Effects of Regression of Atherosclerotic Lesions on the Content and Ester if ication of Cholesterol by Cell-Free Preparations of Pigeon Aorta

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1 Effects of Regression of Atherosclerotic Lesions on the Content and Ester if ication of Cholesterol by Cell-Free Preparations of Pigeon Aorta By Richard W. St. Clair, Thomas B. Clarkson, and Hugh B. Lofland ABSTRACT This study was designed to determine the effect of regression of atherosclerotic lesions on the incorporation of l- 14 C-oleic acid into phospholipids, triglycerides, and cholesteryl esters and to compare these metabolic alterations with changes in the extent of atherosclerosis and the content of cholesterol and cholesteryl esters in the lesions. Aortic atherosclerosis was produced in White Carneau pigeons by feeding them an atherogenic diet for 1-8 months. The birds were then switched to a cholesterol-free diet for 6 months for the regression phase of the experiment. Following the regression phase, no changes were noted in the atherosclerotic index or the freecholesterol content of aortas from pigeons that had received the atherogenic diet for 1 5 months; however, a reduction in both of these parameters was seen in aortas from pigeons fed the atherogenic diet for 8 months. There was a marked reduction in the content of cholesteryl esters in the aortas following the regression phase paralleled by a decrease in the rate of cholesterol esterification. No change was seen in the rate of incorporation of fatty acid into phospholipids or triglycerides. These studies suggest that local cholesterol esterification might be of considerable importance in maintaining the large amount of cholesteryl esters found in the atherosclerotic lesion and that changes in the rate of cholesterol esterification are associated with the early events in both progression and regression of atherosclerosis. KEY WORDS arterial metabolism cholesterol esterification triglycerides phospholipids fatty acid esterification oleic acid Atherosclerosis can be produced experimentally in a variety of animal species by feeding them diets containing relatively large amounts of cholesterol. Subsequent removal of this dietary cholesterol reportedly causes changes in the atherosclerotic lesions generally considered to represent regression. Such changes have been reported in arteries from several species, including the dog (1), the From the Arteriosclerosis Research Center, The Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina This investigation was supported by U. S. Public Health Service Grant HL from the National Heart and Lung Institute and by a grant from the American Heart Association. The work was done during Dr. St. Clair's tenure as an Established Investigator of the American Heart Association. Received February 14, Accepted for publication September 7, chicken (2), the pigeon (3), and the rhesus monkey (4, 5), but apparently no regression occurs in the rabbit (6, 7) except when relatively short-term cholesterol feeding is used (8). In all of these experiments, morphologic changes were noted in the lesions after several months of regression regardless of the change in the lipid content of the lesions. In the atherosclerotic artery, there is an accumulation of several classes of lipids, including primarily free and esterified cholesterol. Although little cholesterol is synthesized by the arterial wall (9), substantial quantities of fatty acids are synthesized and incorporated into phospholipids, triglycerides, and cholesteryl esters (10-12). This synthesis is markedly stimulated in the atherosclerotic 664 R*i*<rcb, Vol. XXXI, Noprrxt* 1972

2 REGRESSION OF ATHEROSCLEROTIC LESIONS 665 artery, and a large portion of the newly synthesized fatty acid is esterified to cholesterol (10). The increase in the esterification of fatty acids to cholesterol is one of the earliest metabolic changes in the arterial wall associated with the development of atherosclerosis and can be demonstrated even before the appearance of grossly visible lesions (13). The absolute contribution of local cholesterol esterification to the accumulation of cholesteryl esters in the atherosclerotic lesion is unknown, but it has been estimated to account for as much as one-half of the accumulated cholesteryl esters (14). In the animal studies reported to date, with the exception of those in the rabbit, the regression of atherosclerosis is associated with a loss of lipid, particularly cholesteryl esters, from the lesions. We are not aware, however, of any reports dealing with the metabolic changes that take place in the lesion during regression. The present study was designed to determine the effect of regression of atherosclerotic lesions in pigeons on the incorporation of fatty acids into phospholipids, triglycerides, and cholesteryl esters and to compare these metabolic alterations with the