REGRESSION OF ATHEROMA AND PUTATIVE ROLE OF CETP IN CHOLESTERYL ESTER REMOVAL.

REGRESSION OF ATHEROMA AND PUTATIVE ROLE OF CETP IN CHOLESTERYL ESTER REMOVAL

Evidence for regression of atherosclerosis induced by cholesterol feeding has been provided by several investigators [1-3]. However, since the extent of atherosclerotic involvement is quite variable, quantitative evaluation of regression is difficult. We have used 3H-cholesteryl linoleyl ether (3H-CLE), a nonhydrolyzable analog of cholesteryl easter as a stable marker for the quantitation of atherosclerotic involvement [4] and evaluat­ ed the potential usefulness of 3H-CLE in the evaluation of regression of atheromatosis [5]. To that end, 20 rabbits were kept on a purina diet en­ riched with 1% cholesterol for 1 month and then on alternate weeks for an additional 2 months. The animals were randomized into two groups according to their plasma cholesterol levels and injected with autologous plasma labeled with 3H-CLE [5]. The baseline group was killed 10-12 days after injection, while the regression group was fed purina fortified with 3% cholestyramine and killed 8-11 months after injection of the 3H-CLE. We investigated the following: Will the 3H-CLE remain in the aorta during the 11-month period of regression? If 3H-CLE is retained, then the specific activity expressed as 3H-CLE/CE mass should rise with CE loss during regression; 3. Is the loss of CE during regression similar from the different parts of the aorta?

At the end of the cholesterol feeding period, the mean plasma chol­ esterol was 1298 mg/dl. The amount of labeled 3H-CLE in the aorta varied markedly among the individual rabbits, but was highly correlated (r =0.875) with the amount of aortic cholesteryl ester determined in the base- line group (Fig. 1). The results presented in Fig. 2 compare the total and esterified cholesterol in the entire aorta of the baseline (10-12 days) and regression groups (11 months). In the baseline group, the mean total cholesterol was 13.2 ± 2.1 mg/aorta and the esterified cholesterol was 7.6 ± 1.3 mg/aorta. After 11 months of the regression regimen, the total cholesterol was 9.5 ± 1.9 mg/aorta, while cholesteryl ester decreased to

3.1 ± 0.7 mg/aorta. The loss of cholesteryl ester was significant (p < 0.01). On the other hand, the amount of 3H-CLE in the regression group was not different from that seen in the baseline group (Fig. 2). The mean specific activity of 3H-CLE/CE was compared in the arch, thoracic and abdominal aorta of the baseline and regression groups (Fig. 3). As can be seen in the baseline group, the specific activity in the three regions examined was quite similar. A much higher specific activity of 3H-CLE/CE was found in the regression group, the highest being seen in the region of the aortic arch.

These results permitted us to conclude that 3H-CLE injected into cholesterol fed rabbits was retained in the aorta for up to 11 months of the regression period, while cholesteryl ester content decreased. The retention of 3H-CLE in the rabbit aorta, in face of high plasma CETP, would not favour the role of CETP in CE removal from the aorta. This could have been due to several possibilities, among them that the 3H-CLE was not accessible to the transfer protein. Indeed, in a model system in culture [6], we have shown that while 3H-CLE present in lipoproteins and bound to

extracellular matrix was accessible to CETP and could be released into the culture medium; once the lipoprotein had become ingested by a cell such as a macrophage it became inaccessible to CETP [6]. Recently, Morton [7] has presented evidence that CETP is able to remove cholesteryl ester from intact macrophages. We proposed to test the putative role of CETP in CE egress from reticuloendothelial cells in vivo in an animal model in which one can modulate plasma CETP levels by dietary means. Son and Zilversmit [8] have shown that cholesterol feeding in rabbits is accompanied by an increase in plasma CETP. We looked, therefore, for a smaller animal which would respond to cholesterol feeding with a rise of plasma CETP in analogy to the rabbit. In view of the studies of Dietschy et al. [9, 10], the hamster appeared to be a suitable model, but there were no data in the literature with respect to plasma CETP in the hamster. Therefore, we have examined hamsters for CETP activity and found measurable activity under

control dietary conditions. We were able to modulate this activity by feeding diets enriched in cholesterol and fat [11]. As seen in Table 1, the hamsters responded to a high fat-high cholesterol diet with a significant increase in CETP activity. Therefore, we decided to use hamsters to evaluate the role of CETP in cholesteryl ester efflux from cells in vivo. The approach was based on our previous findings [12] that when acetylated LDL is labeled with 3H-CLE and injected into rats, it disappears from the liver at a very slow rate. Since the rat does not have measurable CETP

under normal dietary conditions or even after feeding of high fat and cholesterol [11], a comparison of loss of 3H-CLE labeled acetylated LDL from rat and hamster liver could provide some information with respect to the role of CETP in cholesteryl ester removal from cellular elements in vivo. These experiments are now in progress and preliminary results suggest that under these experimental conditions, loss of 3H-CLE from the liver is not increased by CETP.

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