Effects of hypoxia on macrophage function in atherosclerosis

Abstract: Atherosclerosis is a complex disease, which affects large and medium sized arteries and is characterized by an arterial wall thickening. A constant supply of oxygen is needed to maintain cell functions in the atherosclerotic plaque. The oxygen distribution to the cells in the arterial wall depends on diffusion. During the progression of the atherosclerotic plaque, the diffusion distance increases due to the accumulation of immune cells and smooth muscle cells in the arterial wall. The increase in the arterial wall thickness affects the diffusion distance, which promotes hypoxia in the inner part of the vessel wall. This event could explain why hypoxic areas are found in atherosclerotic lesions. LDL oxidation is a key factor for the development and progression of atherosclerosis. This oxidation is suggested to be partly mediated by lipoxygenases expressed by macrophages. Macrophages in the atherosclerotic lesion, which contain oxysterols, an LDL oxidation product, co-localize with hypoxic areas. Oxysterols in macrophages increase interleukin-8 (IL-8) secretion, which stimulates recruitment of T-lymphocytes and smooth muscle cells found in atherosclerotic lesions. The aim of this thesis was to investigate the effects of hypoxia on oxysterol-induced IL-8 secretion and on LDL oxidation in human macrophages. The most pronounced effect on IL-8 secretion was found when hypoxic macrophages were incubated with a low concentration of 25-hydroxycholesterol (25-OH-chol). In human atherosclerotic plaques only low levels of 25-OH-chol are found. Both hypoxia and 25-OH-chol increased the intracellular level of hydrogen peroxide and the binding of the transcription factor activator protein-1 (AP-1) to the IL-8 gene promoter. Hypoxia had an additive effect on the 25-OH-chol-induced IL-8 secretion. These observations suggest that both 25-OH-chol and hypoxia activate the same transcription factor, AP-1, and that hypoxia in the atherosclerotic plaque may increase the secretion of IL-8 from oxysterol-containing macrophages. Hypoxia also increased the macrophage mediated LDL oxidation and the expression of 15-lipoxygenase-2 (15-LOX-2). In contrast to normoxic cells, 15-LOX-2 mRNA and protein expression as well as the enzyme activity were increased in hypoxic macrophages. This lipoxygenase has not previously been found in human macrophages. Interestingly, 15-LOX-2 was also identified in human carotid plaques, but was only found at low levels in non-diseased mammary artery. Overexpression of 15-LOX-2 in the human monocyte cell line THP-1 and human monocyte-derived macrophages showed increased metabolization of arachidonic acid. However the oxidation of LDL was not increased. In conclusion, macrophages incubated at hypoxia increased the 25-OH-chol-induced IL-8 secretion and the LDL oxidation. The expression and the activity of 15-LOX-2 were also increased in hypoxic macrophages. These observations show that hypoxia regulates various biological events, which could contribute to progression of atherosclerosis.

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