(Received for publication, January 30, 1997, and in revised form, March 21, 1997)
From the Department of Pathology and Laboratory
Medicine, University of North Carolina, Chapel Hill, North Carolina
27599-7525 and the ¶ Département
d'Athérosclérose U-325, Institut Pasteur, 59019 Lille,
France
The effect of hepatic lipase (HL) deficiency on
the susceptibility to atherosclerosis was tested using mice with
combined deficiencies in HL and apoE. Mice lacking both HL and apoE
(hhee) have a plasma total cholesterol of 917 ± 252 mg/dl (n = 24), which is 184% that of mice lacking
only apoE (HHee; 497 ± 161 mg/dl, n = 20, p < 0.001). The increase in cholesterol was
mainly in -migrating very low density lipoproteins, although high
density lipoprotein cholesterol (HDLc) was also increased (53 ± 37 versus 20 ± 13 mg/dl, p < 0.01).
Despite the increase in plasma cholesterol, we found that HL deficiency
significantly decreased aortic plaque sizes in female mice fed normal
chow (31 × 103 ± 22 × 103
µm2 in hhee versus 115 × 103 ± 69 × 103 µm2 in
HHee, p < 0.001). Reduction of plaque
sizes was also observed in female heterozygous apoE-deficient mice fed
an atherogenic diet (2 × 103 ± 2.5 × 103 µm2 in hhEe versus 56 × 103 ± 49 × 103 µm2 in
HHEe, p < 0.01). Changes in aortic lesion
size were not apparent in the small number of male mice studied. In
HHee females, both HDLc and the capacity of high density
lipoprotein (HDL) particles to promote cholesterol efflux from cultured
cells were 26% of the wild type. The absence of HL in hhee
females partially restored HDLc levels to 57% and cholesterol efflux
to 55% of the wild type. Circulating pre-
1-migrating
HDL were present in all mutants, suggesting that there are alternative
pathways in the formation of these pre-
-HDL not involving apoE, HL,
or cholesteryl ester transfer protein. The improved capacity to promote
cholesterol efflux, together with increased HDL, may explain why these
animals can overcome the increase in atherogenic lipoproteins.
In circulation, nascent lipoproteins are remodeled by removal of
core lipids and transfer of surface proteins and lipids prior to their
removal through receptor-mediated mechanisms (1). Hepatic lipase
(HL)1 is a 60-kDa lipolytic enzyme involved
in the processing of chylomicrons, intermediate density lipoproteins
(IDL), and high density lipoproteins (HDL) (2). HL efficiently
hydrolyzes both triglycerides (TG) and phospholipids, while lipoprotein
lipase is mainly responsible for hydrolysis of plasma TG. The preferred
enzymatic substrates for HL are intermediate-size particles, such as
IDL, and HDL2. Changes observed in lipoprotein profiles in
humans with congenital HL deficiencies are the presence of
-migrating very low density lipoproteins (
-VLDL) and larger HDL
(3). The
-VLDL that accumulate in HL deficiency are more TG-rich
than
-VLDL in typical type III hyperlipoproteinemia subjects (4). We
previously reported that HL-deficient mice have a mild dyslipidemia
with increased cholesterol and phospholipid, and the plasma contains
large HDL floating in the 1.02-1.04 g/ml density range (5). Consistent with these observations, overexpression of HL decreases HDL cholesterol and HDL particle size in mice (6) and decreases HDL cholesterol and IDL
in rabbits (7).
Both HL and cholesteryl ester transfer protein (CETP) participate in
the metabolism of HDL (8). Both enzymes decrease HDL core lipids,
apparently making lipid-poor apoA-I available for dissociation or
transfer to other particles (8-10). Addition of HL to native plasma or
during perfusion through isolated liver from rat produces small
pre--migrating HDL (11). HL-mediated lipolysis may be the primary
source of the circulating HDL subfractions, pre-
-HDL, with a pre-
electrophoretic mobility on agarose gel. Pre-
1-HDL
particles are thought to be the acceptors of cellular cholesterol in
the early steps of reverse cholesterol transport (12, 13). Disruption
of this pathway could be detrimental to cholesterol homeostasis in
peripheral tissues.