changes in the extent of atherosclerosis and the content of cholesterol and cholesteryl esters in the lesions. Methods For this study, 280 White Carneau pigeons of both sexes, 2 4 months of age, were obtained from our stock colony. All birds were fed a cholesterolfree mixed-grain diet from the time of hatching. Twenty birds were selected at random for baseline determinations, and the remaining 240 birds were fed an atherogenic diet consisting of Purina pigeon pellets coated with cholesterol and lard to a final concentration of 0.5 g cholesterol and 10 g lard/100 g ration. Plasma cholesterol concentrations were determined (15) 1 month after the initiation of a new dietary regimen and at monthly intervals thereafter. At monthly intervals up to 5 months and at 8 months, 40 birds were selected at random from the 240 started on the atherogenic diet; 20 were killed for analysis, and the remaining 20 were kept on a cholesterol-free mixed-grain diet for an additional 6 months, after which they were killed and their aortas analyzed. Circulation Rtstrcb, Vol. XXXI, Noptmktr 1972 The design of the experiment is shown diagrammatically in Figure 1. Birds were killed by decapitation, and their aortas were rapidly removed, cleaned of blood and periadvenritial tissue, and graded for degree of grossly visible atherosclerosis (16). To have enough tissue for analysis, the 20 aortas from each subgroup were divided into two pools of 10 aortas each, and the aortas of each pool were homogenized in 0.1M potassium phosphate buffer (ph7.5). The content of free and esterified cholesterol was determined from a sample of the homogenate. The remaining homogenate was centrifuged at 1,000 g for 30 minutes to remove cellular debris and most of the connective tissue; the cellfree supernatant fluid was used as the enzyme preparation for the determination of incorporation of l- 14 C-oleic acid into phospholipids, triglycerides, and cholesteryl esters. These procedures have been described in detail elsewhere (13). All samples were homogenized for the same length of time and centrifuged under identical conditions to reduce, as much as possible, any variability in enzyme activity due to these procedures. Results Pigeons consuming the atherogenic diet had an average plasma cholesterol concentration of 1170 ±87 (SE) mg/100 ml. This value represents the mean plasma cholesterol concentration determined at monthly intervals for the duration of the experiment. The plasma cholesterol content reached a plateau within 1 month after initiation of the atherogenic diet. Likewise, removal of dietary cholesterol resulted in a decrease in plasma cholesterol concentrations to normal levels within 1 month. The average plasma cholesterol concentration of birds fed cholesterol-free mixed grain over the 6 months of the regression phase of the experiment was 348 ±9 mg/100 ml. The extent of grossly visible atherosclerosis in aortas of pigeons fed the atherogenic diet for up to 8 months compared with that in pigeons fed the atherogenic diet for the same duration but then given the cholesterol-free diet for 6 months is shown in Figure 2. Only in the aortas from birds fed the atherogenic diet for 8 months did removal of dietary cholesterol result in a significant reduction in the aortic atherosclerotic index. This is not to say,

3 666 ST. CLAIR, CLARKSON, LOFLAND however, that there was no regression in the groups on the atherogenic diet for 1-5 months: the atherosclerotic index measures the percent of the intimal surface covered with grossly visible lesions, but it does not consider the thickness or the morphology of the lesions. There was little change in the gross appearance of the lesions after 6 months of regression among those groups previously fed the atherogenic diet for up to 4 months. However, among those groups fed the atherogenic diet for longer periods and then switched to the cholesterol-free diet, there were changes in the gross appearance of the lesions. The plaques appeared flattened and harder, and they were more often light brown rather than bright yellow. The content of free and esterified cholesterol in the aortas is shown in Figure 3. Somewhat to our surprise, the free-cholesterol 260 White Cameau Pigeons Cholesterol-free Diet Aortal from 20 birds analyzed for baseline determination Remaining 240 bird* given atherogentc diet 1 month 2 month 3 month 4 month 5 month Aortas front 20 birds analyzed o month FIGURE 1 Design of lesion regression study. See text for details on diets and methods of analysis. Circulation Ktsesrcb, Vol XXXI, November 1972

