Abstract
Objective-Endothelial lipase (EL) is expressed by macrophages within atherosclerotic lesions. We investigated the influence of EL expression on cholesterol efflux in macrophages. Methods and Results-The present study used lentivirus to introduce either EL shRNA for loss-of-function studies or EL cDNA for gain-of-function studies to investigate the role of EL in apoAI-mediated cholesterol efflux. ApoAI-mediated cholesterol efflux was decreased after EL suppression, but increased with EL overexpression in free cholesterol labeled and acLDL loaded THP-1 macrophages. Similar findings were observed in THP-1 macrophages after exogenous EL addition and in transfected 293 cells. EL-related apoAI-mediated cholesterol efflux decreased after treatment with heparin or catalytic inactivation (S149A mutation or tetrahydrolipstatin) alone, and completely inhibited in combination. Furthermore, EL expression did not change ABCA1 expression, but was positively correlated with apoAI binding to macrophages and 293 cells. This effect was mitigated after heparin treatment but not influenced by catalytic inactivation via tetrahydrolipstatin or the S149A mutation. Moreover, EL expression was positively associated with lysophosphatidylcholine production and inversely with phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin levels. Lysophosphatidylcholine treatment dose-dependently stimulated apoAI-mediated cholesterol efflux in THP-1 macrophages.
Abstract
appears to promote apoAI-mediated cholesterol efflux through catalytic and noncatalytic-dependent mechanisms. (Arterioscler Thromb Vasc Biol. 2009;29:84-91.)

A therosclerosis is a multifactorial disease and is considered to be an inflammatory process where macrophages and consequent foam cell formation play a pivotal role. [1] [2] [3] [4] Cellular lipid homeostasis is dominated by the balance between lipid accumulation and efflux. It is well known that, when activated, macrophages acquire an enhanced ability to internalize lipoproteins and accumulate cholesterol and ultimately transform themselves into lipid-laden foam cells. Lipid efflux is a regulatory pathway which responds to an increased intracellular lipid content. The impairment of lipid efflux in macrophages may aggravate lipid accumulation and accelerate the progression of atherosclerosis.

Cholesterol is mosaiced in the phospholipid bilayer of cell membranes with an uneven distribution between exo-and endo-facial membranes. 5 The interaction of cholesterol with phospholipids such as phosphatidylcholine and sphingomyelin is critical for the maintenance of cholesterol integrity in the membrane. 6, 7 Free diffusion, receptor-facilitated efflux, and ATP-binding cassette (ABC) transporters are the principal pathways for cholesterol efflux in macrophages. The active removal of cholesterol via ABC transporters, mainly ABCA1, assumes a major role in cholesterol efflux, 8 and extracellular apolipoprotein AI (ApoAI) is recognized as the primary acceptor of ABCA1-mediated cholesterol efflux.

Endothelial lipase (EL) is one of several lipases secreted by human macrophages, and its expression in macrophages has been observed in human atherosclerotic lesions. 9 -11 Several clinical studies suggest that EL could be implicated in atherosclerosis. For example, an increased level of plasma EL has been associated with visceral obesity, metabolic syndrome, inflammation, and premature coronary heart disease. [12] [13] [14] However, inconsistent outcomes have been described using 2 animal studies. One study in apoE knockout mice found that EL deficiency significantly decreased atherosclerosis, 15 whereas no significant changes were observed in another study where both apoE and low density lipoprotein receptor (LDLR) knockout mice were investigated. 16 Therefore, the role of EL in atherogenesis still remains unclear, and investigation of EL effects on macrophage function such as cholesterol efflux may provide useful information to help clarify the role of macrophage-derived EL in atherosclerosis.

In the present study, we used lentivirus to suppress or overexpress EL in THP-1 macrophages and found that EL promoted apoAI-mediated cholesterol efflux through mechanisms related to its catalytic and noncatalytic functions.
