apoB Transgenic Mice Research Articles

The NH2-terminal Region of Apolipoprotein B Is Sufficient for Lipoprotein Association with Glycosaminoglycans

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. The co-localization of apolipoprotein (apo) B and proteoglycans within lesions has suggested that retention is due to lipoprotein interaction with these highly electronegative glycoconjugates. Both apoB100- and apoB48-containing lipoproteins, i.e. low density lipoproteins (LDLs) and chylomicron remnants, are atherogenic. This suggests that retention is due to determinants in the initial 48% of apoB. To test this, the interaction of an apoB fragment (apoB17), and apoB48- and apoB100- containing lipoproteins with heparin, subendothelial matrix, and artery wall purified proteoglycans was studied. ApoB100-containing LDL from humans and human apoB transgenic mice and apoB48-containing LDLs from apoE knockout mice were used. Despite the lack of the carboxyl-terminal 52% of apoB, the apoB48-LDL bound to heparin-affinity gel as well as did apoB100-LDL. An NH2-terminal fragment containing 17% of full-length apoB was made using a recombinant adenovirus; apoB17 bound to heparin as well as did LDL. Monoclonal antibodies against the NH2-terminal region of apoB decreased apoB100 LDL binding to heparin, whereas antibodies against the LDL receptor-binding region did not alter LDL-heparin interaction. The role of the NH2-terminal region of apoB in LDL interaction with matrix molecules was also assessed. Media containing apoB17 decreased LDL binding to subendothelial matrix by 42%. Moreover, removal of the apoB17 by immunoprecipitation abrogated the inhibitory effect of these media. Antibodies to the NH2-terminal region decreased LDL binding to matrix and dermatan sulfate proteoglycans. Purified apoB17 effectively competed for binding of LDL to artery derived decorin and to subendothelial matrix. Thus, despite the presence of multiple basic amino acids near the LDL receptor-binding domain of LDL, the NH2-terminal region of apoB is sufficient for the interaction of lipoproteins with glycoconjugates produced by endothelial and smooth muscle cells. The presence of a proteoglycan-binding site in the NH2-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.

Low expression of the apolipoprotein B mRNA-editing transgene in mice reduces LDL levels but does not cause liver dysplasia or tumors.

Hepatic expression of apolipoprotein (apo) B mRNA-editing enzyme catalytic polypeptide 1 (APOBEC-1) has been proposed as a gene therapy approach for lowering plasma low density lipoprotein (LDL) levels. However, high-level expression of APOBEC-1 in transgenic mouse and rabbit livers causes liver dysplasia and hepatocellular carcinoma. To determine the physiological and pathological effects of low-level hepatic expression of APOBEC-1, we used a 52-kb rat APOBEC-1 genomic clone (RE4) to generate transgenic mice expressing low levels of APOBEC-1 (2 to 5 times those in nontransgenic mice). Liver function, liver histology, editing of apoB mRNA at the normal editing site (C6666), and abnormal editing at multiple sites (hyperediting) in these mice were compared with those in transgenic mice expressing intermediate (I-20) or high (I-28) levels of APOBEC-1 in the liver. Hyperediting of mRNA coding for the novel APOBEC-1 target 1 (NAT1) was also examined. In the high-expressing I-28 line, 50% of the mice had palpable tumors at 15 weeks of age, whereas in the intermediate-expressing I-20 line, 50% of the mice had evidence of liver tumors after 1 year. In contrast, low-expressing RE4 mice had normal liver function and histology and did not develop liver tumors when examined at 3 to 17 months of age. Moreover, hyperediting of apoB and NAT1 mRNA in the liver was robust in the I-20 mice but barely detectable in the RE4 mice. The low-level expression resulted in sufficient APOBEC-1 to edit essentially all apoB mRNA at the normal editing site, virtually eliminating apoB-100 and LDL in the plasma of RE4 mice. When RE4 mice were crossed with human apoB transgenic mice, which possess high plasma LDL concentrations, plasma LDL levels in the offspring were reduced to very low levels. These results indicates that long-term hepatic expression of APOBEC-1 at low levels sufficient to eliminate LDL does not cause apparent liver damage or liver tumors in transgenic mice. RE4 APOBEC-1 transgenic mice should prove valuable for studying the roles of apoB-containing lipoproteins in lipid metabolism and atherosclerosis.

