A novel approach to measuring macrophage-specific reverse cholesterol transport in vivo in humans
2017; Elsevier BV; Volume: 58; Issue: 4 Linguagem: Inglês
10.1194/jlr.m075226
ISSN1539-7262
AutoresMarina Cuchel, Anna Raper, Donna Conlon, Daniel A. Pryma, R. Freifelder, Rahul Poria, Debra Cromley, Xiaoyu Li, Richard L. Dunbar, Benjamin French, Liming Qu, William Farver, Ching‐Chiang Su, Sissel Lund‐Katz, Amanda Baer, Giacomo Ruotolo, Peter Åkerblad, Carol S. Ryan, Lan Xiao, Todd G. Kirchgessner, John S. Millar, Jeffrey T. Billheimer, Daniel J. Rader,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoReverse cholesterol transport (RCT) is thought to be an atheroprotective function of HDL, and macrophage-specific RCT in mice is inversely associated with atherosclerosis. We developed a novel method using 3H-cholesterol nanoparticles to selectively trace macrophage-specific RCT in vivo in humans. Use of 3H-cholesterol nanoparticles was initially tested in mice to assess the distribution of tracer and response to interventions known to increase RCT. Thirty healthy subjects received 3H-cholesterol nanoparticles intravenously, followed by blood and stool sample collection. Tracer counts were assessed in plasma, nonHDL, HDL, and fecal fractions. Data were analyzed by using multicompartmental modeling. Administration of 3H-cholesterol nanoparticles preferentially labeled macrophages of the reticuloendothelial system in mice, and counts were increased in mice treated with a liver X receptor agonist or reconstituted HDL, as compared with controls. In humans, tracer disappeared from plasma rapidly after injection of nanoparticles, followed by reappearance in HDL and nonHDL fractions. Counts present as free cholesterol increased rapidly and linearly in the first 240 min after nadir; counts in cholesteryl ester increased steadily over time. Estimates of fractional transfer rates of key RCT steps were obtained. These results support the use of 3H-cholesterol nanoparticles as a feasible approach for the measurement of macrophage RCT in vivo in humans. Reverse cholesterol transport (RCT) is thought to be an atheroprotective function of HDL, and macrophage-specific RCT in mice is inversely associated with atherosclerosis. We developed a novel method using 3H-cholesterol nanoparticles to selectively trace macrophage-specific RCT in vivo in humans. Use of 3H-cholesterol nanoparticles was initially tested in mice to assess the distribution of tracer and response to interventions known to increase RCT. Thirty healthy subjects received 3H-cholesterol nanoparticles intravenously, followed by blood and stool sample collection. Tracer counts were assessed in plasma, nonHDL, HDL, and fecal fractions. Data were analyzed by using multicompartmental modeling. Administration of 3H-cholesterol nanoparticles preferentially labeled macrophages of the reticuloendothelial system in mice, and counts were increased in mice treated with a liver X receptor agonist or reconstituted HDL, as compared with controls. In humans, tracer disappeared from plasma rapidly after injection of nanoparticles, followed by reappearance in HDL and nonHDL fractions. Counts present as free cholesterol increased rapidly and linearly in the first 240 min after nadir; counts in cholesteryl ester increased steadily over time. Estimates of fractional transfer rates of key RCT steps were obtained. These results support the use of 3H-cholesterol nanoparticles as a feasible approach for the measurement of macrophage RCT in vivo in humans. Although an association between plasma concentrations of HDL cholesterol (HDL-C) levels and CVD has been found in epidemiological studies, the causal basis of this association has been recently questioned by the negative results observed in human genetics studies and clinical trials (1Voight B.F. Peloso G.M. Orho-Melander M. Frikke-Schmidt R. Barbalic M. Jensen M.K. Hindy G. Holm H. Ding E.L. Johnson T. Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study.Lancet. 2012; 380: 572-580Abstract Full Text Full Text PDF PubMed Scopus (1648) Google Scholar, 2Rader D.J. Hovingh G.K. HDL and cardiovascular disease.Lancet. 