© 1997 American Heart Association, Inc.
Articles |
Effective Lowering of Plasma, LDL, and Esterified Cholesterol in LDL Receptor–Knockout Mice by Adenovirus-Mediated Gene Delivery of ApoB mRNA Editing Enzyme (Apobec1)
From the Department of Medicine, Baylor College of Medicine, Houston, Tex (B.B.T., S.B., L-Z.S., A.M.G., L.C.); the Department of Medicine, University of California, San Francisco (B.I.); and the Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, Calif (T.M.F.).
Correspondence to BaBie Teng, PhD, Department of Medicine, Section of Atherosclerosis and Lipoprotein Research, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail teng{at}bcm.tmc.edu
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Abstract Adenovirus-mediated gene delivery of apolipoprotein (apo)B mRNA editing enzyme (AvApobec1) was used to study the effect of apoB mRNA editing on apoB production in homozygous LDL receptor–deficient (LDLR-/-) mice. Intravenous injection of AvApobec1 into these mice resulted in a >80% decrease in plasma apoB-100 with a concomitant increase in plasma apoB-48 level. The plasma apoE level also increased. In all cases, total plasma apoB (apoB-100 + apoB-48) decreased by 60% at day 5 and remained
40% lower in AvApobec1-treated compared with control vector Av1LacZ4–treated animals at day 12. On day 12, total plasma cholesterol decreased by 29% in male mice and 18% in female mice that were transduced with AvApobec1. This was reflected in a reduction in apoB-containing lipoprotein cholesterol, which decreased by 34% and 27% in male and female mice, respectively. Apobec1 gene transfer also decreased the cholesteryl ester contents in the LDL fraction, which were 16%, 22%, and 22% in female and 20%, 20%, and 15% in male animals on days 5, 7, and 12, respectively, compared with Av1LacZ controls with 29%, 32%, and 33%, respectively, in female and 29%, 38%, and 36%, respectively, in male animals. Nondenaturing gradient gel electrophoresis indicated almost complete elimination of LDL particles of 29, 27, and 25 nm at days 7 and 12. We conclude that in the absence of a functioning LDL receptor, hepatic overexpression of Apobec1 is highly efficient in lowering plasma apoB-100 levels, leading to the almost complete elimination of LDL particles and a reduction in LDL cholesterol and cholesteryl ester content.
Key Words: gene therapy • LDL receptor–deficient mouse • apoB mRNA editing • hypercholesterolemia • lipoproteins
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Apolipoprotein B-100 (ApoB-100) is an essential protein component of VLDL and LDL.1 2 ApoB-48 is the translation product of apoB-48 mRNA, which is produced from apoB-100 mRNA by a novel genetic mechanism known as apoB mRNA editing.3 4 In humans, apoB-48 is a structural component of chylomicrons and chylomicron remnants and is secreted from the small intestine only.1 2 In some mammals, including the rat and mouse, the liver also produces apoB-48 in the form of VLDL.5 The main function of these lipoprotein particles is to transport lipid to cells, where it is utilized or stored.
It is generally recognized that the steps involved in the assembly of apoB-100– and apoB-48–containing lipoproteins are distinct. Nascent apoB-100–containing particles are formed through the cotranslational association of apoB-100 with lipid, whereas nascent apoB-48–containing VLDLs are formed in the rough endoplasmic reticulum by a two-step process.6 7 Some apoB-48 particles are assembled into larger TG-rich particles that delay the movement of apoB-48 secretion. Additionally, a higher proportion of apoB-48 than apoB-100 particles is degraded before secretion.8 Furthermore, it has been shown that in primary rat hepatocytes, the secretion rate of apoB-48 is slower than that of apoB-100.9 10 In terms of catabolism, the absence of the carboxyl-terminal half of apoB-100 in apoB-48 has profound functional consequences.2 Both the LDL receptor–binding domain, which is essential for cellular uptake of LDL, and the attachment site for apo(a), which is required for the formation of Lp(a) (a highly atherogenic lipoprotein), are missing in apoB-48. In the circulation, VLDL is converted to LDL, a cholesterol-enriched lipoprotein with apoB-100 as its only apoprotein. LDL apoB-100 binds to LDL receptors with relatively low affinity, and LDL has a relatively long half-life in the circulation. ApoE in apoB-48–containing particles is recognized by both the LDL receptor and the LDL receptor–related protein. Chylomicrons and their remnants have a very short half-life in the circulation (for a review, see Reference 1111 ). Thus, there are distinct differences in the biogenesis and catabolism of apoB-100– and apoB-48–containing lipoproteins.
