Lipid Depletion in Atheromatous Coronary Arteries in Rhesus Monkeys after Regression Diets

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1 Lipid Depletion in Atheromatous Coronary Arteries in Rhesus Monkeys after Regression Diets By Mark L. Armstrong and Marjorie B. Megan ABSTRACT Lipids were measured in the coronary arteries of monkeys on an atherogenic diet and in the arteries of matched monkeys on the atherogenic diet followed by regression diets. Cholesterol content was 51 mg/g dry weight in the arteries of monkeys with atheromatosis; after 40 months on the regression diets it was 18 mg/g. Cholesteryl ester was 69% lower and free cholesterol 53% lower after the regression diets. Decreases in triglycerides and phospholipids were not significant. The cholesterol content of the arteries of two monkeys autopsied after 20 months on the regression diets was close to the mean value after 40 months. The data show that cholesterol in both its free form and its ester form is depleted from experimentally induced coronary atheromatosis by dietary regression regimens. The data also suggest that most of the cholesterol depletion occurs during the first half of regression; the susceptibility of the residual excess arterial cholesterol to mobilization from the vessel wall by dietary means is questionable. KEY WORDS experimental atherosclerosis hypercholesterolemia regression of atherosclerosis arterial cholesterol mobilization coronary artery lipids hyperbetalipoproteinemia Regression of experimental coronary atheromatosis in rhesus monkeys was reported from this laboratory in a previous morphologic study (1). Regression was accomplished by substituting for the atherogenic diet a cholesterol-free diet either (1) low in fat or (2) of fat content equal to that of the atherogenic diet, but containing a large amount of linoleic acid. Widening of the arterial lumen and a decrease in intimal mass the principal findings indicating regression were accompanied From the Cardiovascular Division, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa This study was supported by U. S. Public Health Service Grants HE-8408 and HE and Research Career Development Award HE-K3-25, 408 (Dr. Armstrong) from the National Heart and Lung Institute and by the Iowa Heart Association. The study was presented in part at the 43rd Annual Meeting of the American Heart Association, November, Mail reprint requests to Mark L. Armstrong, M.D., Department of Internal Medicine, University Hospitals, Iowa City, Iowa Received January 19, Accepted for publication April 18, by a decrease in mural lipid content, as judged by sudanophilic staining. Use of Sudan dyes, however, may lead to a distorted view of lipid changes in a tissue. Free cholesterol cannot be shown by any of these dyes (2). We have noted that broad differences exist in the uptake of red Sudan dyes by other lipids commonly found in arterial tissue; this finding concurs with the variable staining among lipid classes reported by others (3, 4). This characteristic of sudanophilic stains impairs their usefulness in the interpretation of changes in lipid classes in histologic material. Histochemical techniques can provide additional information about the lipid composition of tissue, but this information is only of a semiquantitative nature. Morphologic regression data require more precise answers as to whether lipid has been lost from the artery wall or merely masked, e.g., hydrolysis of cholesteryl ester to free cholesterol causes loss of sudanophilia. If lipid is in fact depleted from atheromatous coronary arteries during regression regimens, what 675

