Atherosclerosis,

31 (1978)

@ Elsevier/North-Holland

117-124

Scientific

Publishers,

Ltd

REGRESSION OF NATURALLY OCCURRING ATHEROSCLEROTIC LESIONS IN PIGEON AORTA BY INTESTINAL BYPASS SURGERY Early Changes in Arterial Cholesteryl

M.T. RAVI SUBBIAH DAC M. DINH Cardiovascular (L7.S.A.J

Ester Metabolism

*, BETTY A. DICKE,

Research

Unit, Mayo

BRUCE A. KOTTKE,

Clinic and Mayo

Foundation,

IVETTE

A. CARLO

Rochester,

MN 55901

and

(Received 20 September, 1977) (Revised, received 15 June, 1978) (Accepted 21 June, 1978)

Summary Early changes in cholesteryl ester metabolism of the aorta during the regression of naturally occurring atherosclerotic lesions in pigeon aorta by ileal bypass surgery were examined. Three months after surgery, there was a decrease (50%) in the content of cholesteryl esters in the aorta. Increases in the activity of cholesteryl ester hydrolase in the lysosomal (P < 0.05) and the supernatant (P < 0.01) fractions of the aorta also occurred at this time. There were no differences in the activity of cholesteryl ester synthetase and in the plasma levels of cholesterol and triglycerides between the ileal bypass group and the controls. These results suggest that ileal bypass surgery decreases the level of cholesteryl esters in the aorta, probably because of enhanced cholesteryl ester hydrolysis. Key words:

Atherosclerosis - Cholesteryl Intestinal bypass - Regression

ester

hydrolase

-

Chotesteryl

ester

synthetase

-~

_---

Introduction The reversibility of atherosclerosis has considerable significance for the potential medical treatment of coronary heart disease. Experiments with animals have shown that some types of cholesterol-induced atherosclerotic lesions are reversible. Studies by Anitschkow [l] showed that atherosclerotic lesions in This Health, * Reprint Center.

investigation Public

was

Health

requests Cincinnati.

supported

in Part

by Research

Grant

HL-14196

from

the National

Center,

University

Institutes

Service.

to: OH

Dr.

M.T.

45229.

Ravi U.S.A.

Subbiah,

Lipid

Research

of

Cincinnati

Mediva

of

rabbits regressed when the serum cholesterol level returned to normal. Vesselinovitch et al. [Z] noted that regression of atherosclerotic lesions in rabbits can be enhanced by oxygen and cholestyramine. Studies of cholesterol-induced atherosclerosis in chicks indicated that fatty streaks regressed while fibrous plaques did not [3,4]. Tucker et al. [ 51 noted the regression of cholesterolThese authors found that. the induced fatty streaks in the rhesus monkey. regressed lesions contained only a few lipid-laden monocytes and that the lipid content of smooth muscle cells was decreased. The most detailed studies of regression of coronary atherosclerosis were done by Armstrong et al. [6,7] in rhesus monkeys. These authors noted that, after 40 months on regression diets, the stenosis of coronary arteries decreased, as did the content of cholesterol and cholesteryl esters. Structural studies showed that the regression procedure resulted in a collagenous lesion. Regression of cholesterol-induced atherosclerotic lesions in the White Carneau pigeon also was reported by Clarkson et al. [8]. A novel study by Buchwald [9] showed that the severity of atherosclerosis and hypercholesterolemia in the cholesterol-fed rabbits could be decreased by ileal bypass surgery. This was subsequently confirmed by Okuboye et al. [lo] in rabbits and by Scott and his co-workers in the dog [ 111 and the rhesus monkeys [12]. In all of these studies of severity regression, atherosclerosis was induced by cholesterol feeding, and regression was attributed to the changes that occurred after withdrawal of cholesterol. Very few studies were concerned with the regression of naturally occurring atherosclerotic lesions in an effort to study the sequence of events that are independent of the effects of cholesterol feeding and withdrawal. Our laboratory showed, for the first time, that naturally occurring atherosclerotic lesions in the aorta of White Carneau pigeons can regress after ileal bypass surgery [13]. Preliminary studies [ 141 of the aorta after regression indicated a substantial decrease in the content of cholesteryl esters and a decrease in extracellular lipids. One of the unusual findings was that ileal bypass had no effect on the fecal excretion of bile acids and neutral sterols or on the plasma levels of cholesterol in the pigeons [15,16]. This is different from what has been noted in humans [1’7], in whom ileal resection causes a marked increase in the excretion of bile acids and a lowering of plasma cholesterol level. Therefore, we examined the mechanism of this regression, particularly in reference to arterial lipid metabolism. We examined the changes in the concentration of cholesterol and cholesteryl esters in the aorta during the early stages of regression (3 months) and the activit.ies of the key arterial enzymes concerned with the synthesis and hydrolysis of cholesteryl esters. The properties of these enzymes in the pigeon aorta have been described [ 18-201 by others as well as from our laboratory. Methods [4-‘4C]Cholesteryl oleate (sp. act. 55 mCi/mmol), act. 50 mCi/mmol), and [I-14C]oleic acid (sp. act. 50 chased from New England Nuclear, Boston, MA. All checked for purity by thin-layer chromatography and

