Complications of atherosclerosis are the most common cause of death in Western societies. Among the many risk factors identified by epidemiological studies, only elevated levels of lipoproteins containing apolipoprotein (apo) B can drive the development of atherosclerosis in humans and experimental animals even in the absence of other risk factors. However, the mechanisms that lead to atherosclerosis are still poorly understood. We tested the hypothesis that the subendothelial retention of atherogenic apoB-containing lipoproteins is the initiating event in atherogenesis. The extracellular matrix of the subendothelium, particularly proteoglycans, is thought to play a major role in the retention of atherogenic lipoproteins. The interaction between atherogenic lipoproteins and proteoglycans involves an ionic interaction between basic amino acids in apoB100 and negatively charged sulphate groups on the proteoglycans. Here we present direct experimental evidence that the atherogenicity of apoB-containing low-density lipoproteins (LDL) is linked to their affinity for artery wall proteoglycans. Mice expressing proteoglycan-binding-defective LDL developed significantly less atherosclerosis than mice expressing wild-type control LDL. We conclude that subendothelial retention of apoB100-containing lipoprotein is an early step in atherogenesis.

Abstract-Direct binding of apolipoprotein (apo)B-containing lipoproteins to proteoglycans is the initiating event inatherosclerosis, but the processes involved at later stages of development are unclear. Here, we investigated the importance of the apoB-proteoglycan interaction in the development of atherosclerosis over time and investigated the role of lipoprotein lipase (LPL) to facilitate low-density lipoprotein (LDL) retention at later stages of development. Atherosclerosis was analyzed in apoB transgenic mice expressing LDL with normal (control LDL) or reduced proteoglycan-binding (RK3359-3369SA LDL) activity after an atherogenic diet for 0 to 40 weeks. The initiation of atherosclerosis was delayed in mice expressing RK3359-3369SA LDL, but they eventually developed the same level of atherosclerosis as mice expressing control LDL. Retention studies in vivo showed that although higher levels of 131 I-tyramine cellobiose-labeled control LDL ( 131 I-TC-LDL) were retained in nonatherosclerotic aortae compared with RK3359-3369SA 131 I-TC-LDL, the retention was significantly higher and there was no difference between the groups in atherosclerotic aortae. Lower levels of control 125 I-TC-LDL and RK3359-3369SA 125 I-TC-LDL were retained in atherosclerotic aortae from ldlr Ϫ/Ϫ mice transplanted with lpl Ϫ/Ϫ compared with lpl ϩ/ϩ bone marrow. Uptake of control LDL or RK3359-3369SA LDL into macrophages with specific expression of human catalytically active or inactive LPL was increased compared with control macrophages. 1 We tested the response-toretention hypothesis 2 in an earlier study using genetically modified mice that expressed human recombinant lowdensity lipoproteins (LDLs) with reduced proteoglycanbinding activity and provided direct evidence showing that subendothelial retention of apolipoprotein (apo)B100-containing lipoproteins is the initiating event in atherogenesis. 3 Furthermore, we demonstrated that the atherogenicity of LDL is linked to their proteoglycan-binding activity. 3 Lipoproteins associate with artery wall proteoglycans via both direct and indirect interactions. Direct binding between LDL and proteoglycans involves an ionic interaction between basic amino acids in apoB100 (Site B; residues 3359 to 3369) and negatively charged sulfate groups on the glycosaminoglycan (GAG) chains of proteoglycans. 4,5 Indirect binding between LDL and proteoglycans is facilitated by apoE, 3 which is found in human atherosclerotic plaques together with apoB. 6 ApoE binds vascular proteoglycans, and apoEenrichment of proteoglycan-binding-defective LDL (in which Site B has been inactivated by mutagenesis) partly restores proteoglycan binding. 3 Mouse LDL contains significant amounts of apoE, and consequently proteoglycan-bindingdefective LDL isolated from mouse plasma displays Ϸ30% of normal proteoglycan binding. 3 Subendothelial retention of LDL via indirect binding to GAGs can also be facilitated by lipoprotein lipase (LPL). 7 We have shown previously that the binding between LPL and LDL is mediated throug...

Inflammation in the vascular wall is important for development of atherosclerosis. We have shown previously that arachidonate 15-lipoxygenase type B (ALOX15B) is more highly expressed in human atherosclerotic lesions than in healthy arteries. This enzyme oxidizes fatty acids to substances that promote local inflammation and is expressed in lipid-loaded macrophages (foam cells) present in the atherosclerotic lesions. Here, we investigated the role of ALOX15B in foam cell formation in human primary macrophages and found that silencing of human ALOX15B decreased cellular lipid accumulation as well as proinflammatory cytokine secretion from macrophages. To investigate the role of ALOX15B in promoting the development of atherosclerosis in vivo, we used lentiviral shRNA silencing and bone marrow transplantation to knockdown mouse Alox15b gene expression in LDL-receptor-deficient (Ldlr −/−) mice. Knockdown of mouse Alox15b in vivo decreased plaque lipid content and markers of inflammation. In summary, we have shown that ALOX15B influences progression of atherosclerosis, indicating that this enzyme has an active proatherogenic role.

Background Statins have antiinflammatory and antiatherogenic effects that have been attributed to inhibition of RHO protein geranylgeranylation in inflammatory cells. The activity of protein geranylgeranyltransferase type I (GGTase-I) is widely believed to promote membrane association and activation of RHO family proteins. However, we recently showed that knockout of GGTase-I in macrophages activates RHO proteins and proinflammatory signaling pathways, leading to increased cytokine production and rheumatoid arthritis. In this study, we asked whether the increased inflammatory signaling of GGTase-I–deficient macrophages would influence the development of atherosclerosis in low-density lipoprotein receptor–deficient mice. Methods and Results Aortic lesions in mice lacking GGTase-I in macrophages (Pggt1b∆/∆) contained significantly more T lymphocytes than the lesions in controls. Surprisingly, however, mean atherosclerotic lesion area in Pggt1b∆/∆ mice was reduced by ≈60%. GGTase-I deficiency reduced the accumulation of cholesterol esters and phospholipids in macrophages incubated with minimally modified and acetylated low-density lipoprotein. Analyses of GGTase-I–deficient macrophages revealed upregulation of the cyclooxygenase 2–peroxisome proliferator-activated-γ pathway and increased scavenger receptor class B type I– and CD36-mediated basal and high-density lipoprotein–stimulated cholesterol efflux. Lentivirus-mediated knockdown of RHOA, but not RAC1 or CDC42, normalized cholesterol efflux. The increased cholesterol efflux in cultured cells was accompanied by high levels of macrophage reverse cholesterol transport and slightly reduced plasma lipid levels in vivo. Conclusions Targeting GGTase-I activates RHOA and leads to increased macrophage reverse cholesterol transport and reduced atherosclerosis development despite a significant increase in inflammation.