© 1996 American Heart Association, Inc.
Articles |
Transgenic Rabbits Expressing Human Apolipoprotein A-I in the Liver
Rhone-Poulenc Rorer-Gencell, Cell and Gene Therapy Division, Atherosclerosis Department, Centre de recherche de Vitry-Alfortville, Vitry sur Seine (N.D., L.B., F.E., P.D.); Institut Pasteur, INSERM 325, Lille (A.T., L.P.-N., B.L., C.F., G.C., J.C.F.); and Institut National de la Recherche Agronomique, Jouy en Josas (C.V., L.M.H.), France.
Correspondence to Dr Nicolas Duverger, Atherosclerosis Department, Centre de Recherche de Vitry-Alfortville, 13, Quai Jules Guesde-BP 14, 94403 Vitry sur Seine, Cedex, France. E-mail nicolas.duverger@rp.fr.
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Abstract |
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Human apolipoprotein A-I (apo A-I) transgenic rabbits were created by use of an 11-kb genomic human apo A-I construct containing a liver-specific promoter. Five independent transgenic lines were obtained in which human apo A-I gene had integrated and was expressed. Plasma levels of human apo A-I ranged from 8 to 100 mg/dL for the founder and up to 175 mg/dL for the progeny. Rabbit apo A-I levels were substantially decreased in the transgenic rabbits. HDL cholesterol (HDL-C) levels were higher in two of the five transgenic rabbit lines than in controls (line 20 versus nontransgenic littermate, HDL-C=80±7 versus 37±6 mg/dL; line 8 versus nontransgenic littermate, HDL-C=54±16 versus 35±6 mg/dL). This resulted in less atherogenic lipoprotein profiles, with very low (VLDL+LDL-C)/HDL-C ratios. HDL size and protein and lipid compositions were similar between transgenic and littermate nontransgenic rabbits. However, a large amount of pre-ß apo A-I–containing lipoproteins was observed in the plasma of the highest human apo A-I expressor. Cell cholesterol efflux was evaluated with the incubation of whole serum from transgenic and control rabbits. Cell cholesterol efflux was highly correlated with HDL cholesterol, with apo A-I, and with the presence of pre-ß apo A-I–containing lipoproteins. These rabbits will be an extremely useful model for the evaluation of the effect of increased hepatic apo A-I expression on atherosclerosis.
Key Words: New Zealand White rabbit • apolipoprotein A-I • transgenic • cholesterol • lipoprotein
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Introduction |
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Prospective epidemiological studies in humans have suggested that high levels of HDL and apo A-I protect against the progression of atherosclerosis.1 2 HDL and its major protein component, apo A-I, are thought to directly limit the development of atherosclerosis. Badimon et al3 reported that repeated infusions of human HDL into cholesterol-fed rabbits result in less atherosclerosis after 9 weeks compared with controls. More recently, Rubin et al4 reported that transgenic mice overexpressing human apo A-I have less aortic atherosclerosis than controls when they are maintained on an atherogenic diet. However, more data from studies with animal models of atherosclerosis resembling the human disease are necessary to demonstrate the precise role of apo A-I in limiting the progression of this disease.
Rabbits, like humans, are "LDL mammals," in that the LDL fraction is the major carrier of cholesterol in plasma.5 In addition, rabbits, like humans, have cholesteryl ester transfer protein, which plays a central role in cholesterol distribution.6 An extensive body of information about lipoprotein metabolism and pathophysiology in rabbits is available. Considerable genetic variation in lipoprotein levels occurs among various lines of rabbits; there are strains having an LDL receptor defect (Watanabe rabbits)7 and others that overproduce apo B–containing lipoproteins (St Thomas rabbits).8 Furthermore, rabbits develop a human type of atherosclerotic lesion characterized by a large number of smooth muscle cells, necrotic and acellular lesion cores, extracellular matrix deposition, and occasionally, in older animals, superimposed thrombi.9 10 11 12 13 14
The effects of apo A-I overexpression on atherosclerosis in an animal model in which lipoprotein metabolism and atherosclerosis resemble those of humans remain to be established. Therefore, we undertook the generation of transgenic rabbits overexpressing human apo A-I. We report here the successful generation of rabbits expressing high levels of human apo A-I and the modification of cholesterol distribution in the lipoprotein fractions and endogenous apolipoproteins in these animals.