ApoB mRNA editing involves site-specific deamination of cytidine in a CAA codon encoding Gln residue 2153 in apoB-100 mRNA to a uridine, thereby changing CAA to UAA, a stop codon. Translation of the edited apoB mRNA produces the prematurely terminated protein apoB-48.3 4 The catalytic component for the apoB mRNA editing enzyme complex is a cytidine deaminase–like enzyme designated Apobec112 13 and is an essential component for the multicomponent editing enzyme complex.14 Apobec1 contains two putative functional domains, an N-terminal catalytic region that coordinates zinc for activity,15 16 and a C-terminal Leu-rich region thought to be important for protein-protein interaction.12 Apobec1 by itself does not edit apoB mRNA. It requires other proteins to complement the editing activity.12 17 Using in vivo gene transfer techniques, we18 and others19 have shown that Apobec1 confers editing activity to the liver in vivo. The adenovirus-mediated delivery of Apobec1 in C57BL/6 mice increased editing activity in the liver, resulting in a marked reduction of apoB-100 and the almost complete elimination of LDL.18 Hughes et al19 showed that adenovirus-mediated transfer of Apobec1 in human apoB/apo(a)–transgenic mice resulted in a lowering of the concentration of human apoB-100 and Lp(a) in these animals. The normal rabbit liver does not have apoB mRNA editing activity. Hughes et al19 showed that they could induce apoB mRNA editing in the rabbit liver by Apobec1 gene transfer. Yamanaka et al20 showed that transgenic rabbits with the integrated Apobec1 cDNA driven by the apoE promoter expressed editing activity in the liver, and a substantial portion of apoB mRNA was edited. All of these observations indicate that introduction of Apobec1 is an efficient method to downregulate apoB-100 production.
LDLR-/- mice are an animal model for FH. They have delayed clearance of VLDL, IDL, and LDL from the plasma and elevated levels of plasma TC, due mainly to increases in plasma IDL and LDL.21 Thus, LDLR-/- mice have elevated apoB-100 levels compared with wild-type animals and are an excellent model in which to study the effect of augmented hepatic apoB mRNA editing on plasma lipids and apoB concentration against a background of delayed LDL catabolism.
We previously transduced normal C57BL/6 mice with recombinant adenoviral Apobec1 cDNA and demonstrated that Apobec1 reduced plasma apoB-100 levels and normal LDL production.18 In the current study, we show that in vivo transduction of AvApobec1 into LDLR-/- mice leads to decreases in plasma TC and apoB and elimination of large LDL particles. Furthermore, we report that increased hepatic apoB mRNA editing is correlated with a decrease in the EC content of plasma LDL.
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Construction of Recombinant Adenoviral Vectors
Construction of recombinant adenovirus AvApobec1 has been described previously.18 In brief, AvApobec1 contains the full-length cDNA of Apobec1 driven by the Rous sarcoma virus promoter and adenovirus tripartite leader, which replaces the E1a and E1b genes at the left end of the virus. AvApobec1 was purified by plaque assay on 293 cells. Recombinant adenovirus Av1LacZ4 was supplied by Genetic Therapy, Inc. It had the same structure as AvApobec1 except that it contained a 3.1-kb nuclear-targeted ß-galactosidase cDNA insert instead of an Apobec1 cDNA insert. Large-scale production of high-titer recombinant adenovirus was performed by amplification of recombinant adenovirus on 293 cells as described previously.18 Adenoviral vectors were titered by plaque-forming assay on 293 cells. The titer ranged between 1010 and 1011 pfu/mL.