2014; 384: 618-625Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). However, substantial interest remains in the concept that measures of HDL function may be more closely tied mechanistically to atherosclerosis (3Rosenson R.S. Brewer Jr., H.B. Davidson W.S. Fayad Z.A. Fuster V. Goldstein J. Hellerstein M. Jiang X.C. Phillips M.C. Rader D.J. Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport.Circulation. 2012; 125: 1905-1919Crossref PubMed Scopus (681) Google Scholar). One of the biological functions of HDL is its ability to promote cellular cholesterol efflux and the return of that cholesterol to the liver for excretion in the feces in a process known as reverse cholesterol transport (RCT) (4Cuchel M. Rader D.J. Macrophage reverse cholesterol transport: key to the regression of atherosclerosis?.Circulation. 2006; 113: 2548-2555Crossref PubMed Scopus (439) Google Scholar). By using an in vivo assay in mice that we developed to measure the rate of RCT from macrophages to liver and feces, a large number of studies from multiple laboratories have demonstrated that the rate of in vivo macrophage RCT is a much better predictor of the impact of a genetic or pharmacologic perturbation on atherosclerosis than is the simple measure of HDL-C itself (5Rader D.J. Alexander E.T. Weibel G.L. Billheimer J. Rothblat G.H. Role of reverse cholesterol transport in animals and humans and relationship to atherosclerosis.J. Lipid Res. 2009; 50: S189-S194Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar). Furthermore, using an ex vivo method developed to assess the cholesterol efflux capacity of HDL from a specific individual, we showed that this was strongly inversely associated with prevalent atherosclerosis, even after adjusting for HDL-C levels (6Khera A.V. Cuchel M. de la Llera-Moya M. Rodrigues A. Burke M.F. Jafri K. French B.C. Phillips J.A. Mucksavage M.L. Wilensky R.L. Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.N. Engl. J. Med. 2011; 364: 127-135Crossref PubMed Scopus (1532) Google Scholar). Similarly, cholesterol efflux capacity of HDL has been shown to be inversely associated with incident CVD (7Rohatgi A. Khera A. Berry J.D. Givens E.G. Ayers C.R. Wedin K.E. Neeland I.J. Yuhanna I.S. Rader D.R. de Lemos J.A. HDL cholesterol efflux capacity and incident cardiovascular events.N. Engl. J. Med. 2014; 371: 2383-2393Crossref PubMed Scopus (969) Google Scholar, 8Saleheen D. Scott R. Javad S. Zhao W. Rodrigues A. Picataggi A. Lukmanova D. Mucksavage M.L. Luben R. Billheimer J. Association of HDL cholesterol efflux capacity with incident coronary heart disease events: a prospective case-control study.Lancet Diabetes Endocrinol. 2015; 3: 507-513Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar) These results suggest that intervention to promote macrophage efflux and RCT might be an effective approach to prevent and treat atherosclerotic CVD. An approach to assess RCT in humans was reported (9Turner S. Voogt J. Davidson M. Glass A. Killion S. Decaris J. Mohammed H. Minehira K. Boban D. Murphy E. Measurement of reverse cholesterol transport pathways in humans: in vivo rates of free cholesterol efflux, esterification, and excretion.J. Am. Heart Assoc. 2012; 1: e001826Crossref PubMed Google Scholar), but this method is not macrophage-specific. A method to measure macrophage-specific RCT in humans would be useful, not only in assessing the role of key proteins regulating RCT in humans, but also in evaluating the potential of novel therapeutic approaches targeting cholesterol efflux and RCT. Nilsson and Zilversmit demonstrated that intravenous (i.v.) injection of rats with a saturated solution of radiolabeled unesterified cholesterol mixed with albumin rapidly disappeared from the blood compartment, primarily due to the uptake by reticuloendothelial (RE) cells (10Nilsson A. Zilversmit D.B. Fate of intravenously administered particulate and lipoprotein cholesterol in the rat.J. Lipid Res. 1972; 13: 32-38Abstract Full Text PDF PubMed Google Scholar). Schwartz et al. used a similar preparation to study cholesterol metabolic pathways in humans. Their results were consistent with the rapid clearance of the cholesterol-albumin complexes from the blood compartment and subsequent reappearance of the tracer on circulating HDL as free cholesterol (FC), suggesting that this approach may specifically measure the efflux of cholesterol from macrophage cells to HDL as sole acceptor (11Schwartz C.C. Berman M. Vlahcevic Z.R. Halloran L.G. Gregory D.H. Swell L. Multicompartmental analysis of cholesterol metabolism in man. Characterization of the hepatic bile acid and biliary cholesterol precursor sites.J. Clin. Invest. 