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Methods |
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Transgenic Animals
The production of transgenic rabbits was reported by Massoud et al15 and Knight et al.16 An approach similar to theirs was used for our project. New Zealand White adult female rabbits were superovulated by injection of 0.375 mg porcine follicle-stimulating hormone on day 1, 0.782 mg on day 2, and 0.375 mg on day 3. Rabbits were mated on the same day that luteinizing hormone was injected. Embryos were collected 17 hours later. The human apo A-I fragment of genomic DNA used for the microinjection was obtained from a partially digested fragment EcoRI DNA bank of K562 cells and inserted into bacteriophage Charon 4a. The human apo A-I fragment of genomic DNA was previously described by Rubin et al.17 This fragment contains the entire apo A-I gene and, in addition, contains 3.5 kb downstream from the 3' end of the apo A-I gene (1.8 kb) and 5.5 kb of 5'-flanking sequence. About 2 pL DNA solution containing the human apo A-I genomic fragment was injected into the male pronuclei. The injected embryos were transferred to pseudopregnant females.
Detection of the Transgene
DNA was extracted from rabbit tail fragments. The presence of the transgene was detected by polymerase chain reaction using 5'-TGGTTTCTCCAAGTGTCTTCAGGTGG-3' and 5'-GACAGCGGCAGAGACTATGTGTCCCAGTTTGAA-3' oligonucleotide primers specific for the human apo A-I gene. To isolate DNA, tissues were digested in lysis buffer (1% SDS, 100 mmol/L NaCl, 100 mmol/L EDTA, 50 mmol/L Tris, pH 8.0, and 200 µg/mL proteinase K) at 42°C overnight, extracted with phenol/chloroform (1:1 vol/vol), and precipitated in 2 vol ethanol. The copy number of the human apo A-I transgenes in each line of rabbits was estimated by quantitative Southern blot analysis. Genomic DNA (10 µg) was digested with Kpn I, subjected to electrophoresis in a 0.8% agarose gel, and transferred to a nylon membrane (Hybond-N, Amersham). Membranes were hybridized in 50% formamide at 42°C overnight with the 2.2-kb BamHI-BlgII fragment of the human apo A-I gene, which had been labeled by random priming. Filters were washed with 0.5xSSC and 0.1% SDS for 10 minutes at room temperature and twice for 30 minutes at 65°C, then exposed to x-ray film (Kodak X-OMAT-AR, Eastman Kodak Co) at -80°C.
Preparation and Analysis of RNA
Total RNA was extracted from transgenic and control rabbit liver, intestinal epithelium, and heart by the guanidinium isothiocyanate–phenol-chloroform procedure.18 Tissue RNA was further purified by an additional precipitation with 1 vol 8 mol/L LiCl for 3 hours at -20°C. For Northern blots, 10 µg denatured total RNA was subjected to electrophoresis in a 1.2% agarose/formaldehyde gel and transferred to a nylon membrane (Hybond-N, Amersham). The oligonucleotides 5'-GCCGACTGCTGGAAGGAGCGGA-3' and 5'-AGTGACCTGCCCCGCCTCCTGGAGC-3', specific for the rabbit apo A-I cDNA, were used as probe. Human apo A-I cDNA probe19 and GAPDH cDNA clone used as control probe were radiolabeled by a random-primer labeling kit (Boehringer Mannheim), and membranes were hybridized and washed as described for Southern blotting. The above-mentioned mixture of oligonucleotides specific for the rabbit apo A-I cDNA was radiolabeled by phosphorylation, and membranes were hybridized without formamide at 42°C and washed with 0.5xSSC and 0.1% SDS for 10 minutes at room temperature and twice for 30 minutes at 42°C.
