s
The role of abca1 in atherosclerosis: lessons from in vitro and in vivo models
Singaraja, R.R.
Publication date
2003
Link to publication
Citation for published version (APA):
Singaraja, R. R. (2003). The role of abca1 in atherosclerosis: lessons from in vitro and in vivo
models.
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Chapter r
Alterationss of plasma lipids in mice via adenoviral
mediatedd hepatic overexpression of human ABCA1
Cheryll L. Wellington
1, Liam R. Brunham
1, Steven Zhou
1, Roshni R. Singaraja',
Henkk Visscher
1, Allison Gelfer
1, Colin Ross
1, Erick James
1, Guoqing Liu
1, Mary
T.. Huber
2, Yu-Zhou Yang
1, Robin J. Parks
3, Albert Groen
4,
Jamilaa Fruchart-Najib
5, and Michael R. Hayden
1211
Centre for Molecular Medicine and Therapeutics, British Columbia Children's and Women's Hospital,, University of British Columbia, Vancouver, BC, Canada
zz
Xenon Genetics, Inc., Vancouver, BC, Canada
11
Molecular Medicine Program, Ottawa Health Research Institute, Ottawa, ON, Canada 44
Academic Medical Centre, Amsterdam, the Netherlands ::
Institute Pasteur, Lille, France
Abstract t
ABCA11 is a widely expressed lipid transporter essential for the generation of HDL. ABCA1 is particularlyy abundant in the liver, suggesting that the liver may play a major role in HDL homeostasis.. To determine how hepatic ABCA1 affects plasma HDL-C levels, we treated mice withh an adenovirus expressing human ABCA1 under the control of the CMV promoter. Treated micee showed a dose-dependent increase in hepatic ABCA1 protein, ranging from 1.2-fold to 8.3-foldd using doses from 5 x 10" - 1.5 x 10' pfu, with maximal expression observed on day 3 posttreatment.. A selective increase in HDL-C occurred at day 3 in mice treated with 5 x 10: pfu Ad-ABCA1,, but higher doses did not further elevate HDL-C levels. In contrast, TC, TG, PL, non-HDL-CC and apoB levels all increased in a dose-dependent manner, suggesting that excessive overexpressionn of hepatic ABCA1 in the absence of its normal regulatory sequences altered totall lipid homeostasis. At comparable expression levels, BAC transgenic mice, which express ABCA11 under the control of its endogenous regulatory sequences, showed that a greater and moree specific increase in HDL-C than Ad-ABCA1-treated mice. Our results suggest that appropriate regulationn of ABCA1 is critical for a selective increase in HDL-C levels.
Thee contribution of hepatic ABCA1 to lipoprotein homeostasis
Introduction n
ABCA11 is an ATP-ase binding cassette transporter that is essential for the transfer of phospholipid andd cholesterol to lipid-free apoA1 to form pre-(VHDL (1-3). This is the first step in reverse cholesteioll tianspuit ( K U ; , whereby excess cholesterol is removed from cells and transported ass HDL to the liver for eventual excretion in bile (4-8). Absence of ABCA1 activity results in Tangierr Disease (TD), which is characterized by a nearly complete loss of circulating HDL, an accumulationn of cholesterol ester particularly in cells of the reticuloendothelial system, and an increasedd risk of coronary artery disease (9-14).
Sincee the discovery of a highly significant inverse relationship between HDL levels and coronary arteryy disease, the metabolic origin of HDL has been a subject of intense investigation. The discoveryy that ABCA1 is essential for HDL biosynthesis has provided a unique opportunity to investigatee RCT in detail. The prior hypothesis of RCT proposes that the majority of plasma HDL originatess from peripheral tissues (4-8). However, ABCA1 is abundantly expressed in the liver comparedd to other tissues that participate in RCT (15,16), suggesting that hepatocytes may themselvess may play a major role in HDL biosynthesis. This is supported by the observation thatt hepatocytes are the site of greatest accumulation of cholesterol ester in the Wisconsin Hypo-Alphaa mutant (WHAM) chicken, a naturally occurring animal model of ABCA1 deficiency (17).. Furthermore, reconstitution of ABCA1-deficient mice with bone marrow from wild-type micee does not result in restoration of plasma HDL-cholesterol levels, suggesting that monocytes andd macrophages contribute only marginally to HDL-C levels in vivo (18). Finally, hepatocytes aree centrally involved in lipid homeostasis, and the mass of the liver relative to other organs is sufficientt to make a major contribution to plasma HDL-C.
Thesee observations suggested that manipulation of ABCA1 in the liver may be a promising therapeuticc strategy aimed at increasing plasma HDL-C levels and protecting from atherosclerosis. However,, three crucial interrelated questions remain to be addressed. Firstly, it is important to determinee if there may be an optimal level of hepatic ABCA1 expression that results in a selectivee increase in plasma HDL-C. Secondly, because overall lipid homeostasis is significantly coordinatedd by the liver, it is important to understand not only how the regulation of ABCA1 inn the liver affects lipid efflux and formation of HDL particles, but also how hepatic ABCA1 mayy affect overall plasma lipids Finally, it is essentia! to establish how HDL-C levels may be influencedd by ABCA1 in extrahepatic tissues such as the intestine.
Wee recently used transgenic technologies to produce bacterial artificial chromosome (BAC) transgenicc mice expressing human ABCA1 (19). Importantly, the use of a BAC ensures that endogenouss regulatory elements within the ABCA1 transgene are present and that ABCA1 is expressedd with normal temporal, tissue-, and cell-specific patterns. We have shown that activation off LXREs by oxysterols in vivo directly contributes to an increase in human-specific ABCA1 mRNAA and leads to increased expression of the human ABCA1 protein in an identical pattern
ass the endogenous gene (19). This appropriate regulation of ABCA1 results in a 1.6-fold increasee in plasma HDL-C levels without affecting other lipoprotein classes (19). Additionally, byy crossing our BAC transgenic mice onto the well-established apoE -/- model of atherosclerosis, wee have demonstrated that increased ABCA1 activity is associated with markedly reduced lesionn size as well as the appearance of less complex lesions that lack fibrous caps and exhibit reducedd foam cell involvement (20). These studies validate the BAC transgenic model as an excellentt research tool to compare the impact of increased ABCA1 expression due to endogenous versuss other promoters on plasma lipid levels.
