Expression of Human Apolipoprotein A-I/C-III/A-IV Gene Cluster in Mice Induces Hyperlipidemia but Reduces Atherogenesis

Abstract

Abstract-The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis.Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257Ϯ9, 7.1Ϯ0.5, and 1.0Ϯ0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (meanϮSEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB 48 -containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination. Key Words: transgenic mice Ⅲ hypertriglyceridemia Ⅲ cholesterol Ⅲ lipoproteins Ⅲ atherosclerosis P lasma lipoproteins influence the development of atherosclerosis, and their concentrations are associated with the risk of coronary heart disease. 1 The genes for 3 apolipoproteins, apoA-I, apoC-III, and apoA-IV, are grouped together in a cluster on 17 kb of human chromosome 11. 2 ApoA-I is the major protein component of HDL. Through its ability to promote cholesterol efflux from cultured cells and to activate lecithin:cholesterol acyltransferase (LCAT), it is involved in reverse cholesterol transport (RCT). 1 Expression of the human apoA-I ( h apoA-I) gene decreases the development of fatty lesions in cholesterol-fed transgenic (Tg) mice 3 and in apoE-deficient (apoE Ϫ/Ϫ ) mice. 4 ApoA-I deficiency in mice did not increase atherogenesis in a normal background 5 but did so in hypercholesterolemic mice expressing human apoB. 6 ApoC-III is a component of HDL and triglyceride-rich lipoproteins (TGRLs). Plasma apoC-III concentration is positively correlated with triglyceride concentration. By inhibiting TGRL catabolism, apoC-III induces hypertriglyceridemia in Tg mice. 7 Expression of the gene was not atherogenic in apoE Ϫ/Ϫ mice. 8 In contrast, h apoC-III expression, in normal or in LDL ...

The Expression of Intact and Mutant Human apoAI/CIII/AIV/AV Gene Cluster in Transgenic Mice

“…BAC DNA was suc- cessfully introduced into the mouse genome, and four stable transgenic mouse lines containing intact human apoAI/CIII/ AIV/AV cluster constructs were generated. In contrast to the studies on transgenic mice carrying a single apo gene or short 33-kb apoAI/CIII/AIV cluster constructs (20,(23)(24)(25), the apo gene expression exhibited an integrated position-independent pattern in our intact apoAI cluster transgenic mice. Moreover, the transgenes showed a closer approximation of copy numberdependent expression in our transgenic mice (Fig.…”

“…This apoAI expression level is similar to those in the report of transgenic mice carrying a 33-kb human apoAI/CIII/AIV in which intestinal apoAI mRNA did not exceed 20% of liver apoAI mRNA (20). However, in apoAI/CIII transgenic mice carrying only the apoCIII enhancer, 2.1-kb 5Ј region and 1.6-kb 3Ј region of the human apoAI gene, the apoAI expression level in the intestine was almost as high as that seen in the liver (23).…”

“…This observation is consistent with the tissue-specific expression of apoAIV in humans and in human apoAIV gene transgenic mice (30). In contrast, apoAIV was expressed mainly in the liver and at relatively low levels in the intestine in 33-kb human apoAI/CIII/AIV cluster transgenic mice that had the same 3Ј-flanking sequence as the human apoAIV gene transgenic mouse (20). Excluding possible differences in methods used to measure human apoAIV mRNA and the physiological variability of apoAIV mRNA levels, we think this discrepancy is worth noting, and it suggests that our large fragment used to establish the transgenic mice recapitulates the endogenous expression pattern more authentically.…”

“…In our intact apoAI gene cluster transgenic lines, plasma triglyceride level increased ϳ3-fold compared with control mice. This value is higher than that from the apoAI/CIII/AIV/AV transgenic mouse generated by Baroukh et al (21) but is less than that in single human apoCIII transgenic mice (5-20-fold) (6) and in the 33-kb human apoAI/CIII/ AIV transgenic mice (4 -10-fold) (20). In addition, in our mutant transgenic mouse, deletion of the apoCIII enhancer did not affect human apoAV expression but significantly reduced human apoCIII expression, thus leading to a lower triglyceride level compared with control mice (Table I).…”

“…Transgenic lines containing a 33-kb human apoAI/CIII/AIV gene cluster construct were recently generated, in which the expression of transgenes induced hyperlipidema and reduced atherosclerosis (20). This model carried only a relatively short genomic fragment and did not contain the newly identified apoAV.…”