1978; 61: 408-423Crossref PubMed Scopus (133) Google Scholar, 12Schwartz C.C. Vlahcevic Z.R. Berman M. Meadows J.G. Nisman R.M. Swell L. Central role of high density lipoprotein in plasma free cholesterol metabolism.J. Clin. Invest. 1982; 70: 105-116Crossref PubMed Scopus (38) Google Scholar, 13Schwartz C.C. Zech L.A. Vandenbroek J.M. Cooper P.S. Cholesterol kinetics in subjects with bile fistula. Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate.J. Clin. Invest. 1993; 91: 923-938Crossref PubMed Scopus (47) Google Scholar, 14Schwartz C.C. VandenBroek J.M. Cooper P.S. Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans.J. Lipid Res. 2004; 45: 1594-1607Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Based on these data, we hypothesized that cholesterol nanoparticles made of albumin-bound 3H-cholesterol could be used to target 3H-cholesterol selectively to macrophages in vivo and permit the assessment of macrophage-specific cholesterol efflux in vivo in humans. We present here the results of preclinical validation studies and a feasibility study in humans that were conducted to test the validity and feasibility of such approach. Animal studies were generally conducted in C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) fed a standard chow diet (catalog no. 5020, Research Diets, New Brunswick, NJ) ad libitum, unless otherwise stated. Cholesterol nanoparticles were prepared similarly to that for human studies (see below), unless otherwise stated, and injected via tail vein (100 μl solution containing 30 μCi/ml 3H-cholesterol). Blood and/or tissue were obtained at several time points postinjection. Plasma radioactivity was counted directly, and tissue radioactivity was determined after extraction by using SOLVABLE (PerkinElmer, Waltham, MA) according to the manufacturer's directions. Reconstituted HDL was prepared as previously described (15Cuchel M. Lund-Katz S. de la Llera-Moya M. Millar J.S. Chang D. Fuki I. Rothblat G.H. Phillips M.C. Rader D.J. Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 526-532Crossref PubMed Scopus (40) Google Scholar) and administered intraperitoneally at a dose of 3 mg apoA-I/200 μl PBS to age- and gender-matched mice. Validation studies using the liver X receptor (LXR) agonist BMS-779788 were conducted at Bristol-Myers Squibb (Princeton, NJ). Male C57Bl/6J (Jackson Laboratory) and LXR α (−/−), LXR β (−/−), and LXR α/ β double (−/−) (Bristol-Myers Squibb) mice on C57Bl/6 background were single-housed for acclimation and throughout the compound administration phase of the study. Water and standard rodent chow (Harlan Teklad, Frederick, MD) were provided ad libitum. Age-matched mice (12–24 weeks of age; n = 2 or 3 per group) were randomly assigned into the following treatment groups: vehicle [0.5% carboxymethyl cellulose and 0.2% Tween 80 (Sigma-Aldrich, St. Louis, MO)], or 30 mg/kg/d BMS-779788 and dosed at 9 AM, once a day, for 7 days by oral gavage. At 5 h after final dose, animals were anesthetized with inhaled 4% isofluorane and 0.06 ml of BSA (Sigma-Aldrich): 3H-cholesterol nanoparticles (60 μl per animal) were injected intravenously via the orbital plexus of 30 g nonfasted animals. Cholesterol nanoparticles were prepared by mechanical stirring of 972 µl 3% BSA in 0.9% saline with drop-wise addition of 108 µl (1.08 mCi, 2 Ci/mmol) of [3H]cholesterol in ethanol (PerkinElmer) at 600 rpm for 15 min at room temperature. Anesthesia concentration was reduced to 1% after injection and maintained throughout the study period in a humidified anesthesia chamber with tail access ports. Blood samples (25 µl) were collected into microvette plasma isolation tubes (Sarstedt, Nümbrecht, Germany) by venous tail nick from each mouse at 5, 15, 30, 40, 50, 60, 70, 90, 105, 120, 135, and 150 min postinjection. After