Abstract-Cholesterol efflux from macrophage foam cells is a rate-limiting step in reverse cholesterol transport. In this process cholesterol acceptors like high-density lipoproteins (HDL) and apolipoprotein (apo)A-I must cross the endothelium to get access to the donor cells in the arterial intima. Previously, we have shown that apoA-I passes a monolayer of aortic endothelial cells (ECs) from the apical to the basolateral side by transcytosis, which is modulated by the ATP-binding cassette transporter (ABC)A1. Here, we analyzed the interaction of mature HDL with ECs. ECs bind HDL in a specific and saturable manner. Both cell surface biotinylation experiments and immunofluorescence microscopy of HDL recovered Ϸ30% of the cell-associated HDL intracellularly. Cultivated on inserts ECs bind, internalize, and translocate HDL from the apical to the basolateral compartment in a specific and temperature-dependent manner. The size of the translocated particle was reduced, but its protein moiety remained intact. Using RNA interference, we investigated the impact of SR-BI, ABCA1, and ABCG1 on binding, internalization, and transcytosis of HDL by ECs. HDL binding was reduced by 50% and 30% after silencing of SR-BI and ABCG1, respectively, but not at all after diminishing ABCA1 expression. Knock down of SR-BI and, even more so, ABCG1 reduced HDL transcytosis but did not affect inulin permeability. Cosilencing of both proteins did not further reduce HDL binding, internalization, or transport. In conclusion, ECs transcytose HDL by mechanisms that involve either SR-BI or ABCG1 but not ABCA1. cholesterol show an inverse association with the incidence of coronary artery disease. The cardioprotective effect of HDL and its major apolipoprotein (apo)A-I are, in part, related to the ability to promote the reverse transport of cholesterol from macrophage foam cells in the arterial intima to the liver for excretion into the bile. [1][2][3][4] An early step in the reverse transport of cholesterol is the transfer of excess cholesterol from the lipid-laden macrophages to HDLs. Importantly, the loading of cellular cholesterol to HDL does not take place in the plasma compartment but in the subendothelial space of arteries. 1 Consequently, HDLs must cross the endothelium to get into close proximity to the cholesterol donor cells. This passage is not well understood. 5 The endothelium lines the vasculature as a single layer of endothelial cells (ECs). As a semipermeable barrier, it regulates the flux of liquid, solutes, and cells between blood and interstitial space. Two principal pathways are known for transendothelial macromolecule translocation, the transcellular transport, including transcytosis, and the paracellular transfer between adjacent cells. 6,7 The paracellular pathway is formed by gaps between ECs, but regulated adherence and tight junctions restrict and control the free passage of macromolecules larger than 6 nm. 7 Endothelial transcytosis, which is defined as vesicle-mediated transport of proteins, has been best investigated ...

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 ...

High density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) must leave the circulation and pass the endothelium to exert their atheroprotective actions in the arterial wall. We previously demonstrated that the transendothelial transport of apoA-I involves ATP-binding cassette transporter (ABC) A1 and re-secretion of lipidated particles. Transendothelial transport of HDL is modulated by ABCG1 and the scavenger receptor BI (SR-BI). We hypothesize that apoA-I transport is started by the ABCA1-mediated generation of a lipidated particle which is then transported by ABCA1-independent pathways. To test this hypothesis we analyzed the endothelial binding and transport properties of initially lipid-free as well as prelipidated apoA-I mutants. Lipid-free apoA-I mutants with a defective carboxyl-terminal domain showed an 80% decreased specific binding and 90% decreased specific transport by aortic endothelial cells. After prior cell-free lipidation of the mutants, the resulting HDL-like particles were transported through endothelial cells by an ABCG1-and SR-BI-dependent process. ApoA-I mutants with deletions of either the amino terminus or both the amino and carboxyl termini showed dramatic increases in nonspecific binding but no specific binding or transport. Prior cell-free lipidation did not rescue these anomalies. Our findings of stringent structure-function relationships underline the specificity of transendothelial apoA-I transport and suggest that lipidation of initially lipid-free apoA-I is necessary but not sufficient for specific transendothelial transport. Our data also support the model of a twostep process for the transendothelial transport of apoA-I in which apoA-I is initially lipidated by ABCA1 and then further processed by ABCA1-independent mechanisms.