The ß-chain of cell surface F(0)F(1) ATPase modulates apoA-I and HDL transcytosis through aortic endothelial cells.

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 ß-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 ß-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 ß-ATPase and ABCA1 had no additive effects on the cell association and transport of apoA-I. Reduced cell association of HDL by ß-ATPase inhibition was not further decreased by additional knock-down of ABCG1 or SR-BI. CONCLUSIONS: 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.

Apolipoprotein A-I but not high-density lipoproteins are internalised by RAW macrophages: roles of ATP-binding cassette transporter A1 and scavenger receptor BI.

Accumulation of lipid-loaded macrophages (foam cells) within the vessel wall is an early hallmark of atherosclerosis. High-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) can efficiently promote cholesterol efflux from macrophages. Therefore, the interaction of HDL and apoA-I with macrophages appears to be important in the initial steps of reverse cholesterol transport, i.e. the transport of excess cholesterol from foam cells to the liver. However, although several cellular apoA-I and HDL receptors and transporters have been identified, it is as yet controversial how these interactions lead to cholesterol efflux from foam cells. In this study, we show that RAW264.7 macrophages bind HDL and apoA-I in a compatible manner. Furthermore, cell surface biotinylation experiments revealed that apoA-I but not HDL is specifically internalised. Binding of HDL to macrophages is decreased by reducing the expression of scavenger receptor BI (SR-BI) with cyclic adenosine monophosphate (cAMP), acetylated low-density lipoprotein (acLDL) or RNA interference. In contrast, apoA-I cell association and internalisation is modulated in parallel with ATP-binding cassette transporter A1 (ABCA1) expression which is altered by stimulating cells with cAMP and acLDL or expressing short hairpin RNA (shRNA) against ABCA1. Consistent with this, cell surface trapping of ABCA1 with cyclosporin A (CsA) results in increased apoA-I binding but reduced internalisation. Furthermore, blocking apoA-I uptake inhibits cholesterol efflux to apoA-I but not to HDL. Taken together, these data suggest that apoA-I- but not HDL-mediated cholesterol efflux may involve retroendocytosis.

ATP-Binding cassette transporter A1 modulates apolipoprotein A-I transcytosis through aortic endothelial cells.

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.

Lipid efflux by the ATP-binding cassette transporters ABCA1 and ABCG1.

Plasma levels of high-density lipoproteins (HDL) and apolipoprotein A-I (apoA-I) are inversely correlated with the risk of cardiovascular disease. One major atheroprotective mechanism of HDL and apoA-I is their role in reverse cholesterol transport, i.e., the transport of excess cholesterol from foam cells to the liver for secretion. The ATP-binding cassette transporters ABCA1 and ABCG1 play a pivotal role in this process by effluxing lipids from foam cells to apoA-I and HDL, respectively. In the liver, ABCA1 activity is one rate-limiting step in the formation of HDL. In macrophages, ABCA1 and ABCG1 prevent the excessive accumulation of lipids and thereby protect the arteries from developing atherosclerotic lesions. However, the mechanisms by which ABCA1 and ABCG1 mediate lipid removal are still unclear. Particularly, three questions remain controversial and are discussed in this review: (1) Do apoA-I and HDL directly interact with ABCA1 and ABCG1, respectively? (2) Does cholesterol efflux involve retroendocytosis of apoA-I or HDL? (3) Which lipids are directly transported by ABCA1 and ABCG1?

Binding, internalization and transport of apolipoprotein A-I by vascular endothelial cells.

High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I), exert potentially anti-atherogenic properties within the arterial wall. However, it is unknown how they are transported from the blood stream into the vascular wall. Here we investigated the interaction of apoA-I with endothelial cells. At 4 degrees C endothelial cells bound 125I-apoA-I with high affinity, Kd = 2.1 microg/ml and in a saturable manner (Bmax of 35 ng/mg cell protein). At 37 degrees C, the cell association of apoA-I revealed similar affinity as at 4 degrees C (Kd = 2.2 microg/ml) but the maximum specific cell association was much enhanced (Bmax = 360 ng/mg cell protein). Binding and cell association was competed by excess unlabeled apoA-I and HDL but not by albumin. Biotinylation experiments and electron microscopy studies showed that endothelial cells internalize labeled apoA-I. Only minor amounts of the internalized apoA-I were degraded. Cultivated in a Transwell system, the cells transported a fraction of 125I-apoA-I from the apical to the basolateral compartment in a competable and temperature-sensitive manner. Furthermore, after specific transport the originally prebeta-mobile and lipid-free apoA-I was recovered as particles which have electrophoretic alpha-mobility. We conclude that endothelial cells transcytose and lipidate lipid-free apoA-I.