© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Role of Macrophage-Derived Lipoprotein Lipase in Lipoprotein Metabolism and Atherosclerosis
From the Division of Biopharmaceutics (M.V.E., T.J.C.V.B.), Leiden/Amsterdam Center for Drug Research, Sylvius Laboratories, Leiden University, Leiden, The Netherlands; the Department of Biochemistry (R.Z., R.Z.), University of Graz, Graz, Austria; and the Department of Vascular Biology (P.H.E.G.), SmithKline Beecham Research and Development, Harlow, UK.
Correspondence to M. Van Eck, PhD, Division of Biopharmaceutics, Sylvius Laboratories, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands. E-mail M.Eck{at}LACDR.LeidenUniv.nl
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Abstract—Lipoprotein lipase (LPL) synthesis by macrophages is upregulated in early atherogenesis, implicating the possible involvement of LPL in plaque formation. However, it is still unclear whether macrophage-derived LPL displays a proatherosclerotic or an antiatherosclerotic role in atherosclerotic lesion development. In this study, the role of macrophage-derived LPL on lipid metabolism and atherosclerosis was assessed in vivo by transplantation of LPL-deficient (LPL-/-) and wild-type (LPL+/+) bone marrow into C57BL/6 mice. Eight weeks after bone marrow transplantation (BMT), serum cholesterol levels in LPL-/-
C57BL/6 mice
were reduced by 8% compared with those in LPL+/+C57BL/6 mice
(P<0.05, n=16), whereas triglycerides were
increased by 33% (P<0.05, n=16). Feeding the mice a
high-cholesterol diet increased serum
cholesterol levels in LPL-/-C57BL/6 and
LPL+/+C57BL/6 mice 5-fold and 9-fold, respectively, resulting in a
difference of 
50% (P<0.01) after 3 months on the diet.
No effects on triglyceride levels were observed under these
conditions. Furthermore, serum apolipoprotein E levels were reduced by
50% in the LPL-/-C57BL/6 mice compared with controls under both
dietary conditions. After 3 months on a high-cholesterol
diet, the atherosclerotic lesion area in LPL-/-C57BL/6 mice was
reduced by 52% compared with controls. It can be concluded that
macrophage-derived LPL plays a significant role in the
regulation of serum cholesterol, apolipoprotein E, and
atherogenesis, suggesting that specific blockade of macrophage
LPL production may be beneficial for decreasing atherosclerotic
lesion development.
Key Words: atherosclerosis • transplantation • blood cells • enzymes • lipids
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Introduction |
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Lipoprotein lipase (LPL) is a 60-kD glycoprotein that plays a central role in plasma triglyceride metabolism by hydrolysis of triglyceride-rich chylomicrons and VLDL.1 2 3 LPL is bound to the capillary endothelium by interactions with heparan sulfate proteoglycans and is present in organs with a high demand for free fatty acids (FFAs), like the heart, skeletal muscle, and adipose tissue.4 5 Small amounts of LPL are also present in other types of tissues, including the adrenals, brain, lung, and spleen.4 Furthermore, in atherosclerotic lesions, LPL is produced by monocytes, macrophages, and smooth muscle cells.6 7 8 9 Foam cells in atherosclerotic lesions are mainly derived from macrophages, and LPL secretion by this cell type may thus have a direct effect on the formation of atherosclerotic lesions. Several functions for LPL in the atherosclerotic process have been postulated, including a proatherosclerotic or an antiatherosclerotic role. Previously, it has been demonstrated that pharmaceutical interventions intended to increase LPL expression protect against atherosclerosis due to elevations of HDL, indicating that LPL displays an antiatherogenic function.10 11 12 Furthermore, overexpression of LPL in the liver is highly effective in normalizing the atherosclerotic lipoprotein profiles of both apoE-deficient and LDL receptor–deficient mice13 14 and protects wild-type mice against diet-induced hyperlipidemia.15 Alternatively, LPL may induce the retention and/or uptake of atherogenic lipoproteins by cells in the arterial wall, thereby promoting the atherosclerotic process.16 17 Furthermore, LPL has been demonstrated to increase the retention of LDL and VLDL by proteoglycans of the subendothelial matrix in the arterial wall,18 19 20 increase the permeability of the endothelium by formation of lipolysis products,21 and facilitate proteoglycan-mediated monocyte adhesion to the endothelium.22