Mice provide a useful model for studying both HDL metabolism and the role of HDL in atherogenesis. Because mice lack CETP (14), one pathway for reverse cholesterol transport, e.g. transfer of cholesteryl ester from HDL to apoB-containing particles, is not operating. Also, specific components involved in lipoprotein remodeling, such as HL, can be genetically eliminated in mice by gene targeting. This allows specific mutations affecting lipoprotein metabolism to be related to atherogenesis. Certain genetically engineered mice have atherosclerotic plaques similar to those found in humans. For example, mice completely lacking apoE develop atherosclerosis on a regular chow diet (15-18), while mice carrying a single copy of the apoE gene are susceptible to diet-induced atherosclerosis (19).
In this study, we evaluated the effect of HL deficiency on plasma lipoprotein distribution and atherogenesis in mice with apoE deficiency. We found that HL deficiency increases plasma cholesterol levels in female apoE-deficient mice, but their aortic plaque sizes were reduced compared with mice deficient in apoE, but normal in HL.
HL- and apoE-deficient mice were bred to generate double mutants (5, 15, 16). The parental apoE mutants were a seventh generation backcross of F1 (129/Ola: C57BL/6J) to C57BL/6J, and the HL mutants were a second generation backcross to C57BL/6J. Seven groups of animals were compared for lipid concentration in plasma: 1) the wild type, normal gene for HL and apoE (HHEE); 2) homozygous for HL deficiency, normal apoE gene (hhEE); 3) homozygous for HL deficiency and heterozygous for apoE (hhEe); 4) normal for HL and heterozygous for apoE deficiency (HHEe); 5) normal for HL and homozygous for apoE deficiency (HHee); 6) homozygous for apoE and heterozygous for HL deficiency (Hhee); and 7) homozygous for both HL and apoE deficiencies (hhee). Four groups, HHEE, hhEE, HHee, and hhee, were further characterized for lipid and lipoprotein distribution.
To test the effects of HL on lipoproteins and atherosclerosis, three experiments were designed. In the first experiment, female 129/B6 F2 mice deficient in HL (hhEE; n = 10) and their wild-type littermates (HHEE; n = 10) were fed the atherogenic diet for 4 months. In the second experiment, female mice heterozygous for apoE deficiency, which develop atherosclerosis only when fed an atherogenic diet (18), were tested in the presence (HHEe; n = 7) or absence (hhEe; n = 10) of a normal HL gene. The atherogenic diet contained 15.8% (w/w) fat, 1.25% (w/w) cholesterol, and 0.5% (w/w) sodium cholate ad was from Teklad Premier (Madison, WI). This diet was fed to the mice for 4 months, and the mice were killed at 6 months of age. In the third experiment, mice homozygous for apoE deficiency in the presence (HHee; n = 14, 5 males and 9 females) or absence (hhee; n = 18, 4 males and 14 females) of HL expression were maintained on normal chow and killed at 4 months of age.
Mice were maintained on 12-h dark/light cycles and allowed access to food and water ad libitum. The animals were handled following the National Institutes of Health guidelines for the care and use of experimental animals.
Lipids and Lipoprotein AnalysisFollowing an overnight fast, 200-300 µl of blood were collected by retro-orbital bleeding into tubes containing EDTA (final concentration of 2 mM). Plasma samples obtained by centrifugation (8000 × g for 10 min at 4 °C) were supplemented with aprotinin and gentamicin (final concentrations of 0.1 IU and 1 µg/ml, respectively). Plasma was kept at 4 °C until use. Total cholesterol and TG were determined immediately using reagents from Sigma. HDL cholesterol and phospholipids were determined within 3 days by using a kit from Wako Bioproducts (Richmond, VA).
