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 6
MACROPHAGE PHOSPHOLIPID TRANSFER PROTEIN CONTRIBUTES SIGNIFICANTLY TO TOTAL PLASMA PHOSPHOLIPID TRANSFER ACTIVITY AND ITS DEFICIENCY LEADS TO DIMINISHED ATHEROSCLEROTIC LESION DEVELOPMENT
Riikka Vikstedt1, Dan Ye2, Jari Metso1, Reeni B. Hildebrand2, Theo J.C.
Van Berkel2, Christian Ehnholm1, Matti Jauhiainen1, Miranda Van Eck2
1National Public Health Institute, Department of Molecular Medicine, Biomedicum, Helsinki, Finland
2Division of Biopharmaceutics, LACDR, Leiden University, The Netherlands
Arterioscler Thromb Vasc Biol. 2007; 27(3):578-586.
ABSTRACT
Systemic phospholipid transfer protein (PLTP) deficiency in mice is associated with a decreased susceptibility to atherosclerosis, whereas overexpression of human PLTP in mice increases atherosclerotic lesion development. PLTP is alsoexpressed by macrophage-derived foam cells in human atheroscleroticlesions, but the exact role of macrophage PLTP in atherosclerosis is unknown. To clarify the role of macrophage PLTP in atherogenesis, PLTP was selectively disrupted in hematopoietic cells, including macrophages, by transplantation of bone marrow from PLTP knockout (PLTP–/–) mice into irradiated low-density lipoprotein receptor knockout mice. Selective deficiency of macrophage PLTP (PLTP–M/–M) resulted in a 29%(P<0.01 for difference in lesion area) reduction in aortic root lesion area as compared with mice possessing functionalmacrophage PLTP (384±36*103 µm2 in the PLTP–M/–M group (n=10), as compared with 539±35*103 µm2 inthe PLTP+M/+M group (n=14)) after 9 weeks of Western- type diet feeding. The decreased lesion size in the PLTP–M/–M group coincided with significantly lower serum total cholesterol,free cholesterol, and triglyceride levels in these mice. Furthermore,plasma PLTP activity in the PLTP–M/–M group was2-fold (P<0.001) lower than that in the PLTP+M/+M group. In conclusion, macrophage PLTP is a significant contributor to plasma PLTP activity and deficiency of PLTP in macrophages leads to lowered atherosclerotic lesion development in low-density lipoprotein receptor knockout mice on Western-type diet.
INTRODUCTION
PLTP is a lipid transfer protein with a wide tissue distribution.1 In human plasma two forms of PLTP can be distinguished, one with high phospholipid transfer activity (HA-PLTP) and a low active form (LA-PLTP).2,3 PLTP transfers surface phospholipids from chylomicrons and very low-density lipoproteins (VLDL) to high-density lipoprotein (HDL) during lipolysis4 and also participates in HDL conversion.5,6 PLTP modifies HDL3 particles and concomitantly generates large particles resembling HDL2 and a population of small poorly lipidated preß-HDL particles which are efficient acceptors of cholesterol from peripheral cells.7,8
The function of PLTP in the development of atherosclerosis is far from resolved, as PLTP has been reported to be a pro-atherogenic factor, but also anti-atherogenic properties have been associated with PLTP.9 Increased PLTP activity in plasma is a risk factor for coronary heart disease (CHD)10, while serum total PLTP mass protects against CHD in humans.11 In studies using genetically modified mice, primarily pro-atherogenic effects of PLTP on atherosclerosis have been reported. Mice overexpressing human PLTP displayed decreased plasma HDL levels,12-15 increased VLDL levels13,16 and elevated susceptibility to atherosclerosis13,17,18. Conversely, PLTP-deficiency is associated with decreased apolipoprotein B secretion from mouse hepatocytes19,20 with a concomitant decrease in atherosclerotic lesion size.20
The role of PLTP in atherosclerosis is complex and functions that affect its atherogenicity include: i) enhancement of cholesterol efflux via generation of preß-HDL particles12,14,15; ii) determination of plasma HDL levels21,22 by mediating the transfer of post-lipolytic surface remnants of chylomicrons and VLDL into HDL; iii) influencing the production of apoB-containing lipoproteins by the liver19,20; iv) influencing the accumulation of anti- oxidative vitamin E in LDL and VLDL23 and v) transferring lipopolysaccharide (LPS) from HDL to LDL.24
Recently, PLTP was demonstrated in lipid-laden macrophage-derived foam cells in human atherosclerotic lesions.25-27 This observation raised the question whether PLTP has a direct role in cholesterol retention or removal from foam cells present in atherosclerotic lesions. The expression of PLTP in macrophages is upregulated by liver X receptor (LXR) and retinoid X receptor (RXR) agonists.26,28 Furthermore, in vitro studies have demonstrated that PLTP mRNA and protein expression as well as activity is increased upon cholesterol loading of macrophages25,27 and that exogenously added PLTP promotes cholesterol and phospholipid removal from murine macrophages via an ATP-binding cassette transporter A1 (ABCA1) mediated pathway.29 Currently, however, the function of PLTP production by macrophages in atherosclerosis in vivo is unknown.