Inn order to evaluate how hepatic ABCA1 expression in the absence of its appropriate regulatory signalss affects plasma HDL-C levels, we developed an adenovirus expressing full-length human ABCA11 under the control of the CMV promoter (Ad-ABCA1). This vector allowed exogenous ABCA11 to be specifically overexpressed in the liver such that it is uncoupled from its normal regulatoryy circuits, and these results could be compared to results of ABCA1 overexpression in thee BAC transgenic mice. In addition, mice could be treated with various doses of Ad-ABCA1 inn order to determine how increasing hepatic dosage of ABCA1 affected plasma HDL-C levels. Ourr results suggest that the abundance and regulation of ABCA1 in the liver and extrahepatic tissuess are crucial parameters in the regulation of plasma HDL-C levels in vivo.
Materialss and Methods
Recombinantt adenoviral vectors
Thee full-length human ABCA1 cDNA was subcloned into an E1/E3 -deleted recombinant adenoviruss (Ad5) vector backbone containing the CMV promoter-enhancer to generate Ad-ABCA1.. Plaques positive for ABCA1 expression were identified and used for large-scale amplificationn in 293 cells g r o w n in 150 mm culture dishes. Similarly, a recombinant Ad5 vectorr carrying the reporter alkaline phosphatase gene (Ad-AP, (21)) was used for concurrent controll infections. Purification of these recombinant adenoviral vectors was performed by t w o sequentiall rounds of CsCI density gradient ultracentnfugation. The purified virus stocks were desaltedd over a Sephadex G-25 column (Amersham Pharmacia) and eluted in sterile Hepes Bufferedd Saline (HBS). Glycerol (15%) was added and the viral stock was stored at - 8 0 ' C until use.. Infectious viral titres were determined using the Adeno-X Rapid Titer Kit (Clontech) as suggestedd by the manufacturer. The replication incompetence of the E1/E3-deleted recombinant viruss was verified by the lack of cytopathic effect after infection of COS-1 cells, which, unlike 2933 cells, do not complement the gene deletion.
Celll culture
HeLaa and HepG2 cells were cultured in DMEM high glucose (Canadian Life Technologies) supplementedd with 10% fetal calf serum, 50 U/ml penicillin-streptomycin, and 2 mmol glutamine
Thee contribution of hepatic ABCA1 to lipoprotein homeostasis
(Canadiann Life Technologies). For in vitro infection, cells were seeded in 24-well dishes at a densityy of 20,000 cells/well. After 16 h, the conditioned media was removed and cells were incubatedd with various dilutions of stock virus in DMEM with 2% FBS for one hour at 37 C, afterr which the conditioned media was replaced. After an additional 16-24 h, cells were used forr efflux assays or Western analysis.
Animalss and in vivo delivery of recombinant adenovirus
Malee wild-type C57BI/6 mice obtained from Jackson Laboratories were maintained on a standard choww diet (PMI Feeds). Adenoviral vectors were diluted in sterile PBS to deliver doses of 5 x 10" pfu/mouse,, 7.5 x 10" pfu/mouse, 1 x 10'' pfu/mouse, and 1.5 x 109 pfu/mouse for Ad-ABCA1, andd 1.5 x 10' pfu/mouse for Ad-AP. The dose of Ad-AP was matched to the maximum dose of Ad-ABCAII in order to control for any adverse effects due to high-dose adenovirus infection in vivo.. A total of 200 ml of diluted virus was injected into the tail vein of immobilized mice. Additionall mice received 200 ml of PBS only as a vehicle control. Preinjection biood samples weree collected from the saphenous vein or retroorbital plexus. After 3 or 7 days posttreatment, animalss were sacrificed by CO inhalation, blood was collected by cardiac puncture, and liver, spleen,, and small intestine were isolated and immediately frozen at - 8 0:C . BAC transgenic and
wild-typee control mice were maintained on chow or atherogenic diets as previously described (19).. All procedures involving experimental animals were performed in accordance with protocols formm the Canadian Council of Animal Care and the University of British Columbia Animal Care Committees. .
W e s t e r nn blot and immunohistochemistry
Tissuess were homogenized as previously described (22) and protein concentration was determinedd by Lowry assay. Equal amounts of protein were electrophoresed through 7.5% SDSS polyacrylamide gels, electrophoretically transferred to polyvinylidene fluoride (PVDF) membranee (Millipore) and immunodetected using a monoclonal anti-ABCA1 antibody raised againstt the second nucleotide binding domain (NBD2 of ABCA1) or anti-GAPDH (Chemicon) ass a loading control (22). Blots were developed using ECL (Amersham) according to the manufacturer'ss recommendations. Bands were quantitated by densitometry using NIH Image 6.1.. In each lane, ABCA1 levels were normalized to GAPDH levels to control internally for proteinn loading. Data represent the mean and standard deviation from tissue from 3-4 animals, eachh run at least in duplicate.
Forr histochemicaf analysis, tissues were fixed for 16 h in 4 % paraformaldehyde in phosphate bufferedd saline (PBS) prior to paraffin embedding. Hematoxylin-eosin and Giemsa staining weree performed according to standard procedures by the UBC Histology Core Service.
RNAA analysis
RNAA from mouse liver was extracted using Trizol (Canadian Life Technologies) and purified usingg RNeasy reagents (Clontech) as recommended by the manufacturer. The relative levels of humann and murine ABCA1 were determined by semiquantitative RT-PCR from 3 mg of total RNAA as described (15).
