Serum albumin acts as a shuttle to enhance cholesterol efflux from cells

Abstract

An important mechanism contributing to cell cholesterol efflux is aqueous transfer in which cholesterol diffuses from cells into the aqueous phase and becomes incorporated into an acceptor particle. Some compounds can enhance diffusion by acting as shuttles transferring cholesterol to cholesterol acceptors, which act as cholesterol sinks. We have examined whether particles in serum can enhance cholesterol efflux by acting as shuttles. This task was accomplished by incubating radiolabeled J774 cells with increasing concentrations of lipoprotein-depleted sera (LPDS) or components present in serum as shuttles and a constant amount of LDL, small unilamellar vesicles, or red blood cells (RBC) as sinks. Synergistic efflux was measured as the difference in fractional efflux in excess of that predicted by the addition of the individual efflux values of sink and shuttle alone. Synergistic efflux was obtained when LPDS was incubated with cells and LDL. When different components of LPDS were used as shuttles, albumin produced synergistic efflux, while apoA-I did not. A synergistic effect was also obtained when RBC was used as the sink and albumin as shuttle. The previously observed negative association of albumin with coronary artery disease might be linked to reduced cholesterol shuttling that would occur when serum albumin levels are low. An important mechanism contributing to cell cholesterol efflux is aqueous transfer in which cholesterol diffuses from cells into the aqueous phase and becomes incorporated into an acceptor particle. Some compounds can enhance diffusion by acting as shuttles transferring cholesterol to cholesterol acceptors, which act as cholesterol sinks. We have examined whether particles in serum can enhance cholesterol efflux by acting as shuttles. This task was accomplished by incubating radiolabeled J774 cells with increasing concentrations of lipoprotein-depleted sera (LPDS) or components present in serum as shuttles and a constant amount of LDL, small unilamellar vesicles, or red blood cells (RBC) as sinks. Synergistic efflux was measured as the difference in fractional efflux in excess of that predicted by the addition of the individual efflux values of sink and shuttle alone. Synergistic efflux was obtained when LPDS was incubated with cells and LDL. When different components of LPDS were used as shuttles, albumin produced synergistic efflux, while apoA-I did not. A synergistic effect was also obtained when RBC was used as the sink and albumin as shuttle. The previously observed negative association of albumin with coronary artery disease might be linked to reduced cholesterol shuttling that would occur when serum albumin levels are low. acetylated LDL coronary artery disease egg L-α-phosphatidylcholine free cholesterol human serum albumin lipoprotein-depleted sera n-ethylmaleimide red blood cell reverse cholesterol transport reconstituted HDL scavenger receptor class B type I small unilamellar vesicle There are a number of mechanisms that have been shown to contribute to the flux of cholesterol molecules between cells and extracellular particles. These include pathways mediated by scavenger receptor class B type I (SR-BI), ATP binding cassette transporter A1 (ABCA1), ATP binding cassette transporter G1 (ABCG1), and unmediated aqueous transfer. All of these mechanisms have been extensively studied, and a number of reviews discuss them in detail (1Rothblat G.H. Phillips M.C. High-density lipoprotein heterogeneity and function in reverse cholesterol transprot.Curr. Opin. Lipidol. 2010; 21: 229-238Crossref PubMed Scopus (271) Google Scholar–3Rosenson R.S. Brewer H.B. Davidson W.S. Fayad Z.A. Fuster V. Goldstein J. Hellerstein M. Jiang X-C. Phillips M.C. Rader D.J. et al.Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport.Circulation. 