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
Macrophagee ABCA1 over-expression inhibits
atheroscleroticc lesion progression
Mirandaa Van Eck*, Roshni R. Singaraja
1, Reeni B. Hildebrand*, Erick R.
James
1,, Michael R. Hayden
T§, Theo J.C. Van BerkeP
'Divisionn of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories,, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Centree for Molecular Medicine and Therapeutics, Children's and Women's Hospital,
Universityy of British Columbia, Vancouver, Canada 'Thesee groups contributed equally to this work.
Summary y
ATP-bindingg cassette transporter 1 (ABCA1) is a key regulator of cellular cholesterol and phospholipidd transport, which is defective in familial HDL deficiency syndromes such as Tangier disease.. Recently, we have shown that inactivation of macrophage ABCA1 induces atherosclerosis inn LDL receptor knockout (LDLr-/-) mice. Currently, the possibly therapeutic effect of specific up-regulationn of macrophage ABCA1 on atherogenesis is unknown.
Chimerass that specifically overexpress ABCA1 in macrophages were generated by transplantation off bone marrow f r o m human ABCA1 BAC transgenic mice into LDLr-/- mice. To induce atherosclerosis,, the mice were fed a Western-type diet, containing 0.25% cholesterol and 15% fatt for 9 and 12 weeks, allowing analysis of effects on initial lesion development as well as advancedd lesions.
Peritoneall macrophages isolated from the ABCA1 BAC -* LDLr-/- chimeras exhibited a 6 0 % (P<0.001)) increase in cholesterol efflux to apoAl as compared to controls. No significant effect off macrophage ABCA1 over-expression was observed on atherosclerotic lesion size (245*36x10 urn-- in ABCA1 BAC -» LDLr-/- mice versus 21 1 0 urn- in controls) after 9 weeks challenge withh the Western-type diet. After 12 weeks, however, the mean atherosclerotic lesion area in ABCA11 BAC -» LDLr-/- mice was only 1 5x10' urn (P<0,001) as compared to : urn-- in controls.
Thesee results show that ABCA1 up-regulation does not prevent the initiation of atherosclerosis, butt inhibits the progression of initial lesions to advanced atherosclerotic plaques. Therefore, thee development of therapeutic targets that increase macrophage ABCA1 expression is important forr the inhibition of atherosclerotic lesion progression.
Introduction n
Atheroscleroticc cardiovascular disease is the major cause of morbidity and mortality in Western societies.. The genesis and progression of atherosclerotic lesions involves a complicated sequence off events in winch various ceil types in the arterial wall, including macrophages, play an importantt role (1). Deposition of excessive amounts of cholesterol in macrophages leading to thee transformation into foam cells is a pathological hallmark of atherosclerosis. Macrophages cannott limit their uptake of cholesterol via scavenger receptors (2). Therefore, cholesterol effluxx is an important mechanism to maintain cholesterol homeostasis in macrophages and to preventt atherosclerotic lesion development. Epidemiological studies have shown a strong inverse relationshipp between low plasma cholesterol levels and coronary artery disease (3-5). It is currentlyy generally accepted that high plasma levels of high-density lipoprotein (HDL) protect againstt the development of atherosclerosis, HDL exerts its protective effect through its role in reversee cholesterol transport, a process by which excess cholesterol from peripheral tissues is transferredd via the plasma to the liver for either recycling or excretion from the body as bile (6). AA key regulator of cholesterol efflux from macrophages is ATP-binding cassette transporter 1 (ABCA1).. Mutations in the human ABCA1 gene are the underlying molecular defect in familial HDL-deficiencyy syndromes such as Tangier disease (TD) (7-9). TD is an autosomal recessive disorderr that is characterised by severe HDL-deficiency, deposition of cholesteryl esters in cells off the r e t i c u l o e n d o t h e l i a l system, and increased susceptibility t o the development of atherosclerosiss (10,11).