Diet, Lipoprotein Measurements, and Cell Cholesterol Efflux Studies
All animals were maintained routinely on rabbit chow (Bonaliment), which contains 
2.4% fat, 16% protein, and 16.5% crude fiber. Blood from fasting animals was collected after an overnight fast into a tube containing 1 mg/mL EDTA, 50 mg/L gentamicin sulfate, and 0.1% sodium azide. Plasma was separated by centrifugation at 2000g for 15 minutes and kept at 4°C until analysis. All biochemical measurements were performed in triplicate.
Human apo A-I was quantified by rocket immunoelectrophoresis with rabbit polyclonal antibodies (Sebia). Total apo A-I was measured by immunonephelometric assay, for which five monoclonal antibodies (Diagnostics Pasteur Production) to apo A-I were used.20 These antibodies cross-reacted with rabbit apo A-I. This mixture of monoclonal antibodies precipitated 100% of rabbit HDL as well as human HDL in a radioimmunoassay.21 The reproducibility of apo A-I determinations was assessed from control quality data. The coefficients of variation between assays were 5.8% and 7.4% for human and total apo A-I, respectively. Plasma lipids (cholesterol, phospholipids, and triglycerides) and HDL cholesterol were measured colorimetrically with a microtiter plate reader and commercially available reagents (Boehringer Mannheim). All reaction products were spectrophotometrically quantified at 490 nm on a microplate reader (Bio-Teck Instruments EL 311).
Lipoprotein fractions were isolated by sequential ultracentrifugation in a Beckman TL 100 centrifuge.22 Particle sizes were determined by nondenaturing gradient polyacrylamide gel electrophoresis (PAGE).23 The specific protein composition of HDL was analyzed by 4% to 20% SDS-PAGE.24 The distribution of lipoprotein fractions containing apo A-I was determined by two-dimensional gel electrophoresis as previously described by Castro and Fielding.25 Apo A-I was identified by blot analysis with polyclonal antibodies to apo A-I and then with protein A labeled with 125I. For quantification of the distribution of apo A-I, radioactivity was detected by autoradiography at -70°C. Individually labeled membrane areas identified by film were subsequently cut out and counted by a Beckman 
-scintillation counter.
Cellular cholesterol efflux studies using whole sera and the Fu5AH rat hepatoma cell line were performed according to the system established by de la Llera Moya et al.26
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Results |
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Generation of Transgenic Rabbits
Five hundred microinjected embryos were transferred into 24 pseudopregnant females, 3 of which did not become pregnant. Forty-four rabbits were born (8.2% of the transferred embryos), 41 of which survived until after weaning; these were tested for the presence of the transgene. The transferred human DNA was detected by polymerase chain reaction in 6 founder rabbits, 5 males and 1 female (founders 8, 20, 31, 32, 38, and 40). The female founder, rabbit 40, died. Separate transgenic lines from the other 5 founder animals were established. Animals born to transgenic rabbits 20, 8, 31, 32, and 38 contained 5, 2, 1, <1, and 1 copies, respectively, of the human apo A-I transgene, as calculated from densitometric scanning of the Southern blot autoradiograph (data not shown).
Human and Rabbit Apo A-I Expression
To examine the expression of the human apo A-I transgene in the rabbits, total RNA was isolated from several tissues of 4-month-old transgenic (n=3 of each line) and littermate nontransgenic (n=3) female rabbits. Northern blot analysis (Fig 1
) with a human apo A-I probe showed that human apo A-I mRNA was present only in the liver of transgenic rabbits. The expression of rabbit apo A-I gene in the liver and intestine of transgenic and control rabbits is shown in Fig 2. The presence of rabbit apo A-I mRNA was detected in the intestine but not in the liver for both transgenic and control rabbits. In addition, no differences in rabbit apo A-I mRNA levels in the intestine were observed between transgenic and control rabbits.