For lipoprotein analysis, blood was collected in the morning from fasted mice under anesthesia with a lethal dose of Avertin (2,2,2-tribromoethanol). Plasma samples (100 µl) were subjected to gel filtration using a Superose 6HR column (Pharmacia, Uppsala), and lipid analysis of the fractions was carried out as described (20). Lipoproteins from 1 ml of plasma combined from three mice were fractionated by sequential density ultracentrifugation (20) at 70,000 rpm at 4 °C in a Beckman TL-100 ultracentrifuge. Lipoprotein fractions in the density ranges of d < 1.006, d = 1.006-1.02, d = 1.02-1.04, d = 1.04-1.06, d = 1.06-1.08, d = 1.08-1.10, and d = 1.10-1.12 g/ml were obtained following successive centrifugation for 5 h. The d = 1.12-1.21 g/ml fraction was isolated following overnight centrifugation. Lipoproteins from each fraction were recovered by tube slicing and dialyzed in 10 mM Tris buffer, pH 7.4, 150 mM NaCl, and 1 mM EDTA. Lipoproteins were separated by agarose gel electrophoresis using commercially available precast gels (Ciba Corning, Palo Alto, CA). The apolipoprotein composition of each fraction was determined by SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining of the proteins.
HDL Analysis by Two-dimensional ElectrophoresisApoA-I distribution in plasma lipoproteins was determined by two-dimensional nondenaturing gel electrophoresis (13). The first dimension was carried out on 0.75% agarose in 50 mM barbital buffer, pH 8.6, on GelBond (FMC Corp. BioProducts) at 4 °C (200 V) for 100 min. The second dimension was carried out on 2-15% gradient polyacrylamide gels at 120 V for 16 h. Thereafter, the gel content was transferred to a nitrocellulose sheet in buffer containing 0.025 M Tris and 0.192 M glycine, pH 8.3, for 100 min at 392 mA under semidry conditions. ApoA-I-containing fractions were detected by chemiluminescence using an anti-mouse apoA-I antibody labeled with peroxidase and an ECL kit (Amersham Life Science, Inc.).
Cholesterol Efflux by HDLCellular cholesterol efflux from rat Fu5AH hepatoma cells was determined following the procedure described (21). Briefly, 2 ml of medium containing cells at a concentration of 25,000 cells/ml were plated in 2.4-cm multiwell plates and grown until confluence in minimal essential medium supplemented with 5% calf serum. Two days after plating, cellular cholesterol was labeled during a 48-h incubation with [3H]cholesterol (1 µCi/well). To allow equilibration of the label, the cells were rinsed and incubated for 24 h in minimal essential medium containing 0.5% bovine serum albumin. For determination of cholesterol efflux, the cells were washed with 0.1 M phosphate-buffered saline and incubated at 37 °C for 4 h with 100 µg of HDL protein that had been isolated by fast protein liquid chromatography gel filtration. After recovery of the incubation medium, the remaining cellular cholesterol was then extracted. Radioactivity was measured in both medium and cells, and percentage of cholesterol efflux was calculated.
Evaluation of Atherosclerotic LesionsThe mice were killed with an overdose of Avertin. The heart and vascular tree were perfused with 4% phosphate-buffered paraformaldehyde, pH 7.4. Segments containing aortic sinus area were embedded, serially sectioned, and stained. Four sections were used for morphologic evaluations (16, 19). The average of the sizes of lesions in the four sections was used to represent lesion size of each animal's aorta. Morphometric evaluation of the lesions was conducted using the Image measure/IP IM 2500 morphometry system (Phoenix Technology, Federal Way, MA).
Statistical AnalysisPaired and unpaired Student's t tests were performed for the plasma lipid concentrations. The Mann-Whitney U test was used to compare lesion sizes.
