Bh~chimica et Bh~physica Acta. 1085 ~I9'H) 273-2t~,~

273

~ 1991 Elsevier Science Publishers B.V. All rights reserved (1t11)5-2761~/91/$(}3.5q ADONIS 11t105276t)91t11)2665

Review

BBALIP 53740

Cholesterol transport between cells and high-density iipoproteins William J. Johnson, Florence H. Mahlberg, George H. Rothblat

and MichaelC. Phillips F_,t,partrucnt of Physiolog), and Biochemistry. Medical ('ollege o] l>~'nnsyhania. Philadelphia. PA (U.S.A.) (Received 13 March lttt~l)

Key words: Cholesterol transp~rt: Reverse cholesterol transport: IIDI_: Surfact' transfer: Pk~sma membrane

Contents I.

In~r.~duction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

274

II.

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. H D L metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Reverse cholesterol transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cellular cholesterol homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

274 274 276 276

IlL

Mechanisms of surface transfer of cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Model systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cholesterol transfer and exchange between H D L iln3 cells . . . . . . . . . . . . . . . . . . . . . . . . . . D. Effects of cbolestert, I/phospholipid ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Effects of H D L binding and the H D L receptc~r . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

278 278 278 279 280 282

IV.

Efflux of iutraeellular cholesterol to I-IDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Cholesterol oxidase and the assessment of plasma membrane cholesterol . . . . . . . . . . . . . . . . B. Newly synthesized cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Lysosomal cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Cholesterol accumulation in lysosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. T h e endocytic pathway and lysosom '1 cholesterol metabolism . . . . . . . . . . . . . . . . . . . . . . 3. Transport of lysosomal cholesterol to cellular membranes . . . . . . . . . . . . . . . . . . . . . . . . . D. Cytoplasmic cholesteryl ester . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Role of apolipoprotein E in remtwal of intracellular cholesterol . . . . . . . . . . . . . . . . . . . . . F. H D L retroendocytosis and sterol flux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

283 283 284 285 285 286 286 287 289 290

V.

H D L subfractions and eMux of cellular cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

291

VI.

Selective uptake of cholesteryl ester from H D L to cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

292

VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

293

Abbreviations: ACAT, acyI-CoA:cholesterol acyltransferase; a C E | l , acid cholesteryl ester hydrolase; ape, apolipoptotein; cAMP. cyclic 3',5'-adenosine monophosphate; CE, cholesteD'l ester; CHO, chinese hamster ovtlry; DMS, dimethylsube:~midate; FC, free (unesterified) eholesteroh HDL, high-density liooprotein; k¢. first order rate constant for efflux of cellular cholesterol; k,, first order rate constant for influx of extraeellular cholesterol to cells; LCAT. le,dhin-cholesterol acyltransferase; LDL, low-density lipoprotein; nCEH, neutral cholesteryl ester bydrolase; NM R, nuclear magnelic resonance; NPC, Niemann-Pick C; PC, phosphatidylcholint:; SLIV, small unilamellar vesicle; tt/2, halftime: V L D L , very-low-density lipoproteins. Correspondence: M.C. Phillips. Dept. Physiology and Biochemistry, Medical College of Pennsylvania, Philadelphia, PA 19129, U.S.A.

27'* Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

294

Rel~:rcnces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

294

1. Introduction The subject of this review, cholester, .t transpo~'t between cells and high-density lipoproteins (HDL), is believed to bc of physiological relevance because it is central to 'reverse cholesterol transport' in vivo. As discussed in detail below, reverse cholesterol transport is the process whereby cholesterol is transported from peripheral (non-hepatic) cells to the liver for catabolism and removal from the body. Efflux of cholesterol from pcriphcral cells to HDL is the first step in reverse cholcsterol transport and it is critical for cholesterol homeostasis because these cells cannot catabolize any exogenous cholesterol they accumulate. The reverse cholesterol transport process is complex and there are several pathways tor the uhimate~ delivery of cholesterol originating in peripheral celts to the liver. It is known that HDL particles deliver cholesterol to liver and endocrine cells in vivo. The reverse cholesterol transport process alsc has pathophysiological significance because it is critical for prevention of atherogencsis; indeed elevated H D L levels have been shown to lead to regression of atherosclerosis in animal models. This review gives background information on the essential features of the pathways of HDL metabolism and cholesterol homeostasis before summarizing current understanding of the mechanisms of cholesterol movement between cells and HDL. The topics which arc discussed include: (1) the mechanism of efflux of cholesterol from the plasma membrane pool of various cells and the role of H D L binding; (2) efflux of intracellular pools of cholesterol and cholesteryl ester; (3) the properties of various HDI. subspecies as acceptors of cellular cholesterol; and (4) the delivery of H D L cholesteryl ester to cells. The above topics are the subject of active investigation in many laboratories around the world. Measurements of the eftlux of cholesterol from cells in culture have been used frequently both to support the concept and understand the mechanisms of reverse cholesterol transport. Such experiments are appealing because they are straightforward in principle. However, determinatkms of cellular cholesterol efflux to HDL can be confounded by problems such as the following: (1) cells can continue to grow during the efflux period so that the increase in cell number (cell protein) leads to an apparent decrease in cell cholesterol although there is no net movement of cholesterol to HDL; (2) provision of radiolabelled precursors to label the synthesized cholesterol pool leads to formation of several labelled sterols in addition to choles-

terol; (3) different pools of cholesterol and cholesteryl ester in cells are often radiolabelled to different specific activities; (4) enrichment of cells with cholesterol by addition to the medium of cholesterol dissolved in ethanol can lead to a nonphysiological distribution of choleste"ol with microcrystals both inside the cells and stuck to the cell surface; and (5) the continuous esterification of cholesterol and hydrolysis of cholesteryl ester in a futile cycle complicates the determination of the sizes of the cell free cholesterol l~ool. Failure to deal adequately with these difficulties had led to incorrect conclusions being drawn about the mechanisms by which HDL participates in reverse eholesterGl transport. II. Background II-A. HDL metabolism All animal cells require cholesterol to support membrane biosynthesis. This cholesterol can be provided from endogenous (biosynthetic) or exogenous (dietary) sources and the body has several mechanisms for maintaining adequate tissue cholesterol levels (for a review see Ref. 1). Low-density lipoprotein (LDL) particles are the primary carriers of cholesterol (originating from the diet) in the blood plasma and they deliver cholesterol to peripheral ceils via apolipoprotein B / E receptor-mediated endocytosis [2]. The same receptor also functions in hepatocytes to clear LDL and other remnant particles formed by lipolysis of VLDL; the liver is the major catabolic site of apo B- and apo E-containing lipoproteins [3]. Cholesterol which is returned to the liver can be secreted in the bile either unmodified or in oxidized form as bile acids; a fraction of this mixture is then lost from the body in the feces. Since peripheral cells (apart from steroidogenie cells) are not able to degrade cholesterol, a pathway by which intact cholesterol molecules can be removed from the cell is essential for cholesterol homeostasis. The process by which cholesterol from peripheral tissues is returned to the liver for catabolism has been termed 'reverse cholesterol transport' [4] and H D L is believed to be critical in this pathway [3-5]. The major apolipoproteins of the HDL class of lipoproteins are A-I and A-II; these proteins are synthesized and secreted by the liver and intestine (Fig. 1). In the case of liver cells, it seems that both intra- and extracellular assembly of these proteins with lipids occurs. For instance, hepatoma cells secrete lipid-poor apo A-I and lipid-protein particles containing either

,2. I ~ )

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~

I

~/

L_~

Nascent HDL or Pre I] HDL

, /-LCAT

®~

VLDL GE~~ V L D L (TG) LDL LDL

Fig. 1. HDL metabolism and cholesterul transfer. The major steps of HDL metabolismare indicated by the dotted arrows. The transfer of cholesterol between HDL and tissues arc indicated by the solid arrows. (I) Nascent HDL is produced either by secretion from the liver and intestine or by extracellular assembly in the interstitial fluid. (2) Sequential maturation of nascent IIDL into }IDL and HDL z through the esteriflcation of {IDL free cholesterol (Ft') by LCAT. (3) Exchange of cholestery.I ester ICE) from |IDL with triglyceride (TG) from VLDL and LDL, resulting in the lk,rmationof TG-rich HDL,. (4) TG-rich I-IDL_, particles serve as substratc for hepatic lipase (HL) and/or lily,protein lipase ILPL). Throughout these metabolic steps the bidirectional flux of free cholesterol occurs between the lipoprotein and cells. In some tissues there is also a net transfer of HDL cholesteryl ester (CE) to cells without the internalization of the entire lipoprotein particle. See text for futihcr details.

apo A-I or apo A-It alone, or a mixture of both [6,7]. The lipid-poor apoproteins probably associate with additional phospholipid derived from either cell membranes or plasma lipoproteins [8]. In addition to the direct secretion of particles that are precursors of •~lasma HDL, the excess apolipoprotein and phospholipid surface components created by the action of lipoprotein lipase on the triglyceride-rich lipoproteins also contribute to the H D L pool; these particles arc probably discoidal apoprotein-phospholipid complexes [3]. In the plasma compartment, cholesterol in the various precursor H D L particles (some of which originates from peripheral cells-see below) is converted to cholesteryl ester ( £ E ) by the action of lecithincholesterol acyltransferase (LCAT) for which apo A-I is a cofactor [9]. This formation of CE is associated with the maturation of the precursor H D L to spherical plasma H D L particles [10]. As depicted in Fig. 1, inter-conversion of H D L particles between the H D L , and H D L 3 classes occurs by the combined actions of LCAT and hepatic lipase [8] and a transfer protein which exchanges CE and triaeylglycerols [11]. The smaller, denser H D L 3 particle is the preferred substrate for LCAT and the formation of CE by this

enzyme leads to the formation of larger H D L , particles. The lipid transfer protein is th~)ugh~ a~ exchange CE in H D L , with triacylglycerol in LDL or VLDL. The CE is transferred to LDL and then delivered to the liver by whole particle catabolism (see Section II-B for more discussion of this process as part of reverse cholesterol transport). The lriacylglycerol exchanged into lh,." H D I . : particle become'~ a substrate fur hepatic lipase to that the H D L , is reconverted to an HDL~ particle where it can again participate in the cycle of delivc~ ,,f cholesterol to LCAT. Thus, it is apparent that ti~e plasma CE and triacylglycerol transfer protein plays a key role in reverse cholesterol transport: indeed, the overall rate of cholesterol clearante from phtsma correlates with the level of transport protein activity [I 1]. The enrichment of H D L particles with CE can lead to the formation of larger particles which bind apo E; these particles are than taken up by the liver via the apolipoprotein B / E receptor (see Section IV-El. Reflecting the u~verse pathways for formation and intcrconversion of H D L particles, circulating plasma H D L comprises a heterogeneous population of spherical lipid-protein particles with diameters in the range 7-12 nm [12]. Centrifugal studies have demonstrated heterogeneity in density and size: several subclasses of H D L such as H D L z and HDL3 have been defined by this technique [13]. Non-denaturing gradient gel electrophoresis has resolved H D L on the basis of particle sizt into even more subspecies [12,14]. Recently, immunoaffinity chromatography and gradient gel dectrophoresis have demonstrated that there are apoprotcin-spccific populations of plasma H D L [ 15]: one populatkm contains apo A-i only and another contains both apo A-I and apo A-ll. Electrophoresis of plasma on agarose gels shows the presence of a small population of H D L particles containing only apo A-I which exhibit prc-13 mobility ([16]; see Section V). Clearly, a complete understanding of reverse cholesterol transport requires definition of the roles of the various H D L subclasses in cholesterol transport. This question is the focus of much current research on H D L Spherical H D L particles contain roughly equal masses of lipid and protein: they comprise a core of neutral CE and triacylglycerol molecules encapsulated by a surface morn)layer of phospholipid and apolipoprotein [3]. Free cholesterol is able to partition between the core and surfac:: with most of it being in the surface [17]; the cholesterol/phospholipid tool ratio in the surface of human H D L is about {I.2. The apo A-i and apo A-II molecules stabilize the particles and their interaction with lipid is mediated by amphipathic helical segments [18]. The structure is dynamic in the sense that both the lipid and protein components of H D L particles can exchange with other lipoproteins in the plasma compartment. The circulating halftime (11/2) of H D L pro-