Materials and Methods

Cell Culture
THP-1 human monocytes (ATCC) were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic (Invitrogen), 2 mmol/L L-glutamine, 1.5 g/L sodium bicarbonate, and 1.0 mmol/L sodium pyruvate. 293T human embryonic kidney cells were cultured in Dulbecco modified Eagle medium (DMEM), supplemented with 10% FBS, and 1% antibioticantimycotic. FLP-In 293 Cells (Invitrogen) were cultured in DMEM with 10% FBS, 1% antibiotic-antimycotic, and 100 g/mL Zeocin. All cells were incubated in humidified incubator at 95% air, 5% carbon dioxide, and 37°C and used within 20 passages.
Site-Directed Mutagenesis and Lentivirus Construction for Gain-of-Function and Loss-of-Function Studies
Wild-type EL cDNA (EL-WT) was transferred from pcDNA5-FRT-EL (gift from Dr Howard Wong, UCLA, Calif.) into lentiviral vector pWPI-eGFP by Pme I digestion (positive construct is designated as pWPI-eGFP-EL). A catalytically inactive EL (EL-S149A) was generated by site-directed mutation of S149A using template plasmid pWPI-eGFP-EL, primers (sense: 5Ј-ACTTGATCGGCTA-CGCCCTCGGAGCGCACG-3Ј; antisense: 5Ј-CGTGCGCTCCGA-GGGCGTAGCCGATCAAGT-3Ј), and QuikChange XL site-directed mutagenesis kit (Stratagene). A lentiviral expression system (lentiviral vector pWPI-eGFP plus packaging vectors psPAX2 and pMD.2G, TronoLab) was used for EL gain-of-function (GOF) studies. The generation of shRNA-containing lentivirus for loss-offunction (LOF) studies has been described previously. 17
ApoAI-Mediated Cholesterol Efflux
ApoAI-mediated cholesterol efflux was performed in either free 3 H-cholesterol labeled or ac LDL-baded macrophages. THP-1 monocytes were transformed into macrophages by 1ϫ10 Ϫ7 mol/L PMA stimulation after 48-hour lentiviral transduction. Thereafter, macrophages (5ϫ10 5 cells) or FLP-IN 293 EL cells (2ϫ10 5 ) were incubated for 24 hours with 1Ci/mL 3 H-cholesterol with or without 20 g/mL acLDL in base medium (culture medium plus 1% antibiotics-antimycotic and 0.2% BSA; for macrophages, 2% bicarbonate, 1% pyruvate, and 1ϫ10 Ϫ7 mol/L PMA were supplemented additionally) for cholesterol labeling and loading, respectively. After 2-hour equilibration with base medium, cholesterol efflux was induced with 10 g/mL apoAI (Sigma) in base medium for 4 hours. Cholesterol efflux is represented as the percentage of medium cpm to total cpm (medium plus cellular cpm), with the subtraction of basal cholesterol efflux which is defined as the percent of cholesterol in the absence of apoAI. In addition, THL (50 g/mL) or 10units/mL heparin was added into the cholesterol efflux medium to eliminate the noncatalytic function of EL during cholesterol efflux. Furthermore, the apoAI-mediated cholesterol efflux was repeated by measuring cholesterol mass in conditioned medium and cellular compartments using 5ϫ10 6 cells in 2 mL culture medium and a 36-hour efflux period.
ApoAI Binding Assay
ApoAI was labeled with fluorescent dye Alexa Fluor 532 (Invitrogen) following the manufacturer's instructions. Optimal labeling of 4 moles of dye per mole of protein was obtained. The apoAI binding protocol was modified from the literature, 18 10 g/mL fluorescencelabeled apoAI was added into cell culture in base medium for 4 hours at 37°C, then washed with 0.2% BSA-PBS and PBS twice each. Cells were lysed in RIPA buffer, 200 L supernatants were measured for fluorescence at the excitation/emission wavelength of 531/554 nm, and bound apoAI was calculated from a standard curve, and normalized for total cellular protein.