This potentially proatherosclerotic function of LPL is supported by the findings of Renier et al,23 who demonstrated that the susceptibility to atherosclerosis in inbred mouse strains is associated with high LPL expression, whereas resistance is associated with low LPL levels. Analysis of the effects of homozygous LPL deficiency on lipoprotein metabolism or atherosclerosis in mice is not possible, because LPL deficiency leads to death of the animals shortly after birth.23 24 In heterozygotes, however, both triglycerides and cholesterol are only slightly increased compared with corresponding levels in wild-type animals.23 24 Recently, Semenkovich et al26 demonstrated that feeding the heterozygous LPL-deficient mouse an atherogenic diet results in a more profound dyslipidemia due to an increase in VLDL triglycerides and VLDL and LDL cholesterol. Despite this increase in fasting lipids, no effect on atherosclerotic lesion area could be demonstrated in these animals, indicating that the detrimental effects of dyslipidemia may be influenced by the positive effects of decreased LPL expression in the vascular wall. The role of LPL in atherogenesis is thus still in dispute, although it is most likely that its role may depend on the tissue in which it is expressed. In the arterial wall, LPL may be atherogenic, whereas in muscle and adipose tissue it may be protective.
To elucidate the role of macrophage-derived LPL in atherogenesis, LPL-deficient bone marrow from mice expressing LPL exclusively in muscle was transplanted into irradiated C57BL/6 mice. Using this technique, we created chimeric animals that were deficient in LPL in cells derived from the transplanted stem cells, including monocytes and macrophages. Our results indicate that the absence of macrophage-derived LPL profoundly influences serum cholesterol and apoE levels, whereas atherosclerotic lesion formation is reduced by 52%.
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Animals
Transgenic knockout mice expressing LPL exclusively in muscle (L0-MCK) were generated by breeding transgenic mice, containing the muscle-specific creatine kinase promotor driving a human LPL minigene, onto the LPL knockout background as described earlier.27 28 29 Littermates with normal LPL expression were used as controls (L2-MCK). All mice were hybrids of the C57BL/6 and CBA strains. C57BL/6NCrlBR mice, purchased from Broekman Institute BV, Someren, The Netherlands, were used as recipients for bone marrow transplantation (BMT).
All mice used for BMT experiments were housed in sterilized, filter-top cages and given free access to food and water. Animals were maintained on sterilized regular chow (SRM-A) containing 5.7% (wt/wt) fat (Hope Farms) or were fed a high-cholesterol diet that contained 15% (wt/wt) fat, 1% (wt/wt) cholesterol, and 0.5% (wt/wt) cholic acid formulated according to Nishina et al.30 Drinking water was supplied with antibiotics (83 mg/L ciprofloxacin and 67 mg/L polymyxin B sulfate) and 6.5 g/L sugar.
A cocktail of ketamine, fentanyl, droperidol, and fluanisone (10 µL/g mouse) was used to anesthetize the mice before all lethal experiments. Animal procedures were performed at the Sylvius Laboratories of the Leiden/Amsterdam Center for Drug Research in accordance with national laws. All experimental protocols were approved by the Ethics Committee for Animal Experiments of Leiden University.
Irradiation and BMT
To induce bone marrow aplasia, female C57BL/6 mice (aged 5 to 6
weeks) were exposed to a single dose of 13 Gy (0.28 Gy/min, 200 kV, 4
mA) total-body irradiation by using an Andrex Smart 225 Röntgen
source (Andrex Radiation Products AS) with a 4-mm aluminum filter 1
day before transplantation, as previously described.31
Bone marrow cell suspensions were prepared by flushing the femurs and
tibias from LPL-/- or LPL+/+ male donor mice with PBS. Irradiated
recipients received 107 LPL-/- or LPL+/+ bone
marrow cells by intravenous injection into the tail
vein.
Serum Cholesterol, Triglyceride, and
ApoE Analysis
After an overnight fast, 100 µL of blood was drawn from
each mouse by tail bleeding. The concentrations of total
cholesterol and triglycerides in serum were
determined by using enzymatic procedures (Boehringer Mannheim).