276 teir, in humans is 3-~') days but the t~/, for exchange of lipids arc much shorter man this [8]. The transfer ,ff the phospholipid, CE and triacylglycerol componcm~ of HDL is protcin-mcdiated, as discussed above, while the exchange of cholesterol molecules between HDL and either other lipoprotcins or red blood cells is unmcdiated. The tt/2 for exchange of cholesterol molcculcs out of human HDL 3 to excess acceptor particles is about 3 min [19]. The major sites of catabolism of apo A-I arc the kidney and liver while lhc HDL cholesterol is degraded in either liver or steroidogcnic cclls [10]. H-B. I¢;ecetwechoh'sterol tran.sport The fact that the free cholesterol content of plasma HDL particles is regulated, in part, by the action of LCAT led Glomset [20] to propose that this esterification of cholesterol in HDL enhances the transport of cholesterol from membranes of 9eriphere, l ceils to the liver. The concept of HDL playing a key role in reverse cholesterol transport is supported by cell culture experiments showing that HDL can clear excess cholesterol from some cells (see Section l i d and epidemiological evidence showing that low levels of circulating H D L chole:,tcrol are associated with increased coronary artery disease rates [21] and mortality [22]. Clinical studies of drugs with HDL-raising activity and of individuals with genetic disorders of H D L metabolism are also generally consistent with HDL playing a key role in reverse cholesterol transport (for a review, see Ref. 23). It is likely that different genetic defects of H D L metabolism perturb the reverse cholesterol transport of cells in various tissues to different degrees. The HDL particles that are involved in the initial transport of cholesterol away from peripheral cells are not simply those present in plasma because these cells arc bathed by interstitial fluid which is separated from the plasma compartment by the capillary endothelium. Interstitial HDL particles arise both by filtration of plasma HDL across this barrier after which they are rcm,~dclcd, and b~, direct secretion of apolipoproteins by some peripheral cells ([24,25] and see Section IV-El. The lymph HDL particles are relatively enriched in free cholesterol relative to CE [25] and the HDL concentration in human peripheral lymph is about 10% of that in plasma [26]. These interstitial fluid lipoprotcins ~,vhich arc precursors of plasma HDL (see above) seem to be effective acceptors of cell cholesterol and good substratcs for LCAT [25,26]. Besides their normal function of transporting cholesterol away from peripheral cells, higher concentrations of these lymph HDL 13article~ presumably promote clearance ~f cholesterol from the cholesterol-loaded cells of atherosclerotlc plaque. C0nsistcnt wilh this conceot, it has been demonstrated recently that intravenous administration

of 5() mg protein/weck of h(,mologous HDL during cholesterol feeding of rabbits for 8 weeks inhibits the dcwq~)pment of fatty streaks and lipid deposition ill the aortic wall [27]. Furthermore, administration of HDL after the development of athcrosclerotic lesions induces regression of established aortic lesions [28]. More generally, studies of animal models show that lowering plasma cholesterol levels leads to gradual removal of intra- and extracellular lipid deposits and regression of plaque [29]. The physical states of the lipids that accumulate in the arterial intima critically affect the progression and regression of lesio,~s [30]. To prove that reverse cholesterol transport occurs ~n vivo, there is a need for direct demonstrations that cholesterol can be picked up from peripheral tissues by HDL and then delivered to liver and converted to bile acid. As summarized below, a body of experimental evidence supporting these concepts has been generated. Kinetic studies of H O L free cholesterol in humans are consistent with this pool playing a central role in exchange reactions and in the vasculature-tissue cholesterol transport system [31]. In agreement with this idea, the primary acceptol" of unesterified cholesterol from the spleen of rats is H D L [32]. Transfer of cholesterol from peripheral tissue to H D L changes the structure of the lipoprotein particles; increases in size and decrease in density have been observed in the circulating H D L of rabbits when the peripheral tissues are loaded with cholesterol by intravenous injection of acetylated or native human LDL [33]. The fact that these changes are similar to those observed when H D L is incubated with cholesterol-loaded macrophages (see Section IV-El suggests that H D L is involved in reverse cholesterol transport in vivo. Cholesterol is more efficiently removed than CE from H D L by the perfused liver of African green monkeys; furthermore, the free cholesterol from H D L appears more rapidly in bile than HDL CE [34]. It has been shown that H D L is involved in the transport of cholesterol from the stnusoidal endothelial cells to the parenchymal cells in rat liver [35], providing what may be a useful single-organ model for reverse cholesterol transport. II.C. Celhdar cholesterol homeostasis It is apparent from the above information, that reverse cholesterol transport involves situations where the flux of cholesterol between cells and HDL is critical. To understand the latter process fully, the transport from the various metabolic and physical pools of cholesterol within cells needs to be considered. Generally, the homeostasis of cholesterol in a cell involves a balance of the fluxes of cholesterol into and out of the cell and the rate of de novo synthesis of cholesterol.

The pathways involved in cellular cholesterol metabolism are summarized in Fig. 2; the receptor-mediated

277

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AOAT/~L,,c, " - - - - ~ j L ° \

~ ~

"

l

Hot_ Efflux

Fig. 2. Cholesterol metabolism in mammalian ,.'ells. (.'ells acquire cholesterol by endocytic uptake of lipoproteins and extracenular lipid droplets (Influx), and by biosynthesis (conversion of acetate: carbon to cholesterol). Phagocytosis of extracellular lipid droplets (which

arise from the lysisof dead foam cells) may be an important process in atheroselerotic lesions. Biosynthesisand the lysosomaldegradation of exogenouseholesterylester contribute to internal (non-plasma membrane) pools of free cholesterol (FC). There is continual exchange of cholesterol between the internal pools and cytoplasmic cholesteryl ester droplets via a cycle of reacfiutts catalyzed by acyl CoA:eholesterol acyltransferase(ACAT) and neutral chnlesteryl ester hydrolase(nCEH). Cellular cholesterol homeostasisis maintained by the effluxof cholesterol from~he plasmamembraneto particlesin the HDL fraction of plasma and interstitial fluid. The removal of cholesterol from internal pools probably requires preliminary transport to the plasmamembrane.See text 0.~rI'urtherdetails.

uptake of modified LDL occurs only in certain cell types such as maerophages (see below). Exogenous cholesterol can be deiivered to macrophages by the three influx pathways as indicated (Fig. 2). The endocytic pathway for catabolism of the LDL ~artie!e after it binds to the LDL (apolipoprotein B / E ) receptor has been elucidated in detail [2]. Macrophages are unusual among peripheral cells because, in addition to the LDL receptor, they have at least two other classes of receptors which recognize modified LDL. Macrophages possess a scavenger receptor [36] which recognizes both acetyl LDL and oxidized LDL and an oxidized LDL receptor which recognizes ortly oxidatively modified LDL [37]. A recent report suggests that smooth muscle cells and fibroblasts may also express the scavenger receptor [38]. In all cases, the receptormediated endocytosis of normal or modified LDL leads to the ultimate delivery of the lipoprotein particle via a series of endocytic vesicles [39] to mature lysosomes. However, a vital difference is that expression of the modified LDL receptors is not down-regulated by cell sterol levels. Consequently, maerophages exposed to modified LDL ovcraccumulate cholesterol and eventually become foam cells [40]. An additional pathway by

which macrophages and smooth muscle cells can become foam cells involves the phagocytosis of CE-rich lipid droplets [41,42]. As far as endogenous cholesterol is concerned, the rate-limiting step in the biosynthetic pathway for cholesterol is the reduction of ¢¢-hydroxy/3-mcthylglutarTI coenzyme A (HMG-CoA) to mcvalonic acid [2,43]. The transcription of the gcnc for HMG-CoA rcductase is repressed when cells are loaded with sterols. The promoter for this gone involves a stcrol regulatory element which is common to the promoter foe the LDL receptor gene [44], This leads to coordinate regulation of the expression of both proteins with both being subject to end-prodnct feedback rcguhttion by cholesterol so that as endocytosis of LDL leads to a rise in cell cholesterol levels, the expression oi both proteins is suppressed. As depicted in Fig. 2, CE delivered to ~'sosomcs by the various influx pathways is hydrolyzed to free cholesterol by an acid CE hydrolasc (aCEH) [45]. This free cholesterol then leaves lysosomes by ill-defined pathways (scc Section IV-C.3) and becomes part of the intraccllular free cholesterol pool(s). Some of thts free cholesterol comprises a pool that activates acylCoA:cholcstcrol acyhransferase (ACAT) which is located in the rough endoplasmic reticulum [46]. Frec cholesterol that is in excess of cellular requirements is esterificd by ACAT and the CE product is stored in cytoplasmic inclusions. This CE is the substrate for a neutral CE hydrolase (nCEH), and there is it cycle involving the hydrolase and ACAT by which the stored CE is cuntinuously turned over with expenditure of metabolic energy (for a review, see Ref. 45). Free cholesterol molecules can move between the intracclluhtr pool(s) and the plasma membrane where they become available for efflux from the cell to extracellular acccptor particles (see Section IV-C.3.). For most types of cells this is the only mechanism available for removal of celiular cholesterol, in addition to the free cholestero! efflux pathway, steroidogenic cells can oxidize cholesterol to steroid hormones while hepatocytes can either convert intracellular cholesterol to bile acids by a pathway in which cholesterol 7-c~-hydroxylase is rate-limiting or secrete cholesterol in lipoprotein particles. tin sui.tiYlary, cells possess several mechanisms to maintain the unesterified cholesterol located in membranes at appropriate levels. While cells which acquire exogenous cholesterol only by the LDL receptor are able to regulate cholesterol influx by this route, macrophages and other cells which leave modified-LDL receptors that are not regulated by intracellular sterol levels can cweraccumulate exogenous cholesterol and become foam ccll~. Thc,~c peripheral ceils can only release cholesterol by cfflux to cxtracclluhtt acccptor particles. Subsequent sections in this review address the details of the movement of cholesterol i'rom the

278 ¢~lasma membrane a:ld from w~rious intracellular pools to cxtracellular HDL particles. Ill. Mechanisms of surface transfer of cholesterol

IliA. Background It is well known that unesterified cholesterol can exchange between plasma and cells. The original obscrvation was made with red blood cells by Hagerman and Gould in 1951 [47]. It has also been established that exchange of labelled cholesterol occurs between extracellular lipoproteins and cultured cells which have intracellular organelles; notable early observations were made with mouse L-cells [48,49] and macrophages [5052]. The exchange of cholesterol between lipoproteins and cells is a surface transfer process in the sense that interna[izatlon of the lipoprotein by the cell is not required. Since these early findings which have been reviewed by Bruckdorfer and Graham [53], there have been many studies elucidating more of the details of the processes of surface transfer and exchange between membranes (for recent reviews, see Refs. 54-57). It is apparent that free cholesterol exchange by surface transfer is a physical-chemical phenomenon which does not require metabolic energy and which occurs independently of lipoprotein endocytosis and secretion. Here, we give a flavor of this work while focusing on the major features of the transport of cholesterol between the cell plasma membrane and HDL particles

HI-B. Model systems in light of the above information, it is unsurprising that the exchange of free cholesterol not only occurs between lipoproteins and cells but also between different lipoprotein classes. A bidirectional flux of unesterifled cholesterol molecules occurs between HDL and LDL particles when they are incubated in vitro [19]. A detailed analysis of the exchange kinetics in this relatively well defined system showed that at 37 °C the tt/2 values for cholesterol transfer from human HDL3 to LDL and from LDL to HDL3 particles are about 3 and 45 rain, respectively. Also, when excess acceptor particles are present the t~/2 is independent of the ratio of doaor/acceptor particles indicating that the exchange process is not mediated by collisions between donor and acceptor particle. NMR studies showed that the physical states of cholesterol molecules in the phospholipid/water interfaces of serum lipoprotein particles and small unilamellar vesicles (SUV) are similar [17,58,59]. Consistent with this, the overall kinetics of cholesterol exchange in the HDL-LDL [19] and SUVSUV [60] systems are similar. Studies from several laboratories have demonstrated that exchange of free cholesterol molecules between SUV occurs by an aque-

@ MEMBRANE

Fig, 3. Aqueous diffusion rr~chanism for efflux of cellular cholesterol. Schematic representation of the mechanismby which cholesterol moleculestransfer from the plasma membraneof a cell to an acceptor particle such as a phospholipidvesicleor lipoprotein in the extracellular medium. The solid arrow represents the rate-limiting step which is the desorption of a cholesterol molecule from the donor membrane into the aqueousphase. The dissolved cholesterol molecule diffuses through the unstirred water layer around the cell unti~ it collides with an acceptor particle (see text for details). From Phillips et al. [67].

pus diffusion mechanism [60-62]. In this process, cholesterol molecules desorb from the donor lipidwater interface and diffuse through the intervening aqueous layer until they collide with and are absorbed by an acceptor particle; when excess aceeptor particles are present, the rate-limiting step is the desorption o f cholesterol molecules from the donor particle (Fig. 3). We have reviewed the aqueous diffusion mechanism comprehensively [54] and pointed out that the following four kinetic criteria are characteristic of the process: (1) the rate of free cholesterol exchange is first order with respect to the concentration of free cholesterol in the donor particles; (2) the tl/2 for transfer of phosphatidylcholine (PC) molecules (containing palmitate and oleate chains)from a given P C / c h o l e s t e r o l / water interface is about 20-times longer than that for free cholesterol; (3) the free cholesterol exchange process is strongly temperature dependent and the activation energy for SUV is about 70 kJ/mol; and (4) at high acceptor/donor particle ratios, the rate of exchange is zero order with respect to the concentration of acceptor particles. The rate of desorption of cholesterol molecules is not affected by transient vesiclevesicle associations such as may occur in concentrated solutions of SUV [63]. The transfer of cholesterol molecules to the aqueous phase proceeds through a transition-state complex where the cholesterol molecule is attached to the donor