2 676 ARMSTRONG, MEGAN lipid classes are depleted? Biochemical studies bearing on these issues are presented. Methods Thirty adult male rhesus monkeys were fed a semipurified diet containing 40% egg-yolk fat (5) for 17 months; they were then divided into three groups matched for hypercholesterolemia. One group was autopsied for base-line coronary atherosclerosis. The other two groups were fed regression diets for 40 months. One group received a cholesterol-free, low-fat (4% of total calories) diet; the other was fed the same diet enriched with corn oil to 40% of the total calories. Ten control animals were fed the low-fat diet throughout the study. At autopsy the heart was removed and fixed in formalin before the extramural coronary arteries were dissected free for study. After arterial tissue was taken for morphologic studies at fixed points (1), the remaining coronary segments were prepared for biochemical studies. The validity of lipid data from tissue subjected to formalin fixation has been evaluated by Dayton et al. (6), who found no change in the values for major lipid classes up to 8 weeks after the fixation. In preparatory experiments identical to those used in this investigation we also found no changes after formalin fixation, and extraction of 4-14 C- cholesterol from atherosclerotic and regression vessels in isotope-fed animals was complete. The vessel segments were magnified for complete removal of adventitia and clots. The specimens were dried to a constant weight at 41 C, pulverized as previously described (7). and extracted three times with 20 ml of chloroform-methanol (2:1) (8). The combined lipid extracts were evaporated in a stream of nitrogen. Separation of the lipid classes was carried out on Florisil thin-layer plates, 0.5 mm, in a solvent system of heptane-diethyl ether-acetic acid (70:30:0.5). The cholesterol, cholesteryl TABLE 1 Lipid Content (nig/g dry wgt) of Coronary Arteries Cholesterol Total Ester* Free Triglycerides Phospholipids Control (10) 6.S * * =±= =* * 2.7 ester, and triglyceride fractions were visualized with rhodamine 6-G and eluted with chloroform. Phospholipids were eluted with methanol-o.ln HC1 (20:1). The cholesteryl ester fraction was saponified by mild ethanolic alkali at 100 C (9). (Hydrolysis of labeled cholesteryl ester averaged 98%.) The cholesterol moiety in the ester and free fractions was measured by gas liquid chromatography as the trimethylsilyl ether (9). Triglycerides were measured by the method of Van Handel and Zilversmit (10) and lipid phosphorus by the method of Bartlett (11). Recovery of 4-14 C-cholesterol added to the lipid extracts before the analytic procedures was 89-97% and averaged 93%. It was assumed that the other lipid fractions were recovered in the same proportion. The methods used to determine serum lipids and lipoproteins have been described (5, 7) and the results reported (1). Results ARTERIAL LIPID CHANGES The mural content of the major lipid classes in the coronary arteries is shown in Table 1 for control, atherosclerotic, and regression arteries. The regression data are presented as a whole, because there was no significant difference in total arterial cholesterol, free cholesterol, or cholesteryl ester between the two groups on regression diets; morphologic regression was also similar (1). In atherosclerosis total cholesterol was sevenfold higher than it was in controls. Increased cholesteryl ester in atherosclerotic coronary arteries accounted for most of this change, but there was a twofold rise in free cholesterol as well. Coronary triglyceride Atherosclerotic (10) 51.2 =t =<= S = t = «= = «= 3.9 Regression (20) 18.1 ± ± ± ± ± 3.1 All values are means *Cholesteryl moiety. SE; number of monkeys in each group is given in parentheses.

3 'JS1 L1PID LOSS IN CORONARY ATHEROSCLEROSIS 677 content in the monkeys with atherosclerosis was nearly twice that of controls (see Discussion). Phospholipids also increased significantly in atherosclerosis (P < 0.05). At the end of regression, average total cholesterol was 65% lower than the value in base-line atherosclerosis, but it was still significantly above control levels (P<0.01). Cholesteryl ester was reduced 69% and free cholesterol 53%. Indeed, free cholesterol approached control levels, and cholesteryl ester remained higher than free cholesterol as in base-line atherosclerosis. Triglycerides were lower than in atherosclerotic arteries, but the difference was not significant (P = 0.1). Phospholipid content showed a minimal and nonsignificant decrease. The total cholesterol content for all coronary segments in the atherosclerotic and regression groups is shown by decile ranking in Figure 1. It indicates both the variability of cholesterol accumulation among segments in experimental atherosclerosis and the retention of cholesterol after the regression period. The ratios of free cholesterol and of cholesteryl ester to total cholesterol change, and these I 1 1 CC\J D a D = Atherosclerotic group = Regression group m D D D D " l- 1--* i i i 9 RANKED CORONARY SEGMENTS FIGURE 1 Decile distribution of cholesterol content of coronary segments from atherosclerotic and regression groups. Differences between ranked subset points are indicated by boundaries of the stippled region. i changes may also be used to depict either the accumulation of or the decrease in the two moieties in arterial tissue (12). Figure 2 shows this relationship for atherosclerotic coronary arteries. The accumulations of free cholesterol and of cholesteryl ester are represented as simple linear functions over a wide range of total cholesterol content in the intimamedia. As total cholesterol increased, cholesteryl ester became more preponderant. Figure 3 shows the comparable data from monkeys on the regression diets. Although total cholesterol content was much lower in regression segments, residual cholesteryl ester over a tenfold range still remained preponderant over free cholesterol, in contrast to levels found in normal arterial tissue. Of particular note was the higher level of cholesteryl ester in regression vessels whose total cholesterol content fell within the normal range. The product-moment correlation coefficients between total cholesterol and cholesteryl ester and between total and free cholesterol for the coronary segments from monkeys with atherosclerosis were 0.99 (ester) and 0.85 (free). For the coronary segments from monkeys on the regression diets the corresponding correlation coefficients were 0.88 (ester) and 0.78 (free). It is not known whether the regression n=ester =Free = 1 SEIvalues <1mq/q not shown) TOTAL CHOLESTEROL(mq/g) 180 FIGURE 2 Decile-ranked coronary segments from atherosclerotic monkeys. Content of free cholesterol and cholesteryl ester (ordinate) is shown for each rank, four segments per point.

