(Received for publication, October 24, 1995; and in revised form, January 16, 1996 )
From the
We have used the technique of adenovirus-mediated gene transfer
to study the in vivo function of the very low density
lipoprotein receptor (VLDLR) in low density lipoprotein receptor (LDLR)
knockout mice. We generated a replication-defective adenovirus
(AdmVLDLR) containing mouse VLDLR cDNA driven by a cytomegalovirus
promoter. Transduction of cultured Hepa (mouse hepatoma) cells and
LDLR-deficient CHO-ldlA7 cells in vitro by the virus led to
high-level expression of immunoreactive VLDLR proteins with molecular
sizes of 143 kDa and 161 kDa. Digestion of the cell extract with the
enzymes neuraminidase, N-glycanase, and O-glycanase
resulted in the stepwise lowering of the apparent size of the 161-kDa
species toward the 143-kDa species. LDLR (-/-) mice fed a
0.2% cholesterol diet were treated with a single intravenous injection
of 3 10
plaque-forming units of AdmVLDLR. Control
LDLR (-/-) mice received either phosphate-buffered saline
or AdLacZ, a similar adenovirus containing the LacZ cDNA instead of
mVLDLR cDNA. Comparison of the plasma lipids in the 3 groups of mice
indicates that in the AdmVLDL animals, total cholesterol is reduced by
50% at days 4 and 9 and returned toward control values on day 21.
In these animals, there was also a
30% reduction in plasma
apolipoprotein (apo) E accompanied by a 90% fall in apoB-100 on day 4
of treatment. By FPLC analysis, the major reduction in plasma
cholesterol in the AdmVLDLR animals was accounted for by a marked
reduction in the intermediate density lipoprotein/low density
lipoprotein (IDL/LDL) fraction. Plasma VLDL, IDL/LDL, and HDL were
isolated from the three groups of animals by ultracentrifugal
flotation. In the AdmVLDLR animals, there was substantial loss
(
65%) of protein and cholesterol mainly in the IDL/LDL fraction on
days 4 and 9. Nondenaturing gradient gel electrophoresis indicates a
preferential loss of the IDL peak although the LDL peak was also
reduced. When
I-IDL was administered intravenously into
animals on day 4, the AdmVLDLR animals cleared the
I-IDL
at a rate 5-10 times higher than the AdLacZ animals.
We conclude that adenovirus-mediated transfer of the VLDLR gene induces high-level hepatic expression of the VLDLR and results in a reversal of the hypercholesterolemia in 0.2% cholesterol diet-fed LDLR (-/-, mice. The VLDLR overexpression appears to greatly enhance the ability of these animals to clear IDL, resulting in a marked lowering of the plasma IDL/LDL. Further testing of the use of the VLDLR gene as a therapeutic gene for the treatment of hypercholesterolemia is warranted.
Lipoprotein metabolism and cholesterol homeostasis are mediated
in part by receptor-mediated uptake of lipoproteins from the
circulation via specific cell-surface receptors. The very low density
lipoprotein receptor (VLDLR) ()is the most recently
identified apolipoprotein (apo) E receptor that belongs to the low
density lipoprotein receptor (LDLR) gene family. This gene family
includes LDLR,
-macroglobulin receptor/LDLR-related
protein, and glycoprotein 330, which all share some common structural
features including: 1) cysteine-rich repeats consisting of
40
amino acid residues in the ligand binding domain or in complement-type
domain; 2) epidermal growth factor precursor-type repeats; 3) modules
of
50 amino acid residues with a consensus tetrapeptide, YWTD; 4)
a single transmembrane domain; and 5) a cytoplasmic domain containing
an NPXY sequence, thought to be important for the clustering of the
receptor into coated
pits(1, 2, 3, 4) . Structurally, the
VLDLR is most similar to the LDLR. A major difference appears to be
that the VLDLR N-terminal domain contains eight, instead of seven,
cysteine-rich repeats as in LDLR. The similarities in the structure of
VLDLR and LDLR are reflected in their genomic organization(5) .