Cholesteroll and phosphatidylcholine efflux
Ad-ABCA11 or mock infected HeLa cells and fibroblasts were labelled with 1 mCi/ml of 3H-cholesteroll (New England Nuclear) or 3PTcholine (Amersham) for 16 h. Labelled cells were then washedd and equilibrated in DMEM with 0.2% BSA for 1 h, after which 20 mM of lipid-free apoA1 (Calbiochem)) was added for 4 h. Efflux assays were performed as described previously (23). For cholesteroll efflux, media and ceil lysates were mixed with sontillant and counted directly (23). Forr phosphatidylcholine efflux, lipids from the media and cell lysates were first extracted with chloroform:methanoll (2:1) prior to detection (24). Data are representative of the mean and standardd deviations from at least t w o independent experiments, each performed in triplicate.
Analysiss of bile and fecal sterol levels
Galll bladder bile was aspirated at sacrifice using a 30G needle and frozen immediately at -80:CC until use. Feces generated over a 24 h period were collected pre- and post-injection and storedd at - 8 0 C until use. Biliary bile salt and cholesterol concentrations were determined as previouslyy described (25). Neutral and acidic fecal sterol content was determined by gas liquid chromatographicc analysis as described (25).
Plasmaa lipid and lipoprotein analysis
Alll blood samples were collected following a 4 h fast. After centrifugation for 10 min at 12,0000 rpm at 4 C, plasma was frozen at - 8 0 ' C until use. Serum and lipoprotein lipid (cholesterol, triglycerides,, phospholipids) concentrations were determined by enzymatic assays adapted to microtiterr plates using commercially available reagents (Boehringer Mannheim, Germany) as previouslyy described (20). Serum HDL-cholesterol levels were determined after precipitation of ApoB-containingg lipoproteins w i t h phosphotungstic a c i d / M g (Roche Diagnostics GmbH, Mannheim).. Serum levels of apoB were measured by an immunonephelemetnc assay using a specificc mouse polyclonal antibody as previously described (20). Tabulated data represent the meanss and standard deviations of 3-4 mice per group. Relative changes in day 3 plasma lipids inn Ad-ABCAI or Ad-AP treated mice were determined by normalizing values from treated animalss to PBS-only controls. Data was then expressed as the percent change relative to the baselinee observed in PBS-treated mice. Similarly, relative changes in plasma lipids in BAC transgenic micee were determined by normalized values in BAC transgenic mice, on either a chow or atherogenicc diet (19), to those observed in nontransgenic controls on the same diet. Data was
Thee contribution ot hepatic ABCA1 to lipoprotein homeostasis
thenn expressed as percent change relative to the baseline in wild-type animals. Data represent thee means and standard deviations from at least 4 BAC or 4 wild-type mice. Statistical analysis wass by one-way ANOVA with a Neuman-Keuls posttest, using Graph-Pad Prism 3.03.
6 6 5 5 x x 22 4 it t
55
3
2 2 1 1 0 0 HeLa a 1 1 1 1 Ad-ABCA11 (MOI = 25) phosphocholine e cholesterol l ABCA1 1 GAPDH H HepG2 2 -apoA1 1 ++ apoA1 7.5 5 75 5 750 0 MOI I ABCA1 1 mockk 7.5 Ad-ABCA11 MOI. .
GAPDH HFiguree 1. Functional activity of Ad-ABCA1 in cultured cells. (A) ApoA1-dependent cholesterol and phosphatidylcholine effluxx are specifically increased in HeLa cells infected with Ad-ABCA1. Cells were either mock infected (-) or infected withh Ad-ABCA1 at an MOI of 25 (+), labelled with 3H-cholesterol or 3H-choline, and exposed to 20 mg/ml lipid-free apoA11 (black bars) or left untreated (stippled bars) for4h. Media and cells were collected separately and used to determinee percent efflux. Data represent the mean and standard deviations of two independent, pooled experiments, eachh performed in triplicate. Western blot analysis (inset) demonstrates high expression of ABCA1 only in infected cells.. (B) Dose-dependent increase in cholesterol efflux in HepG2 cells by Ad-ABCA1. Cells were either mock infected (-)) or infected with Ad-ABCA1 at MOIs of 7.5, 75, and 750, labelled with 3H-cholesterol, and exposed to 20 mg/ml lipid-freee apoA1 (black bars) or left untreated (clear bars) for 4h. Media and cells were collected separately and used too determine percent efflux. Data represent the mean and standard deviations of triplicate measurements. Western blott analysis (inset) demonstrates high expression of ABCA1 only in infected cells.
Results s
Adenovirall delivery of human ABCA1 promotes cholesterol and phospholipid efflux in culturedd cells
Expressionn and physiological activity of the recombinant adenovirus expressing human ABCA1 (Ad-ABCA1)) was first confirmed in vitro. HeLa cells express very little endogenous ABCA1 and havee a correspondingly low level of basal cholesterol or phosphatidylcholine efflux. HeLa cells infectedd with Ad-ABCA1 at a multiplicity of infection (MOI) of 25 demonstrated enhanced
Dayy 3 Ad-ABCA11 Ad-AP OO O O O O OO LO O Dayy 7 Ad-ABCA11 Ad-AP oo o o o o Liver r ABCA1 1 GAPDH H Spleen n ABCA1 1 GAPDH H
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xx x x x -n -oo m o m ^ ^ 55 h - ^ ^ dayy 3 dayy 7Figuree 2. Dose-dependent increase in ABCA1 expression specifically in the livers of Ad-ABCA1-treated mice. (A) Westernn blots of liver and spleen following injection of mice with the indicated doses of Ad-ABCA1 (5.0 x 10a -1.55 x 10" pfu/mouse), 1.5 x 109 pfu/mouse of Ad-AP, or PBS as a vehicle only control. Total protein lysates preparedd from tissues harvested 3 or 7 days postmjection were immunodetected with antibodies specific for ABCA11 and GAPDH to serve as a loading control. (B) Quantitation of ABCA1 expression ABCA1 abundance was normalizedd to GAPDH levels using densitometry. Pooled data (means and standard deviations) were generated fromm at least two independent gels for each of the 3-4 mice per group, and expressed relative to endogenous ABCA11 levels in PBS-treated mice.
