This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/5/678    most recent 01.RES.0000260202.79927.4fv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Van Eck, M. Articles by Van Berkel, T. J.C. Search for Related Content PubMed PubMed Citation Articles by Van Eck, M. Articles by Van Berkel, T. J.C. Related Collections Animal models of human disease Gene expression Genetically altered mice Lipid and lipoprotein metabolism

(Circulation Research. 2007;100:678.)
© 2007 American Heart Association, Inc.

Molecular Medicine

Important Role for Bone Marrow–Derived Cholesteryl Ester Transfer Protein in Lipoprotein Cholesterol Redistribution and Atherosclerotic Lesion Development in LDL Receptor Knockout Mice

Miranda Van Eck^*, Dan Ye^*, 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 (M.V.E., D.Y., R.B.H., J.K.K., M.H., T.J.C.V.B.), Leiden University, Leiden, The Netherlands; Department of Molecular Medicine (C.E., M.J.), National Public Health Institute, Biomedicum, Helsinki, Finland; Department of General Internal Medicine, Endocrinology and Metabolic Diseases (W.d.H., P.C.N.R.), Leiden University Medical Center, Leiden, The Netherlands.

Correspondence to M. Van Eck, Division of Biopharmaceutics, Gorlaeus Laboratories, Einsteinweg 55, 2333 CC Leiden, The Netherlands. E-mail M.Eck{at}LACDR.LeidenUniv.nl

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 µg/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 a 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 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.

Key Words: atherosclerosis • macrophages • lipoproteins • bone marrow transplantation • mouse models

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 concomitant equimolar transfer of triglycerides to HDL.¹ High HDL levels protect against the development of atherosclerosis by virtue of its essential role in the removal of excess cholesterol from peripheral cells and its antioxidative, antiinflammatory, and antithrombotic 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 increased susceptibility to atherosclerosis.^4–6 Furthermore, humans possessing a genetic deficiency for CETP have higher HDL cholesterol levels and a reduced prevalence of coronary artery disease (CAD).^7,8 However, in certain populations CETP deficiency appears to have deleterious effects on CAD risk.^9,10

CETP mRNA shows a widespread tissue distribution, with the highest levels found in liver, spleen, and adipose tissue.^11–13 Interestingly, CETP is also expressed locally in the arterial wall.^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 cholesteryl esters from HDL to apoB-containing lipoproteins CETP remodels the HDL particle, which is accompanied by a reduction in size and by the dissociation of preß-migrating, lipid poor apoAI,^16,17 which is an important acceptor of ABCA1-mediated cholesterol efflux from macrophages.^18,19 Locally, in the arterial wall the action of CETP might thus be implicated in the conversion of 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 cholesterol efflux from COS cells¹⁴ and J774 macrophages.²¹ The in vivo effects of macrophage CETP production, however, are currently unknown.