ApoAI Binding Assay
Please see the supplemental materials (available online at http:// atvb.ahajournals.org) for details of the construction of EL-expressing FLP-IN 293 cell line, EL purification by fast protein liquid (FPLC), total phospholipase activity assay, ABCA1 suppression, real-time quantitative RT-PCR, Western blot for evaluation of EL and ABCA1 expression, cellular lipid composition analysis by high-performance liquid chromatography (HPLC), lysophosphatidylcholine (lyso-PC) measurement, the effect of lyso-PC on apoAI-mediated cholesterol efflux, and statistical analyses.
Results

Lipase Suppression and Overexpression
As reported previously, lentivirus containing EL-shRNA reduced EL mRNA and protein by Ͼ70% in comparison to the scrambled shRNA-containing lentivirus at a MOI of 20 and 100% transduction efficiency. 17 Macrophages overexpressing either wild-type (EL-WT) or mutant (EL-S149A) EL were generated by lentivirus. The transduction of gain-offunction (GOF) lentivirus markedly increased EL mRNA by Ͼ100 fold at MOI of 20 for both wild-type and mutant EL (supplemental Figure IA) . Correspondingly, both wild-type and mutant EL protein were markedly elevated in conditioned medium of EL transduced macrophages in comparison with an undetectable level in control cells (supplemental Figure  IB) . Most of the EL in lentivirus-transduced macrophages was secreted into the culture medium as the EL protein in cellular compartment only increased by 4.4-and 3.8-fold for EL-WT and EL-S149A, respectively (supplemental Figure  IB) . Furthermore, in wild-type EL transduced macrophages, total phospholipase activity was elevated by 1.5-fold, and no apparent increase in total phospholipase activity was observed in mutant EL transduced macrophages (supplemental Figure IA) . The EL cDNA was stably transfected into FLP-IN-293 cells with 2000-and Ͼ4-fold increases in EL mRNA and total phospholipase activity, respectively (supplemental Figure IC ). In addition, EL protein was only detected in EL-transfected cells and cell-associated EL was increased by 6-fold in comparison to control 293 cells (supplemental Figure ID) .
Lipases Promote ApoAI-Mediated Cholesterol Efflux
The ABCA1 specificity of apoAI-mediated cholesterol efflux was evaluated by assessing apoAI-mediated efflux after ABCA1 suppression. ABCA1 mRNA was reduced by 57% and apoAI-mediated cholesterol efflux decreased by 67% at 100 nM Stealth RNAi duplex (supplemental Figure II ), suggesting the apoAI-mediated cholesterol efflux in our macrophage model is highly ABCA1-specific.
Lipases Promote ApoAI-Mediated Cholesterol Efflux
In free cholesterol labeled THP-1 macrophages, apoAImediated cholesterol efflux was significantly decreased by 20% after EL suppression (EL-LOF), but increased by 1.5fold with wild-type EL overexpression (EL-WT-GOF; Figure  1A ). Furthermore, we assessed EL effect on apoAI-mediated cholesterol efflux in acLDL-labeled macrophages, and similar results were observed where an 11% decrease in efflux was observed for EL suppression and 1.6-fold increase for wild-type EL overexpression ( Figure 1B) . In both models, the basal cholesterol efflux in the absence of apoAI did not change significantly.
Lipases Promote ApoAI-Mediated Cholesterol Efflux
FLP-IN 293 cells have a relatively low level of apoAImediated cholesterol efflux attributable to a low expression of ABCA1 protein. However, the stimulatory effect of EL on apoAI-mediated cholesterol efflux was still observed in FLP-IN 293 cells, with a 2.3-fold increase for wild-type ELoverexpression compared with an empty vector control ( Figure  1C ). Using exogenous semipurified EL, a dose-dependent increase in apoAI-mediated cholesterol efflux was observed in THP-1 macrophages with a 25% increase in efflux at a protein concentration of 0.5 g protein/mL ( Figure 1D ).