Recently,, we have shown that specific disruption of ABCA1 in macrophages results in an increasedd susceptibility to atherosclerotic lesion development, providing evidence that macrophagee ABCA1 plays a critical role in the protection against atherosclerosis (12). Activation off this ABC transporter is thus a potentially attractive target for therapeutic interventions to preventt atherosclerosis. The expression of ABCA1 is tightly controlled by intracellular cholesterol levelss (13,14). Its activity is dramatically increased upon cholesterol loading of macrophages andd the subsequent transformation into foam cells. It is therefore conceivable that cholesterol effluxx via ABCA1 is already maximally activated in macrophages in the atherosclerotic lesion. Too study the therapeutic potential of specific up-regulation of macrophage ABCA1 to prevent atherosclerosis,, we have determined atherosclerosis susceptibility of chimeras that specifically over-expresss ABCA1 on macrophages, created by transplantation of bone marrow from human ABCA11 over-expressing bacterial artificial chromosome (BAC) transgenic mice to an established modell of atherosclerosis. The findings from these studies revealed that specific up-regulation off macrophage ABCA1 does not affect initial lesion development, but prevents further progression off atherosclerosis.
Methods s
Mice.. ABCA1 BAC 269 transgenic mice hemizygous for the human ABCA1 gene, lacking its upstreamm promoter and exon 1, and backcrossed to the C57BI./6 background (N6) have been generatedd as previously described (15,16). Non-transgenic littermates were used as controls. Homozygouss LDL receptor knockout (LDLr ) mice (17) were obtained from The Jackson Laboratoryy (Bar Harbor, ME, USA) as mating pairs and bred at the Gorlaeus Laboratory, Leiden, Thee Netherlands. Mice were housed in sterilised filter-top cages and given unlimited access to foodd and water. They were maintained on sterilised regular chow, containing 4.3 % ( w / w ) fat andd no cholesterol (RM3, Special Diet Services, Witham, UK), or were fed a semi-synthetic high cholesteroll Western-type diet, containing 15% ( w / w ) fat and 0.25% ( w / w ) cholesterol (Diet W,, Hope Farms, Woerden, The Netherlands). Drinking water was supplied with antibiotics (83 mg/LL ciprofloxacin and 67 mg/L polymyxin B sulphate) and 6.5 g/L sucrose.
Bonee m a r r o w transplantation. To induce bone marrow aplasia, male LDLr mice were exposedd to a single dose of 9 Gy (0.19 Gy/min, 200 kV, 4 mA) total body irradiation, using an Andrexx Smart 225 Röntgen source (XYLON International, Copenhagen, Denmark) with a 6-mm aluminiumm filter, one day before the transplantation. Bone marrow was isolated by flushing the femurss and tibias from male ABCA1 BAC mice or male wildtype littermates with phosphate-bufferedd saline. Single-cell suspensions were prepared by passing the cells through a 30-mm nylonn gauze. Irradiated recipients received 0.5 x 10' bone marrow cells by intravenous injection intoo the tail vein.
Assessmentt of chimerism. The hematologic chimensm of the LDLr mice was determined in genomicc DNA from bone marrow by PCR at 20 weeks post transplant. The forward and reverse primerss 5'-GGCTGGATTAGCAGTCCTCA-3' and 5'-ATCCCCAACTCAAAACCACA-3' for human ABCA11 and 5'-TGGGAACTCCTAAAAT-3' and 5'-CCATGTGGTGTGTAGACA-3' for the mouse ABCA1 genee were used and resulted in 304bp and 750bp amplification products, respectively
Macrophagee cholesterol efflux studies. Thioglycollate-elicited peritoneal macrophages were harvestedd from mice transplanted with control and human ABCA1 BAC bone marrow at 20 weekss after transplantation and were seeded on 24-well plates at a density of 0.5 x 10 cells in
5000 pi DMEM, supplemented with 10% (w/v) bovine calf serum (BCS), 2 mmol/l L-glutamine, 1000 pg/ml streptomycin, and 100 lU/ml penicillin. After 4 hours, non-adherent cells were removedd by washing. After 2 days in culture, the cells were washed and incubated with 0.5 pCi/mLL H-cholesterol in DMEM/0.2% BSA for 24 hours at 37 C. Subsequently, the medium wass removed and the cells were washed 3 times with DMEM/0.2% BSA. To determine cholesterol loading,, cells were washed three times with washing buffer (50 mmol/l Tris, containing 0.9%
NaCI,, 1 mmol/l EDTA, 5 mmol/l CaCI , and 0.2% (w/v) BSA, pH 7.4), followed by t w o washing stepss without BSA. The cells were lysed in 0.1 mol/l NaOH and the radioactivity was determined byy liquid scintillation counting. Cell protein was measured according to Lowry et al. (18). Cholestctull c-ffluA was studied by incubation of the cells with D M b M / 0 . 2 % BSA alone, or supplementedd with either 10 ug/ml apolipoprotein Al (Calbiochem) or 50 ug/ml human HDL. Radioactivityy in the medium was determined by scintillation counting after 24 hours of incubation.. To analyse liver X receptor (LXR) responsiveness, efflux was determined in the absencee and presence of 10 pM LXRa agonist T-0901317.