Precipath (standardized serum, Boehringer Mannheim) was used as
an internal standard. The distribution of cholesterol and
triglycerides among the different lipoproteins in serum was
determined by loading 30 µL of serum from each mouse onto a Superose
6 column (3.2x30 mm; Smart-system, Pharmacia). Serum was
fractionated at a constant flow rate of 50 µL/min with PBS. Total
cholesterol and triglyceride contents in the
effluent were determined enzymatically. ApoE was measured with a
sandwich ELISA specific for mouse apoE, as described
earlier.31 Pooled sera from C57BL/6 mice, with a known
apoE level, was used as the standard.
Liver Function Test
A liver function test was performed by analysis of
alanine aminotransferase (ALAT) enzyme activity in serum after a
1-month feeding with the high-cholesterol diet N (3 months after
BMT). The analysis was performed by the Central Clinical
Chemical Laboratory of the Leiden University Medical Center on an
automated analyzer and according to the recommendations of the
International Federation of Clinical Chemistry.32
Determination of Lipolytic Enzyme Activity
To determine plasma LPL activity, blood was drawn 5 months after
BMT after an overnight fast both before and after an
intravenous bolus injection of heparin (100 U/kg).
Lipolytic activity was measured in several tissues, including heart
muscle, straight femoral muscle, brain, spleen, lung, and liver 5
months after BMT, as described previously.27 28 In brief,
tissue specimens of 100 mg were incubated in Dulbecco’s
modified Eagle’s medium/2% bovine serum albumin (BSA) and 2
U/mL heparin (Leo Pharmaceutical Products BV) for 1 hour at
37°C after the tissue was minced with scissors. The lipolytic
activity of the tissue supernatant as well as that in preheparin and
postheparin plasma was measured by using a radiolabeled
triolein emulsion as described by Zechner.33 In brief, the
substrate consisted of a radiolabeled triolein emulsion prepared by
sonication (4x 1 minute on ice at 100 W) of a mixture of 50 µCi
glycerol-tri[9,10(n)-3H]oleate, 4 mg of
unlabeled glycerol trioleate, 0.1 mol/L Tris-HCl (pH 8.6), 0.1% Triton
X-100, 2% BSA, and 2 mL of heat-inactivated human serum (a
source of apoC-II, an LPL activator). Subsequently,
LPL-containing samples (10 to 100 µL) were added to 0.2 mL of
substrate and incubated for 30 minutes at 37°C. The reaction was
stopped by addition of 3.25 mL of a mixture of
chloroform/methanol/n-heptane (1:1.28:1.37, vol/vol/vol) and
1 mL of 0.1 mol/L
K2CO3/H3BO3
(pH 10.5). FFAs were extracted by vortexing this mixture for 15
seconds, phases were separated by centrifugation at
3000 rpm at 4°C for 20 minutes, and 1 mL of the upper phase was
counted for radioactivity. The lipolytic activity was calculated from
the amount of FFAs released per tissue weight per hour. Liver lipase
activity was determined in the presence or absence of 1 mol/L NaCl to
differentiate between LPL and hepatic lipase activity. LPL activity was
calculated as the portion of total lipase activity inhibited by 1 mol/L
NaCl.
VLDL Isolation and Labeling
VLDL was isolated from sera of fasted, healthy volunteers by
discontinuous KBr gradient ultracentrifugation at
250 000g for 18 hours, as described by Redgrave et
al.34 The fraction of d<1.006 g/mL was
isolated and dialyzed against PBS/1 mmol/L EDTA. VLDL was labeled
with 125I at pH 10.0 according to
McFarlane,35 modified as described
earlier.36
Isolation and Culture of Murine Peritoneal Macrophages
Thioglycollate-elicited macrophages were harvested from
control LPL+/+C57BL/6 and LPL-/-C57BL/6 mice and plated in
24-well plates at a density of 0.5x106 cells/500
µL in Dulbecco’s modified Eagles medium, supplemented with 10%
(wt/vol) bovine calf serum, 2 mmol/L
L-glutamine, 100 µg/mL streptomycin, and 100 IU/mL
penicillin. After 4 hours, nonadhering cells were removed by washing.
