macrophage cholesterol homeostasis and atherosclerosis
Ye, D.
Citation
Ye, D. (2008, November 4). ABC-transporters and lipid transfer proteins : important players in macrophage cholesterol homeostasis and atherosclerosis. Retrieved from https://hdl.handle.net/1887/13220
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Chapter 7
IMPORTANT ROLE FOR BONE MARROW-DERIVED CHOLESTERYL ESTER TRANSFER PROTEIN IN LIPOPROTEIN CHOLESTEROL REDISTRIBUTION AND ATHEROSCLEROTIC LESION DEVELOPMENT IN LDL RECEPTOR KNOCKOUT MICE
Dan Ye*, Miranda Van Eck*, Reeni B. Hildebrand, J. Kar Kruijt, Willeke de Haan, Menno Hoekstra, Patrick C.N. Rensen, Christian Ehnholm, Matti Jauhiainen, Theo J.C. Van Berkel.
From the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden University, Leiden, The Netherlands (M.V.E., D.Y., R.B.H., J.K.K., M.H., Th.J.C.V.B.). Department of Molecular Medicine, National Public Health Institute, Biomedicum, Helsinki, Finland (C.E., M.J.). Department of General Internal Medicine, Endocrinology and Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands (W.d.H., P.C.N.R.). *The first two authors contributed equally to this work.
Circ Res. 2007; 100(5):678-85.
ABSTRACT
Abundant amounts of cholesteryl ester transfer protein (CETP) are found in macrophage-derived foam cells in the arterial wall, but its function in atherogenesis is unknown. To investigate the role of macrophage CETP in atherosclerosis, LDL receptor knockout mice were transplanted with bone marrow from CETP transgenic mice, which express the human CETP transgene under control of its natural promoter and major regulatory elements. CETP production by bone marrow-derived cells induced a 1.8-fold (P<0.01) increase in atherosclerotic lesion development. The increase in lesion size coincided with an increase in VLDL/LDL cholesterol and a decrease in HDL cholesterol. The cholesterol redistribution in serum was a direct effect of the substantial serum CETP activity and mass (38±3 nmol/mL/h and 4.8±0.5 mg/mL, respectively) induced by CETP production by bone marrow-derived cells. Conversely, specific disruption of CETP production by bone marrow-derived cells in CETP transgenic mice resulted in an approximately 2-fold (P<0.0001) reduction in serum CETP activity and mass, demonstrating the quantitative relevance of bone marrow-derived CETP. Finally, we show that in liver Kupffer cells, hepatic macrophages, contribute approximately 50% to the total hepatic CETP expression. In conclusion, bone marrow-derived CETP induces a proatherogenic lipoprotein profile and promotes the development of atherosclerotic lesions in LDL receptor knockout mice. Most importantly, we show for the first time that bone marrow-derived CETP is an important contributor to total serum CETP activity and mass.
INTRODUCTION
Cholesteryl ester transfer protein (CETP) is a 74-kDa glycoprotein that facilitates the transfer of cholesteryl esters from antiatherogenic HDL to proatherogenic apoB-containing lipoproteins and a concomitantequimolar transfer of triglycerides to HDL.1 High HDL levels protect against the development of atherosclerosis by virtueof its essential role in the removal of excess cholesterol from peripheral cells and its antioxidative, antiinflammatory, andantithrombotic properties.2,3 Several lines of evidence indicate that CETP is proatherogenic. Introduction of the simian or the human CETP gene in mice naturally lacking CETP, results in a dose- dependent reduction of HDL cholesterol and an increasedsusceptibility to atherosclerosis.4-6 Furthermore, humanspossessing a genetic deficiency for CETP have higher HDL cholesterollevels and a reduced prevalence of coronary artery disease (CAD).7,8 However, in certain populations CETP deficiency appears to havedeleterious effects on CAD risk.9,10
CETP mRNA shows a widespread tissue distribution, with the highestlevels found in liver, spleen, and adipose tissue.11–13 Interestingly, CETP is also expressed locally in the arterialwall.14,15 Abundant amounts of CETP mass are found in macrophage-derived foam cells in human atherosclerotic lesions, but not in the healthy arterial wall. By promoting the transfer of cholesterylesters from HDL to apoB-containing lipoproteins CETP remodels the HDL particle, which is accompanied by a reduction in sizeand by the dissociation of preß-migrating, lipid poorapoAI,16,17 which is an important acceptor of ABCA1-mediated cholesterol efflux from macrophages.18,19 Locally, in the arterialwall the action of CETP might thus be implicated in the conversionof large cholesteryl ester enriched HDL into lipid-poor preß- HDL and thus may also have an antiatherogenic function. Interestingly, macrophage cholesterol loading results in a dose-dependent increase in macrophage secretion of CETP activity.20 Furthermore, in vitro studies indicated a direct role for CETP in cholesterolefflux from COS cells14 and J774 macrophages.21 The in vivoeffects of macrophage CETP production, however, are currentlyunknown.
