© 1995 American Heart Association, Inc.
Articles |
Phenotypic Correction of Hypercholesterolemia in ApoE-Deficient Mice by Adenovirus-Mediated In Vivo Gene Transfer
From the Department of Molecular and Cell Biology, Genetic Therapy Inc, Gaithersburg, Md.
Correspondence to Alan McClelland, Department of Molecular and Cell Biology, Genetic Therapy, Inc, 938 Clopper Rd, Gaithersburg, MD 20878.
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Abstract |
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Abstract To investigate the potential use of apoE in gene therapy of hyperlipidemias, an adenoviral vector was constructed that contained the human apoE3 cDNA under the control of the RSV promoter (Av1RE). Transduction of HepG2 cells resulted in the overexpression of human apoE secreted into the culture medium. Intravenous injection of 5x1011 Av1RE vector particles into apoE-deficient mice resulted in expression of human apoE3 in mouse plasma at levels of 1.2±0.4 µg/mL (mean±SEM, n=5) 7 days after injection. Mice injected with the control vector Av1Lacz4 did not express detectable levels of human apoE. Average plasma cholesterol concentrations were reduced approximately eightfold from 737.5±118 mg/dL (mean±SEM, n=6) to 98.2±4.4 mg/dL (mean±SEM, n=5) and were unaffected in the control vector group. Expression of human apoE resulted in a shift in the plasma lipoprotein distribution from primarily VLDL and LDL in the control mice to predominantly HDL in the Av1RE-treated group. Western blot analysis of fast protein liquid chromatography–fractionated mouse plasma showed that the human apoE protein was associated with VLDL, LDL, and HDL. Correction of the hyperlipidemic condition found in the apoE-knockout mouse strain by direct in vivo gene transfer establishes the potential of this approach for treatment of hyperlipidemia caused by apoE deficiency or malfunction in human disease.
Key Words: atherosclerosis • apolipoprotein E • gene therapy • lipoproteins
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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ApoE is a component of several plasma lipoproteins, including chylomicrons, VLDL, and HDL. Receptor-mediated catabolism of these lipoprotein particles is mediated through the interaction of apoE with the LDL receptor (LDLR) or with the LDLR-related protein (LRP).1 2 Injection of exogenous apoE into normal and Watanabe heritable hyperlipidemic (WHHL) rabbits resulted in a decrease in plasma cholesterol concentrations.3 4 These studies demonstrated that elevation of apoE levels in plasma results in the increased clearance of lipoproteins from the circulation. However, the cholesterol-lowering effect resulting from the intravenous injection of apoE was transient, lasting approximately 20 hours. In apoE-transgenic mice, stable overexpression of apoE resulted in a sustained reduction of plasma cholesterol concentrations and a resistance to dietary elevation of plasma cholesterol concentrations.5 Kinetic studies of VLDL, LDL, and chylomicron uptake in apoE-transgenic mice showed that overexpression of apoE enhanced the clearance of these lipoproteins from the circulation.6 7 Taken together, these studies support the hypothesis that apoE overexpression will reduce plasma cholesterol and/or triglyceride concentrations by increasing the clearance of plasma lipoproteins from the circulation.
ApoE-deficient mice are severely hypercholesterolemic, with average plasma cholesterol concentrations of 400 to 800 mg/dL on a regular chow diet.8 9 10 11 Triglyceride levels in these mice are not severely elevated compared with those in control animals.8 These mice also develop atherosclerotic lesions at approximately 11 weeks of age with the appearance of foam cells that progress to a more involved, complex lesion consisting of cholesterol clefts and fibrous caps.10 The severe hypercholesterolemia found in apoE-deficient mice and the profound effect on lipoprotein metabolism resulting from the deletion of the apoE gene demonstrate that apoE plays a key role in the receptor-mediated clearance of plasma lipoproteins through its interaction with either the LDLR or the LRP.
