T he inner half of the arterial wall has few vasa vasorum, and the oxygen supply to this area depends on direct diffusion from the arterial lumen. Therefore, hypoxia in the arterial wall could easily be induced by a deterioration of this diffusion process. For example, Heughan et al. 1 reported that the oxygen tension in the arterial walls of normal rabbits was 30 to 40 mm Hg, while that of cholesterol-fed rabbits was 10 mm Hg. These results suggest that atherosclerosis induced by cholesterol feeding affects the oxygen supply to the arterial walls.Arterial tissue hypoxia could be induced by systemic hypoxia. Kjeldsen et al. 2 reported that systemic hypoxia promoted atherosclerosis in cholesterol-fed rabbits, and we also reported similar results with Watanabe heritable hyperlipidemic (WHHL) rabbits.3 Hypoxia reportedly affects lipid metabolism in aortic tissue cultures 46 and in cultured aortic smooth muscle cells.6 Hypoxia may, therefore, play a role in the initiation or promotion of atherosclerosis.We recently reported that, under hypoxic conditions, there was an increased cholesterol accumulation in cultured rabbit aortic smooth muscle cells. 7 The present study was designed to further elucidate the mechanisms of cholesterol accumulation in cells under hypoxic conditions. We tested the effects of hypoxia on the following: de novo synthesis of sterol, acyl-CoA:cholesterol acyltransferase (ACAT) activity, and the efflux of cholesterol in cultured rabbit skin fibroblasts. oleate, free cholesterol, and oleate were purchased from Sigma (St. Louis, MO). Dulbecco's modified Eagle's minimum essential medium and fetal bovine serum were obtained from GIBCO (Grand Island, NY). Culture flasks (25 cm 2 and 75 cm 2 ) were purchased from Coming (Iwaki Glass, Tokyo, Japan). All other chemicals were of commercially available reagent grade.
Methods Materials
SerumNormolipemic rabbit serum (NRS) was obtained from male Japanese White rabbits fed a normal rabbit chow diet (ORC-4 Oriental Yeast, Tokyo, Japan). Hyperiipemic rabbit serum (HRS) was obtained from rabbits fed a rabbit chow diet supplemented with 1% cholesterol for at least 1 month. Blood was collected from unanesthetized rabbits by venous puncture of the marginal ear following overnight fasting. Serum was obtained by centrifugation and was sterilized by filtration through 0.45 /im filters (Millipore Japan, Tokyo, Japan). Serum cholesterol was measured by an enzymatic technique with Determiner TC '5' (Kyowa Hakko Kogyo, Tokyo, Japan).
Preparation of Lfpoproteln-deffclent SerumUpoprotein-deficient serum (LPDS) (d>1.21 g/ml) from NRS was isolated by ultracentrifugation, as described by Havel et al. 8 The fraction was dialyzed against 0.15 M NaCI containing 0.01% ethylenediarninetetraacetJc acid (EDTA), pH 7.4, and sterilized by passage through 0.45 ym filters.
CellsSkin fibroblasts were cultured from tissue explants derived from male Japanese White rabbits as described previously. 9 We used Dulbecco's modified Eagle's minimal essential medium, supplemented with penicill...
