Apolipoprotein A-I induces translocation of cholesterol, phospholipid, and caveolin-1 to cytosol in rat astrocytes.

Intercellular cholesterol transport in the brain is carried by high density lipoprotein (HDL) generated in situ by cellular interaction with the apolipoprotein apoE, which is mainly synthesized by astrocytes, and with apoA-I secreted by cells such as endothelial cells. Rat astrocytes in fact generate HDL with extracellular apoA-I in addition to releasing HDL with endogenously synthesized apoE, seemingly by the same mechanism as the HDL assembly for systemic circulation. Relating to this reaction, apoA-I induced translocation of newly synthesized cholesterol and phospholipid to the cytosol prior to extracellular assembly of HDL, accompanied by an increase of caveolin-1 in the cytosol, activation of sterol regulatory element-binding protein, and enhancement of cholesterol synthesis. The lipid translocated into the cytosol was recovered in the fraction with a density of 1.09-1.16 g/ml as well as caveolin-1 and cyclophilin A. Cyclosporin A inhibited these apoA-I-mediated reactions and suppressed apoA-I-mediated cholesterol release. The findings suggest that such translocation of cholesterol and phospholipid into the cytosol is related to the apo A-I-mediated HDL assembly in astrocytes through functional association with caveolin-1 and a cyclosporin A-sensitive cyclophilin protein(s).     

Intracellular cholesterol mobilization involved in the ABCA1/apolipoprotein-mediated assembly of high density lipoprotein in fibroblasts

Differential regulation has been suggested for cellular cholesterol and phospholipid release mediated by apolipoprotein A-I (apoA-I)/ABCA1. We investigated various factors involved in cholesterol mobilization related to this pathway. ApoA-I induced a rapid decrease of the cellular cholesterol compartment that is in equilibrium with the ACAT-accessible pool in cells that generate cholesterol-rich HDL. Pharmacological and genetic inactivation of ACAT enhanced the apoA-I-mediated cholesterol release through upregulation of ABCA1 and through cholesterol enrichment in the HDL generated. Pharmacological activation of protein kinase C (PKC) also decreased the ACAT-accessible cholesterol pool, not only in the cells that produce cholesterol-rich HDL by apoA-I (i.e., human fibroblast WI-38 cells) but also in the cells that generate cholesterol-poor HDL (mouse fibroblast L929 cells). In L929 cells, the PKC activation caused an increase in apoA-I-mediated cholesterol release without detectable change in phospholipid release and in ABCA1 expression. These results indicate that apoA-I mobilizes intracellular cholesterol for the ABCA1-mediated release from the compartment that is under the control of ACAT. The cholesterol mobilization process is presumably related to PKC activation by apoA-I.     

Apolipoprotein-mediated cellular cholesterol/phospholipid efflux and plasma high density lipoprotein level in mice

Helical apolipoprotein(apo)s generate pre-beta-high density lipoprotein (HDL) by removing cellular cholesterol and phospholipid upon the interaction with cells. To investigate its physiological relevance, we studied the effect of an in vitro inhibitor of this reaction, probucol, in mice on the cell-apo interaction and plasma HDL levels. Plasma HDL severely dropped in a few days with probucol-containing chow while low density protein decreased more mildly over a few weeks. The peritoneal macrophages were assayed for apoA-I binding, apoA-I-mediated release of cellular cholesterol and phospholipid and the reduction by apoA-I of the ACAT-available intracellular cholesterol pool. All of these parameters were strongly suppressed in the probucol-fed mice. In contrast, the mRNA levels of the potential regulatory proteins of the HDL level such as apoA-I, apoE, LCAT, PLTP, SRB1 and ABC1 did not change with probucol. The fractional clearance rate of plasma HDL-cholesteryl ester was uninfluenced by probucol, but that of the HDL-apoprotein was slightly increased. No measurable CETP activity was detected either in the control or probucol-fed mice plasma. The change in these functional parameters is consistent with that observed in the Tangier disease patients. We thus concluded that generation of HDL by apo-cell interaction is a major source of plasma HDL in mice.     

Efflux of lipid from fibroblasts to apolipoproteins: dependence on elevated levels of cellular unesterified cholesterol.