Macrophages, present in atherosclerotic lesions, primarily depend on infiltration from bone marrow-derived monocytes into the arterial wall.
Therefore, to clarify the role of macrophage PLTP in atherogenesis, we created a mouse model with selective deficiency of PLTP in hematopoietic cells, including macrophages, by using the bone marrow transplantation (BMT) technique. Our results demonstrate that macrophage PLTP is a significant modulator of plasma PLTP activity and that PLTP-deficiency in
macrophages leads to lowered atherosclerotic lesion development in LDL receptor knockout mice on WTD.
MATERIALS AND METHODS
For detailed methodology, please see http://atvb.ahajournals.org.
Chimeric mice with a selective deficiency of PLTP in hematopoietic cells, including macrophages, were generated by using the BMT technique.
Hereto, female LDLr-/- mice (C57Bl/6J strain; 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) one day before transplantation.
Irradiated recipients were transplanted by intravenous injection of 0.5x107 bone marrow cells, isolated from male wildtype C57Bl/6J PLTP+/+ mice or male PLTP-/- mice on the C57Bl/6J background21. After that, transplanted LDLr-/- mice (from now indicated as PLTP+M/+M and PLTP-M/-M mice, respectively) were maintained on sterilised regular chow diet (RM3, Special Diet Services, Witham, UK) for 8 weeks to allow the mice to recover from the BMT. To induce the development of atherosclerosis, themice 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 after which the mice were sacrificed and atherosclerotic lesion development and the composition of the lesions was quantified.
At 8 weeks posttransplant when the mice were on regular chow diet and at 17 weeks posttransplant when the animals were fed WTD, blood was drawn after an overnight fasting-period for determination of serum cholesterol, triglycerides, and phospholipids. In addition, the distribution of lipids between the different lipoproteins in serum was determined. Preβ-HDL and α-HDL levels12, mouse apoA-I12, PLTP activity5,30, hepatic lipase (HL) activity31, and lecithin-cholesterol acyltransferase (LCAT) activity32 were determined as previously described.
PLTP mRNA expression was determined in whole livers of transplanted mice at 17 weeks posttransplant and in parenchymal, endothelial, and Kupffer cells isolated from livers of wildtype C57Bl/6J mice33 using real time-quantitative PCR. Furthermore, PLTP protein levels were determined immunohistochemically in livers and lungs of the transplanted mice at 17 weeks posttransplant.
Animal experiments were performed at the Gorlaeus laboratories of the Leiden/Amsterdam Center for Drug Research in accordance with the National Laws. All experimental protocols were approved by the Ethics Committee for Animal Experiments of Leiden University.
Statistical analyses were performed utilising the unpaired Student’s t-test (Instat GraphPad software, San Diego, CA).
RESULTS
Generation of LDLr–/– Mice With a Specific Disruption of PLTP in Bone Marrow–Derived Cells
To evaluate the role of macrophage PLTP in lipoprotein metabolism and in the development of atherosclerotic lesions, PLTP gene expression was specifically disrupted in cells of hematopoietic origin by transplantation of bone marrow from C57Bl/6J mice lacking functional PLTP21 into LDLr-/- mice (Figures I and II, available online at http://atvb.ahajournals.org).