Lipases Promote ApoAI-Mediated Cholesterol Efflux
Furthermore, we analyzed the absolute mass of cholesterol in conditioned media and the cellular compartment in ELoverexpression models. As shown in supplemental Figure III, overexpression of both wild-type (EL-WT) and mutant (EL-S149A) EL significantly reduced the cellular cholesterol by 34% and 25%, respectively. Furthermore, the wild-type EL was associated with a greater concentration of medium cholesterol (5% increase) compared to inactive EL. The medium cholesterol levels for both wild-type and mutated EL were not significantly different from the control. Overall, the total cholesterol (medium plus cellular cholesterol) was 21% and 17% lower for both the wild-type and mutated EL in comparison with the control, implying the presence of enhanced efflux of cholesterol in EL-overexpressing macrophages. Therefore, the apoAI-mediated cholesterol efflux calculated as the percentage from medium cholesterol of total cholesterol appeared to be 2.3-and 1.8-fold higher than the control for wild-type and mutant EL (supplemental Figure  III) . It was noted that the cholesterol efflux calculated by measuring cholesterol mass was higher than that by the radiolabeling method. This difference may be partially explained by the fact that the unlabeled cholesterol, particularly from intracellular compartments, was not measurable by the radiolabeling method and that a longer incubation time was used for this experiment to facilitate cholesterol measurement. All of these results suggest that EL overexpression promotes apoAI-mediated cholesterol efflux.
The Differential Role of the Catalytic and Noncatalytic Functions of EL in Cholesterol Efflux
Like other lipase family members, EL is conferred with catalytic and noncatalytic (bridging) functions. To differentiate the relative role of each function in cholesterol efflux, a lipase inhibitor, tetrahydrolipstatin (THL; the trioleinase and in-well phospholipase activities of EL were completely inhibited, data not shown), or heparin were used to eliminate either or both of the catalytic and noncatalytic functions of EL in free cholesterol labeled macrophages.
The Differential Role of the Catalytic and Noncatalytic Functions of EL in Cholesterol Efflux
In EL-overexpressing macrophages, the inhibition of the catalytic activity by THL resulted in a 59% reduction of EL-dependent apoAI-mediated cholesterol efflux (Figure 2A) . Similarly, the elimination of the noncatalytic cell surface function by heparin treatment led to a 29% decrease in EL-related apoAI-mediated cholesterol efflux (Figure 2A ). With the inhibition of both catalytic and noncatalytic functions of EL, 93% of the EL effect on apoAI-mediated cholesterol efflux was removed ( Figure 2A ). The same experiment was also repeated in FLP-IN 293 cells and similar results were observed with reductions of 40%, 54%, and 88% in EL-related cholesterol efflux after THL, heparin, and combination treatments, respectively ( Figure 2B) .
The Differential Role of the Catalytic and Noncatalytic Functions of EL in Cholesterol Efflux
To avoid the potential of off-target effects of THL treatment, apoAI-mediated cholesterol efflux was repeated in macrophages overexpressing mutant EL (EL-S149). Compared to wild-type EL, the catalytic inactivation of EL strikingly reduced the EL-related apoAI-mediated cholesterol efflux by 74%, whereas the addition of heparin completely eliminated any increase in cholesterol efflux mediated by mutant EL (Figure 2C ). Taken together, both catalytic and noncatalytic functions of EL participate in the apoAI-mediated cholesterol efflux in THP-1 macrophages and 293 embryonic kidney cells.
The Effect of EL Expression on Cholesterol Efflux Is Independent of ABCA1 Expression
As shown in this study, the apoAI-mediated cholesterol efflux is largely mediated through the ABCA1 transporter, thus we investigated whether the expression of EL altered ABCA1 expression. However, neither EL suppression nor overexpression (wild-type or mutant EL) altered ABCA1 mRNA and protein levels in macrophages and FLP-IN 293 cells when compared to the control condition (supplemental Figure IV) .