The generation and characterization of the apoE-deficient mouse strain provide a useful animal model to test various treatments for hyperlipidemias. To investigate the potential of apoE gene delivery to affect hypercholesterolemia, we constructed an adenoviral vector that contains the human apoE3 cDNA. Adenovirus-mediated expression of human apoE in apoE-deficient mice resulted in a complete phenotypic correction of the hypercholesterolemic state.
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Methods |
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Animals
C57BL/6J-Apoem1Unc apoE-deficient mice were obtained from The Jackson Laboratory. These 10-week-old mice were homozygous for disruption of the apoE gene.8 12 The mice were fed laboratory mouse chow and allowed to acclimate for approximately 3 weeks before vector administration. Each mouse was treated by tail vein injection with 5x1011 recombinant adenovirus vector particles, corresponding to approximately 5x109 plaque-forming units (pfu) in 400 µL using Hanks' buffered saline solution as diluent.
Lipoprotein and Protein Analysis
Plasma cholesterol concentrations were determined before and
after treatment with enzymatic methods (Sigma Chemical Co). Blood
collected from either the retro-orbital plexus or the tail vein was
immediately transferred to heparinized tubes. Plasma was collected
after centrifugation at 7000g for 5 minutes. EDTA, Pefabloc,
and aprotinin were added to all plasma samples at final concentrations
of 2 mmol/L, 1 mmol/L, and 10 µg/mL, respectively.
A 1-µL aliquot of plasma was denatured and applied to a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel (Novex). The proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by use of a small transblot apparatus (Biorad) for 30 minutes at 100 V. After the transfer was completed, the PVDF membrane was transiently stained with Ponceau red, and the molecular weight standards were marked directly on the membrane. Molecular weight markers used ranged from 200 to 14 kD (Biorad). The PVDF membrane was blocked in 10 mmol/L Tris, pH 7.4, containing 150 mmol/L NaCl, 2 mmol/L EDTA, 0.04% Tween 20, and 5% milk. The blocked membrane was first incubated for 1 hour at room temperature in a 1:3000 dilution of the primary antibody, the anti–human apoE monoclonal clone 3H1 (obtained from Dr Y. Marcel, University of Ottawa Heart Institute). The membrane was developed with a secondary goat anti–mouse IgG1 horseradish peroxidase (HRPO)–conjugated antibody (Southern Biotechnology Associates, Inc) by use of an enhanced chemiluminescence system (Amersham Lifesciences). The membrane was exposed to film for approximately 1 to 10 seconds. Purified human apoE (Calbiochem) was used as a positive control on all Western blot analyses.
Equivalent volumes of plasma from each mouse were pooled per treatment group, and 200 µL was applied to a Superose 6 gel filtration fast protein liquid chromatography (FPLC) column (Pharmacia). The column was equilibrated in 10 mmol/L Tris, pH 7.4, containing 150 mmol/L NaCl, 2 mmol/L EDTA, and 0.02% sodium azide at a flow rate of 0.35 mL/min, and 0.5-mL fractions were collected. Cholesterol was determined on 100 µL of each fraction by enzymatic methods. Purified human VLDL and HDL (Calbiochem) were used to calibrate the column.