Macrophage PLTP Deficiency Decreases Atherosclerosis in LDLr–/–
Mice
To induce atherosclerotic lesion development, the transplantedmice were fed WTD, containing 0.25% cholesterol and 15% fat,starting at 8 weeks after transplantation. After 9 weeks ofWTD feeding, lesion development was analyzed in the aortic rootof the PLTP+M/+M and PLTP–M/–M mice. As shown inFig. 1A, macrophage PLTP deficiency leads to a 29% (P<0.01) decrease in the mean atherosclerotic lesion area (PLTP+M/+M,539±35x103 µm2, n=14 versus PLTP–M/–M, 384±36x103 µm2, n=10). The relative macrophage content of the lesions of WT → LDLr–/– mice was 32±3%, whereas the collagen content was 7±1% (Fig. 1B and 1C).No significant effect of macrophage PLTP deficiency was observed on the relative macrophage content of the lesions (35±3%).However, a trend to a reduced collagen content was observed(3±1%, P=0.07) in absence of macrophage PLTP production(Fig. 1C). Analysis of the average thickness of the caps of the lesions showed that the caps were smaller in animals transplantedwith PLTP–/– bone marrow (7±2 µm2, P<0.01) as compared with control transplanted animals (21±4µm2). The trend to reduced collagen content as well asthe smaller cap thickness observed in PLTP–M/–M mice is most likely a direct effect of the smaller and thusless advanced lesions observed in these animals.
Fig. 1. Atherosclerotic lesion formation in LDLr–/– mice with macrophage PLTP deficiency. Formation of atherosclerotic lesions was determined at 17 weeks posttransplantation, ie, after 9 weeks WTD, in PLTP+M/+M and PLTP–M/–M mice. A, The mean lesion area (µm2) was calculated from 10 oil red O-stained sections of the aortic root at the level of the tricuspid valves. B, Immunohistochemical quantification of lesion macrophages using MOMA-2. The macrophage content of the lesions is indicated as % of the total lesion area. C, Masson trichrome staining; blue staining indicates collagen. The collagen content of the lesions is indicated as % of the total lesion area. Values represent the means±SEM calculated from 14 PLTP+M/+M mice (white column) and 10 PLTP–M/–M mice (black column).
Statistically significant differences between PLTP+M/+M and PLTP–M/–M mice are indicated.
**P<0.01. Original magnification x50.
Macrophage PLTP Deficiency Influences Serum Lipid Levels and Lipoprotein Distribution
During the course of the experiment, the effects of PLTP deficiency in hematopoietic cells (including macrophages) on serum lipid, lipoprotein, and apoA-I concentrations were determined. At 8 weeks posttransplantation, when the mice had been on a chow diet, serum total cholesterol(TC) and free cholesterol (FC) concentrations were similar inboth groups (Table 1).
No significant differences were observedbetween groups in VLDL and LDL cholesterol levels (Fig. 2A). However, HDL cholesterol was significantly higher in the PLTP–M/–M group (P<0.01). On challenging the mice with WTD, the concentrationsof TC (PLTP+M/+M from 7.14 mmol/L to 29.13 mmol/L, PLTP–M/–M from 7.59 mmol/L to 21.67 mmol/L) and FC (PLTP+M/+M from 1.44 mmol/L to 8.42 mmol/L, PLTP–M/–M from 1.64 mmol/L to 6.71 mmol/L) increased dramatically in both groups (Table 1).However, in the PLTP–M/–M group, TC and FC levelswere significantly lower than in PLTP+M/+M group (P<0.05for both TC and FC). The increases in FC and TC were the resultof a marked increase in VLDL and LDL cholesterol, which wassignificantly lower (P<0.05) for the PLTP–M/–M group (Fig. 2B). Under these feeding conditions, HDL cholesterolwas decreased in both groups but there was no significant differencebetween the groups.