ApoA-I deficiency causes both hypertriglyceridemia and increased atherosclerosis in human apoB transgenic mice

To study the role of low levels of high density lipoprotein (HDL) and apolipoprotein (apo) A-I in atherosclerosis risk, human apoB transgenic mice (HuBTg) were crossed with apoA-I-deficient (apoA-I–/–) mice. After a high fat challenge, total cholesterol levels increased drastically due to an increase in the non-HDL cholesterol as confirmed by FPLC analysis. In addition, total cholesterol levels in A-I–/– HuBTg mice were lower than the control HuBTg mice, due mainly to decreased HDL-C in A-I–/– HuBTg mice. Analysis of atherosclerosis in the proximal aorta in mice fed a high-fat Western-type diet for 27 weeks revealed a 200% greater lesion area in female apoA-I–/– HuBTg mice (49740 ± 9751 μm2) compared to control HuBTg mice (23320 ± 4981 μm2, P = 0.03). Lesion size (12380 ± 3281 μm2) in male A-I–/– HuBTg mice was also about 200% greater than that in the control HuBTg mice (5849 ± 1543 μm2), although not statistically significant. Very few and small lesions were observed in both apoA-I–/– HuBTg and control HuBTg animals fed a chow diet. Therefore, the adverse effect of low HDL on atherosclerosis in mice was only evident when LDL-cholesterol was markedly elevated by high-fat challenge. Male apoA-I–/– HuBTg mice exhibited hypertriglyceridemia when challenged with a high-fat diet. This correlated with both a reduction in lipoprotein lipase activity and a decrease in lipoprotein lipase activation by HDL. In summary, low high density lipoprotein levels due to apolipoprotein A-I deficiency exacerbated the development of atherosclerotic lesions in mice with elevated atherogenic lipoproteins. This mouse model mimics human conditions associated with low HDL levels and provides additional evidence for the anti-atherogenic role of apoA-I.—Voyiaziakis, E., I. J. Goldberg, A. S. Plump, E. M. Rubin, J. L. Breslow, and L-S. Huang. ApoA-I deficiency causes both hypertriglyceridemia and increased atherosclerosis in human apoB transgenic mice.

Overexpression of Hepatic Lipase in Transgenic Mice Decreases Apolipoprotein B-containing and High Density Lipoproteins: EVIDENCE THAT HEPATIC LIPASE ACTS AS A LIGAND FOR LIPOPROTEIN UPTAKE