Serumm lipid analyses. After an overnight fasting-period, approximately 100 pi blood was drawnn from each individual mouse by tail bleeding. The concentrations of total cholesterol, triglycerides,, and phospholipids in serum were determined using enzymatic colorimetric assays (Rochee Diagnostics, Mannheim, Germany). The distribution of lipids over the different lipoproteins inn serum was determined by fractionation of 30 ml serum of each mouse using a Superose 6 columnn (3.2x30mm, Smart-system, Pharmacia, Uppsala, Sweden). Total cholesterol, triglyceride, andd phospholipid contents in the effluent were determined using enzymatic colorimetric assays (Rochee Diagnostics, Mannheim, Germany).
Histologicall analysis of the aortic root. To analyse the development of atherosclerosis at thee aortic root, transplanted mice were sacrificed at 17 and 20 weeks post transplant and after 99 and 12 weeks of feeding the high cholesterol Western-type diet, respectively. The arterial treee was perfused in situ with phosphate-buffered saline (100 mm Hg) for 20 minutes via a cannulaa in the left ventricular apex. The heart plus aortic root and descending aorta were excisedd and stored in 3.7% neutral-buffered formalin (Formal-fixx, Shandon Scientific Ltd., England).. The atherosclerotic lesion areas in oil red O-stained cryostat sections of the aortic roott were quantified using the Leica image analysis system, consisting of a Leica DMRE microscopee coupled to a video camera and Leica Qwin Imaging software (Leica Ltd., Cambridge, England).. Mean lesion area (in p m ) was calculated from 10 oil red O-stained sections, starting att the appearance of the tricuspid valves.
Statisticall analyses. Statistical analyses were performed utilising the unpaired student's t-test (Instatt GraphPad software, San Diego, USA).
Results s
Generationn of LDL receptor knockout mice over-expressing macrophage ABCA1 Too assess the therapeutic potential of increasing macrophage ABCA1 to prevent atherosclerotic
lesionn development, we used the technique of bone marrow transplantation to selectively up-regulatee ABCA1 in hematopoietic cells. Bone marrow from previously generated human ABCA1 over-expressingg BAC transgenic mice was transplanted into LDLr-/- mice, which represent an establishedd model for the development of atherosclerosis. Successful reconstitution of recipients withh hematopietic donor cells was established at 20 weeks post transplant by PCR assisted amplificationn using primers specific for human and mouse ABCA1 (Figure 1A). Genomic DNA isolatedd from the LDLr-/- mice transplanted with bone marrow from ABCA1 BAC transgenic micee contained both the human and the mouse ABCA1 transcript whereas the control transplantedd group only contained mouse ABCA1, indicating that the bone marrow transfer wass successful. Furthermore, ABCA1 activity was determined by means of cholesterol efflux assayss on peritoneal macrophages isolated from the transplanted mice at 20 weeks after transplantation.. As previously shown for the ABCA1 BAC transgenic mice (15), macrophages isolatedd from LDLr-/- mice transplanted with ABCA1 BAC bone marrow exhibited a 6 0 % (n=3, P<0.001)) increase in cholesterol efflux to apoAl compared with cells from control transplanted LDLr-/-- mice (Figure 1 B).