Table 1 The effect of macrophage PLTP-deficiency on serum lipids and apoA-I concentrations
Serum lipids and apoliprotein A-I concentrations in PLTP+M/+M and PLTP-M/-M mice maintained on a chow diet for 8 weeks and on a high-cholesterol Western-type diet (WTD) for 9 weeks (17 weeks after BMT). Data present means±SEM.
ND, not determined
*analyzed from plasma samples
aP<0.05, bP<0.01, cP<0.001 difference between PLTP+M/+M and PLTP-M/-M mice at the indicated times posttransplant.
dP<0.05, eP<0.001difference between 8 and 17 weeks posttransplant in the indicated mice.
WTD feeding increased triglycerides (TG) in the PLTP+M/+M group,whereas TG levels in the PLTP–M/–M group were decreased(Table 1). This resulted in significantly lower levels of TGobserved in mice transplanted with PLTP–/–
bonemarrow after 9 weeks WTD feeding (PLTP+M/+M, 2.08 mmol/L versus PLTP–M/–M 1.16 mmol/L, P<0.01). Size-exclusionchromatography analysis
showed a trend to lower levels of TGin the VLDL and LDL fractions of PLTP–M/–M mice(data not shown).
The concentration of total phospholipids (PL) was higher inthe PLTP–M/–M group after 8 weeks on chow diet (P<0.01) (Table 1), however HDL phospholipids did not differ significantlybetween the groups (Fig. 2C). After 9 weeks on WTD, no differencesin total PL were observed, although HDL- associated PL was higherin PLTP–M/–M mice (P<0.01) (Fig. 2D).
In addition, the effects of disruption of PLTP in bone marrow-derivedcells on serum apoA-I levels were determined both on chow diet and after feeding WTD (Table 1). In PLTP+M/+M mice WTD feeding resulted in a significant reduction in the apoA-I concentrationcompared with the values on chow diet (from 1.44 g/L to 0.98g/L, P<0.001), whereas in PLTP–M/–M mice a trendto increased (P=0.09) apoA-I levels was observed (from 1.30 g/L to 1.63 g/L). As a result, on WTD the serum apoA-I concentrationwas significantly higher in the PLTP–M/–M group (PLTP+M/+M, 0.98 g/L versus PLTP–M/–M, 1.63 g/L;P<0.001).
As PLTP acts as an important factor in the production of preß-HDL particles, it was also of interest to study preß-HDL levels in this experimental setting. At 8 weeks posttransplantation,no significant effect of macrophage PLTP deficiency was observedon circulating preß-HDL levels.
However, after 9 weeksof WTD feeding, the preß-HDL levels were 24%
lower in PLTP–M/–M mice as compared with PLTP+M/+M mice (PLTP+M/+M, 20.7% versus PLTP–M/–M, 15.7%; P<0.05).
0 100 200 300 400 500
1 5 9 13 17 21
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PLTP+M/+M PLTP-M/-M Chow
VLDL+LDL HDL**
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PLTP+M/+M PLTP-M/-M Chow
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PLTP+M/+M PLTP-M/-M Chow
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PLTP+M/+M PLTP-M/-M Chow
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0 200 400 600 800 1000 1200
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PLTP+M/+M PLTP-M/-M WTD
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0 200 400 600 800 1000 1200
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PLTP+M/+M PLTP-M/-M WTD
VLDL+LDL* HDL
A C
B D
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PLTP+M/+M PLTP-M/-M WTD
VLDL+LDL* HDL**
0 50 100 150 200
1 5 9 13 17 21
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Phospholipids (µmol/L)
PLTP+M/+M PLTP-M/-M WTD
VLDL+LDL* HDL**
Fig. 2. The effect of macrophage PLTP-deficiency on plasma cholesterol and phospholipid distribution. Blood samples were drawn after 8 weeks feeding regular chow diet and at 17 weeks posttransplant after 9 weeks feeding of Western-type diet (WTD). Serum from individual mice was fractionated on a Superose 6 PC column and fractions 2-14 represent
VLDL and LDL and fractions 15-22, HDL. The distribution of cholesterol (A, B) and phospholipids (C, D) between the different lipoproteins of PLTP+M/+M and PLTP-M/-M mice was determined. Values shown in the figures are the means of 10-14 mice. Statistically significant differences between PLTP+M/+M and PLTP-M/-M mice are indicated, *P<0.05 and **P<0.01.