Fasted plasma lipid levels of mice with both HL and apoE deficiencies (hhee) were significantly elevated compared with mice with only apoE deficiency (HHee), as shown in Table I. Total cholesterol levels in hhee mice were nearly twice those in HHee controls. While a significant reduction in HDLc levels was observed in apoE-deficient animals (HHee) compared with wild-type mice (HHEE), HDLc levels in hhee mutants were similar to those in wild-type mice. HDLc levels in hhee mice showed a 2-fold increase over those in HHee mice. Triglycerides were also significantly higher in hhee compared with HHee mice. Male mice heterozygous for HL and homozygous for apoE (Hhee) had intermediate levels of plasma cholesterol (765 ± 110 mg/dl). An ~75% increase in total cholesterol was observed in hhee compared with HHee mice. In contrast, only a 26% increase in total cholesterol was observed in hhEE compared with HHEE mice. Therefore, the increase in total cholesterol levels seen in HL deficiency is enhanced by the absence of apoE.
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Female mice heterozygous for apoE with and without HL (HHEe and hhEe, respectively) responded to an atherogenic diet with a significant increase in total plasma cholesterol. Degrees of increase in HHEe and hhEe mice were similar, suggesting that apoE (but not HL) is the primary factor affecting the plasma lipid increase in response to dietary change. The atherogenic diet reduced TG by 65-77% and HDLc by 17-23% in HHEe and hhEe mice, respectively.
Plasma from different mutants was fractionated by gel filtration
chromatography (Fig. 1). In contrast to HHEE
and hhEE mice, in which most of the cholesterol was in the
HDL range, the cholesterol in apoE-deficient mutants (HHee
and hhee) was distributed mostly among larger lipoproteins
in the VLDL and IDL/LDL range. The hhee mutants had further
increased cholesterol content in these potentially atherogenic
particles compared with HHee mice. In addition, the HDL
fractions in hhee mice were increased, resulting in similar HDLc levels as observed in wild-type mice (HHEE). Consistent
with previous results (5), HL deficiency increased the size of HDL particles as revealed in the earlier elution of HDL fractions from the
gel permeation column (Fig. 1). However, the shift of the HDL fraction
peak was not obvious in hhee compared with HHee mice. Fractionation of lipoproteins by sequential ultracentrifugation also showed that the distribution of cholesterol and phospholipids is
determined mainly by apoE genotype (Fig. 2). Most of the
cholesterol in apoE-deficient mice was within the density range of
1.006-1.04 g/ml. In contrast, the cholesterol in HHEE and
hhEE mice was present primarily within the density range of
1.06-1.21 g/ml. The distribution of cholesterol in HL-deficient mice
was shifted toward lower buoyant density compared with that in
wild-type mice. In contrast, no significant difference among different
groups of mice was observed in TG distribution, which was mostly in
VLDL fractions (Fig. 2).
Agarose gel electrophoresis of lipoproteins in density fractions
revealed a dramatic increase in the d < 1.02 g/ml
fractions (-VLDL) in HHee mice, which was further
increased in hhee mutants (Fig. 3).
Consistent with gel permeation data, there was also a significant
increase in the
-migrating particles in hhee mice, similar to the amount observed in HHEE mice. In
hhee mice, lipoproteins with
-mobility were present in
fractions with d < 1.06 g/ml. Similar particles were
present at much lower levels in hhEE mutants, but they were
absent in HHEE and HHee mutants. Previous studies showed that the d = 1.02-1.04 g/ml fraction of
hhEE mice contains
-migrating HDL that reacts with
anti-apoE and anti-apoA-I antisera, but not anti-apoB antiserum (5).
The absence of apoE is associated with increased apoB-48 and apoB-100
in the d < 1.006 g/ml fraction. SDS-polyacrylamide gel
electrophoresis of lipoproteins showed that the amount of apoB in the
various density fractions from hhee and HHee mice
was not altered (data not shown), suggesting that the particle number
had not significantly changed, but that the increase in size was mostly
due to enrichment of cholesterol.