Macrophages, present in atherosclerotic lesions primarily depend on infiltration from bone marrow (BM)-derived monocytes into the 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-containing lipoproteins, is highly atherogenic because of its significant contribution to the serum cholesteryl ester transfer capacity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 single dose of 9 grays (Gy) (0.19 Gy/min, 200 kV, 4 mA) X-ray total body 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 regulatory elements,²² or from wild-type littermates. To induce the development of atherosclerosis, the mice were fed Western-type diet (WTD), containing 15% (w/w) total fat and 0.25% (w/w) cholesterol (Diet W, Special Diet Services, Witham, UK) for 9 weeks, starting at 8 weeks after transplantation after which the mice were euthanized and atherosclerotic lesion development and the composition of the 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 CETP mass was determined using a CETP ELISA (Daiichi). In addition, CETP mRNA expression was determined in whole livers and spleens of transplanted mice at 17 weeks posttransplant and in parenchymal, endothelial, and Kupffer cells isolated from livers of CETP Tg mice using real time-quantitative PCR.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 selectively introduce CETP in hematopoietic cells. Hereto, bone marrow from CETP transgenic mice was transplanted into LDLr^–/– mice (CETP TgLDLr^–/–), which represents an established animal model for the development of atherosclerosis. LDLr^–/– mice transplanted with bone marrow from nontransgenic littermates devoid of CETP expression were used as controls (WTLDLr^–/–). For our experiments we used CETP transgenic mice expressing human CETP under control of its own promoter and natural flanking regions.²² These mice display a tissue distribution pattern of human CETP mRNA expression that is similar to those observed in humans (liver, spleen, small intestine, kidney, and adipose tissue) 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, plasma CETP 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 transplanted mice were fed a Western-type diet, containing 0.25% cholesterol and 15% fat, starting at 8 weeks after transplantation. After 9 weeks on Western-type diet atherosclerotic lesion development was analyzed in the aortic root of WTLDLr^–/– mice and in CETP TgLDLr^–/– animals. As shown in Figure 1a, production of CETP by BM-derived cells induced a 1.8-fold increase in lesion size (561±52x10³ µm² in CETP TgLDLr^–/– mice versus 309±36x10³ µm² in WTLDLr^–/– mice, n=9, P<0.01). The macrophage content of the lesions of WTLDLr^–/– mice was 64±6%, while the collagen content was 4±1%. No significant effect of CETP expression by BM-derived cells was observed on the relative macrophage and 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 WTLDLr^–/– mice and 17±4% in CETP TgLDLr^–/– animals). Thus, CETP production by BM-derived cells induces the growth of the atherosclerotic lesions without markedly affecting lesion composition.

View larger version (35K): [in this window] [in a new window]   Figure 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-loaded peritoneal macrophages from human CETP transgenic mice and wild-type littermates that do not produce CETP was compared (Figure 1b). No effect of macrophage CETP expression was observed on cholesterol efflux to lipid-free apoAI. After 24 hours, 11.7±1.7% of the cholesterol was effluxed from wild-type macrophages to apoAI 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 macrophages after 24 hours) or mouse HDL devoid of CETP (39.4±1.2% for wild-type and 37.7±2.7% for CETP producing macrophages after 24 hours). It is thus unlikely that the increase in lesion development is caused by effects of macrophage CETP production on 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-containing lipoproteins. Therefore, next we analyzed the effect of CETP production by BM-derived cells on serum lipid levels. On regular chow diet, no effect of CETP production by BM-derived cells on the concentration of free cholesterol and cholesteryl esters in the circulation was observed (Table). CETP, however, did induce a prominent redistribution of cholesteryl esters from HDL to apoB-containing lipoproteins (Figure 2a). As a result, HDL cholesterol was reduced from 180±10 mg/dL in control WTLDLr^–/– animals to 145±9 mg/dL (P<0.05) in CETP TgLDLr^–/– mice (Table).

View this table: [in this window] [in a new window]   Table 1. Effect of CETP Production by BM-Derived Cells on Serum Lipid Levels in LDLr–/– Mice

View larger version (26K): [in this window] [in a new window]   Figure 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 -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.

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 TgLDLr^–/– mice because of an increase in VLDL and LDL cholesterol levels (Table, Figure 2a). HDL cholesterol levels were reduced from 76±7 mg/dL in control WTLDLr^–/– animals to 53±7 mg/dL (P<0.05) in CETP TgLDLr^–/– 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 (Figure 2b). On Western-type diet, the amount of preß-HDL particles was 3-fold (P<0.001) increased in serum of CETP TgLDLr^–/– mice (40±4%) as compared with control WTLDLr^–/– animals (14±2%). BM-derived CETP is thus capable of transferring cholesterol from HDL to apoB-containing lipoproteins, thereby transforming HDL and generating metabolically more preß-HDL. Despite the increased antiatherogenic potential of HDL as a result of CETP production by BM-derived cells, the dramatic increase in proatherogenic VLDL and LDL levels still resulted in an increased susceptibility to lesion development.