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Lipoprotein Profiles of 3-Month-Old Rabbits
Plasma was analyzed to study the consequences of the transgene expression. High levels of human apo A-I were found in the plasma of the transgenic animals, as indicated in Table 1 for the founders and in Table 2 for the F1 progeny. Rabbit apo A-I plasma levels were calculated from the difference between total (human+rabbit) apo A-I and human apo A-I levels. As shown in Table 1, rabbit apo A-I levels were lower in transgenic rabbits than in controls. The plasma of rabbits expressing high levels of human apo A-I (founders 8 and 20) contained almost exclusively human apo A-I, whereas founder 32, which expressed less human apo A-I, had both human and rabbit apo A-I in its plasma. The rabbit expressing the highest amount of human apo A-I, rabbit 20, had a plasma level of human apo A-I twice the level of rabbit apo A-I usually found in the control rabbits. The plasma levels of total cholesterol and cholesterol of lipoprotein fractions in 3-month-old transgenic and control rabbits are presented in Table 1. Cholesterol-associated lipoprotein fractions in rabbits are highly variable. However, transgenic rabbits 8 and 20, which expressed the most human apo A-I, had a level of HDL cholesterol twofold higher than that of controls. These data are also found for the F1 progeny (Table 2). Using data from 27 transgenic rabbits (lines 31, 20, and 8, founder and F1 progeny), we found a correlation between HDL-C and human apo A-I (r=.770; P<.015).
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HDL Structure, Composition, and Size
Size distribution of isolated HDL from transgenic and control rabbits was assessed by nondenaturing gradient PAGE (Fig 3). HDL isolated from normal rabbit consists of one major population with a Stokes diameter of 10.2±0.8 nm. HDL isolated from transgenic rabbits had the same size distribution pattern as HDL from normal rabbits. This HDL distribution for transgenic rabbits was different from that obtained with HDL isolated from transgenic mice for human apo A-I, which consisted of two distinct and equal HDL subfractions with Stokes diameters of 10.6±0.4 and 8.5±0.3 nm, identical to the pattern of apo A-I without apo A-II–containing lipoproteins found in humans.27 17 The protein composition of HDL isolated from transgenic and nontransgenic rabbits was determined by SDS-PAGE. No difference in protein components was observed, except that human apo A-I replaced the endogenous apo A-I in transgenic rabbits (data not shown). Lipid and protein compositions of HDL were analyzed. The composition by weight for transgenic line 8 (n=3) was protein 51.8±10.2%, phospholipid 23.1±6.2%, cholesteryl ester 8.0±2.3%, free cholesterol 2.1±1.3%, and triglyceride 15.0±2.2%; for transgenic line 20 (n=16), it was protein 50.0±11.2%, phospholipid 22.9±6.5%, cholesteryl ester 7.9±2.4%, free cholesterol 2.0±1.4%, and triglyceride 17.2±3.2%; and for control rabbits (nonlittermates of lines 8 and 20, n=24), it was protein 51.8±9.0%, phospholipid 24.0±7.2%, cholesteryl ester 8.1±3.0%, free cholesterol 2.2±1.7%, and triglyceride 14.9±3.4%. There were no statistical differences in lipid and protein compositions between these three groups of animals.
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The presence of human apo A-I in pre-ß particles was determined by two-dimensional gel electrophoresis. As illustrated in Fig 4, the amount of apo A-I in pre-ß particles in the serum of transgenic rabbit 20 (pooled sera from three animals) was high and represented 23.4% of the total apo A-I in this serum. In contrast, the amount of apo A-I in pre-ß particles in the serum of transgenic rabbit 31 (pooled sera from three animals) was low and represented only <0.15% of the total apo A-I in this serum. The amount of apo A-I in pre-ß particles in the serum of transgenic rabbit 8 (pooled sera from three animals) was intermediary and represented 8.90% of the total apo A-I in this serum.