isolation by centrifugation at room temperature, 10 µl plasma was added to 5.0 ml Ecolite scintillation fluid (MP Biomedical, Santa Ana, CA), and radioactivity was determined by liquid scintillation counting. Kupffer cells (KCs) were isolated from mouse liver as previously described (16Nagelkerke J.F. Barto K.P. van Berkel T.J. In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells.J. Biol. Chem. 1983; 258: 12221-12227Abstract Full Text PDF PubMed Google Scholar). Briefly, mice were anesthetized, and the inferior vena cava was cannulated. Subsequently, the vena porta was ligated, and the liver was perfused for 5 min with Liver Perfusion Buffer (Invitrogen, Tokyo, Japan) until the liver turned pale, then digested in situ with Liver Digest Solution (Invitrogen) for approximately 10 min. Parenchymal cells were isolated after mincing the liver in Hepatocyte Wash Medium (Invitrogen), filtering through nylon gauze, and centrifuging for three times 5 min at 50 g. The pellets consisted of pure (>99%) parenchymal cells as judged by phase-contrast light microscopy. The supernatants were centrifuged for 10 min at 500 g in order to harvest the nonparenchymal cells. By means of centrifugal elutriation, the endothelial cells (ECs) and KCs were separated according to the method of Nagelkerke et al. (16Nagelkerke J.F. Barto K.P. van Berkel T.J. In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells.J. Biol. Chem. 1983; 258: 12221-12227Abstract Full Text PDF PubMed Google Scholar). KCs were depleted in mice by using clodronate as previously described (17Van Rooijen N. Sanders A. Kupffer cell depletion by liposome-delivered drugs: comparative activity of intracellular clodronate, propamidine, and ethylenediaminetetraacetic acid.Hepatology. 1996; 23: 1239-1243Crossref PubMed Google Scholar). Briefly, mice were i.v. injected with 200 µl of liposome-encapsulated clodronate (1 mg), which is known to deplete KCs, or PBS encapsulated in multilamellar liposomes (ClodronateLiposomes.com, Amsterdam, The Netherlands). KC depletion was assessed by using quantitative RT-PCR to quantify liver F4/80 and Nramp1 mRNA. Results were normalized by mRNA levels of β-actin. After 48 h, the mice were injected via tail vein with 100 µl of 3H cholesterol nanoparticles and were bled and euthanized either 1 or 10 min after injection. Radioactivity was measured in plasma, liver, and red blood cells as described above. The cholesterol efflux of isolated KCs from C57BL/6 mice was assessed according to an established protocol using cis-retinoic acid and 22-hydroxycholesterol to upregulate the ABCA1 transporter and either apoA-I or the HDL fraction obtained by precipitation of ApoB using polyethylene glycol (PEG), as an acceptor (18Asztalos B.F. de la Llera-Moya M. Dallal G.E. Horvath K.V. Schaefer E.J. Rothblat G.H. Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux.J. Lipid Res. 2005; 46: 2246-2253Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). The University of Pennsylvania or the Bristol-Myers Squibb Institutional Animal Care and Use Committee approved the animal experiments, and all experiments were conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Albumin-bound 3H-cholesterol (cholesterol nanoparticles) was prepared in the Cyclotron Facility of the University of Pennsylvania under strict operating procedures that aimed to create complexes with uniform size characteristics. GMP cholesterol was obtained from Avanti Polar Lipids (Alabaster, AL), and 3H-cholesterol (40–60 Ci/mmol) was from Perkin Elmer. Cholesterol nanoparticles were prepared by adding 15 ml of 1% human serum albumin to a solution of cholesterol in a vial (150 µl unlabeled cholesterol, 1 mg/ml in ethanol, and 75–200 μl 3H-cholesterol, 1 mCi/ml). The solution was stirred for 15 min at 600 rpm and then filtered through a 0.22 µM filter to obtain the final product. The preparation, containing approximately 50–75 μCi 3H-cholesterol and 150 μg FC, was administered i.v. as a bolus within 1 h of being prepared. To determine the size of the cholesterol nanoparticles, analyses were performed on an unlabeled solution by using a laser-diffraction particle size distribution analyzer (catalog no. LA-950A, software