Cloning of the ApoE cDNA
The apoE cDNA was constructed by use of gene overlap extension
polymerase chain reaction (PCR) methods13 with Pfu DNA
polymerase (Stratagene) in the presence of 10% dimethyl sulfoxide. The
5' end of the apoE cDNA, nucleotides -39 to 292, was generated with
liver cDNA (Clonetech) as the template with the following primers: P1,
5'-ACTCAGCCCCAGCGGAGGTGAAGGACGTCCTTCCCCAGGAGCCG-3';
P2, 5'-TTCCTCCAGTTCCGATTTGTAGGCCTTCAACTCCTTCATGGTCTCGTC-3'. The primer
P1 was designed to start at the major transcription initiation
site in exon 1.14 The PCR was carried out with the
following conditions: 95°C for 10 minutes, followed by 30 cycles of
95°C for 30 seconds, 60°C for 1 minute, 72°C for 2 minutes, and
finally a 72°C extension for 10 minutes. The 3' end of the apoE cDNA
was amplified from the cloned apoE fragment15 EB4
(obtained from Dr Steve Humphries) with the primers P3
(5'-GCCTACAAATCGGAACTGGAGGAA-3') and P4 (5'-AGGCTTCGGCGTTCAGTGATTGT-3')
to produce a 696–base pair (bp) fragment. The 5' and 3' PCR apoE
fragments were gel-purified. The PCR was performed with equal volumes
of the melted fragments and the end primers P1 and P4. The expected
full-length apoE cDNA, 1025 bp, was amplified and ligated directly into
the pCRII vector (Invitrogen). Several clones were screened by
restriction enzyme analysis and sequenced. A clone matching the
expected sequence (GenBank accession No. K00396) was selected.
Recombinant Adenoviruses
A recombinant adenovirus vector containing the human apoE3 cDNA
was constructed by use of the vector system described
previously.16 The apoE3 cDNA was placed downstream of the
RSV promoter in an adenoviral backbone deleted for E1a, most of the E1b
region, and the E3 region. The 293 cells were cotransfected with
Kpn I–linearized pAvS6E and the large Cla
I fragment of Ad5dl327. This transfection produced the recombinant
adenovirus containing the apoE3 cDNA and was called Av1RE. Recombinant
adenoviral plaques were identified with PCR to detect the apoE3 cDNA
and were expanded in 293 cells. The adenovirus titers (particles per
milliliter) were determined spectrophotometrically17 18
and compared with the biological titer (pfu per milliliter). The ratio
of total particles to infectious particles (particles per pfu) was
usually 100 or less. Fig 1
schematically shows maps of
Av1LacZ4 and Av1RE vectors.
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Cell Culture
HepG2 cells were cultured in Eagle's minimum essential medium
(EMEM) containing 10% fetal bovine serum (FBS). Transductions were
carried out in EMEM containing 2% FBS, 100 U/mL penicillin, and 10
µg/mL streptomycin when the cells had reached approximately 90%
confluency. The adenoviral vector was diluted in 0.5 mL of the
transduction medium and was placed on the cell monolayer for 1.5 hours
at 37°C. The medium was removed, and 1 mL of fresh transduction
medium was then added. After 24 hours, the medium was collected, and
Western blot analysis was carried out on a 10-µL aliquot.
Human ApoE Enzyme-Linked Immunosorbent Assay
Human apoE in mouse plasma was measured with a sandwich-type
enzyme-linked immunosorbent assay (ELISA) with a mouse monoclonal,
9-H8, as the capture antibody and a goat polyclonal as the detecting
antibody. Values were determined by use of a standard curve obtained by
the inclusion of varying amounts of purified recombinant human apoE3
(Calbiochem) into plasma from apoE-deficient mice. Microtiter plates
(Immulon 4, Dynatech) were coated with the 9-H8 antibody (5 µg
protein/mL, Cappel) in 100 mmol/L sodium bicarbonate, pH 9.6, overnight
at room temperature. The monoclonal antibody solution was removed, and
the plate was washed five times with phosphate-buffered saline (PBS)
containing 0.05% Tween 20. Unless otherwise noted, all subsequent
incubations were carried out for 1 hour at room temperature. The
nonspecific protein binding sites in each well were blocked with PBS
containing 10% milk. The blocking buffer was removed, and the plates
were washed as described previously. The standards and samples were
diluted in PBS containing 2% bovine serum albumin (BSA) and allowed to
incubate at 4°C. A 100-µL aliquot of each sample or standard
dilution was placed in individual wells and allowed to incubate. The
plates were washed as described and were then incubated with 100 µL
of a 1:4000 dilution of the secondary anti–human apoE goat polyclonal
antibody (Calbiochem) in PBS containing 2% BSA. After washing, the
plates were incubated with 100 µL of a 1:3000 dilution of the
tertiary swine anti–goat IgG-HRPO polyclonal antibody (Caltag). The
reaction was developed with 100 µL of 0.2 g/L
3,3',5,5'-tetramethylbenzidine and 0.01% H2O2
and was stopped by the addition of 100 µL of 1 mol/L phosphoric acid.
