 |
INTRODUCTION |
Plasma remnant lipoproteins are the metabolic end products of
intestine-derived chylomicrons and hepatocyte-derived very low density lipoprotein (1-3). Apolipoprotein
(apo)1 E mediates the uptake
of remnants in the liver by binding to the LDL receptor (LDLR), the
LDLR-related protein (LRP), and heparan sulfate proteoglycans (HSPG)
(4-7). Inefficient clearance leads to the accumulation of remnants in
plasma and contributes to premature atherosclerosis (8, 9).
The liver is the major source of plasma apoE; extrahepatic tissues,
primarily macrophages, contribute up to 10% of plasma levels (10).
Hepatocyte-derived apoE has been proposed to promote remnant clearance
and uptake through a two-step process referred to as secretion-capture
(3, 11, 12). In the first step, a portion of newly secreted apoE
interacts with HSPG and remains bound to hepatocyte cell surfaces; the
remainder is released into the space of Disse, where it serves to
enrich lipoproteins (11-13). The presence of apoE on hepatocytes is
thought to promote the trapping, or sequestration, of remnants (6, 11).
In support of this mechanism, distinct dynamic pools of apoE on
hepatocyte cell surfaces have recently been reported in
vitro (14). In the second step, sequestered remnants are further
enriched with hepatocyte-derived apoE and are internalized into
hepatocytes through processes mediated by receptors, including the LDLR
and the LRP (7, 15-19), or through interactions with HSPG alone (11,
20, 21). When apoE/HSPG interactions are disrupted in vivo
by intravenous heparinase infusion, remnant clearance is severely
inhibited, supporting the importance of the secretion-capture role of
apoE in remnant clearance (22, 23).
The importance of hepatically synthesized and localized apoE, and thus
the secretion-capture role of apoE, has recently been questioned (24).
Moreover, evidence from mouse models expressing apoE exclusively from
extrahepatic sources suggests that hepatic apoE expression is not
absolutely required for remnant clearance or for normal plasma
cholesterol levels (25-27). Finally, it is not clear whether hepatic
apoE expression is required for remnant clearance in mice lacking the
LDLR (24, 28, 29).
We previously reported (30) the generation of hypomorphic apoE (hypoE)
mice that express reduced levels of Arg-61 apoE, which is similar to
human apoE4 in that it displays apoE4 domain interaction. The reduction
in apoE levels is caused by a neomycin (neo) cassette
flanked by loxP sites in Apoe intron 3. However, the expression pattern of the hypomorphic allele remains normal, with
the liver producing the majority of apoE in these mice (30). Excision
of the neo cassette by Cre-mediated gene repair restores normal expression of the allele in all tissues (31). Despite having
only 2-5% of wild-type plasma apoE levels, hypoE mice display a
nearly normal lipoprotein profile. However, in
Apoe
/
mice with similar plasma apoE levels
after transplantation with varying amounts of wild-type bone marrow,
remnants accumulate (32). It was not until plasma apoE levels exceeded
10% of wild-type levels that remnant levels approached normal.
The more efficient remnant clearance in hypoE mice suggests the
importance of hepatocyte-derived apoE and the secretion-capture role of apoE.
This study was designed to address the controversy concerning the
importance of both hepatically derived and localized apoE in remnant
clearance in in vivo models. To this end, remnant clearance was assessed in hypoE mice (a model of hepatocyte-derived apoE) and in
Apoe
/
mice expressing similar levels of
Arg-61 apoE after bone marrow transplantation (a model of
non-hepatocyte-derived apoE). Our results demonstrate that remnant
clearance is more efficient in the hepatocyte-derived apoE model and
provide in vivo evidence for the importance of hepatically
derived and localized apoE in remnant clearance.
 |
EXPERIMENTAL PROCEDURES |
Hepatocyte-derived ApoE Mouse Model--
The generation of hypoE
mice, or the hepatocyte-derived apoE model, has previously been
described (30). Briefly, a neo cassette flanked by
loxP sites was inserted into Apoe intron 3 by
gene targeting in embryonic stem cells to help follow the replacement of the human equivalent of Thr-61 by an arginine, aimed at creating a
model of apoE4 (31). The presence of the neo cassette in
Apoe intron 3 results in reduced apoE mRNA levels in all
tissues and organs (30). The apoE mRNA levels in the liver, brain,
and spleen in targeted mice are ~5% of those in wild-type mice,
resulting in plasma apoE levels equal to 0.05-0.12 mg/dl (~2-5% of
wild-type levels) as determined by Western blot analysis using mouse
apoE as standards, with the liver remaining the primary
source of apoE. However, following Cre-mediated excision of the
neo cassette in targeted mice, normal expression of the
Arg-61 Apoe allele is restored. The mice were weaned at 21 days of age, housed in a barrier facility with a 12-h light/12-h
dark cycle, and fed a chow diet containing 4.5% fat (Ralston
Purina, St. Louis, MO).