After 2 days in culture, the cells were washed and incubated with 10
µg/mL 125I-VLDL in Dulbecco’s modified
Eagle’s medium/2% BSA for 3 hours at 37°C. At the indicated times,
cells were washed and lysed in 0.1 mol/L NaOH, and cell protein content
was determined according to Lowry et al.37 Cell-associated
radioactivity and degradation products of VLDL in the supernatant
were determined.
The effect of LPL deficiency on macrophage apoE secretion was analyzed in thioglycollate-elicited macrophages from LPL-/- and LPL+/+ littermates. After 24 hours in culture in Dulbecco’s modified Eagle’s medium/2% BSA, apoE secretion in the medium was measured with a sandwich ELISA for mouse apoE as described previously.31 Furthermore, the lipolytic activity of the supernatant was measured by using a radiolabeled triolein emulsion as described by Zechner.33 The lipolytic activity was calculated from the amount of FFAs (µmol) released per milligram of cell protein per minute.
Histological Analysis of Hearts and Aortas
for Atherosclerosis
To analyze the development of
atherosclerosis, transplanted mice were killed after 3
months of feeding with a high-cholesterol diet (15% fat,
1% cholesterol, and 0.5% cholic acid). Hearts and aortas
were perfusion-fixed, and atherosclerotic lesion area in oil red
O–stained cryostat sections of the aortic root was quantified, as
described previously.31 38 Mean lesion area was calculated
(in µm2) from 10 sections, starting at the
appearance of the tricuspid valves as described
previously.31 38
Immunocytochemistry
Macrophages were detected in atherosclerotic lesions by
immunolocalization of the MOMA-2 macrophage marker.
Formaldehyde-fixed, cryostat sections were washed with washing buffer
(100 mmol/L Tris, 150 mmol/L NaCl, pH 7.5) and incubated for
1 hour in blocking buffer (100 mmol/L Tris, 150 mmol/L NaCl,
1% blocking reagent for ELISA, and 5% normal goat serum, pH 7.5).
Subsequently, sections were incubated with rat anti-mouse MOMA-2
antiserum (dilution 1:10 in blocking buffer) for 1 hour at room
temperature and overnight at 4°C. After being washed, the sections
were exposed for 1 hour to goat anti-rat IgG conjugated to alkaline
phosphatase (Sigma Immuno Chemicals, dilution 1:200 in blocking
buffer). Thereafter, sections were extensively washed with washing
buffer, and MOMA-2–positive macrophages were visualized by
incubation with 5-bromo-4-chloro-3-indolyl phospate/nitro blue
tetrazolium alkaline phosphatase substrate (Sigma Chemicals) in a 0.2
mol/L Tris-HCl/10 mmol/L MgCl2 buffer,
pH 9.6.
The presence of LPL in atherosclerotic lesions was assessed
immunohistochemically with a rabbit anti-human LPL polyclonal antibody
(kindly provided by Dr S. Vilaro, University of Barcelona, Barcelona,
Spain). Formaldehyde-fixed, cryostat sections were washed with PBS and
subsequently incubated for 0.5 hour with 0.3%
H2O2 and for 1 hour with
blocking buffer (PBS, 1% BSA, and 10% normal goat serum, pH 7.4).
Subsequently, the sections were incubated with rabbit anti-human LPL
polyclonal antibody (1:25 in blocking buffer) for 1 hour at room
temperature and overnight at 4°C. After being washed, the sections
were exposed for 2 hours to biotin 
-rabbit immunoglobulin (Amersham,
dilution 1:200 in blocking buffer), washed again, and exposed for 1
hour to biotinylated streptavidin, conjugated to horseradish peroxidase
(Amersham, dilution 1:200 in blocking buffer). Thereafter the sections
were extensively washed with PBS, and LPL-positive lesion areas were
visualized by incubation with 3,3'-diaminobenzidine (Sigma Chemicals)
as the horseradish peroxidase substrate in 0.05 mol/L Tris-HCl (pH
7.4), 7% sucrose, and 0.03%
H2O2.
Statistical Analysis
Statistically significant differences among the means of
populations in repeated measurements of cholesterol and
triglyceride levels were tested in time by ANOVA with the
Student-Newman-Keuls post test (Instat Graphpad software).