Earlier work from this laboratory showed that enrichment of cells with free cholesterol enhanced the efflux of phospholipid to lipoprotein acceptors, suggesting that cellular phospholipid may contribute to high density lipoprotein (HDL) structure and the removal of sterol from cells. To test this hypothesis, we examined the efflux of [3H]cholesterol (FC) and [32P]phospholipid (PL) from control and cholesterol-enriched fibroblasts to delipidated apolipoproteins. The percentages of [3H]cholesterol and [32P]phospholipid released from control cells to human apolipoprotein A-I were 2.2 +/- 0.5%/24 h and 0.8 +/- 0.1%/24 h, respectively. When the cellular cholesterol content was doubled, efflux of both lipids increased substantially ([3H]FC efflux = 14.6 +/- 3.6%/24 h and [32P]PL efflux = 4.1 +/- 0.3%/24 h). Phosphatidylcholine accounted for 70% of the radiolabeled phospholipid released from cholesterol-enriched cells. The cholesterol to phospholipid molar ratio of the lipid released from cholesterol-enriched cells was approximately 1. This ratio remained constant throughout an incubation time of 3 to 48 h, suggesting that there was a coordinate release of both lipids. The concentrations of apoA-I, A-II, A-IV, E, and Cs that promoted half-maximal efflux of phospholipid from cholesterol-enriched fibroblasts were 53, 30, 68, 137, and 594 nM, respectively. With apoA-I and A-IV, these values for half-maximal efflux of phospholipid were identical to the concentrations that resulted in half-maximal efflux of cholesterol. Agarose gel electrophoresis of medium containing apoA-I that had been incubated with cholesterol-enriched fibroblasts revealed a particle with alpha to pre-beta mobility. We conclude that the cholesterol content of cellular membranes is an important determinant in the ability of apolipoproteins to promote lipid removal from cells. We speculate that apolipoproteins access cholesterol-phosphatidylcholine domains within the plasma membrane of cholesterol-enriched cells, whereupon HDL is generated in the extracellular compartment. The release of cellular lipid to apolipoproteins may serve as a protective mechanism against the potentially damaging effects of excess membrane cholesterol.     

Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL

Helical apolipoproteins of high density lipoprotein (HDL) remove phospholipid and cholesterol from cells and generate HDL particles being mediated by ATP binding cassette transporter A1 (ABCA1). In murine macrophage cell line RAW264 cells, cAMP induced expression of ABCA1, release of cellular phospholipid and cholesterol by apolipoprotein A-I (apoA-I), and reversible binding of apoA-I to the cell. The apoA-I-dependent lipid release was directly proportional to the cAMP-induced binding of apoA-I, and was inhibited 70% by a monoclonal antibody selective to lipid-free apoA-I, 725-1E2. In contrast, apparent cellular cholesterol release to HDL was substantial even without ABCA1 induction, and it was increased only by 27% after the cAMP treatment. The antibody inhibited this increment by 70%. Lipid-free apoA-II liberated apoA-I from HDL by displacement and thereby markedly expanded the cAMP-induced part of the cholesterol release to the HDL-containing medium, and the antibody inhibited this part also by 70%. Binding experiments of the double-labeled reconstituted HDL showed that cAMP induced reversible binding of apoA-I but not the association of cholesteryl ester with the cells. The effect of the antibody on the cellular cholesterol release to the reconstituted HDL was similar to that of the HDL-mediated release. The data implicated that the ABCA1-dependent cholesterol release to HDL is mediated by apoA-I dissociated from HDL.     

On the hepatic mechanism of HDL assembly by the ABCA1/apoA-I pathway

The mechanism for the assembly of HDL with cellular lipid by ABCA1 and helical apolipoprotein was investigated in hepatocytes. Both HepG2 cells and mouse primary culture hepatocytes produced HDL with apolipoprotein A-I (apoA-I) whether endogenously synthesized or exogenously provided. Probucol, an ABCA1 inactivator, inhibited these reactions, as well as the reversible binding of apoA-I to HepG2. Primary cultured hepatocytes of ABCA1-deficient mice also lacked HDL production regardless of the presence of exogenous apoA-I. HepG2 cells secreted apoA-I into the medium even when ABCA1 was inactivated by probucol, but it was all in a free form as HDL production was inhibited. When a lipid-free apoA-I-specific monoclonal antibody, 725-1E2, was present in the culture medium, production of HDL was suppressed, whether with endogenous or exogenously added apoA-I, and the antibody did not influence HDL already produced by HepG2 cells. We conclude that the main mechanism for HDL assembly by endogenous apoA-I in HepG2 cells is an autocrine-like reaction in which apoA-I is secreted and then interacts with cellular ABCA1 to generate HDL.     