Non-hepatocyte-derived ApoE Mouse Model--
Mice expressing
Arg-61 apoE primarily from a peripheral source were generated by
transplanting Cre-deleted Arg-61 mouse bone marrow into lethally
irradiated Apoe
/
mice. Bone marrow was
collected by flushing femurs and tibias with RPMI 1640 containing 2%
fetal bovine serum and 10 units/ml heparin (Sigma). Cells were washed,
counted, resuspended in RPMI, and used immediately for transplantation
into lethally irradiated Apoe
/
recipient
mice. The cells (5 × 106 in a volume of 300 µl)
were injected into the tail vein 4 h after irradiation with 900 rads from a cesium gamma source.
Immunohistochemistry--
Mice were fasted for 4 h,
anesthetized with avertin, and flush perfused with PBS, pH 7.2, and 3%
paraformaldehyde in PBS, pH 7.2, at room temperature for 5 min.
Hardened liver lobes were cut into slices 2-3 mm thick and further
fixed overnight by immersion in 3% paraformaldehyde in PBS, pH 7.2, at
4 °C. Slices were briefly washed in PBS, pH 7.2, drained, placed in
molds with Tissue-Tek compound (Sakura Finetek, Torrance, CA), and
frozen in liquid nitrogen. Blocks were held at
70 °C until cut
into 6-8-µm thick sections on a Leica Frigocut 2800 cryostat and
mounted on glass slides.
Slides were immunostained as follows at room temperature unless
otherwise indicated. Sections were incubated in sequence with rabbit
polyclonal anti-mouse apoE antiserum (31) (1:1000) overnight at 4 °C
with biotinylated goat anti-rabbit IgG (Zymed Laboratories Inc., South San Francisco, CA) at 0.4 µg/ml, with
streptavidin-horseradish peroxidase conjugate from a tyramide signal
amplification kit (TSA Fluorescein System, NEL 701, PerkinElmer Life
Sciences) (1:500), and finally with fluorescein tyramide from
the same kit (1:100). Slides were coverslipped after application of
Vectashield anti-fade mounting medium (Vector H-1000, Vector
Laboratories, Burlingame, CA) and imaged by epifluorescence using a
Nikon E600 microscope equipped with a SPOT 2 digital camera (Diagnostic
Instruments, Sterling Heights, MI).
Lipid and Lipoprotein Determination--
Lipids and lipoproteins
were measured in age-matched male mice that had been fasted for 4 h, anesthetized, and bled by retro-orbital puncture. Lipoproteins were
fractionated by fast-performance liquid chromatography (FPLC) on a
Superose 6 column (Amersham Biosciences), and plasma and lipoprotein
fractions were examined by agarose gel electrophoresis (Universal
Gel/8, Helena Laboratories, Beaumont, TX). Cholesterol and triglyceride
levels in plasma and FPLC fractions were determined with colorimetric
assays (Spectrum, Abbott, Irving, TX, and Triglycerides, Roche
Molecular Biochemicals, respectively). The statistical significance of
differences in lipid levels was determined by the Student's
t test.
ApoE and ApoB Quantitation--
Fasted mouse plasma was
subjected to SDS-PAGE with 10-20% or 4-15% gels and transferred to
nitrocellulose. Western blotting was performed with rabbit antiserum
against mouse apoE (31) and apoB (30). Western blots were incubated
with primary antibodies (1:2000), and bound primary antibodies were
detected with a horseradish peroxidase-conjugated anti-rabbit antibody
(Invitrogen). Signals were generated by incubating membranes with
chemiluminescent reagent (Amersham Biosciences) and exposing them to
x-ray film (Kodak, Rochester, NY). Signals were quantified with
phosphorimaging and quantification software (Quantity One,
Bio-Rad).