27 t) particle by the tip of its hydrophobic tail. The height of the activation energy barrier for formation of this transition state is affected by the interaction energy of the cholesterol molecule with neighbouring molecules in the surface of the donor p::rticle [54]. Thus, if the Van der Waals attraction betwecr., cholesterol and the host phospholipid is relatively low, the rate of desorption of cholesterol molecules is enhanced [64]. For this reason, decreases in: (1) the degrees of saturation of the host phospholipid molecules; (2) the sphingomyelin content of the l i p i d / w a t e r interface; and ( 3 ) t h e size of P C / c h o l e s t e r o l SUV, are accompanied by increases in the cholesterol exchange rate (for a review, see Rcf. 54). Current u n d e r s t a n d i n g of the effects of proteins on the kinetics of cholesterol exchange are limited. Two recent studies have shown that apo A-I can enhance the rate of cholesterol exchange from mitochondria [65] and SUV [66]. In the latter case, it was suggested that the presence of the amphipathic alphahelices of apolipoprotein molecules in the donor l i p i d / w a t e r interface creates packing defects which facilitate the desorption of cholesterol molecules. This effect of apolipoprotein molecules of the A and C classes probably underlies the short t~/,_ for cholesterol exchange observed with h u m a n HDL.~ c o m p a r e d to egg P C / cholesterol SUV [19]. III-C. Cholesterol transfer a n d e.rchange between H D L a n d cells

M e a s u r e m e n t s in this laboratory of the exchange of cholesterol between cells and extracellular acceptor particles have shown that the kinetics are consistent with the aqueous diffusion model as outlined above [67,68]. W h e n the cxtracellular particles contain cholesterol, a bidirectional flux of cholesterol occurs between the particles and the cell plasma m e m b r a n e (Fig. 4). Efflux is defined as the rate of movement of free cholesterol from the cells to the medium, and influx is the rate of movement in the opposite direction: each process has a characteristic rate constant, designated here as k¢ and k~ for cfflux and influx, respectively. The methods for d e t e r m i n i n g k~, and k i have been described elsewhere [69,70]. In the red blood cell which contains only a single membrane, cell cholesterol behaves as a single kinetic pool and exchanges to equilibrium with exces,~ extracellular human H D L 3 with a t t / , - of 4.6 + (1.6 h [71]. This t i t , " is characteristic of the rate-limiting desorption of cholesterol molecules from the h u m a n red blood cell into the extracellular aqueous phase and, as expected from the earlier discussion of the aqueous diffusion model, the t i t z is longer for different species of red blood cells which contain e n h a n c e d levels of sphingomyelin. The fact that red blood cell m e m b r a n e cholesterol behaves as a single kinetic pool indicates that the rate of

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%\ ["c]~.o,.,...,

:.~

.2 o

'

..o~O----'- o - ' ~ ' ~

['"1~"o',,'°,o'

I o 'o

3 0 0'

,oo

time(mini Fig. 4. The bidirectional flux of free cholesterol (F(_')between human IIDL ;rod rat FuSAH hcpatoma cells. The IIDL and cells were prelabeled, respectively,with [7- ~11]cholesteroland [4-t~(']cholesterol undt-r conditions that prevented esterification of the tracers. The | IDL was diluted into tissue culture medium and applied to cells. At intervals, the cells and medium were separated and analyzed fit)r isotopic composition by liquid scintillation counting. The fractional retetttion of [t~('lcholesterol IX) and the fractional uptake of [ ~tl]cholcstcrol (~) by the cells are plotted against incubation time in min. The ,,m~th cul~'eswere obtained by the computerized fitting tff each ~,cl ol' kinetic measurements to a model for cholesterol equillpration between two i~fls. This model predicts that the timecourse for [la(']cholesterol rctentiem by cells should conform to the relationship tz(- cells = - k- c s4("cells, t = II k t + k c "e

I~,.~,lr +

k, k,+k~.

and that for [ ~ll]frce cholesterol uptake by the cells to: ~11cells k. (~ .g.u k. ............ c , , 4- . . . . . ~|1 med, t = It k.+k c k.+k¢. where la(, cells ;'cS~.~.;:ii

~ll cells and

'ttm~d.,=O

are, respectively, the fractional retention ol[laC]cholcsterol and the fractional uptake of [3H]cholesterol. and /,¢ and k. are the rate constants h~r cholesterol efflux and influx, respectively. (For cumplete details, see Johnson ct al. [69]).

translocation of cholesterol molecules between the inner and outer monolayers of the membrane is faster tha~ the rate of transfer from the membrane [71,72]. ,'is summarized in Fig. 2, cells that can synthcsizc frcc cholesterol and C E have internal pools of cholestcroL Consequently, there is the possibility of multiple kinetic pools of cell-free cholesterol when the bidirectional flux of cholesterol between such cells and H D L is examined. More than one kinetic pool of cell cholesterol has often been observed [54], but, as summarized in Fig. 4, the bidirectional flux of cholesterol between

280 human HDL and rat Fu5AH hcpatoma cells containing normal levels of cholesterol can be described quantitatively by assuming homogenous cell and HDL free cholesterol pools [69]. It follows that both translocation of cholesterol molecules in the plasma membrane and mo;emcnt of cholesterol from internal sites to the plasma membrane are rapid relative to the rate of tran,;fcr of cholesterol molecules to the HDL particles. The t:/2 values observed for efflux of cholesterol from a variety of cells are typically in the range 1-24 h (reviewed in Ref. 54). In the presence of excess extracellu!ar accepter particles, variations in tl/z for efflux of cellular cholesterol from different types of cells are duc to differences in plasma membrane structure because intact cells and isolated plasma membrane vesicles release cholesterol at similar rates [73]. The dependence of k~ on the concentration of extracellular HDL is consistent with the aqueous diffusion mechanism [54,69]. At low HDL concentration, tw2 for efflux is relatively long because the overall rate of transfer of cholesterol molecules to HDL particles is affected by the collision frequency of desorbed cholesterol molecules and HDL particles in the unstirred water layer surrounding the cell. At high H D L concentrations, the rate-limiting step for efflux is the rate of dcsorption of cholesterol molecules from the cell plasma membrane (see Fig. 3). A variation of this model has been proposed in which partially desorbed cholesterol molecules arc induced to transfer to an accepter particle by collisions with the accepter particle [74]. This implies that direct contact between the plasma membrane and the accepter iipoprotein particles is required for transfer of a cholesterol molecule; this model is not consistent with the results from several laboratories showing that cholesterol molecules can diffuse between donor and accepter particles which arc physically separated by either a dialysis membrane or a polymer phase [60,61,75]. As discussed in detail elsewhere [54], the influx and efflux of frec cholesterol mass between cells and HDL can bc expressed in terms of k i and k~ and the concentrations of free cholesterol in the HDL and cell pools. Free cholesterol surface transfer processes drive the cell free cholesterol content towards a steady-state level defined by the external concentration of free cholesterol and the ratio of k i and k c. The net movement of free cholesterol between cells and HDL seems to involve the diffusion of cholesterol molecules down their free energy gradient l'rom regions with a high free cllolcsterol/phospholipid ratio to regions with a low ratio. Enrichment of the plasma membrane with cholesterol does not alter k~. very much [76]; the flux of cholesterol out of the cell is increased because of the enlarged pool of plasma membrane cholesterol. The concept of the partition of cholesterol between HDL and cell being primarily governed by the free choles-

te-ol/phospholipid ratio is supported by experiments :,hewing that the free cholesterol/phespholipid ratio of red blood cells can be increased or decreased in direct proportion to the free cholesterol/phospholipid ratio of extracellular liposomes [77] (see, Section Ill-D). HDL seems to participate in simple exchange (i.e., efflux = influx) with cells containing normal amounts of cholesterol, whereas HDL can cause the net release of cholesterol from cells enriched in cholesterol (see Section IV-D). It should be noted that simple observation of the release of labelled cholesterol from cells by HDL does not imply that net efflux of cholesterol is occurring; either the bidirectional flux of cholesterol must be monitored or the decrease in cell cholesterol ~nass determined directly [69,70,78].

III-D. Effects of cholesterol/phospholipid ratio The direction of net cholesterol flux between cells and HDL is affected by H D L cholesterol and phospholipid composition. Two general approaches have been used to modify lipoprotein lipid composition, one in which native particles are selectively enriched with cholesterol or depleted in phospholipid, and a second in which artificial particles of known composition are reassembled from H D L components or pure lipids. Both experimental systems have proved satisfactory and have yielded generally similar results. In one of the earliest investigations of the effect of H D L free cholesterol content on cellular cholesterol metabolism, sonicated mixtures of free cholesterol PC, together with serum, were incubated with cells in culture [79]. These studies demonstrated that particles with molar ratios of cholesterol/PC > I stimulate cellular cholesterol accumulation whereas particles with mole ratios < 1 generally deplete cells of cholesterol. It was noted in these studies that if the cholesterol-enriched particles are present in the culture medium in the absence of serum much less cholesterol accumulates in the cells [79]. Subsequently, it has been demonstrated that cholesterol-enriched dispersions promote cholesterol deposition by different mechanisms, depending on the cells used and the lipoproteins that are co-incubated with the dispersions. For example, McCloskey and colleagues [80] showed that the receptormediated uptake of cholesterol/PC liposomes complexed with either LDL or acetyl LDL results in enhanced cholesterol accumulation in macrophages. In contrast, the incubation of H D L with cholesterol/PC mixtures with molar ratios > 1 selectively enriches the HDL with cholesterol, and the cholesterol-enriched HDL then stimulates net cholesterol uptake in the absence of any appreciable H D L internalization [81]. This observation is consistent with the hypothesis that the cholesterol/phospholipid molar ratio of HDL is critical in determining whether the lipoprotein particle

281 will be either a donor or an acceptor of free cholesterol when exposed to cells (cf. Section Ill-C). The variations observed in the net movement of cholesterol between cells and H D L preparations having different cholesterol contents arc a reflection of the size of the lipoprotein free cholesterol pool and are not a result of changes in either k i and k¢. Thus, Johnson and colleagues [76] showed that the rate constants for flux remain unchanged when fibroblasts arc incubated with H D L preparations that are either enriched or depicted in cholesterol. They also observed that influx and efflux were equal (i.e., exchange occurs) when fibroblasts are exposed to H D L 3 having a normal free cholesterol content (e.g., approx. 5 0 - 6 0 p . g / m g protein) [76]. Besides setting up a concentration gradient favoring efflux of cholesterol by surface transfer, enrlc'tmcnt of cells with free cholesterol also enhances I- nding of H D L to cells (see Section I l l - E l . The possible enh a n c e m e n t of sterol efflux by the binding e l H D L to the plasma m e m b r a n e has been ruled out by studies showing: ( I t that there is no relationship between the concentration d e p e n d e n c i e s of efflux and H D L binding; and (2) that the inhibition of H D L binding has little or no effect on the ability of H D L to promote efflux from the plasma m e m b r a n e of either normal or sterol-enriched cells [82,83]. Studies on the role of H D L phospholipid composition in the modulation of cholesterol flux primarily have involved the depletion of H D L phospholipids by t r e a t m e n t with phospholipases. Such an approach results in an increase in the free cholesterol to phospholipid molar ratio without changing the cholesterol content of the lipoprotein. One of the first studies by B a m b e r g e r and eoworkers [84] d e m o n s t r a t e d that the depletion of H D L phospholipids by t r e a t m e n t with snake v e n o m phospholipase A , , or with the more physiologically relevant hepatic lipasc, produces a lipoprotein that stimulates the net accumulation of cholesterol in h e p a t o m a cell~ ~s d e t e r m i n e d by an increase in cell cholesterol mass and the s t i m u l a t k m of cholesterol esterification. Since the net uptake of free cholesterol can result from e i t h e r e n h a n c e d influx from H D L or decreased efflux from the cells, bidirectional flux studies were conducted to establish the mechanism responsible for the accumulation of cholesterol in cells exposed to phospholipid-depleted H D L ,,t the hepatoma system, it was established that cell free cholesterol efflux is reduced and that influx is not significantly changed [85]. Subsequently, other cell types were studied using essentially similar experimental systems. As shown in Table I, the results differ d e p e n d i n g upon the type of cell. Although many cells increase their cholesterol content upon exposure to phospholipasetreated HDL, no changes in cholesterol contents are observed in erythrocytes or endothelial cells [86-88]. Even in those cells that exhibit an increase in the net

TABLE

I

Effects of pt*o.~ph~,lipid-deph'thm of IIDL on the distribution of choh'stcrtd between cell~ and tlDI,