Serum amyloid A generates high density lipoprotein with cellular lipid in an ABCA1- or ABCA7-dependent manner

Serum amyloid A (SAA) is an amphiphilic helical protein that is found associated with plasma HDL in various pathological conditions, such as acute or chronic inflammation. Cellular lipid release and generation of HDL by this protein were investigated, in comparison with the reactions by apolipoprotein A-I (apoA-I) and several types of cells that appear with various specific profiles of cholesterol and phospholipid release. SAA mediated cellular lipid release from these cells with the same profile as apoA-I. Upregulation of cellular ABCA1 protein by liver X receptor/retinoid X receptor agonists resulted in an increase of cellular lipid release by apoA-I and SAA. SAA reacted with the HEK293-derived clones that stably express human ABCA1 (293/2c) or ABCA7 (293/6c) to generate cholesterol-containing HDL in a similar manner to apoA-I. Dibutyryl cyclic AMP and phorbol 12-myristate 13-acetate, which differentiate apoA-I-mediated cellular lipid release between 293/2c and 293/6c, also exhibited the same differential effects on the SAA-mediated reactions. No evidence was found for the ABCA1/ABCA7-independent lipid release by SAA. Characterization of physicochemical properties of the HDL revealed that SAA-generated HDL particles had higher density, larger diameter, and slower electrophoretic mobility than those generated by apoA-I. These results demonstrate that SAA generates cholesterol-containing HDL directly with cellular lipid and that the reaction is mediated by ABCA1 and ABCA7.     

An inhibitor of acylCoA: cholesterol acyltransferase increases expression of ATP-binding cassette transporter A1 and thereby enhances the ApoA-I-mediated release of cholesterol from macrophages

The effect of inhibition of acylCoA: cholesterol acyltransferase (ACAT) was studied on high density lipoprotein (HDL) metabolism. An inhibitor of ACAT, MCC-147, was given mouse peritoneal macrophages and expression of ATP-binding cassette transporter A1 (ABCA1) was examined. ABCA1 was increased both at the mRNA and protein levels, only when the cells are cholesterol-loaded and thereby the inhibitor decreased esterified cholesterol and increased unesterified cholesterol. In this condition, the ACAT inhibitor increased reversible binding of apoA-I to the cells and enhanced apoA-I-mediated release of cellular cholesterol and phospholipid, but did not influence nonspecific cellular cholesterol efflux to lipid microemulsion. It was therefore concluded that the ACAT inhibitor increased the release of cholesterol from the cholesterol-loaded macrophages by increasing the expression of ABCA1, putatively through shifting cholesterol distribution from the esterified to the free compartments.     

Structural modification of plasma HDL by phospholipids promotes efficient ABCA1-mediated cholesterol release

It has been suggested that ABCA1 interacts preferentially with lipid-poor apolipoprotein A-I (apoA-I). Here, we show that treatment of plasma with dimyristoyl phosphatidylcholine (DMPC) multilamellar vesicles generates prebeta(1)-apoA-I-containing lipoproteins (LpA-I)-like particles similar to those of native plasma. Isolated prebeta(1)-LpA-I-like particles inhibited the binding of (125)I-apoA-I to ABCA1 more efficiently than HDL(3) (IC(50) = 2.20 +/- 0.35 vs. 37.60 +/- 4.78 microg/ml). We next investigated the ability of DMPC-treated plasma to promote phospholipid and unesterified (free) cholesterol efflux from J774 macrophages stimulated or not with cAMP. At 2 mg DMPC/ml plasma, both phospholipid and free cholesterol efflux were increased ( approximately 50% and 40%, respectively) in cAMP-stimulated cells compared with unstimulated cells. Similarly, both phospholipid and free cholesterol efflux to either isolated native prebeta(1)-LpA-I and prebeta(1)-LpA-I-like particles were increased significantly in stimulated cells. Furthermore, glyburide significantly inhibited phospholipid and free cholesterol efflux to DMPC-treated plasma. Removal of apoA-I-containing lipoproteins from normolipidemic plasma drastically reduced free cholesterol efflux mediated by DMPC-treated plasma. Finally, treatment of Tangier disease plasma with DMPC affected the amount of neither prebeta(1)-LpA-I nor free cholesterol efflux. These results indicate that DMPC enrichment of normal plasma resulted in the redistribution of apoA-I from alpha-HDL to prebeta-HDL, allowing for more efficient ABCA1-mediated cellular lipid release. Increasing the plasma prebeta(1)-LpA-I level by either pharmacological agents or direct infusions might prevent foam cell formation and reduce atherosclerotic vascular disease.     