The VLDLR is highly expressed in heart, muscle, and adipose tissue that are active in fatty acid metabolism. Interestingly, the amount of VLDLR mRNA in the liver is very small(3, 6, 7, 8, 9, 10) , although the liver is a primary tissue for clearance of circulating lipoproteins. The tissue-specific expression of the VLDLR is very similar to, but not identical with that of lipoprotein lipase. Lipoprotein lipase resides on the endothelial surface of capillaries and hydrolyzes triglycerides of circulating triglyceride-rich lipoproteins such as VLDL, intermediate density lipoprotein (IDL), and chylomicrons, thus playing a pivotal role in lipoprotein metabolism. Given the structural features and ligand specificity of VLDLR, and the tissue distribution of the VLDLR mRNA, it was hypothesized that the primary role of the VLDLR is the delivery of triglycerides in triglyceride-rich apoE-containing lipoproteins to extrahepatic tissues for energy source or storage(3, 11) .
Apart from
its postulated physiological role, the VLDLR has been implicated in the
pathogenesis of atherosclerosis. Incubation of -VLDL with
LDLR-deficient CHO-ldlA7 cells transfected with rabbit VLDLR enabled
these cells to accumulate cholesteryl ester resulting in foam cell
formation(12) . However, in human monocytic leukemia cell line
THP-1 and rabbit resident alveolar macrophages, the mRNA level of VLDLR
was not affected by incubation with
-VLDL(5, 9, 12) . Macrophages secrete
apoE and lipoprotein lipase, which might facilitate cellular uptake of
VLDL(13) . However, although arterial smooth muscle cells
express VLDLR mRNA, human monocyte-derived macrophages do not have any
detectable VLDLR mRNA. Therefore, the physiological and possible
pathological role of this member of the LDLR gene family remain to be
elucidated.
As a first step toward understanding the functional role of the VLDLR in vivo, we have examined the effect of adenovirus-mediated transfer of the VLDLR gene in LDLR knockout mice fed a 0.2% cholesterol diet. Since these mice do not express LDLR, changes in lipoprotein metabolism following experimental perturbations cannot be attributed to changes in LDLR expression, a fact which simplifies our interpretation of the experimental data. We have selected the in vivo administration of a VLDLR adenoviral vector as the method for gene delivery because previous experiments using similar techniques have resulted in the transgene being delivered preferentially to the liver where it is expressed at a high level(14, 15, 16) . Since the normal liver expresses little VLDLR, we can readily monitor the changing level of VLDLR mRNA and protein expression in this organ. Finally, if VLDLR gene transfer is proven to be effective in lowering plasma cholesterol in cholesterolfed LDLR (-/-) mice, it provides a possible alternative form of gene therapy for the treatment of familial hypercholesterolemia.
pAvCvSv was constructed
by addition of promoter and polyadenylation signal in pXCJL-1. Human
cytomegalovirus promoter and a translation enhancer sequence which acts
by decreasing the requirements for initiation factors in protein
synthesis was inserted upstream of multiple cloning sites, and the SV40
polyadenylation signal was added downstream of multiple cloning sites.
The recombinant adenovirus was prepared by cotransfection of pAvCvSv
containing full-length mouse VLDLR cDNA (10) and pJM17 (17) into 293 cells(18) . In brief, a 3.0-kilobase cDNA
encoding mouse VLDLR was subcloned into the blunt-ended BglII
site of pAvCvSv, and the resulting shuttle plasmid (10 µg) was
cotransfected with 10 µg of supercoiled pJM17 (Microbix Biosystems
Inc.) into 293 cells plated onto a 60-mm culture dish the day before
transfection at a density of 2 10
cells/dish by
calcium phosphate coprecipitation method using a kit (Promega). Two
weeks after transfection, infectious recombinant adenoviral vector
plaques were picked, propagated, and screened for VLDLR sequences by
polymerase chain reaction. Adenoviral vectors that contained VLDLR were
purified on 293 cells.
Large scale production of high titer
recombinant adenovirus was performed as described(16) . AdLacZ
contained -galactosidase cDNA instead of the mouse VLDLR cDNA.