4 REGRESSION OF ATHEROSCLEROTIC LESIONS I _ I I Cholesterol-fed Cholesterol -fed then 6mos. without Dietary Cholesterol Standard error of the mean I Duration of Cholesterol Feeding (mos.) FIGURE 2 Effect of removal of dietary cholesterol on extent of aortic atherosclerosis in pigeons. Each bar represents the mean extent of grossly visible atherosclerosis in the 20 pigeons in each subgroup. content did not change after 6 months on a cholesterol-free diet in aortas from pigeons fed the atherogenic diet for up to 5 months. Only in the group fed the atherogenic diet for 8 months was there a reduction in the freecholesterol content of the aortas following removal of dietary cholesterol. On the other hand, after 6 months on the cholesterol-free diet, the content of cholesteryl esters was substantially less in the aortas from all but the 1-month group. Unfortunately, because of the necessity of pooling aortas to have enough tissue for metabolic studies, it was not possible to determine the statistical variability among the aortas from these pools. As can be seen from Figure 4 and as has been reported previously (13), the esterification of l- 14 C-oleic acid to cholesterol appeared to be stimulated in the aortas from pigeons fed the atherogenic diet for only 1 month, and the esterification increased some fiftyfold in severely diseased aortas from pigeons fed the atherogenic diet for 4-8 months. In addition, there was a marked decrease in the rate of cholesterol esterification in all groups 6 months after switching to the cholesterol-free diet. Along with this decrease in the rate of cholesterol esterification, there was a drop in CtrcnUtien Reiesrcb, Vol. XXXI, Sonemier 1972 the content of cholesteryl esters in the lesions associated with the regression (Fig. 3). There was considerable variability in the incorporation of l- 14 C-oleic acid into phospholipids and triglycerides throughout the duration of the experiment. Despite this variability, there was no apparent correlation of the mean rate of incorporation of l- 14 C-oleic acid into phospholipids and triglycerides with either the duration of the the atherogenic diet or the subsequent removal of dietary cholesterol. The effect of duration of the atherogenic diet on incorporation of l- 14 C-oleic acid into phospholipids and triglycerides has been published elsewhere (13), and this previous paper shows the extent of the variability of these results. We were interested in knowing whether the reduction in the rate of cholesterol esterification was specific for esterification with cholesterol or whether there was a similar reduction in incorporation of l- 14 C-oleic acid into phospholipids and triglycerides. For this comparison, we expressed the rate of incorporation of l- 14 C-oleic acid into phospholipids, triglycerides, and cholesteryl esters at the end of the atherogenic diet as 100*. The incorporation of l- 14 C-oleic acid into these

5 668 ST. CLAIR, CLARKSON, LOFLAND "> 7 i Free Cholesterol Cholesterol-fed m Cholesterol - fed then 6 mos. " without Dietary Cholesterol ffli Control I 4 1 ' a? o 6 *b 5 try 3A f<o 2 I Control Duration of Cholesterol Feeding (mos.) FIGURE 3 Changes in cholesterol content in aortas from pigeons 6 months after switching to a cholesterol-free diet. Each bar represents the average cholesterol content of 20 aortas. The aortas were analyzed in two pooled samples of 10 aortas each; thus, it was not possible to determine the statistical variability among individual aortas. The content of free or esterified cholesterol in the two pools of aortas from the 1-3-month samples did not differ from the mean by more than 6%, and for pools of aortas from the 4 8-month samples it differed from the mean by less than 20%. The control represents a pool of 20 aortas from young birds fed cholesterol-free mixed grain. lipids 6 months after switching to the cholesterol-free diet was then expressed as a percent of the rate observed at the end of the atherogenic diet. As can be seen in Figure 5, the reduction in incorporation of l- 14 C-oleic acid into cholesteryl esters was specific for esterification to cholesterol. Because of the variability of the results, the moderate increases in incorporation of l-^c-oleic acid into phospholipids and triglycerides after the regression phase of the experiment were not statistically significant. On the other hand, the decrease in the rate of cholesterol esterification in these same lesions was significantly less (P < 0.05) after the regression phase of the experiment. Discussion From the early work of Horlick and Katz (2) in chickens and the more recent studies in nonhuman primates by Armstrong et al. (4) and Tucker et al. (5), it is apparent that atherosclerotic lesions can regress, at least to some extent. It is difficult to define precisely what is meant by regression, since the definition depends somewhat on the criteria for evaluation. This point is clearly demonstrated by considering the apparent lack of regression in the groups fed the atherogenic diet for up to 5 months when the changes in free-cholesterol content or in the percent of the intimal surface covered with lesions were evaluated (Figs. 2 and 3). On the other hand, some regression was obvious in these same arteries when the decrease in the content of cholesteryl esters was considered. It is apparent, however, from both the chemical data and the gross appearance of the lesions, that some change took place during the regression phase of the experiment, even when the total area covered with lesions did jiot change. Circulation Research, Vol. XXXI, November 1972