Animal Experiments
All animal experiments were conducted in accordance with the guidelines of the Animal Protocol Review Committee of the Baylor College of Medicine. Mice lacking the LDL receptor, which were hybrids of a C57BL/6J–129Sv cross,21 were maintained on a normal chow diet that contained 4% (wt/wt) animal fat and <0.04% (wt/wt) cholesterol (Harlan Teklad).
Recombinant adenovirus stock was diluted with PBS to the appropriate concentration. Aliquots (0.5 mL) of diluted adenoviral vector were injected into a tail vein. After adenoviral vector transduction, the animals were fasted for 6 hours before blood was drawn and collected into EDTA at the times indicated. Plasma was stored at 4°C before analysis for lipids and apolipoproteins or lipoprotein fractionation by ultracentrifugation.
At the times indicated, the animals were anesthetized and exsanguinated and the liver, stomach, small intestine, kidney, lung, spleen, and heart removed. For the liver, 1-mm slices were taken for histochemical analysis, a piece was snap-frozen for preparation of RNA and DNA, and one piece was rinsed briefly in PBS and processed for cytosolic S-100 extraction for apoB mRNA editing activity. Samples from other tissues were fixed for histochemical analysis or snap-frozen for DNA preparation.
Southern Blot Analysis of Liver DNA
Genomic DNA was prepared from mouse liver as described.22 DNA was digested with the appropriate restriction enzymes under conditions recommended by the suppliers, fractionated by electrophoresis on 1% agarose gels, and transferred to ZETA probe membranes (Bio-Rad). Filters were hybridized to a 32P-labeled full-length Apobec1 cDNA probe, and radiolabeled bands were quantified by a PhosphorImager SF (Molecular Dynamics).
Preparation of Cytosolic S-100 Extracts From Mouse Liver
Mouse liver fragments were homogenized in a Dounce homogenizer with a type B pestle in Dignam buffer A containing protease inhibitors as described previously.23 After centrifugation for 10 minutes at 2000 rpm in a Beckman J2-21 centrifuge, we added 0.11 volume of Dignam buffer B to the supernatant fraction, which was then centrifuged for 1 hour at 100 000g. The supernatant was dialyzed against Dignam buffer D. Protein concentration was determined colorimetrically24 and the final cytosolic S-100 extract stored at -80°C until use.
In Vitro ApoB mRNA Editing Assay
Synthetic apoB mRNA was prepared from pRBF-CAA, a rat apoB cDNA fragment of 470 bp (nucleotides 6512 to 6982) spanning the RNA editing region. pRBF-CAA was linearized at a BamHI site. In vitro transcription was performed with Sp6 RNA polymerase, producing a 364-base-long synthetic RNA fragment. The in vitro editing assay was carried out as described previously25 with 2 ng of synthetic RNA substrate in the presence of the indicated amount of mouse liver cytosolic S-100 extracts. The primer-extension products were fractionated on an 8% polyacrylamide-urea gel (National Diagnostics), and radiolabeled bands were quantified by a PhosphorImager SF.
Lipid Analysis of Plasma Samples
Plasma samples were separated by centrifugation at 13 000 rpm at 3°C for 5 minutes and subjected to quantitative lipid and apolipoprotein measurements. Plasma TC was determined by using a Boehringer Mannheim enzymatic reagent kit (Boehringer Mannheim Corp). Plasma TG was determined enzymatically by using a commercial kit from Sigma Chemical Co. Both methods were modified for analysis by a microtiter plate reader (model 340, BioTek). Both mouse and human plasma controls were assayed simultaneously to normalize samples for interassay variations (correlation variation was <10%).
