Targeted Replacement of Mouse Apolipoprotein A-I with Human ApoA-I or the Mutant ApoA-IMilano: EVIDENCE OF APOA-IM IMPAIRED HEPATIC SECRETION

Cinzia Parolini,Giulia Chiesa,Yiwen Zhu,Trudy Forte,Silvia Caligari,Elisabetta Gianazza,Maria Grazia Sacco,Cesare R Sirtori,Edward M Rubin

doi:10.1074/jbc.m207335200

Abstract

Despite a pro-atherogenic profile, individuals carrying the molecular variant (R173C) of apolipoprotein (apo)A-I, named apoA-I(Milano) (apoA-I(M)), appear to be at reduced risk for cardiovascular disease. To develop an in vivo system to explore, in a controlled manner, the effects of apoA-I(M) on lipid metabolism, we have used the gene targeting technology, or "gene knock-in" (gene k-in), to replace the murine apoA-I gene with either human apoA-I or apoA-I(M) genes in embryonic stem cells. As in human carriers, mice expressing apoA-I(M) (A-I(M) k-in) are characterized by low concentrations of the human apolipoprotein and reduced high density lipoprotein cholesterol levels, compared with A-I k-in animals. The aim of the present study was to investigate the basic mechanisms of hypoalphalipoproteinemia associated with the apoA-I(M) mutation. ApoA-I and apoA-I(M) mRNA expression, as assessed by Northern blot analysis and quantitative real time reverse transcription-PCR, did not exhibit significant differences in either liver or intestine. Moreover, human apolipoprotein synthesis rates were similar in the k-in lines. When the secretion rate of the human apolipoproteins was assessed in cultured hepatocytes from the mouse lines, secretion from apoA-I(M)-expressing cells was markedly reduced (42% for A-I(M) k-in and 36% for A-I/A-I(M) k-in mice) as compared with that of A-I k-in hepatocytes. These results provide the first evidence that the hypoalphalipoproteinemia in apoA-I(M) human carriers may be partially explained by impaired apoA-I(M) secretion.

Highlights

Despite a pro-atherogenic profile, individuals carrying the molecular variant (R173C) of apolipoproteinA-I, named apoA-IMilano, appear to be at reduced risk for cardiovascular disease
These results provide the first evidence that the hypoalphalipoproteinemia in apoA-IM human carriers may be partially explained by impaired apoA-IM secretion
In contrast to classical transgenic approaches, gene targeting replacement strategies [52] for manipulating the mouse genome allow precise location of the transgene, permitting direct comparisons between different genes at the same chromosomal location. This procedure has allowed, for the first time, the generation of two animal models that differ only in the biochemical nature of the apoA-I, i.e. carrying in one case the human wild type apoA-I gene (A-I k-in) and, in the other, the apoA-IM gene (A-IM k-in). These mice provide a means to study the molecular mechanisms responsible for the lipoprotein abnormalities noted in apoA-IM human carriers

Summary

Introduction

Despite a pro-atherogenic profile, individuals carrying the molecular variant (R173C) of apolipoprotein (apo)A-I, named apoA-IMilano (apoA-IM), appear to be at reduced risk for cardiovascular disease. When the secretion rate of the human apolipoproteins was assessed in cultured hepatocytes from the mouse lines, secretion from apoA-IM-expressing cells was markedly reduced (42% for A-IM k-in and 36% for A-I/A-IM k-in mice) as compared with that of A-I k-in hepatocytes These results provide the first evidence that the hypoalphalipoproteinemia in apoA-IM human carriers may be partially explained by impaired apoA-IM secretion. Several naturally occurring mutations associated with reduced plasma HDL-C and apoA-I concentrations have been described for human apolipoprotein (apo)A-I [22, 23], the major protein constituent of HDL Some of these hypoalphalipoproteinemic states are associated with an increased risk of atherosclerotic vascular disease, others do not seem to predispose to accelerated premature disease [24]. The presence of a cysteine residue results in the formation of homodimers and heterodimers with apoA-II

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