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High-density lipoproteins can be separated into distinct particles based on their apolipoprotein content. In the present study, the in vivo metabolism of apoE within the apoE-containing HDL particles LpE, LpE:A-I, LpE:A-II and LpE:A-I:A-II was assessed in control subjects and in patients with abetalipoproteinemia (ABL), in whom HDL are the sole plasma lipoproteins. The metabolism of apoE within these HDL subspecies was investigated in three separate studies which differed by donor or recipient status: (1) particles purified from normolipidemic plasma and reassociated with 125I or 131I-labeled apoE injected into normolipidemic subjects (study 1); (2) particles purified from ABL plasma injected into normolipidemic subjects (study 2); and (3) particles purified from ABL plasma injected into ABL subjects (study 3). The plasma residence times (RT, hours) in study 1 were 14.3+/-2.9, 11.3+/-3.4, and 9.1+/-1.2 for apoE within LpE:A-I:A-II, LpE:A-II and LpE:A-I, respectively, while those in study 2 were 10.1+/-2.2, 9.7+/-2.4, 7.9+/-1.0 and 7.3+/-0.8 for apoE within LpE:A-I:A-II, LpE:A-II, LpE:A-I and LpE, respectively. In study 3, RTs for apoE within LpE:A-I:A-II and LpE were 8.7+/-0.9 and 6.8+/-0.9, respectively. In comparison, RT for apoA-I on LpA-I:A-II has been reported to be 124.1+/-5.5 h and that for apoA-I on LpA-I 105.8+/-6.2 h. Thus, apoE within the different apoE-containing HDL particles was metabolized rapidly and at a similar rate in control and ABL subjects. The plasma RT of apoE was longest when injected on LpE:A-I:A-II particles and shortest when injected on LpE. In summary, our data show that: (1) the plasma RT of apoE within HDL is approximately ten times shorter than that of apoA-I within HDL, and (2) apoE within HDL is metabolized at a slower rate when apoproteins A-I and A-II are present (LpE:A-I:A-II RTLpE:A-IILpE:A-ILpE). These differences were related to the lipid and apolipoprotein composition of the HDL subspecies, and, in control subjects, to the transfer of apoE from HDL subspecies to apoB-containing lipoproteins as well.

In order to evaluate the coordinate role that he- patic lipase (HL) and 1ecithin:cholesterol acyltransferase (LCAT) play in modulating HDL particle heterogeneity and function in vivo we utilized recombinant adenovirus to ex- press HL in control and LCAT transgenic mice. Adenovirus- mediated expression of human HL in control (n = 4, LCAT activity = 42 2 1 nmol/ml per h) and LCAT-tg mice (n = 4, LCAT activity = 3566 I 93 nmol/ml per h) resulted in post heparin HL activities of 24358 5 6080 and 27266 2 7985 nmol/ml per min, respectively. Overexpression of HL led to significant reductions in total cholesterol, phospholipids, and HDL cholesterol in both LCAT-tg (62,62, and 63%, P< 0.05) and control mice (68,63, and 78%, P< 0.01) as well as to the formation of more homogenous HDL. However, compared to control animals, the reductions in the plasma concentrations of HDL-cholesterol and apoA-I were less in LCAT-tg mice (HDL-cholesterol: -62 t 15% vs. -78 +- 15%, P = 0.18; apoA-I: -36 2 7% vs. -76 2 8%, P < 0.0005). Gel filtration analysis revealed that in LCAT-tg mice the apoE-rich HDL, was preferentially reduced by expression of HL in vivo. Com- pared to control mice the reduction in the apoA-I/A-I1 HDL in transgenic mice was significantly less indicating that a sub- set of HDL in LCAT transgenic mice are resistant to the action of HL.M These combined data support a role for both HL and LCAT in modulating HDL heterogeneity and function, properties which may ultimately affect the ability of LCAT transgenic mouse HDL to function in the process of reverse cholesterol transport.-Dugi, K. A, B. L. Vaisman, N. Sakai, C. L. Knapper, S. M. Meyn, H. B. Brewer, Jr., and S. Santama- rina-Fojo. Adenovirus-mediated expression of hepatic lipase in LCAT transgenic mice. ,J Lipid Res. 1997. 38: 1822-1832.