Macrophages, present in atherosclerotic lesions primarily depend on infiltration from bone marrow (BM)-derived monocytes intothe arterial wall.
Therefore, in the present study, we investigated the effects of selective introduction of CETP in BM-derived cells and thus macrophages on lipoprotein metabolism and atherosclerotic lesion development by using bone marrow transplantation. We show that CETP production by macrophages and other BM-derived cells, under conditions of impaired clearance of apoB-containinglipoproteins, is highly atherogenic because of its significantcontribution to the serum cholesteryl ester transfer capacity.
MATERIALS AND METHODS
For detailed methodology, please refer to the online data supplement available at http://circres.ahajournals.org. Bone marrow transplantation (BMT) was used to selectively introduce CETP in hematopoietic cells, including macrophages. Briefly, female LDL receptor knockout (LDLr–/–)
mice (C57Bl/6J N5) were exposed to a singledose of 9 grays (Gy) (0.19 Gy/min, 200 kV, 4 mA) X-ray totalbody irradiation, using an Andrex Smart 225 Röntgen source(YXLON International, Copenhagen, Denmark) 1 day before transplantation. Irradiated recipients were transplanted by intravenous injection of 0.5x107 bone marrow cells, isolated from male CETP transgenic mice (CETP Tg; strain 5203; C57Bl/6J N10), overexpressing human CETP under the control of its own promoter and other major regulatoryelements,22 or from wild-type littermates. To induce the developmentof atherosclerosis, the mice were fed Western-type diet (WTD),containing 15% (w/w) total fat and 0.25% (w/w) cholesterol (DietW, Special Diet Services, Witham, UK) for 9 weeks, startingat 8 weeks after transplantation after which the mice were euthanized and atherosclerotic lesion development and the composition ofthe lesions was quantified. For determination of serum lipid levels blood was drawn after an overnight fasting-period. CETP activity in serum was measured as the transfer/exchange of 14C-cholesterol oleate between exogenously added human LDL and HDL, while CETPmass was determined using a CETP ELISA (Daiichi). In addition,CETP mRNA expression was determined in whole livers and spleensof transplanted mice at 17 weeks posttransplant and in parenchymal,endothelial, and Kupffer cells isolated from livers of CETPTg mice using real time-quantitative PCR.
RESULTS
CETP Production by BM-Derived Cells Induces Atherosclerotic Lesion Development in LDLr–/– Mice
To assess the role of CETP production by BM-derived cells in atherosclerotic lesion development, we used BMT to selectivelyintroduce CETP in hematopoietic cells. Hereto, bone marrow fromCETP transgenic mice was transplanted into LDLr–/– mice (CETP Tg → LDLr–/–), which represents an established animal model for the development of atherosclerosis. LDLr–/– mice transplanted with bone marrow from nontransgenic littermatesdevoid of CETP expression were used as controls (WT → LDLr–/–). For our experiments we used CETP transgenic mice expressing human CETP under control of its own promoter and natural flankingregions.22 These mice display a tissue distribution patternof human CETP mRNA expression that is similar to those observedin humans (liver, spleen, small intestine, kidney, and adiposetissue) and the expression is regulated by dietary cholesterol. Plasma CETP levels in the CETP transgenic mice are similar to the levels in normolipidaemic humans.23 Furthermore, plasmaCETP activity in the CETP transgenic animals (45±6 nmol/mL/h)is comparable to the activity that we have previously described for healthy Finnish subjects (35±7 nmol/mL/h24).