Tablee 1: Lipid values in Ad-ABCA1-treated mice (N=4, p relative to PBS-treated mice)
Virus s Ad-ABCA1 1 Ad-ABCA1 1 Ad-ABCA1 1 Ad-ABCA1 1 Ad-AP P PBS S Dose e pfu/mouse e 5x10= = 7.55 x 10= 11 x 10= 1.55 x 10' 1.55 x 10-Day y 3 3 7 7 3 3 7 7 3 3 7 7 3 3 7 7 .: : 7 7 3 3 7 7 Mean n 134,37 7 108,25 5 121,95 5 143,38 8 148,90 0 131,08 8 183,03 3 155,18 8 104,35 5 96,43 3 100,03 3 82,83 3 TC(mg/dl) ) Stdd dev 3,95 5 4,92 2 6,88 8 9,26 6 14,28 8 16,80 0 10,73 3 5,12 2 4,09 9 21,00 0 15,92 2 8,35 5 P P <00 01 <0.05 5 >0.05 5 <0.00' ' <0.00' ' <0.00 0 <0.00 0 <0.00 0 >0.05 5 >0.05 5 Mean n 122,13 3 136,78 8 142,70 0 162,10 0 150,87 7 143,78 8 190,03 3 155,85 5 139,68 8 112,27 7 154,35 5 116,63 3 TG(mg/dl) ) Stdd dev 15,25 5 15,05 5 24,71 1 11,42 2 45,20 0 19,72 2 18,11 1 3,85 5 12,33 3 11,04 4 12,77 7 19,54 4 P P >0.05 5 >0.05 5 >0.05 5 >0.05 5 >0.05 5 >0.05 5 >0.05 5 >0.05 5 >0.05 5 >0.05 5 Mean n 258,30 0 239,25 5 240,90 0 304,43 3 284,63 3 273,50 0 347,78 8 310,55 5 234,50 0 228,77 7 220,55 5 189,50 0 PL(mg/dl) ) Stdd dev 6,35 5 17,93 3 25,47 7 10,88 8 7,54 4 32,62 2 24,21 1 7,81 1 7,77 7 65,77 7 30,35 5 23,69 9 P P >0.05 5 >0.05 5 >0.05 5 <0.001 1 <0.05 5 O . 0 1 1 <0.001 1 <0.001 1 >0.05 5 >0.05 5
Thee contribution of hepatic ABCA1 to lipoprotein homeostasis
Humann ABCA1 is expressed specifically in liver following injection of Ad-ABCA1 Havingg validated the functional activity of Ad-ABCA1, male wild-type C57BI/6 mice received dosess of Ad-ABCA1 ranging from 5.0 x 10" - 1.5 x 10'' pfu/mouse, or a control virus expressing alkalinpp phosphatase (Ad-AP) M 1 5 x 10' pfu/mcuse, cr PBS as a .-chicle only Luntiul, Ly Ictil veinn injection. Liver, spleen and small intestine were harvested 3 or 7 days after injection and probedd with an ABCA1-specific antibody to monitor expression levels in multiple tissues. As expectedd by intravenous delivery, exogenous ABCA1 expression was observed only in the liver (26),, which exhibited a dose-dependent response of ABCA1 expression (Figure 2). For example, aa 8.3-fold increase in ABCA1 protein levels was observed in animals injected with the highest dosee of Ad-ABCA! (1.5 x 10"' pfu/mouse) by 3 days and declined by approximately 4 0 % by dayy 7 (Figure 2A). No increase in ABCA1 expression was observed in spleen (Figure 2) or small intestinee (data not shown). Furthermore, RT-PCR analysis of RNA extracted from livers of Ad-ABCA1-injectedd mice confirmed that the increase in ABCA1 protein levels resulted from expression off exogenous human ABCA1 and not by upregulation of endogenous murine ABCA1 (data nott shown). These results demonstrated that ABCA1 abundance increased in a dose-dependent mannerr specifically in the livers of Ad-ABCA1 infected mice.