EL Increases ApoAI Binding via its Bridging Function
The stimulatory effect of EL on apoAI-mediated cholesterol efflux was mitigated by heparin treatment in macrophages and 293 cells, suggesting the noncatalytic function of EL may Figure 2 . The relative role of catalytic and noncatalytic functions of EL in apoAI-mediated cholesterol efflux. A, ApoAI-mediated cholesterol efflux in free cholesterol labeled EL-overexpressing macrophages without (EL-WT) or with treatment ofwith either no treatment (EL-WT), 50g/mL THL (EL-WTϩTHL), 10 U/mL heparin (EL-WTϩheparin), or a combination of THL and heparin (EL-WTϩTHLϩheparin). B, ApoAI-mediated cholesterol efflux in free cholesterol labeled transfected EL-overexpressing 293 cells with either no treatmentwithout or with treatment of, 50 g/mL THL, 10 U/mL heparin, or a combination of THL and heparin. C, ApoAI-mediated cholesterol efflux in free cholesterol labeled Figure 2 (Continued) . macrophages transduced with either a wild-typen EL cDNA insert (EL-WT) or, a mutant EL cDNA insert containing the S149A substitution (EL-S149A) in the absence or presence of heparin. Data are expressed as the mean of the relative proportion of EL-mediated percent cholesterol efflux as 100%ϮSEM, *PϽ0.05, **PϽ0.01, ***PϽ0.001. play a role in apoAI accessibility to membrane cholesterol. Therefore, we investigated the influence of EL expression on apoAI binding. When EL expression was suppressed, total apoAI binding was reduced by 34% in macrophages ( Figure  3A) . By contrast, wild-type EL overexpression significantly increased apoAI binding by 1.2-fold in comparison to the control condition. This EL-related increase in apoAI binding (the clear portion of each bar) was not abolished after the catalytic inhibition by THL but significantly reduced by heparin treatment (80% reduction, Figure 3B ). A similar relationship was also seen in transfected 293 cells: EL increased apoAI binding by 34%, and this increase was inhibited by heparin (40%) but not THL treatment ( Figure  3C ). Furthermore, the ability of EL to enhance apoAI binding was still retained after catalytic inactivation by the S149A mutation but reduced by 60% by heparin addition ( Figure  3D) . These data suggest that the noncatalytic function of EL can mediate apoAI binding to macrophages.
EL Expression Alters Membrane Phospholipid Composition
The observation that the catalytic inhibition of EL decreased apoAI-mediated cholesterol efflux raised a question whether EL could modify the membrane lipid composition and thus influence cholesterol efflux. Therefore, cellular lipids were extracted for the analysis of phospholipid composition. As reported previously, 19 the major phospholipid component, phosphatidylcholine (PC), was increased by 39% in ELsuppressed macrophages where lysophosphatidylcholine (lyso-PC), the metabolite of phosphatidylcholine, was significantly decreased by 20%. The level of phosphatidylethanolamine (PE) was also modestly increased in EL-suppressed macrophages. Currently, a 10% decrease in PC and 26% increase in lyso-PC were observed in wild-type EL overexpressing macrophages. In addition, wild-type EL overexpression reduced the level of PE and sphingomyelin (SPH) ( Figure 4A) . In parallel to the findings in EL-overexpressing macrophages, a 14% reduction in PC shaded bar) . B, ApoAI binding in EL-overexpressing macrophages transduced with wild-type EL (EL-WT-GOF) and or empty vector (control) in the absence or presence of heparin or THL treatments. C, ApoAI binding in transfected 293 cells transfected with either wild-type EL cDNA insert (293 EL-WT-GOF) or empty vector (control) in the absence or presence of heparin or THL treatments. D, ApoAI binding in macrophaged tranduced with a mutant EL-overexpressing cDNA (EL-S149A-GOF) macrophages and or empty vector (control). Data are expressed as meansϮSEM and statistical comparisons were made between control and experimental conditions, (**PϽ0.01, ***PϽ0.001) whereas statistical comparisons in EL-related apoAI binding (top portion of clear bar) are indicated as: †PϽ0.05, † † PϽ0.01, † † †PϽ0.001 in the figure. and a corresponding 31% increase in lyso-PC were detected in 293 cells transfected with wild-type EL ( Figure 4B ).