Remnant Lipoprotein Clearance Study--
Remnant lipoproteins
were prepared from plasma, adjusted to a density of d = 1.04 g/ml, from fasted Apoe
/
mice. Plasma
was centrifuged in a Beckman ultracentrifuge in a TL-100.3 rotor at
80,000 rpm for 16 h at 8 °C. Remnants were isolated and
recentrifuged for two additional 16 h periods at d = 1.04 g/ml to ensure removal of plasma albumin. The purity of the
remnant preparation was examined by SDS-PAGE and staining with
Coomassie Blue, which revealed apoB48 as the major protein component
followed by apoAI; apoB100 was present in trace amounts. Remnants were
labeled with Na125I (Amersham Biosciences) by a
modification of the iodine monochloride method first described by
McFarlane (33). 125I-labeled remnants were
extensively dialyzed against PBS, pH 7.2, and adjusted to a specific
activity of 65,000 cpm/µg protein. Labeled remnants (24 µg of
protein in a volume of 400 µl of PBS) were injected into the tail
vein of recipient mice (wild-type, hepatocyte-derived apoE model,
non-hepatocyte-derived apoE model, and Apoe
/
mice, n = 3/group). Blood (50 µl) was collected from
the retro-orbital venous plexus into heparinized tubes 1, 5, 15, 30, 60, and 180 min after injection of 125I-labeled remnants.
Aliquots of plasma were analyzed for radioactivity on a gamma counter
(Packard). Total counts were calculated assuming that plasma represents
3.5% of total body weight (34). At 180 min, the mice were anesthetized
with avertin and flush perfused with PBS, pH 7.2. Whole livers were
collected, and the amount of 125I in the liver was determined.
 |
RESULTS |
Detection of ApoE on Hepatocyte Cell Surfaces in the
Liver--
The impact of the tissue source of apoE on remnant
clearance was assessed by studying two mouse models expressing low
levels of plasma apoE derived primarily from liver hepatocytes or
primarily from a peripheral source. We first examined liver-associated
apoE in both mouse models. Like wild-type mice, the hepatocyte-derived apoE model had detectable levels of apoE bound to hepatic sinusoidal surfaces, although at reduced levels (Fig.
1). In contrast, and similar to
Apoe
/
mice, the non-hepatocyte-derived apoE
model had little if any detectable apoE bound to hepatic sinusoidal
surfaces. However, unlike Apoe
/
mice, the
non-hepatocyte-derived apoE model had some apoE-immunoreactive cells in
liver sections, which likely represent macrophage-derived Kupfer cells
expressing apoE or cells that have taken up apoE from the
circulation. Thus, the enrichment of apoE on hepatic sinusoidal
surfaces required local apoE expression, present only in the
hepatocyte-derived apoE model.

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Fig. 1.
Localization of apoE in mouse liver.
Livers from wild-type, the hepatocyte-derived apoE model, the
non-hepatocyte-derived apoE model, and Apoe /
mice were perfused with saline, fixed with paraformadehyde, and
prepared for cryosectioning. ApoE was detected by
immunofluorescence and viewed by an inverted light microscope. The
scale bar is 100 µm.
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|
Plasma ApoE Levels in the Hepatocyte-derived and in the
Non-hepatocyte-derived ApoE Models--
Despite having different
levels of liver-associated apoE, both the hepatocyte-derived and the
non-hepatocyte-derived apoE models expressed similarly low levels of
apoE in plasma, corresponding to ~2-5% of wild-type apoE levels in
the hepatocyte-derived apoE model and ~2-fold more in the
non-hepatocyte-derived apoE model (Fig.
2).

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Fig. 2.
Relative levels of apoE in mouse plasma.
Plasma (1 µl) from fasted mice was resolved by SDS-PAGE, and apoE was
detected by Western blotting. Lane 1, recombinant mouse
apoE; lanes 2 and 3, plasma from two
hepatocyte-derived apoE mice; lanes 4 and 5,
plasma from two non-hepatocyte-derived apoE mice.
|
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Plasma Lipid and Lipoprotein Levels in the Hepatocyte-derived and
in the Non-hepatocyte-derived ApoE Models--
The tissue source of
apoE resulted in a marked difference in plasma lipid and lipoprotein
levels. Plasma cholesterol levels in the non-hepatocyte-derived apoE
model were 2-fold higher than those in the hepatocyte-derived apoE
model (230 ± 50 versus 98 ± 18 mg/dl,
n = 6, p = 0.02) and plasma
triglyceride levels were 4-fold higher in the non-hepatocyte-derived
apoE model than in the hepatocyte-derived apoE model (176 ± 27 versus 45 ± 12 mg/dl, n = 6, p = 0.03). Fractionation of mouse plasma showed a
marked difference in the lipoprotein profiles (Fig.