2012; 125: 1905-1919Crossref PubMed Scopus (684) Google Scholar). The earliest cholesterol flux mechanism that was identified was aqueous transfer, in which there is a desorption of free cholesterol (FC) molecules from a donor, such as a cell membrane, into the aqueous phase followed by diffusion of the molecules and their subsequent collision with and incorporation into an acceptor particle (such as a lipoprotein) (4Phillips M.C. Johnson W.J. Rothblat G.H. Mechanism and consequence of cellular cholesterol exchange and transfer.Biochim. Biophys. Acta. 1987; 906: 223-276Crossref PubMed Scopus (424) Google Scholar). This diffusional movement of FC can be enhanced by what has been described as a “shuttle and sink” mechanism in which some compounds present in the aqueous phase can serve as FC shuttles, enhancing the flux of cholesterol between donor and acceptor particles (“sinks”) (5Kilsdonk E.P.C. Yancey P. Stoudt G. Bangerter F.W. Johnson W.J. Phillips M.C. Rothblat G.H. Cellular cholesterol efflux mediated by cyclodextrins.J. Biol. Chem. 1995; 270: 17250-17256Abstract Full Text Full Text PDF PubMed Scopus (705) Google Scholar, 6Atger V.M. de la Llera Moya M. Stoudt G.W. Rodrigueza W.V. Phillips M.C. Rothblat G.H. Cyclodextrins as catalysts for the removal of cholesterol from macrophage foam cells.J. Clin. Invest. 1997; 99: 773-780Crossref PubMed Scopus (160) Google Scholar). Perhaps the best examples of shuttles are cyclodextrins, which act as catalysts that bind FC, greatly increasing the bidirectional flux of FC through the aqueous phase (6Atger V.M. de la Llera Moya M. Stoudt G.W. Rodrigueza W.V. Phillips M.C. Rothblat G.H. Cyclodextrins as catalysts for the removal of cholesterol from macrophage foam cells.J. Clin. Invest. 1997; 99: 773-780Crossref PubMed Scopus (160) Google Scholar). Although the studies using cyclodextrins clearly demonstrated the feasibility of shuttles and sinks enhancing FC flux, there was no evidence that synergistic efflux could be the result of the interaction of physiologically relevant particles and that such a process was taking place in serum. Little research has been done on the shuttle/sink mechanism since the initial reports. However, a recent publication by Hoang et al. (7Hoang A. Drew B.G. Low H. Remaley A.T. Nestel P. Kingwell B.A. Sviridov D. Mechanism of cholesterol efflux in humans after infusion of reconstituted high-density lipoprotein.Eur. Heart J. 2012; 33: 657-665Crossref PubMed Scopus (61) Google Scholar) studying the flux of cholesterol in human sera after infusion of reconstituted HDL (rHDL) yielded data that were consistent with a shuttle/sink mechanism. The results of this study indicated that although rHDL are good cholesterol acceptors, they have only a limited capacity to store cholesterol. This property of rHDL would result in rHDL performing the role of a cholesterol shuttle. In contrast, the data from this infusion study indicated that the plasma reservoirs with a large capacity to hold cholesterol are the apoB-containing LDL and VLDL, thus playing the role of cholesterol sinks. In the present study, we have examined the possibility that a synergistic enhancement of cholesterol flux occurs through the interaction of components that are present in serum. For these studies, we have employed J774 mouse macrophages labeled with [3H]cholesterol as cholesterol donors, plasma components as shuttles, and LDL, small unilamellar vesicles (SUV), or red blood cells (RBC) as cholesterol sinks. To investigate synergistic effects, we quantitated the FC efflux from cells incubated with a constant amount of LDL, or other potential sinks, and increasing concentrations of lipoprotein-depleted sera (LPDS), or components present in LPDS. Synergy was measured as the difference in fractional efflux in excess of that predicted by the addition of the individual efflux values of sink and shuttle alone. Tissue culture plasticware was purchased from Thermo Fisher (Rochester, NY) and Corning (Corning, NY). Minimum essential medium Eagle (MEM), RPMI 1640, and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Mediatech Cellgro (Manassas, VA). MEM buffered with 10 mM HEPES (pH 7.4) was prepared in the laboratory using HEPES purchased from BioWhittaker (Walkersville, MD). Fetal bovine serum (FBS), gentamicin, trypsin EDTA, acyl-CoA cholesterol acyltransferase (ACAT) inhibitor (Sandoz 58-035), Cpt-cAMP, ovalbumin, and fatty acid-free human serum albumin (HSA) were all purchased from Sigma-Aldrich (St. Louis, MO). Fatty acid-free BSA was purchased from Millipore (Kankakee, IL) and