BM-Derived Cells Significantly Contribute to Serum CETP Activity and Mass
As shown in Figure 3a, CETP production by BM-derived cells resulted in substantial levels of CETP activity in the serum of CETP TgLDLr^–/– mice (38±3 nmol/mL/h). This level of serum CETP activity is comparable to the activity in the donor CETP Tg mice, expressing CETP in all endogenous tissues (45±6 nmol/mL/h) as well as the activity that we have previously described for healthy Finnish subjects (35±7 nmol/mL/h²⁴). In addition, a CETP mass of 4.81±0.51 µg/mL was determined in sera of the LDLr^–/– animals reconstituted with CETP Tg bone marrow. BM-derived cells overall are thus an 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 TgLDLr^–/– mice. No effect of introduction of CETP in BM-derived cells was observed on serum phospholipid transfer protein (PLTP) activity (23±1µmol/mL/h and 22±1µmol/mL/h for WTLDLr^–/– and CETP TgLDLr^–/– mice, respectively).

View larger version (10K): [in this window] [in a new window]   Figure 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 CETP mRNA levels and hepatic CETP output.²⁵ Therefore, CETP mRNA expression was determined in the transplanted mice both in liver and in spleen (Figure 3b). As expected, reconstitution of LDLr^–/– mice with CETP Tg bone marrow resulted in the appearance of CETP mRNA in spleens. Interestingly, CETP mRNA levels in livers of the transplanted mice were 5.2-fold higher as compared with the levels in spleens (0.393±0.121 in liver as compared with 0.074±0.024 in spleen, P<0.05).

The liver contains several different cell types including parenchymal cells, endothelial, and Kupffer cells. Kupffer cells are liver-specific macrophages that develop from BM-derived monocytes and account for 15% of all liver cells and 2.5% of liver protein.²⁶ To analyze the replacement of Kupffer cells after BMT, LDLr^–/– mice were transplanted with bone marrow from mice, expressing enhanced green fluorescent protein (EGFP). As shown in Figure 4a, blood cells are quantitatively replaced after transplantation by 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 were EGFP-positive and thus of donor-origin (Figure 4b). Kupffer cells are thus suspected to be a predominant source of CETP in livers of LDLr^–/– mice reconstituted with CETP-expressing bone 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 red pulp, which is rich in macrophages as well as in the marginal zone that is rich in B-cells, while only limited replacement of T-cells in the white pulp was observed. In lesions EGFP was expressed by macrophage foam cells, but not by endothelial cells overlying the lesion.

View larger version (41K): [in this window] [in a new window]   Figure 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 Kupffer cells of livers from total-body CETP Tg mice that express CETP in all endogenous tissues (Figure 5a). CETP mRNA expression in parenchymal liver cells was 0.006±0.001. Expression in 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 to 2.5% of the total liver protein, they do contain at least 48% of the total liver CETP expression, as compared with 38% and 14% for parenchymal cells and endothelial cells, respectively. Immunohistochemical staining of liver sections from CETP Tg mice also indicated the predominant Kupffer cell localization of CETP in the liver (Figure 5b).

View larger version (56K): [in this window] [in a new window]   Figure 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. b, 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 to macrophages contribute to the circulating CETP activity levels, CD4+ T-helper cells, CD8+ cytotoxic T-cells, B-cells, and macrophages/neutrophils were isolated from spleens of CETP Tg mice. As shown in Figure 5c, CETP mRNA was found in all splenocytes analyzed, with no significant difference in the level of expression between the different cell 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 surrounding the white pulp, which is rich in B-cells (Figure 5d). Thus, other BM-derived cells also contribute to CETP production besides macrophages.