To induce atherosclerotic lesion development, the transplantedmice were fed a Western-type diet, containing 0.25% cholesteroland 15% fat, starting at 8 weeks after transplantation. After 9 weeks on Western-type diet atherosclerotic lesion developmentwas analyzed in the aortic root of WT → LDLr–/– miceand in CETP Tg → LDLr–/– animals. As shown in Fig. 1a, production of CETP by BM-derived cells induced a 1.8-fold increase in
lesion size (561±52x103 µm2 in CETP Tg → LDLr–/– mice versus 309±36x103 µm2 in WT → LDLr–/– mice, n=9, P<0.01). The macrophage content of the lesionsof WT → LDLr–/– mice was 64±6%, while the collagencontent was 4±1%. No significant effect of CETP expressionby BM-derived cells was observed on the relative macrophageand collagen contents of the lesions (60±5% and 7±2%,respectively). A predominant part of the lesions also consisted of acellular necrotic areas which did not differ significantly between the 2 groups (11±6% in WT → LDLr–/– mice and 17±4% in CETP Tg → LDLr–/– animals).Thus, CETP production by BM-derived cells induces the growthof the atherosclerotic lesions without markedly affecting lesion composition.
Fig. 1. CETP expression in BM-derived cells promotes atherosclerotic lesion development in LDLr–/– mice, but does not influence macrophage cholesterol efflux.
a, Formation of atherosclerotic lesions was determined at the aortic root of LDLr–/– mice reconstituted with wild-type (WT) or CETP transgenic (CETP Tg) bone marrow that were fed a Western-type diet for 9 weeks. Representative photographs of oil red O-stained cross sections of the aortic root at the level of tricuspid valves (left). The lesion area was calculated from 10 consecutive sections (right). **P<0.01 vs mice transplanted with WT bone marrow. b, Effect of macrophage CETP production on cholesterol efflux. Thioglycollate-elicited macrophages derived from WT mice (open bars) and from CETP Tg mice (filled bars) were loaded with 3H- cholesterol for 24 hour. 3H-cholesterol loading was 5.7±0.9 nmol/mg cell protein and 5.3±0.8 nmol/mg for WT and CETP Tg macrophages, respectively. After washing, cholesterol efflux was subsequently determined for the indicated times at 37°C in DMEM/0.2% BSA (BSA) or in the presence of 5 µg/mL lipid-free apoAI (apoAI) or 50 µg/mL human HDL (hHDL) and mouse HDL devoid of CETP (mHDL). Values are means±SEM of 4 individual mice.
Effect of Macrophage CETP Production on Cholesterol Efflux
To study the effect of endogenous CETP production on macrophage cholesterol efflux, efflux of cholesterol from 3H-cholesterol-loadedperitoneal macrophages from human CETP transgenic mice and wild-typelittermates that do not produce CETP was compared (Fig. 1b). No effect of macrophage CETP expression was observed on cholesterolefflux to lipid- free apoAI. After 24 hours, 11.7±1.7%of the cholesterol was effluxed from wild-type macrophages toapoAI as compared with 9.1±1.1% from CETP producing macrophages.In addition, no effect was observed on efflux to human HDL (27.2±0.6%for wild-type and 28.3±1.8% for CETP producing macrophagesafter 24 hours) or mouse HDL devoid of CETP (39.4±1.2%for wild-type and 37.7±2.7% for CETP producing macrophagesafter 24 hours).
It is thus unlikely that the increase in lesiondevelopment is caused by effects of macrophage CETP productionon cholesterol efflux.
CETP Production by BM-Derived Cells Induces Redistribution of Cholesteryl Esters From HDL to ApoB-Containing Lipoproteins The primary function in which CETP is implicated is the transfer of cholesteryl esters from HDL to proatherogenic apoB-containinglipoproteins.