Recombinant adenovirus
stock was diluted with phosphate-buffered saline (PBS) to the
appropriate concentration, and 0.5 ml of diluted recombinant adenovirus
was injected via tail vein. Following adenoviral transduction, animals
were fasted for 6 h before blood was collected in a tube containing
EDTA by puncturing the retro-orbital plexus. Plasma was stored at 4
°C prior to lipid analysis or lipoprotein fractionation. At the
times indicated, animals were anesthetized and killed by cervical
dislocation. Prior to turnover studies, animals were fasted for 6 h and
anesthetized by intraperitoneal Avertin injection. The external jugular
vein was laid open by a skin incision and the indicated I-lipoproteins were slowly injected in a total volume of
200 µl. The wound was closed by stapling. Blood (50-100
µl) was obtained at the indicated times by puncture of the
retro-orbital plexus from the anesthetized animals and collected into
EDTA-treated Pasteur pipettes. Turnover studies were performed as
described by Ishibashi et al.(15) . For turnover
studies of VLDL, IDL, and LDL, plasma collected was diluted to 0.25 ml
with saline containing human LDL (0.5 mg/ml) as carrier, and 0.25 ml of
isopropyl alcohol was added to precipitate apoB. For turnover studies
of HDL, the plasma content of trichloroacetic acid-precipitable
I radioactivity was measured(20) .
Apolipoprotein B-48 and
B-100 were analyzed by quantitative scanning densitometry of Coomassie
Blue R-250 (Bio-Rad)-stained polyacrylamide gels. ApoB-containing
lipoproteins were isolated by density gradient ultracentrifugation
(Beckman 42.2Ti rotor, 40,000 rpm, 10 °C, 8 h) after adjusting
25-µl plasma samples to a density of 1.063 g/ml with a KBr solution (d = 1.35 g/ml) in tubes containing a KBr overlay
solution (d = 1.063 g/ml). The top 50-µl
lipoprotein fractions were dialyzed against SalEN and enzymatically
assayed for cholesterol. Cholesterol recoveries were compared to VLDL
+ LDL cholesterol measurements of identical plasma samples treated
with polyethylene glycol(28) . For analysis, purified
lipoproteins (2-10 µg of cholesterol) were solubilized in SDS
sample buffer at 60 °C for 30 min. ApoB-48 and -B-100 were resolved
in a 2-20% linear polyacrylamide gel containing dilutions of
purified mouse -VLDL previously quantified for apoB-48 and
apoB-100 protein content by densitometry to purified human LDL
apoB-100. Scanned peak areas in the range of 0.125 to 2.00 µg were
linear (r = 0.989, n = 12), and
chromogenicities were similar for both proteins.
Figure 1: Immunoblots of extracts of cultured cells infected with AdmVLDLR. A, Hepa, a mouse hepatoma cell line. B, CHO-ldlA7, an LDLR-deficient cell line. C, effect of digestion with N-glycanase, neuraminidase, and O-glycanase on the mobility of immunoreactive mVLDLR on SDS-polyacrylamide gel. For A and B, the amount of virus used is as indicated for individual lanes. For C, doses of 50 pfu/cell and 200 pfu/cell were used for Hepa and CHO-ldlA7, respectively. In cell cases, cells were harvested 24 h after infection, and membranes were prepared as described under ``Experimental Procedures.''
Figure 2:
Analysis of LDLR (-/-) mouse
liver DNA and RNA following AdmVLDLR administration. A,
Southern blot at various times after injection of PBS (Mock), AdLacZ,
or AdmVLDLR (3 10
pfu). *, endogenous mVLDLR
genomic band;**, AdmVLDLR-specific band. B, Northern blot of
the same animals.
