title: Macrophage-released proteoglycans enhance LDL aggregation: studies in aorta from apolipoprotein E-deficient mice.
abstract: Aggregated low-density lipoprotein (LDL) was shown to be present in the atherosclerotic lesion, but the mechanism responsible for its formation in vivo is not known yet. To find out whether LDL aggregation occurs in the arterial wall during atherogenesis, LDLs were extracted from the aortas of apolipoprotein E-deficient (E(0)) mice during their aging (and the development of atherosclerosis), and were analyzed for their aggregation states, in comparison to LDLs isolated from aortas of control mice. LDL isolated from aortas of E(0) mice was already aggregated at 1 month of age and its aggregation state substantially increased with age, with 3-fold elevation at 6 months of age compared to younger, 1-month-old, mice. Only minimal aggregation could be detected in LDL derived from control mice. Electron microscopy examination revealed that LDL particles from aortas of the E(0) mice were heterogeneous in their size, ranging between 20 and 300 nm. The mouse aortic LDL contained proteoglycans (PGs) and their content increased with the age of the mice, with about 2-fold higher levels than those found in LDLs derived from aortas of control mice. Macrophage-released PGs were previously demonstrated to enhance LDL aggregation in vitro. However, their involvement in LDL aggregation in vivo has not been studied yet. Thus, we next studied the effect of arterial macrophage-released PGs on the susceptibility of plasma LDL to aggregation by Bacillus cereus sphingomyelinase (SMase). Foam cell macrophages were isolated from aortas of the atherosclerotic E(0) mice at 6 months of age and were found to be loaded with cholesterol and to contain oxidized lipids. To analyze the effect of macrophage-released PGs on LDL aggregation, PGs were prelabeled by cell incubation with [35S]sulfate, followed by incubation of macrophage-released PGs with E(0) mouse plasma LDL (200 microg protein/ml) for 1 h at 37 degrees C. [35S]Sulfated PGs were found to be LDL-associated and the susceptibility of PG-associated LDL to aggregation by SMase was increased by up to 45% in comparison to control LDL. Similar results demonstrating the involvement of PGs in LDL aggregation were obtained upon incubation of LDL with increasing concentrations of PGs that were isolated from the entire aorta of E(o) mice (rather than the isolated macrophages). The stimulatory effect of macrophage-released PGs on LDL aggregation was markedly reduced when the PGs were pretreated with the glycosaminoglycan-hydrolyzing enzymes, chondroitinase ABC or chondroitinase AC, and to a much lesser extent with heparinase. We thus conclude that macrophage-released chondroitin sulfate PG can contribute to the formation of atherogenic aggregated LDL in the arterial wall.
authors.affiliation: The Lipid Research Laboratory, The Bruce Rappaport Faculty of Medicine, Technion, The Rappaport Family Institute for Research in the Medical Sciences and Rambam Medical Center, Haifa, Israel.
authors.forename: I
authors.initials: I
authors.lastname: Maor
authors.forename: T
authors.initials: T
authors.lastname: Hayek
authors.forename: M
authors.initials: M
authors.lastname: Hirsh
authors.forename: T C
authors.initials: TC
authors.lastname: Iancu
authors.forename: M
authors.initials: M
authors.lastname: Aviram
mesh_terms: D000375:Aging / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000378:metabolism* / Q000648:ultrastructure; D001057:Apolipoproteins E / Q000172:deficiency*; D001161:Arteriosclerosis / Q000378:metabolism* / Q000473:pathology; D002809:Chondroitin Sulfates / Q000494:pharmacology; D006497:Heparan Sulfate / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000378:metabolism* / Q000648:ultrastructure; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D008854:Microscopy, Electron; D010316:Particle Size; D011509:Proteoglycans / Q000096:biosynthesis / Q000502:physiology*
chemical_list: D001057:Apolipoproteins E; D002809:Chondroitin Sulfates; D006497:Heparan Sulfate; D008077:Lipoproteins, LDL; D011509:Proteoglycans