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Cellular Cholesterol Efflux
A time course of labeled cholesterol efflux from Fu5AH cells exposed to 2.5% serum of transgenic and control rabbits was performed. The data shown in Fig 5 represent the release of labeled cholesterol from Fu5AH cells incubated 2 or 4 hours with serum from transgenic rabbits of lines 8, 20, and 31 and nontransgenic littermate rabbits. All serum promoted cholesterol efflux. However, cell cholesterol effluxes obtained with the incubation of serum (n=3 for each line) from transgenic rabbit lines 8 and 20 (+19% and 30% of control at 2 hours, respectively) were significantly higher than those of controls. Relations between cholesterol efflux and the parameters non-HDL cholesterol level, HDL cholesterol level, concentration of apo A-I in pre-ß particles, and human apo A-I level were analyzed. Very high correlations were obtained between cholesterol efflux with HDL cholesterol levels (r=.926; P<.0035), human apo A-I levels (r=.885; P<.005), and the concentration of apo A-I in pre-ß particles (r=.973; P<.02), whereas no relation was found between cholesterol efflux and non-HDL cholesterol level.
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Discussion |
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The potential role of apo A-I and HDL in preventing atherosclerosis is of intense scientific interest and clinical importance. However, this role has been studied with transgenic technology only in mice. New models that are more similar to humans are needed for investigating this important question. We report here the generation of transgenic rabbits expressing human apo A-I, a model that will substantially advance our understanding of the role of apo A-I in lipoprotein metabolism and atherosclerosis.
In contrast to mice, rabbits have been extensively used as an animal model for the study of human lipoprotein metabolism and atherosclerosis. Rabbits are "LDL mammals" like humans, rather than "HDL mammals" like mice. Furthermore, rabbits produce cholesteryl ester transfer protein, and their HDL metabolism is similar to that of humans. Finally, both cholesterol-fed and genetically mutant (Watanabe and St Thomas strain) rabbits develop a form of atherosclerosis that resembles the human disease.9 10 11 12 13 14 In this study, we describe apo A-I transgenic rabbits, a potential new "antiatherogenesis" animal model, and report significant differences in human apo A-I metabolism and cholesterol distribution between rabbits transgenic for human apo A-I and observations reported in mice transgenic for human apo A-I.17 28
Rabbit endogenous apo A-I is secreted primarily from the intestine. In contrast, most species, including humans, secrete apo A-I from both the intestine and the liver.29 30 Reisher et al31 showed that rabbit liver contains the necessary trans-acting factors for apo A-I gene expression. In rabbits, hepatic apo A-I gene expression is downregulated by nonparenchymal cell paracrine factor.32 However, the genomic DNA fragment of human apo A-I we used to produce transgenic rabbits resulted in its hepatic expression.17 28 These data were consistent with the study of Walsh et al,33 which reported that the intestinal expression of the human apo A-I gene is not directed by this genomic DNA fragment but rather is controlled by a DNA region 3' to the gene in the promoter of the adjacent convergently transcribed apo C-III gene. Given that only one to five copies of the human apo A-I gene were integrated in the rabbit, human apo A-I is apparently not influenced by the same factors that limit the secretion of rabbit apo A-I to the intestine. The 5' flanking region of human apo A-I has been demonstrated to function as a liver transcriptional enhancer.34 Therefore, despite a high sequence homology (>78%) of the 200 bp of the 5' upstream flanking region of the rabbit and human apo A-I genes, the nonparenchymal cell–secreted factor that attenuates rabbit hepatic apo A-I gene expression may not bind to this human apo A-I gene fragment.
We observed a reduction of endogenous rabbit apo A-I plasma levels in the transgenic rabbits. This has also been noted in transgenic mice overexpressing human apo A-I.17 However, in contrast to mice, rabbit apo A-I is derived primarily from the intestine, whereas human apo A-I in rabbits is derived solely from the liver. The level of intestinal rabbit apo A-I mRNA was not reduced, indicating that the rabbit apo A-I plasma level is subject to posttranscriptional or translational regulation. All steps in the pathways from endogenous apo A-I synthesis to catabolism could be altered in the transgenic animals. However, preliminary kinetic studies of human and rabbit apo A-I in transgenic rabbits did not show differences in catabolic rates (data not shown), and this may suggest a default in rabbit apo A-I production.