Reverse Bone Marrow Transplantation of Wild-Type Bone Marrow to CETP Tg Mice
To confirm the quantitative importance of hematopoietically-derived CETP for determining plasma CETP levels, the effect of specific disruption of BM-derived CETP in CETP Tg mice was determined. Hereto a reverse BMT experiment was performed, in which bone marrow from wild-type littermates was transplanted into CETP Tg mice having a functional LDL receptor. At 8 weeks after transplantation, CETP activity in control transplanted CETP TgCETP Tg animals was 50.1±3.2 nmol/mL/h, as compared with only 23.1±2.5 nmol/mL/h in WTCETP Tg mice (P<0.0001) (Figure 6a). CETP mass was reduced from 2.26±0.35 µg/mL to 1.31±0.23 µg/mL (P<0.0001). Specific disruption of CETP production by BM-derived cells thus resulted in a 53.8% reduction in CETP activity and a 41.9% reduction in CETP mass, confirming our conclusions about the quantitative importance of BM-derived CETP. The observed reduction in plasma CETP activity and mass levels also coincided with 2-fold lower (P=0.029) CETP mRNA expression levels in the liver (0.18±0.03 for control CETP TgCETP Tg animals and 0.09±0.02 for WTCETP Tg mice).

View larger version (26K): [in this window] [in a new window]   Figure 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 TgCETP Tg animals and WTCETP 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 diet and after feeding the high-cholesterol diet, no effect of disruption of 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±10.0*10³ µm² for CETP TgCETP Tg animals [n=9] and 74.7±10.0*10³ µm² for WTCETP Tg mice [n=8]) (Figure 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 that cholesterol efflux from macrophages was not affected by CETP expression, also no local effects of disruption of macrophage CETP expression on atherosclerotic lesion development were found in CETP Tg mice with a functional LDL receptor.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has already been known for almost two decades that CETP is produced by macrophages.²⁰ In addition, abundant amounts of CETP protein and mRNA are detected in macrophage-derived foam cells in human aortic and coronary atherosclerotic lesions, indicating that CETP production by macrophages might play an important role in cholesterol homeostasis locally in the arterial wall.^14,15 Until now, however, no in vivo studies were performed on 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 human CETP in BM-derived cells, including arterial intima macrophages, by transplantation of bone marrow from human CETP transgenic mice into LDLr^–/– mice. Interestingly, we found that CETP production by BM-derived cells promoted the development of atherosclerosis, whereas efflux of cholesterol from peritoneal macrophages to lipid-free apoAI or HDL was not affected. Thus, in contrast to previous in vitro studies suggesting an antiatherogenic function for macrophage CETP^14,15,20,21 in the current study we provide strong in vivo evidence that CETP production by BM-derived cells, 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 significant redistribution of cholesteryl esters from HDL to pro-atherogenic apoB-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 the circulation. The large increase in plasma cholesteryl ester levels in the CETP TgLDLr^–/– mice is primarily the consequence of the circulating CETP activity levels on the cholesteryl ester transfer process in these mice. In absence of the LDL receptor, the removal of triglyceride-rich apoB-containing lipoproteins from the circulation is severely impaired, leading to their accumulation. 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 disruption of CETP production by BM-derived cells in CETP Tg mice we established that BM-derived cells contribute for 50% to the total circulating CETP 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 receptor deficiency is a major contributor to the observed effects of BM-derived CETP in LDL receptor knockout mice. In addition, these studies implicate that macrophage CETP does not affect cholesterol homeostasis locally in the arterial wall.

Macrophages are an important source of BM-derived cells that reside within almost all tissues. Important sources of serum CETP 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 provide evidence that Kupffer cells are an important source of CETP in livers of total-body CETP transgenic mice and that disruption of CETP expression in BM-derived cells results in a 50% decrease in hepatic CETP mRNA expression because of the absence of Kupffer cell CETP expression. In agreement with this, nonparenchymal cells of the hepatic sinusoids are the principal source of CETP in livers of cynomolgus monkeys that like humans naturally express CETP.²⁷ Thus, although it still remains to be determined whether and 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 that our findings apply to species that naturally express CETP.