Macrophage PLTP Is an Important Contributor to Plasma PLTP Activity
Size-exclusion chromatographic analysis demonstrated that PLTPactivity was almost exclusively associated with HDL lipoproteins with a similar distribution pattern in both groups (supplementalFigure III). On chow diet, plasma PLTP activity was 1.4-fold lower in the PLTP–M/–M group as compared with thePLTP+M/+M group (PLTP+M/+M, 19.5 µmol/mL per hour versusPLTP–M/–M, 14.3 µmol/mL per hour; P<0.01)(Table 2). WTD feeding increased plasma PLTP activities in bothgroups of mice and were 2.5-fold higher in PLTP+M/+M mice andonly 1.7-fold higher in PLTP–M/–M mice after 9 weeks on WTD as compared with the values on chow diet. As a consequence,plasma PLTP activity in mice of the PLTP–M/–M groupwas 2- fold lower as compared with the activity in mice of thePLTP+M/+M group (PLTP+M/+M, 48.3 µmol/mL per hour versus PLTP–M/–M 24.0 µmol/mL per hour; P<0.001).These results demonstrate that macrophage PLTP is an importantcontributor to plasma total PLTP activity. The activity of hepatic lipase and lecithin-cholesterol acyltransferase in plasma did not differ between the 2 groups (Table 2).
Table 2 Effect of Macrophage PLTP Deficiency on Plasma PLTP, HL, and LCAT Activities
Plasma PLTP, HL, and LCAT activities in PLTP+M/+M and PLTP–M/–M mice maintained on a chow diet for 8 weeks and on a high-cholesterol WTD for 9 weeks (17 weeks after BMT). Data present means±SEM.
ND, not determined.
aP<0.01, bP<0.001 difference between PLTP+M/+M and PLTP–M/–M mice at the indicated time posttransplant.
cP<0.001 difference between 8 and 17 weeks posttransplant in the indicated mice.
PLTP is a ubiquitously expressed protein with a moderate level of expression in liver.1 However, liver as a large organ can contribute to a relatively high level to circulating PLTP. Therefore, PLTP mRNA expression was determined in livers of the transplanted mice (Fig. 3A). Interestingly, a trend to reduced PLTP expression was evident in livers from PLTP-M/-M mice (0.007±0.002) as compared to livers from PLTP+M/+M animals (0.010±0.003).
In addition we performed experiments in which the PLTP activity levels were analyzed in liver homogenates of the transplanted PLTP+M/+M and PLTP-M/-M animals. In accordance with the mRNA data, PLTP activity levels in the liver homogenates were 26 % lower in PLTP-M/-M mice (31±2 nmol/mg protein vs. 42±2 nmol/mg; PLTP-M/-M vs. PLTP+M/+M, P=0.0019) confirming the importance of PLTP production by bone marrow-derived cells for hepatic PLTP activity.
A
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0.000 0.005 0.010 0.015
PLTP mRNA expression (A.U.)
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EC KC
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**
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Absolute PLTP mRNA expression (A.U.)
PC EC KC
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**
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PLTP mRNA expression (A.U.)
PLTP+M/+MPLTP-M/-M
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**
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Relative PLTP mRNA expression (A.U.)
EC KC
PC
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**
**
0.05 0.10 0.15 0.20 0.25
**
0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035
Absolute PLTP mRNA expression (A.U.)
PC EC KC
*
**
**
Fig. 3. PLTP mRNA expression and activity in liver samples. A) Left, PLTP mRNA expression in whole liver tissue samples taken from PLTP+M/+M (white column, n=8) and PLTP-
M/-M
(black column, n=9) mice at 17 weeks posttransplant. Right, PLTP activities in liver homogenates of the transplanted PLTP+M/+M (white column, n=14) and PLTP-M/-M (black column, n=11) mice at 17 weeks posttransplant. B) Left, relative expression of PLTP mRNA in parenchymal cells (PC; n=12), endothelial cells (EC; n=6) and Kupffer cells (KC; n=6) isolated from livers of wildtype C57Bl/6J mice. Right, absolute contribution of different cell types to hepatic PLTP mRNA expression calculated based on the protein contribution of the different cell types in livers of C57Bl/6J mice. Data represent the means±SEM. Statistically significant differences are indicated, *P<0.05 and **P<0.01.
