharide because IdceA(2-OSO₃)-GlcNSO₃(6-OSO₃) units have been implicated in the binding of HS to PDGF-AA_L. By contrast, the expression of FGF-2-binding HS domains was not affected by aging, as might be expected because regardless of the subject age the HS species were rich in IdceA(2-OSO₃) residues and had proportions similar to those of N-sulfate groups.

	DISCUSSION

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The present study demonstrates that human aging is accompanied by specific alterations in the fine structure of HS in the aortic wall. The increased degree of 6-O-sulfate substitution of GlcNSO₃ residues in old subjects is reflected by an elevated proportion of trisulfated, "heparin-type" IdceA(2-OSO₃)-GlcNSO₃(6-OSO₃) disaccharide units and enhanced binding of the HS to PDGFs. Such progressive, age-dependent structural alteration represents a previously unrecognized type of functional modulation of a mammalian macromolecule. Unlike rapid and reversible modifications such as phosphorylation, the alterations in sulfation described here occur during an extended time span of several decades. The remarkable structural and functional similarities of HS specimens from different age-matched subjects suggest a strictly controlled process of HS assembly.

Our findings point to 6-O-sulfation of GlcNSO₃ units as a key regulator of the biological properties of HS in the human aortic wall. The 2-O- and 6-O-sulfation reactions appear to be separately regulated during HS biosynthesis, because the former sulfation type is largely confined to the contiguous N-sulfated sequences, whereas the latter modification occurs within as well as outside these domains (4). Intriguingly, the enhanced 6-O-sulfation appeared to selectively involve the contiguous N-sulfated domains, because the sulfation of alternating domains (recovered as tetrasaccharides after cleavage of HS with HNO₂ at pH 1.5) was essentially similar in HS from young and old subjects. This finding suggests the involvement of two (or more) HS GlcN 6-O-sulfotransferase species that differ in substrate specificity and mode of regulation. Unfortunately, our knowledge regarding the regulatory mechanisms in HS biosynthesis is still fragmentary (3). Could the abundance of a given sulfate group simply reflect the expression level for a corresponding sulfotransferase?

Atherosclerotic lesions are frequently encountered in human aorta, and HS-binding growth factors such as FGFs and PDGFs are thought to contribute to the pathological smooth muscle cell migration and proliferation characterizing the disease (16). The expression level of the PDGF-A_L chain is high in human arterial smooth muscle cells and increases markedly during the conversion of monocytes into macrophages (32). It has been shown that PDGF isoforms containing the A_L or B chains are retained at cell surfaces or in the extracellular matrix by HS (20, 21). In the arterial wall, increased binding of PDGF to HS could thus result in extracellular accumulation of PDGF. Such early changes might facilitate later pathophysiological processes such as aberrant smooth muscle cell migration and growth in individuals prone to develop atherosclerotic disease. Moreover, we note that the trisulfated IdceA(2-OSO₃)-GlcNSO₃(6-OSO₃) disaccharide units found to promote binding of PDGF to HS has also been implicated in binding of lipoprotein lipase (6), a cell surface-bound enzyme that catalyzes the breakdown of triglycerides and affects the cellular uptake of lipids (17). The observed change in HS structure thus is likely to have more widespread functional implications than those emphasized in this study.