Plasmaa HDL-C levels are elevated in Ad-ABCA1 -treated mice, but not in a dose-dependent manner r
Ass expected, HDL-C levels were increased in Ad-ABCA1 infected mice compared t o both PBS andd Ad-AP controls, and maximal elevation of HDL-C was typically observed at day 3 in all AD-ABCA1-treatedd mice (Table 1). By day 7, HDL-C levels tended to decrease, as expected due to thee decline in ABCA1 expression in the liver at that time (Figure 2). Mice receiving the lowest dosee of Ad-ABCA1 (5.0 x 10" pfu), which resulted in a 1.2-fold increase in ABCA1 expression (Figuree 2), had a significant 27% increase in plasma HDL-C levels (Table 1, p < 0.05). Intermediate
HDL-CC (mg/dl) non-HDL-C (mg/dl) apoB (mg/dl) Mean n 1Q1 ,83 3 76,70 0 91,22 2 97,45 5 98,17 7 85,65 5 111,25 5 94,98 8 82,65 5 67,93 3 79,83 3 75,65 5 Stdd dev 77 75 5,42 2 12,64 4 3,47 7 2,73 3 11,15 5 7,32 2 7,02 2 5,55 5 9,00 0 16,58 8 5,79 9 P P ""00 05 >00 05 >0.05 5 ' 0 . 0 5 5 >0.05 5 > 0 0 5 5 ' 0 0 0 1 1 >0.05 5 > 0 0 5 5 >0.05 5 Mean n ?->?-> C 1 31,53 3 30,73 3 45,95 5 50,00 0 45,45 5 71,75 5 60,18 8 21,70 0 28,50 0 20,20 0 7,13 3 Stdd dev oo n t ; 5,09 9 7,40 0 7,93 3 8,23 3 10,45 5 12,83 3 4,60 0 6,81 1 14,80 0 10,17 7 3,55 5 P P >> U . U J ' 0 . 0 1 1 >0.05 5 ' 0 . 0 0 1 1 ' 0 . 0 1 1 ' 0 0 0 1 1 ' 0 0 0 1 1 ' 0 . 0 0 1 1 >00 05 ' 0 . 0 5 5 Mean n 31,04 4 39,72 2 39,00 0 44,48 8 39,36 6 41,28 8 57,32 2 52,32 2 36,24 4 23,57 7 38,20 0 18,72 2 Stdd dev 2,50 0 6,48 8 5,91 1 1,36 6 5,69 9 4,46 6 3,27 7 4,95 5 3,43 3 7,26 6 6,94 4 3,29 9 P P -*0.05 5 <0.001 1 >0.05 5 <0.001 1 >0.05 5 ' 0 . 0 0 1 1 <0.001 1 <0.001 1 >0.05 5 >0.05 5
dosess of exogenous Ad-ABCA1 (7.5 x 10" and 1.0 x 10' pfu) did not result in further significant elevationss of plasma HDL-C, despite a 2.6-fold or 6.5-fold increase in hepatic ABCA1 levels, respectivelyy (Figure 2). HDL-C levels were, however, significantly increased by 39% in mice treatedd with the highest dose of Ad-ABCA1 (1.5 x 10' pfu) that resulted in an 8.3-fold overexpressionn in hepatic ABCA1 levels (Table 1, p < 0.001). These results confirm that increased hepaticc ABCA1 raises HDL-C, in agreement with our previously published findings in chow-fed ABCA11 BAC transgenic mice (19).
However,, these results also give the first indication that the normal regulation of ABCA1 is likelyy to be important for selectively and maximally increasing plasma HDL-C. Compared to the Ad-ABCA11 treated mice, the greatest increase in plasma HDL-C levels was observed in BAC transgenicc animals in which a 1.6-fold increase in appropriately expressed ABCA1 protein in thee liver was sufficient to increase plasma HDL-C levels by 65% (19). We note that because the
CHOLESTEROLL day-3 15 5 10 0
A .. . ^
100 20 30 40 50 60 fractionn number Ad-ABCA11 (5X108) Ad-ABC11 (7.5x108| Ad-ABC11 (1X109| Ad-ABC11 (1,5X109) Ad-ALKK phos (1.5X109) PBS S 70 0 C H O L E S T E R O LL day-7 15 5 10 0 5 5 £ £& &
10 0 200 30 40 50 fractionn number 60 0 70 0 -Ad-ABCAII (5X108) Ad-ABC11 (7.5x108) Ad-ABC11 (1X109) Ad-ABC11 (1,5X109) -Ad-ALKK phos (1.5X109) PBS SFiguree 3. Cholesterol content of lipoproteins fractionated by FPLC. Plasma samples were collected 3 or 7 days afterr injection with the indicated dose (pfu/mouse) of Ad-ABCA1, Ad-AP, or PBS as indicated. Fraction numbers aree indicated at the bottom.
Thee contribution of hepatic ABCA1 to lipoprotein homeostasis
BACC transgenic mice express the ABCA1 transgene using endogenous regulatory sequences containedd within the BAC, we cannot exclude the possibility that increased ABCA1 in other tissuess may also contribute to the increase in plasma HDL-C levels. For example, intestinal ABCA11 may participate in the regulation ot lipoprotein homeostasis, and increased intestinal ABCA11 expression in BAC transgenic mice may also contribute to the increase in HDL-C levels observedd in these animals. However, it is likely that ABCA1 in the liver plays a major role in elevationn of HDL-C levels in the BAC transgenic mice due not oniy to tissue mass as well as the highh level of ABCA1 expression compared to other tissues (15).
Excessivee hepatic ABCA1 via adenoviral delivery results in a non-selective increase in otherr plasma lipoprotein levels
Inn addition to increased HDL-C levels, we observed increased TC, TG, PL, non-HDL-C, and apoB levelss in Ad-ABCA1 injected mice compared to controls (Table 1). In mice receiving the highest dosee of Ad-ABCA1 (1.5 x 10'' pfu), the greatest alterations in plasma lipids were for non-HDL-CC levels, which were nearly triple those of control mice at day 3 (p<0.001) and at least double thatt of control mice at day 7 (Table 1, p<0.001). Additionally, we observed a dose-dependent increasee in apoB concentration that resulted in a nearly two-fold increase in apoB levels on dayss 3 and 7 in mice treated with 1,5 x 1 0!' pfu of Ad-ABCA1 (Table 1, pO.001), whereas there weree no significant changes in the levels of apoCIII, apoE, and apoAII (data not shown). Finally, FPLCC analysis of cholesterol distribution revealed that the greatest changes in cholesterol due to Ad-ABCA11 treatment were in the LDL range of fractions (Figure 3). Together, these results suggest thatt the 8.3-fold overexpression of exogenously regulated ABCA1 in mice receiving 1.5 x 10'' pfu off Ad-ABCA1 resulted in increased cholesterol in apoB-containing lipoproteins.
Figuree 4. Comparison of ABCA1 expression levels in thee livers of chow-fed BAC transgenic or Ad-ABCA1-treatedd mice. ABCA1 levels in total protein lysates preparedd from livers of BAC transgenic, Ad-ABCA1-treatedd (5.0 x 10" or 7.5 x 10; pfu/mouse as indicated), andd PBS-treated mice were determined by Western blot analysiss from gels from at least three animals per group. Alll of the mice were maintained on a leguiar chow diet.. ABCA1 levels were quantitated by densitometry andd normalized to GAPDH levels in the same lane to controll for equal protein loading. The 1 6 fold increase inn ABCA1 expression in the BAC transgenice was determinedd relative to wild-type control mice, in which ABCA11 levels were set to 1.0. Similarly, the 1.2 fold and 2.66 fold increases in ABCA1 expression in the Ad-ABCA1 treatedd mice were determined relative to PBS-treated controll mice, in which ABCA1 levels were set to 1.0. Barss represent the mean and standard deviations of measurementss from at least three mice.