3). The non-hepatocyte-derived apoE mice
transported 60-80% of plasma cholesterol as remnant lipoproteins. In
contrast, the hepatocyte-derived apoE mice transported only 30-40% of
plasma cholesterol as remnant lipoproteins (30), which is more like
wild-type mice that transport the majority of their plasma cholesterol
as high density lipoprotein (17). Thus, the non-hepatocyte-derived apoE
model transports 2-3-fold more remnant lipoprotein-associated
cholesterol and less high density lipoprotein cholesterol than the
hepatocyte-derived model. Agarose gel electrophoresis of mouse plasma
confirmed the more normal lipoprotein profile in the hepatocyte-derived
apoE model and the accumulation of remnants in the
non-hepatocyte-derived model (data not shown).

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Fig. 3.
Plasma lipoprotein profiles of
hepatocyte-derived and non-hepatocyte-derived apoE models. Plasma
(100 µl) from four fasted mice was pooled and fractionated by FPLC.
Fractions corresponding to the remnant and high density lipoprotein and
the distribution of plasma cholesterol levels are indicated.
|
|
The distribution of apoE among the classes of plasma lipoproteins in
both mouse models was determined by pooling plasma FPLC fractions into
lipoprotein classes, followed by Western blotting. As shown in Fig.
4, apoE was present in the remnant
fractions in both mouse models and demonstrates that the distribution
pattern of apoE among plasma lipoprotein classes in both mouse models is identical.

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Fig. 4.
Distribution of apoE in various lipoprotein
classes from hepatocyte-derived and non-hepatocyte-derived apoE
models. Plasma (100 µl) from four fasted mice was pooled and
separated into lipoprotein classes by FPLC: very low density
lipoprotein (fractions 5-8), intermediate density lipoprotein
(IDL) (fractions 9-12), LDL (fractions 13-16), and HDL
(fractions 17-24), see Fig. 3. ApoE was detected by SDS-PAGE Western
blotting.
|
|
SDS-PAGE Western blot analysis of mouse plasma demonstrated that both
models accumulate apoB48. The non-hepatocyte-derived apoE model had
~8-fold more plasma apoB48 than the hepatocyte-derived apoE model,
but both models had similar levels of plasma apoB100, which was
lower than in wild-type mice (Fig. 5).
These results are consistent with the more rapid removal of
apoB48-containing remnants in the hepatocyte-derived apoE model than
the non-hepatocyte-derived apoE model. Taken together, these results
demonstrate that remnant clearance is more effective in the
hepatocyte-derived apoE model than in the non-hepatocyte-derived model
due to hepatically derived and localized apoE.

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Fig. 5.
Relative levels of apoB100 and apoB48 in
mouse plasma. Plasma (1 µl) from fasted mice was resolved by
SDS-PAGE, and apoB was detected by Western blotting. Lane 1,
wild-type mouse plasma; lane 2,
Apoe / mouse plasma; lanes 3 and
4, plasma from two hepatocyte-derived apoE mice; lanes
5-7, plasma from three non-hepatocyte-derived apoE mice.
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Uptake of I125-labeled Remnant Lipoproteins in
Mice--
The importance of the source of apoE synthesis for remnant
uptake was further assessed by measuring the plasma clearance and liver
uptake of 125I-labeled apoE-deficient mouse remnants (Fig.
6A). Although both the
hepatocyte-derived apoE model and the non-hepatocyte-derived apoE model
had an overall delay in remnant clearance relative to wild-type mice,
only the hepatocyte-derived apoE model reached wild-type levels at
3 h. Moreover, the livers of the hepatocyte-derived apoE model
(n = 3) contained 70 ± 5% of the total
radioactivity found in wild-type mouse livers, whereas the livers of
the non-hepatocyte-derived apoE model (n = 3) contained
only 38 ± 4% after 3 h.

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Fig. 6.