version 5.00, Horiba, Kyoto, Japan) and via dynamic light-scattering analysis (Wyatt DynaPro plate reader, Wyatt Technology, Santa Barbara, CA; software Dynamics version 6.12.0.3 for data collection and evaluation). The two methods gave comparable results. A mean hydrodynamic radii (RH) of the cholesterol nanoparticles was determined to be 80–100 nm. We enrolled men and women ages 18–70 years (n = 30) in good overall health and with a range of HDL-C levels. Female subjects were included only if of nonchildbearing potential. Use of lipid-lowering drugs within the 6 weeks before dosing or during the study and/or prescription or nonprescription drugs within 2 weeks before dosing was exclusionary. Eligible subjects were admitted to the clinical research center within 30 days of screening. Subjects were asked to fast for a minimum of 12 h before their research unit admission and remain in the research unit for a total of 27 h. Upon admission, cannulation of the antecubital vein was performed in both arms (one was used for radiolabeled cholesterol administration and the other for blood sampling). Subjects were provided with a nonfat breakfast 3 h before the start of the tracer administration and a low-fat lunch and dinner at scheduled times after the injection of the tracer. Cholesterol nanoparticles were prepared as described above and administered within 1 h of preparation as a rapid i.v. bolus. Blood samples (18 ml) were collected in EDTA-containing vacutainer tubes before and several times after tracer administration and immediately placed on ice. Subjects were discharged from the research unit after the 24 h blood draw and returned to the research unit at 48 h, 72 h, 96 h, and 8 days after dosing for blood sampling and stool collection. The study was conducted under Food and Drug Administration Investigational New Drug Application 115,938 and approved by the University of Pennsylvania Institutional Review Board, and all subjects signed the approved informed consent. Blood samples were placed in ice and centrifuged within 20 min. A 2 ml aliquot of plasma was added in a 15 ml glass extraction tube containing 2 ml of HDL-precipitating solution which contains phosphotungstic acid (Thermo Scientific, Middletown, VA), vortexed, and centrifuged. The supernatant (HDL fraction) was collected and flash frozen in a 4 ml cryovial on dry ice and stored at −80°C. The nonHDL pellet was washed with 2 ml of PBS, vortexed, and centrifuged again. The wash was discarded, and the pellet was frozen in the glass tube at −80°C. The remaining plasma was aliquoted, flash frozen in dry ice, and stored at −80°C for further analysis and as backup. Measurement of a lipid panel was performed at several time points to evaluate the stability of cholesterol concentration during the study and to be used for calculation of specific activity. The blood cell pellet was retained and stored at 4°C. Stools were refrigerated at 4°C until analysis. Lipids were extracted by using standard Bligh-Dyer methodology (19Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42681) Google Scholar). Plasma, HDL, and nonHDL lipid extracts were then further processed to separate FC and cholesterol ester (CE) fraction with silica acid column chromatography. Red blood cells were washed with saline and reisolated, and lipids were extracted as described above. Stool collections were weighed and homogenized. Samples of >400 g from 25 individuals were homogenized, and a 3 g aliquot was saponified, lipids were extracted with hexane, and the cholesterol fraction was obtained by silica acid column chromatography. The presence of bile pigment prevented the determination of tracer in the stool bile acid fraction. Radioactivity in cholesterol extracts was determined by using liquid scintillation counting. Lipid and lipoproteins were measured by using standard commercial kits. Ex vivo cholesterol efflux capacity of the HDL fraction was measured as previously described (6Khera A.V. Cuchel M. de la Llera-Moya M. Rodrigues A. Burke M.F. Jafri K. French B.C. Phillips J.A. Mucksavage M.L. Wilensky R.L. Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.N. Engl. J. Med. 2011; 364: 127-135Crossref PubMed Scopus (1532) Google Scholar). The multicompartmental analysis was conducted by using the WinSAAM modeling program. The model (Fig. 1), based on the cholesterol model developed by Schwartz et al. (14Schwartz C.C. VandenBroek J.M. Cooper P.S. Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans.J. Lipid Res. 2004; 45: 1594-1607Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), features efflux of FC to HDL from cells of the RE system as a key measure of the RCT process. Because our protocol used a single tracer and did not involve bile sampling, the Schwartz model was modified so that all parameters could be identified with confidence. Previously published results provided estimates of the initial parameters utilized during the fitting process (13Schwartz C.C. Zech L.A. Vandenbroek J.M. Cooper P.S. Cholesterol kinetics in subjects with bile fistula. Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate.J. Clin. Invest. 1993; 91: 923-938Crossref PubMed Scopus (47) Google Scholar, 15Cuchel M. Lund-Katz S. de la Llera-Moya M. Millar J.S. Chang D. Fuki I. Rothblat G.H. Phillips M.C. Rader D.J. Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 526-532Crossref PubMed Scopus (40) Google Scholar). FC on HDL can transfer to nonHDL particles, red blood cells, or peripheral tissue cholesterol (a composite of cholesterol from all nonliver tissues, including steroidogenic tissues) or be esterified, at which point it either transfers to nonHDL particles or is taken up by liver. Cholesterol taken up by liver is either resecreted into plasma as FC onto HDL or nonHDL, accounting for recycling of the labeled cholesterol tracer or excreted in feces. The statistical analysis of data obtained in the human study was performed with R (Version 3.0.1; R Development Core Team, Vienna, Austria). Subject characteristics were summarized by using standard descriptive statistics. The slope of appearance between 60 and 240 min postinjection was estimated for each subject by using a linear model for relevant parameters. Pearson coefficient was used to assess correlation between variables of interest. Statistical significance was defined as P ≤ 0.05. After the bolus administration of nanoparticle cholesterol in WT mice, the tracer rapidly disappeared (half-life < 1min) from circulation, reaching a nadir within 10 min postinjection, followed by reappearance in plasma that plateaued by 2 h and then began declining at 6 h postinjection (Fig. 2A). Ten minutes after injection, the cholesterol tracer was distributed primarily in the liver and the spleen (Fig. 2B). By 6 and 24 h after injection, the total counts in the liver and spleen were decreased, and counts in other tissues, such as adrenal, kidney, and heart, were detectable (Fig. 2B). Isolation of liver parenchymal and nonparenchymal cells showed that, on a milligram of protein basis, the amount of tracer in nonparenchymal cells was >20 times that of parenchymal cells (8.7 ± 3.8 vs. 0.4 ± 0.1%/mg protein) (Fig. 3A). Six h after injection, the tracer amount present in the nonparenchymal cells had decreased to 1.8 ± 0.5%/mg protein (Fig. 3A), whereas that in parenchymal cells remained essentially unchanged (0.4 ± 0.08% injected dose/mg).Fig. 3A: 3H-cholesterol distribution, as percentage injected counts per mg of protein, in liver parenchymal (open bars) and nonparenchymal (filled bars) cells at 10 and 360 min after administration of a saline solution containing 3H-cholesterol. B–D: Mice were injected with PBS or clodronate-encased liposomes and injected with 3H-cholesterol 2 days later. Mice were euthanized at either 1 or 10 min postinjection, and 3H counts were measured in plasma, red blood cell pellet, and liver. Data are a percentage of total injected dose. n = 5 mice/group/time point.