Abstract 825: Impaired ABCA-1 Mediated Cholesterol Efflux In Hypoxic Macrophages
Atherosclerotic plaques contain large numbers of metabolically active cells that can be deprived of normal oxygen (02) tension, with levels documented below 10mm Hg (~1% O2) in rabbit and human plaques (vs. normal tissue; 20 –70mmHg, 2.5%–9% O2). Hypoxia has been assumed, but not documented in mouse plaques. Hypoxia stabilizes transcription factor HIF-1α, the master regulator of the cellular response to hypoxia and also involved in reduction-oxidation and inflammatory pathways. Atherosclerotic plaques of apoE knockout mice were immunostained and areas + for HIF-1α as well as two downstream targets (VEGF and Glut-1) were present in increased levels in deeper plaque regions. We next investigated the effect of hypoxia on macrophage function in vitro by incubating murine J774 macrophages in 1% 02 in DMEM containing 15% FBS for 24 h and found a 30% increase in cellular total cholesterol (TC) associated with hypoxia. In cells loaded by cyclodextrin-cholesterol, a ~400% increase in TC was observed in hypoxic relative to normoxic cells. Assays using apoAI as a cholesterol acceptor showed that in 1% 02 there was a ~90% decrease in ABCA-1 mediated efflux relative to normoxia. In spite of this, ABCA-1 mRNA was increased ~2.5 fold in hypoxic vs. normoxic macrophages. ABCA-1 protein levels by Western analysis remained equal in the 2 conditions. However, by immunofluorescence and confocal microscopy, in sharp distinction to the plasma membrane and cytoplasmic distribution seen in normoxic cells, ABCA-1 localized mainly in a juxtanuclear location in hypoxic macrophages, suggesting impaired trafficking to the membrane.
HIF-1α is present in murine plaques, suggesting that they have true hypoxic areas;
Hypoxia in vitro promotes macrophage cholesterol accumulation concurrent with ABCA-1 dysfunction; and
Hypoxia redistributes ABCA1 from the plasma membrane to the juxtanuclear region.