Therefore, next we analyzed the effect of CETPproduction by BM-derived cells on serum lipid levels. On regular chow diet, no effect of CETP production by BM-derived cellson the concentration of free cholesterol and cholesteryl estersin the circulation was observed (Table). CETP, however, didinduce a prominent redistribution of cholesteryl esters fromHDL to apoB- containing lipoproteins (Fig. 2a). As a result,HDL cholesterol was reduced from 180±10 mg/dL in control WT → LDLr–/– animals to 145±9 mg/dL (P<0.05)in CETP Tg → LDLr–/– mice (Table).
Table 1. Effect of CETP Production by BM-Derived Cells on Serum Lipid Levels in LDLr–/–
Mice a
Mice Diet Free cholesterol
(mg/dL)
Cholesteryl esters (mg/dL)
HDL cholesterol (mg/dL)
WT → LDLr-/- Chow 71±5 407±24 180±10
CETP Tg → LDLr-/- Chow 64±5 404±19 145±9 **
b
Mice Diet Free cholesterol
(mg/dL)
Cholesteryl esters (mg/dL)
HDL cholesterol (mg/dL)
WT → LDLr-/- WTD 204±31 1520±302 76±7
CETP Tg → LDLr-/- WTD 498±60** 2593±280* 53±7*
Blood samples were drawn after an overnight fast at 8 weeks post transplant while feeding regular chow diet (Chow) (a) and at 17 weeks after BMT after 9 weeks feeding of a high-cholesterol Western-type diet (WTD) (b). Sera from individual mice were fractionated using a superose 6 column to determine HDL cholesterol levels. The amount of free and esterified cholesterol, and HDL cholesterol in LDLr-/- mice transplanted with wild-type or CETP Tg bone marrow is shown. Values are the mean±SEM of at least 9 mice. *P<0.05,
**P<0.001 vs control animals transplanted with wild-type bone marrow.
On challenging the mice with the Western-type diet, the effect of CETP production by BM-derived cells was even more pronounced. Under this condition serum free cholesterol and cholesteryl esters were 2.4-fold (P<0.001) and 1.7-fold (P<0.05),respectively, higher in CETP Tg → LDLr–/–
mice becauseof an increase in VLDL and LDL cholesterol levels (Table, Fig. 2a).HDL cholesterol levels were reduced from 76±7 mg/dL incontrol WT → LDLr–/– animals to 53±7 mg/dL (P<0.05)in CETP Tg → LDLr–/– mice.
CETP production by BM-derived cells thus markedly influences serum cholesterol levels and the distribution of cholesterol among the different lipoproteins.To investigate changes in HDL subclass distribution, serum of the transplanted mice was also analyzed by crossed immunoelectrophoresis (Fig. 2b). On Western-type diet, the amount of preß-HDLparticles was 3- fold (P<0.001) increased in serum of CETPTg → LDLr–/– mice (40±4%) as compared with controlWT → LDLr–/– animals (14±2%). BM-derived CETPis thus capable of transferring cholesterol from HDL to apoB-containing lipoproteins, thereby transforming HDL and generating metabolicallymore preß-HDL. Despite the increased antiatherogenic potential of HDL as a result of CETP production by BM-derivedcells, the dramatic increase in proatherogenic VLDL and LDL levels still resulted in an increased susceptibility to lesiondevelopment.
Fig. 2. CETP expression by BM-derived cells increases VLDL and LDL cholesterol levels, reduces total HDL cholesterol, and increases preß-HDL in LDLr–/– mice. Blood was collected after an overnight fast at 8 weeks post transplantation when the mice had been on regular chow diet (chow) and at 17 weeks after BMT when the mice had been on Western-type diet (WTD) for 9 weeks. a, The amount of free cholesterol (left) and esterified cholesterol (right) and their distribution over the different lipoproteins in LDLr–/– mice transplanted with wild-type (open circles) or CETP Tg (closed circles) bone marrow is shown. Inserts show free cholesterol distributions at a smaller scale. Fractions 3 to 7 represent VLDL; fraction 8 to 14, LDL; and fractions 15 to 19, HDL, respectively. Values are the mean±SEM of 9 mice. b, The amount of preß-HDL and α-HDL was determined by apoAI crossed immunoelectrophoresis (top) in the sera of LDLr–/– mice transplanted with wild-type or CETP Tg bone marrow. The bottom panel illustrates the quantification of the changes observed. Open bars represent LDLr–/– mice transplanted with wild-type bone marrow and filled bars mice transplanted with CETP Tg bone marrow. Values are the mean±SEM of 9 to 10 mice. *P<0.001 vs mice transplanted with wild- type bone marrow.