Plasma HDL cholesterol was measured after precipitation of apoB-containing lipoproteins with an equal volume (25 µL) of 200 mmol/L Gly-HCl, pH 10.0 solution containing 20% (wt/vol) polyethylene glycol 8000 (Polysciences). The supernatant was obtained by centrifugation at 13 000 rpm at 3°C for 5 minutes and cholesterol content measured enzymatically (Boehringer Mannheim).
Quantification of Plasma Apolipoproteins
ApoAI and apoE levels in plasma were determined by radial immunodiffusion assay as previously described.26 Monospecific antisera of anti-mouse apoAI and anti-mouse apoE were used for the assay. ApoB-100 and apoB-48 levels were determined by quantitative scanning densitometry of Coomassie Blue R-250–stained SDS polyacrylamide gels as described previously.27 In brief, plasma samples (25 to 50 µL) were adjusted to d=1.063 g/mL by adding KBr/NaCl solution (d=1.3500 g/mL); VLDL and LDL lipoprotein particles were isolated by density ultracentrifugation in a Beckman 42.2Ti rotor and spun at 40 000 rpm at 10°C for 8 hours. The top 50-µL lipoprotein fractions were collected and dialyzed overnight at 4°C against saline/EDTA buffer (150 mmol/L NaCl, 1 mmol/L EDTA [pH 7.4], and 0.5% NaN3). The lipoprotein fraction of d<1.063 g/mL was assayed for cholesterol by enzymatic method as described above. Sample recovery was compared with the VLDL plus LDL cholesterol of the same plasma sample treated with polyethylene glycol as described above; recovery typically ranged from 75% to 95%.
For analysis of the two forms of apoB, purified lipoprotein fractions (2 to 10 µg cholesterol) were solubilized in SDS sample buffer at 60°C for 30 minutes. They were electrophoresed on 2% to 20% linear-gradient SDS polyacrylamide gels and the mass of apoB bands determined by quantitative scanning densitometry at 590 nm (model R/D, Helena Labs). To standardize the assay, the content of apoB-100 and apoB-48 in purified mouse ß-VLDL was determined by quantitative SDS-PAGE gels and densitometry scanning. Human LDL apoB-100 was used as the standard. The areas under the peak scanned in the range 0.125 to 2.00 µg were linear (r=.989, n=12), and chromogenicities were similar for the two proteins.
Fractionation of Lipoprotein Particles by Ultracentrifugation
Plasma from control (untreated), Av1LacZ4-treated, and AvApobec1-treated mice at days 5, 7, 12, and 31 after treatment was fractionated by sequential ultracentrifugation at d=1.006 (VLDL), 1.063 (LDL), and 1.21 (HDL) g/mL as described.28 Lipoprotein fractions were collected and dialyzed extensively. Protein concentration was measured by a colorimetric assay.24 Each fraction was used for lipid analysis and nondenaturing gradient gel electrophoresis.
Lipid Analysis of Lipoprotein Fractions
TG, TC, nonesterified FC, and phospholipid contents of each lipoprotein fraction were determined with reagents obtained from Wako Chemicals USA, Inc. The esterified cholesterol content of each lipoprotein fraction was determined by subtracting the FC from the TC value.
Nondenaturing Gradient Gel Electrophoresis
To measure their particle size distributions, VLDL and LDL were separated by electrophoresis on 2% to 16% nondenaturing polyacrylamide gels; HDL profiles were obtained on 4% to 30% nondenaturing gels as described by Nichols et al.29 Gels of VLDL were stained with oil red O and those of LDL and HDL with Coomassie Blue G-250. Particle size distribution was assessed by scanning densitometry.
Statistical Methods
One-way ANOVA and Fisher's protected least significant difference tests from the StatView 4 computer program were used to evaluate differences between groups of same-day samples, as indicated in the footnotes to the tables.