Lesion Development
To investigate the effect of HL deficiency on atherosclerosis development, three experiments were carried out. In the first experiment, hhEE mice and their wild-type littermates were fed an atherogenic diet for 4 months. No significant plaques were found in animals of either group, suggesting that hepatic lipase deficiency does not by itself increase susceptibility to diet-induced atherosclerosis in mice (data not shown).
In the second experiment, we evaluated diet-induced atherosclerosis
using animals heterozygous for apoE deficiency. We fed the atherogenic
diet to HHEe and hhEe females for 4 months.
Animals were killed at 6 months of age, and aortic plaque sizes were
evaluated (Fig. 4a). We found that the extent
of lesions in hhEe mice was only 3% (2.0 × 103 ± 2.5 × 103 µm2,
n = 7) of that in HHEe mice (56 × 103 ± 49 × 103 µm2,
n = 10) despite the higher levels of plasma cholesterol
in HHEe mice, shown in Table I.
In the third experiment, we evaluated the effect of HL deficiency on spontaneous atherosclerosis caused by the complete lack of apoE. Mice homozygous for apoE deficiency in the presence or absence of HL were maintained on a regular chow diet and killed at 4 months of age. All apoE null mutants developed atherosclerotic lesions (Fig. 4b). Strikingly, the plaque sizes in hhee females were 25% (31 × 103 ± 21 × 103 µm2, n = 14) of those in HHee control females (115 × 103 ± 69 × 103 µm2, n = 9, p < 0.001). In contrast, no significant difference in plaque size was observed in the small number of males examined (20 × 103 ± 5.5 × 103 µm2 (n = 4) versus 21 × 103 ± 12 × 103 µm2 (n = 5)) despite higher plasma cholesterol levels as compared with HHee mice (Table I). Lesion sizes in male and female hhee mice were not significantly different. Maturity of plaques was directly correlated with their size, and no particular differences in plaque components between mice with and without HL were apparent by light microscopic evaluation.
Cholesterol Efflux and HDL ParticlesTo determine if HDL
function is altered in HL-deficient mice, we assessed their capacity to
promote efflux of cellular cholesterol (Fig. 5). HDL
from both hhee and HHee mice had reduced capacity to promote cholesterol efflux (55 and 26%, respectively) compared with
HDL from wild-type mice (100%). Both male and female HHee mice had equal capacity to promote cholesterol efflux, but HDL from
male hhee mice had 50% higher capacity compared with HDL from female hhee mice, consistent with the fact that male
hhee mice have a higher plasma HDLc level compared with
female hhee mice (Table I).
We then examined whether the effect on cholesterol efflux was related
to qualitative changes in HDL particles. Plasma samples from different
mice were separated by two-dimensional nondenaturing gel
electrophoresis and blotted with an apoA-I antibody (Fig. 6). The general electrophoretic pattern of HDL was
similar in all genotypes and very close to that observed in humans
(13). However, pre-1-HDL from all mutant mice displayed
increased levels and slightly larger sizes compared with wild-type
mice. The
1-HDL fraction was markedly reduced in
HHee mutants and less in hhee mutants. A new
fraction of
-mobility,
2, was found in
hhee mice and much less so in HHee mice. This
fraction is larger than
1 in size. Electrophoretic
patterns of male and female mutants were indistinguishable (data not
shown).
The most striking change in the lipid profiles of mice deficient
in both HL and apoE is a marked increase in cholesterol present mostly
in -VLDL. Although this increase primarily results from the lack of
apoE, the comparison between hhee and HHee mice
clearly demonstrates additional and independent effects of HL
deficiency. The absence of HL therefore increases the accumulation of
atherogenic
-VLDL already present in apoE deficiency. The increase
in lipids is, however, not associated with an apparent increase in
apoB-48 or apoB-100, suggesting that hhee mice accumulate
larger apoB-containing particles. This is consistent with a role of HL
in the remodeling of remnant particles.