egg L-α-phosphatidylcholine (egg PC) was from Avanti Polar Lipids (Alabaster, AL). Red blood cells were purchased from Innovative Research, Inc. (Novi, MI) and [1, 2-3H]cholesterol was from PerkinElmer Analytical Sciences (Waltham, MA). All other reagents and organic solvents were purchased from Fisher Scientific (Pittsburgh, PA). Human LPDS, HDL, LDL, and VLDL were isolated by sequential ultracentrifugation from serum obtained with approved consent from healthy, normolipemic individuals as previously described (HDL, d = 1.06–1.21; LDL, d = 1.01–1.06; and VLDL, d = 0.95–1.01) (8Hatch F.T. Lees R.S. Practical methods for plasma lipoprotein analysis.Adv. Lipid Res. 1968; 6: 1-68Crossref PubMed Google Scholar). To obtain acetylated low-density lipoprotein (acLDL), LDL was modified using acetic anhydride, as previously described (9McCloskey H.M. Rothblat G.H. Glick J.M. Incubation of acetylated low-density lipoprotein with cholesterol-rich dispersions enhances cholesterol uptake by macrophages.Biochim. Biophys. Acta. 1987; 921: 320-332Crossref PubMed Scopus (33) Google Scholar). ApoA-I was purified from delipidated HDL using ethanol/diethyl ether followed by anion-exchange chromatography on a Q-Sepharose column. The apoA-I fractions were pooled, dialyzed against 5 mM NH4HCO3, lyophilized, and stored at −20°C. The apoA-I was resolubilized in 6 M guanidine hydrochloride and dialyzed extensively against saline (0.15 M NaCl, pH 7.4) prior to incubating with the cells. Prior to use, LPDS, HDL, LDL, and VLDL were all extensively dialyzed against 0.15 M NaCl (pH 7.4) and sterilized by filtration using a 0.45 µm Millipore filter. Egg PC SUVs were made as previously described (10McLean L.R. Phillips M.C. Mechanism of cholesterol and phosphatidylcholine exchange or transfer between unilamellar vesicles.Biochemistry. 1981; 20: 2893-2900Crossref PubMed Scopus (275) Google Scholar). J774 mouse macrophage stock cells were maintained in RPMI growth medium supplemented with 10% FBS and gentamicin. For experiments, J774 cells were plated in RPMI growth medium in 24-well plates at a density of 150,000 cells/well for 24 h. Cholesterol-normal cells were labeled for 24 h with 1 μCi/ml [3H]cholesterol in medium supplemented with 5% FBS and 2 μg/ml ACAT inhibitor. In experiments using cholesterol-enriched cells, J774 cells were incubated with the same labeling medium plus 25 μg/ml acLDL. After labeling plus or minus cholesterol enrichment, the cells were washed 2× with MEM HEPES and equilibrated with RPMI medium containing 0.2% BSA and cAMP (0.3 mmol/l) for 18 h. After this equilibration period, the cells were washed 2× with MEM-HEPES buffer and incubated with MEM-HEPES media containing exogenous acceptors alone or in combination (shuttle and sink) for 4 h, or as indicated in the figures, to measure the labeled FC efflux. Aliquots of media were collected to measure the of radiolabeled cholesterol. J774 cell at were washed 2× with and cholesterol was using The efflux is on the [3H]cholesterol in the cells at The ACAT inhibitor was used in all of experiments labeling to efflux to of radiolabeled cholesterol would be present as this could the of the fractional of labeled cholesterol from the cells. the of the efflux the cell were washed with and cell was using of containing 5 of ether Louis, as an for chromatography A of the was used to measure cholesterol incorporated into cellular using The was prepared for to measure cholesterol as previously described Yancey Johnson W.J. Glick J.M. by of Lipid Res. 1995; Full Text PDF PubMed Google Scholar). The of of cell FC was on the cholesterol of the cells at prior to the efflux was measured by the described by et al. A of the to in and lipoprotein PubMed Scopus Google Scholar). were measured the and after the there were no in cell growth between the of equilibration and at the of efflux All were using Inc. were as was by was at The earliest mechanism demonstrated to a role in the movement of FC molecules between cells and was aqueous In this molecules of FC from the cell and are incorporated into a such as a or into the plasma (4Phillips M.C. Johnson W.J. Rothblat G.H. Mechanism and consequence of cellular cholesterol exchange and transfer.Biochim. Biophys. Acta. 1987; 906: 223-276Crossref PubMed Scopus (424) Google Scholar, L.R. Phillips M.C. Mechanism of cholesterol and phosphatidylcholine exchange or transfer between unilamellar vesicles.Biochemistry. 