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Hepatic Uptake and Expression of AvApobec1 in the Liver
As shown previously, AvApobec1 was efficiently transduced and expressed in C57BL/6 mouse hepatocytes.18 F and M LDLR-/- mice were injected with 1x1010 pfu of AvApobec1 (F=15, M=15) or Av1LacZ4 (as controls; F=15, M=15) into a tail vein. Tissue and blood samples were obtained at different times thereafter. The amount of AvApobec1 DNA in the transduced liver was quantified by Southern blot analysis. As shown in Fig 1
at day 7, an average of 2.6 copies of AvApobec1 DNA per cell was detected (n=3). The copy number decreased to 1.7 copies per cell by day 12 (n=2). There was no difference between M and F animals (data not shown). These data are concordant with observations in wild-type C57BL/6 mice, which have shown that after gene delivery, the amount of AvApobec1 DNA in the liver decreases gradually. By day 31 AvApobec1 DNA was barely detectable.
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Effect of AvApobec1 Treatment on In Vitro ApoB mRNA Editing Activity
To determine the effect of Apobec1 gene transfer on apoB mRNA editing activity, we prepared mouse liver S-100 extracts from the experimental animals on days 5, 7, 12, and 31. As shown in Fig 2, in vitro editing activity was performed with liver S-100 extracts and synthetic rat apoB RNA as the template. Under these assay conditions, untreated control (n=4) and Av1LacZ4-treated (n=4) mouse liver S-100 extracts edited 6.9±4.1% (F), 10±6.9% (M), 8.2±3.3% (F), and 11±5.6% (M), respectively, of synthetic apoB RNA in vitro; in contrast, AvApobec1-treated liver extracts on days 5 (n=4), 7 (n=4), and 12 (n=4) edited 58±4.9% (F), 62±10% (M), 68±7.6% (F), 69±8.5% (M), 49±7.6% (F), and 56±6.6% (M), respectively, of apoB RNA in the same experiment. In vitro editing activity returned to control levels of 13±3.2% (F) and 10±6.1% (M) by day 31. Taken together, these data indicate that adenovirus-mediated Apobec1 gene transduction increases in vivo editing activity in the liver of LDLR-/- mice.
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Effect of AvApobec1 Transduction on Plasma Lipids
Plasma TC, HDL cholesterol, and TG were measured in untreated control, Av1LacZ4-treated, and AvApobec1-treated LDLR-/- mice. Blood samples were collected on days 5, 7, 12, and 31 after transduction from both F and M mice. In agreement with the findings of other investigators,21 the basal plasma TC level in LDLR-/- mice was 3-fold higher than in C57BL/6 mice when they were fed regular chow. Compared with other animals (Table 1) on day 12 after AvApobec1 transduction, plasma TC in F LDLR-/- mice decreased by 18% (P<.03), mainly due to a marked decrease in VLDL and LDL cholesterol (27%, P<.01). A more dramatic reduction in cholesterol level was observed in M LDLR-/- mice. In comparison with Av1LacZ4-treated mice, plasma TC levels of AvApobec1-treated animals decreased by 29% (P<.005), and this change was also reflected in a 34% reduction in VLDL and LDL cholesterol levels in these animals (P<.04). There was no significant difference in HDL cholesterol among all treatment groups. The plasma TC level returned to the control value by day 31. In conclusion, in LDLR-/- mice, AvApobec1 treatment reduced plasma TC, mainly due to a reduction in apoB-containing lipoprotein cholesterol. There was a significant increase in plasma TG concentration at day 7 after transduction with both types of adenovirus in F and M LDLR-/- mice. This effect of viral transduction was not present on day 12 (Table 2).