The indicated t~,peso f cells were incubated in the presence of c~ther control human |II)L or tlDL that had been partially depleted of phospholipids by treatment with phospholipase. Following incubation. the lrce cholesterol IF(') mass contents of the cells were dcterminctl to establish if the exposure to Ihe depleled |IDL modified the ecll cholesterol content relative tt~ that of cells exposed to control IIDL An increase in cell cholesterol content can result from either enhanced ( T) influx of HDL cholesterol or reduced ( l ) efflux of cell cholesterol. The changes in flux produced by reducing IIDL phospholipid content were established by monitoring the mtwement of radiolahelcd cholesterol between the cell and medium compartments. Cell type

Fu5AII hepatoma Iluman eDthroe~,tes Rat hepatocytcr, l luman cndot hclizd Iluman granulosa I Inman Iibrobla~,ts (nOrlllHlt | hlman fihrtlhlaMs (lram,l~)rmcd)

Net cell

FC flux

Reference

uptake .~J F(" yes no y¢~. no yes

change efflux L none influx T none influx T

[6t)-$51 [,~tq 1871 [S7.SS] [87]

yes

illfltlx T

[St)l

yes

efflux ,~

Is'~l

uptake of cholesterol from the phospholipid-depleted HDL, the underlying mechanism for the accumulation differs, in some systems a reduction in efflux appears to be responsible whereas in other cells an increase in influx has been observed (Table !). A mechanistic e×planation for the differences in flux observed between cells exposed to control and phospholipiddepicted H D L has not been developed. The studies described above on phospholipid-dcpleted H D L used lipoprotcins that were depicted in PC. Attempts to modify the concentrations of other phospholipids have been limited. A recent investigation by Sloite and collcagues [9(1] demonstrated that the bidirectional flux of labeled cholesterol between H D L and fibroblasts does not change even when the H D L sphingomyclin is extensively depleted by exposure to sphingomyelinase. W h e t h e r this is a reflection of the general unresponsiveness of normal fibroblasts to changes in H D L composition, or indicates that cholesterol flux is in some way influenced to a greater extent by lipoprotcin PC content remains to be determined. Two enzymes that arc thought to play an important role in the reverse cholesterol transport process by modulating H D L lipid composition are LCAT and hepatic lipase (see Section II-B). As first proposed by Glomsct [20], LCAT may provide a driving forcc in reverse cholesterol transport by esterifying the ,-:llular cholesterol removed by HDL, thereby maintaining a cholesterol gradient favoring net cfflux. Cell culture

282 studies have documented the ability of LCAT to modulate thc movcmcnt of free cholesterol between HDL and cells. Thus, exposure of cells to control HDL results in cholesterol exchange without a change in content, whereas incubation of cells with HDL that has prcviously been modified by exposure to LCAT produccs a decrease in cell cholesterol content [91]. A series of studies by Fielding and colleagues [92,93] have documented the need for LCAT to achieve continued net cfflux of cholesterol from cells incubated in serum. Just as LCAT functions to produce a cholesterol gradient that stimulates cholesterol efflux from peripheral cells, so might hepatic lipase function to establish a cholcstcrol gradient within the liver that favors the net movement of frec cholesterol from H D L to hepatocytes. The hypothesis that hepatic lipase, through its phospholipasc activity, depletes H D L of phospholipid and enhances cholesterol uptake was first proposed by Jansen and Hulsman [94]. Subsequent free cholesterol flux studies using phospholipid-depleted HDL, as described above, have demonstrated the feasibility of this hypothesis. However, validation of the role of hepatic lipasc in reverse cholesterol transport in vivo remains to be established. The intriguing studies demonstrating that elevated concentrations of lipoprotcin free cholesterol constitute a significant risk factor for atherosclerosis [95] can be explained by a disruption of the normal free cholesterol gradients between cells and lipoprotcins resulting in an inhibition of reverse cholesterol transport ([93] and see Section Ill-C). The importancc of HDL cholesterol/phospholipid ratios in regulating cholesterol movement between cells and lipoprotcins is well documented, and the recent studies by Slotte and colleagues have now demonstrated that the maintenance of proper cholesterol/ phospholipid ratios in the plasma membrane is also critical in regulating the movement of cholesterol between plasma membrane and intracellular sterol pools. These investigators showed that the rapid depletion of fibroblast plasma membrane sphingomyelin by treatmcnt with sphingomyclinase rcstllts in a shift of plasma membrane cholesterol to internal pools as demonstrated by a marked increase in the activity of ACAT [96]. This redistribution of cellular cholesterol is reversed as normal sphingomyelin levels are restored in cells [97]. it should bc noted that although depletion of plasma membrane sphingomyelin produces a rapid movcmcnt of membrane cholesterol into the ceils, such as depletion has no effect on the cfflux of cellular cl]olestcrol to HDL [9{)]. Thus, the flow of cholesterol to thc intracellular compartments is maintained and the cfflux of cholesterol to extracellular acceptors is unaffcctcd. A number of explanations can be proposed for this rather unexpected observation; these include selective movement of cholesterol from the cytoplasmic side of thc plasma membrane, membrane lipid rcorga-

nization in depleted cells, a n d / o r stimulation of iPtracellular transport mechanisms. III-E. Effects of HDL binding and the HDL receptor

As reviewed previously [54], HDL binds specifically to cells and isolated plasma ;.,embrane. Characteristics of the binding include: (l) Kd in the range 2-approx. 100/zg protein/ml (21ll)-1000 nM); (2) specificity for apolipoproteins A-i, A-11 and A-IV [98,99]; (3) lack of dependence on Ca 2+ or LDL (apo B / E ) receptor; (4) reversibility by way of either desorption from the plasma membrane or retroendocytosis; (5) inhibition by pretreatment of H D L with agents that cause covalent cross-linking of apoproteins within HDL particles [100]; (6) stimulation by enrichment of cells with sterol or treatment of cells with 25-hydroxycholesterol [99,1(11]; and ( 7 ) o t h e r possible forms of regulation induced by peptide hormones [102,103]. Recently, it was found in experiments with enterocytes that Lovastatin, an inhibitor of HMG-CoA reductase, blocks the up-regulation of H D L binding induced by enrichment of the cells with exogenous sterol [104]. This suggests that the regulation of binding may be mediated by an endogenous isoprenoid. Proposed functions of H D L binding include the promotior, of sterol efflux from cells (see Section Ill-C) and the delivery of H D L CE to cells (see Section VI). An important focus of recent investigations in this area has been the identification of the structure responsible for H D L binding. Ligand blotting methods have been used to identify several proteins with high affinity for either apo E-free H D L or isolated apo A-I (the major protein of HDL) (Table II). These include a 78 kDa protein from membranes of sheep adrenal cortex [105], a II0 kDa protein from membranes of human fibroblasts, bovine endothelial cells, and other mammalian cells [106], a 110 kDa protein from the post-nuclear supernatant of J774 mouse macrophages [107], a 120 kDa protein (dissociable into 50 kDa and 30 kDa fragments by reducing conditions) from homogenates of human term placenta [108], 100 kDa and 120 kDa proteins from plasma membranes of rat and human liver [109], and a 58 kDa protein from membranes of rat ovaries [110]. A bifunctional cross-linking reagent has been used to demonstrate the presence of HDL-binding proteins (maximum molecular weight of approx. 100 kDa) on the surface of a variant of the Ob17 mouse adipocyte [111]. More recently, two binding proteins with molecular weights of 80-92 kDa (possibly differentially glycosylated forms of the same peptide) have been isolated in high yield and purity from another variant of the Ob17 cell [112]. Additional characterizations of the HDL binding proteins have shown that under reducing conditions, the binding activity is lost from the 120 kDa proteins

283 TABLE II HDL binding protehls isolated or identified by ligand bhming procedures Cell or membrane source Sheep adrenal cortex plasma membranes

Kd (nM) Not reported

Mol. wt. (kDa) 78

Reference [105]

Fibroblast, smtmth musclecell. endothelial cell. and J774 macrophage microsomal membranes

approx. 20

I I0

IIOH

J774 macrophagepost- 2.2 nuclear supernatant

I IO

[107t

Human term placenta

not reported

1211

tt0sl

Rat and human liver plasma membranes

not reported

)00 1211

[109l

Rat ovary lOOOOxg membranes

15 (#g protein/ml)

5S

ltt01

OBI7MTI8 mouseadipocytes

I tlO0

80 92

[1121

from liver and placenta, whereas the activity is retained in the 110 kDa proteins from fibroblasts, macrophages, and other sources, and in the 100 kDa and 58 kDa proteins from liver and ovary, respectively. The 100 kDa and 120 kDa proteins of rat liver also appear to be distin~,t from each other as judged by amino acid composition and immunoreactivity. For many of these proteins, it has been shown that the binding of HDL to the blots is not dependent on Ca z+, and that it changes in parallel with changes in HDL binding to whole cells when the latter is modified by hormones, growth status, or sterol enrichment [106,109,110,112]. As yet, there are no published data on the molecular biology or genetics of the putative HDL receptors. In contrast to the above view, Mendel et al. [ 113,114] have reported that the binding of HDL to fibroblasts and hepatocytes appears to be mediated by structures having very low molecular weights (16 kDa and Ic~,~ than 10 kDa, respectively), as indicated by sensitivit~ ~o ionizing radiation. Based on this and other results, they argue that HDL binding probably is not mediated by a 'classical' protem receptor, but rather by a set of heterogeneous low molecular weight structures. IV. E m u x of intracellular cholesterol to HDL

As summarized in F:g. 2, cellular unesterified cholesterol is dis:ributed among several compartments: these include the lysosomal, microsomal and plasma membranes. When ret.roendocytosis of HDL particles is not involved, efflux of cholesterol from intracellular

sites requires transport to the plasma membrane prior to transfer to extracellular HDL particles. A major difficulty in the analysis of cholesterol flux from intracellular sites is the selective tracing of each of these pools. IV-A. (Ttolesterol oxidase and the assessment o f plasma m o n b r a n e cholesterol

Over the last 15 years, cholesterol oxidase has been used extensively to study the distribution and transport of cellular eholesterc!. "[his enzyme (EC 1.1.3.61, usually isolated from species in the genus Brevibacterium, reacts with unesterified cholesterol to produce cholestenonc. Gottlieb first used cholesterol oxidase to study the distribution of cholesterol within human erythrocyte membranes [115]. He reported that cholesterol from intact ceils is not oxidized by the enzyme, and complete oxidation requires either hemolysis or exposure of the inner surface of the membrane to the enzyme. Subsequently, Lange and colleagues [116] modified the assay for the selective measurement of plasma membrane cholesterol in intact cultured cells. These authors reported that cellular cholesterol is not oxidized in physiological buffer. However, when the reaction is pcrfo,"mcd in h.~, ionic strength buffer (310 mM sucrose, 5 mM sodium phosphate), or when the cells are enriched with exogenous cholesterol, almost all the cellular cholesterol reacts with the enzyme [I 17]. Prior treatment of the cells with glutaraldehyde accelerates the reaction [116]. Based on these observations, the conditions usually employed for the assay include glutaraldehyde fixation and subsequent incubation of the cells for 45 rain with the enzyme in low salt buffer. When treated under these conditions, 80 to 95% of the cellular cholesterol is oxidized in fibroblasts, CHO cells, hepatocytes [i16] and erythrocytes [117]. Based on these rcsults, and assuming that cholesterol oxidase reacts selectively with the plasma membrane cholesterol, it has been concluded that 80 to 95% of cellular cholesterol is located in the plasma membrane. These values are much higher than those reported by Van Meer [118], who, by combining data obtained from ,~ubcellular fractionation and morphometric measurements~ estimated that less than 4(1% of cellular cholesterol is located in the plasma membrane of rat liver. However, the general concept and conditions of the cholesterol oxidase assay as developed by Lange and colleagues have been accepted and used by other investigators to study cholesterol trafficking in cells [83,119]. Various investigations have been conducted to further characterize the cholesterol oxidase reaction and validate its use as a probe for plasma membrane cholesterol. When it was first used to study the transbilayer movement ('flip-flop') of cholesterol within the

284 plasma membrane of human crythrocytes, a very rapid

tt/2 of 3 s was reported [117]. However, Brasaemle and colleagues [72] demonstrated that, under the conditions used by Lange and coworkers, extensive lysis of crythrocytc occurs. When lysis is prevented by the addition of magnesium, the measured tl/2 for flip-flop is much longer, ranging from 51) to 1311 min; the data also suggest that there is an asymmetric distribution of cholesterol across the membrane with 1(1 to 30% of the membrane cholesterol being located in the outer monolayer [72]. Thurnhofcr and colleagues [120] showed that the oxidation of cholesterol in brush border membrane vesicles from rabbit small intestine requires prior disruption of the membrane by a detergent such as Triton X-100; this is also the case for cholesterol present in phospholipid SUV. After glutaraldehyde fixation, incubation in low salt buffer and treatmcnt with cholesterol oxidase, the permeability of CHO cells is rapidly increased, as indicated by the release of intracellular K + which parallels the increased reactivity of cholesterol to the oxidase [121]. Similar results have bccn obtained in preliminary experiments in this laboratory when the release of 5tCr from J774 macrophages was monitored. The availability of cholesterol in membranes for oxidation is also influenced by the composition of the surrounding lipids. For exampie, in mixed lipid monolayers, cholesterol oxidase activity depends on the lateral surface pressure [122]. Sphingomyclin condenses the monolaycr and the denser lateral packing protects cholesterol from oxidation by the enzyme [122]. Taken together, the above results indicate that the cholesterol oxidasc assay cannot be used indiscriminately for the reliable quantitation of plasma membrane cholesterol. Because membrane disruption appears to be a prerequisite for cellular cholesterol oxidation, the large amount of cholesterol oxidized under the 'standard assay conditions' may well be a consequence of artifacts, such as enzyme penetration into the cells or rapid rearrangement of cholesterol among cellular membranes during the assay, rather than a selective reaction of the plasma membrane cholesterol with the enzyme. Therefore, estimates of plasma membrane cholesterol levels obtained with this assay should bc interpreted with caution and any changes in )he experimental conditions should be validated and justified. However, if used under well controlled conditions which dr> not alter the cell permeability, cholesterol oxidasc can bc useful for studying other aspects of cholesterol metabolism, such as transport from intracellular pools to the plasma membrane [121]. In addition, since minor differences in sterol structure may markedly influence cellular sterol metabolism, it is important that commercial preparations of radiolabelled cholesterol bc critically evaluated prior to use. Cholesterol oxidasc has proved to be a useful tool for

RER

SER

..'" "...