EL Expression Increases the Production of Lysophosphatidylcholine Which Stimulates ApoAI-Mediated Cholesterol Efflux
EL displays a predominant phospholipase activity, and one of its lipolytic products is lyso-PC which has been shown to be able to increase cholesterol efflux in murine macrophages 20 ; therefore, we further hypothesized that EL may stimulate cholesterol efflux via its ability to produce lyso-PC. After being normalized for cellular protein, we found that EL overexpression significantly increased the production of lyso-PC by 48%, and the elimination of the lipase activity of EL by S149A mutation almost completely relinquished this effect ( Figure 4C ). The effect of lyso-PC on apoAI-mediated cholesterol efflux was also repeated in THP-1-derived macrophages. After lyso-PC administration, apoAI-mediated cholesterol efflux was increased in a dose-dependent manner; at the concentration of 25 mol/L, there was an 86% increase in apoAI-mediated cholesterol efflux (supplemental Figure V) .
Discussion
In the present study, two complementary strategies of lossof-function and gain-of-function were used to elucidate the role of EL in cholesterol efflux in THP-1 macrophages. Clinical evidence supports a proatherogenic role for plasma derived EL, so we initially hypothesized that the expression of EL may impair cholesterol efflux in macrophages. Surprisingly, EL expression is positively associated with apoAImediated cholesterol efflux in THP-1 macrophages based on the results from LOF and GOF studies implying a multifaceted role of EL in macrophages and atherogenesis. Furthermore, the roles of the catalytic and noncatalytic functions of EL appear to be additive because the inhibition of each function alone only partially decreased EL-related, apoAImediated cholesterol efflux, whereas the removal of both catalytic and noncatalytic functions of EL had the greatest effect. However, it remains unclear whether EL expression in macrophages in vivo can influence intracellular cholesterol concentration through this mechanism in an effort to maintain cholesterol homeostasis.
Discussion
Cellular cholesterol is contained within both fast and slow metabolic pools where the fast pool of cholesterol is mainly situated on the phosphatidylcholine-rich domains of the outer membrane leaflet whereas the slow pool of cholesterol is interdigitated in sphingomyelin-rich domains of inner membrane leaflet originating from intracellular cytoplasmic organelles such as endosomes and lysosomes. 5, [21] [22] [23] [24] In the current study, EL increased the cholesterol desorption from the fast pool as the cholesterol efflux from free H 3 -cholesterol labeled macrophages was increased by EL during a 4-hour incubation with apoAI. Ac-LDL loading of macrophages delivers cholesterol toward intracellular compartments, and the efflux assay in this model would reflect the mobilization of the slow pool of cholesterol. EL also promoted the cholesterol efflux in ac-LDL-loaded macrophages, suggesting EL can stimulate the cholesterol efflux from both fast and slow metabolic pools.
