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Objective-Both HDLs and their major protein constituent apolipoprotein A-I (apoA-I) are transported through aortic endothelial cells. The knock-down of the ATP-binding cassette transporters A1 (ABCA1), G1 (ABCG1), and of the scavenger receptor-BI (SR-BI) diminishes but does not completely block the transport of apoA-I or HDL, so that other receptors appear to be involved. The ectopic ␤-chain of F 0 F 1 ATPase has been previously characterized as an apoA-I receptor, triggering HDL internalization in hepatocytes. Methods and Results-The ectopic presence of the ␤-chain of F 0 F 1 ATPase on the surface of endothelial cells was confirmed by cell surface biotinylation. RNA-interference and the F 0 F 1 ATPase inhibitory peptide IF 1 reduced cell binding of apoA-I but not HDL, as well as association and transendothelial transport of both apoA-I and HDL. Furthermore, apoA-I stimulated F 0 F 1 ATPase catalyzed ATP hydrolysis. The generated ADP as well as apoA-I stimulated the binding, cell association, and internalization of HDL. Both in the presence and absence of ADP inhibition of the purinergic receptor P2Y 12 but not P2Y 1 decreased the cell association of apoA-I and HDL. Coinhibition of ␤-ATPase and ABCA1 had no additive effects on the cell association and transport of apoA-I. Reduced cell association of HDL by ␤-ATPase inhibition was not further decreased by additional knock-down of ABCG1 or SR-BI. Conclusion-Binding of apoA-I to ectopic F 0 F 1 ATPase triggers the generation of ADP, which via activation of the purinergic receptor P2Y 12 stimulates the uptake and transport of HDL and initially lipid-free apoA-I by endothelial cells. Key Words: apolipoproteins Ⅲ endothelium Ⅲ lipoproteins Ⅲ F0F1 ATPase Ⅲ transcytosis P lasma levels of HDL cholesterol as well as apolipoprotein A-I (apoA-I) are inversely correlated with the risk of atherosclerosis. In addition, both apoA-I and HDL exert several atheroprotective properties within the arterial wall rather than in the blood stream, including cholesterol efflux from macrophage foam cells. 1 HDLs are indeed the most abundant lipoproteins in the extravascular space. 2-5 Recently, we provided evidence that endothelial cells bind, internalize, and transcytose apoA-I and HDL in a saturable and temperature-dependent manner. 6 By siRNA interference we also showed that the ATP-binding cassette transporter (ABC) A1 modulates endothelial transport of apoA-I, 7 whereas ABCG1 and the scavenger receptor BI (SR-BI) modulate the transport of HDL. 8 In addition, we showed that the transendothelial apoA-I transport is a 2-step process in which apoA-I is initially lipidated by ABCA1 and then further processed by mechanisms that are independent of ABCA1 but involve Previously, the ectopically expressed ␤-chain of F 0 F 1 ATPase (␤-ATPase) has been identified as a hepatic receptor for apoA-I. 10 F 0 F 1 ATPase is an enzymatic complex responsible for the synthesis of ATP in mitochondria, prokaryote membranes, and chloroplasts. The mitochondrial F 0 F 1 ATPase (about 600 kDa) is composed of 2 ...

Abstract-High-density lipoproteins and their major protein constituent apolipoprotein A-I (apoA-I) possess diverse atheroprotective properties. Most of them must be exerted within the arterial wall. Actually, high-density lipoproteins are the most abundant lipoproteins within the arterial intima. We have recently reported that apoA-I is transcytosed through aortic endothelial cells. In the present study, we evaluate the role of ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor BI (SR-BI) in this process. Using pharmacological interventions and RNA interference, we investigated whether ABCA1 and SR-BI modulate apoA-I binding, internalization and transcytosis in endothelial cells. Upregulation of ABCA1 with oxysterols increased apoA-I binding and internalization. Trapping ABCA1 on the cell surface with cyclosporin A enhanced apoA-I binding but decreased its internalization and transcytosis. In addition, apoA-I binding, internalization, and transcytosis were reduced by at least 50% after silencing ABCA1 but not after knocking down SR-BI. The integrity of the endothelial cell monolayer was affected neither by cyclosporin A treatment nor by ABCA1 silencing, as controlled by measuring inulin permeability. Finally, in ABCA1-GFP-expressing cells, fluorescently labeled apoA-I colocalized intracellularly with ABCA1-GFP. However, apoA-I-containing vesicles did not colocalize with the late endosome marker LAMP-1 (lysosome-associated membrane protein-1). In conclusion, ABCA1, but not SR-BI, modulates the transcytosis of apoA-I through endothelial cells.