~.

CL~vOL

O--L]

•

NUCLEUS •

~P~A

~,

Fig. 5. G protein of vesicular stomatitisvirus and biosyntheticcholesterol (CHOL) are transported to the plasma membrane (PM) in separate vesicles. G protein vesiclesmay bud off from the rough ER (RER). move through the Golgi. and be sorted to the PM. Cholesterol-rich vesicles may be derived from smooC'l ER (SER) and proceed directly to the PM. LRVF: lipid-rich vesicle traction. From Urbani and Simoni[129].

validating the authenticity of such cholesterol preparations [123].

IV-B. Newly synthesized cholesterol The synthesis of cholesterol in mammalian cells occurs in the endoplasmic reticulum [124], peroxisomes [125], and possibly other internal membranes [126] (also see Section ll-C). The efflux of this sterol requires at least two distinct steps: (1) translocation to the plasma membrane; and (2) desorption to an extracellular acceptor such as HDL (see Section III-C). In studies devoted to the intracellular translocation step, it has been found that this transport is rapid (tt/2 = 10-60 min, depending on cell type), temperature-sensitive, energy-dependent and appears to be mediated by lipid-rich intracellular vesicles (reviewed by Billheimer and Reinhart, [127]). The transport is unaffected by the drugs monensin and Brefeldin A, implying that it does not involve transit through the Golgi apparatus [12g,129]. Thus, the mechanism (Fig. 5) is distinct from that for newly synthesized sphingolipids [130] and membrane proteins [131]. In studies addressing both intracellular translocation and desorption of sterol from the plasma membrane, Slotte and colleagues [83] reported that in stcrol-enriched fibroblasts and endothelial cells, native HDL 3 promotes effici,_'nt effl~tx of both plasma membrane and internal newly synthesized cholesterol, whereas HDL 3 that has been modified with tetranitromethane (to prevent specific binding of the lipoprotein to the cell surface) promotes efficient efflux of only plasma

285 membrane cholesterol. They proposed that the specific binding of HDL to cells stimulates the transport of internal cholesterol to the cell '~urface, thereby making it available for efflux. In support of this finding, it was reported that HDL specificahy stimulates the transport of newly synthesized sterol into a cholesterol oxidaseaccessible pool. Other studies from the same laboratory reported that the treatment of sterol-enriched fibroblasts with gamma-interferon (an inhibitor of cell proliferation) causes parallel increases in: (1) the specific binding of HDL to cells; (2) the presence of a 110 kDa membrane protein thought to be responsible for specific HDL binding; and (3) the ability of HDL to remove newly synthesized sterol from the cells [11)2]. It was concluded that cells respond to the inhibition of cell proliferation by the upregulation of HDL binding, which in turn permits more efficient elimination of unneeded internal cholesterol. This paper did not contain data on whether interferon alters the desorption of plasma membrane cholesterol or the efflux of biosynthetic sterol to acceptors that do not interact with HDL-specific binding sites. More recent work from the same laboratory indicates that in sterol-enriched human monocyte-derived macrophages, HDL stimulates the movement of newly synthesized stcrol into a cholesterol oxidase-accessible pool [132]. This translocation precedes the availability of the sterol for efflux, and correlates with the extent of HDL binding; translocation is not induced if HDL is treated with tetranitromcthane. Thus, the results are consistent with the hypothesis that the binding of HDL to the cell stimulates cholesterol transport from sites of synthesis to the plasma membrane. In these studies [132], the ~Hlabeled biosynthetic sterol was analyzed by reversephase thin-layer chromatography and found to consist mostly of material comigrating with desmosterol, with relatively little migrating as cholesterol. The two forms of sterol behaved similarly in efflux and cholesterol oxidase-sensitivity studies. It was also reported that in non-enriched maerophages, both native and chemically modified HDL stimulate the apparent internalization of plasma membrane sterol to a cholesterol oxidase-inaccessible pool. This result may be related to the observation by Tabas and co-workers [119] that LDL and acetyl LDL stimulate the internalization of plasma membrane sterol in maerophages. The hypothesis that the binding of HDL to cells regulates the transloeation of newly synthesized cholesterol ot the plasma membrane deserves further careful scrutiny. Several important issues remain to be resolved. Firstly, the identities of the labeled biosynthetic sterols obtained under various labeling conditions need to be examined carefully. The potential for complex product mixtures was demonstrated by Aviram and colleagues [132], as described above. That this may be a serious concern is suggested by the recent demonstra-

tion that thc efflux of plasma membrane zymosterol from fibroblasts to blood plasma is more than twice as efficient as the effiux of plasma membrane cholesterol [133]. The complete characterization of biosynthetic sterols is best done by reverse phase high-performance liquid chromatography [134]. Secondly, conclusions drawn from measurements of the accessibility of cell cholesterol to cholesterol oxidase must be viewed cautiously. As discussed in Section IV-A, the conditions used for the cholesterol oxidase assay often cause disruption of the plasma membrane. Finally, it has not been established that the transport of biosynthetic sterol reflects the transport of either lysosomal sterol or sterol originating in cytoplasmic CE inclusions. A relationship to lysosomal or inclusion sterol needs to be shown in order for studies on the efflux of biosynthetic cholesterol to have direct bearing on the process of athcrosclerosis. The importance of tiffs issue is underscored by the fact that in sterol-rich foam cells there is probably negligibie sterol synthesis.

IV-('. Lysosomal cholesterol IV-C. 1. Choh'sterol accunndation in lysosontes Both free and esterified cholesterol are delivered constantly to lysosomes by the internalization of exogenous particles and by the turnover of cellular membranes and organelles. A significant deposition of cholesterol in lysosomes has been observed in a number of pathological conditions such as lysosomal storage diseases and atherosclerosis. The defect in lya~somal processing of cholesterol which is responsible for cholesterol accumulation differs among these conditions, as does the form of the cholesterol which is deposited (i.e., free, ester or both). Two related conditions, CE storage disease and Wolman's disease [135] are associated with a genetic deficiency of aCEH which results in a very significant reduction in the hydrolysis of CE incorporated by lipoprotein uptake. Essentially all other aspects of sterol metabolism in cells from these patients are normal. In these patients, extensive lysosomal accumulation of CE occurs which provokes severe and ultimately lethal symptoms, in the foam cells present in atherosclerotic plaque, free and esterifled cholesterol accumulate in both cytoplasmic and lysosomal lipid dronlets [136]. The distribution of excess cholesterol between these two compartments depends upon the stage of development of the atherosclerotic lesion [137,138]. In early lesions, CE accumulates primarily in cytoplasmic droplets whereas in older areas of plaque increased proportions of both free and esterified cholesterol are localized within membranebound (i.e.. lysosomal) lipid droplets. This appears to be the case for foam cells derived from both monocyte/macrophages and smooth m,:scle cells. This pattern of intraccllular deposition of cholesterol in foam

286 cells has been observed in experimental as well as in human athcrosclerosis [30,136,139] and has stimulated extensive experimentation and speculation. Initially. it was proposed that insufficient lysosomal aCEH activity contributes to the lysosomal accumulation of cholesterol in foam cells [140], but a number of observations do not favor this hypothesis. For instance, lysosomal enzyme activities are markedly increased in atherosclerotic foam cells [141-143] and in lipid-loaded cells in culture [144]. In addition, the lysosomal accumulation of significant amounts of cholesterol in the unesterified form indicates that aCEH is active [145]. The recent development ot a tissue culture macrophage foam cell model in which lysosomal accumulation of both estcrificd and uncstcrified cholesterol can be induced should provide valuable information about the biochemical events involved in the progressive lysosomal lipid loading observed in vivo [42]. Preliminary studies suggest that the physical state a n d / o r the lipid composition of lipid droplets that recycle through the lysosome may markedly influence the trapping of cholesterol within these organcllcs ([42], and F.H. Mahlberg et al., unpublished observations).

11/-(22. The endo~Ttic pathway and lysosomal cholesterol metabolism Recent studies indicate that the cellular trafficking and utilization of lysosomal cholesterol may be influenced by the endocytic pathway through which the lipoprotein carrying the cholesterol is internalized. Tabas et al. [146] showed that human LDL and rabbit /3-VLDL are both internalized by mouse peritoneal macrophages via the apo B / E receptor. However, the rate of hydrolysis of/3-VLDL-CE is slower than that of LDL-CE, and even when equivalent masses of CE are hydrolyzed, LDL-derived cholesterol is less efficient in stimulating ACAT activity than fl-VLDL-derived cholesterol. Examination by fluorescence microscopy indicates that the endocytic vesicles formed following uptake of these lipoproteins differ both in morphology and cellular location [146]. These observations using lipoprotcins are consistent with other studies showing that cndocytic vesicles in macrophages exhibit a high degree of plasticity, with lysosomal morphology and cellular transport of metabolites being influenced by a number of factors such as cellular pH and the size of the ingested materials [147-149]. Although LDL and /3-VLDL differ in composition and size, the factor responsible for the observed differences in utilization seems to be apo E, since /3-VLDL containing mutant forms of apt) E elicits a response similar to that of LDL [146]. Thcsc results suggest that apo E acts as a signal for thc specific processing of lipoprotcin particles and th,'. effici,:nt delivery of cholesterol to A C A T (also see Section IV-El. This concept is supported by the studies of Adclman and St. Clair [150] who compared the

efficiencies of pigeon VLDL and rabbit /3-VLDL in stimulating ACAT activity in mouse peritoneal macrophages. In this system, ACAT activation is higher when cholesterol is delivered in apo E-rich rabbit /3-VLDL compared to pigeon VLDL which lacks apo E. Although the studies described above on lipoproteinlysosome trafficking are still limited, they do clearly indicate a selectivity in the utilization of lysosomal cholesterol by ACAT. Since there is almost always a reciprocal relationship between efflux of cellular cholesterol and ACAT activity, it follows that a selective sorting of cholesterol, mediating by apolipoproteins, may inflt~nee the availability of lysosomal cholesterol for efflux.