In our BMT chimeras also significant expression of CETP was found in spleen. In agreement, CETP expression was previously shown in spleens of adult total-body CETP transgenic mice,²⁸ hamsters,¹³ and humans.²⁹ In the current study, we show that B-cells, T-cells, and macrophages/neutrophils isolated from spleens of total-body CETP Tg mice all express CETP mRNA. CETP protein colocalized with macrophages in the red pulp of the spleen. In addition, substantial CETP protein expression was found in the marginal zone, the interphase between the nonlymphoid red pulp and the lymphoid white pulp, an area rich in B-cells. In hamsters, CETP is primary localized at the periphery of germinal follicles of the spleen.¹³ Also in human spleens CETP protein was found in white pulp germinal centers and colocalized with B-cells and a proportion of marginal zone B-cells.²⁹ Thus, CETP is not only restricted to macrophages, but is also produced by other cells from bone marrow origin.

An important question remains about the function of CETP production by macrophages. Macrophage foam cells are primarily restricted to atherosclerosis, whereas activated macrophages are a common feature of many inflammatory diseases. Interestingly, CETP belongs to the family of lipid transfer/lipopolysaccharide-binding proteins.³⁰ Furthermore, administration of lipopolysaccharide to hamsters with endogenous CETP expression or to transgenic mice expressing human CETP induces a rapid decrease in serum CETP concentration,^31,32 suggesting that CETP with its molecular mimicry to lipopolysaccharide-binding proteins might be able to modulate the lipopolysaccharide response and thus can play a role in innate immunity in addition to its role in cholesterol metabolism.

In conclusion, the current study strongly suggests that CETP produced by BM-derived cells, including macrophages, is an important contributor to total serum CETP activity. It modulates lipoprotein metabolism by mediating the redistribution of cholesteryl esters from HDL to apoB-containing lipoproteins, and promotes the development of atherosclerosis in LDL receptor knockout mice with impaired clearance of apoB-containing lipoproteins. The substantial contribution of BM-derived cells to circulating total CETP activity implicates an important role for CETP in macrophage function.

	Acknowledgments

 
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 2001T041 to M.V.E.), the Finnish Foundation for Cardiovascular Research, and the Sigrid Juselius Foundation.

Disclosures

None.

	Footnotes

 
*These authors contributed equally to this work.

Original received September 7, 2006; revision received January 12, 2007; accepted January 31, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Tall AR. Plasma lipid transfer proteins. Ann Rev Biochem. 1995; 64: 235–257.[CrossRef][Medline] [Order article via Infotrieve]

2. Barter PJ, Nicholls S, Rye KA, Anantharamaiah GM, Navab M, Fogelman AM. Antiinflammatory properties of HDL. Circ Res. 2004; 95: 764–772.[Abstract/Free Full Text]

3. Assmann G, Gotto AM Jr. HDL cholesterol and protective factors in atherosclerosis. Circulation. 2004; 109: III8–14.[Medline] [Order article via Infotrieve]

4. Marotti KR, Castle CK, Boyle TP, Lin AH, Murray RW, Melchior GW. Severe atherosclerosis in transgenic mice expressing simian cholesteryl ester transfer protein. Nature. 1993; 364: 73–75.[CrossRef][Medline] [Order article via Infotrieve]

5. Plump AS, Masucci-Magoulas L, Bruce C, Bisgaier CL, Breslow JL, Tall AR. Increased atherosclerosis in apoE and LDL receptor gene knockout mice as a result of human cholesteryl ester transfer protein gene expression. Arterioscler Thromb Vasc Biol. 1999; 19: 1105–1110.[Abstract/Free Full Text]