1981; 20: 2893-2900Crossref PubMed Scopus (275) Google Scholar, G.H. Phillips M.C. Mechanism of cholesterol efflux from cells. of acceptor and Biol. Chem. Full Text PDF PubMed Google Scholar). This mechanism is and to the exchange of FC between cells and as as the exchange of FC between Phillips M.C. and mechanism of free cholesterol exchange between human serum and low-density 21: PubMed Scopus Google Scholar). of the limited of FC in an aqueous the FC flux between cells and acceptors by aqueous transfer is although a number of recent studies have demonstrated that aqueous transfer contribute to cell FC efflux M. of efflux pathways in cholesterol efflux from macrophage foam cells in Opin. Lipidol. 2010; 21: PubMed Scopus Google Scholar, Rader D.J. Phillips M.C. Rothblat G.H. The role of different pathways in the of cholesterol from Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). The fractional of aqueous transfer to efflux is by the of other flux pathways present in the The with aqueous transfer could be enhanced a cholesterol were present that the to with FC and the flux between cholesterol donor and A number of this concept to a “shuttle and sink” in which a cell would act as a FC donor, and a lipoprotein or vesicle would serve as a FC acceptor (6Atger V.M. de la Llera Moya M. Stoudt G.W. Rodrigueza W.V. Phillips M.C. Rothblat G.H. Cyclodextrins as catalysts for the removal of cholesterol from macrophage foam cells.J. Clin. Invest. 1997; 99: 773-780Crossref PubMed Scopus (160) Google Scholar, W.V. Rothblat G.H. Phillips M.C. and mechanisms for the synergistic between different acceptor particles in the of cellular Biol. 1997; PubMed Scopus Google Scholar). these prior studies used compounds as shuttles and sinks and we the present study to whether there are compounds in serum that as shuttles, increasing the movement of FC between cells and that serve as sinks. The that to and the of shuttles to efflux employed human LDL as a sink and human LPDS as a of potential shuttles. The fractional efflux of FC from J774 cells produced by of these alone and in was efflux obtained from the combination of LDL and LPDS that was the values of the LDL and LPDS alone from to the of a shuttle in the LPDS This was on the LPDS and can be as the fractional efflux of the shuttle or as the of the efflux the synergistic efflux values are small on LPDS the of this efflux to the efflux is on LPDS In experiments, the LDL was with human VLDL which also as a cholesterol although the of was that obtained with obtained from J774 cells when LPDS is used as a shuttle and LDL as a J774 cells were incubated with increasing concentrations of LPDS alone or in combination with 25 LDL, and cholesterol efflux was measured after 4 h. Synergy was as the fractional efflux between efflux values from the combination of and the of LPDS and LDL are as the efflux that is to or the of to efflux methods for were LPDS are as = The are the A of experiments was in an to the in serum that to the in A was HDL, and when as a potential shuttle at concentrations from to levels of shuttling was produced at concentrations between and no shuttling was at levels of HDL The of shuttling at HDL concentrations and the of this effect as HDL was be to the cholesterol efflux of as HDL is and efflux is the of shuttling to efflux becomes could serve as shuttles, we the LPDS with increasing concentrations of human apoA-I from 2 to 10 a synergistic we observed a consistent in efflux that the efflux obtained in a with apoA-I and LDL was the values of apoA-I and LDL alone data The for this is be to a interaction of apoA-I with the LDL sink or the donor cells since these J774 cells A was by and albumin is a in cholesterol transport between cells and PubMed Scopus Google Scholar) in which cholesterol efflux from to media containing serum albumin and HDL was the of the efflux by alone. has been demonstrated that the of can the flux of FC between cells and by the of acceptor and thus the FC between cells and Stoudt G. Rothblat