IV-C.3. Transport of lysosomal cholesterol to cellular membranes The transport of cholesterol from lysosomes to other cellular membranes involves at least three steps: (1) transfer to the lysosomal membrane from an intralysospinal site of hydrolysis, (2) translocation to the cytosol; and (3) transport through the cytosol to other cellular membranes. The mechanisms involved in each of these steps are not yet identified. Efficient transfer of cholesterol from the site of hydrolysis to the lysosomal membrane may be achieved by diffusion since the distances are small and no competing membrane systems are present [151]. However, the availability of free cholesterol for movement to tl,e lysosomai membrane could be influenced by the presence of lipid stores within the lysosome. For example, a cholesterol trapping phenomenon may contribute to the deposition of cholesterol in the lysosomes of foam cells within the atheroscletotic lesion [137,138]. Preliminary cell culture studies have indicated that cholesterol can be solubilized within a lipid phase present in model foam cells, thus limiting the transport of the cholesterol out of the lysosomes [42]. Only a few studies have specifically addressed the transport of cholesterol from lysosomes to other cellular membranes. Recent studies demonstrate that transport of LDL-cholesterol from lysosomes to the plasma membrane is very rapid. Kinetic studies with FuSAH rat hepatoma cells indicate that there is a lag of some 40-50 min between the lysosomal hydrolysis of LDLCE and the appearance of the resulting free cholesterol in the extracellular medium [152]. Other experimental approaches with different cell systems have provided similar findings. In CHO cells, data obtained using a modified cholesterol oxidase assay show that LDL-derived lysosomal cholesterol reaches the plasma membrane within 2 min [121]. In MAI0 Leydig cells, LDL-derived cholesterol moves to the plasma membrane with a tt/2 about 32 min [153]. The rapid movement of lysosomal cholesterol to the plasma membrane most likely reflects a facilitated transpor: mechanism

287 rather than an unmediated diffusion process. Facilitated transport may involve apolipoproteins, sterol carrier proteins, sphingomyelin lamellar bodies [154] or vesicular transport of the type described for the movement of newly-synthesized cholesterol. Niemann-Pick Type C (NPC) disease is characterized by a mild accumulation of sphingomyelin and the deposition of large amounts of free cholesterol in lysosomes. It has been revealed recently that this disorder is not associated with a defect in phospholipid metabolism but rather is specifically linked to a defect in the transport of cholesterol out of the lysosomes [155,156]. Since this discovery, animal and tissue culture models of NPC have been used extensively to further characterize the mechanisms of lysosomal cholesterol transport. The transport of LDL-derived cholesterol out of the lysosomes of NPC fibroblasts is sluggish, and the regulatory responses normally elicited to maintain cellular cholesterol homeostasis are delayed [157,158]. However. the transport of newly synthesized cholesterol in these cells is normal, and the usual regulatory responses (i.e., ACAT stimulation and down regulation of HMG-CoA reductase and LDL receptor activities) are induced by exogenous mevalonate and 25-hydroxycholesterol [158]. The cellular function impaired in this disease specifically concerns the transport of free cholesterol from the lysosomcs to other cell membranes, resulting in extensive accumulation in the lysosomes [155,159]. Interestingly, the abnormal metabolism of LDL-derived cholesterol in NPC fibroblasts can be reversed by the addition of 2% dimethyl sulfoxide to the culture medium [160]. These data indicate that membrane permeability or other factors affecting cholesterol solubilization in the cell may be critical in the cellular transport of cholesterol. Alteration of lysosomal cholesterol transport has also been obtained after chemical [161,162] and genetic [163] manipulations of cells and these approaches may provide valuable tools for future studies. It has been proposed that the translocation of internal pools of r.ewly synthesized cholesterol to the plasma membrane is catalyzed by the interaction of HDL with its specific receptor (see Section lit-El. However, there is no evidence for a common mechanism of transport of cholesterol among the various intracellular compartments, and the limited data currently available do not indicate a role for HDL binding in lysosomal cholesterol movement. Studies by Johnson and colleagues [152], using both hepatoma cells and skin fibroblasts, have shown that the short transit time for the movement of lysosomally generated free cholesterol to the plasma membrane is independent of the type and concentration of a number of different extracellular acceptors, including PC SUV, HDL3 or DMS-HDL.a. Neither the vesicles not the dimethyl suberimidate (DMS) cross-linked HDL bind to the HDL receptor

TABLE I11 Efflrt~ of merrlhrane and lysosomal cholesterol from intact cells to immunopurified subfractions of human HDL

Experiments were performed as dc~ribed by Johnsonet ::!. [15--.,]. Membrane cholcster()lwas labeled by incubatingcells 1-2 days in a medium that contained lipoprotein-deficient ~erum and [14C]cholesterol. The effluxof this tracer is representative of sterol dcsorption from the plasma mctnbranc. Lysosomalcholesterol was labeled by incubating cells with reconstituted LDL containing [31-1]cholesteryl oleate. After labeling, cells were incubated with LP-AI or LP-AI/A-IIat a concentration of 50 tzg phospholipid/ml. After 4 h incubation,cells and media were recovered and analyzed to determine percentage efflux of [l"aCl and l~l-I] free cholesterol. Each value is the mean+_I S.D. of at least three determinations in a ~ingle experiment. ('t:ll

Acccptor

Cholesterolefflux membrane lysosomal Effluxratio (%/4 h) (%/4 h) (Lysos./ Membrane) FuSAH LP-AI 10.h+_ll.h 9.6+_0.6 0.91 rat hcpatoma LP-AI/AII IO.1+-t).8 8.8+(I.5 I).87 GM34~S human fibroblast

LP-AI 3.0+-O.1 5.9+_11.4 L P - A I / A I I 3.2+_0.3 6.2+_11.5

1.9¢D 1.94

Rabbit smooth muscle

LP-AI 5.2 _+0.5 LP-AI/AII 8.I +_0.7

11.92 11.811

4.8 _+(I.4 6.5 _+0.8

[ 1(~)]. In addition, no differences in transport of lysosomal cholesterol occur in cells exposed to HDL subclasses having either apo A-i alone or in combination with apo A-ll (Table lilt. The influence of specific FiDL-apolipoproteins on the utilization of lysosomal cholesterol has also been investigated using a J774 macrophage foam cell model in which efflux of lysosomal and membrane cholesterol was monitored in the presence of either phospholipid liposomes or purified apolipoproteins (human apo A-l, apo A-II or apo C) complexed with phospholipids [164]. As shown in Table IV, the ratio between effiux of lysoso-nal cholesterol and efflux of membrane cholesterol is independent of the type and concentration of acceptor particles. This demonstrates that the apoproteins A-I, A-II and C's do not selectively stimulate efflux of lysosomal cholesterol relative to plasma membrane cholesterol. Although the studies described above have failed to demonstrate a specific regulatory role for HDL in the metabolism of cholesterol originating in the lysosomes, HDL may play such a role in other cell types or in the regulation and translocation of other pools of cellular cholesterol (see Section IV-D). IV-D. Cytoplasm& cholesteryl ester

A large number of studies have demonstrated that different cell types in culture are capable of clearing cytoplasmic esterified cholesterol if an acceptor of free

288 TABLE IV Efflt~ of (vsosomal and membrane cholesterol from J774 macrophages IO reconstitttted tlDL particles

J774 macrophages were prepmcd as described previously[42]. Briefly. J774 cells were plated on glass covcrslips and membrane cholesterol was radiolabcled for 2d with [4-14C]cholesterol.Confluent monolayers were then loaded for 2 h with sonicated droplets containing [311]cholesterollinoleate [42]. Cells were then washed and incubated with RPMI medium containing lt7, BSA. 100 p.g/ml oleic acid and various concentrations of acceptors. The acceptors were either egg phosphatidyleholinc (PCI vesicles or recombinant particles of egg PC and purilicd haman apt~lipoproteinsprepared by the eholale dialysis technique !9], After 9 It, cells and media were harvested and analyzed to determine the cfflux of [14C] and ['~H] free cholesterol. Results are expressed as the ratio of the fraction of lysosomal free cholesterol tt) the fracli:~l of membrane eholesle,'.l removed from the cells over 9 h. (n = number of concentrations of acceptors tested, each of them being determined in four dishes). Acceptor

(kmeentration range (/xg PC/ml)

Apo A-I/P(' Apo A-II/PC Apo C's/PC PC vesicles

25- I 5tit) 25-1500 25-1500 10[1-3ill]0

Cholesterol emux ratio (lysosomal/membrane) 0.41 ± 0.03 (n = 9) 0.40 ± o.n2 (n = 9) {1~2_+0.03 (n = 9l 0.41 _+0.02 (;1 = 4)

cholesterol is present in the extracellular medium. Most of these investigations have used exogenous aceeptors which contain either no cholesterol or reduced levels of cholesterol. Such acceptors are the p h o s p h o l i p i d / apoprotein complexes remaining in lipoprotein-deficient serum, partially delipidated lipoproteins, recombinant particles assembled from H D L apoproteins and phospholipid, or phospholipids alone. U n d e r these conditions a free cholesterol gradient is estab!ished between cells and acceptors leading to the net movement of free cholesterol from the cells (see Section Ill-D), which results in the diversion of cholesterol from the C E cycle and a subsequent reduction in C E stores. Although it is frequently accepted that native H D L can also induce the clearance of C E in all cells, the actual data supporting this generalization are limited and based large':,, on studies using mouse peritoneal m~rcrophages. It is a g r e e d that CE-Ioaded mouse pcritoncal macrophages decrease their ester stores when incubated with H D L [165-167]. In contrast to mouse peritoneal macrophages, other cell types a p p e a r to be more resistant to H D L - m e d i a t e d clearance of CE. In Fu5AH rat h e p a t o m a cells, which accumulate large pools of C E which can be depleted by growth in the presence of a p o l i p o p r o t e i n / p h o s p h o l i p i d complexes [168], addition of native H D L to the incubation medium results in an additional accumulation of esterifled cholesterol rather than clearance [167,169]. In comparative studies using mouse peritoneal macrophages and the J774 mouse m a c r o p h a g e cell line, Bernard and colleagues [167] d e m o n s t r a t e d that the

same preparations of either total H D L or H D L 3 that stimulated clearance from the peritoneal cells were unable to produce significant reductions in the C E content of the J774 cells. This inability of H D L to efficiently clear the C E from the J774 cells could not be linked to e i t h e r the physical state of the CE, the m e t h o d of loading the cells or the origin and t r e a t m e n t of the HDL. Based on the currently available data it can be suggested that the ability of H D L to mediate cellular C E depletion is not a generalized p h e n o m e n o n and that the mouse peritoneal m a c r o p h a g e may be more responsive than other celt types. Such a conccpt is supported by the morphological observations of Pitas and co-workers [170] who d e m o n s t r a t e d that foam cells obtained from lesions in rabbits retain C E molecules much longer than mouse peritoneal m a c r o p h a g e s w h e n both types of cells are exposed to H D L A further indication that mouse peritoneal m a c r o p h a g e s may be particularly vigorous in their ability to clear cytoplasmic C E was o 0 t a i n e d by M a t h u r and colleagues [166] who c o m p a r e d the clearance of esters from rabbit and mouse m a c r o p h a g e s and observed a significantly more rapid depletion of these lipids from the mouse cells. Thus, the extensive deposition of C E in both the maerophage- and smooth muscle-derived foam cells of atherosclerotic plaque may be, at least in part, a reflection of sluggish C E clearance that occurs in the presence of H D L or subclasses of HDL, and mouse m a c r o p h a g e s may not be the most representative exp e r i m e n t a l syst,~m for the study of foam cell C E metabolism. The clearance of c~,,toplasmie pools of esterified cholesterol involves a n u m b e r of a p p a r e n t l y independ e n t steps. Since C E molecules are not released intact from peripheral cells, the first step in ester clearance is hydrolysis to free cholesterol (see Fig. 2). If the free cholesterol g e n e r a t e d by hydrolysis is not removed efficiently from the C E cycle it u n d e r g o e s re-esterification resulting in the turnover of C E without a net change in cellular C E mass [165,167,171]. Two different models have been suggested for the subsequent removal of free cholesterol g e n e r a t e d from stored CE: (1) the translocation of the liberated free cholesterol to the plasma m e m b r a n e where it u n d e r g o e s effiux to H D L [167,168,172]; or (2) the transfer of intracellular free cholesterol to H D L that has been internalized and is then released through a process of retroendocytosis ([173], see Section 1V-F). A l t h o u g h all cells apparently will clear C E if a sufficiently large gradient is produced by exposure t s cholesterol-depleted acceptors, the clearance to native H D L or H D L subclasses is more complex since only a modest cholesterol gradient is produced upon incubation of H D L with most cells (see Section ill-D). Thus, for H D L to stimulate C E depletion it should both enhance esterified cholesterol conversion to free cholesterol and influence the intra-

289 cellular location of free cholesterol pools. How HDL participates in, and modulates, these metabolic events remains a subject of intensive investigation. An increase in the activity of the neutral cholesteryl ester hydrolase (nCEH) could lead to enhanced HDLmediated clearance of cellular CE. lii ~ome macrophages [174,175] and endocrine cells [176,177] the activity of nCEH has been shown to be hormonally regulated and linked to cAMP. levels. This enzyme appears to be ide,-tical to the hormone-sensitive lipase that has been extensive:y characterized in adipose tissue [178]. There are conflicting reports on the importance of hormonal regulation of nCEH in both the liver [178-1801 and in ~fnooth muscle cells [181,182] (for a review of CE hydrolases, see [45]). A primary mechanism by which H D L induces CE clearance may be promoting an increase in cAMP and thus up-regulating nCEH activity. HDL may increase cellular cAMP levels either through an interaction with specific HDL receptors or through less specific interactions of the lipoprotein, apoprotein or certain HDL lipids with the plasma membrane. Either specific or non-specific cellH D L interactions could trigger second messenger pathways resulting in CE clearance. Although HDL mediation of cAMP levels is an attractive hypothesis, currently there is no experimental evidence to demonstrate a relationship between H D L and the cAMP stimulation of CE hydrolysis. To obtain net esterified cholesterol clearance, even under conditions where CE hydrolase activity is increased, it is still necessary to remove the liberated free cholesterol from the CE cycle. Studies in J774 mouse maerophages have demonstrated that the exposure of these cells to cAMP produces a 2-3-fold increase in the rate of CE hydrolysis; however, this increase in hydrolysis is accompanied by a proportional increase in A C A T activity if the cells are incubated in the absence of a cholesterol acceptor [183]. Thus, the cycle simply turns over faster and there is no net change in CE content [183]. Although the mechanism(s) responsible for the intraceUular transport of cholesterol has not been established definitely (see Sections IV-B and -C), the diversion of free cholesterol from the CE cycle may also be linked to cAMP levels. Studies using J774 cells have demonstrated that increased concentrations of free cholesterol produced by the hydrolysis of CE when ACAT is inhibited are not as efficiently removed as excess free cholesterol generated upon the up-regulation of nCEH by cAMP [183]. Even more convincing evidence for a dual role of cAMP is the observation that exposure of J774 cells to cAMP and H D L stimulates both nCEH activity and net cholesterol clearance whereas P'e phorbol ester, TPA, produces a similar increase in hydrolase activity without the accompanying depletion of cellular cholesterol [184]. These observations are consistent with a

model in which cAMP both increases hydrolase activity and the subsequent efflux of free cholesterol. The enhanced efflux could result either from an increase in rctrocndocytosis [185,186] or from the enrichment of cholesterol in the plasma membrane, which in turn would result in the net movement of free cholesterol from cells to the H D L acceptor by an aqueous diffusion mechanism (see Sections llI-C and -D).