[4-‘4C]cholesterol (sp. mCi/mmol) were purthe compounds were by scanning the thin-

layer plates using a Packard Radiochromatogram Scanner. White Carneau pigeons were obtained from the Palmetto Pigeon Plant, Sumter, SC. The pigeons were maintained on a cholesterol-free grain diet during the experiments. Six-year-old adult pigeons (100% incidence of aortic atherosclerosis at this age) (211 were used for the study and were divided into two groups of 20 birds each for chemical studies. A separate study consisting of 40 birds (20 sham-operated and 20 with ileal bypass) was used for the enzyme studies. One group from each study was subjected to ileal bypass surgery, as described by Gomes et al. [ 131 based on the procedure of Buchwald [ 91. Ileal bypass was accomplished by side-to-side anastomosis of the small intestine and the cloaca (cloaca in pigeons is equivalent to colon in mammals). The products of digestion were excluded from the bypassed segment by a ligature. The other group was subjected to a sham operation (abdominal exploration) and was used as a control. One bird with bypass in the first study and two birds with bypass in the second study died during the first week after surgery. These birds were not replaced. At the end of 3 months, samples of blood were taken, and the pigeons were killed by an overdose of barbital. The aortas were dissected quickly, and the area of the lesion at the celiac branch of the aorta [22] was photographed and estimated by planimetry, as previously described [ 131. The area of the lesion at the celiac branch of the aorta was used for chemical and enzymatic studies. Chemical analysis was carried out on three separate pools of aortas (each pool comprising 6 or 7 aortas) from each group. Total lipids from each pool of aortas were extracted with 20 volumes of chloroform : methanol (2 : I), as described by Folch et al. [ 231. Various lipids were separated by thin-layer chromatography on silica gel using a solvent system of heptane : isopropyl ether : acetic acid (65 : 40 : 4, v/v/v), as described by Kuksis [24]. In this solvent system, all the phospholipids remain at the origin. Bands corresponding to standards of free cholesterol and cholesteryl esters were marked and scraped into vials. Both sterols and steryl esters were measured from the silica gel G by 3 extractions, each with 4 ml of chloroform. The extracts were pooled and evaporated to dryness. The steryl ester fraction was saponified with 1 N NaOH in 20% ethanol [22], The sterol fraction was recovered by the use of petroleum ether. The sterols were then quantitated by gas-liquid chromatography. The sterols (both free and esterified) were converted to trifluoroacetate derivatives and separated by gas-liquid chromatography using 5a-cholestane as an internal standard [22]. The sterols were quantitated on the basis of the internal standard peak areas. St,erols were analyzed in a gas-liquid chromatograph (Packard-Becker model 409) on 4-ft, 4-mm ID glass columns of 10% SP-2401 on Supelport (100 to 120 mesh). Column conditions were as follows: oven, 220°C; injector, 220°C; detector, 330°C; and carrier gas, helium, 50 ml/min. Sterols were identified by their retention times as compared with those of authentic standards. The plasma cholesterol level was measured by the method of Levine and Zak [25]. Plasma triglyceride levels were measured by the method described by Ellefson and Caraway [ 261. The method used to measure cholesteryl ester synthetase activity was that of St. Clair et al. [ 181, with a slight modification as reported previously [20]. The