678 ARMSTRONG, MEGAN 40 30 20 10 D=Ester 'Free (volues<1mg/g not shown] 10 20 30 40 TOTAL CHOLESTEROL(mg/g) FIGURE 3 Decile-ranked coronary segments from monkeys fed the sequence of atherogenic and

4 678 ARMSTRONG, MEGAN D=Ester 'Free (volues<1mg/g not shown] TOTAL CHOLESTEROL(mg/g) FIGURE 3 Decile-ranked coronary segments from monkeys fed the sequence of atherogenic and regression diets. Content of free cholesterol and cholesteryl ester (ordinate) is shown for each rank, eight segments per point. slopes apply to time intervals other than those used for end-point atherosclerosis and endpoint regression. MIDREGRESSION ARTERIAL LIPID CHANGES The death of an animal after 20 months on the low-fat regression diet (1) required autopsy of the matching animal on the linoleate-enriched diet to preserve comparability of regression periods (1). This circumstance fortuitously provided arterial lipid values halfway through the regression trials. These data were well within the range observed at the end of the 40-month regression period. In the animal that died arterial free cholesterol and cholesteryl ester were 5.7 and 14.9 mg/g, respectively (total cholesterol 20.6); the cholesterol content in the matching animal was 3.8 for free cholesterol and 11.8 for cholesteryl ester (total cholesterol 15.6). SERUM LIPID CHANGES Total serum cholesterol decreased from average levels slightly over 700 mg/dliter at the end of the atherogenic period to control levels near 140 mg/dliter within 60 days of the start of regression diets (1). Phospholipids fell correspondingly. Serum triglycerides showed little change in response to dietary cholesterol 50 content in these primates and consequently did not decrease. Lipoprotein electrophoresis showed a marked increase in staining of the beta band in animals fed the atherogenic diet, but control appearance returned in parallel with the decline in serum cholesterol. Discussion Diet-induced hyperbetalipoproteinemia increased serum cholesterol fivefold and the cholesterol content of the coronary arteries by an average of sevenfold. More than three years after serum cholesterol had returned to its original level in prolonged dietary regression trials, the coronary artery cholesterol per unit tissue weight was less than 40% of the level found in monkeys with base-line atherosclerosis. It is important to distinguish whether the decrease in arterial cholesterol content in monkeys on regression diets was caused by depletion or by dilution by reparative tissue. The 65% difference in total cholesterol content between atheromatous and regression arteries is inexplicable by dilution for the following reason. The coronary arteries were measured in situ from their ostia to the posterior descending branches. Cholesterol content in the vessels of monkeys with atherosclerosis was ±0.032 mg/cm length of artery, and it was ±0.008 mg/cm in monkeys on regression diets; this is conclusive evidence against dilution. In this type of experimental atherosclerosis (13) the expression of cholesterol in terms of unit weight would tend to underestimate changes, because both cholesterol content and tissue weight are simultaneously changing in the artery. This is a case in point of the general issue of shifts in vascular weight related to changes in the content of the vascular wall noted by Wolinsky (14). In contrast to control arteries, in which most of the cholesterol mass was in the media, the excess cholesterol in atherosclerotic tissue occurred chiefly in intimal lesions. Whether the intimal location of atheromas is favorable for the depletion that occurred during the regression period is uncertain. One mode of depletion, destruction of sterol in situ, may be