Clearance of apoE-deficient remnants in
mice. A, 125I-labeled apoE-deficient mouse
remnants were injected into mice via the tail vein, and blood samples
were collected at various time points. The presence of remnants in
plasma is presented as the percentage of the injected dose remaining in
the plasma. Values represent the mean ± S.D. of three mice at
each time point. B, the same experiment was performed in the
hepatocyte-derived apoE model as well as in
Apoe / and wild-type mice.
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|
Contrary to wild-type mice, the initial rates of remnant clearance in
the hepatocyte-derived apoE and apoE null mice were identical,
indicating that the initial rates in these models were not affected by
the levels of hepatically localized apoE or by the number of apoE
molecules per remnant (Fig. 6, A and B). Rather, the difference in the rate of remnant clearance between the two models
was apparent only at the later time points diverging after the first 30 min. These results demonstrate that apoE synthesized by liver
hepatocytes favors an overall greater capacity for liver-mediated remnant uptake than apoE synthesized by extrahepatic tissue. Moreover, these results support a role for apoE-enrichment of remnants
sequestered at the surface of hepatocytes and are consistent with the
plasma lipoprotein profiles of fasted mice that show a significant
accumulation of remnants in the non-hepatocyte-derived apoE model, but
not in the hepatocyte-derived apoE model (Fig. 3).
 |
DISCUSSION |
This study presents in vivo evidence that demonstrates
that the site of apoE synthesis significantly influences remnant
clearance and, consequently, plasma cholesterol and triglyceride
levels in mice. Hepatically derived and localized apoE in the
hepatocyte-derived apoE model was found to be more effective than
macrophage-derived apoE in the non-hepatocyte-derived apoE model in
promoting the plasma clearance and liver uptake of apoB48-containing
remnants. Moreover, the low levels of hepatically derived and localized apoE in the hepatocyte-derived apoE model influenced the late but not
the initial part of remnant clearance.
Because the hepatocyte-derived apoE model expresses Arg-61 apoE, the
non-hepatocyte-derived apoE model was generated by using Cre-deleted
Arg-61 bone marrow to allow for a direct comparison with the
hepatocyte-derived apoE model. In these mice, Cre-mediated excision of
the neo cassette in Apoe intron 3 results in
normal apoE expression levels in all tissues, including macrophages
(31). The normal lipid and lipoprotein levels in Cre-deleted Arg-61 mice suggest that Arg-61 apoE and wild-type apoE are equally effective in remnant clearance (31).
Immunohistochemical analysis of liver sections demonstrated that the
hepatocyte-derived apoE model contained significant levels of
hepatically localized apoE, whereas the non-hepatocyte-derived apoE
model did not. The hepatically localized apoE in the hepatocyte-derived apoE model likely originated mostly from hepatocytes, as the expression pattern of the hypomorphic Apoe allele in the
hepatocyte-derived apoE model is normal, with the liver producing the
majority of apoE in these mice (30). However, we cannot exclude the
possibility that some of the hepatically localized apoE in the
hepatocyte-derived apoE model originated in the periphery.
The higher plasma lipid and lipoprotein levels in the
non-hepatocyte-derived apoE model relative to those in the
hepatocyte-derived apoE model, despite similarly low apoE plasma
levels, is consistent with a more efficient clearance of remnants in
the hepatocyte-derived model. The non-hepatocyte-derived apoE model
accumulated 2-fold more plasma cholesterol and 4-fold more plasma
triglycerides than the hepatocyte-derived apoE model. The 8-fold
greater accumulation of apoB48-containing remnants in the
non-hepatocyte-derived apoE model is also consistent with the site of
apoE synthesis influencing remnant clearance, despite both models
having similar levels of plasma apoE. In the hepatocyte-derived apoE
model, low levels of hepatic apoE synthesis overcome to a significant
degree the deficiency in remnant clearance seen in the
non-hepatocyte-derived apoE model. In contrast to wild-type mice and
similar to Apoe
/
mice, both models had
equally low levels of plasma apoB100. The low levels of plasma apoE in
the two models apparently failed to sufficiently enrich
apoB48-containing remnants with apoE, causing them to become poor
competitors with apoB100-containing LDL for binding to the LDLR.