120

activity of cholesteryl ester hydrolase was measured as described previously [ 191. For the enzyme studies, the second group of pigeons were killed by exsanguination, and their aortas were removed quickly, sliced, and homogenized in 0.1 M phosphate buffer (pH 7.4) for 1 min. The homogenate was subjected to conventional centrifugation procedures previously standardized for pigeon aortic homogenates [20]. The homogenate was centrifuged at 1,000 Xg for 20 min to remove the cell debris. Next, the supernatant was centrifuged at 12,000 Xg for 15 mm, and the pellet was used as a source of “lysosome-rich” fraction. This supernatant was then centrifuged at 100,000 X g for 30 min using an ultracentrifuge (Beckman LK-75). The pelleted microsomes were suspended in 0.1 M phosphate buffer to give an approximate protein concentration of 1 mg/ml. Aliquots of the supernatant fraction also were used for the enzyme studies. The protein concentrations were measured by the method of Lowry et al. [27]. For determining the activity of cholesteryl ester synthetase, 1 mg of microsomal suspension in phosphate buffer (pH 7.4) was incubated with ATP 15 pmol, CoA 15 pmol, NaF 15 pmol, MgCl, 12 pmol, and [‘4C]oleic acid 5 nmol in 25 ~1 of acetone. The incubation was carried out for 1 h at 37°C. The isolation of the end products of incubation by extraction and thin-layer chromatography is as described below. Incubation for the determination of cholesteryl ester hydrolase was set up as described previously [ 191. It consisted of incubating 1 ml of microsomal or supernatant fraction or 0.2 ml of lysosomal fraction with [ 14C]cholesteryl oleate (3-4 nmol in 25 ~1 of acetone) for an hour at 37°C in a metabolic shaker bath. The pH of the incubation mixture for microsomal and supernatant cholesteryl ester hydrolase was 7.4, while lysosomal incubations were carried out at apH of 4.4. If the substrate is added in acetone, virtually no acid cholesteryl hydrolase activity is found in the supernatant fraction. The blank tubes contained boiled suspension. : methanol (2 : 1) were At the end of all incubations, 20 ml of chloroform added to stop the reaction; 50-100 pug of the carrier cholesteryl oleate and cholesterol were then added to each tube. The organic layer was removed, evaporated to a small volume, and subjected to thin-layer chromatography on silica gel G using the solvent system heptane : isopropyl ether : acetic acid (65 : 40 : 4, vol/vol/vol) to separate free and esterified cholesterol [22]. The bands corresponding to the free and esterified sterols were then scraped and eluted four times with 4 ml of chloroform. The extracts were dried and assayed for radioactivity in a liquid scintillation counter (Packard model 3385). Enzyme activities were calculated as picomoles of product formed per mg in 1 h. All calculations were made on the assumption that there was no equilibration with the endogenous substrate. Our previous studies [ 191 found no equilibration of added substrate with the endogenous compound. Aortic subcellular fractionation studies by Brecher et al. [28] showed that acid cholesteryl ester hydrolase activity (presumably of lysosomal origin) was located mainly in the 100,000 Xg supernatant rather than in the 10,000 Xg precipitate. To test this unequal distribution of lysosomal enzymes in various cell fractions, acid phosphatase was used as a marker enzyme for lysosomes. Acid phosphatase activity was determined as described by Kwak et al. [ 291.

121 TABLE 1 BODY

WEIGHTS.

PLASMA

LIPIDS, AND

AREA

OF ATHEROSCLEROTIC

PIGEONS ___

LESIONS

(M i SE) IN

__-

Group

Body weight

(n)

(g)

Area of lesion

Plasma lipids (mg/dl) --

(mm2)

Cholesterol

Triglycerides

302 ? 19 291 * 11

156 + 33 151 +_35

___

---~ Sham (20) Ileal bypass (19)

556.9 f 9.6 541.2 f 10.6

a Significant difference between groups (P < 0.01). Methods section).

11.7 k 1.3 a 6.1 ? 1.2 a

Determined from the first study group of pigeons (see

Significance of the difference between the groups Student’s t test, with P < 0.05 considered significant.

was evaluated

using

Results There were no differences in body weights or in plasma cholesterol and triglyceride levels between the two groups, but the surface area of atherosclerosis in the aorta of bypass birds was significantly (P< 0.01)lower when these birds were compared with the sham birds (Table 1). There was no difference in the content of aortic free cholesterol between the two groups (Table 2). The content of cholesteryl esters, however, was significantly (P< 0.05) reduced in the pigeons with ileal bypass. The total sterol content of the aorta from pigeons with ileal bypass also was reduced, although the difference was not statistically significant. The activity of the cholesteryl ester synthetase was not different between the two groups (Table 3). The cholesteryl hydrolase activity present in the microsomal fraction of the aorta did not differ between the two groups. The activity of the cholesteryl ester hydrolase in the lysosomal fraction.was threefold higher (P< 0.05) in the aorta of ileal bypass birds. The cholesteryl ester hy(P< 0.01) drolase activity in the supernatant fraction also was significantly higher in the ileal bypass pigeons. Unlike the observations of Brecher et al. 1281, cholesteryl ester hydrolase activity at pH 4.4 in the supematant fraction of aorta was small (