6. Westerterp M, van der Hoogt CC, de Haan W, Offerman EH, Dallinga-Thie GM, Jukema JW, Havekes LM, Rensen PC. Cholesteryl ester transfer protein decreases high-density lipoprotein and severely aggravates atherosclerosis in APOE*3-Leiden mice. Arterioscler Thromb Vasc Biol. 2006; 26: 2552–2559.[Abstract/Free Full Text]

7. Moriyama Y, Okamura T, Inazu A, Doi M, Iso H, Mouri Y, Ishikawa Y, Suzuki H, Iida M, Koizumi J, Mabuchi H, Komachi Y. A low prevalence of coronary heart disease among subjects with increased high-density lipoprotein cholesterol levels, including those with plasma cholesteryl ester transfer protein deficiency. Prev Med. 1998; 27: 659–667.[CrossRef][Medline] [Order article via Infotrieve]

8. Brousseau ME, O’Connor JJ Jr., Ordovas JM, Collins D, Otvos JD, Massov T, McNamara JR, Rubins HB, Robins SJ, Schaefer EJ. Cholesteryl ester transfer protein TaqI B2B2 genotype is associated with higher HDL cholesterol levels and lower risk of coronary heart disease end points in men with HDL deficiency: Veterans Affairs HDL Cholesterol Intervention Trial. Arterioscler Thromb Vasc Biol. 2002; 22: 1148–1154.[Abstract/Free Full Text]

9. Hirano KS, Yamashita S, Kuga Y, Sakai N, Nozahi S, Kihara S, aria T, Yanagi K, Takami S, Menju M, Ishigami M, Yoshida Y, Kameda-Takemura K, Hayashi K, Matsuzawa Y. Atherosclerotic disease in marked hypolipoproteinemia: combined reduction of cholesteryl ester transfer protein and hepatic triglyceride lipase. Arterioscler Thromb Vasc Biol. 1995; 15: 1849–1856.[Abstract/Free Full Text]

10. Zhong S, Sharp DS, Grove JS, Bruce C, Yano K, Curb JD, Tall AR. Increased coronary heart disease in Japanese-Am Men with mutation in the cholesteryl ester transfer protein gene despite increased HDL levels. J Clin Invest. 1996; 97: 2917–2923.[Medline] [Order article via Infotrieve]

11. Drayna D, Jarnagin AS, Mclean J, Henzel W, Kohr W, Fielding C, Lawn R. Cloning and sequencing of human cholesteryl ester transfer protein cDNA. Nature. 1987; 327: 632–634.[CrossRef][Medline] [Order article via Infotrieve]

12. Nagashima M, McLean JW, Lawn RM. Cloning and mRNA tissue distribution of rabbit cholesteryl ester transfer protein. J Lipid Res. 1988; 29: 1643–1649.[Abstract]

13. Jiang XC, Moulin P, Quinet E, Goldberg IJ, Yacoub LK, Agellon LB, Compton D, Schnitzer-Polokoff R, Tall AR. Mammalian adipose tissue and muscle are major sources of lipid transfer protein mRNA. J Biol Chem. 1991; 266: 4631–4639.[Abstract/Free Full Text]

14. Zhang Z, Yamashita S, Hirano K, Nakagawa-Toyama Y, Matsuyama A, Nishida M, Sakai N, Fukasawa M, Arai H, Miyagawa J, Matsuzawa Y. Expression of cholesteryl ester transfer protein in human atherosclerotic lesions and its implication in reverse cholesterol transport. Atherosclerosis. 2001; 159: 67–75.[CrossRef][Medline] [Order article via Infotrieve]

15. Ishikawa Y, Ito K, Akasaka Y, Ishii T, Masuda T, Zhang L, Akishima Y, Kiguchi H, Nakajima K, Hata Y. The distribution and production of cholesteryl ester transfer protein in the human aortic wall. Atherosclerosis. 2001; 156: 29–37.[CrossRef][Medline] [Order article via Infotrieve]

16. Rye KA, Barter PJ. Formation and metabolism of prebeta-migrating, lipid-poor apolipoprotein A-I. Arterioscler Thromb Vasc Biol. 2004; 24: 421–428.[Abstract/Free Full Text]

17. Rye KA, Hime NJ, Barter PJ. Evidence that cholesteryl ester transfer protein-mediated reductions in reconstituted high density lipoprotein size involve particle fusion. J Biol Chem. 1997; 272: 953–3960.