To determine the mechanisms by which human hepatic lipase (HL) contributes to the metabolism of apolipoprotein (apo) B-containing lipoproteins and high density lipoproteins (HDL) in vivo, we developed and characterized HL transgenic mice. HL was localized by immunohistochemistry to the liver and to the adrenal cortex. In hemizygous (hHLTg+/0) and homozygous (hHLTg+/+) mice, postheparin plasma HL activity increased by 25- and 50-fold and plasma cholesterol levels decreased by 80% and 85%, respectively. In mice fed a high fat, high cholesterol diet to increase endogenous apoB-containing lipoproteins, plasma cholesterol decreased 33% (hHLTg+/0) and 75% (hHLTg+/+). Both apoB-containing remnant lipoproteins and HDL were reduced. To extend this observation, the HL transgene was expressed in human apoB transgenic (huBTg) and apoE-deficient (apoE-/-) mice, both of which have high plasma levels of apoB-containing lipoproteins. (Note that the huBTg mice that were used in these studies were all hemizygous for the human apoB gene.) In both the huBTg,hHLTg+/0 mice and the apoE-/-,hHLTg+/0 mice, plasma cholesterol decreased by 50%. This decrease was reflected in both the apoB-containing and the HDL fractions. To determine if HL catalytic activity is required for these decreases, we expressed catalytically inactive HL (HL-CAT) in apoE-/- mice. The postheparin plasma HL activities were similar in the apoE-/- and the apoE-/-,HL-CAT+/0 mice, reflecting the activity of the endogenous mouse HL and confirming that the HL-CAT was catalytically inactive. However, the postheparin plasma HL activity was 20-fold higher in the apoE-/-,hHLTg+/0 mice, indicating expression of the active human HL. Immunoblotting demonstrated high levels of human HL in postheparin plasma of both apoE-/-,hHLTg+/0 and apoE-/-,HL-CAT+/0 mice. Plasma cholesterol and apoB-containing lipoprotein levels were approximately 60% lower in apoE-/-,HL-CAT+/0 mice than in apoE-/- mice. However, the HDL were only minimally reduced. Thus, the catalytic activity of HL is critical for its effects on HDL but not for its effects on apoB-containing lipoproteins. These results provide evidence that HL can act as a ligand to remove apoB-containing lipoproteins from plasma.

Vascular accumulation of Lp(a): in vivo analysis of the role of lysine‐binding sites using recombinant adenovirus

Despite the importance of lipoprotein(a) [Lp(a)] as an atherogenic risk factor, very little information, especially from in vivo studies, is available concerning which structural features of apo(a) contribute to the interactions of Lp(a) with the vessel wall and its proatherogenic properties. Nearly all the proposed and proven activities of apolipoprotein(a) [apo(a)] focus on its high degree of sequence homology with plasminogen and the possibility that structural features shared by these two molecules contribute to the atherogenesis associated with high Lp(a) plasma levels in humans. In these studies, we examined the properties of three forms of Lp(a) differing at postulated lysine-binding domains contained in the constituent apo(a). We used the recombinant adenoviral gene delivery system to produce apo(a) in the plasma of human apoB transgenic mice, resulting in high levels of Lp(a) similar to those found in the plasma of humans. By comparison of in vitro lysine-binding properties of these forms of Lp(a) with measurements of Lp(a) vascular accumulation in the mice, we have demonstrated that lysine-binding defective forms of Lp(a) have a diminished capacity for vascular accumulation in vivo.

Lipoprotein(a) vascular accumulation in mice. In vivo analysis of the role of lysine binding sites using recombinant adenovirus.

Although the mechanism by which lipoprotein(a) [Lp(a)] contributes to vascular disease remains unclear, consequences of its binding to the vessel surface are commonly cited in postulated atherogenic pathways. Because of the presence of plasminogen-like lysine binding sites (LBS) in apo(a), fibrin binding has been proposed to play an important role in Lp(a)'s vascular accumulation. Indeed, LBS are known to facilitate Lp(a) fibrin binding in vitro. To examine the importance of apo(a) LBS in Lp(a) vascular accumulation in vivo, we generated three different apo(a) cDNAs: (a) mini apo(a), based on wild-type human apo(a); (b) mini apo(a) containing a naturally occurring LBS defect associated with a point mutation in kringle 4-10; and (c) human- rhesus monkey chimeric mini apo(a), which contains the same LBS defect in the context of several additional changes. Recombinant adenovirus vectors were constructed with the various apo(a) cDNAs and injected into human apoB transgenic mice. At the viral dosage used in these experiments, all three forms of apo(a) were found exclusively within the lipoprotein fractions, and peak Lp(a) plasma levels were nearly identical (approximately 45 mg/dl). In vitro analysis of Lp(a) isolated from the various groups of mice confirmed that putative LBS defective apo(a) yielded Lp(a) unable to bind lysine-Sepharose. Quantitation of in vivo Lp(a) vascular accumulation in mice treated with the various adenovirus vectors revealed significantly less accumulation of both types of LBS defective Lp(a), relative to wild-type Lp(a). These results indicate a correlation between lysine binding properties of Lp(a) and vascular accumulation, supporting the postulated role of apo(a) LBS in this potentially atherogenic characteristic of Lp(a).