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 prebeta-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) is an enzyme involved in the intravascular metabolism of high density lipoproteins (HDLs). Overexpression of human LCAT (hLCAT) in transgenic rabbits leads to gene dose-dependent increases of total and HDL cholesterol concentrations. To elucidate the mechanisms responsible for this effect, 131I-HDL apoA-I kinetics were assessed in age- and sex-matched groups of rabbits (n=3 each) with high, low, or no hLCAT expression. Mean total and HDL cholesterol concentrations (mg/dl), respectively, were 162+/-18 and 121+/-12 for high expressors (HE), 55+/-6 and 55+/-10 for low expressors (LE), and 29+/-2 and 28+/-4 for controls. Fast protein liquid chromatography analysis of plasma revealed that the HDL of both HE and LE were cholesteryl ester and phospholipid enriched, as compared with controls, with the greatest differences noted between HE and controls. These compositional changes resulted in an incremental shift in apparent HDL particle size which correlated directly with the level of hLCAT expression, such that HE had the largest HDL particles and controls the smallest. In vivo kinetic experiments demonstrated that the fractional catabolic rate(FCR, d(-1)) of apoA-I was slowest in HE (0.328+/-0.03) followed by LE (0.408+/-0.01) and, lastly, by controls (0.528+/-0.04). ApoA-I FCR was inversely associated with HDL cholesterol level (r=-0.851,P

Plasma concentrations of low-density lipoproteins (LDLs) and high-density lipoproteins (HDLs) are inversely related in several dyslipoproteinemias. To elucidate the interactions between these lipoproteins, we used a recombinant adenovirus (hLDLR-rAdV) to express human LDL receptors (hLDLRs) in LDL receptor-deficient rabbits. hLDLR-rAdV administration resulted in hepatocyte expression and a reduction of total, intermediate-density lipoprotein (IDL), and LDL cholesterol. In addition, we found that hLDLR-rAdV treatment induced (1) increased very-low-density lipoprotein (VLDL) cholesterol, (2) increased VLDL, IDL and LDL triglycerides, (3) decreased alpha- and pre-beta-migrating apolipoprotein E (apo E) and decreased pre-beta-migrating apo A-I at 2 to 4 days posttreatment, and (4) increased total plasma apo A-I and pre-beta-migrating apo A-I beginning 8 to 10 days posttreatment. Virtually all plasma apo A-I was present on alpha- and pre-beta-HDL. Pre-beta-HDL particles with size and electrophoretic properties consistent with nascent HDL demonstrated the greatest relative apo A-I enrichment following hLDLR-rAdV treatment. In summary, enhanced expression of hepatocyte LDLRs by hLDLR-rAdV treatment markedly altered apo A-I-containing lipoproteins and IDL and LDL. The use of recombinant viruses to express physiologically relevant genes in intact animals, analogous to transfection of cells in culture, provides a new strategy for the evaluation of effects of specific gene products on metabolic systems in vivo.

Hepatic lipase (HL) is an endothelial-bound lipolytic enzyme which functions as a phospholipase as well as a triacylglycerol hydrolase and is necessary for the metabolism of IDL and HDL. To evaluate the feasibility of replacing an enzyme whose in vivo physiologic function depends on its localization on the vascular endothelium, we have infused recombinant replication-deficient adenovirus vectors expressing either human HL (HL-rAdV; n = 7) or luciferase cDNA (Lucif-rAdV; n = 4) into HL-deficient mice with pretreatment plasma cholesterol, phospholipid, and HDL cholesterol values of 176 +/- 9, 314 +/- 12, and 129 +/- 9, respectively. After infusion of HL-rAdV, HL could be detected in the postheparin plasma of HL-deficient mice by immunoblotting and postheparin plasma HL activities were 25,700 +/- 4,810 and 1,510 +/- 688 nmol/min/ml on days 5 and 15, respectively. Unlike the mouse HL, 97% of the newly synthesized human HL was heparin releasable, indicating that the human enzyme was virtually totally bound to the mouse vascular endothelium. Infusion of HL-rAdV in HL-deficient mice was associated with a 50-80% decrease in total cholesterol, triglyceride, phospholipids, cholesteryl ester, and HDL cholesterol (P < 0.001) as well as normalization of the plasma fast protein liquid chromatography lipoprotein profile by day 8. These studies demonstrate successful expression and delivery of a lipolytic enzyme to the vascular endothelium for ultimate correction of the HL gene defect in HL-deficient mice and indicate that recombinant adenovirus vectors may be useful in the replacement of endothelial-bound lipolytic enzymes in human lipolytic deficiency states.

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 an 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 +/- 18mg/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 10(3) microns2) when compared with control mice infused with rAdv.luc (161 +/- 10 x 10(3) microns2; 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.