Murine e
ABCA1 1
Human n
ABCA1 1
W T ^^
LDLr-/-_
_
_ _
ABCA11 BAC -*
LDLr-/-—— m
P P 1 0 0 00 b p 8 0 00 b p 6 0 00 b p 4 0 00 b p o o at at to o o o J = = O O 15 5 10 0 5 5 E22 W T ^ ABCA1 BAC -> LDLr-/"" ^
ns svzm vzm
1 1
ns sFiguree 1. Verification of success of bone marrow transplantation.. A) Verification of successful reconstitution with donorr hematopoietic cells by PCR amplification of the human ABCA11 and murine ABCA1 gene at 20 weeks post transplant usingg genomic DNA isolated from bone marrow. B) ApoAl and HDLL induced cellular cholesterol efflux from -H-cholesterol-labeled peritoneall macrophages isolated from mice transplanted with eitherr ABCA1 BAC (n=3) or control (n=3) bone marrow at 20 weekss post transplant. Efflux to ApoA-l from BAC ->LDLr-/-micee showed a statistically significant difference of ***P<0.001 comparedd to WT -> LDLr mice.
BSAA ApoAl HDL
Effectt of macrophage ABCA1 over-expression on plasma lipid levels
Duringg the course of the experiment, the effects of macrophage over-expression of ABCA1 on serumm lipid levels were carefully monitored. On regular chow diet, the majority of the cholesterol inn LDLr-/- mice is transported by LDL and HDL, phospholipids by HDL, and triglycerides by
VLDLL and LDL (Figure 2). In contrast to the ABCA1 BAC transgenic mice that displayed mildly increasedd HDL cholesterol levels, no significant effect on HDL cholesterol, triglyceride, or phospholipidd levels was observed when ABCA1 was over-expressed solely in macrophages. Inn ordei to induce atherosclerotic lesion development, the transplanted mice were fed a high cholesteroll Western-type diet, containing 0.25% cholesterol and 15% fat, starting at 8 weeks afterr transplantation. Upon challenging the mice with a high cholesterol diet, serum cholesterol levelss increased approximately 3-fold in both groups of mice due to an increase in VLDL and LDLL cholesterol (Table I). The increase in VLDL and LDL cholesterol coincided with an increase inn phospholipids. Also under these conditions, no significant effect of macrophage ABCA1 over-expressionn on serum lipid levels or lipid distribution over the different lipoproteins was observedd (Figure 2).
Cholesteroll Phospholipids Triglycerides
1000 0 E E O) ) n n in in "D D O O _ l l _J J E E en n Z L L
</> >
T3 3 O O _ l l 750 0 500 0 250 0 0 0 1000 0 750 0 500 0 250 0 0 0 Chow Chow ,, r., H D L LDLL o V L D L // 8 r \We. .
y y
WTD WTD VLDL L jj LDL \\ HDLU U
U t t t
VLDL L 1 Chow Chow HDL L WTD WTD WTD WTD VLDL Ll\ l\
II ' ^ H D L 00 5 1 0 1 5 20 0 5 1 0 1 5 20 0 5 1 0 1 5 20 Fractionn number WTT . ABCA11 BAC ,LDLr-/-Figuree 2. Effect of macrophage ABCA1 over-expression on serum cholesterol, phospholipids, and triglyceride
distribution.. Blood samples were drawn after an overnight fast at 8 weeks post transplant while feeding regular choww diet (CHOW) and at 17 weeks after bone marrow transplantation after 9 weeks of feeding a high cholesteroll Western-type diet (WTD). Sera from individual mice were loaded onto a Superose 6 column and fractionss were collected. Fractions 3 to 7 represent VLDL; fraction 8 to 14, LDL, and fractions 1 5 to 19, HDL, respectively.. The distribution of cholesterol, phopholipids, and triglycerides over the different lipoproteins in WT —>> LDLr ( )) and ABCA1 BAC —> LDLr chimeras is shown. Values represent the mean+SEM of at least 12 micee No statistically significant differences were observed.