Individual comparisons were made by using unpaired Student’s
t test. A P value of <0.05 was regarded as
significant.
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Effect of BMT on Serum Cholesterol and Triglyceride Levels in Chow-Fed Mice
To assess the role of macrophage-derived LPL in atherosclerosis, C57BL/6 mice were transplanted with LPL-deficient bone marrow, giving rise to animals that were deficient for LPL in all cell types of hematopoietic origin. The effect of transplantation on serum cholesterol and triglyceride levels was evaluated (Figures 1A and 1B). At 8 weeks after BMT, total cholesterol levels in LPL-/-
C57BL/6 mice were
reduced by 8% (P<0.05, n=16) compared with
LPL+/+C57BL/6 mice (Figure
1A). As shown in Figure
1B, serum triglyceride levels were increased by 33%
(P<0.05, n=16) in the LPL-/-C57BL/6 animals at 8 weeks
after BMT. The cholesterol and triglyceride
distribution among the different lipoprotein fractions was
analyzed by liquid chromatography 8 weeks after
BMT (Figures 2A and 2B).
Analysis of the cholesterol distribution in serum
revealed that it had not significantly changed by 8 weeks after BMT on
a normal chow diet (Figure 2A). The increase in serum
triglyceride levels in LPL-/-C57BL/6 mice 8 weeks
after BMT seemed to be due to an increase in VLDL
triglycerides, although no statistically significant
difference was achieved (Figure 2B).
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Effect of BMT on Serum ApoE Levels
Macrophages in the atherosclerotic lesion produce LPL as
well as apoE.7 8 39 Because cholesterol
homeostasis in macrophages may be regulated by both LPL and
apoE, the effect of the introduction of LPL-deficient bone marrow on
serum apoE levels was determined. Strikingly, serum apoE levels were
approximately 50% (P<0.001, n=16) reduced, to 3.52±0.33
mg/dL, in the LPL-/-C57BL/6 mice compared with 7.16±0.78 mg/dL in
the control transplanted animals 8 weeks after transplantation. No
difference between the 2 groups was observed before
transplantation.
The metabolic cause of the reduced serum apoE levels was
explored through in vitro incubation studies with LPL-/- and LPL+/+
macrophages (Table 1).
Macrophages were isolated from L0-MCK (LPL-/-) and L2-MCK
(LPL+/+) littermates, and apoE secretion by these macrophages
into the supernatant was measured. As shown in Table 1, LPL
activity in the medium from control LPL+/+ macrophages was
97.8±28.4 mU/mg cell protein (n=5), whereas the LPL activity in
macrophages from LPL-/- mice was nearly undetectable. The
absence of LPL production was found to be associated with a
marked decrease in apoE secretion into the supernatant in the
LPL-deficient macrophages compared with control cells,
indicating that in the absence of macrophage LPL
production, apoE secretion is markedly lower.
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Effect of BMT on Plasma and Tissue Lipase Activity
Analysis of the lipolytic activity of preheparin and
postheparin plasma (Table 2)
revealed that macrophage LPL production did not
contribute significantly to the lipolytic activity located either in
plasma or on the endothelial surface. Furthermore,
tissue LPL activity was determined in cardiac muscle, skeletal muscle,
brain, spleen, lung, and liver. As depicted in Figure
3, heparin-releasable lipase activity
was significantly reduced in organs containing high amounts of
macrophages, like the lungs (10-fold, P=0.04) and
spleen (2-fold, P=0.03). Unexpectedly, heparin-releasable
lipase activity in cardiac muscle was also found to be reduced by 69%
(P=0.0009) in the mice transplanted with LPL-deficient bone
marrow compared with controls. Compared with control C57BL/6 mice,
irradiation and the BMT procedure itself did not influence either the
postheparin plasma or the tissue LPL activity (data not
shown).
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Effect of BMT on Association and Degradation of
Iodinated Human VLDL by Macrophages
The association and degradation of iodinated human
VLDL by macrophages isolated from control transplanted animals
was compared with the activity of macrophages from animals
transplanted with LPL-deficient bone marrow. From the data shown in
Figure 4A, it is evident that the
association, including binding and uptake, was significantly higher
(P=0.04, n=3) in control macrophages compared with
macrophages that lacked LPL. The association of
125I-VLDL with control macrophages after
3 hours of incubation was 888±138 ng/mg cell protein compared with
584±119 ng/mg cell protein in macrophages from
LPL-/-C57BL/6 animals. No effect was observed on the degradation
of iodinated human VLDL, indicating that
macrophage-derived LPL is important for the association of
VLDL but not for its degradation (Figure 4B).
