|[Frontiers in Bioscience 2, d298-308, June 15, 1997]|
TRANSGENIC RABBIT MODELS FOR THE STUDY OF ATHEROSCLEROSIS
John M. Taylor and Jianglin Fan
Gladstone Institute of Cardiovascular Disease, the Department of Physiology, and the Cardiovascular Research Institute, University of California, San Francisco, CA
The rabbit is naturally deficient in hepatic lipase, having about 10% the activity found in other animals such as the rat (40). Hepatic lipase occupies a central role in plasma lipoprotein metabolism, with probable functions (Fig. 2) in the conversion of IDL to LDL, the remodeling of large triglyceride-rich HDL2 to smaller dense HDL3 (reviewed in ref. (41)), the transfer of HDL cholesterol to the liver (42, 43), and the clearance of chylomicron and VLDL remnants (44). Thus, the overexpression of hepatic lipase in the rabbit offered a unique opportunity to understand the mechanisms by which this enzyme modulates plasma lipid metabolism.
Figure 2. Metabolic pathways of lipoproteins. The chylomicron, VLDL, and HDL pathways for plasma lipoprotein metabolism are shown with major sites of action for principal enzymes and factors. The separation densities of individual classes of lipoprotein fractions are indicated, as well as the major apolipoprotein components of each lipoprotein class. Abbreviations are explained in the text.
A total of nine transgenic founders were generated initially using a human hepatic lipase cDNA construct that directed expression only to the liver. Protein expression was detected subsequently in six of the founders (13). There was no hepatic lipase found normally in plasma, but the intravenous administration of heparin released substantial activity into the circulation of the transgenic animals, ranging from 7- to 83-fold above nontransgenic control values. The amount of human hepatic lipase corresponded approximately to the transgene copy number, and the enzymatic activity was comparable to that reported for post-heparin human plasma. Immunocytochemical studies at the ultrastructural level have demonstrated that the transgenic hepatic lipase is concentrated at cell surfaces in the space of Disse in the liver, consistent with its metabolic action (45).
The hepatic lipase transgenic rabbits had reductions in plasma lipid levels compared to normal animals, with total cholesterol decreased up to 40% and total triglycerides decreased by 60%. An 80% average decrease in HDL cholesterol accounted for most of the total decrease in plasma cholesterol. The changes in plasma lipid levels were similar in both male and female transgenic rabbits.
To examine the effects of transgene expression on individual lipoprotein classes, different density fractions were isolated by sequentially adjusting plasma to various densities with potassium bromide, then floating them by ultracentrifugation. Each fraction was resolved further by agarose gel electrophoresis. Then, neutral lipid in lipoproteins was detected by staining the gels with Fat Red 7B, and their protein components were identified by blotting the gels to membrane filters and reacting the filters with specific antibodies to rabbit apolipoproteins.
The detailed analysis of plasma lipoproteins confirmed that the overexpression of hepatic lipase in the transgenic rabbit reduced the plasma content of HDL. All major classes of HDL decreased in quantity. These decreases were reflected in a substantial reduction in the content of apoA-I, a major characteristic component of HDL. The reduction in HDL1 and HDL2 levels is consistent with a role for hepatic lipase in HDL remodeling, whereby large, triglyceride-rich HDL are converted to smaller dense HDL by lipolytic activity. However, the decrease in HDL3 suggests that the action of hepatic lipase on this class of lipoproteins is an important function. By its location on liver cell surfaces, hepatic lipase may act on the surface of HDL through its phospholipase activity, altering the lipid environment to favor cholesterol transfer to cell membranes. Thus, hepatic lipase may facilitate the ability of HDL to deliver cholesterol directly to the liver and contribute to the function of HDL in reverse cholesterol transport. The overall reduction in HDL levels may reflect an increased catabolism, an increased clearance from plasma, or a decreased rate of production; but metabolic studies are required to determine the key mechanism.
The amount and distribution of apoB-containing lipoproteins also were affected by hepatic lipase expression. There was a reduction in IDL levels that was accompanied by a slight increase in the content of small LDL. These findings are consistent with roles for hepatic lipase in the conversion of IDL to LDL and in the increased uptake of remnant lipoproteins by the liver. However, additional studies are needed to determine if the changes in apoB-containing lipoproteins in transgenic rabbit plasma reflect increased clearance or increased catabolism. Thus, changes in the distribution of plasma lipoproteins were observed in the transgenic rabbit that could have opposing effects on the development of atherosclerosis: a decrease in atherogenic IDL, and a decrease in lipoproteins (HDL) that are thought to be protective against lesion development.
To determine the susceptibility of the hepatic lipase transgenic rabbit to atherosclerosis, a study of the effects of transgene expression on the response of the rabbit to dietary cholesterol was undertaken (J. Taylor et al., manuscript in preparation). Both transgenic and normal control rabbits, beginning at three months of age, were maintained on a diet supplemented with 0.3% cholesterol and 3% soybean oil. In the normal rabbit, plasma cholesterol levels increased from a baseline of about 50 mg/dl to reach peak levels of about 1200-1500 mg/dl by 4-5 weeks, after which there was no further increase. ApoB-containing particles, migrating as beta-VLDL, were the most abundant class of lipoproteins as expected. In the cholesterol-fed hepatic lipase transgenic rabbits, plasma cholesterol levels were raised only to about one-third the level of the control rabbits. Relatively large, apoE-deficient VLDL were the major lipoproteins found in the transgenic rabbits. There was also a striking reduction in the content of IDL and LDL compared to the controls, consistent with the increased expression of hepatic lipase in the transgenic rabbits.
The development of atherosclerosis in hepatic lipase transgenic rabbits was examined after a period of 10 weeks on the 0.3% cholesterol-supplemented diet. A comparison was made to normal rabbits that had been maintained on this diet, but with the lipid content varied to match plasma cholesterol levels of the normal controls with that of the transgenic animals. Aortas were harvested, trimmed of external fat, spread out, and stained with Sudan IV to detect lipid deposits. The area of the aortic surface that was covered with lesion was quantified by digitized image analysis, and lesion sections were examined to determine thickness, involvement of the artery wall, and pathology. As expected, most of the lesion involvement was in the aortic arch with additional lesions found at intercostal artery ostia and little involvement in the abdominal region. In both types of animals, about 8% of the aortic surface was covered with thick, raised lesions. However, the lesions in the ascending aorta and the aortic arch of the transgenic rabbits were significantly thicker than in the normal rabbits. Further studies will be needed to determine the basis for this remarkable difference in the atherosclerotic response of the vascular wall in the hepatic lipase transgenic rabbit to dietary cholesterol.