Tablee 1: Effect of leukoc Mice e WTT -» ABCA11 BAC -» LDLr-/-ytee ABCA1 Time e (weeks) ) baseline e 8 8 baseline e 8 8 over-expr r DD e1 Chow w Chow w WTD D Chow w Chow w WTD D
essionn on serum lipid levels Totall Cholesterol (mg/dl) ) 322+13 3 0 0 5 5 2 2 7 7 8 8 HDLL Cholesterol (mg/dl) ) ND D 7 7 118+5 5
'.: :
125+19 9 117+4 4 Phospholipids s (mg/dl) ) .. : =: : 475+65 5 7 7 4 4 9 9 '.. ' '_42 Triglycerides s (mg/dl) ) 4 4 2 2 1 1 296+11 1 1 1 1 1Serumm lipids were measured in LDLr-/- mice before transplantation (baseline) and at 8 and 17 weeks after transplantationn with control bone marrow or ABCA1 BAC over-expressing bone marrow. At 8 weeks after transplantation,, the regular chow diet was switched to a high cholesterol Western-type diet (WTD). Data representt mean+SEM of at least 12 mice. No statistically significant differences were observed between the controll transplanted group and the mice transplanted with ABCA1 BAC bone marrow.
Over-expressionn of macrophage ABCA1 prevents atherosclerotic lesion progression. Inn order to investigate the therapeutic potential of increasing macrophage ABCA1 expression ass a means of preventing atherosclerosis, we assessed whether and to what degree up-regulation off ABCA1 in macrophages affected lesion formation in the arterial wall. Atherosclerotic lesion developmentt was analysed in the aortic root of WT -> LDLr-/- mice and in ABCA1 BAC —> LDLr-/-- chimeras after 9 and 12 weeks of Western-type diet feeding. After 9 weeks on the Westernn diet, no significant effect of macrophage ABCA1 over-expression on the atherosclerotic lesionn size was observed (245+36x103 urrr in ABCA1 BAC -> LDLr-/- mice versus "
WTT -> LDLr ABCA1 BAC -> LDLr
-/-EE 750
V W TT ABCA1 BAC >LDLr-/-99 12
Timee (weeks)
Figuree 3. Macrophage ABCA1 over-expression prevents atherosclerotic lesion progression Formation of atheroscleroticc lesions was determined at 17 and 20 weeks post transplant at the aortic root of WT —> LDLr and ABCA11 BAC 7 LDLr chimeras that were fed a high cholesterol Western-type diet for 9 and 12 weeks, respectively. Thee mean lesion area was calculated from oil red O-stamed cross-sections of the aortic root at the level of the tricuspidd valves. Values represent the mean of at least 12 mice mice. Original magnification 50x. Lesions in BAC —*LDLr-/-- mice showed a statistically significant difference of ***P<0.001 when compared to WT—> LDLr mice.
pirn-- in controls), indicating that macrophage ABCA1 over-expression does not prevent the initiationn of atherosclerosis. From week 9 until week 12 of diet feeding, atherosclerosis in the micee transplanted with control bone marrow progressed further to advanced lesions with a meann lesion aiea of - prrv. In the ABCA1 BAC - ^ LDLr-/- mice, however, the mean atheroscleroticc lesion area was only 164+15x10"' urn2 (P<0.001), indicating that lesion progressionn was markedly inhibited by up-regulation of ABCA1 in macrophages. Raising macrophagee ABCA1 expression is thus a promising therapeutic strategy to inhibit progression off premature atherosclerotic lesions. The identification of the nuclear receptor liver X receptor (LXR)) as an activator of ABCA1 has raised the possibility of using LXR agonists to raise ABCA1 inn macrophages thereby increasing cholesterol efflux (19). As shown in Figure 4, incubation of peritoneall macrophages isolated from the control transplanted mice with the synthetic LXRa agonistt T-0901317 increased cholesterol efflux to ApoAl 1.8-fold (PO.05). Although cholesterol effluxx from macrophages isolated from the ABCA1 BAC -H> LDLr-/- chimeras was already increased duee to the over-expression of ABCA1, incubation with the LXR agonist further increased cholesteroll efflux 2.1-fold (PO.001).