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Effect of BMT on the Response to a High-Cholesterol
Diet and Atherosclerosis
To analyze the effect of the introduction of LPL-deficient
macrophages in C57BL/6 mice on atherosclerosis,
the mice were challenged with a high-cholesterol diet. As
shown in Figure 5A, after 8 weeks on this
diet LPL-/-C57BL/6 mice and control LPL+/+C57BL/6 animals
responded with a 5-fold and a 9-fold increase in serum
cholesterol levels, respectively, thereby increasing the
difference in serum cholesterol levels between the groups
to 52% (P<0.01). On feeding the mice a
high-cholesterol diet, serum triglyceride
levels were slightly reduced in both groups. However, no significant
difference in serum triglyceride levels could be
demonstrated between LPL-/-C57BL/6 and LPL+/+C57BL/6 mice under
these conditions (Figure 5B).
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Fractionation of serum lipoproteins by liquid
chromatography revealed that the increase in
cholesterol levels after feeding the mice a
high-cholesterol diet was associated with an increase in
cholesterol in VLDL and LDL, whereas HDL
cholesterol was unchanged (Figure 6A). The difference in response to the
high-cholesterol diet between LPL-/-C57BL/6 mice
and LPL+/+C57BL/6 animals was due to a difference in the increase in
VLDL and LDL levels. Under these dietary conditions, no significant
differences in triglyceride levels in VLDL between
LPL-/-C57BL/6 mice and LPL+/+C57BL/6 animals were found (Figure
6B). The combined increase in VLDL cholesterol and
reduction in VLDL triglycerides indicates that feeding the
mice a high-cholesterol diet induces both an increase in
VLDL and LDL levels and replacement of triglycerides in
VLDL by cholesteryl esters.
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Analysis of serum apoE levels after the mice were fed the
high-cholesterol diet for 2 and 8 weeks revealed that the
diet induced a large increase in serum apoE levels in both groups of
mice (Figure 7). However, the reduction
in serum apoE levels in mice transplanted with LPL-deficient bone
marrow, as observed on the chow diet at 8 weeks after BMT, also
remained during the period when the mice were fed the
high-cholesterol diet (38% reduction; P<0.05,
n=16).
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After 3 months on the high-cholesterol diet, the hearts and
aortas were perfused, fixed, and examined
histologically. Quantification of atherosclerotic
lesion area demonstrated that transplantation of LPL-deficient bone
marrow into C57BL/6 mice reduced the susceptibility of these mice to
diet-induced atherosclerosis (Figure
8). The mean atherosclerotic lesion
area in LPL-/-C57BL/6 mice (6898±1560
µm2, n=13) was 52% (P<0.01)
smaller than that found in LPL+/+C57BL/6 mice (14 652±2441
µm2, n=13). Representative
photomicrographs of lipid-rich atherosclerotic lesions are shown in
Figure 9. Staining of the
atherosclerotic lesion area for MOMA-2, a macrophage marker,
revealed that in both transplantation groups the atherosclerotic lesion
area consisted primarily of lipid-laden macrophages (Figures 10A and 10B).
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The extent of reconstitution of arterial wall
macrophages by LPL-/- macrophages after BMT is
important when analyzing the effects on
atherosclerosis. Therefore, localization of the LPL
protein in atherosclerotic lesions was determined by using an antibody
against human LPL that cross-reacts with murine LPL (kindly provided by
Dr S. Vilaro, University of Barcelona, Barcelona, Spain). In mice
transplanted with LPL+/+ bone marrow, LPL was abundantly present in
macrophage-rich regions of atherosclerotic lesions (Figure
10C) as well as the smooth muscle cells in the aortic root. In
mice transplanted with LPL-/- bone marrow, however, the
macrophage-rich lesion area did not stain for LPL (Figure 10D), whereas some staining of the smooth muscle cells of the
aortic root was still visible.