IV-E. Role of apolipoprotein E h~ hztracelhdar d~olesterol remot'al The hypothesis that apolipoprotein E plays a role in reverse cholesterol transport is based on the following observations: (I) apo E shares common receptors with al:o B. both in peripheral tissues and in the liver [187], (2) HDL~. a CE-enriched lipoprotein containing apo E. is often present in cholesterol-fed animals [188], (3) the enrichment of HDL 3 with CE results in the binding of apo E to the particle [189], (4) smooth muscle cells [ 190,191 ] and macrophages synthesize and secrete apo E [192,193]; and (5)cholesterol-loading of macrophages results in an increase in apo E synthesis and secretion [192,194]. These facts, taken together, form the basis of a model in which the secretion of apo E from cells facilitates the release of excess cholesterol from peripheral cells to HDL, and that subsequently apo E directs the return of the cholesterol-enriched HDL to thc liver (see Fig. 6). Since the first observation that macrophages have the capacity to synthesize apo E [192], such synthesis has been demonstrated in a variety of cell types in different tissues, including smooth muscle cells [190,191], kidney [195], endocrine cells [195,196], glial cells [197], as well as the livcr [195]. The first studies on mouse macrophages by Basu and co-workers [1921 revealed that the production of ado E increases by 3-8fold if the macrophages are enriched with cholesterol. This upregulation of apo E synthesis and secretion is associated with increases in cell free cholesterol content [198] and is a reflection of an increase in apo E m R N A [194,199]. The link between cell cholesterol accumulation and increased apo E secretion prompted the obvious speculation that macrophages are capable of releasing a nascent lipoprotein that carries excess cell cholesterol with it. However, it was soon demonstrated that the secretion of apo E and the efflux of cholesterol represent two independent phenomena which can be dissociated [200]. Thus, apo E release from macrophages is inaintain~d in ~he absence of serum, under which conditions there is no efflux of cholesterol [200]. Conversely, monensin effectively blocks apo E secretion and has no effect on the efflux of cholesterol from cells [200]. in the absence of exogenous lipoproteins a large fraction of the apo E that is released from either macrophages, apo E-transfected

290

-'IF

D "< eE

~ ' ~

~

LIVER Fig. 6. Possible rolcs of apolipoprotein E in reverse cholesterol transport. ( l ) Apo E on lipoprotein particles serves as a ligand for

receptor-mediated uptake of the particlesby peripheralcells. (2) The presence of apo E on the particle serves to target the internalized lipoprotcin to specificclassesof lyosomes.(3) In cells that synthesize apo E the protein participates in the intracellular transport of cholesterol, regulating its transport to different intraeellular pools such as the plasma membraneand ACAT substrate pools. (4) Free apo E can be secreted from cells and subsequentlyinteract with the cell plasma membranefirst acquiring phospholipid,after which the apo E/phnspholipidcomplexcan serve as ~cholesterol acceptor. (5) Apo E may be secreted as part of a nascent~lipoproteinconsistingof apoprotein and phospholipid, after which it would acquire cholesterol by provokingcellular cholesterol efflux. Through the action of LCAT the nascent particle is converted to a mature lipoprotein. (6) Plasma HDL acquires both cholesterol and apo E through the interaction with peripheral cells. After conversionof the excessfree cholesterol to eholesteryl ester through the action of LCAT, the cholesteryl ester-rich particle (HDL(.) can be removed by the liver usingthe apo E as a ligand. See text for furtherdetails. L-cells or smooth muscle cells has a density < 1.21 g / m l [191,201,202], indicating that the nascent particle contains lipid, presumably phospholipid. The apo Econtaining nascent particles produced by macrophages have been shown to be primarily discs [201]. further confirming the association of secreted apo E with phospholipid. It has not been established if this particle is assembled within the cell and is then released with phospholipid, or if free apo E is secreted and subsequently acquires cell phospholipid. The apo E/phospholipid particle that is produced by macrophages may play a number of roles in reverse cholesterol transport (see Section I1-B). One possibility is that the nascent particle serves directly as an aeeeptor of cell cholesterol (Fig. 6). The small size and high phospholipid content of the lipoprotein would make it an efficient acceptor of cholesterol. In addition, its secretion from cholesterol-loaded cells would result in a high concentration of the acceptor in the unstirred water layer surrounding the cells, an environment that is particularly important in influencing the rate of cholesterol cfflux from cells (see Section Ill-C). However, the ability of the particle to function as an efficient acceptor within the unstirred water layer would be influenced by the presence of apo E receptors on the cell. The presence of receptors would result in the

re-uptake of the lipoprotein and thus limit the net removal of cellular cholesterol. One of the most convincing roles proposed for apo E in the reverse cholesterol transport pathway is in the remodeling of HDL (see Section II-A) and the provision of a ligand which is recognized by receptors in the liver [189,200]. This model is supported by substantial in vitro data. The incubation of canine serum with cholesterol-loaded macrophages results in the enrichment of HDL with CE accompanied by an increase in the size of the lipoprotein and the association of apo E with the particle [203]. These apo E-containing H D L t / H D L c lipoproteins are only formed if apo E is available from other lipoproteins or by secretion by macrophages [203]. LCAT is necessary for the conversion of smaller HDL particles to the larger apo E-containing HDLc, and the binding of apo E is linked to the expansion of the CE core of the lipoprotein particle [203]. Thus, the following sequence of four events can be proposed in this model: (1) subfraction of HDL consisting of small, phospholipid-rich particles acquires cholesterol from cholesterol-loaded cells within the interstitial space. (2) Through the action of LCAT the excess free cholesterol derived from the cells is converted to esterified cholesterol and this CE contributes to the expansion of the lipoprotein core. It can be anticipated that the continued removal of cell free cholesterol by these particles requires the conversion of the free cholesterol to esterified cholesterol to maintain a cholesterol gradient (see Section III-C and -D). However, LCAT would also deplete the H D L phospholipids which then have to be replenished from either other iipoprotein pools or by efflux of phospholipids from cells. (3) The expansion of the core of the lipoprotein results in changes in the surface structure of the particle that favors the association of apo E. (4) The apo E on the HDL then serves as a ligand for the uptake of the HDL by the liver. It should be noted that the regulatory role proposed for apo E in the reverse cholesterol transport pathway may also be operating in other tissues for the redistribution of cholesterol between cells [197].

IV-F. HDL retroendocytosis and sterol flux Data from various sources indicate that in some types of cells a portion of HDL that binds to the plasma membrane is endocytosed and then resecreted (retroendocytosed) without undergoing hydrolytic degradation. Two proposed functions of this process are removal of sterol from macrophage foam cells and delivery of CE to hepatocytes. Two types of observations support the reality of HDL retroendocytosis. One consists of electron-microscopic studies demonstrating that HDL tagged in various ways to permit its visualization is endocytosed and

291 then delivered either to an endosomal or trans-Golgi compartment rather than to lysosomes [173,185,186, 204]. In rat macrophages, HDL-ferritin and HDL-horse radish peroxidasc conjugates ultimately are re-packaged into what appear to be secretory vesicles [204]. The second type of result is obtained by incubating cells with HDL that contains tasl-labeled apoproteins, and then monitoring the fate of the label during incubation in an unlabeled chase medium, in hepatoma ceils, a large fraction of cell-associated tZ~l-l-tDL is delivered to a trypsin-inaccessible compartment and then released from cells in a form that is precipitable with triehloroaeetic acid [186,205]. The release of acid preeipitable H D L has also been observed with human monocyte-derived macrophages [206], although d r a m and colleagues [172] could find no evidence for the entry of H D L into trypsin-inaccessible compartments in mouse peritoneal macrophages. The release of exogenously derived H D L from hepatoma cells is inhibited by energy poisons [186] and monensin [205], consistent with active secretion of the lipoprotein, rather than simple desorption from the cell surface. Sehmitz and colleagues [207] have reported that HDL is not retroendocytosed properly in monocytes from patients with the Tangier lipid-storage disease, but rather is retained and degraded in lysosomes. This may contribute to the low plasma level of HDL and the abnormal storage of lipids in these patients. Biochemical demonstrations of HDL retroendocytosis, which rely on a low temperature pulse with HDL followed by rinsing and .hen a chase at physiological temperature, imply that at least a portion of the HDL traversing the retroendocytic pathway first binds to the cell surface. This binding probably is not mediated by the LDL receptor, since normal human HDL (which is nearly devoid of apo B and apo E) does not bind to this receptor [208], yet it does undergo retroendocytosis [205]. Is the binding mediated by the high-affinity HDL-speeifie binding site(s) described by Fidge, d r a m , Sehmitz, and others? Possibly, but this does not seem to be well established. Retroendocytosis and specific H D L binding may be related in Hep G2 cells, since both increase when cells are enriched with sterol [205]. Similar data are lacking in other cell systems. There are no clear demonstrations that binding and retroendoeytosis have similar dependencies on H D L concentration, or can be inhibited in parallel. Reported modifications of H D L during retroendocytosis include: (1) increased particle size (as indicated by increased precipitability with dextran sulfate) and the possible acquisition of apo E during interaction with human macrophages ([206] and see Section IV-E); and (2) the generation of smaller, apo E-depleted particles during transport through Fu5AH rat hepatoma cells [186]. These observations are consistent with the proposed functions of retroendocytosis in macrophages

and hepatocytes, respectively, but do not establish them with certainty. As yet, there are no careful quantitative comparisons between: (1)predictions of lipid movemerit either into or out of cells based on the measured flux of HDL through a retroendocytic pathway; and (2) the measured changes in either cell lipids or HDL lipids that occur during interaction of HDL with cells. It should be possible to obtain correlations of this sort, if retroendocytosis is a significant factor in the movement of sterol between HDL and cells. V. HDL subfractions and efilux of cellular cholesterol

Using apolipoprotein-specific immunoaffinity chromatography to remove selected lipoproteins from blood plasma, Fielding and Fielding [209] found that most of the cfflux of cholesterol from fibroblasts to the plasma can be attributed to a minor subfraction of HDL that contains LCAT, apo A-I and apo D, but no apo A-II. In more recent studies, which relied on two-dimensional gel electrophoresis (agarose gel clectrophoresis followed by nondenaturing gradient polyacrylamide gel eleetrophoresis), H D L particles that ~ppear to comprise this subfraction have been isolated and characterized [16,211)]. The subfraetion appears to consist of three particle types (designated LP-A1 pre-/31, pre-/32, and pre-/33) that resemble each other in containing apo A-i (but no apo A-II) and in having ore-/3 mobility in the first gel dimension. The particles differ in size, with apparent particle weights of 71 kDa, 325 kDa, and greater than 325 kDa, respectively. In addition, the largest of the particles, LP-A1 pre-/33, contains immunoreactive LCAT, CE transfer protein and apo D. Castro ~md Fielding [210] reported that after incubation c ~ plasma for 1 min with fibroblasts containing [3H]cholesterol, a large fraction (approx. 39%) of the labeled sterol in the plasma is associated with LP-A-I pre-fll. Incubation for an additional 2 min in the presence of unlabeled cells leads to the transfer of labeled sterol into LP-A! pre-/32, as well as into the bulk (alpha-migrating) H D L fraction. In incubations extending 3-15 min, labeled CE appeared in LP-AI pre-/33 and alpha-HDL [16]. It appears that particles in the minor ore-/3 subfractions of HDL are highly efficient in removing cholesterol from cells. These results are consistent with a pathway for the removal of cell sterol, involving initial transfer to LP-AI pre-/31, followed by transfer to LP-AI pre-/32, and then on the sites of LCAT-mediated esterification, (LP-AI pre-/33 and alpha-HDL) (Fig. 7). It is not yet clear to what extent efflux through this pathway is balanced by influx from pre-B HDL particles. Nor has it been determined whether this pathway is quantitatively more intportant than direct c~flux :.~ ;?~e much higher capacity alphaH D L fraction.

292

PreB3HDL F C ~ C E

FC "~-FC

I

FC

!o

FC ~

/ FC

-.