Relativee ABCA1 expression
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Ü Ü < < Ad-ABCA1 1Theree were no marked overall differences in plasma lipids in PBS or Ad-AP injected mice, suggestingg that the adenovirus infection per se did not alter plasma lipids. Histological examinationn of liver sections prepared from Ad-ABCA1 or Ad-AP-treated mice revealed no significantt differences at equivalent doses (data not shown). Finally, we observed no change inn bile and fecal sterol cholesterol levels in Ad-ABCA1-treated mice compared to Ad-AP or PBS controlss (data not shown). These observations suggest that the altered lipid profiles in mice thatt received high-dose Ad-ABCA1 are unlikely to have arisen from non-specific changes including liverr damage upon adenoviral infection. Rather, these results suggest that the high levels of ABCA11 overexpression together with the dissociation of ABCA1 from its normal regulatory circuits,, are responsible for the general perturbation in plasma lipids.
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Figuree 5. Relative plasma lipid changes in BAC compared to Ad-ABCA1 -treated mice. The percent change in total cholesterol,, triglycerides, HDL-C and nonHDL-C levels were compared among chow-fed BAC transgenic mice, Ad-ABCA11 -treated mice, and Ad-AP treated mice. Plasma lipids in BAC transgenic mice were determinedd relative to nontransgenicc controls and expressed as percent change from the baseline observed in the wild-type control animals.. Plasma lipids in Ad-ABCA1 or Ad-AP-treated mice were determined relative to those observed mice injectedd with PBS only, and expressed as percent change from the baseline in PBS-treated mice. Viral dose in pfu/ mousee is given below the bars, and the fold overexpression of ABCA1 is given above the bars. Data represent the relativee means and standard deviations from at least 3 mice per group.
Thee contribution of hepatic ABCA1 to lipoprotein homeostasis
AA selective increase in HDL-C is best achieved by appropriate regulation of hepatic ABCA1 1
Itt is not possible to directly compare the changes in plasma lipids in mice treated with high dosess of Ad-ABCA1 (1 0 - 1,5 x 10J pfu} to those observpd in thn RAr tM nr gCn i c mice,
becausee ABCA1 expression is increased 6.5 - 8.3 fold in high-dose Ad-ABCA1 treated mice comparedd to 1.6 fold in BAC transgenic mice (Figure 2). However, mice treated with low doses off Ad-ABCA1 (5.0 - 7.5 x 10'" pfu) have a 1.2 - 2.6 fold increase in ABCA1 expression (Figure 2),, making them roughly comparable to the BAC transgenic mice in terms of hepatic ABCA1 levelss (Figure 4). This observation gave us the opportunity to determine the relative importance off appropriate regulation of ABCA1 in the liver compared to the simple abundance of hepatic ABCA11 in mediating a selective increase in plasma HDL-C levels. We therefore determined the relativee changes in plasma lipids observed in BAC transgenic mice compared to nontransgenic controls,, and compared these to relative changes in day 3 plasma lipids observed in Ad-ABCAI-treatedd or Ad-AP-treated mice, which were normalized to PBS-treated controls (Figure 5). Thiss analysis revealed that at roughly comparable expression levels in the liver, total cholesterol levelss in the BAC transgenic mice tended to be higher than in Ad-ABCA1-treated mice, which wass fully accounted for by a selective increase in HDL-C levels (Figure 5, Table 2). For example, considerr a comparison between chow-fed BAC transgenic mice that have a 1.6-fold increase of ABCA11 with mice treated with 7.5 x 10"- pfu of Ad-ABCA1 that exhibit a 2.6-fold increase in ABCA1.. The BAC transgenic mice have a 4 2 % increase in total cholesterol (p<0.0003) and a 65%% increase in HDL-C levels (p<0.005) compared to wild-type animals (19), whereas the Ad-ABCA1-treatedd mice have a 2 2 % increase in total cholesterol (p<0.01) and a 14% increase in HDL-CC levels (p<0.05) compared to controls (Table 1). These results suggest that despite higher hepaticc ABCA1 levels in the Ad-ABCA1 treated mice compared to the BAC transgenic mice, adenovirall delivery of ABCA1 was less effective than the endogenously regulated transgene in mediatingg a selective increase in plasma HDL-C levels in vivo. Moreover, HDL-C levels were consistentlyy higher in the BAC transgenic mice than in Ad-ABCA1 -treated at any dose tested, furtherr supporting the hypothesis that ABCA1 expressed under the control of endogenous regulatoryy signals is more likely to selectively increase HDL-C than ABCA1 expressed from an exogenouss promoter. The Ad-ABCA1-treated mice are obviously not completely comparable too the BAC transgenic mice even at comparable expression levels in the liver, because transgenic ABCA11 is also overexpressed in nonhepatic tissues in the BAC transgenic animals. Therefore, we cannott exclude the possibility that appropriately regulated ABCA1 expression in hepatic as well ass extrahepatic tissues may play a role in selectively increasing plasma HDL-C levels in the BAC transgenicc mice. Conditional transgenic approaches will be required to definitively elucidate the relativee importance of hepatic compared to extrahepatic ABCA1 in controlling HDL-C levels. Inn addition to a less marked impact on plasma HDL-C levels, Ad-ABCA1 treatment resulted in
Tablee 2: Percent changes in plasma lipids in BAC transgenic compared to Ad-ABCA1 treated mice Mouse e pfu/mouse e BAC/WT T Ad-ABCA1/PBS S Ad-ABCA1/PBS S Ad-ABCA1/PB5 5 Ad-ABCA1/PBS S Ad-AP/PBS S Viruss dose ABCA1 1 na a 5.00 x 10" 7.55 x 10= 1.00 x 10: ' 1.5xx 10-1.55 x 10' Foldd increase 1.66 x 1.22 x 2.66 x 6.55 x 8.33 x 1.00 x TC C 422 +/ 10 344 +/- 16 222 +/- 1 7 499 +/- 18 833 +/- 1 7 44 V - 16 Percent t TG G 11 +/-33 -- 21 +/- 1 5 -- 8 +/- 19 -- 2 +/- 31 233 +/- 13 -11 +/- 10 change e HDL-C C 655 +/ 19 277 y- 22 144 +/- 25 233 +/-21 399 +/ 22 33 +/ 22 non-HDL-C C 111 +/ 20 77 +/- 59 522 +/- 56 1477 +/- 53 2555 +/ 53 77 +/- 6
naa = not applicable. Fold increase was calculated relative to ABCA1 expression in WT or PBS-treated mice, each normalizedd to GAPDH levels, N=3. Percent change was calculated by determining the relative means and standardd deviations of BAC relative to WT mice, or Adenovirus-treated relative to PBS-treated mice.