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Plasma ApoB-100 and apoB-48 Concentrations After AvApobec1 Transduction
Plasma apoB-100 and apoB-48 concentrations were determined on days 5, 7, and 12 after adenoviral vector transduction. In LDLR-/- mice, >80% of plasma apoB was apoB-100. At day 5 after AvApobec1 transduction, the plasma concentration of apoB-100 decreased by >80% (P<.0001) compared with Av1LacZ4-treated mice for both F and M animals, whereas plasma apoB-48 increased significantly (F=33%, P<.0001; M=110%, P<.004; Table 3). This marked reduction in apoB-100 concentration was still present on day 12, at which time the apoB-48 concentration remained at the same elevated level in F LDLR-/- mice but decreased from 110% to 60% in M mice. Compared with Av1LacZ4-treated animals on days 5, 7, and 12 after AvApobec1 transduction, the total plasma apoB concentration (apoB-100 and apoB-48) decreased 60%, 56%, and 42%, respectively, in F mice and 50%, 49%, and 49%, respectively, in M mice. Concomitant with this reduction in total apoB content was a profound change in the ratio of apoB-100 to apoB-48. On day 5 after adenovirus administration, the ratio of apoB-100 to apoB-48 was 10-fold lower in AvApobec1-treated (0.49 in F and 0.3 in M) than in Av1LacZ4-treated (4.36 in F and 3.71 in M; P<.0001) animals. A marked difference in this ratio between the two groups of mice persisted at day 12 (Table 3). An even more pronounced difference in the ratio of apoB-100 to apoB-48 was evident when AvApobec1-treated animals were compared with untreated controls (Table 3). As previously noted by us and others,18 19 Av1LacZ4 actually increased apoB-100 and apoB-48 concentrations, which gradually returned toward normal control levels on day 12. Taken together, the data show that AvApobec1 treatment has a profound effect on apoB-containing lipoprotein production.
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Effect of AvApobec1 Transduction on Plasma ApoE and AI
Plasma apoE and AI levels were measured on days 5, 7, and 12 (Table 4). By day 7 after AvApobec1 transduction, the plasma apoAI concentration decreased by 26% and 22% (P<.01) in F and M animals, respectively, compared with Av1LacZ4-treated animals. In general, comparison of Av1LacZ4-treated animals with untreated controls revealed that adenovirus treatment per se seemed to cause an increase in apoE concentration, although this change was not significant. Compared with Av1LacZ4-treated animals, plasma apoE levels on day 5 after AvApobec1 transduction increased by 94% (P<.001) and 56% (P<.001) in F and M mice, respectively. Plasma apoE concentrations remained elevated on day 12 in both F and M animals (Table 4).
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Analysis of Lipoprotein Composition After AvApobec1 Transduction
To study the effects of AvApobec1 transduction on lipoprotein composition, we pooled equal volumes (50 µL) of plasma from groups of 5 animals on days 5, 7, 12, and 31 from untreated control, Av1LacZ4-treated, and AvApobec1-treated mice and fractionated the pooled plasma into VLDL, LDL, and HDL (d=1.006, 1.063, and 1.21 g/mL, respectively) by sequential ultracentrifugal flotation. TC, unesterified cholesterol, TG, phospholipid, and protein contents were determined. The lipoprotein composition of each density fraction is shown in Table 5. The relative amount of each component (ie, FC, EC, TG, phospholipid, and protein) was calculated as the percentage of total mass in each fraction. In untreated control, Av1LacZ4-treated, and AvApobec1-treated groups, VLDL consisted of TG-rich particles, and 50% of the cholesterol was in the esterified form. In contrast, LDL was cholesterol rich but TG poor: 45% of the total mass was cholesterol and only 5% was TG. In control untreated and Av1LacZ4-treated groups, EC content was >30% of the total mass, and these results were similar for M and F animals. However, on day 5 after AvApobec1 transduction, in F LDLR-/- animals the EC level decreased to 16% and remained low at 22% and 22%, respectively, on days 7 and 12. By day 31, EC had returned to the control level of 32%. This was also true in M LDLR-/- mice after AvApobec1 transduction, in which EC contents decreased to 20%, 20%, and 15%, respectively, on days 5, 7, and 12. By day 31, EC had returned to the control level of 34%. Interestingly, the FC content after Apobec1 treatment increased to 18%, 17%, and 16% on days 5, 7, and 12, respectively, compared with untreated control or Av1LacZ4-treated F animals, which had an average of 12% FC. The results were similar in M animals after Apobec1 treatment. The lipoprotein composition of the HDL fraction in all three groups in both F and M animals was similar: >80% of the cholesterol was in the esterified form (Table 5). Taken together, these data indicate that adenovirus-mediated Apobec1 gene transduction increases in vivo editing activity in the liver and is accompanied by a decrease in apoB-100 production together with a decrease in EC in the LDL fraction.