18. Van Eck M, Pennings M, Hoekstra M, Out R, Van Berkel ThJC. Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis. Curr Opin Lipidol. 2005; 16: 307–315.[Medline] [Order article via Infotrieve]

19. Jessup W, Gelissen IC, Gaus K, Kritharides L. Roles of ATP binding cassette transporters A1 and G1, scavenger receptor BI and membrane lipid domains in cholesterol export from macrophages. Curr Opin Lipidol. 2006; 17: 247–257.[Medline] [Order article via Infotrieve]

20. Faust RA, Tollefson JH, Chait A, Albers JJ. Regulation of LTP-I secretion from human monocyte-derived macrophages by differentiation and cholesterol accumulation in vitro. Biochim Biophys Acta. 1990; 1042: 404–409.[Medline] [Order article via Infotrieve]

21. Morton RE. Interaction of plasma-derived lipid transfer protein with macrophages in culture. J Lipid Res. 1988; 29: 1367–1377.[Abstract]

22. Jiang XC, Agellon LB, Walsh A, Breslow JL, Tall A. Dietary cholesterol increases transcription of the human cholesteryl ester transfer protein gene in transgenic mice. Dependence on natural flanking sequences. J Clin Invest. 1992; 90: 1290–1295.[Medline] [Order article via Infotrieve]

23. Gautier T, Masson D, Jong MC, Pais de Barros JP, Duverneuil L, Le Guern N, Deckert V, Dumont L, Bataille A, Zak Z, Jiang XC, Havekes LM, Lagrost L. Apolipoprotein CI overexpression is not a relevant strategy to block cholesteryl ester transfer protein (CETP) activity in CETP transgenic mice. Biochem J. 2005; 385: 189–195.[CrossRef][Medline] [Order article via Infotrieve]

24. Söderlund S, Soro-Paavonen A, Ehnholm C, Jauhiainen M, Taskinen MR. Hypertriglyceridemia is associated with prebeta-HDL concentrations in subjects with familial low HDL. J Lipid Res. 2005; 46: 1643–1651.[Abstract/Free Full Text]

25. Quinet E, Tall A, Ramakrishnan R, Rudel L. Plasma lipid transfer protein as a determinant of the atherogenicity of monkey plasma lipoproteins. J Clin Invest. 1991; 87: 1559–1566.[Medline] [Order article via Infotrieve]

26. Blouin A, Bolender RP, Weibel ER. Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study. J Cell Biol. 1997; 72: 441–455.

27. Pape ME, Ulrich RG, Rea TJ, Marotti KR, Melchior GW. Evidence that the nonparenchymal cells of the liver are the principal source of cholesteryl ester transfer protein in primates. J Biol Chem. 1991; 266: 12829–12831.[Abstract/Free Full Text]

28. Yang TP, Agellon LB, Walsh A, Breslow JL, Tall AR. Alternative splicing of the human cholesteryl ester transfer protein gene in transgenic mice. Exon exclusion modulates gene expression in response to dietary or developmental change. J Biol Chem. 1996; 271: 12603–12609.[Abstract/Free Full Text]

29. Ito K, Ishikawa F, Kanno T, Ishikawa Y, Akasaka Y, Akishima Y, Ishii T, Terayama Y, Sugimoto M, Watanabe T, Mori S. Expression of cholesteryl ester transfer protein (CETP) in germinal centre B cells and their neoplastic counterparts. Histopathology. 2004; 45: 73–81.[CrossRef][Medline] [Order article via Infotrieve]

30. Bingle CD, Craven CJ. Meet the relatives: a family of BPI- and LBP-related proteins. Trends Immunol. 2005; 25: 53–55.