G.H. of acyltransferase on cholesterol in cells and Biophys. Acta. PubMed Scopus (33) Google H. acyltransferase on cellular by free 1995; PubMed Scopus Google we the LPDS with n-ethylmaleimide at a that of the present in LPDS. When to the cell at increasing the LPDS shuttling capacity was by of LPDS that could be for FC shuttling is A number of studies have demonstrated that some FC can with albumin albumin is a in cholesterol transport between cells and PubMed Scopus Google Scholar, for the of and mechanisms of cholesterol efflux from into human Biol. Chem. Full Text PDF PubMed Google Scholar) and that albumin can serve as a cholesterol although the fractional efflux produced by the incorporation of albumin in the media is that obtained with HDL or LPDS albumin is a in cholesterol transport between cells and PubMed Scopus Google Scholar, Rothblat G.H. by culture effect of serum Biophys. Acta. PubMed Scopus Google Scholar, The of serum albumin and on in culture Biophys. Acta. PubMed Scopus Google Scholar). has been demonstrated that albumin the bidirectional flux of cellular FC and as a capacity cholesterol transporter albumin is a in cholesterol transport between cells and PubMed Scopus Google Scholar). These of albumin are to of cyclodextrins, which have been shown to be shuttles FC between and obtained with albumin when as or of efflux to that obtained with LPDS 2 with There was no difference in when BSA was with of BSA with demonstrated the of serum albumin since no was produced by This result could be as and albumin is a in cholesterol transport between cells and PubMed Scopus Google Scholar) previously demonstrated that could the efflux of FC from when with there are no binding for FC on albumin of cholesterol in aqueous PubMed Scopus Google Scholar) and the association of FC with purified albumin is removal of albumin from plasma by chromatography efflux by as as for the of and mechanisms of cholesterol efflux from into human Biol. Chem. Full Text PDF PubMed Google Scholar). purified albumin to the culture medium alone is a of efflux, the of with as is the with enhances efflux. In addition to studies in which cell cholesterol efflux to albumin was we an to the in fractional efflux removal of albumin from LPDS. The removal of albumin in a in efflux and a in the efflux to data for LDL as the cholesterol sink in the in LDL FC, the shuttling capacity of albumin to the of cholesterol into cells and the efflux of FC, FC these movement of cholesterol of the donor cells would be by the of cholesterol into the cells W.J. Phillips M.C. Rothblat G.H. The bidirectional flux of cholesterol between cells and Biol. Chem. Full Text PDF PubMed Google Rothblat G.H. of cholesterol bidirectional flux between cells and Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). This bidirectional flux results in the of [3H]cholesterol in the no in cell cholesterol This was the in a number of to in the cholesterol of donor J774 cells incubated with albumin and LDL that were To measure the of albumin acting as a shuttle to cell cholesterol was to a cholesterol sink that thus the of the bidirectional flux In the present study, LDL was with which as a In such a in cell FC could be measured after a as shown in FC and cells, there was a of cell cholesterol with the combination of albumin as a shuttle and as a that the shuttling capacity of albumin can contribute to of cholesterol FC are the used in this in donor cell cholesterol were observed with or albumin alone of albumin on the of cholesterol from J774 cells when incubated with with cells were incubated for 6 h with media supplemented with of or a combination of BSA plus cholesterol from cell was measured using and is as different = with their for cells = for cells with 25 of = in a J774 cells were incubated for 6 h with media supplemented with of or a combination of BSA plus cholesterol from cell was measured using and is as different = with their for cells = for cells with 25 of = The data collected using LDL or as a sink are consistent with the role of albumin acting as a shuttle and enhancing the aqueous diffusion of FC, and flux a To the capacity of albumin to function as a we an in which radiolabeled J774 cells were incubated with