Effect of BMT on Liver Function
To exclude the possibility that the observed effects on serum
cholesterol levels were caused by liver damage as a result
of the BMT procedure and the subsequent consumption of an atherogenic
diet, serum ALAT activity was measured and the liver was
analyzed histologically. As indicated in Table 3,
no significant difference in serum
ALAT activity was observed between both groups of transplanted mice and
control C57BL/6 mice of the same age. These data indicate that the
liver parenchyma and biliary system were not seriously damaged by the
BMT procedure and subsequent feeding with the atherogenic diet at 3
months after BMT and 1 month of feeding with the
high-cholesterol diet. Histological
analysis of the livers after 3 months of feeding the
high-cholesterol diet revealed no
histological evidence of hepatocyte
necrosis or fibrosis.
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Discussion |
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In the present study, we investigated the in vivo role of macrophage-derived LPL in lipoprotein metabolism and atherogenesis by using BMT. BMT provides a powerful technique to produce an animal model that lacks LPL specifically in all types of macrophages. Transplantation of LPL-deficient hematopoietic stem cells into lethally irradiated C57BL/6 mice resulted in a significant reduction in tissue lipase activity in organs containing high amounts of macrophages, like the spleen and lungs, indicating that these tissue macrophages were successfully replaced by cells of donor origin.
Macrophage LPL deficiency induced a significant reduction in serum cholesterol levels on both a chow diet and a high-cholesterol diet. Fractionation of serum lipoproteins revealed that this decrease in cholesterol levels was confined to the VLDL- and LDL-size fractions, while no significant effect on HDL cholesterol could be demonstrated. Thus, the reduction in tissue LPL activity due to the absence of LPL production by macrophages is not critical for the regulation of serum HDL levels in mice. These results are consistent with those of Coleman et al,25 who demonstrated that LPL activity has a limited role in determining HDL cholesterol levels in mice. Surprisingly, VLDL and LDL cholesterol levels were reduced in the absence of macrophage-derived LPL. In contrast, heterozygous LPL deficiency in all LPL-expressing organs is associated with an increase in VLDL and LDL cholesterol levels.26 These contradictory results support the hypothesis that the physiological function of LPL depends on the tissue in which it is expressed. It is unlikely that the observed effects on serum cholesterol levels were caused by liver damage as a result of the BMT procedure and the subsequent consumption of a high-cholesterol diet, because no effect on serum ALAT activity was observed, indicating that the liver parenchyma and biliary system were not damaged. Furthermore, no histological evidence of hepatocyte necrosis or fibrosis was found. Analysis of postheparin lipolytic activity in the plasma of transplanted mice revealed that macrophage LPL deficiency did not significantly affect the activity of the LPL pool located on the endothelial surface of the vascular tree. Thus, the effect of macrophage LPL deficiency on cholesterol levels in the circulation also appears not to be mediated by effects on the functional LPL pool on the endothelial surface. Another mechanism that can be postulated that may explain the observed reduction in cholesterol levels as induced by macrophage LPL deficiency might be reduced VLDL production by the liver. This alternative mechanism is currently under investigation. Although the exact mechanism of the cholesterol lowering remains to be determined, our data clearly provide evidence that macrophage LPL production significantly affects the metabolism of lipoproteins in the circulation.
Despite the decrease in serum cholesterol levels, serum
triglyceride levels were increased 8 weeks after BMT in
LPL-/-C57BL/6 mice, probably due to reduced
triglyceride hydrolysis in the periphery. Feeding the mice
a high-cholesterol diet reduced triglyceride
levels again, which is consistent with data from Semenkovich et
al26 and LeBoeuf et al,40 who demonstrated
that increased dietary fat decreases serum triglycerides in
mice.
