Heparan sulfate in perlecan promotes mouse atherosclerosis: roles in lipid permeability, lipid retention, and smooth muscle cell proliferation

Abstract

Heparan sulfate (HS) has been proposed to be antiatherogenic through inhibition of lipoprotein retention, inflammation, and smooth muscle cell proliferation. Perlecan is the predominant HS proteoglycan in the artery wall. Here, we investigated the role of perlecan HS chains using apoE null (ApoE0) mice that were cross-bred with mice expressing HS-deficient perlecan (Hspg2(Delta3/Delta3)). Morphometry of cross-sections from aortic roots and en face preparations of whole aortas revealed a significant decrease in lesion formation in ApoE0/Hspg2(Delta3/Delta3) mice at both 15 and 33 weeks. In vitro, binding of labeled mouse triglyceride-rich lipoproteins and human LDL to total extracellular matrix, as well as to purified proteoglycans, prepared from ApoE0/Hspg2(Delta3/Delta3) smooth muscle cells was reduced. In vivo, at 20 minutes influx of human (125)I-LDL or mouse triglyceride-rich lipoproteins into the aortic wall was increased in ApoE0/Hspg2(Delta3/Delta3) mice compared to ApoE0 mice. However, at 72 hours accumulation of (125)I-LDL was similar in ApoE0/Hspg2(Delta3/Delta3) and ApoE0 mice. Immunohistochemistry of lesions from ApoE0/Hspg2(Delta3/Delta3) mice showed decreased staining for apoB and increased smooth muscle alpha-actin content, whereas accumulation of CD68-positive inflammatory cells was unchanged. We conclude that the perlecan HS chains are proatherogenic in mice, possibly through increased lipoprotein retention, altered vascular permeability, or other mechanisms. The ability of HS to inhibit smooth muscle cell growth may also influence development as well as instability of lesions.

Figure 1

Quantification of atherosclerosis in ApoE0 and ApoE0/Hspg2^Δ;3/Δ3 mice. Oil-RedO/Htx staining of aortic root sections from female mice at 15 (A and B; n=10 and 9) and 33 weeks (C; n=11 and 10). D and E shows en face analysis of whole aortas at 33 weeks (n=12 and 10).

Figure 2

Size fractionation of lipoproteins. Males 15 weeks (A), females 15 weeks (B), males 33 weeks (C), and females 33 weeks (D). ApoE0 (●), ApoE0/Hspg2^Δ3/Δ3 mice (○).

Figure 3

Gradient gel electrophoresis of proteoglycans produced by aortic SMCs from ApoE0 (A) and ApoE0/Hspg2^Δ3/Δ3 (Δ3) mice. Proteoglycans were labeled with ³⁵S-sulfate, DEAE-purified and digested with chondroitinase and/or heparinase I and II as described in Methods. Note lack of smear in the stacking gel of chondroitinase-digested samples from ApoE0/Hspg2^Δ3/Δ3 SMC cultures indicating lack of large HS proteoglycans.

Figure 4

Binding of mouse triglyceride-rich lipoproteins to total ECM prepared from aortic SMCs from ApoE0 (■) and ApoE0/Hspg2^Δ3/Δ3 (□) mice (A; n=4) and binding of human LDL to proteoglycans purified from ApoE0 (■) and ApoE0/Hspg2^Δ3/Δ3 (□) SMCs (B; one of two experiements with identical results).

Figure 5

ApoB staining of aortic roots (female 15 weeks; n=10 and 9) with adjacent sections stained with Oil Red-O (A–F). Quantification of apoB staining (G).

Figure 6

Qualitative differences between aortic root lesions from ApoE0 (A, D, and G) and ApoE0/Hspg2^Δ3/Δ3 (B, E and H) mice. Immunohistochemical demonstration and quantification of α-actin for SMCs (A, B and C; n=9 and 7) and CD68 for macrophages (G, H and I; n=7). Quantification of staining (C,F and I) expressed as % of total lesion area. The arrowhead in B indicates α-actin staining in a fibrous cap region. Also note prominent medial thinning underneath large lesions. D, E and F show picrosirus red staining for collagen (n=10 and 13) analyzed and quantified using polarized light.