LIPID LOSS IN CORONARY ATHEROSCLEROSIS 679 considered to be highly improbable (15); whether the remaining mechanism of depletion, efflux of cholesterol from the vessel wall, occurs chiefly into the

5 LIPID LOSS IN CORONARY ATHEROSCLEROSIS 679 considered to be highly improbable (15); whether the remaining mechanism of depletion, efflux of cholesterol from the vessel wall, occurs chiefly into the lumen or transmurally into adventitial vessels (16) is not entirely clear (15). The change from high to low /3- lipoprotein concentrations in the plasma bathing the endothelial surfaces probably contributed to arterial loss of excess cholesterol just as the reverse situation contributed to its accumulation. The more specific presumption that plasma /3-lipoprotein provided the chief source of excess cholesterol in the atheromatous coronary wall also seems probable, but both points are of inferential nature in this study. Free cholesterol rather than cholesteryl ester is considered to be the preferred, if not the exclusive, form in which this sterol is exchanged between plasma and cells, including those of the normal arterial wall (15, 17, 18). In uncomplicated atheromas, however, the relative and the absolute amount of cholesteryl ester is increased along with the absolute amount of free cholesterol (18). The large accumulation of mural cholesterol in the ester form is in accord with studies of human coronary atherosclerosis (19-21); this study establishes a marked example of the same trend in the coronary arteries of a nonhuman primate. The absolute reduction of cholesteryl ester in the coronary walls in regression indicates the possibilities of significant loss either as the ester form or after hydrolysis to free cholesterol. Data suggesting that substantial efflux of cholesteryl ester may occur from the walls of plaques but not from normal arteries have been reported by Lofland and Clarkson (18). Whatever mechanisms may be available for depletion of cholesteryl ester, in the present investigation the residual content of cholesteryl ester remained higher than that of free cholesterol in the coronary arteries as a biochemical "scar" of the atherosclerotic process. The triglyceride content of the coronary arteries nearly doubled in monkeys with atherosclerosis and was at an intermediate level in regression. In this study the coronary arteries were prepared by meticulous adventitial stripping. We assume that even after microscopic dissection a minimal amount of fat-filled adventitia was present as a source of systematic error in estimating the triglyceride content of the intima-media. Systematic error would not explain the difference between the control monkeys and those with atherosclerosis. Furthermore, the triglyceride values of the atherosclerotic arteries are comparable to the values for human atherosclerotic arteries found by Bottcher et al. (19), who took samples of the coronary intima-media by a needle or scalpel to avoid adventitial contamination. Scott et al. (20) employed careful adventitial stripping of human atherosclerotic vessels and also found comparable triglyceride values. Also pertinent to this argument is the report of Dayton et al. (6) that atheroma lipid from human coronary arteries has a high triglyceride content and that atheroma lipid from aorta has a low triglyceride content. Increased phospholipid in the intimamedia together may reflect either increased cellularity of the wall or increased membrane formation in the absence of an increased cell concentration. The atheromatous arteries were more cellular than control or regression arteries, and it is probable that the higher phospholipid content in the atherosclerotic arteries resulted from a combination of more cells plus membrane elaboration but that in regression the high residual phospholipid content reflected more purely an increased membrane content. To the extent that the dietary regression regimens indicate the possibility of mobilization of arterial lipid from coronary atheromas, it would seem that cholesterol might be most successfully mobilized. Data obtained after 20 months on regression diets suggest that the major depletion of excess cholesterol may be relatively early; the residual excess cholesterol may not be as susceptible to depletion by dietary means. Furthermore, the proportion of arterial lipid accessible to depletion by any regression technique may well vary inversely with the severity and the complicating features of atheromatous lesions.