Hypolipidemic and Hyperlipidemic Phenotypes in Transgenic Mice Expressing Human Apolipoprotein E2

Transgenic mice were produced that expressed different plasma levels (3-60 mg/dl) of human apolipoprotein (apo) E2(Arg158 --> Cys), which is associated with the recessive form of human type III hyperlipoproteinemia (HLP). In transgenic mice fed a normal chow diet, low levels of apoE2 (<10 mg/dl) did not significantly alter the lipid phenotype. Mice expressing intermediate levels of apoE2 (10-30 mg/dl) had a 50-60% decrease in total cholesterol compared with nontransgenic mice. The decrease was almost entirely due to a reduction in high density lipoprotein (HDL) cholesterol. These hypolipidemic apoE2 transgenic mice were cross-bred with human apoB transgenic mice, which have increased total cholesterol and low density lipoprotein (LDL) levels. The apoE2/apoB double transgenics revealed that expression of apoE2 on the background of human apoB overexpression resulted in a substantial decrease in LDL and HDL cholesterol and a corresponding accumulation of very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL). Thus, the double transgenics had a lipid phenotype resembling human type III HLP. In contrast to the hypolipidemic mice, mice expressing high levels of apoE2 (>50 mg/dl) were hyperlipidemic. The VLDL and IDL in these mice were significantly increased and cholesterol-enriched and had other characteristics of remnant lipoproteins. Upon agarose gel electrophoresis, the VLDL and IDL from both intermediate and high expressers migrated more slowly toward the beta position compared with the pre-beta-mobility of nontransgenic mouse VLDL and IDL. Thus, depending on plasma apoE2 levels, the expression of human apoE2 in the transgenic mice leads to either a hypolipidemic or hyperlipidemic phenotype. This animal model provides the opportunity to study the factors that cause hypolipidemia and those that precipitate the hyperlipidemia of type III HLP.

Complete Phenotypic Characterization of apobec-1 Knockout Mice with a Wild-type Genetic Background and a Human Apolipoprotein B Transgenic Background, and Restoration of Apolipoprotein B mRNA Editing by Somatic Gene Transfer of Apobec-1