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We assumed that the liver nonparenchymal cells responsible for uptake of the cholesterol nanoparticles were cells of the RE system, namely KCs. To further substantiate our assumption, KCs were depleted from mice by using clodronate liposomes. After 48 h of clodronate treatment, KCs were depleted by >90%, as judged by decrease in mRNA of macrophage-specific markers F4/80 and Nramp1 (data not shown). The removal of cholesterol nanoparticle from the plasma of mice that received PBS is extremely rapid with a half-life of <1 min and with <3% of plasma counts remaining after 10 min (Fig. 3B). The depletion of KCs in mice that received clodronate resulted in a reduced clearance of the tracer, with 21% of plasma counts remaining after 10 min (Fig. 3B). This was accompanied by a 2- to 3-fold increase in tracer in the red blood cell fraction (Fig. 3C) at both 1 min (8.1 ± 0.9% vs. 4.5 ± 0.8%) and 10 min (4.5 ± 1.5% vs. 1.3 ± 0.2%). Importantly, the uptake by the liver at 1 min was reduced by 50% (40 ± 2% vs. 19 ± 5% of injected dose) and remained decreased at 10 min (54 ± 2% vs. 30 ± 3%) (Fig. 3D). To test their ability to efflux cholesterol to known acceptors, isolated murine KCs demonstrated physiological cholesterol efflux to HDL and apoA-I that was responsive to LXR activation with 9-cis-retinoic acid/22-R hydroxycholesterol to stimulate ABCA1 expression comparable with that seen in macrophages (Fig. 4). To validate the method, we conducted studies using two interventions expected to increase cholesterol efflux and RCT. The effect of LXR agonism, a mechanism known to stimulate RCT in other assays, was evaluated first. C57Bl/6 mice were treated with the LXR agonist BMS-779788 (20Kirchgessner T.G. Martin R. Sleph P. Grimm D. Liu X. Lupisella J. Smalley J. Narayanan R. Xie Y. Ostrowski J. Pharmacological characterization of a novel liver X receptor agonist with partial LXRalpha activity and a favorable window in nonhuman primates.J. Pharmacol. Exp. Ther. 2015; 352: 305-314Crossref PubMed Scopus (29) Google Scholar) or vehicle for 7 days, followed by the administration of [3H]cholesterol nanoparticles. Within 30 min, 98% of the injected label was cleared from the plasma in both treatment groups. Label subsequently reappeared in the vehicle-treated group, and this rate was increased in BMS-779788-treated animals (Fig. 5A). Similar results were obtained in LXRβ KO animals (Fig. 5C). However, no reappearance of label in plasma was apparent in either vehicle or LXR agonist-treated groups in LXRα KO and LXRα/β double-KO mice (Fig. 5B, D). This suggests that in this model, both basal and LXR agonist stimulated macrophage cholesterol efflux is LXRα-dependent, but not LXRβ-dependent. Second, mice were infused with apoA-I/phospholipid complexes (rHDL) or saline as we previously described (15Cuchel M. Lund-Katz S. de la Llera-Moya M. Millar J.S. Chang D. Fuki I. Rothblat G.H. Phillips M.C. Rader D.J. Pathways by which reconstituted high-density lipoprotein mobilizes free cholesterol from whole body and from macrophages.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 526-532Crossref PubMed Scopus (40) Google Scholar). As expected, rHDL infusion resulted in a significant increase in cholesterol mass in plasma (Fig. 6A). Notably, rHDL infusion also resulted in a significant increase in the rate of reappearance of cholesterol tracer after nanoparticle injection, with plasma counts at 2 h 3-fold higher in the rHDL group compared with the saline control group (Fig. 6B). A total of 30 healthy subjects were enrolled into the study. Demographic characteristics and baseline lipid profiles are shown in Table 1.TABLE 1Demographic characteristics and lipid profile of study subjects (n = 30)Age, years36.1 ± 14.5Male, n (%)28 (93)RaceCaucasian, n (%)19 (63)Black, n (%)9 (30)Other, n (%)2 (7)BMI (kg/m2)25.6 ± 4.1Systolic BP (mmHg)120 ± 10Diastolic BP (mmHg)73 ± 9Lipid profile (mg/dl)Total Cholesterol173 ± 42.0LDL-Cholesterol105 ± 33HDL-Cholesterol50 ± 16Triglycerides70 (56–110)Apo A-I128 ± 28ApoB76 ± 23Lp(a)21 (9–54)Cholesterol efflux0.698 ± 0.1556Data are expressed as mean ± SD or median (interquartile range), if not otherwise specified. Cholesterol efflux: ex vivo cholesterol efflux capacity is reported as value normalized to the value of a pooled control serum, as previously described (8Saleheen D. Scott R. Javad S. Zhao W. Rodrigues A. Picataggi A. Lukmanova D. Mucksavage M.L. Luben R. Billheimer J. Association of HDL cholesterol efflux capacity with incident coronary heart disease events: a prospective case-contr
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