6 REGRESSION OF ATHEROSCLEROTIC LESIONS 669 I i Control Duration of Cholesterol Feeding (mos.) FIGURE 4 Changes in cholesterol esterification in aortas from pigeons 6 months after switching to a cholesterol-free diet. The incubation mixture consisted of the 1000-% supernatant fluid of the aortic homogenates made up to a total volume of 3 ml with 0.1M potassium phosphate buffer (ph 7.5). To this mixture was added 15 ntnoles of ATP, 1.5 /xmoles of acetyl coenzyme A, 15 fimoles of NaF, and 12 fimoles of MgCl t. 1-HC-oleic acid, 2.2 X 10 6 dpm (specific activity 127,670 dpm/nmole), was added as substrate. Results represent the mean and range of duplicate determinations on two pools of 10 aortas each. The control represents the mean of 12 determinations done at intervals throughout the experiment on pools of aortas from young birds fed cholesterol-free mixed grain. We were somewhat surprised to see the substantial difference in lability of the free and the esterified cholesterol in the atherosclerotic aortas after switching the birds to the cholesterol-free diet. In the atherosclerotic aortas, the cholesteryl-ester fraction decreased most readily during the regression of the atherosclerotic lesions. This finding is consistent with those from another study from our laboratory (3). Portman et al. (17) also found that the cholesteryl-ester fraction decreased most readily in atherosclerotic aortas from squirrel monkeys previously fed an atherogenic diet and then switched to a cholesterolfree diet. Armstrong and Megan (18) reported similar findings in a lesion-regression study in rhesus monkeys. In the pigeon aorta, the cholesteryl-ester fraction appeared to increase before the free-cholesterol fraction in the progression of the atherosclerotic lesion (13), suggesting an important role for choles- CircaUlion Relttrcb, Vol. XXXI, Norembs* 1972 teryl esters in the early phase of progression as well as regression of atherosclerosis. It has been well established that, in atherosclerotic aortas, the esterification of cholesterol is markedly stimulated (12, 13, 19). The present study demonstrated for the first time that the regression of atherosclerotic lesions is also associated with a relatively specific decrease in the rate of cholesterol esterification in the lesions themselves. The decrease in cholesterol esterification corresponded well with the decrease in the content of cholesteryl esters during lesion regression. This finding suggests, but does not prove, that local cholesterol esterification may be responsible for maintaining a substantial amount of the cholesteryl esters that accumulate in the atherosclerotic vessel. This conclusion has been reached by others as well (14). It is difficult to explain the lack of change in the content of aortic free cholesterol seen in

7 670 ST. CLAIR, CLARKSON, LOFLAND Standard error of the mean Phospholipid Triglyceride Cholesteryl Ester FIGURE 5 Percent change in esterification of l- 1 *C-oleic acid in aortas from pigeons 6 months after switching to a cholesterol-free diet compared with esterification before removal of dietary cholesterol. Incubation conditions were the same as those in Figure 4. The mean rate of incorporation of l- 1 ic-oleic acid into phospholipids, triglycerides, and cholesteryl esters in the 24 determinations on 12 pools of 120 aortas analyzed during the time when the pigeons were fed the atherogenic diet is expressed as 100%. The bars represent the percent change in incorporation of l-*lc-oleic acid into these lipids in a similar number of analyses on aortas 6 months after switching to the cholesterol-free diet. Results were analyzed statistically using a paired t-test. all but the 8-month group during the regression phase of the experiment. Several studies have indicated that radioactive free cholesterol from the blood is readily incorporated into the arterial wall (20, 21) and would suggest that the free cholesterol of the arterial wall should be quite labile. Much of this radioactivity, however, may not represent net uptake of free cholesterol but rather exchange (22). Consequently, the free cholesterol that accumulates in the early stages of lesion development may be bound, perhaps to membranes, in a manner that prevents its net removal but still allows exchange with another molecule of free cholesterol. Obviously, there are differences in the susceptibility to regression of individual lesions, depending on their severity and perhaps on their morphologic characteristics, as can be seen by comparing regression in aortas from birds fed the atherogenic diet for 8 months with that in birds fed the diet for 5 months or less. Unfortunately, it was necessary for us to pool aortas to have enough tissue with which to work. It would have been interesting to know if there were morphologic differences between lesions from pigeons fed the atherogenic diet for up to 5 months and those from birds fed the diet for 8 months that would explain the difference in the response of these lesions during the regression phase of the experiment. Circulation Rtstarcb, Vol. XXXI, Nottmbtr 1972