~ r e ~ l -

aHDL HDL

Cell sudace Fig. 7. Genesis of HDL in plasma from cell-derived cholesterol. Human plasma was incubated with cells containing labeled cholesterol and then analyzedby two-dimensionalgel electrophoresis.The results suggestthat pre-,61-HDLparticles are the initial acceptor of cell cholesterol. This is followed by the indicated sequential m(wement to mature alpha-HDL particles. See text for details. From Francone and Fielding[240]. The role of H D L apolipoproteins in controlling intracellular cholesterol movement and efflux has not been clearly established. Early studies of DeLamatre and colleagues [211] demonstrated that reconstituted particles containing phospholipids and either apo A-I or apo C differ in their ability to stimulate cholesterol efflux from a variety cf cells, including J774 macrophages and human fibroblasts. In contrast, this differential effect was not observed with other cell types, such as Fu5AH rat hepatoma cells and HEPG2 cells. Stein and coworkers [212] have shown that proteoliposomes containing either apo A-I, apo A-IV or apo E are equally efficient in inducing cholesterol efflux from fibroblasts, whereas apo C liposomes are less efficient. More recently, using a J774 macrophage foam cell model, it has been shown that particles containing apo C's, apo A-II or apo A-1 stimulate efflux of both lysosomal and membrane cholesterol; these apolipoproteins do not exert any selective effect on the efflux of either meml:rane or lysosomal cholesterol ([164] and Table IV). However, the particles containing apo A-I seem to be more efficient in stimulating efflux from both pools [164]. In contrast, Barbaras and colleagues [213] demonstrated with a line of cultured preadipose cells (OB17) that immunopurified LpA-I or proteoliposomes containing only apo A-I or apo A-IV [214] reduce cell cholesterol content, whereas proteoliposomes containing only apo A-II or apo E and immunopurified LpA-I/A-II particles do not induce any cellular cholesterol depletion. These results have been attributed to a difference in the abilities of apo A-! and apo A-II to stimulate cholesterol efflux. These investigators postulated that cellular cholesterol efflux is mediated through the interaction of the apoproteins with a specific H D L receptor (see Section Ill-El, and that although both apo A-I and apo A-II are recog-

nized as ligands, apo A-II is an antagonist of apo A-I [213]. However this model has to be reconciled with the following results: (1)proteoliposomes containing a r,tixture of apo A-I and apo A-II are as efficient as LpA-I and as apo A-l-containing liposomes in reducing the cholesterol content of OBI7 cells [213]; and (2) HDL 3 is an acceptor of cellular cholesterol in OBI7 cells [215] and fibroblasts [216,217], even though these particles are relatively rich in apo A-II [15,218]. In contrast, Johnson and co-workers have shown that LP-AI and L P - A I / A I I have very similar abilities to remove radiolabeled cholesterol from human fibroblasts and rat Fu5AH hepatoma cells (Table III). The role of each of the H D L apolipoproteins in the regulation of cholesterol metabolism and movement still remains to be clarified. The discrepancies in the reported results strongly suggest that responses may vary depending on the cell type being studied. This may be a reflection of differences in the distribution and organization of cholesterol within the various cells.

VI. Selective uptake of cholesteryl ester from HDL to cells Numerous investigations have examined the possibility of direct, non-endocytie transfer of CE from H D L to cells. In an early study, Quarfordt and coworkers [219] perfused the apo A-l-rich fraction of rat H D L through rat livers and found that the protein and CE of the lipoprotein were taken up non-preferentially. This early report contrasts sharply with several subsequent reports of the highly preferential transfer of CE from H D L to various tissues and cells. Glass and colleagues [220] injected rats with H D L containing t2Sl-tyramine cellobiose-labeled aim A-I and [3Hkholesteryl linoleyl ether (non-hydrolyzable tracers for apoprotein and CE, respectively), and found that organs active in cholesterol metabolism (liver, adrenal and ovary) absorb the CE tracer 2 to 7-fold more efficiently than the apoprotein tracer. A similar 'selective uptake" of H D L CE has been demonstrated in cultures of hepatocytes and adrenal cells [221], Currently, there are reports of selective CE uptake by a wide variety of other cells, including macrophages [222] fibroblasts [223], adipocytes [224], and hepatoma cells [85,225J. Support for the biological significance of selective uptake of CE comes mostly from studies with adrenocortical cells. In these cells, adrenocorticotropin (ACTH) stimulates selective uptake several fold, in parallel with the stimulation of steroidogenesis [221,226]. In studies with ACTH-treated rat adrenal cells, Gwynne and Mahaffee [227] found that H D L induces a substantial stimulation of steroidogenesis, which is mostly accounted for by the conversion of selectively absorbed CE to steroids. In ACTH-stimu-

293 lated YI-BSI adrenal tumor cells, selective uptake results in the net delivery of CE mass to cells, and, under appropriate conditions is accompanied by an increase of 0.1)8 g / m l in the modal density of HDL [223]. These findings are consistent with the net transfer of CE to the cells, accompanied by shrinkage of H D L particles. Similar, although less dramatic, changes in HDL density or size have been reported during the perfusion of HDL through rat livers [228], and during the incubation of HDL with adipocytes [224], and rabbit hepatocytes [229]. The mechanisms associated with selective uptake of CE have been studied in a number of cell systems. As described above, ACTH stimulates selective uptake in adrenocortical cells. In other cell types, selective uptake is increased by depletion of cell sterol [230,231]. These effects suggest regulation (and thus some form of mediation) of selective uptake. Using a variety of cell types in culture, Pittman and colleagues [223,232] found that selective uptake does not depend on cellular metabolic energy, the LDL (apo B / E ) receptor, the endocytosis of HDL, or on any specific apoprotein of HDL, although uptake is enhanced by the presence of apoproteins that result in smaller, more highly curved H D L particles. Thus, the uptake appears to involve the passive transfer of CE from H D L into or through the plasma membrane by a mechanism that probably does not depend on apoprotein-specific binding of H D L to the cell surface. The lack of importance of HDL binding is supported by whole-animal studies showing that the inhibition of H D L binding by apoprotein cross-linking has no effect on the clearance of H D L CE from blood [233]. The fact that isolated bovine adrenal membranes and canine adipocyte plasma membranes exhibit selective uptake suggests that cell membranes can accomplish selective uptake without assistance from cytoplasmic components [234,235]. Plasma CE transfer protein (CETP) has been reported to mediate the uptake of H D L CE by Hop G2 cells [236]. However, Rinninger and Pittman [237] presented data suggesting that this apparent mediation ol CE uptake resulted from the CETP-dependent transfer of H D L CE to apo B- and E-containing lipoproteins (that are secreted by the Hep G2 cells) followed by endocytosis of these particles. The removal of H D L phospholipids (by treatment with phospholipase A 2) enhances the selective uptake of CE by rat hepatocytes in culture [238]; this suggests that hepatic lipase in the liver and similar lipases in steroidogenic glands may facilitate the selective delivery of H D L CE to these organs. However, these lipases are not essential for selective uptake, as this process also occurs in fibroblasts and other cells lacking the abil;ty to lipolyze extraceUular phospholipids. Selective uptake by YI-BSI adrenocortieal cells involves the movement of CE into two cellular pools, one subject to removal by incubation with unlabeled

HDL, and the other not subject to removal [239]. The quantities of CE tracer in the two pools are correlated with each other, and the presence of CE in the desorbable pool precedes its presence in the nondesorbable pool, suggesting a precursor-product relationship. it is proposed [239] that the desorbable CE is solubilized in the plasma membrane bilayer, whereas the nondesorbablc CE has been transferred to internal cellular compartments. Following selective uptake by adrenal cells, a large fraction of HDL-derived CE is hydrolyzed, mostly by mechanisms that are insensitive to chloroquine and thus may be nonlysosomal [226,227]. In adrenal cells, another fraction of internalized CE appears to I;e incorporated directly into cytoplasmic CE droplets [226]. The mechanism by which CE is transferred into the plasma membrane, and the exact itinerary of this CE within cells remain to be established. ¥11. Summary Various types of studies in humans and animals suggest strongly that HDL is anti-atherogenie. The anti-atherogenic potential of HDL is thought to be due to its participation in reverse cholesterol transport, the process by which cholesterol is removed from nonhepatic cells and transported to the liver for elimination from the body. Extensive studies in cell culture systems have demonstrated that H D L is an important mediator of sterol transport between cells and the plasma compartment. The topic of this review is the mechanisms that account for sterol movement between HDL ~ancl cells. The most prominent and easily measured aspect of sterol movement between H D L and cells is the rapid bidirectional transfer of cholesterol between the lipoprotein and the plasma membrane. This movement occurs by unmediated diffusion, and in most situations its rate in each direction is limited by the rate of desorption of sterol molecules from the donor surface into the adjacent water phase. The net transfer of sterol mass out of cells occurs when there is either a relative enrichment of sterol within the plasma membrane or a depletion of sterol in HDL. Recent studies suggest that certain minor subfractions of HDL (with pro-beta mobility on agarose gel electrophoresis and containing apoprotein A-I but no apo A-ll) are unusually efficient at promoting efflux of cell sterol. To what extent efflux to these HDL fractions is balanced by influx from the lipoprotein has not yet been established clearly. The prevention and reversal of atherosclerosis require the mobilization of cholesterol from internal (non-plasma membrane) cellular locations. To some extent, this may involve the retroendocytosis of H D L However, most mobilization probably involves the

294 transport of internal sterol to the plasma membrane, followed by desorption to extracellular HDL. Several laboratories are investigating the transport of sterol from intracellular locations to the plasma membrane. Studies on biosynthetic sterol (probably originating mostly in the smooth endoplasmic reticulum) suggest that there is rapid transport to the plasma membrane in lipid-rich vesicles. Important features of this transport are that it bypasses the Golgi apparatus and may be positively regulated by the specific binding of HDL to the plasma membrane. LDL-derived lysosomal cholesterol is also transported very rapidly to the plasma membrane but the transport intermediates have not been identified and the process does not appear to be regulated by HDL. Cholesterol originating from intracellulat lipid inclusions and phagocytosed lipid droplets is transported to the plasma membrane and thereby made available for removal from cells, but the transport to the cell surface does not appear to be as efficient as that for lipoprotein-derived lysosomal cholesterol. Limiting factors may include ACAT-mediated reesterification of cholesterol, the solubilization of sterol into the unhydrolyzed portions of lipid droplets, and the saturation of transport mechanisms by the large quantities of sterol potentially available from these sources. Although the mechanisms responsible for sterol transport from lysosomes and intraeellular inclusions to the cell surface remain undefined, recent work suggests the possibility of cAMP-mediated regulation of the transport from intracellular inclusions. HDL may also contribute to reverse cholesterol transport by the delivery of cholesterol to the liver. HDL particles containing apo E can be subjected to receptor-mediated endocytosis by hepatocytes. Another well established aspect of this delivery is the selective uptake of HDL eholesteryl ester into hepatic cells. The quantitative importance of this uptake in comparison to the endocytosis and degradation of LDL and related lipoproteins has not been established. Current knowledge and techniques have opened a number of opportunities for further investigation of lipid transport between HDL and cells. It has become clear that a large variety of qualitatively distinct particles exist within the HDL fraction. Further heterogeneity arises from quantitative differences in lipid content, dietary effects, and the differences in HDL metabolism in different locations in the body. Very little work has been done on defining sterol flux between cells and the various HDL subfractions and on how each of these subfractions affects the mobilization of intracellular sterol. There has been very little direct experimentation on the mechanisms and regulation of sterol transport from intracellular compartments to the plasma membrane. Knowledge on this aspect of sterol transport may benefit greatly from attempts to reconstitute specific pathways in cell-free systems. These studies

could be modeled on methods for reconstituting intracellular protein and sphingolipid transport, which are well established and are producing rapid progress in these areas of cell biology. Another largely unexplored area consists of the genetics of sterol transport. To date there is only one well established genetic defect in the process of sterol elimination from cells (the naturally occurring Niemann-Pick C defect) and there have been few attempts to produce cell mutants that are defective in releasing sterol to extracellular acceptors [163]. Additional mutants in this process will aid in the identification of proteins required for sterol transport between cells and lipoproteins and will also provide important tools for defining the molecular biology of sterol transport.

Aelmowledgments We are indebted to the following faculty, postdoctoral fellows and graduate students at the Medical College of Pennsylvania who have made valuable contributions to the studies performed in this laboratory: Catherine Benedict, David Bernard, John Bielieki, Geo-ge Chaeko, Jane Giiek, Jennifer Gold, Joan Karlin, Joan Letizia, Sissel Lund-Katz, Holly MeCloskey, Lisa Minor and Rajendra Tangirala. Dr. Arie van Tol (Erasmus University, Rotterdam, The Netherlands) has collaborated in the studies on the efflux of cell cholesterol to immunopurified subfractions of HDL. The expert preparation of this review by Barbara Engle is gratefully acknowledged. The research from this laboratory cited in the review was supported by NIH Program Project Grant HL22633, Institutional Training Grant HL07443, and a NATO Grant for International Collaboration in Research (SA.5-2-05RG).

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