Alternatively, the reduced levels of apoB100 in both models may result
from decreased apoB100 secretion by the liver. However, hepatic apoE
expression has been reported to influence the production rate of both
apoB48 and apoB100 in mice (35). The finding that the levels of apoB48
are far greater in the non-hepatocyte-derived apoE model than in the
hepatocyte-derived apoE model, emphasizes the importance of hepatically
derived and localized apoE in remnant clearance, as the
non-hepatocyte-derived apoE model likely secretes reduced levels of
apoB48, similar to Apoe
/
mice (35). Thus, a
direct assessment of apoB secretion rates in both models will be
necessary to confirm this possibility. Lastly, remnant clearance in
both models may have been influenced by the recently described
recycling pathway of apoE in liver hepatocytes (36). However, as only
6% of internalized apoE was reported to be re-secreted by hepatocytes,
the pathway likely plays a minor role in mediating remnant clearance in
our models that have very low levels of plasma apoE.
Directly comparing remnant clearance in both the hepatocyte-derived and
non-hepatocyte-derived apoE models confirmed the conclusions drawn from
the steady-state plasma lipid and lipoprotein levels, which indicate
that hepatocyte-derived apoE is more effective than
non-hepatocyte-derived apoE in remnant clearance. When compared with
wild-type mice, the initial phase of remnant clearance was similarly
reduced in both mouse models and comparable with apoE null mice. As the
initial phase likely reflects the binding and sequestration of remnants
on hepatocyte cell surfaces in the space of Disse, the levels of apoE
molecules per remnant as well as the levels of hepatically localized
apoE in the hepatocyte-derived apoE model do not appear to contribute
significantly to this process. Moreover, the differences in lipoprotein
pool sizes in the models did not affect the rapid initial clearance of
remnants. However, the near normal level of remnant clearance after
3 h only in the hepatocyte-derived apoE model, suggests that
hepatically derived and localized apoE contribute substantially to a
slower component of remnant clearance, which likely represents
liver-uptake. Indeed, livers from the hepatocyte-derived apoE model
contained 70 ± 5% of normal 125I levels, whereas
livers from the non-hepatocyte-derived apoE model contained only
38 ± 4%.
In the hepatocyte-derived apoE model, apoE-poor remnants sequestered on
hepatocytes likely become enriched with newly secreted apoE in the
space of Disse or by the passive exchange from existing hepatically
localized apoE, allowing for accelerated receptor-mediated internalization through the LDLR and the LRP or through HSPG
alone. However, in the non-hepatocyte-derived apoE model, a large
proportion of sequestered apoE-poor remnants likely redistribute to and
accumulate in the circulation due to the absence of apoE-enrichment in
the space of Disse and inefficient receptor-mediated uptake.
Studies of remnant clearance in an isolated mouse liver perfusion model
have recently questioned the importance of hepatic synthesis and
localization of apoE and thus the importance of the secretion-capture
role of apoE in remnant clearance (24). Livers from
Apoe
/
mice cleared an infused bolus of
apoE-containing rat chylomicron remnants as efficiently as those from
wild-type mice, which, unlike livers from
Apoe
/
mice, had abundant levels of apoE
localized on hepatocyte cell surfaces (24). More recent data from this
model suggested that apoE/HSPG interactions on hepatocyte cell surfaces
are not required for efficient remnant uptake (29). Rather, apoE/LRP
interactions were proposed to mediate the direct sequestration and
internalization of remnants.
Interestingly, and in parallel to the results observed in the mouse
liver perfusion model (24), the absence of hepatically localized apoE
in both the non-hepatocyte-derived apoE model and in
Apoe
/
mice did not influence the early phase
of remnant clearance relative to the hepatocyte-derived apoE model.
Indeed, all three of these mouse models displayed similarly delayed
initial rates of remnant clearance relative to wild-type mice. However,
a shortcoming of the liver-perfusion model is that the results cannot
necessarily be extrapolated to explain the steady-state levels of
remnants in mice. For example, in apoE null mice, the perfused liver
effectively cleared an infused bolus of remnants, whereas at
steady-state the mice accumulate remnants in plasma. In contrast, our
in vivo models directly focus on the steady-state levels of
remnants in mice.
In conclusion, this in vivo study in mice expressing apoE
from hepatic versus extrahepatic sources demonstrates and
underscores the importance of hepatically derived and localized apoE
for efficient remnant clearance by the liver. Because remnant clearance
operates close to its sub-optimal level in the hepatocyte-derived apoE model, the model should be informative in revealing the relative contributions of the LDLR and the LRP in remnant clearance in the
context of low levels of plasma apoE. The hepatocyte-derived apoE model
can also serve to determine the contribution of other proteins known to
be ligands for remnant clearance such as hepatic lipase (37, 38).