In addition to lipid abnormalities in HL-deficient humans, several
studies have suggested that HL plays a role in chylomicron remnant
removal (22, 23). Acute inhibition of HL activity by antibodies in
primates or in a rat liver perfusion system causes the accumulation of
TG-rich lipoproteins (22). Ji et al. (23) have shown
in vitro that cell surface-bound HL enhances binding of
remnant lipoproteins to the cells. This process does not require apoE
since binding of -VLDL isolated from apoE-deficient mice and binding
of apoE-containing
-VLDL from rabbits were equally enhanced. HL has
been shown to bind to the LDL receptor-related protein and promote
-VLDL uptake (24). These interactions are inhibited by heparin,
suggesting that HL may act as a bridge between remnant lipoproteins and
cell-surface heparan sulfate proteoglycans. However, HL-deficient mice
display no apparent impairment in chylomicron clearance (5), suggesting
that this pathway is not crucial for their clearance in mice under
normal physiological conditions. In contrast, in apoE deficiency, when
the major ligand mediating removal is not operating, the absence of HL
causes a further increase in the accumulation of cholesterol-rich
remnants. The increase in remnant lipoproteins in hhee mice
may result from an impairment in HL-facilitated remnant uptake by the
liver or delayed conversion of remnants to smaller particles,
preventing their effective removal.
Another effect related to HL deficiency is an increase in HDL levels.
Large -migrating HDL enriched in apoE but devoid of apoB were
observed on agarose gel electrophoresis of d = 1.02-1.04 g/ml fractions in HL-deficient mice. These particles contain
apoA-I, cholesterol, and phospholipids (5). In this study, we found larger HDL in HL-deficient mouse plasma fractionated by gel permeation chromatography or ultracentrifugation. In addition, two-dimensional nondenaturing gel electrophoresis showed the presence of large HDL
particles in the
2-fraction in the hhee
mutants. We also observed a slight increase in apoA-I in the
1-migrating fraction of hhee mutant HDL
compared with HHee mutant HDL. In contrast to results
reported by Koo et al. (25) suggesting that apoE is required
for the formation of large HDL, our observations indicate that apoE is
not essential for this process.
The origin of large 2-migrating HDL in HL-deficient mice
is not clear. The most simple explanation is that these particles are
generated as a consequence of sequential lecithin:cholesterol acyltransferase-mediated enlargement of small HDL3 first to
HDL2 and then to HDL1 (1). HDL1 are
normally enriched in apoE and are thought to be removed through
apoE-mediated LDL receptor interaction (26). The absence of apoE in
hhee mutants may contribute to the accumulation of these
particles by delaying their removal by hepatic receptors. Further
studies are necessary for determining the exact origin of these large
-migrating fractions.
It has been proposed that only a small portion of apoA-I-containing
particles are active in cholesterol removal from peripheral cells and
transport back to liver, a process known as reverse cholesterol
transport (27). These lipid-poor HDL have pre- electrophoretic
mobility, and HL and CETP are thought to be important in their
formation (8). HL activity has been found to contribute to the
formation of pre-
-migrating HDL as observed in in vitro and liver perfusion experiments (8, 11). However, the distributions of
pre-
-migrating HDL in the plasma of hhee,
HHee, and hhEE mice are similar to that in
wild-type mice. Because hhee mice lack CETP, HL, and apoE,
other enzymes must be sufficient for the formation of pre-
-HDL. One
alternative mechanism to generate these particles involves phospholipid
transfer protein, which is believed to promote fusion of HDL particles,
liberating small lipid-poor particles analogous to
pre-
1-HDL (28). The absence of HL may favor this process
by increasing phospholipid transfer protein substrate. Another possible
mechanism may involve the recently described HDL receptor or scavenger
receptor type BI, which has been reported to be expressed in a variety
of tissues including the liver (29). This receptor mediates selective
uptake of cholesteryl esters without endocytosis, perhaps making apoA-I
available to form smaller particles similar to
pre-
1-HDL.