Tablee 3: Comparison of results of adenoviral delivery of ABCA1 studies in vivo
Variablee Basso et al Wellington et al Mousee strain Age e Adenovirall promoter Speciess of ABCA1 Construct t Dose e Timee of analysis ABCA11 expression TC C HDL-C C non-HDL-C C C57BI/6 6 2-44 months TRE/minCMV V murine e ABCA1-GFP P 1-55 x 10" pfu/mouse
66 hours to 4 days posttreatment 1.66 fold 2-33 fold 2-33 fold nott reported C57BI/6 6 6-88 months CMV V human n ABCA1 1 5x10"-- 1.5x10' pfu/mouse 33 and 7 days posttreatment 1.22 - 8.3 fold, increasing by dose 1.3-- 1.8 fold, increasing by dose 1.3-- 1.4 fold, increasing by dose 1.66 - 3.5 fold, increasing by dose
changess in other lipids that were not observed in the BAC transgenic mice. For example, chow-fedd BAC transgenic mice have no significant changes in non-HDL-C levels compared to wild-typee controls (Figure 5, p>0.05). In comparison, non-HDL-C levels were significantly elevated inn mice that received 1.0 - 1 . 5 x 10' pfu of Ad-ABCA1 (Figure 5, Table 1). Although non-HDL-CC levels were not significantly elevated 3 days after treating mice with the lowest dose of Ad-ABCA11 (5.0 x 10:" pfu), there was a significant increase in non-HDL-C in these mice by day 7
comparedd to either vehicle or Ad-AP-treated controls (Figure 5, p<0.002). These results again suggestt that even at relatively low expression levels, inappropriate regulation of ABCA1 can leadd to changes in plasma lipids other than HDL-C.
Discussion n
Thee discovery of ABCA1 as a critical protein in the generation of HDL led to the hypothesis that ann increase in ABCA1 will elevate plasma HDL-C levels and decrease atherosclerosis ( 1 , 9,14). Thiss hypothesis has been confirmed in the BAC transgenic animal model, where human ABCA1
Thee contribution of hepatic ABCA1 to lipoprotein homeostasis
iss expressed from its endogenous regulatory signals on a bacterial artificial chromosome. In thesee mice, ABCA1 overexpression in chow-fed animals is increased 1.6-fold over endogenous ABCA1,, which results in a 6 5 % increase in plasma HDL-C (19). When crossed onto the apoE-knockoutt model of atherosclerosis, appropriately regulated ABCA1 was found to be dramatically protectivee against the formation of atherosclerotic plaques (20). In contrast, an ABCA1 cDNA transgenicc model showed mixed results with respect to protection from atherosclerosis. In the ABCA11 cDNA transgenic mice, exogenous ABCA1 expression is driven by the apoE promoter thatt directs ABCA1 overexpression to the liver and macrophages (27). Two lines of mice were generated,, with approximately 30 or 40 copies of the human ABCA1 transgene, which results inn a similar two-fold elevation of plasma HDL-C on a chow diet (27). When these mice were fed aa high fat diet, increased ABCA1 was found to strengthen the atheroprotective lipid profile andd reduce atherosclerosis (28). However, increased atherosclerosis was observed when these micee were crossed onto an apoE-deficient background (28). These different results underscore thee importance of investigating relationships among ABCA1 regulation, abundance, and expression inn selected tissues to plasma HDL-C levels and protection from atherosclerosis in vivo.
Too specifically address how hepatic ABCA1 expression contributes to plasma lipids, we used ann adenovirus to selectively overexpress human ABCA1 in the liver and determined the dose-dependentt effect of exogenous ABCA1 expression on plasma lipid and lipoprotein profiles. Thee adenoviral construct used in this study was first validated in vitro by demonstrating efficient apoA1-dependentt cholesterol and phospholipid efflux in both HeLa and HepG2 cells. Inn vivo adenoviral delivery of ABCA1 resulted in an increase in plasma HDL-C levels as expected, demonstratingg that hepatic ABCA1 can participate in HDL-C homeostasis. However, at comparable expressionn levels, HDL-C was increased by 14-27% in Ad-ABCA1 treated mice compared with aa 65% increase in BAC transgenic animals, suggesting that transgenic expression of exogenous ABCA11 from the BAC is more effective in raising plasma HDL-C levels than delivering exogenous ABCA11 with an adenovirus. In addition, we also observed a striking effect of exogenous ABCA11 on the entire lipid profile, which was most pronounced at high doses of Ad-ABCA1 treatmentt that resulted in a 8.3-fold overexpression of hepatic ABCA1. For example, the levels off TC, TG, PL, and non-HDL-C, and apoB were all increased in Ad-ABCA1-treated mice in a doss-dspsridcritt manner, and the magnitude of these changes often exceeded that of the increasee in HDL-C levels. Interestingly, a similar dose-dependent increase in apoB and non-HDL-CC levels were also observed in chow-fed apoE-ABCA1 cDNA transgenic mice, although these increasess did not reach significance and it is not possible to distinguish whether these effects aree due to nonphysiologic regulation of ABCA1 or high-level ABCA1 expression driven by the apoEE promoter (27). Nevertheless, these observations suggest that hepatic ABCA1 can greatly influencee overall plasma lipids.