To determine whether the transduced mVLDLR mRNA was efficiently translated into VLDLR protein, we performed immunoblot analysis of hepatic membrane preparations from mice treated with AdmVLDLR at days 4, 9, and 21 following virus administration. Control samples consist of preparations from day 4 Mock (PBS-injected) and AdLacZ-injected mice. Twenty µg of total membrane protein was separated on a 7.5% SDS-polyacrylamide gel and transferred to an Optitran membrane, and immunoreactive VLDLR protein was detected by chemiluminescence (see ``Experimental Procedures''). As shown in Fig. 3, PBS-treated and AdLacZ-treated day 4 samples failed to show the immunoreactive VLDLR bands, whereas in the AdmVLDLR-treated animals, there was a very high level expression of the VLDLR bands in the day 4, and to a lesser extent, in the day 9 samples. Even on day 21, when the VLDLR bands had largely disappeared, they were still somewhat higher than the control samples. Some lower molecular weight bands were also detected in the day 4 samples which account for <1% of the total and were not investigated further.
Figure 3:
Immunoblot of LDLR (-/-) mouse
liver membrane preparations following AdmVLDLR administration. Animals
were injected intravenously with PBS (Mock), AdLacZ, or AdmVLDLR (3
10
pfu). Membranes were prepared from the liver on
the days indicated and processed for immunoblot analysis as described
under ``Experimental
Procedures.''
Figure 4: FPLC profiles of total plasma cholesterol in LDLR (-/-) mice 4 days after receiving PBS (Mock), AdLacZ (LacZ), or AdmVLDLR (VLDLR). Values are mean ± S.D. The first peak (fractions 5-8) represents VLDL, second peak (fractions 11-25), mainly IDL/LDL, and third peak (fractions 30-38), HDL.
Figure 5: Profiles of IDL/LDL fractions from control, AdLacZ, and AdmVLDLR mice at days 4, 9, and 21 obtained from nondenaturing gradient gel scans. The particle sizes, in nanometers, of major peaks and shoulders are indicated by the numbers over these components. Equal volumes of each fraction were applied to the gel. Note that IDL/LDL from the AdmVLDLR mice at days 4 and 9 appears to have diminished mass compared with controls. It is also apparent that the IDL component (30-36 nm) compared with the LDL component (25-29 nm) is dramatically reduced at days 9 and 21.
In a separate experiment, IDL (d 1.006-1.019 g/ml) and LDL (d 1.019-1.063 g/ml) were isolated from LDLR (-/-) mice ultracentrifugally to establish the size boundaries for each. The nondenaturing gradient gel scan of each fraction (Fig. 6A) indicates that there is little or no overlap of particle size between these two fractions. IDL banded between 30 and 35 nm, whereas LDL had a major component at approximately 28-29 nm and minor components between 25 and 27 nm. The apolipoprotein composition of the IDL and LDL fractions is shown in Fig. 6B where it is apparent that apoB-100, apoB-48, and apoE are the major proteins of each; however, the gel also indicates that apoB-100 is more pronounced in the LDL fraction than in the IDL fraction.
Figure 6: The nondenaturing gradient gel profiles of ultracentrifugally separated IDL (d 1.006-1.019 g/ml) (solid line) and LDL (d 1.019-1.063 g/ml) (dashed line) are seen in A, while the corresponding SDS-PAGE of the fractions are seen in B. The IDL encompass particles in the size range of 30-35 nm and have little or no overlap with the LDL particles which band between 25 and 29.5 nm. The LDL are more heterogeneous in particle size with approximately 5 components in evidence than the IDL which possess approximately 3 distinct components. The IDL and LDL fractions were electrophoresed on SDS-PAGE to determine protein composition of the particles; 5 µg of protein was applied to each lane. The major proteins in each fraction were apoB-100, apoB-48, and apoE; however, it appears that the LDL fraction has relatively more apoB-100 compared to apoB-48 than the IDL fraction. The LDL fraction also contains minor amounts of apoA-I and small molecular mass proteins.
The major VLDL species in control mice is a particle which peaks at 38-39 nm (Fig. 7); the size and distribution were little altered in the AdLacZ-treated mice. AdmVLDLR-treated mice have a major peak in this region, but at day 4 and 9 this fraction also possesses a small quantity of smaller sized particles seen as a shoulder at 35 nm. At these time points, there is also an apparent decrease in total mass of VLDL in the AdmVLDLR-treated mice. At day 21, the VLDL profile of the AdmVLDLR-treated mice, with the appearance of an intermediate component at 37.7 nm, begins to show signs of reversing to that of AdLacZ-treated or control mice.