31. Hardardóttir I, Moser AH, Fuller J, Fielding C, Feingold K, Grünfeld C. Endotoxin and cytokines decrease serum levels and extra hepatic protein and mRNA levels of cholesteryl ester transfer protein in syrian hamsters. J Clin Invest. 1996; 97: 2585–2592.[Medline] [Order article via Infotrieve]

32. Masucci-Magoulas L, Moulin P, Jiang XC, Richardson H, Walsh A, Breslow JL, Tall A. Decreased cholesteryl ester transfer protein (CETP) mRNA and protein and increased high density lipoprotein following lipopolysaccharide administration in human CETP transgenic mice. J Clin Invest. 1995; 95: 1587–1594.[Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				D. Lakomy, C. Rebe, A.-L. Sberna, D. Masson, T. Gautier, A. Chevriaux, M. Raveneau, N. Ogier, A. T. Nguyen, P. Gambert, et al. Liver X Receptor-Mediated Induction of Cholesteryl Ester Transfer Protein Expression Is Selectively Impaired in Inflammatory Macrophages Arterioscler Thromb Vasc Biol, November 1, 2009; 29(11): 1923 - 1929. [Abstract] [Full Text] [PDF]

				M. Hoekstra, D. Ye, R. B. Hildebrand, Y. Zhao, B. Lammers, M. Stitzinger, J. Kuiper, T. J. C. Van Berkel, and M. Van Eck Scavenger receptor class B type I-mediated uptake of serum cholesterol is essential for optimal adrenal glucocorticoid production J. Lipid Res., June 1, 2009; 50(6): 1039 - 1046. [Abstract] [Full Text] [PDF]

				D. Masson, X.-C. Jiang, L. Lagrost, and A. R. Tall The role of plasma lipid transfer proteins in lipoprotein metabolism and atherogenesis J. Lipid Res., April 1, 2009; 50(Supplement): S201 - S206. [Abstract] [Full Text] [PDF]

				N. J. Hime, A. S. Black, J. J. Bulgrien, and L. K. Curtiss Leukocyte-derived hepatic lipase increases HDL and decreases en face aortic atherosclerosis in LDLr-/- mice expressing CETP J. Lipid Res., October 1, 2008; 49(10): 2113 - 2123. [Abstract] [Full Text] [PDF]

				M. Lee-Rueckert, R. Vikstedt, J. Metso, M. Jauhiainen, and P. T. Kovanen Association of cholesteryl ester transfer protein with HDL particles reduces its proteolytic inactivation by mast cell chymase J. Lipid Res., February 1, 2008; 49(2): 358 - 368. [Abstract] [Full Text] [PDF]

				W. D. Nelson, A. G. Zenovich, H. C. Ott, C. Stolen, G. J. Caron, A. Panoskaltsis-Mortari, S. A. Barnes III, X. Xin, and D. A. Taylor Sex-Dependent Attenuation of Plaque Growth After Treatment With Bone Marrow Mononuclear Cells Circ. Res., December 7, 2007; 101(12): 1319 - 1327. [Abstract] [Full Text] [PDF]

				H. Tanigawa, J. T. Billheimer, J.-i. Tohyama, Y. Zhang, G. Rothblat, and D. J. Rader Expression of Cholesteryl Ester Transfer Protein in Mice Promotes Macrophage Reverse Cholesterol Transport Circulation, September 11, 2007; 116(11): 1267 - 1273. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/5/678    most recent 01.RES.0000260202.79927.4fv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Van Eck, M. Articles by Van Berkel, T. J.C. Search for Related Content PubMed PubMed Citation Articles by Van Eck, M. Articles by Van Berkel, T. J.C. Related Collections Animal models of human disease Gene expression Genetically altered mice Lipid and lipoprotein metabolism