criticallyy affects lipid homeostasis. We observed no impact on non-HDL-C levels in either the BACC transgenic mice or in mice treated with the lowest dose of Ad-ABCA1, which result in relativelyy modest levels of ABCA1 overexpression in the liver (1.6-fold and 1.2-fold, respectively). However,, increasing Ad-ABCA1 dose resulted in a clear and dose-dependent increase in non-HDL-CC levels. This observation suggests that although a modest increase in hepatic ABCA1 mayy preferentially increase HDL-C, excessive ABCA1 may perturb overall lipid homeostasis, perhapss by permitting the transfer of excess free cholesterol t o lipoproteins of different subclasses.. This effect may only be observed when ABCA1 is highly overexpressed in the absencee of appropriate physiological regulatory signals.
AA second interpretation is that appropriate regulation of ABCA1 may be critical for selectively increasingg HDL-C. In this interpretation, lipid homeostasis is regulated through exquisite control off multiple hepatic genes including ABCA1. Because the Ad-ABCA1 vector used in this study containedd only the full-length human ABCA1 cDNA, it is likely that this construct lacked elementss that ultimately regulate the appropriate targeting of ABCA1 to the basolateral membrane (29,30)) or to direct efficient posttranslational modification of ABCA1 (24). Appropriate subcellular localizationn and/or modification of ABCA1 may play important roles in appropriate lipidation off apoA1.
Itt is also possible that coordinated ABCA1 expression in multiple tissues may be required to observee a selective increase in plasma HDL-C levels. In the BAC transgenic mice, the presence off endogenous regulatory signals ensures that transgenic ABCA1 is overexpressed in all tissues wheree it is normally expressed, therefore resulting in a coordinated overexpression of ABCA1 throughoutt the body. It is possible that the combined effects of hepatic and extrahepatic ABCA11 contribute to the efficient increase in HDL-C levels in the BAC transgenic mice compared too Ad-ABCA1-treated mice at comparable expression levels. Because macrophage-specific ABCA1 hass been shown to have a minimal contribution to plasma HDL-C levels in vivo (18), it is possiblee that other extrahepatic tissues such as the intestine may impact HDL-C homeostasis in vivoo through ABCA1 activity.
Whilee this manuscript was in preparation, Basso et al. reported their findings on adenoviral deliveryy of ABCA1 to mice (31) (Table 3). Although both groups used the same mouse strain (C57BI/6),, the constructs were different. Basso et al. used a murine ABCA1 cDNA fused to GFP,, whereas we used an unmodified human ABCA1 cDNA. The dose used by Basso et al. (1-55 x 10H pfu/mouse), which resulted in an increase in ABCA1 protein by 1.6-1.7 fold within 24h (31),, is most comparable to the lowest dose used in our study (5 x 10' pfu/mouse), which resultedd in a 1.2 fold increase in ABCA1 protein by 3 days.
Bassoo et al. observed maximal changes in plasma lipids 2 days after treatment, with the major effectss being a 2-3 fold increase in HDL-C, TC, PL, FC, and CE levels and a significant increase inn a-HDL particles (31). In their study, there was little apparent effect on non-HDL-C levels, in
Thee contribution of hepatic ABCA1 to lipoprotein homeostasis
contrastt to their previous observations in the apoE-ABCA1 cDNA transgenic mice (27,31). Additionally,, Basso et al. observed significant increases in HMG CoA reductase, LDLr, and LRP expressionn levels that were detectable tn as little as 6 h after Ad-ABCA1-GFP treatment (31). W LL detected maximal changes in plasma lipids at day 3 after treatment, suggesting that the changess in plasma lipid levels appeared to follow similar dynamics in the t w o studies. Importantly,, comparison of the relative changes in plasma lipids in the BAC transgenic mice andd Ad-ABCA1-treated mice at roughly comparable expression levels showed HDL-C levels were selectivelyy increased more efficiently in the BAC transgenic model. This observation suggests that regulationn of ABCA1, in the liver and in other tissues, may be critical for effectively raising HDL-CC levels. ABCA1 may also function coordinateiy with other genes help to maintain specificity to HDL.. In the Ad-ABCAI-treated mice, increased ABCA1 expression is uncoupled from other hepatic geness as well as other tissues that may also contribute to regulating HDL-C levels.
Endogenouss ABCA1 is under exquisite regulation at transcriptional and posttranslational levels, andd appropriate levels in multiple tissues, subcellular location within cells of particular tissues, andd interaction with other cellular proteins are likely to be critical parameters for the ability of ABCA11 to selectively increase HDL-C levels. Furthermore, ABCA1 activity in multiple tissues mayy also affect HDL-C homeostasis in vivo. Our results suggest that therapeutic approaches basedd on increasing ABCA1 expression will likely have the greatest beneficial effect if they targett endogenous pathways that maintain appropriate regulation of ABCA1,
Acknowledgements s
Wee are grateful to Alan Attie, Angie Tebon, and Sherrie Tafuri for their many insightful comments duringg the course of this study. This work is supported by the Canadian Institutes of Health Researchh (MRH, RJP), the Heart and Stroke Foundation of Canada (MRH), the Canadian Networks off Centres of Excellence (NCE Genetics, MRH), and Xenon Genetics Inc. RJP is a CIHR New Investigator,, and MRH is a holder of a Canada Research Chair.
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