Discussion
Gauster et al have reported that EL reduced cholesterol efflux in Cos-7 cells when HDL was modified by EL and then used as the cholesterol recipient, however no apparent effect of EL-modified HDL on cholesterol efflux was detected in THP-1 macrophages. 25 In the present study we have focused on the role of apoAI-mediated cholesterol efflux which uses different mechanisms, but predominantly the ABCA1 pathway. The phospholipid content in HDL is an important determinant for HDL and cell surface interaction, 26 and EL has been shown to be capable of hydrolyzing phospholipids. Thus, it was proposed that the depletion of phospholipids in HDL by EL compromised the HDL affinity for the cell membrane, leading to decreased cholesterol efflux. The phospholipid content in apoAI may influence its ability to mediate cholesterol efflux as well. In one study, it was reported that the association of apoAI with phospholipids reduced its ability to interact with ABCA1. 18 Here, we speculate that the overexpression of EL may deplete phospholipids from lipidated apoA-I producing a form of apoAI that can more readily interact with ABCA1 and thus enhance cholesterol efflux. Moreover, the perturbation of membrane phospholipids by EL, despite the unchanged level of ABCA1 mRNA and protein after EL suppression or overexpression, may alter the ABCA1 function and subsequently contribute to EL-related apoAI-mediated cholesterol efflux.
Discussion
In the present study, EL stimulated apoAI-mediated cholesterol efflux by increasing apoAI binding on the cell surface which was dependent on the noncatalytic function of the lipase. EL bears a lipid-binding domain in the C terminus and can interact with apoB100. 27 EL was also shown to be able to interact with HDL for the selective uptake of cholesterol, 27 suggesting that EL may also have the ability to interact with apoAI. The observed increased apoAI docking by EL may promote the interaction between ABCA1 and apoAI so as to stimulate cholesterol efflux via ABCA1-mediated active transport.
Discussion
The fact that membrane phospholipid composition was altered in LPL-deficient patients when compared to normal controls 28 aroused our speculation that EL expression may change the membrane phospholipid composition so as to influence apoAI-mediated cholesterol efflux. Phospholipids have been shown to be the preferred substrates for EL. 29, 30 In this study, the relative levels of 2 major phospholipid components, phosphatidylcholine and sphingomyelin, were decreased with the overexpression of EL and increased after EL suppression. Phosphatidylcholine and sphingomyelin present in the plasma membrane are critical to maintain the 2 kinetic pools (fast and slow pools) of cholesterol because of their unique distribution. 23 The decreased membrane phosphatidylcholine content associated with EL overexpression may promote cholesterol desorption. Similarly, sphingomyelin forms strong hydrogen bonds with cholesterol, 31, 32 the degradation of sphingomyelin by EL can then accelerate the cholesterol liberation, as evidenced in fibroblasts, astrocytes, and CHO cells. [33] [34] [35] Other phospholipids like phosphatidylethanolamine, the level of which was changed with the modification of lipase expression, may also impose modulatory effects on membrane cholesterol stability. Throughout this process, apoAI docked on the cell surface by EL may become an immediate acceptor to receive cholesterol released from both fast and slow pools.
Discussion
We also found that the production of lyso-PC in macrophages and transfected FLP-IN 293 cells was positively correlated with lipase expression. Also, the direct measurement of Lyso-PC in EL-overexpressing macrophages supports that EL can hydrolyze phosphatidylcholine, which can originate from either the membrane compartment or the conditioned medium, to produce Lyso-PC. A dose-dependent increase in apoAI-mediated cholesterol efflux by Lyso-PC was demonstrated in macrophages in this study, which is consistent with a previous report. 20 The released Lyso-PC may incorporate into and microsolubilize apoAI to facilitate the acceptance of cholesterol. The metabolite of sphingomyelin, ceramide, was also reported to stimulate apoAImediated cholesterol efflux through an ABCA1 pathway. 36, 37 The decreased level of sphingomyelin observed after EL overexpression in this study may suggest that the production of ceramide may increase, and then enhance apoAI-mediated cholesterol efflux.
Discussion
In summary, endothelial lipase expression in macrophages promotes apoAI-mediated cholesterol efflux. Mechanisms explaining this observation may be related in part to the ability of EL to enhance apoAI binding and modify membrane lipid composition. Further research on the specific nature of the interaction of EL and apoAI should provide insight into these mechanisms. 
Sources of Funding


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