We have produced gene knockout mice by targeted disruption of the apobec-1 gene. As recently reported by Hirano et al. (Hirano, K.-I., Young, S. G., Farese, R. V., Jr., Ng, J., Sande, E., Warburton, C., Powell-Braxton, L. M., and Davidson, N. O. (1996) J. Biol. Chem. 271, 9887-9890), these animals do not edit apolipoprotein (apo) B mRNA or produce apoB-48. In this study we have performed a detailed analysis of the lipoprotein phenotypic effects of apobec-1 gene disruption that were not examined in the previous study. We first analyzed the plasma lipoproteins in knockout animals with a wild-type genetic background. Although there was no difference in plasma cholesterol between apobec-1(-/-), +/-, or +/+ mice, there was a marked (176%) increase in plasma apoB-100, from 1.8 +/- 1.2 mg/dl in apobec-1(+/+) mice to 2.7 +/- 0.6 mg/dl in apobec-1(+/-) and 5.0 +/- 1.4 mg/dl in apobec-1(-/-) mice. Plasma apoE was similar in these animals. By fast protein liquid chromatography (FPLC) analysis, there was a significant decrease in plasma high density lipoprotein (HDL) cholesterol in apobec-1(-/-) mice. We further fractionated the plasma lipoproteins into d < 1.006, 1.006-1.02, 1.02-1.05, 1.05-1.08, 1.08-1.10, and 1.10-1.21 g/ml classes, and found a marked (30-40%) reduction in the cholesterol and protein content in the (d 1.08-1.10 and 1.10-1.21) HDL fractions, corroborating the FPLC data. SDS-gel analysis revealed an absence of apoB-48, an increase in apoB-100 in the very low density lipoprotein (VLDL) and low density lipoprotein (LDL) fractions, and a small decrease in apoA-I in the HDL fractions in the apobec-1(-/-) samples. We next raised the basal plasma apoB levels in the apobec-1(-/-) animals by cross-breeding them with human apoB transgenic (TgB) mice. The plasma apoB-100 was 3-fold higher in apobec-1(-/-)/TgB+/- mice (26.6 +/- 18.3 mg/dl) than in apobec-1(+/+)/TgB+/- mice (9.8 +/- 3.9 mg/dl, p < 0.05). The apobec-1(-/-)/TgB+/- mice had a plasma cholesterol levels of 170 +/- 28 mg/dl and triglyceride levels of 106 +/- 31 mg/dl, which are 80% and 58% higher, respectively, than the corresponding values of 94 +/- 21 mg/dl and 67 +/- 11 mg/dl in apobec+/+/TgB+/- mice. By FPLC, the apobec-1(-/-)/TgB+/- animals developed markedly elevated plasma LDL cholesterol (518.5 +/- 329.5 microg/ml) that is 373% that of apobec1(+/+)/TgB+/- mice (139.0 +/- 87.0 microg/ml) (p < 0.05). The elevated plasma triglyceride was accounted for mainly by a 97% increase in VLDL triglyceride in the apobec1(-/-)/TgB+/- mice. We conclude that apobec-1(-/-) animals have a distinctive lipoprotein phenotype characterized by significant hyperapoB-100 and HDL deficiency in mice with a wild-type genetic background. Furthermore, the abolition of apoB mRNA editing elevates plasma total cholesterol and LDL cholesterol in apobec-1(-/-) animals with a TgB background. Finally, to exclude the possibility that absence of apoB mRNA editing was a secondary effect of chronic Apobec-1 deficiency, we treated apobec-1(-/-) mice with a replication-defective mouse Apobec-1 adenoviral vector and found that we could acutely restore apoB mRNA editing in the liver. These experiments indicate that Apobec-1 is an essential component of the apoB mRNA editing machinery and absence of editing in the knockout animals is a direct consequence of the absence of functional Apobec-1.

Transgenic Mice That Overexpress Mouse Apolipoprotein B: EVIDENCE THAT THE DNA SEQUENCES CONTROLLING INTESTINAL EXPRESSION OF THE APOLIPOPROTEIN B GENE ARE DISTANT FROM THE STRUCTURAL GENE

An 87-kilobase (kb) P1 bacteriophage clone (p649) spanning the mouse apolipoprotein (apo) B gene was used to generate transgenic mice that express high levels of mouse apoB. Plasma levels of apoB, low density lipoprotein cholesterol, and low density lipoprotein triglycerides were increased, and high density lipoprotein cholesterol levels were decreased in the transgenic mice, compared with nontransgenic littermate controls. Although p649 contained 33 kb of 5'-flanking sequences and 11 kb of 3'-flanking sequences, the tissue pattern of transgene expression was different from that of the endogenous apoB gene. RNA slot blots and RNase protection analysis indicated that the transgene was expressed in the liver but not in the intestine, whereas the endogenous apoB gene was expressed in both tissues. To confirm the absence of transgene expression in the intestine, the mouse apoB transgenic mice were mated with the apoB knockout mice, and transgenic mice that were homozygous for the apoB knockout mutation were obtained. Because of the absence of transgene expression in the intestine, those mice lacked all intestinal apoB synthesis, resulting in a marked accumulation of fats within the intestinal villus enterocytes. The current studies, along with prior studies of human apoB transgenic animals, strongly suggest that the DNA sequence element(s) controlling intestinal expression of the apoB gene is located many kilobases from the structural gene.

Using genetically modified mice to study apolipoprotein B.