The liver contains several different types of cells which all have their specific localization and function. The majority of the liver consists of parenchymal cells, which contribute 92.5% to the total liver protein mass. In addition, the liver contains endothelial and Kupffer cells that account for 3.3% and 2.5% of the liver protein mass, respectively.33,34 As Kupffer cells are of hematopoietic origin, it is likely that the observed reduction in hepatic PLTP activity of the PLTP-M/-M group is due to the reconstitution with Kupffer cells derived from the PLTP-/- donor bone marrow. To confirm that Kupffer cells are an important source of PLTP in the liver, the PLTP mRNA expression was determined in purified parenchymal cells, Kupffer cells and endothelial cells isolated from livers of C57Bl/6J mice on normal chow diet (Fig. 3B). The expression of PLTP mRNA was 6-fold (P<0.01) higher in Kupffer cells as compared to parenchymal cells, while endothelial cells produced only a minor amount of PLTP mRNA. Thus, although Kupffer cells only contribute to 2.5% of the total liver protein, they do contain 13.8% of the total liver PLTP expression, as compared to 85.7% and 0.5% for parenchymal cells and endothelial cells, respectively. Immunohistochemical
localization of PLTP protein in livers of the transplanted PLTP+M/+M and PLTP-M/-M mice also clearly demonstrated a reduction in hepatic PLTP protein expression due to disruption of PLTP in bone marrow-derived cells (Fig. 4A). In addition to liver, PLTP is highly expressed in lung.1 Therefore also the expression of PLTP protein in lungs of PLTP+M/+M and PLTP-M/-M mice were compared (Fig. 4B). Interestingly, disruption of macrophage PLTP production also resulted in a drastic decrease in PLTP expression in the bronchioles of the lung.
Fig. 4. Immunohistochemical detection of PLTP protein in liver and lung samples. For detection of PLTP protein, cryostat sections of liver (A) and lung (B) of the transplanted PLTP+M/+M (left) and PLTP-M/-M mice (right) were stained immunohistochemically for PLTP (red) and nuclei (blue). Arrows indicate PLTP positive staining. Note the reduced expression of PLTP protein in both livers and lungs of PLTP-M/-M mice.
Thus, the significant contribution of Kupffer cells to the hepatic PLTP production and lung macrophages to PLTP expression in the lung, combined with that of other resident macrophages, may explain the large effects of PLTP-deficiency in bone marrow-derived cells on lipoprotein metabolism and atherosclerosis.
DISCUSSION
In the present study we show that selective deficiency of PLTP in macrophages in LDLr-/- mice: i) decreased the size of atherosclerotic lesions, ii) decreased the relative preß-HDL levels, iii) decreased serum cholesterol, and iv) lowered plasma PLTP activity. We also demonstrate for the first time that hepatic Kupffer cells express higher PLTP mRNA levels than parenchymal cells and endothelial cells in livers of wildtype C57Bl/6J mice.
How can the observed changes explain the anti-atherogenic effect caused by selective PLTP-deficiency in macrophages? An important process during the early steps of atherosclerotic lesion formation is the accumulation of cholesterol, derived from modified LDL and/or (β-)VLDL, in arterial macrophages transforming them into lipid-laden foam cells. The opposite event, reverse cholesterol transport (RCT), removes excess cholesterol from macrophages in the artery wall and transports it to the liver for excretion,35 thereby preventing excessive cholesterol accumulation. Upon WTD feeding of LDLr-/- mice, the balance between the cholesterol influx and efflux in macrophages is severely compromised leading to excessive accumulation of lipid. Recently, several groups have reported that macrophages in atherosclerotic lesions express PLTP,25-27 but its relation to atherosclerotic lesion formation was unknown. In the current study we show that disruption of PLTP in macrophages reduces atherosclerosis in LDLr-/- mice.