Characterization of nascent HDL particles and microparticles formed by ABCA1-mediated efflux of cellular lipids to apoA-I

The nascent HDL created by ABCA1-mediated efflux of cellular phospholipid (PL) and free (unesterified) cholesterol (FC) to apolipoprotein A-I (apoA-I) has not been defined. To address this issue, we characterized the lipid particles released when J774 mouse macrophages and human skin fibroblasts in which ABCA1 is activated are incubated with human apoA-I. In both cases, three types of nascent HDL containing two, three, or four molecules of apoA-I per particle are formed. With J774 cells, the predominant species have hydrodynamic diameters of approximately 9 and 12 nm. These discoidal HDL particles have different FC contents and PL compositions, and the presence of acidic PL causes them to exhibit alpha-electrophoretic mobility. These results are consistent with ABCA1 located in more than one membrane microenvironment being responsible for the production of the heterogeneous HDL. Activation of ABCA1 also leads to the release of apoA-I-free plasma membrane vesicles (microparticles). These larger, spherical particles released from J774 cells have the same PL composition as the 12 nm HDL and contain CD14 and ganglioside, consistent with their origin being plasma membrane raft domains. The various HDL particles and microparticles are created concurrently, and there is no precursor-product relationship between them. Importantly, a large fraction of the cellular FC effluxed from these cells by ABCA1 is located in microparticles. Collectively, these results show that the products of the apoA-I/ABCA1 interaction include discoidal HDL particles containing different numbers of apoA-I molecules. The cellular PLs and cholesterol incorporated into these nascent HDL particles originate from different cell membrane domains.     

Lateral distribution of phospholipid and cholesterol in apolipoprotein A-I recombinants

The influence of cholesterol on the assembly and structure of model high-density lipoproteins (HDL) has been investigated. Model HDL composed of apolipoprotein A-I (apoA-I) and 1,2-dimyristoylphosphatidylcholine (DMPC) formed spontaneously at the transition temperature (Tc) of the lipid. Those composed of apoA-I and 1-palmitoyl-2-oleoylphosphatidylcholine were formed by a cholate dialysis method. At low cholesterol/phospholipid ratios both lipids and assembly methods yielded a model HDL whose composition was identical with that of the initial mixture; as the cholesterol/phospholipid ratio of the initial mixture was increased, the fraction of cholesterol appearing in the model HDL decreased, and a negative correlation between the cholesterol and protein contents of the model HDL was observed. At high cholesterol/phospholipid ratios the association of apoA-I and phospholipids appeared to be thermodynamically unfavorable. The effects of cholesterol content on the thermal properties of a model HDL composed of DMPC and apoA-I were further investigated by differential scanning calorimetry, fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, fluorescence energy transfer, and excimer fluorescence of pyrenyl derivatives of phosphatidylcholine (PC) and cholesterol. The addition of cholesterol decreased the transition enthalpy of DMPC, raised the midpoint of the transition, and modulated motional freedom in the phospholipid matrix. The amount of cholesterol required to produce these effects was lower in the model HDL than in multilamellar liposomes. In a model HDL composed of DMPC and apoA-I, the lateral diffusion of a pyrene-labeled cholesterol was dramatically changed at the Tc whereas little change was observed in that of a pyrene-labeled PC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1

Mutations of the ABC1 transporter have been identified as the defect in Tangier disease, characterized by low HDL and cholesterol ester accumulation in macrophages. A full-length mouse ABC1 cDNA was used to investigate the mechanisms of lipid efflux to apoA-I or HDL in transfected 293 cells. ABC1 expression markedly increased cellular cholesterol and phospholipid efflux to apoA-I but had only minor effects on lipid efflux to HDL. The increased lipid efflux appears to involve a direct interaction between apoA-I and ABC1, because ABC1 expression substantially increased apoA-I binding at the cell surface, and chemical cross-linking and immunoprecipitation analysis showed that apoA-I binds directly to ABC1. In contrast to scavenger receptor BI (SR-BI), another cell surface molecule capable of facilitating cholesterol efflux, ABC1 preferentially bound lipid-free apoA-I but not HDL. Immunofluorescence confocal microscopy showed that ABC1 is primarily localized on the cell surface. In the absence of apoA-I, cells overexpressing ABC1 displayed a distinctive morphology, characterized by plasma membrane protrusions and resembling echinocytes that form when there are excess lipids in the outer membrane hemileaflet. The studies provide evidence for a direct interaction between ABC1 and apoA-I, but not HDL, indicating that free apoA-I is the metabolic substrate for ABC1. Plasma membrane ABC1 may act as a phospholipid/cholesterol flippase, providing lipid to bound apoA-I, or to the outer membrane hemileaflet.     