The B apolipoproteins, apo-B48 and apo-B100, are key proteins in mammalian lipoprotein metabolism and are components of all classes of lipoproteins considered to be atherogenic. Our laboratory has generated an array of genetically modified mice for studying apo-B biology. Using gene targeting in mouse embryonic stem cells, we have generated apo-B-deficient mice. Heterozygotes had low plasma levels of apo-B and cholesterol; homozygotes died early in embryonic development, most likely because the absence of lipoprotein secretion by the yolk sac interfered with the delivery of lipid nutrients to the developing embryo. We have also generated human apo-B transgenic mice with an 80-kb genomic DNA fragment spanning the entire human apo-B gene; those mice had markedly increased plasma levels of low density lipoprotein cholesterol and exhibited increased susceptibility to atherosclerosis. The human apo-B transgenic mice have also yielded insights regarding the regulation of apo-B expression in different tissues. Although the 80-kb transgene contained nearly 20 kb of 5' and 3' flanking sequences and was expressed at high levels in the liver, no transgene expression was detectable in the intestine. Subsequent transgenic mouse studies have demonstrated that the expression of the apo-B gene in the intestine is controlled by DNA sequences that are very distant from the structural gene. Transgenic mice have also proved useful for studying apo-B structure/function relationships. By expressing mutant forms of human apo B in transgenic mice, we have examined the structural features of the apo-B molecule that are required for lipoprotein (a) formation. We have demonstrated that the carboxyl terminal cystine residue of apo-B100, cysteine-4326, is required for apo-B100's disulfide linkage with apo(a) to form lipoprotein (a). Finally, we have used gene targeting techniques to generate mice that synthesize exclusively apo-B48 (apo B48-only mice) and mice that synthesize exclusively apo-B100 (apo-B100 only mice): These mice have helped to clarify the unique metabolic roles of the two apo-B proteins.

Site-specific Mutagenesis Demonstrates That Cysteine 4326 of Apolipoprotein B Is Required for Covalent Linkage with Apolipoprotein(a) in Vivo

In the formation of the lipoprotein(a) (Lp(a)) particle, apolipoprotein(a) (apo(a)) and apolipoprotein B (apoB) are covalently linked via a disulfide bond in both humans and human-apo(a)/apoB transgenic mice. Studies based upon fluorescent labeling of free cysteine residues have suggested that cysteine 3734 of the 4 carboxyl-terminal cysteines of apoB (Cys-3734, Cys-3890, Cys-4190, and Cys-4326) is the most likely candidate to form a disulfide bond with apo(a). However, other recent studies using truncated apoB molecules suggest that Cys-4326, the terminal cysteine of apoB, may be implicated in the binding to apo(a). In order to definitively show which of apoB's carboxyl-terminal cysteines is essential in interacting with apo(a) we have used RecA-assisted restriction enzyme digestion coupled with site-specific mutagenesis to convert Cys-3734 and Cys-4326 to serine within separate 90-kilobase pair apoB P1 phagemid clones. Transgenic mice containing the normal or mutated apoB transgenes were created, and the covalent association of mutated apoB with apo(a) was assessed in mice transgenic for both apoB and apo(a). Analysis by ultracentrifugation and immunoblotting revealed that Cys-4326, but not Cys-3734, was essential in the formation of the covalent bond between apo(a) and apoB in vivo.

Transgenic mice expressing human apoB100 and apoB48

Transgenic mice that express human apolipoprotein (apo)B have been developed by microinjecting fertilized mouse oocytes with an 80 kb genomic DNA fragment that encompasses the entire human APOB gene. In the transgenic mice expressing the largest amounts of human apoB, the concentration of human apoB100 in the plasma is nearly as high as the levels observed in normolipidemic humans (50 mg/dl). Virtually all of the human apoB100 in the transgenic plasma is located in the LDL fraction, resulting in substantially increased levels of LDL cholesterol. These human apoB-transgenic mice should be useful animal models for understanding various aspects of lipoprotein metabolism and for further delineating the role of LDL in atherogenesis.