Strikingly, reconstitution of C57BL/6 mice with LPL-deficient
macrophages also resulted in a sustained decrease in
circulating apoE levels. Although the liver is the major source of apoE
synthesis, macrophages in different organs are also active in
secreting large quantities of apoE.41 42 43 Recently,
we31 and others44 45 demonstrated, using the
technique of BMT, that introduction of apoE-producing
macrophages in apoE-deficient mice reduced the severe
hypercholesterolemia in these mice, indicating
the important role of macrophage-derived apoE in an
apoE-deficient background. However, we46 and Fazio et
al47 also showed that no effect on serum apoE
concentration was observed in macrophage-specific apoE
knockouts, suggesting that apoE production by
macrophages cannot influence serum apoE levels in the presence
of normal liver apoE production. Therefore, it is most likely
that the 50% reduction in serum apoE concentration in
LPL-/-C57BL/6 mice was not caused by any direct effects
of macrophage LPL deficiency on macrophage apoE
synthesis but was a consequence of the observed reduction in serum
cholesterol levels.
Because local production of apoE by macrophages may influence the process of foam cell formation, the effect of macrophage LPL deficiency on macrophage apoE production was analyzed in vitro. This study revealed that LPL-deficient macrophages were less active in secreting apoE into the medium than were wild-type macrophages, indicating that apoE secretion is influenced by endogenous LPL production. Recently, Lucas et al48 demonstrated that addition of exogenous LPL to mouse J774 cells transfected with human apoE cDNA prevented the release of newly synthesized apoE by these cells. Thus, both absence of endogenous LPL and addition of exogenous LPL reduce macrophage apoE secretion.
Transplantation of LPL-deficient bone marrow into C57BL/6 mice results in the replacement of all tissue macrophages, including those of the arterial wall that are involved in fatty streak formation. To analyze the effect of macrophage LPL deficiency on atherosclerosis susceptibility, the transplanted mice were challenged with a high-cholesterol diet containing cholate, a diet that has been extensively used to evaluate atherosclerotic lesion development in resistant mouse models.49 50 51 After 3 months of feeding this diet, atherosclerotic lesion development was reduced by 52% in C57BL/6 mice transplanted with LPL-deficient bone marrow compared with control transplanted animals. Cholesterol accumulation in macrophages and therefore, foam cell formation depends on the balance between cholesterol influx and cholesterol efflux. The following factors will influence the susceptibility to diet-induced atherosclerosis in C57BL/6 mice transplanted with LPL-deficient bone marrow: (1) decreased influx of cholesterol due to decreased exposure to serum cholesterol (antiatherogenic), (2) decreased influx of cholesterol due to reduced uptake of atherogenic lipoproteins (antiatherogenic), and (3) decreased efflux of cholesterol due to reduced apoE synthesis by macrophages (proatherogenic).
By performing immunohistochemistry, we showed that reconstitution of
C57BL/6 mice with LPL-deficient macrophages resulted in an
almost complete absence of LPL in fatty streak lesions. Thus, most of
the LPL present in fatty streak lesions was derived from local
synthesis by macrophages, rather than from the plasma
compartment. Atherosclerotic lesion area quantification showed that
absence of macrophage LPL production in the
arterial wall resulted in reduced susceptibility to
diet-induced atherosclerosis, indicating that the
observed decreased influx of VLDL cholesterol in
macrophages of LPL-/-C57BL/6 mice is the major determinant
of the effect of LPL on atherosclerotic lesion development.
Furthermore, the lowered apoE production was apparently still
sufficient for cholesterol release from
macrophages.
While our manuscript was under review, a relevant article describing the transplantation of fetal liver cells from LPL-deficient mice to C57BL/6 animals was published by Babaev et al.52 In accordance with our data, they showed that macrophage LPL deficiency resulted in a 55% reduction in mean atherosclerotic lesion area, whereas no effect on serum lipid levels was observed. In summary, we have shown that macrophage LPL deficiency decreases serum cholesterol concentrations, circulating apoE levels, and diet-induced atherosclerosis in C57BL/6 mice, suggesting that specific blockade of macrophage LPL production may be beneficial for lowering atherosclerotic lesion formation.
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Acknowledgments |
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This research was supported by SmithKline Beecham Pharmaceuticals, Harlow, UK, and the Netherlands Heart Foundation (project 98192). The authors wish to thank Dr G.M. Benson for helpful assistance with the image analysis of atherosclerotic lesions.
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Footnotes |
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This work was presented at the 71st Scientific Sessions of the American Heart Association, November 8–11, 1998, Dallas, Tex, and published in abstract form (Circulation. 1998;98[suppl I]:I-663.).
Received June 24, 1999; accepted February 14, 2000.
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