BM-Derived Cells Significantly Contribute to Serum CETP Activity and Mass
As shown in Fig. 3a, CETP production by BM-derived cells resulted in substantial levels of CETP activity in the serum of CETPTg → LDLr–/– mice (38±3 nmol/mL/h). This levelof serum CETP activity is comparable to the activity in thedonor CETP Tg mice, expressing CETP in all endogenous tissues(45±6 nmol/mL/h) as well as the activity that we havepreviously described for healthy Finnish subjects (35±7nmol/mL/h24). In addition, a CETP mass of 4.81±0.51 µg/mL was determined in sera of the LDLr–/–
animals reconstitutedwith CETP Tg bone marrow. BM-derived cells overall are thusan important contributor to serum total CETP activity and mass, thereby explaining the observed redistribution of cholesteryl esters from HDL to proatherogenic apoB-containing lipoproteins and the enhanced formation of preß-HDL in CETP Tg → LDLr–/– mice. No effect of introduction of CETP in BM-derived cellswas observed on serum phospholipid transfer protein (PLTP) activity(23±1 µmol/mL/h and 22±1 µmol/mL/h forWT → LDLr–/– and CETP Tg → LDLr–/– mice, respectively).
Fig. 3. CETP production by BM-derived cells significantly contributes to serum CETP activity and hepatic and splenic CETP expression. a, CETP activity in the sera of LDLr–/–
mice transplanted with bone marrow from wild-type (WT) or CETP transgenic (CETP Tg) mice.
b, CETP mRNA expression in liver and spleen of LDLr–/– mice following BMT (as above), determined by real-time PCR. CETP mRNA expression is shown relative to the average expression of the housekeeping genes HPRT, ß-actin, and GAPDH. Open bars represent LDLr–/– mice transplanted with WT bone marrow and filled bars mice transplanted with CETP Tg bone marrow. Values are the mean±SEM of 9 to 10 mice. *P<0.05 and ***P<0.0001 vs.
mice transplanted with wild-type bone marrow.
Circulating CETP levels are highly correlated with hepatic CETPmRNA levels and hepatic CETP output.25 Therefore, CETP mRNAexpression was determined in the transplanted mice both in the liverand in the spleen (Fig.
3b). As expected, reconstitution of LDLr–/– mice with CETP Tg bone marrow resulted in the appearance ofCETP mRNA in spleens. Interestingly, CETP mRNA levels in livers of the transplanted mice were 5.2-fold higher as compared withthe levels in spleens (0.393±0.121 in liver as comparedwith 0.074±0.024 in spleen, P<0.05).
The liver contains several different cell types including parenchymalcells, endothelial, and Kupffer cells. Kupffer cells are liver-specificmacrophages that develop from BM-derived monocytes and accountfor 15% of all liver cells and 2.5% of liver protein.26 To analyzethe replacement of Kupffer cells
after BMT, LDLr–/– mice were transplanted with bone marrow from mice, expressingenhanced green fluorescent protein (EGFP). As shown in Fig.
4a,, blood cells are quantitatively replaced after transplantationby the donor EGFP-positive cells reaching a level of 99±0.2% at 8 weeks after transplantation. At this time point already 54±4% (n=5) of the F4/80- expressing Kupffer cells wereEGFP-positive and thus of donor-origin (Fig.
4b). Kupffercells are thus suspected to be a predominant source of CETPin livers of LDLr–/– mice reconstituted with CETP-expressingbone marrow. In addition to the liver, EGFP-positive macrophages were found in other organs, including spleen and lung. Interestingly,in spleen EGFP-positive cells were primarily found in the redpulp, which is rich in macrophages as well as in the marginal zone that is rich in B-cells, while only limited replacementof T-cells in the white pulp was observed. In lesions EGFP was expressed by macrophage foam cells, but not by endothelial cellsoverlying the lesion.