or LPDS, and the media collected was used to measure to cells data for from LPDS or was and the the of this were [3H]cholesterol for LPDS and for These results in the of other albumin cholesterol movement into cells and is for a of the by LPDS. To that the flux of FC between cells could be by we used RBC as a cholesterol sink and whether the of albumin in a fractional efflux of FC from J774 cells as and RBC as shown in the fractional efflux of cholesterol from J774 was with the combination of albumin plus RBC RBC consistent with a in which albumin the FC movement by binding cholesterol and increasing the of the in the aqueous and that albumin can enhance movement of FC, only between cells and also between of albumin on exchange of cholesterol between J774 cells and [3H]cholesterol efflux 2 different from BSA or RBC alone = J774 cells were incubated for 2 h with media supplemented with 5 or a combination of BSA plus efflux is on the [3H]cholesterol in the J774 cells at different from BSA or RBC alone = in a J774 cells were incubated for 2 h with media supplemented with 5 or a combination of BSA plus efflux is on the [3H]cholesterol in the J774 cells at Although albumin have binding for cholesterol of cholesterol in aqueous PubMed Scopus Google can small of of the concentrations of albumin in the of this to efflux from cells is This was demonstrated in an study by and for the of and mechanisms of cholesterol efflux from into human Biol. Chem. Full Text PDF PubMed Google Scholar) that the removal of albumin from plasma by in a of in cholesterol efflux from A study by and albumin is a in cholesterol transport between cells and PubMed Scopus Google Scholar) indicated that albumin could in the bidirectional flux of cholesterol between and although this to efflux is that obtained with HDL or reconstituted HDL (rHDL) particles. The of albumin to FC would to function as a to that shown to when cyclodextrins the flux of cholesterol between donor cells and acceptors, such as particles of cyclodextrins to plasma cholesterol and Biophys. Acta. PubMed Scopus Google Scholar). Although there are a number of pathways that can in cell FC flux, albumin acting as a shuttle effect by enhancing aqueous as by the observed when or RBC as FC aqueous diffusion of FC is and can an important role in cell cholesterol flux Rader D.J. Phillips M.C. Rothblat G.H. The role of different pathways in the of cholesterol from Lipid Res. Full Text Full Text PDF PubMed Scopus Google the enhancement of aqueous diffusion by albumin could have a on cholesterol in This might be a at serum albumin levels the synergistic potential of albumin might be and this the of albumin to synergistic efflux. However, in individuals with albumin levels in serum albumin levels synergistic cholesterol efflux. There have been that serum albumin concentrations in humans be with of coronary artery disease D. albumin and of and in the PubMed Scopus Google Scholar, D. M. J. G. albumin as a of coronary J. PubMed Scopus Google and a number of have been to this effects, of blood and association with HDL concentrations of serum albumin as a for and coronary disease in and Google Scholar). is that the association of albumin with is a of the of albumin to shuttle FC between a of shuttling would the movement of FC between the cholesterol present in plasma and the of levels as cholesterol is A important cholesterol that could be by the shuttling of albumin is RBC cholesterol. The cholesterol of RBC is to that in plasma J. Red blood cells a role in reverse cholesterol Biol. 2012; PubMed Scopus Google and has been shown that RBC a role in reverse cholesterol transport J. Red blood cells a role in reverse cholesterol Biol. 2012; PubMed Scopus Google Scholar). FC in the of RBC with other of cholesterol an aqueous diffusion mechanism M. A study of cholesterol between and PubMed Scopus Google Phillips M.C. of on the of cholesterol exchange between and different of red blood Biophys. Acta. PubMed Scopus Google Scholar). the flux of cholesterol between plasma and as as the of RBC cholesterol to J. Red blood cells a role in reverse cholesterol Biol. 2012; PubMed Scopus Google is enhanced by the of albumin to serve as a cholesterol shuttle. The for