Apolipoprotein E (apoE)-deficient mice develop marked hyperlipidemia as well as atherosclerosis and thus are an excellent animal model for evaluating the potential for gene therapy in human genetic dyslipoproteinemias. Recombinant adenovirus containing either human apoE (rAdv.apoE) or the reporter gene luciferase (rAdv.luc) were generated and infused intravenously in apoE-deficient mice with preinfusion plasma total cholesterol of 644±149 mg/dl and cholesterol rich VLDL/IDL. After a single infusion of rAdv.apoE, plasma concentrations of human apoE ranging from 1.5 to 650 mg/dl were achieved. Adenovirus-mediated apoE replacement resulted in normalization of the lipid and lipoprotein profile with markedly decreased total cholesterol (103±18 mg/dl), VLDL, IDL, and LDL, as well as increased HDL. Measurement of aortic atherosclerosis 1 mo after adenoviral infusion demonstrated a marked reduction in the mean lesion area of mice infused with rAdv.apoE (58±8 X 103 ,um2) when compared with control mice infused with rAdv.luc (161±19 X 103 ,um2; P < 0.0001). Thus, apoE expression for 4 wk was sufficient to markedly reduce atherosclerosis, demonstrating the feasibility of gene therapy for correction of genetic hyperlipidemias resulting in atherosclerosis. The combined use of adenovirus vectors and the apoE-deficient mouse represents a new in vivo approach that will permit rapid screening of candidate genes for the prevention of atherosclerosis. (J. Clin. Invest. 1995.

Lecithin cholesterol acyltransferase (LCAT) is a key enzyme which catalyzes the esterification of free cholesterol present in plasma lipoproteins. In order to evaluate the role of LCAT in HDL metabolism, a 6.2-kilobase (kb) fragment consisting of 0.851 and 1.134 kb of the 5'- and 3'-flanking regions, as well as the entire human LCAT gene, was utilized to develop transgenic mice. Three different transgenic mouse lines overexpressing human LCAT at plasma levels 11-, 14-, and 109-fold higher than non-transgenic mice were established. Northern blot hybridization analysis demonstrated that the injected 6.2-kb fragment contained the necessary DNA sequences to direct tissue specific expression of the human LCAT gene in mouse liver. Compared to age- and sex-matched controls, total cholesterol and HDL cholesterol levels were increased in all 3 transgenic mice lines by 124-218 and 123-194%, respectively, while plasma triglyceride concentrations remained similar to that of control animals. Fast protein liquid chromatography analysis of transgenic mouse plasma revealed marked increases in high density liposportin (HDL)-cholesteryl ester and phospholipid as well as the formation of larger size HDL. Thus, the majority of the increase in transgenic plasma cholesterol concentrations was due to accumulation of cholesteryl ester in HDL consistent with enhanced esterification of free cholesterol in mouse HDL by human LCAT. Plasma concentrations of apoA-I, apoA-II, and apoE were increased in high expressor homozygote mice who also demonstrated an accumulation of an apoE-rich HDL1. Like the mouse enzyme, human LCAT was found to be primarily associated with mouse HDL. Our studies demonstrate a high correlation between plasma LCAT activity and total as well as HDL cholesterol levels establishing that in mice LCAT modulates plasma HDL concentrations. Overexpression of LCAT in mice leads to HDL elevation as well as increased heterogeneity of the HDL lipoprotein particles, indicating that high levels of plasma LCAT activity may be associated with hyperalphalipoproteinemia and enhanced reverse cholesterol transport.