The absorbance at 450 nmol/L was measured, with 405 nm as the
reference. Human plasma samples were used as controls in each assay,
and reproducible values within the reported range of 3 to 5
mg/dL19 were obtained.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Adenoviral Expression of Human ApoE In Vitro
An adenoviral vector (Av1RE) containing the human apoE3 cDNA was constructed (Fig 1
). HepG2 cells were transduced with the Av1RE vector
at multiplicities of infection of 10 or 100 or a control vector,
Av1Lacz4, containing a nuclear targeted ß-galactosidase cDNA. The
culture medium was analyzed by Western blot analysis with an
anti–human apoE monoclonal antibody, 3H1 (Fig 2).
Untransduced HepG2 cells20 and Av1LacZ4-transduced HepG2
cells secreted a low level of apoE that was detected in the culture
medium (Fig 2, lanes 3 and 4). However, Av1RE-transduced HepG2 cells
secreted substantially higher levels of apoE, indicating that the
vector directs the overproduction of apoE in vitro. Vector-derived
human apoE had an identical Mr of 34 000 compared with both
purified human apoE (Fig 2, lanes 1 and 2) and the endogenous protein
(Fig 2, lanes 3 and 4)
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Adenoviral Expression of Human ApoE In Vivo
To determine whether the human apoE produced by the Av1RE vector
could influence cholesterol metabolism in vivo, 5x1011
viral particles (5x109 pfu) of the Av1RE or Av1LacZ4 vector
were administered to apoE-deficient mice by tail vein injection. We and
others previously demonstrated that intravenous injection of adenoviral
vectors results in preferential and efficient transduction of liver
hepatocytes.16 21 22 23 At 7 days after injection, blood was
obtained from the tail vein, and plasma was analyzed by Western blot
analysis (Fig 3). Human apoE was detected in the
plasma of the Av1RE-treated mice and was not present in the plasma
of the Av1LacZ4 vector control group. Human apoE concentrations in
mouse plasma were quantified by ELISA. The average apoE concentration
in mouse plasma 7 days after vector administration was 1.2±0.4 µg/mL
(mean±SEM, n=5), which is approximately 4% of normal human apoE
levels.19
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Consistent with previous data, the mean plasma cholesterol value
in the apoE-deficient mice before vector treatment was 737.5±118 mg/dL
(mean±SEM, n=6).8 9 11 24 One week after administration
of the Av1RE vector, plasma cholesterol levels had declined eightfold
to a mean of 98.2±4.4 mg/dL (mean±SEM, n=5) (Fig 4),
which is equivalent to levels found in normal C57BL6 mice fed a chow
diet.5 8 25 Mice that received the control Av1LacZ4 vector
had similar levels of plasma cholesterol before and after treatment,
indicating that the reduction in plasma cholesterol concentrations was
due to the expression of human apoE.
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Fig 5 shows the plasma lipoprotein distributions of the
Av1RE- and Av1LacZ4-treated mice 7 days after vector administration.
Pooled plasma from each treatment group was fractionated with a
Superose 6 gel filtration column, and the cholesterol content was
measured in each fraction across the elution profile. As expected for
untreated apoE-deficient mice, the lipoprotein elution profile of
the Av1LacZ4-treated group showed that the majority of the cholesterol
eluted in the VLDL-LDL region.8 9 In contrast, the
lipoprotein distribution of the Av1RE-treated animals was shifted so
that the cholesterol found in the VLDL-LDL region was reduced and HDL
was the primary cholesterol-containing lipoprotein.