To get a better understanding of the various potential mechanisms contributing to this beneficial effect of macrophage PLTP-deficiency, it is important to recognize the plasma factors affecting lesion formation in the arterial intima, as well as local macrophage-derived arterial effects. In vitro, both wild-type and PLTP-/- macrophages can be converted into foam cells upon incubation with acetylated LDL. No differences in the overall cholesterol uptake between wild-type and PLTP-/- macrophages were observed as recently demonstrated by Lee-Rueckert et al.36 Thus, PLTP- deficiency in the macrophage does not influence cholesterol uptake and deposition once challenged with modified LDL. Exogenous PLTP increases HDL-induced phospholipid and cholesterol removal from macrophage foam cells,29 and therefore via enhancing RCT it may be anti-atherogenic. One of the best characterized lipid exporters from macrophages is ABCA1, which mediates cholesterol efflux to lipid-poor apolipoproteins, including apoA-I and apoE.37 In addition, ABCG138,39 and scavenger receptor BI (SR-BI)40,41 mediate the efflux of cholesterol to mature HDL. Recently, we have shown that absence of endogenous PLTP impairs ABCA1-dependent efflux from macrophage foam cells in vitro.36 Macrophages also synthesize and secrete apoE, which can induce cellular cholesterol efflux and protect against the development of atherosclerosis.42 Interestingly, apoE interacts with human plasma PLTP and activates the low-activity form of PLTP.43 Furthermore, apoE, as well as, PLTP are under positive control of LXR agonists and during cholesterol loading expression levels of both are increased,25-28,44 suggesting that both apoE and PLTP may facilitate cholesterol efflux.45 Here we show that disruption of macrophage PLTP reduces atherosclerotic lesion development and that macrophage PLTP is pro-atherogenic in LDLr-/- mice. Thus, in vivo apparently other pro-atherogenic properties of PLTP, probably related to the observed changes in plasma lipoproteins, override the potential anti-atherogenic function of macrophage PLTP in mediating cholesterol efflux.
Surprisingly, we observed that macrophage PLTP-deficiency significantly reduced plasma PLTP activity. We also demonstrated that PLTP activity in PLTP-M/-M liver homogenates was significantly reduced as compared to livers obtained from PLTP+M/+M animals depicting that hepatic bone marrow- derived cells can provide active PLTP. Kupffer cells, resident macrophages
of the liver, significantly contributed to the hepatic PLTP production and it is thus conceivable that the reduced plasma PLTP activity measured in PLTP-
M/-M
mice is a direct effect of the absence of PLTP production by Kupffer cells of the liver and other resident macrophages in lung, adipose tissue, and spleen. Replacement of Kupffer cells after BMT was assessed by transplantation of LDLr-/- mice with bone marrow from EGFP expressing mice. Already at 8 weeks after transplantation a significant amount of Kupffer cells were EGFP positive and of donor-origin (unpublished data).
These findings are in agreement with a previous study from Paradis et al.46 who showed that already at 21 days after bone marrow transplantation Kupffer cells were predominantly of donor bone marrow origin.
Immunohistochemical localization also confirmed the reduction in PLTP protein expression in livers of PLTP-M/-M mice. In addition, PLTP protein expression was drastically reduced in lungs, one of the organs with the highest expression of PLTP1.
The effect of macrophage PLTP-deficiency on total plasma PLTP activity significantly influenced lipoprotein metabolism in the PLTP-M/-M mice. On WTD, preß-HDL levels were lower in mice lacking PLTP in macrophages.
The low levels of preß-HDL in PLTP-M/-M animals may be due to the lower levels of serum PLTP activity in these mice. Preß-HDL is highly efficient in the removal of cholesterol from cells. However, despite the decreased levels of preß-HDL, lesion development was reduced, implicating that the pro-atherogenic effects of lower preß-HDL levels are overruled by other factors. PLTP-deficiency of macrophages also resulted on WTD in significantly higher plasma apoA-I levels and HDL phospholipids.
Distribution of HDL subclasses and the roles of the different subclasses in reverse cholesterol transport are at present far from resolved. We assume that the elevated apoA-I and HDL-associated phospholipids may result in the formation of HDL subclasses that could contribute to enhanced cholesterol efflux, and provide an explanation for the reduced size of the lesions formed in PLTP-M/-M mice.