Figure 7: Profiles of VLDL fractions from control, AdLacZ, and AdmVLDLR mice at days 4, 9, and 21 obtained from nondenaturing gradient gel scans. The numbers over the peaks and shoulders indicate particle size in nanometers; an equal volume of each fraction was applied to the gel. It is apparent that in all cases the major VLDL particle is approximately 38-39 nm in diameter. In the AdmVLDLR-treated mice, there is a decrease in mass of the 39 nm component compared to controls and there is a concomitant appearance of a smaller sized particle at 35 nm. At day 21, it appears that the VLDL profile may be normalizing since a larger particle at 37.7 nm is also present.
Unlike the less dense lipoproteins, HDL size distribution, as determined by nondenaturing gradient gel electrophoresis, is not altered by the expression of the VLDL receptor. The HDL for control, AdLacZ, and AdmVLDLR mice are homogeneous in size and have peaks at 10.2, 10.3, and 10.0 nm, respectively (data not shown).
Figure 8:
Disappearance of I-lipoproteins from the circulation of LDLR
(-/-) mice 4 days after treatment with 3
10
pfu of AdLacZ (open circles) or AdmVLDLR (closed
circles). A, disappearance of
I-VLDL and
I-IDL. B, disappearance of
I-LDL
and
I-HDL. Each curve represents a separate
animal.
The physiological role of the VLDLR in normal lipoprotein metabolism is unclear. Recently, Frykman et al.(30) showed that mice with targeted disruption of the VLDLR gene have normal plasma lipoproteins. Homozygous VLDLR (-/-) mice had normal lipids and lipoproteins when they were fed normal, high carbohydrate, or high fat diets. Therefore, if the VLDLR plays a significant role in lipoprotein metabolism, its role must be subtle and can be filled by adaptation in these VLDLR (-/-) animals.
To date, the ligand-binding function of
the VLDLR has been tested only in in vitro systems using
transfected cells(3, 13, 31) . Although
non-lipoprotein ligands have been
identified(24, 31, 32) , most of the studies
have concentrated on identifying possible lipoprotein ligands. These
experiments demonstrate that the VLDLR recognizes apoE-containing
lipoproteins. VLDL artificially enriched with apoE in vitro(13) as well as diet-induced apoE-rich -VLDL (3) appear to serve as effective ligands for VLDLR-expressing
cells in culture. In contrast, apoB-100-only LDL is poorly recognized
by these cells(3, 11) . The gene transfer experiments
described in this study allow us to determine what lipoprotein ligands
are recognized by the VLDLR in vivo.
Transfer of the mVLDLR gene to cultured mouse hepatoma cells and LDLR-deficient CHO-ldlA7 cells induced the expression of two species of immunoreactive VLDLR (Fig. 1). By selective glycosidase digestion, we showed that the high molecular mass (161 kDa) species represents a fully processed glycosylated and the low molecular mass (143 kDa) species, the unglycosylated mVLDLR. These results corroborate and extend the observations of Jokinen et al.(8) in rats.
We have chosen LDLR (-/-) mice as the experimental model for a number of reasons. The LDLR knockout mice are a useful model of familial hypercholesterolemia(15) . When they are put on a 0.2% cholesterol diet, they develop significant hypercholesterolemia involving mainly the IDL/LDL fraction(15) . In these animals, any effect of gene transfer on the plasma lipoproteins can be attributed to VLDLR expression because the animals do not express LDLR.