8 REGRESSION OF ATHEROSCLEROTIC LESIONS 671 Data presented by Clarkson et al. (3) suggested that there are regressible and nonregressible pools of plaque cholesterol and that the regressible pool might be in the foam cells of the lesion. Support for this concept has been provided by Eisenberg et al. (23), who indicated that there are different pools of free and esterified cholesterol within the developing lesions of rabbits. We have no information from this present experiment, however, as to whether the decrease in the content of cholesterylesters in the atherosclerotic lesions during regression represents the loss of intracellular or extracellular cholesteryl esters or some combination of the two. An understanding of the mechanism of regression of atherosclerosis may evolve with the increase in knowledge about the distribution and composition of these different pools. References 1. BEVANS, M., DAVIDSON, J.D., AND KENDALL, F.E.: Regression of lesions in canine arteriosclerosis. Arch Pathol 51: , HORLJCK, L., AND KATZ, L.N.: Retrogression of atherosclerotic lesions on cessation of cholesterol feeding in the chick. J Lab Clin Med 34: , CLARKSON, T.B., KING, J.S., LOFLAND, H.B., AND FELDNER, M.A.: Changes in pathologic characteristics and composition of plaques during regression (abstr.). Circulation 44 (suppl. II): H-48, ARMSTRONG, M.L., WARNER, E.D., AND CONNOR, W.E.: Regression of coronary atheromatosis in rhesus monkeys. Circ Res 27:59-67, TUCKER, C, STRONC, J., CATSULIS, C, AND EGGEN, D.: Regression of cholesterol-induced atherosclerotic lesions in rhesus (abstr.). Circulation 44 (suppl. II): 11-48, CONSTANTTNIDES, P., BOOTH, J., AND CARLSON, C: Production of advanced cholesterol atherosclerosis in the rabbit. Arch Pathol 70: , FRIEDMAN, M., AND BYERS, S.O.: Observations concerning the evolution of atherosclerosis in the rabbit after cessation of cholesterol feeding. Am J Pathol 43: , BORTZ, W.M.: Reversibility of atherosclerosis in cholesterol-fed rabbits. Circ Res 22: , ST. CLAIR, R.W., LOFLAND, H.B., JR., PRICHARD, R.W., AND CLARKSON, T.B.: Synthesis of squalene and sterols by isolated segments of Circulation Research, Vol. XXXI, tiovtmbtr 1972 human and pigeon arteries. Exp Mol Pathol 8: , ST. CLAIR, R.W., LOFLAND, H.B., JR., AND CLARKSON, T.B.: Composition and synthesis of fatty acids in atherosclerotic aortas of the pigeon. J Lipid Res 9: , WHEREAT, A.F.: Lipid biosynthesis in aortic intima from normal and cholesterol-fed rabbits. J Athero Res 4: , LOFLAND H.B., MOURY, D.M., HOFFMAN, C.W., AND CLARKSON, T.B.: Lipid metabolism in pigeon aorta during atherogenesis. J Lipid Res 6: , ST. CLAIR, R.W., LOFLAND, H.B., AND CLARKSON, T.B.: Influence of duration of cholesterol feeding on esterification of fatty acids by cellfree preparation of pigeon aorta. Circ Res 27: , DAYTON, S., AND HASHIMOTO, S.: Origin of fatty acids in lipids of experimental rabbit atheroma. J Athero Res 8: , BLOCK, W.D., JARRETT, K.J., JR., AND LEVINE, J.B.: Improved automated determination of serum total cholesterol with a single color reagent. Clin Chem 12: , LOFLAND, H.B., AND CLARKSON, T.B.: Biochemical study of spontaneous atherosclerosis in pigeons. Circ Res 7: , PORTMAN, O.W., ALEXANDER, M., AND MAHUFFO, C.A.: Nutritional control of arterial lipid composition in squirrel monkeys: Major ester classes and types of phospholipids. J Nutr 91:35-46, ARMSTRONG, M.L., AND MEGAN, M.B.: Nature of arterial lipid depletion in regression of experimental coronary atheromas in rhesus monkeys (abstr.). Circulation 42 (suppl. Ill): , DAY, A.J., AND WAHLQVIST, M.L.: Uptake and metabolism of C 14 -labeled oleic acid by atherosclerotic lesions in rabbit aorta: Biochemical and radioautographic study. Circ Res 23: , LOFLAND, H.B., AND CLARKSON, T.B.: Bidirectional transfer of cholesterol in normal aorta, fatty streaks, and atheromatous plaques. Proc Soc Exp Biol Med 133:1-8, NEWMAN, H.A.I., AND ZILVERSMTT, D.B.: Uptake and release of cholesterol by rabbit atheromatous lesions. Circ Res 18: , ST. CLAIR, R.W., AND LOFLAND, H.B., JR.: Uptake and esterification of exogenous cholesterol by organ cultures of normal and atherosclerotic pigeon aorta. Proc Soc Exp Biol Med 138: , EISENBERC, S., RACHMTLEWTTZ, D., STEIN, O., AND STEIN, Y.: Metabolic nonhomogeneity of lecithin and cholesterol in aortae. Biochim Biophys Acta 231: , 1971.