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, n=6) or the Av1RE-treated group
(
, n=5) was fractionated with a Superose 6 gel filtration column,
and the cholesterol content was determined on individual fractions
across the elution profile. The cholesterol concentration (in
micrograms per milliliter) was plotted for each fraction collected
(note the different y axes for each treatment group). The
elution positions of purified human VLDL and HDL are indicated.
The plasma lipoprotein distribution of human apoE in the Av1RE-treated
mice 7 days after vector administration was confirmed by Western blot
analysis of the fractionated plasma samples (Fig 6).
The majority of the human apoE was associated with the VLDL-LDL
fraction, although a smaller proportion of apoE was detected in the HDL
fraction. This result was similar to the plasma lipoprotein
distribution of endogenous apoE from normal C57BL6 mice fed an
atherogenic diet (data not shown).26
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The reduction in total plasma cholesterol levels in the Av1RE-treated
apoE-deficient mice persisted for at least 21 days after
administration of the adenoviral vector (Fig 7A). The
mean plasma cholesterol concentrations in the Av1RE-treated
apoE-deficient mice were 98.2±4.4, 215±61.4, and 161.5±26.3 mg/dL
(mean±SEM, n=5) at 7, 14, and 21 days after vector treatment,
respectively. At 35 days after vector administration, the plasma
cholesterol concentrations in the Av1RE-treated group increased to
approximately 550 mg/dL, but this was still lower than that seen in the
control vector group. A significant change in the plasma cholesterol
concentrations was not observed over the course of the study in the
Av1LacZ4-treated control mice.
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Expression of human apoE in mouse plasma persisted for at least 35 days
after administration of the adenoviral vector (Fig 7B). The
concentration of human apoE varied over the course of the study, with
the highest level of 3.4±0.9 µg/mL (mean±SEM, n=4) found at 14
days. The increase in plasma cholesterol concentrations 35 days after
injection correlated with a decline in the plasma human apoE
concentrations to a level of 0.5±0.1 µg/mL (mean±SEM, n=5) (Fig 7B).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Adenovirus-mediated expression of human apoE in hypercholesterolemic apoE-deficient mice resulted in a transient phenotypic reversion of the hypercholesterolemic state normally found in this mouse strain. The levels of human apoE expression were sufficient to produce a significant lowering of plasma cholesterol concentrations that persisted for at least 21 days after vector administration. Correction of the hypercholesterolemic condition in the apoE-deficient mouse model by direct in vivo gene transfer establishes the potential of this approach for treatment of hyperlipidemias resulting from apoE deficiency27 or malfunction.28
An inverse relation between plasma apoE concentrations and plasma cholesterol levels was reported previously in transgenic mice expressing the rat and human apoE genes.5 29 These studies demonstrated that a plasma apoE concentration of approximately 90 to 100 µg/mL was needed before a decline in cholesterol levels was found. The current study showed that a human apoE concentration of 1 to 4 µg/mL was sufficient to lower plasma cholesterol levels to the normal range in apoE-deficient mice. Taken together, these studies suggest that in normal animals, substantial overexpression of apoE above the endogenous apoE levels may be required to elicit an effect on plasma lipid levels. However, the severe hypercholesterolemia resulting from gene knockout is affected by relatively modest apoE levels. It will be of interest to test the Av1RE vector in various hyperlipidemic animal models such as the LDLR-deficient mice, WHHL rabbits, apoCIII-transgenic mice, or apoE2-transgenic mice to assess whether apoE gene therapy might be of general benefit in human disease. In addition to the treatment of hyperlipidemias, the Av1RE vector also could be used to assess the effects of human apoE3 gene therapy on the development of Alzheimer's disease.