Fig. 4. Reconstitution of blood cells and macrophages in liver, spleen, lung, and lesions with donor-derived EGFP-expressing cells after bone marrow transplantation. a, The appearance of EGFP-expressing blood cells in the circulation determined by FACS analysis at different time points after transplantation of LDLr–/– mice (n=5) with EGFP-expressing bone marrow. b, Representative merged photomicrographs of EGFP fluorescence (green), immunohistochemical staining against mouse macrophage F4/80 (red) and Dapi for nuclei (blue) in liver, spleen, lung at 8 weeks after transplantation as well as atherosclerotic lesions.
Colocalization of macrophages with EGFP is yellow.
To investigate the potential contribution of Kupffer cells to total hepatic CETP production, the expression of CETP was evaluated in purified parenchymal cells, endothelial cells, and Kupffercells of livers from total- body CETP Tg mice that express CETPin all endogenous tissues (Fig. 5a).
CETP mRNA expression in parenchymal liver cells was 0.006±0.001.
Expressionin endothelial cells was ≈10-fold higher (0.062±0.021;P<0.01) as compared with parenchymal liver cells. However, a 47-fold higher expression was found in Kupffer cells (0.277±0.036; P<0.0001). Thus, although Kupffer cells only contribute to2.5% of the total liver protein, they do contain at least 48%of the total liver CETP expression, as compared with 38% and14% for parenchymal cells and endothelial cells, respectively.
Immunohistochemical staining of liver sections from CETP Tgmice also indicated the predominant Kupffer cell localizationof CETP in the liver (Fig.
5b).
Fig. 5. CETP expression by different cell types of the liver and spleen. a, Hepatic CETP mRNA expression was determined in total-body CETP transgenics by real-time PCR after separation of the parenchymal cells, endothelial cells, and Kupffer cells in the liver by collagenase perfusion. c, For determination of CETP mRNA expression in spleen, splenocytes were harvested using magnetic nanoparticles conjugated with anti-mouse CD4, CD8a, CD45R/B220, or CD11b monoclonal antibodies for the isolation of CD4+ T-helper cells, CD8+
cytotoxic T-cells, B-cells, and macrophages/neutrophils. CETP mRNA expression is shown relative to the average expression of the housekeeping genes HPRT, ß-actin, and GAPDH.
Values are the mean±SEM of 6 female CETP Tg mice. *P<0.01, **P<0.0001 vs. parenchymal
cells. b and d, For detection of CETP protein cryostat sections of liver (b) and spleen (d) of CETP Tg mice were stained immunohistochemically for CETP (green), F4/80-positive macrophages (red) and nuclei (blue). Colocalization of CETP and macrophage F4/80 is yellow.
To determine which types of BM-derived cells, in addition tomacrophages contribute to the circulating CETP activity levels,CD4+ T-helper cells, CD8+
cytotoxic T-cells, B-cells, and macrophages/neutrophilswere isolated from spleens of CETP Tg mice. As shown in Fig. 5c,CETP mRNA was found in all splenocytes analyzed, with no significant difference in the level of expression between the differentcell types. Immunohistochemical staining of spleen sections for CETP showed CETP protein expression in macrophages of the red pulp of the spleen and in the marginal zone surroundingthe white pulp, which is rich in B-cells (Fig. 5d). Thus,other BM- derived cells also contribute to CETP production besidesmacrophages.
Reverse Bone Marrow Transplantation of Wild-Type Bone Marrow to CETP Tg Mice
To confirm the quantitative importance of hematopoietically-derivedCETP for determining plasma CETP levels, the effect of specificdisruption of BM- derived CETP in CETP Tg mice was determined.Hereto a reverse BMT experiment was performed, in which bonemarrow from wild-type littermates was transplanted into CETPTg mice having a functional LDL receptor. At 8 weeks after transplantation,CETP activity in control transplanted CETP Tg
→ CETP Tg animals was 50.1±3.2 nmol/mL/h, as compared with only 23.1±2.5nmol/mL/h in WT → CETP Tg mice (P<0.0001) (Fig. 6a). CETP mass was reduced from 2.26±0.35 µg/mL to 1.31±0.23µg/mL (P<0.0001).