Release of cellular cholesterol: molecular mechanism for cholesterol homeostasis in cells and in the body

Most mammalian somatic cells are unable to catabolize cholesterol and therefore need to export it in order to maintain sterol homeostasis. This mechanism may also function to reduce excessively accumulated cholesterol, which would thereby contribute to prevention or cure of the initial stage of atherosclerotic vascular lesion. High-density lipoprotein (HDL) has been believed to play a main role in this reaction based on epidemiological evidence and in vitro experimental data. At least two independent mechanisms are identified for this reaction. One is non-specific diffusion-mediated cholesterol 'efflux' from cell surface. Cholesterol molecules desorbed from cells can be trapped by various extracellular acceptors including various lipoproteins and albumin, and extracellular cholesterol esterification mainly on HDL may provide a driving force for the net removal of cell cholesterol by maintaining a cholesterol gradient between lipoprotein surface and cell membrane. The other is apolipoprotein-mediated process to generate new HDL by removing cellular phospholipid and cholesterol. The reaction is initiated by the interaction of lipid-free or lipid-poor helical apolipoproteins with cellular surface resulting in assembly of HDL particles with cellular phospholipid and incorporation of cellular cholesterol into the HDL being formed. Thus, HDL has dual functions as an active cholesterol acceptor in the diffusion-mediated pathway and as an apolipoprotein carrier for the HDL assembly reaction. The impairment of the apolipoprotein-mediated reaction was found in Tangier disease and other familial HDL deficiencies to strongly suggest that this is a main mechanism to produce plasma HDL. The causative mutations for this defect was identified in ATP binding cassette transporter protein A1, as a significant step for further understanding of the reaction and cholesterol homeostasis.     

Cholesterol-sphingomyelin interaction in membrane and apolipoprotein-mediated cellular cholesterol efflux

Helical apolipoproteins interact with cellular surface and generate high density lipoprotein (HDL) by removing phospholipid and cholesterol from cells. We have reported that the HDL is generated by this reaction with the fetal rat astrocytes and meningeal fibroblasts but cholesterol is poorly available to this reaction with the astrocytes (Ito et al. 1999. J. Neurochem. 72: 2362;-2369). Partial digestion of the membrane by extracellular sphingomyelinase increased the incorporation of cholesterol into thus-generated HDL from both types of cell. This increase was diminished by supplement of endogenous or exogenous sphingomyelin to the cells. The sphingomyelinase treatment decreased cholesterol in the membrane mainly in the detergent-resisting domain. The intracellular cholesterol used by acylCoA:cholesterol acyltransferase increased by the sphingomyelinase treatment in the absence of apoA-I, more remarkably in the fibroblast than in the astrocytes. ApoA-I suppressed this increase completely in the astrocytes, but only partially in the fibroblast. The effect of the sphingomyelin digestion was more prominent for the apolipoprotein-mediated reaction than the diffusion-mediated cellular cholesterol efflux. Thus, cholesterol molecules restricted by sphingomyelin in the domain of the plasma membrane appear to be primarily used for the HDL assembly upon the apolipoprotein;-cell interaction.     

Folded functional lipid-poor apolipoprotein A-I obtained by heating of high-density lipoproteins: relevance to high-density lipoprotein biogenesis

