Novel role of ADAMTS-5 protein in proteoglycan turnover and lipoprotein retention in atherosclerosis

Abstract

Atherosclerosis is initiated by the retention of lipoproteins on proteoglycans in the arterial intima. However, the mechanisms leading to proteoglycan accumulation and lipoprotein retention are poorly understood. In this study, we set out to investigate the role of ADAMTS-5 (a disintegrin and metalloprotease with thrombospondin motifs-5) in the vasculature. ADAMTS-5 was markedly reduced in atherosclerotic aortas of apolipoprotein E-null (apoE(-/-)) mice. The reduction of ADAMTS-5 was accompanied by accumulation of biglycan and versican, the major lipoprotein-binding proteoglycans, in atherosclerosis. ADAMTS-5 activity induced the release of ADAMTS-specific versican (DPEAAE(441)) and aggrecan ((374)ALGS) fragments as well as biglycan and link protein from the aortic wall. Fibroblast growth factor 2 (FGF-2) inhibited ADAMTS-5 expression in isolated aortic smooth muscle cells and blocked the spontaneous release of ADAMTS-generated versican and aggrecan fragments from aortic explants. In aortas of ADAMTS-5-deficient mice, DPEAAE(441) versican neoepitopes were not detectable. Instead, biglycan levels were increased, highlighting the role of ADAMTS-5 in the catabolism of vascular proteoglycans. Importantly, ADAMTS-5 proteolytic activity reduced the LDL binding ability of biglycan and released LDL from human aortic lesions. This study provides the first evidence implicating ADAMTS-5 in the regulation of proteoglycan turnover and lipoprotein retention in atherosclerosis.

FIGURE 1.

Loss of ADAMTS-5 and proteoglycan accumulation in atherosclerosis. A, Oil red O-stained cross sections of WT and apoE^−/− aortic roots. Results are representative of three aortic roots. Scale bar 100 μm. B, immunoblotting of aortic extracts for ADAMTS-5 (TS5), versican, biglycan, ADAMTS-1 (TS1), DPEAAE⁴⁴¹ versican neoepitopes, and FGF-2. β-Actin served as loading control. Three aortas were pooled for each experimental condition. MW, molecular weight. C, densitometry measurements of immunoblots for ADAMTS-5 (red bars), biglycan (blue bars), and versican (green bars). Values were normalized to β-actin. Results are mean ± S.D. from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's post hoc comparisons with WT controls (*, p ≤ 0.05, **, p ≤ 0.01). AU, absorbance units. D, mRNA expression of biglycan and versican as measured by quantitative PCR. Values were normalized to gapdh. Results are mean ± S.D. from n = 4 experiments. E, aortic SMCs derived from WT and apoE^−/− mice were either stimulated with 50 ng/ml FGF-2 or left untreated (CON) for 24 h. Quantitative PCR of adamts5, biglycan, and versican with values normalized to gapdh was performed. Results are mean ± S.D. from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's post hoc test (**, p ≤ 0.01). F, immunoblotting for ADAMTS-5, ADAMTS-1, versican, and biglycan in the conditioned medium of aortic SMCs, incubated with or without FGF-2 (50 ng/ml) for 24 h. MMP-2 was detected by zymography. Cellular lysates were immunoblotted for β-actin. Results are representative of at least 3 independent experiments. G, immunoblotting for ADAMTS-5, ³⁷⁴ALGS aggrecan fragments, and DPEAAE⁴⁴¹ versican fragments in the conditioned medium of ex vivo cultured abdominal aortas incubated with or without FGF-2 (100 ng/ml) for 24 h. Aortic lysates were immunoblotted for β-actin.

FIGURE 2.

ADAMTS-5-mediated cleavage of vascular proteoglycans affects lipoprotein retention. A, aortas from 10-week-old WT mice were incubated either in plain MMP reaction buffer (CON) or in MMP reaction buffer supplemented with either 50 pm murine recombinant ADAMTS-1 (+TS1) or 50 pm ADAMTS-5 (+TS5) for 24 h at 37 °C. The supernatants were analyzed by immunoblotting for ³⁷⁴ALGS and DPEAAE⁴⁴¹ neoepitopes, HPLN1, and biglycan. Each lane had 20 μg of protein. Results are representative of at least 3 independent experiments. B, guanidine extracts from ADAMTS-treated aortas were immunoblotted for versican neoepitopes and β-actin. C, a human aortic specimen was dissected into three equal blocks incubated either in plain MMP reaction buffer or in MMP reaction buffer supplemented with either 100 pm human recombinant ADAMTS-1 or 100 pm ADAMTS-5 for 24 h at 37 °C. The supernatants were immunoblotted for DPEAAE⁴⁴¹, HPLN1, and biglycan. D, immunoblotting of WT, TS5 Δcat, and apoE^−/− aortic extracts for DPEAAE⁴⁴¹ neoepitopes, biglycan, and β-actin. E, densitometry measurements of DPEAAE⁴⁴¹ neoepitopes normalized to β-actin. Results are mean ± S.D. from n = 3 independent experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni's post hoc comparison with the 10-week-old WT control (***, p ≤ 0.001). AU, absorbance units. F, amount of LDL (measured as apoB-100) bound to biglycan after 5 h of digestion with 100 pm ADAMTS-5. Values are mean ± S.D. from n = 4 independent experiments, each performed with six replicates/condition. Statistical analysis was performed using two-tailed, unpaired t test. G, three human aortic specimens with subendothelial lipid accumulation were treated either with plain MMP reaction buffer or with 100 pm ADAMTS-5 for 24 h at 37 °C. The aortic supernatants were used to coat 96-well immunoplates, and the amount of LDL was quantified using HRP-conjugated antibodies against apoB-100. Values are mean ± S.D. from n = 3 independent experiments and six replicates/condition. Statistical analysis was performed using two-tailed, unpaired t test. H, aortic supernatants from two human specimens used in G were immunoblotted for DPEAAE⁴⁴¹, biglycan, apoA1, and apoE. I, consecutive, parallel sections from 48-week-old apoE^−/− abdominal aortas were probed with antibodies to apoB or DPEAAE⁴⁴¹ neoepitope. Note the difference in staining for DPEAAE⁴⁴¹ following incubation with 50 pm ADAMTS-5 for 24 h at 37 °C (right panels) as compared with controls incubated in plain MMP reaction buffer (left panels). Negative controls were probed with secondary antibodies only (−VE). Nuclei were counterstained with Mayer's hematoxylin. Scale bar, 25 μm.