Specific disruption of CETP productionby BM-derived cells thus resulted in a 53.8% reduction in CETPactivity and a 41.9% reduction in CETP mass, confirming ourconclusions about the quantitative importance of BM-derived CETP. The observed reduction in plasma CETP activity and masslevels also coincided with 2-fold lower (P=0.029) CETP mRNAexpression levels in the liver (0.18±0.03 for controlCETP Tg → CETP Tg animals and 0.09±0.02 for WT → CETP Tg mice).
Fig. 6. Disruption of CETP production by BM-derived cells reduces serum CETP activity but does not affect atherosclerotic lesion development in CETP Tg mice with a functional LDL receptor. a, CETP activity in the sera of human CETP transgenic (CETP Tg) mice transplanted with bone marrow from CETP Tg or wild-type (WT) mice. Open bars represent CETP Tg mice transplanted with CETP Tg bone marrow (n=9) and filled bars mice transplanted with WT bone marrow (n=9). ***P<0.0001 vs. mice transplanted with CETP Tg bone marrow. b, Formation of atherosclerotic lesions was determined at the aortic root of CETP Tg mice reconstituted with CETP Tg or WT bone marrow that were fed a high cholesterol/cholate diet for 8 weeks. Representative photographs of oil red O-stained cross sections of the aortic root at the level of tricuspid valves (left). The lesion area was calculated from 10 consecutive sections (right).
The atherosclerosis susceptibilities of CETP Tg → CETP Tg animalsand WT → CETP Tg mice were compared after 8 weeks on a high-cholesterol diet, containing 1% cholesterol, 15% fat, and 0.5% cholate, started at 8 weeks after transplantation. Both on chow dietand after feeding the high- cholesterol diet, no effect of disruptionof CETP production by BM-derived cells was observed on serum cholesterol levels (data not shown). In addition, no differences were observed in atherosclerotic lesion size (73.8±10.0x103 µm2 for CETP Tg → CETP Tg animals [n=9] and 74.7±10.0x103 µm2 for WT → CETP Tg mice [n=8]) (Fig. 6b). In both groups of mice lesions were composed of multiple layers of foam cells.Thus, in agreement with the in vitro studies which showed thatcholesterol efflux from macrophages was not affected by CETPexpression, also no local effects of disruption of macrophage CETP expression on atherosclerotic lesion development were foundin CETP Tg mice with a functional LDL receptor.
DISCUSSION
It has already been known for almost two decades that CETP isproduced by macrophages.20 In addition, abundant amounts of CETP protein and mRNA are detected in macrophage-derived foamcells in human aortic and coronary atherosclerotic lesions, indicating that CETP production by macrophages might play animportant role in cholesterol homeostasis locally in the arterial wall.14,15 Until now, however, no in vivo studies were performedon the role of macrophage-derived CETP in atherosclerotic lesion development. To specifically study the role of macrophage CETP in atherosclerosis, we have created chimeras that express humanCETP in BM-derived cells, including arterial intima macrophages,by transplantation of bone marrow from human CETP transgenic mice into LDLr–/– mice.
Interestingly, we foundthat CETP production by BM-derived cells promoted the development of atherosclerosis, whereas efflux of cholesterol from peritonealmacrophages to lipid-free apoAI or HDL was not affected. Thus,in contrast to previous in vitro studies suggesting an antiatherogenicfunction for macrophage CETP14,15,20,21
in the current studywe provide strong in vivo evidence that CETP production by BM-derivedcells, including macrophages is proatherogenic in mice lacking the LDL receptor. The increased atherosclerosis in these mice expressing CETP in BM-derived cells coincided with a significantredistribution of cholesteryl esters from HDL to pro-atherogenicapoB-containing lipoproteins and a dramatic increase in the levels of proatherogenic apoB-containing lipoproteins as a result of the
appearance of substantial amounts of active CETP in thecirculation. The large increase in plasma cholesteryl esterlevels in the CETP Tg LDLr–/–
mice is primarily theconsequence of the circulating CETP activity levels on the cholesterylester transfer process in these mice. In absence of the LDL receptor, the removal of triglyceride-rich apoB-containing lipoproteinsfrom the circulation is severely impaired, leading to theiraccumulation. Similar effects of CETP expression on the accumulation of apoB-containing lipoproteins have previously been described in apoE and LDL receptor knockout mice.5 By specific disruptionof CETP production by BM-derived cells in CETP Tg mice we establishedthat BM-derived cells contribute for 50% to the total circulatingCETP activity levels. In these animals with a functional LDL receptor, no effects on serum cholesterol levels or atherosclerotic lesion development were observed, indicating that LDL receptordeficiency is a major contributor to the observed effects ofBM- derived CETP in LDL receptor knockout mice. In addition,these studies implicate that macrophage CETP does not affectcholesterol homeostasis locally in the arterial wall.