HL deficiency increased -VLDL cholesterol levels in apoE-deficient
mice. This would be expected to have an atherogenic effect. However,
the female hhee mutants developed smaller atherosclerotic lesions compared with the HHee mutants. No difference was
observed in the small number of males examined. Reduction in plaque
size was also observed in female hhEe mice compared with
HHEe mice when fed an atherogenic diet. Since genetic
background is clearly a factor that contributes to atherosclerosis, it
is important to use experimental and control groups of mice with
comparable genetic backgrounds. The mice used in this study carried
~87% of their genome from the C57BL/6J strain, with the rest being from 129/Ola. Offspring from several families were used to help ensure
that the region from 129/Ola would be randomly distributed. The only
systemic difference between the two groups (HHee and hhee, or HHEe and hhEe) is the region
near the HL gene: this region in hh mice is from 129/Ola,
while in HH mice, it is from C57BL/6J. We cannot therefore
exclude a possibility that some loci linked to HL, but not HL
deficiency itself, are the cause of the protection in hhee
mice. However, since no major loci that affected atherosclerosis are
known to be linked to the HL gene, this systemic difference is unlikely
to have influenced our results.
An increase in -VLDL particles but reduced atherosclerosis was
recently observed in apoE-deficient mice overexpressing human apoA-IV
(30). The reason for these findings is not clear, but they may be the
result of a more favorable balance in extrahepatic cholesterol by
either decreased influx or increased efflux. Although the size of
-VLDL was not directly measured, our data are consistent with an
increase in
-VLDL size. Larger
-VLDL particles have a lesser
ability to enter the artery wall, and their conversion by HL to smaller
particles may increase their atherogenecity, as suggested by previous
studies in rabbits (31, 32). Furthermore, we observed an increase in
HDL cholesterol in hhee mutants and a higher capacity of
hhee mutant HDL to promote cholesterol efflux in
vitro compared with HHee mutant HDL. However,
protection against atherosclerosis in mice may not solely depend on the
capacity to promote cholesterol efflux since male hhee mice
had a higher efflux than female hhee mice, yet did not
appear to display a decrease in atherosclerosis. Further studies
including determination of the sizes of
-VLDL particles in males and
females with HL deficiency are necessary to explain the sex-related
differences.
It is premature to conclude that the inhibition of HL may help reduce formation of atherosclerotic plaques in humans. Physiological differences between the two species, such as the absence of CETP activity in mice and apoB editing in the liver of mice, but not in humans, may influence differently the role of HL in atherogenesis. Consequently, HL deficiency in humans may be associated with premature atherosclerosis (3). In addition, while men are at higher risk for developing atherosclerosis than women, the gender effects appear to be opposite in mice. Previously, we observed that chow-fed female apoE-deficient mice (33) and female heterozygous apoE-deficient mice fed an atherogenic diet (34) develop larger plaques in their aortic sinus area compared with males. Similar observations were made by Paigen et al. (35) in the diet-induced atherosclerosis of inbred C57BL/6J mice. Further investigations in HL-deficient mice may help us to understand the cause of this gender difference.
In conclusion, our study clearly shows that HL is involved in the
catabolism of -VLDL/IDL and HDL by a mechanism independent of apoE.
We found that neither HL nor CETP is essential for the formation of
murine pre-
1-HDL and that HL deficiency is associated with decreased susceptibility to atherosclerosis in female mice despite
higher plasma cholesterol levels. This beneficial effect was associated
with increased cholesterol efflux by HDL from HL-deficient mice.
We thank D. Chappell, M. Hinsdale, S. Quarfordt, H. deSilva, and P. Sullivan for helpful comments on the manuscript. We also thank Julia Tyson, Elena Avdievich, Sunny Zhang, and Shinja Kim for technical help.