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.

Objective-Macrophage-mediated oxidation of low-density lipoprotein (LDL) by enzymes, such as the lipoxygenases, is considered of major importance for the formation of oxidized LDL during atherogenesis. Macrophages have been identified in hypoxic areas in atherosclerotic plaques. Methods and Results-To investigate the role of hypoxia in macrophage-mediated LDL oxidation, we incubated human monocyte-derived macrophages with LDL under normoxic (21% O 2 ) or hypoxic (0% O 2 ) conditions. The results showed that hypoxic macrophages oxidized LDL to a significantly higher extent than normoxic cells. Interestingly, the mRNA and protein expression of 15-lipoxygenase-2 (15-LOX-2) as well as the activity of this enzyme are elevated in macrophages incubated at hypoxia. Both the unspliced 15-LOX-2 and the spliced variant 15-LOX-2sv-a are found in macrophages. In addition, 15-LOX-2 was identified in carotid plaques in some macrophage-rich areas but was only expressed at low levels in nondiseased arteries. Conclusions-In summary, these observations show for the first time that 15-LOX-2 is expressed in hypoxic macrophages and in atherosclerotic plaques and suggest that 15-LOX-2 may be one of the factors involved in macrophage-mediated LDL oxidation at hypoxia. A n early phenomenon in atherosclerosis is the retention, oxidation, and accumulation of low-density lipoprotein (LDL) in the vessel wall. 1,2 Oxidized LDL (oxLDL), one of the key players in atherogenesis, attracts monocytes to the vessel wall where they differentiate into macrophages. 3,4 Oxidation of LDL mediated by macrophages is considered to be of major importance for the formation of oxLDL within the atherosclerotic plaque. Enzymes involved in this process are 15-lipoxygenase (15-LOX), 5,6 myeloperoxidase (MPO), 7 and NADPH oxidase. 8 Macrophages in the arterial wall take up oxLDL through scavenger receptors and accumulate oxLDL as cholesterol esters, which results in foam cell formation.The thickness of the arterial wall increases as the atherosclerotic plaque develops. This leads to an impaired diffusion, which results in oxygen and nutrient deficiency in the deeper portion of the arterial intima and in atherosclerotic plaques. Simultaneously, oxygen consumption by cells within the plaque rises, 9,10 which could be because of the increased number of energy-consuming foam cells. 10 In healthy tissues, oxygen tension is 20 to 70 mm Hg (2.5% to 9% O 2 ). However, in diseased tissue, eg, in atherosclerotic plaques, inadequate perfusion may reduce O 2 tension to below 10 mm Hg (Ͻ1% O 2 ) in some regions. 11 Results from our laboratory have previously shown that areas of hypoxia occur within atherosclerotic plaques in cholesterol-fed rabbits. 12 Hypoxia may lead to retention of macrophages in these areas, because it has been shown that macrophage migration is reduced by hypoxia. 13 The role of hypoxia in the development of atherosclerotic plaques is not known. In this study, we have explored the effect of hypoxia on macrophage-mediated LDL oxidation and the expression o...

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Macrophages are prominent in hypoxic areas of atherosclerotic lesions, and their secreted proteoglycans (PG), such as versican, can modulate the retention of lipoproteins and the activity of enzymes, cytokines, and growth factors involved in atherogenesis. In this study, we report the effects of hypoxia on PG secreted by human monocyte-derived macrophages (HMDM) and the potential regulation by the transcription factor hypoxia-inducible factor (HIF-1alpha and HIF-2alpha). We found that versican co-localized with HIF-1alpha in macrophage-rich areas in human advanced atherosclerotic lesions. Versican and perlecan mRNA expression increased after exposure to 0.5% O(2) (hypoxia) compared with 21% O(2) (control cells). Using precursors to GAG biosynthesis combined with immunoabsorption with a versican antibody an increased versican synthesis was detected at hypoxia. Furthermore, siRNA knockdown of HIF-1alpha and HIF-2alpha in THP-1 cells showed that the hypoxic induction of versican and perlecan mRNA expression involved HIF signaling. Versican expression was co-regulated by HIF-1alpha and HIF-2alpha but expression of perlecan was influenced only by HIF-1alpha and not by HIF-2alpha knockdown. The results show that oxygen concentration is an important modulator of PG expression in macrophages. This may be a novel component of the complex role of macrophages in atherosclerosis.