We have established a mouse model for human LCAT deficiency by performing targeted disruption of the LCAT gene in mouse embryonic stem cells. Homozygous LCAT-deficient mice were healthy at birth and fertile. Compared with age-matched wild-type littermates, the LCAT activity in heterozygous and homozygous knockout mice was reduced by 30 and 99%, respectively. LCAT deficiency resulted in significant reductions in the plasma concentrations of total cholesterol, HDL cholesterol, and apoA-I in both LCAT ؊/؊ mice (25, 7, and 12%; p < 0.001 of normal) and LCAT ؉/؊ mice (65 and 59%; p < 0.001 and 81%; not significant, p ‫؍‬ 0.17 of normal). In addition, plasma triglycerides were significantly higher (212% of normal; p < 0.01) in male homozygous knockout mice compared with wild-type animals but remained normal in female knockout LCAT mice. Analyses of plasma lipoproteins by fast protein liquid chromatography and two-dimensional gel electrophoresis demonstrated the presence of heterogenous pre␤-migrating HDL, as well as triglyceride-enriched very low density lipoprotein. After 3 weeks on a high-fat high-cholesterol diet, LCAT ؊/؊ mice had significantly lower plasma concentrations of total cholesterol, reflecting reduced levels of both proatherogenic apoB-containing lipoproteins as well as HDL, compared with controls. Thus, we demonstrate for the first time that the absence of LCAT attenuates the rise of apoB-containing lipoproteins in response to dietary cholesterol. No evidence of corneal opacities or renal insufficiency was detected in 4-month-old homozygous knockout mice. The availability of a homozygous animal model for human LCAT deficiency states will permit further evaluation of the role that LCAT plays in atherosclerosis as well as the feasibility of performing gene transfer in human LCAT deficiency states. Lecithin:cholesterol acyltransferase (LCAT)1 is a 63-kDa glycoprotein synthesized primarily by the liver, which plays a major role in the metabolism of HDL (1). In plasma, LCAT is preferentially associated with HDL (2) but may also interact with low density lipoproteins (3), where it catalyzes the transfer of a fatty acid from the sn-2 position of phosphatidylcholine to the 3-hydroxyl group of cholesterol, generating cholesteryl esters and lysolecithin. The newly formed cholesteryl esters (CE) are then transferred to the core of the HDL lipoprotein particle, a process that results in the formation and maturation of spherical HDL (4). LCAT-mediated esterification of free cholesterol in HDL helps maintain a concentration gradient for efflux of cholesterol from peripheral cells to the HDL particle surface for ultimate transport to the liver (4, 5). Thus, together with hepatic lipase and cholesteryl ester transfer protein LCAT appears to be essential for the process of reverse cholesterol transport, one of several proposed mechanisms by which HDL may protect against atherosclerosis (6, 7).The important role that LCAT plays in HDL metabolism has been established by the identification and characterization of patients...

Cholesterol esterification within plasma lipoprotein particles is catalyzed by lecithin:cholesterol acyltransferase (LCAT). The impact of the overexpression of this enzyme on plasma concentrations of the different plasma lipoproteins in an animal model expressing cholesteryl ester transfer protein was evaluated by generating rabbits expressing human LCAT. A 6.2-kilobase human genomic DNA construct was injected into the pronuclei of rabbit embryos. Of the 1002 embryos that were injected, 3 founder rabbits were characterized that expressed the human LCAT gene. As in mice and humans, the principal sites of mRNA expression in these rabbits is in the liver and brain, indicating that the regulatory elements required for tissue-specific expression among these species are similar. The alpha-LCAT activity correlated with the number of copies of LCAT that integrated into the rabbit DNA. Compared with controls, the high expressor LCAT-transgenic rabbits total and high density lipoprotein (HDL) cholesterol concentrations were increased 1.5-2.5-fold with a 3.1-fold increase in the plasma cholesterol esterification rate. Analysis of the plasma lipoproteins by fast protein liquid chromatography indicates that these changes reflected an increased concentration of apolipoprotein E-enriched, HDL1-sized particles, whereas atherogenic apolipoprotein B particles disappeared from the plasma. The concentrations of plasma HDL cholesterol were highly correlated with both human LCAT mass (r = 0.93; p = 0.001) and the log LCAT activity (r = 0.94; p < 0.001) in the transgenic rabbits. These results indicate that overexpression of LCAT in the presence of cholesteryl ester transfer protein leads to both hyperalpha-lipoproteinemia and reduced concentrations of atherogenic lipoproteins.