The decrease in plasma cholesterol levels observed in the Av1RE-treated
mice was accompanied by changes in the plasma lipoprotein distribution
(Fig 5). The presence of human apoE in the plasma of the apoE-deficient
mice produced a decrease in VLDL and LDL cholesterol and an increase in
HDL cholesterol. ApoE-deficient mice have both intestine and
liver-derived remnant lipoprotein particles that accumulate in plasma
and result in elevated plasma cholesterol
concentrations.8 9 These remnant particles are normally
cleared from the circulation through the interaction of apoE with the
LDLR, LRP, or both.2 24 Previous studies showed that the
elevation of apoE levels can enhance the clearance of VLDL, LDL, and
chylomicrons from the circulation.6 7 The reduction of
plasma cholesterol concentrations and changes in the plasma lipoprotein
distribution were presumably the result of the association of the human
apoE protein with both apoB48- and apoB100-remnant lipoprotein
particles (Fig 6), thereby increasing their rate of removal from the
circulation. We have used adenovirus-mediated gene delivery to deliver
a functional human apoE cDNA to correct the genetic deficiency found in
the apoE-deficient mouse model. The reduction of plasma cholesterol
concentrations and changes in the plasma lipoprotein distribution
presumably resulted when the human apoE associated with the apoB48- and
apoB100-remnant lipoprotein particles, increasing their removal from
the circulation. The present study demonstrates that the phenotypic
correction of the apoE-deficient mouse can be achieved by transient
delivery of the apoE3 gene by use of an adenoviral vector. This
approach has also been used to produce a transient phenotypic
correction of LDLR-deficient mice and WHHL rabbits with
adenovirus-mediated expression of the human LDLR.30 31
Normal C57BL6 mice on a chow diet have average plasma cholesterol
concentrations that range from 40 to 100 mg/dL.5 8 25 The
Av1RE-treated apoE-deficient mice had plasma cholesterol concentrations
similar to those of normal mice for at least 21 days after vector
administration (Fig 7A). However, the cholesterol-lowering effect was
transient, and the elevation of plasma cholesterol concentrations 35
days after vector administration was correlated with a decline in human
apoE concentrations (Fig 7). The decline in plasma human apoE
concentrations probably resulted from the loss of the vector DNA from
the liver, as found in previous studies.16 32 However, an
immune response to the human apoE3 protein cannot be ruled out at this
point as an explanation of the decreased plasma concentrations.
Atherosclerosis develops readily in the apoE-deficient mouse model.10 11 Evaluation of the effects of apoE gene therapy on the development of atherosclerosis in the apoE-deficient mouse will most likely require a more sustained and persistent gene expression. The current vector, Av1RE, resulted in expression of apoE for at least 21 days, with subsequent cholesterol lowering over that time. However, 21 days may not be long enough to influence the amount and extent of atherosclerosis in these animals. As Yang et al32 described, the current hypothesis to explain the decline in transgene expression over time suggests that a low level of viral gene expression occurs and invokes a cellular immune response against the adenovirus-infected cells. To achieve persistent transgene expression, current efforts are focused on vector modifications designed to reduce functional expression of adenoviral backbone sequences.33 With persistent expression of apoE, the evaluation of apoE gene therapy on the development of atherosclerosis can be further studied.
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Acknowledgments |
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We would like to thank Drs Steve Humphries and Robert Jambou for critically reviewing this manuscript, Dr Paul Tolstoshev for his support and encouragement, and Dr Russette Lyons for her assistance with the tail vein injections.
Received September 13, 1994; accepted January 20, 1995.
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K. C-W. Yu, Y. Jiang, W. Chen, and A. D. Cooper Rapid initial removal of chylomicron remnants by the mouse liver does not require hepatically localized apolipoprotein E J. Lipid Res., November 1, 2000; 41(11): 1715 - 1727. [Abstract] [Full Text]
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T. Athanasopoulos, J. S. Owen, D. Hassall, M. G. Dunckley, J. Drew, J. Goodman, A. D. Tagalakis, D. R. Riddell, and G. Dickson Intramuscular injection of a plasmid vector expressing human apolipoprotein E limits progression of xanthoma and aortic atheroma in apoE-deficient mice Hum. Mol. Genet., October 1, 2000; 9(17): 2545 - 2551. [Abstract] [Full Text] [PDF]
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