Human apo A-I can be incorporated into rabbit HDL complexes. Rabbit plasma contains very little apo A-II, and despite the high homology of human and rabbit apo A-I,35 rabbit apo A-I–containing lipoproteins do not look like apo A-II–free apo A-I–containing lipoproteins in humans.27 Also, unlike human apo A-I distribution in human apo A-I transgenic mice,17 we did not observe an appearance of two distinct subclasses of HDL size. Both transgenic and control rabbits have large HDL particles. Lipoprotein lipase and hepatic lipase (HL) are implicated in changing the size of HDL.36 37 38 Large HDL concentrations in humans correlate negatively with the postheparin activity of HL.37 Rabbits have low HL activity (15% of the corresponding value in human postheparin plasma).39 40 Therefore, the distribution of human or rabbit apo A-I–containing lipoprotein may reflect the low activity of HL in this species. Recently, human HL has been overexpressed in transgenic rabbits, and a marked reduction of HDL size has been observed in these rabbits.41 An increase of HL in human apo A-I transgenic rabbits may restore the human apo A-I–containing lipoprotein distribution, as observed in humans.
HDLs are heterogeneous in composition and structure. The functions of each of these HDL species in the process of reverse cholesterol transport are not well defined. One way that HDL species can be differentiated is on the basis of their electrophoretic mobility.42 Previous studies have shown that HDL can be divided into two major subfractions, those with an 
-electrophoretic mobility (-HDL) and those with pre-ß mobility (pre-ß HDL). Pre-ß HDLs are small, protein-rich lipoproteins that are composed predominantly of apo A-I without apo A-II and are structurally distinct from -HDL. Although the majority of apo A-I–containing lipoproteins in plasma are -HDL, pre-ß HDLs are also normal components of human plasma. The average concentration of pre-ß HDL is about 2% to 5% of total apo A-I in normolipidemic individuals.43 44 45 Pre-ß HDLs have been identified in several species, such as mice, monkeys, and dogs.46 47 48 For human apo A-I transgenic rabbits, we observed the appearance of large amounts of pre-ß HDL that correspond to 9% and 28% of human apo A-I for transgenic rabbit lines 8 and 20, respectively. These pre-ß HDLs were not observed in HDL prepared by the ultracentrifugal flotation method. This technique is known to perturb lipoproteins, at least pre-ß HDLs, which are unstable and rapidly converted to -HDL.49 Also, ultracentrifugation does not isolate apo A-I–containing lipoproteins that fall outside the range of 1.063<d<1.210 g/mL. The high levels of pre-ß HDL found in transgenic rabbits could be a consequence of the lack of apo A-II,50 which is associated primarily with apo A-I in -migrating HDL, reducing the availability of apo A-I for pre-ß HDL. Pre-ß HDLs have been postulated to serve a key role in the process of reverse cholesterol transport by acting as initial acceptors of cellular unesterified cholesterol.25 In the present cell cholesterol efflux studies, we show that the cholesterol efflux was correlated to total apo A-I level, HDL cholesterol concentration, and apo A-I in pre-ß HDL levels.
In conclusion, we have created several lines of transgenic rabbits overexpressing human apo A-I. These rabbits will continue to be a valuable source for investigating the physiological role of apo A-I in atherosclerosis.
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Acknowledgments |
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This work was supported by the BIO/AVENIR program financed by Rhone-Poulenc-Rorer SA and the Ministere de la Recherche l'Enseignement Superieur. We gratefully acknowledge the assistance of Florence Attenot, Isabelle Viry, Bruno Derudas, Catherine De Geitere, Herve Pointu, and Stephan Cuine for excellent technical assistance.
Received July 26, 1995;
revision received April 10, 1996;
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