[Frontiers in Bioscience 2, d298-308, June 15, 1997]
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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

7. APOLIPOPROTEIN E TRANSGENIC RABBITS

Apolipoprotein E (Mr = 35,000 ) participates in diverse metabolic processes (summarized in ref. (46)). As a component of various lipoprotein classes, apoE occupies a central role in cholesterol metabolism (Fig. 1), mediating the clearance of chylomicron and VLDL remnants from plasma via cell-surface receptors, facilitating the delivery and incorporation of cholesterol into HDL, mediating the redistribution of lipid between cells and tissues, and acting as a co-factor for hepatic lipase (47). To determine the contributions that these mechanisms make to the development of atherosclerosis, transgenic rabbits that overexpress human apoE (the E3 phenotype) were generated (J. Fan et al., manuscript submitted). A 14-kb genomic fragment was used that contains the intact human apoE gene ligated to its hepatic control region (construct LE1 in ref. (48)). Three transgenic lines were established: the two highest expressers had human apoE levels of 12-13 mg/dl in circulation with little apparent effect on endogenous rabbit apoE levels. Compared to normal controls, total plasma triglycerides were decreased by nearly 50% in the transgenic animals, with all of this decrease found in the VLDL fraction. Unexpectedly, total plasma cholesterol was increased by up to twofold, with the additional cholesterol found in LDL and in HDL1.

To understand these dramatic effects, the apoB-containing lipoproteins were characterized further. Negative-staining electron microscopy showed VLDL having a median particle diameter of 26 nm in both normal and transgenic animals, but the number of particles larger than 32 nm in diameter was decreased by 75% in the transgenic rabbits. Thus, large VLDL were preferentially decreased as a consequence of apoE overexpression. Studies of LDL receptor-binding competition using cultured human fibroblasts showed that VLDL from transgenic rabbits were about threefold more effective than VLDL from normal rabbits in competing with control LDL for fibroblast receptors. This difference was most likely due to an increase in the content of apoE on the transgenic VLDL. In contrast, LDL from the transgenic rabbit were equivalent to LDL from the normal rabbit in their receptor-binding activity, suggesting that the transgenic LDL were not defective in their receptor-binding capacity. However, radioactively labeled human LDL were cleared more slowly from transgenic rabbit plasma than from normal control rabbit plasma.

Taken together, these studies suggested that the greater content of apoE on the VLDL could make them more effective competitors than LDL for receptors, leading to an accumulation of LDL in plasma. This possibility is supported by the higher affinity of apoE-containing lipoproteins for the LDL receptor than apoB-containing lipoproteins (summarized in ref. (46)), which would favor an increased uptake of apoE-rich particles compared to apoB-only LDL. Alternatively, the accumulation of LDL in apoE transgenic animals might be due to a reduction in LDL receptor activity as a consequence of an increased cholesterol uptake into the liver by VLDL. To distinguish between these two mechanisms, a monoclonal antibody that binds to the LDL receptor was administered intravenously to the rabbits: the antibody was cleared from the plasma of both transgenic and control rabbits at the same rate. Thus, the level of LDL in plasma appeared to be influenced by the content of apoE on large VLDL, giving them a competitive advantage for LDL receptor binding and subsequent clearance.

A study of the response to dietary cholesterol of the apoE transgenic rabbit in comparison to normal control rabbits was initiated according to the protocol described above. Overexpression of apoE limited the increase in plasma cholesterol to about one-third of the level found in the controls. Only low levels of VLDL were present in these transgenic rabbits, and the small IDL/large LDL fraction constituted the dominant class of lipoproteins in these animals. Thus, the lipoprotein profile in the cholesterol-fed apoE transgenic rabbit was almost the inverse of that found in the cholesterol-fed hepatic lipase transgenic rabbit, consistent with the complementary nature of their functions. The proximal aorta of the apoE transgenic rabbit aorta had only thin fatty streaks with little increase in intimal thickening, but about 14% of the vascular surface had lesion involvement significantly greater than controls. Thus, it appears that the predominance of atherogenic IDL might facilitate the initiation of an atherosclerotic process and the increased level of circulating apoE in the transgenic rabbit minimizes the deposition of lipid in the artery wall. While these studies are provocative, a study of the effects of transgene expression on HDL will be necessary to understand the response to dietary cholesterol in both apoE and hepatic lipase transgenic rabbit models.

Transgenic rabbits have been generated that express the human apoE2 phenotypic variants (Y. Huang et al., manuscript submitted). These animals have substantial elevations in plasma cholesterol that are reflected in an increase in beta-VLDL with a markedly different plasma lipoprotein profile. These animals are likely to be excellent models for the disorder type III hyperlipoproteinemia.