6 680 ARMSTRONG, MEGAN References 1. ARMSTRONG:, M.L., WARNER, E.D., AND CONNOR, W.E.: Regression of coronary atheromatosis in rhesus monkeys. Circ Res 27:59-67, ADAMS, C.W.M.: Histochemistry of Atherosclerosis Correlated with Structural and Clinical Pathology in Vascular Histochemistry. Chicago, Year Book Medical Publishers, 1967, p SMITH, E.B., EVANS, P.H., AND DOWNHAM, M.D.: Lipid in the aortic intima: Correlation of morphological and chemical characteristics. J Atherosclerosis Res 7= , ADAMS, C.W.M.: Neurohistochemistry. Amsterdam, Elsevier Publishers, 1965, pp ARMSTRONG, M.L., CONNOR, W.E., AND WARNER, E.D.: Xanthomatosis in rhesus monkeys fed a hypercholesterolemic diet. Arch Pathol 74: , DAYTON, S., HASHIMOTO, S., AND PEARCE, M.L.: Influence of a diet high in unsaturated fat upon composition of arterial tissue and atheromata in man. Circulation 32: , ARMSTRONC, M.L., CONNOR, W.E., AND WARNER, E.D.: Tissue cholesterol concentration in the hypercholesterolemic rhesus monkey. Arch Pathol 87:87-92, FOLCH, J., LEES, M., AND SLOANE-STANLEY, G.H.: Simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226: , MIETTINEN, T.A., AHRENS, E.H., JR., AND GRUNDY, S.M.: Quantitative isolation and gasliquid chromatographic analysis of total dietary and fecal neutral steroids. J Lipid Res 6: , VAN HANDEL, E., AND ZILVERSMIT, D.B.: Micromethod for the direct determination of serum triglycerides. J Lab Clin Med 50: , BARTIJETT, G.R.: Phosphorus assay in column chromatography. J Biol Chem 234: , PORTMAN, O.W.: Atherosclerosis in nonhuman primates: Sequences and possible mechanisms of change in phospholipid composition and metabolism. Ann NY Acad Sci 162: , ARMSTRONG, M.L., AND WARNER, E.D.: Morphology and distribution of diet-induced atherosclerosis in rhesus monkeys. Arch Pathol 92: , WOLINSKY, H.: Response of the rat aortic wall to hypertension: Importance of comparing absolute amounts of wall components. Atherosclerosis 11: , DAYTON, S., AND HASHIMOTO, S.: Recent advances in molecular pathology: A review: Cholesterol flux and metabolism in arterial tissue and in atheromata. Exp Molec Pathol 13: , ADAMS, C.W.M., AND MORGAN, R.S.: Autoradiographic demonstration of cholesterol filtration and accumulation in atheromatous rabbit aorta. Nature (Lond) 210: , ROTHBLAT, G.H., AND KRITCHEVSKY, D.: Metabolism of free and esterified cholesterol in tissue cultured cells. Exp Molec Pathol 8: , LOFLAND, H.B., AND CLARKSON, T.B.: Bi-directional transfer of cholesterol in normal aorta, fatty streaks and atheromatous plaques. Proc Soc Exp Biol Med 133:1-8, BfJTTCHER, C.J.F., BOELSMA-VAN HOUTE, E., TER HAAR ROMENY-WACHTER, C.C., WOODFORD, F.P., AND VAN GENT, CM.: Lipid and fatty-acid composition of coronary and cerebral arteries at different stages of atherosclerosis. Lancet 2: , SCOTT, R.F., DAOUD, A.S., WORTMAN, B., MORRISOX, E.S., AND JARMOLYCH, J.: Proliferation and necrosis in coronary and cerebral arteries. J Atherosclerosis Res 6: , MEYER, B.J., MEYER, A.C., PEPLER, W.J., AND THERON, J.J.: Chemical composition of the aorta, coronary arteries and cerebral arteries of Europeans and Bantu. Am Heart J 71:68-78, 1966.

Intimal Thickening in Normocholesterolemic Rhesus Monkeys Fed Low Supplements of Dietary Cholesterol

Intimal Thickening in Normocholesterolemic Rhesus Monkeys Fed Low Supplements of Dietary Cholesterol By Mark L. Armstrong, Marjorie B. Megan, and Emory D. Warner ABSTRACT Rhesus monkeys were fed a high-fat