_3 3
15 5
"5 5
Ü Ü
Controll LXR agonist
Figuree 4. Induction of ABCA1-mediated cholesterol efflux by LXRa agonist T-090131 7. ApoAI-induced cellular cholesteroll efflux from ;H-cholesterol-labeled peritoneal macrophages isolated from mice transplanted with eitherr ABCA1 BAC (n=3) or control (n=3) bone marrow was analysed for 24 hours with and without pre-incubationn with 10pM LXRa agonist T-0901 31 7. Statistically significant difference of *P<0.05 and ***P<0.001 comparedd to WT —> LDLr mice
Discussion n
Insightss into the roles of ABCA1 in atherogenesis have been gained from both patients affected withh Tangier disease and recently developed animal models. Tangier disease, a result of mutations inn ABCA1, is characterized by extremely low HDL levels, accumulation of cholesterol in macrophages,, and an increased prevalence of coronary artery disease (CAD) (10-11). Interestingly, heterozygotess for ABCA1 mutations are significantly at risk for CAD (20-22). These cardioprotective
15 5 10 0 00 WT ABCA1 BAC
--1 --1
* *
P P
*** *
effectss of ABCA1 have recently been confirmed in animal models. Overexpression of ABCA1 resultedd in increased HDL cholesterol levels and a decreased susceptibility to spontaneous atheroscleroticc lesion development in apolipoprotem E knockout mice (16) and in C57BI/6 mice withh diet-induced atherosclerosis (23). Furthermore, using bone marrow transplantation studies, wee (12) and Aeillo et al. (24) have shown that selective inactivation of ABCA1 in macrophages resultss in markedly increased atherosclerosis in different animal models
Thee cardioprotective effects of ABCA1 are generally expected to be a direct result of its function inn facilitating cholesterol efflux. Cholesterol efflux processes play an important role in prevention off the formation of the intimal macrophage-rich fatty streak, the first stage of atherosclerotic lesionn development. Strikingly, in this study we found that over-expression of ABCA1 in macrophagess did not affect initial lesion development, but rather inhibited the progression of fattyy streaks into advanced lesions. In humans, fatty streaks are ubiquitous. However, not all fattyy streaks evolve into advanced atherosclerotic lesions. In this study, we have demonstrated forr the first time that ABCA1 is an important determinant for this progression of atherosclerosis fromm fatty streaks into advanced lesions
Progressionn of atherosclerotic lesions is characterized by an ongoing chronic inflammatory reactionn and extensive cellular necrosis and apoptosis (25). Accumulation of apoptotic markers duringg atherosclerosis progression indicates that in advanced lesions the removal of apoptotic cellss is impaired. Several lines of evidence have suggested a role for ABCA1 in the engulfment off apoptotic cells (26). ABCA1 expression during development follows strictly the spatial and temporall pattern of programmed cell death (27). Conversely, mice deficient for ABCA1 show aa defective engulfment of apoptotic corpses during development (28). Furthermore, engulfment off apoptotic thymocytes by peritoneal macrophages lacking ABCA1 is impaired (28). It is thereforee conceivable that increased phagocytic activity of lesion macrophages due to ABCA1 over-expressionn may have led to accelerated clearance of apoptotic material thereby preventing excessivee inflammatory responses and further progression of the atherosclerotic lesion. Thee important role for ABCA1 in prevention of lesion progression reported in this study renders thiss transporter an attractive target for the development of novel therapeutic agents designed too prevent the development of advanced atherosclerotic lesions that may result in coronary or cerebrall infarction. Promising candidates for raising macrophage ABCA1 expression are synthetic LXRaa agonists, that induce ABCA1 expression in macrophages and can mitigate atherosclerosis inn animal models (19,29).
Contributors s
M.. Van Eck and R.R. Singaraja were responsible for the design of the study and wrote the paper.. R.B. Hildebrand did all the bone marrow transplantation studies, including lipid analysis andd dtheioscleiusis muiphumetty. L.k. James coordinated the breeding ot the ABCA1 BAG mice.. M.R. Hayden and Th.J.C. Van Berkel are the project leaders.
Conflictt of interest statement Nonee declared
Acknowledgements s
Thiss work was supported by the Netherlands Heart Foundation (grant #2001 D041) and a Pfizerr International HDL Research Award. Dr. M.J. Smit (Department of Pharmacology, Organon BV,, The Netherlands) is thanked for kindly providing us with the LXRa agonist T-0901317.
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