Consistent with the lower plasma PLTP activity, macrophage PLTP- deficiency also resulted in substantially lower concentrations of cholesterol and triglycerides, mainly as a consequence of lower VLDL levels. Since Kupffer cells seem to contribute significantly to PLTP mRNA expression and PLTP activity in the liver it is conceivable that Kupffer cells’ PLTP could directly or indirectly influence VLDL biosynthesis or secretion and could thus provide an explanation for the lower levels of apoB-containing lipoproteins in PLTP-M/-M mice. Plasma PLTP activity reportedly correlates positively with triglyceride levels.47 Furthermore, in apoE-/- and human apoB transgenic mice total PLTP-deficiency decreased serum levels and production of apoB-containing lipoproteins.20 However, total PLTP- deficiency did not influence serum apoB-lipoprotein levels or their production in LDLr-/- mice.20 In our chimeric mouse model, macrophage- specific PLTP-deficiency in LDLr-/- mice did cause a reduction in the levels of apoB-containing lipoproteins, which is probably related to the reduced plasma PLTP activity and provides an important explanation for the reduced susceptibility of the PLTP-M/-M mice to atherosclerotic lesion development.
Recently, using a similar experimental setup as we have used, Valenta et al
48 showed that diet-induced atherosclerosis was increased in LDLr-/- mice
upon disruption of PLTP in hematopoietic cells. The authors postulated that the contribution of PLTP to atherosclerosis is determined by a balance between lesion PLTP activity (antiatherogenic) and plasma PLTP activity (proatherogenic). Under our experimental conditions in which the mice were fed a mild atherogenic Western-type diet, containing 15 % fat and 0.25 % cholesterol for 9 weeks, macrophage PLTP production contributes to plasma PLTP activity, induces higher VLDL cholesterol levels, and is thus pro-atherogenic. In the study of Valenta et al. the effect of macrophage PLTP production on atherosclerosis susceptibility was studied using a high cholesterol diet, composed of 15.8 % fat and 1.25 % cholesterol for 16 weeks. Under these high cholesterol conditions the effect of macrophage- derived PLTP on plasma PLTP activity did not affect VLDL cholesterol levels and thus was not rate-limiting and protected against the development of atherosclerosis probably as a result of the antiatherogenic properties of macrophage PLTP in the lesion. Both studies thus strengthen the postulation that the balance between factors influencing the antiatherogenic lesion PLTP activity and factors affecting the proatherogenic plasma PLTP activity is essential for the eventual outcome of PLTP modulation on atherosclerotic lesion development. This is also confirmed by the data of Valenta et al.28 who showed that in the presence of elevated plasma concentrations of apoAI macrophage PLTP activity was no longer protective for atherosclerotic lesion development and a trend to a reduced lesion size was observed in absence of macrophage PLTP. In addition, to the differences in the cholesterol content of the diet used in the two studies, we also used female recipients, while Valenta et al. used males. Recently, Yang et al (Genome Res. 2006:16:995-1004) reported that in mice many hepatic genes show sexual dimorphism (~70%). Furthermore, the highest changes (> 3 fold) in gene expression between females and males were observed in genes involved in steroid and lipid metabolism. In addition to the differences in the cholesterol content of the diets between our work and that recently published by Valenta et al.28, this sexual dimorphism could contribute to the differential effects of macrophage-derived PLTP on serum VLDL levels.
In conclusion, our study shows that macrophage PLTP significantly contributes to plasma PLTP activity and that deficiency of macrophage PLTP results in increased apoA-I and decreased VLDL/LDL levels, changes that may explain why deficiency of PLTP in macrophages leads to a decrease in atherosclerotic lesion development in LDLr-/- mice.
ACKNOWLEDGEMENTS
We thank Ritva Nurmi and Sari Nuutinen for their excellent technical assistance. The study was supported by the Sigrid Juselius Foundation, the Finnish Foundation for Cardiovascular Research, the Netherlands Heart Foundation (2001T041), and the Netherlands Organisation for Scientific Research (917.66.301).
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