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Nondenaturing Gradient Gel Analysis
The particle size distribution of VLDL, LDL, and HDL fractions was analyzed by nondenaturing gradient gels. VLDLs from both M and F mice were heterogeneous in size with a predominance of particles in the 40- to 44-nm range in M and the 38- to 39-nm range in F animals. There was no significant difference among untreated control, Av1LacZ4-treated, or AvApobec1-treated mice (data not shown). HDL size distribution was similar for both M and F mice, consisting of a monodisperse peak at 11.3 nm in both cases (data not shown).
The LDL size fraction showed the most change after AvApobec1 transduction. Because there was no significant difference in LDL size profiles between M and F mice, only the LDL profile in M mice is shown in Fig 3. The LDL particles in untreated controls consisted of a predominant peak at 29 to 30 nm with additional minor peaks at 25 to 27 nm. The particle distribution of the Av1LacZ4-treated group was similar to that in controls, with the major LDL peak remaining at 29 to 30 nm. AvApobec1 transduction resulted in a profound change in the size distribution of LDL particles on days 7 and 12, when there was almost complete elimination of the 29-, 27-, and 25-nm particles and the concomitant appearance of a minor component in the 20- to 21-nm range. By day 31, the size distribution reverted to that of untreated control or Av1LacZ4-treated animals.
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ApoB-containing lipoproteins constitute a major component of the plasma lipoprotein transport system. The apoproteins involved, apoB-100 and apoB-48, have distinct pathways in lipoprotein assembly, secretion, and metabolism. In this study we examined the metabolic consequences in LDLR-/- mice that were induced to express almost exclusively apoB-48 by adenovirus-mediated transfer of the Apobec1 gene. LDLR-/- mice are an excellent animal model of FH. We also showed that enhanced hepatic expression of Apobec1 in both F and M LDLR-/- mice resulted in a marked decrease in plasma apoB-100 concentration, a significant decrease in plasma TC, and virtual elimination of plasma LDL. Furthermore, there was a significant reduction in the cholesteryl ester content of apoB-containing lipoproteins.
In agreement with our previous observation in wild-type C57BL/6 mice, in vivo gene transfer of Apobec1 resulted in an 6-fold increase in apoB mRNA editing activity in liver extracts (Fig 2
). This increase in hepatic apoB mRNA editing changed the lipoprotein metabolism in these animals markedly. Five days after Apobec1 treatment, there was a pronounced drop of 83% and 87% in plasma apoB-100 concentration in F and M animals, respectively, with a concomitant increase in plasma apoB-48 of 33% and 111%. Plasma apoE concentration also increased (Tables 3 and 4). Despite the rise in apoB-48 level, total plasma apoB (apo-100 and apoB-48) dropped 60% after Apobec1 treatment. This decrease in total apoB content persisted to day 12, when a significant decrease in the plasma TC of 18% and 29% in F and M animals, respectively, was also observed. The decrease in plasma TC was caused mainly by a significant decrease in apoB-containing lipoprotein cholesterol concentration (Table 1). The reduction in apoB-100 and the increase in apoB-48 concentration after Apobec1 treatment was a direct result of increased apoB mRNA editing; the increased apoE was probably a secondary effect of increased output of hepatic apoB-48–containing particles enriched with apoE. As noted in our previous study of Apobec1 gene transfer in wild-type animals,18 we also observed an increase in plasma apoE after administration of the recombinant virus. The exact change in apoE was not quantified in that study. On the other hand, in wild-type C57BL/6 mice, hepatic overexpression of Apobec1 did not result in a change in plasma cholesterol. In LDLR-/- mice 12 days after treatment, the control virus Av1LacZ4 caused a mild elevation in plasma TC, whereas AvApobec1 caused a drop in plasma TC due exclusively to a fall in (VLDL and LDL) cholesterol (Table 1). It is interesting that the decrease in apoB particles was much greater than that in plasma TC. It is possible that apoB-48 particles are able to transport more FC than are apoB-100 particles. We also note that loss of the larger LDL particles on days 7 and 12 after Apobec1 treatment was accompanied by an increase in small particles of 21 nm. These particles may be the apoB-48 particles that account for the increased FC content (Table 5).