Macrophages are an important source of BM-derived cells thatreside within almost all tissues. Important sources of serumCETP are liver and spleen.11–
13 CETP production by the liver has primarily been attributed to the parenchymal cells. However, the liver consists of 15% Kupffer cells, representing the hepatic macrophages.26 In the current study we also provideevidence that Kupffer cells are an important source of CETPin livers of total-body CETP transgenic mice and that disruptionof CETP expression in BM-derived cells results in a 50% decrease in hepatic CETP mRNA expression because of the absence of Kupffercell CETP expression. In agreement with this, nonparenchymalcells of the hepatic sinusoids are the principal source of CETPin livers of cynomolgus monkeys that like humans naturally expressCETP.27 Thus, although it still remains to be determined whetherand how much macrophages and other BM-derived cells contribute to plasma CETP activity in humans, the comparable findings in human CETP transgenic mice and cynomolgus monkeys suggest thatour findings apply to species that naturally express CETP.
In our BMT chimeras also significant expression of CETP wasfound in spleen. In agreement, CETP expression was previouslyshown in spleens of adult total-body CETP transgenic mice,28 hamsters,13 and humans.29 In the current study, we show thatB-cells, T-cells, and macrophages/neutrophils isolated fromspleens of total-body CETP Tg mice all express CETP mRNA.
CETPprotein colocalized with macrophages in the red pulp of thespleen. In addition, substantial CETP protein expression wasfound in the marginal zone, the interphase between the nonlymphoidred pulp and the lymphoid white pulp, an area rich in B-cells.In hamsters, CETP is primary localized at the periphery of germinalfollicles of the spleen.13 Also in human spleens CETP proteinwas found in white pulp germinal centers and colocalized with B-cells and a proportion of marginal zone B-cells.29 Thus, CETPis not only restricted to macrophages, but is also producedby other cells from bone marrow origin.
An important question remains about the function of CETP productionby macrophages. Macrophage foam cells are primarily restricted to atherosclerosis, whereas activated macrophages are a commonfeature of
many inflammatory diseases. Interestingly, CETP belongsto the family of lipid transfer/lipopolysaccharide-binding proteins.30 Furthermore, administration of lipopolysaccharide to hamsterswith endogenous CETP expression or to transgenic mice expressinghuman CETP induces a rapid decrease in serum CETP concentration,31,32 suggesting that CETP with its molecular mimicry to lipopolysaccharide-bindingproteins might be able to modulate the lipopolysaccharide responseand thus can play a role in innate immunity in addition to itsrole in cholesterol metabolism.
In conclusion, the current study strongly suggests that CETPproduced by BM-derived cells, including macrophages, is an importantcontributor to total serum CETP activity. It modulates lipoproteinmetabolism by mediating the redistribution of cholesteryl estersfrom HDL to apoB-containing lipoproteins, and promotes the developmentof atherosclerosis in LDL receptor knockout mice with impaired clearance of apoB-containing lipoproteins. The substantial contributionof BM-derived cells to circulating total CETP activity implicatesan important role for CETP in macrophage function.
ACKNOWLEDGEMENTS
The authors thank Jari Metso, M.Sc. for expert analysis of preß-HDL particles in mice sera and determining CETP activities.
SOURCES OF FUNDING
This work was supported by The Netherlands Organization of Scientific Research (VIDI grant 917.66.301 to M.V.E., VIDI grant 917.36.351 to P.C.N.R.), the Netherlands Heart Foundation (grant 2001T041to M.V.E.), the Finnish Foundation for Cardiovascular Research,and the Sigrid Juselius Foundation.
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