HDL (high-density lipoproteins) remove cell cholesterol and protect from atherosclerosis. The major HDL protein is apoA-I (apolipoprotein A-I). Most plasma apoA-I circulates in lipoproteins, yet ~5% forms monomeric lipid-poor/free species. This metabolically active species is a primary cholesterol acceptor and is central to HDL biogenesis. Structural properties of lipid-poor apoA-I are unclear due to difficulties in isolating this transient species. We used thermal denaturation of human HDL to produce lipid-poor apoA-I. Analysis of the isolated lipid-poor fraction showed a protein/lipid weight ratio of 3:1, with apoA-I, PC (phosphatidylcholine) and CE (cholesterol ester) at approximate molar ratios of 1:8:1. Compared with lipid-free apoA-I, lipid-poor apoA-I showed slightly altered secondary structure and aromatic packing, reduced thermodynamic stability, lower self-associating propensity, increased adsorption to phospholipid surface and comparable ability to remodel phospholipids and form reconstituted HDL. Lipid-poor apoA-I can be formed by heating of either plasma or reconstituted HDL. We propose the first structural model of lipid-poor apoA-I which corroborates its distinct biophysical properties and postulates the lipid-induced ordering of the labile C-terminal region. In summary, HDL heating produces folded functional monomolecular lipid-poor apoA-I that is distinct from lipid-free apoA-I. Increased adsorption to phospholipid surface and reduced C-terminal disorder may help direct lipid-poor apoA-I towards HDL biogenesis.     

ApoA-I induces a preferential efflux of monounsaturated phosphatidylcholine and medium chain sphingomyelin species from a cellular pool distinct from HDL(3) mediated phospholipid efflux

Electrospray ionization tandem mass spectrometry (ESI-MS/MS) was used for a detailed analysis of cellular phospholipid and cholesterol efflux in free cholesterol (FC) loaded human primary fibroblasts and human monocyte-derived macrophages (HMDM) loaded with enzymatically modified LDL (E-LDL). Although both cell models differed significantly in their cellular lipid composition, a higher apoA-I specific efflux was found for monounsaturated phosphatidylcholine (PC) species together with a decreased contribution of polyunsaturated PC species in both cell types. Moreover, medium chain sphingomyelin (SPM) species SPM 14:0 and SPM 16:1 were translocated preferentially to apoA-I in both cell types. In contrast to fibroblasts, HMDM displayed a considerable proportion of cholesteryl esters (CE) in basal and apoA-I specific efflux media, most likely due to secretion of CE associated to apoE. Analysis of HDL(3) mediated lipid efflux from HMDM using D(9)-choline and (13)C(3)-FC stable isotope labeling revealed significantly different D(9)-PC and D(9)-SPM species pattern for apoA-I and HDL(3) specific efflux media, which indicates a contribution of distinct cellular phospholipid pools to apoA-I and HDL(3) mediated efflux. Together with a partial loading of fibroblasts and HMDM with HDL(3)-derived CE species, these data add further evidence for retroendocytosis of HDL. In summary, analysis of apoA-I/ABCA1 and HDL(3) mediated lipid efflux by ESI-MS/MS demonstrated a preferential efflux of monounsaturated PC and medium chain SPM to apoA-I. Moreover, this is the first study, which provides evidence for distinct cellular phospholipid pools used for lipid transfer to apoA-I and HDL(3) from the analysis of phospholipid species pattern in HMDM.     

Composition and ultrastructure of size subclasses of normal human peripheral lymph lipoproteins: quantification of cholesterol uptake by HDL in tissue fluids

Peripheral lymph lipoproteins have been characterized in animals, but there is little information about their composition, and none about their ultrastructure, in normal humans. Therefore, we collected afferent leg lymph from 16 healthy males and quantified lipids and apolipoproteins in fractions separated by high performance-size exclusion chromatography. Apolipoprotein B (apoB) was found almost exclusively in low density lipoproteins. The distribution of apoA-I, particularly in lipoprotein A-I (LpA-I) without A-II particles, was shifted toward larger particles relative to plasma. The fractions containing these particles were also enriched in apoA-II, apoE, total cholesterol, and phospholipids and had greater unesterified cholesterol-to-cholesteryl ester ratios than their counterparts in plasma. Fractions containing smaller apoA-I particles were enriched in phospholipid. Most apoA-IV was lipid poor or lipid free. Most apoC-III coeluted with large apoA-I-containing particles. Electron microscopy showed that lymph contained discoidal particles not seen in plasma. These findings support other evidence that high density lipoproteins (HDL) undergo extensive remodeling in human tissue fluid. Total cholesterol concentration in lymph HDL was 30% greater (P < 0.05) than could be explained by the transendothelial transfer of HDL from plasma, providing direct confirmation that HDL acquire cholesterol in the extravascular compartment. Net transport rates of new HDL cholesterol in the cannulated vessels corresponded to a mean whole body reverse cholesterol transport rate via lymph of 0.89 mmol (344 mg)/day.