Macrophages bind LDL using heparan sulfate and the perlecan protein core

Abstract

The retention of low-density lipoprotein (LDL) is a key process in the pathogenesis of atherosclerosis and largely mediated via smooth-muscle cell-derived extracellular proteoglycans including the glycosaminoglycan chains. Macrophages can also internalize lipids via complexes with proteoglycans. However, the role of polarized macrophage-derived proteoglycans in binding LDL is unknown and important to advance our understanding of the pathogenesis of atherosclerosis. We therefore examined the identity of proteoglycans, including the pendent glycosaminoglycans, produced by polarized macrophages to gain insight into the molecular basis for LDL binding. Using the quartz crystal microbalance with dissipation monitoring technique, we established that classically activated macrophage (M1)- and alternatively activated macrophage (M2)-derived proteoglycans bind LDL via both the protein core and heparan sulfate (HS) in vitro. Among the proteoglycans secreted by macrophages, we found perlecan was the major protein core that bound LDL. In addition, we identified perlecan in the necrotic core as well as the fibrous cap of advanced human atherosclerotic lesions in the same regions as HS and colocalized with M2 macrophages, suggesting a functional role in lipid retention in vivo. These findings suggest that macrophages may contribute to LDL retention in the plaque by the production of proteoglycans; however, their contribution likely depends on both their phenotype within the plaque and the presence of enzymes, such as heparanase, that alter the secreted protein structure.

Keywords: atherosclerosis; chondroitin sulfate; extracellular matrix; glycosaminoglycan; heparan sulfate; low-density lipoprotein; macrophage; perlecan; proteoglycan; quartz crystal microbalance.

Figure 1

CS and HS are localized in the core and cap of late atherosclerotic plaques. Representative micrographs showing the localization of (A) CS and (B) HS compared with the (C) isotype control at (i) low and (ii) higher magnification of the boxed area indicated in (i). HS was probed with the monoclonal anti-HS chain antibody (clone 10E4) and CS was probed with the monoclonal anti-CS antibody (clone CS-56). Antibody binding was visualized with DAB (brown) and nuclei were counterstained with hematoxylin (blue). Scale bar is 1 mm in (i) and 200 μm in (ii).

Figure 2

M1 and M2-polarized primary and THP-1 cells differentially express glycosaminoglycans. A, the presence of CS chains produced by the THP-1 cells using mouse monoclonal antibody clone CS-56 determined by ELISA (n = 3). B, the presence of HS chains produced by the THP-1 cells using mouse monoclonal antibody clone 10E4 determined by ELISA (n = 3). Asterisk indicates significant difference compared with M1. C, proportion of CS disaccharides present in proteoglycan-enriched medium conditioned elaborated by M1 and M2-polarized primary and THP-1 cells. D, proportion of HS disaccharides present in proteoglycan-enriched medium conditioned elaborated by M1 and M2-polarized primary and THP-1 cells. The HS disaccharide structures are indicated by both their abbreviated name and structure (table inset).

Figure 3

THP-1 cells with M1 and M2 phenotypes internalize oxLDL to become foam cells. Representative light microscopy images of oil red O stained M1 or M2-polarized THP-1 cells either untreated or treated with oxLDL to form foam cells. Scale bar represents 20 μm.

Figure 4

Macrophage proteoglycans bind LDL via HS and their protein core. A, determination of LDL binding to M1 and M2 THP-1 proteoglycans by turbidity measurement over a range of proteoglycan doses. Data are presented as mean ± SD (n = 3). B, sample QCM-D experiment of LDL binding to M1 THP-1 proteoglycans displayed as changes in frequency (Δf) and dissipation (ΔD) versus time for the third overtone. The vertical lines indicate the addition of PBS, proteoglycans, albumin, or LDL. C, mass of proteoglycan fractions bound to sensor surface. The mass of proteoglycan (PG) bound was determined by Voigt modeling of the QCM-D data. Data are presented as mean ± SD (n = 4). Mass of LDL bound to proteoglycans elaborated by (D) different primary and THP-1 macrophage subsets or (E) M1-polarized primary and THP-1 cells. The mass of LDL bound was determined by Voigt modeling of the QCM-D data. Data are presented as mean ± SD (n = 4). Selected proteoglycan preparations were treated with C'ase ABC (-CS) and/or HepIII (-HS) prior to immobilization on the sensor surface and compared with the undigested fraction (control). Asterisk indicates significant difference compared with same cell type control or as indicated. F, schematic representation of LDL binding to the HS chains and protein cores present in the macrophage-derived proteoglycans. Mass of LDL bound to proteoglycans elaborated by (G) M1 polarized THP-1 cells following treatment with 1 M NaCl and (H) M2-polarized primary and THP-1 cells. The mass of LDL bound was determined by Voigt modeling of the QCM-D data. Data are presented as mean ± SD (n = 4). Selected proteoglycan preparations were treated with C'ase ABC (-CS) and/or HepIII (-HS) prior to immobilization on the sensor surface and compared with the undigested fraction (control). Asterisk indicates significant difference compared with same cell type control or as indicated. (I) Representative ΔD versus Δf plot (Df plots) for LDL binding to M2 THP-1 proteoglycan preparations. The arrow indicates the time course of the data points.

Figure 5

Perlecan binds LDL via the protein core. A, sample QCM-D experiment of LDL binding to M2 THP-1 proteoglycans displayed as changes in frequency (Δf) and dissipation (ΔD) versus time for the third overtone. The vertical lines indicate the addition of PBS, proteoglycans, albumin, antibody, or LDL. B, mass of LDL bound to proteoglycans elaborated by M2-polarized THP-1 macrophages treated with both C'ase ABC (-CS) and HepIII (-HS) prior to immobilization followed by the addition of anti-perlecan, anti-versican, or anti-biglycan antibodies or no antibody (control). The mass of LDL bound was determined by Voigt modeling of the QCM-D data. Data are presented as mean ± SD (n = 4). Asterisk indicates significant difference compared with same cell type control. C, representative western blot of proteoglycan fraction elaborated by M2-polarized THP-1 macrophages either untreated (control) or treated with either HepIII (-HS) or C'ase ABC (-CS), electrophoresed on an 3–8% Tris-acetate gel, and probed for the presence of perlecan using a mouse monoclonal anti-perlecan antibody (clone E-6). Annotations on the right of the blot indicate the migration position of the lowest point of each band. D, mass of LDL bound to immunopurified perlecan derived from M2-polarized THP-1 cells compared with the M2 THP-1 fraction depleted of perlecan and glycosaminoglycans and immunopurified perlecan derived from human aortic endothelial cells. Selected experiments were performed after treatment of the proteoglycans with C'ase ABC and/or HepIII or addition of the anti-perlecan antibody. The mass of LDL bound was determined by Voigt modeling of the QCM-D data. Data are presented as mean ± SD (n = 4). Asterisk indicates significant difference compared with perlecan for the same cell type. E, schematic representation of LDL binding to the protein core of perlecan but not to the protein cores present in the proteoglycan fraction depleted of perlecan.

Figure 6

Perlecan colocalized with M2 macrophages in late atherosclerotic plaques. Representative micrographs showing the colocalization of perlecan and M2 macrophages at (A) low and (B and C) higher magnification of the boxed areas indicated in (A). Perlecan was probed with the rabbit polyclonal anti-perlecan antibody CCN-1 and visualized with DAB (brown). M2 macrophages were probed with the anti-CD163 antibody (clone 10D6) and visualized with Vina Green (green). Nuclei were counterstained with hematoxylin (blue). Arrows indicate regions of colocalization. Scale bar is 200 μm in (A) and 50 μm in (B and C).