Interestingly, the decrease in plasma apoB-100 and TC and increase in plasma apoE were associated with a decrease in EC in the d=1.006 to 1.063 g/mL LDL fraction (Table 5). In other words, an increase in hepatic apoB mRNA editing activity is associated with a decrease in LDL cholesteryl ester content. Inui et al30 previously demonstrated that in rats fed a cholesterol diet, there was an inverse correlation between hepatic cholesteryl ester content and apoB mRNA editing activity. It has been suggested that cholesteryl ester synthesis regulates apoB secretion in the liver (for a review, see Reference 3131 ). However, we found no difference in hepatic FC, EC, and acylcoenzyme A:cholesterol acyltransferase activity in untreated control, Av1LacZ4-treated, or AvApobec1-treated animals (data not shown). Whether increased hepatic apoB mRNA editing has a direct effect on cholesteryl ester synthesis and whether the altered cholesteryl ester content affects apoB production will be the focus of our future investigation.
The gradient gel analysis of the LDL particles indicates that there was an almost complete disappearance of LDL particles in the 25- to 30-nm range on days 7 and 12 (Fig 3). These data corroborate the apoB protein (Table 3) and apoB-containing lipoprotein cholesterol (Table 1) levels that were found to have decreased at the same time. In LDLR-/- mice, LDL particles tend to be larger (peak size, 29 to 30 nm) than those in wild-type C57BL/6 animals (peak size, 25 to 27 nm18 ). Patients with FH have been noted to have LDL particles that are enriched with cholesterol32 ; our observations in LDLR-/- mice suggest that Apobec1 overexpression can potentially eliminate these particles.
The studies presented herein suggest that Apobec1 may be an effective candidate therapeutic gene that will lower plasma apoB-100 and LDL in patients with FH. Furthermore, Hughes et al19 showed that overexpression of Apobec1 was also effective in removing plasma Lp(a), a highly atherogenic lipoprotein. However, caution should be exercised in the use of Apobec1 for somatic gene therapy. Yamanaka et al20 showed that massive overexpression of Apobec1 led to hepatocellular carcinoma and dysplasia in transgenic animals. They also demonstrated that low-level hepatic expression of Apobec1 is associated with normal hepatocellular histology. Our experience with Apobec1-transgenic mice also indicates that low-level expression is not associated with any hepatocellular neoplasms (P.P. Lau and L. Chan, unpublished observations). In normal mice, Apobec1 expression is very low. Therefore, if Apobec1 is to be used as a form of hepatic gene therapy, one should aim at low-level regulatable expression, which will be the objective in our future experiments.
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Acknowledgments |
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This research was supported by National Institutes of Health grants HL-53441 (to B.B.T.), HL-16512 (to L.C.), and HL-18574 for a program project at Lawrence Berkeley Laboratory. We thank Dr Margret Tiebel from Baylor College of Medicine and Laura Knoff from the University of California for their excellent technical assistance. We are also grateful to Dr Makoto Nakamuta from Baylor College of Medicine for his excellent graphic design and suggestions on statistical analysis. We also thank Dr Bruce Trapenel, Genetic Therapy, Inc, for providing the Av1LacZ4 adenoviral vector.
Received August 12, 1996; accepted November 25, 1996.
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References |
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