We used the technique of adenovirus-mediated gene transfer to
deliver the VLDLR to the liver of LDLR (-/-) mice. In
agreement with previous experiments from this laboratory (16) and other laboratories(14, 15) , most of
the transgene ends up in the liver. The mouse liver normally expresses
very low amounts of VLDLR(7, 10) . Within 4 days of in vivo gene transfer, Western blots (Fig. 3) indicate
that VLDLR was expressed at a high level in the liver. This was
accompanied by a lowering in the plasma apoE concentration, an
observation consistent with apoE-enriched lipoproteins being the
preferred ligand for the VLDLR. Interestingly, there was an even more
pronounced drop in the plasma level of apoB-100. Since apoB-100-only
lipoproteins are poorly recognized by the VLDLR, the clearance of
apoB-100 must involve particles that contain both apoB-100 and apoE.
The fact that the decrease in apoB-100 was substantially greater than
that of apoE suggests that a significant portion of apoE was in a form
(in lipoprotein particles or as the free soluble protein) not
recognized by the VLDLR, or a high production rate of apoE relative to
apoB-100 was able to partially compensate for its greatly enhanced
clearance. In any case, the reduction in apoE and apoB-100 was
accompanied by an 50% fall in plasma cholesterol. The plasma
lipoprotein fraction most affected by the treatment was in the IDL/LDL
class, which lost much of its protein and cholesterol content in
response to treatment (Table 3).
The size distribution of the
IDL/LDL on day 4 was very informative (Fig. 5). There was a
selective lowering of IDL (30-35 nm range), which suggests that
IDL is the preferred ligand for the VLDLR. This interpretation was
supported by the I-lipoprotein disappearance curves (Fig. 8). In comparing AdmVLDLR- and AdLacZ-treated animals, the
greatest difference in clearance rate of the injected
I-labeled lipoprotein was with IDL. The VLDLR animals
cleared the
I-IDL at least 5-10-fold faster than
the LacZ animals. There was no difference between these animals in
their ability to clear
I-HDL and only minor differences
in their ability to clear
I-VLDL. We believe that the
slightly increased clearance of
I-LDL in two of the five
animals tested (Fig. 8B, left panel) was
possibly caused by contamination of the LDL by IDL. The LDL preparation
used for the study contained significant amounts of apoE which could
not be removed by repeated ultracentrifugal flotation (Fig. 6).
The animal that had the fastest clearance of the injected
I-LDL also expressed the highest amount of VLDLR in the
liver as determined by Western blot analysis.
In vitro binding experiments using cells transfected with VLDLR indicate the importance of apoE in receptor recognition. We have extended these observations by demonstrating that among the apoE-containing lipoproteins, IDL appears to be taken up most avidly in vivo. Since IDL is the precursor of LDL, the LDL level is also lowered in the AdmVLDLR-treated LDLR (-/-) animals (Fig. 5; note that equal amounts of protein were loaded on these gels and about 3-fold more of the lipoprotein fraction had to be loaded on the gel for the AdmVLDLR sample).
Our observations raise the possibility that the VLDLR may be an effective therapeutic gene for the treatment of hypercholesterolemia. We have shown here that VLDLR gene transfer to the liver enables the recipient animal to efficiently remove atherogenic IDL/LDL particles from the circulation. Use of this gene is especially attractive for the treatment of familial hypercholesterolemic patients who have total absence of the LDLR or who have major abnormalities in their LDLR. These patients are likely to be resistant to the usual hypolipidemic agents. Replacement of the normal LDLR gene in these patients may appear to be a rational approach and has been found to be efficacious in experimental animals in the short term(15, 33, 34) . However, there are theoretical drawbacks to LDLR gene transfer that may be circumvented by the use of the VLDLR gene instead. The normal LDLR might be recognized as a foreign protein in LDLR-deficient patients and its expression on the liver cell surface could evoke an immunological response which may eventually cause the inactivation of the LDLR protein. The VLDLR, in contrast, is nonimmunogenic, because VLDLR is normally expressed in various tissues of the LDLR (-/-) patient and is not recognized by the host immune system as a foreign protein. Additional experiments comparing the long-term expression of LDLR and VLDLR in LDLR (-/-) animals, and eventually in LDL (-/-) patients, will be needed to determine if this is a valid concern. In any case, VLDLR appears to be a good alternative to LDLR in the treatment of hypercholesterolemia. Further testing of its use in somatic gene therapy is warranted.