Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

Biochimica et Biophysics Acru 958 (1988) 308-312 Elsevier

308

BBA Report

BBA 50224

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells J. Peter Slotte and Edwin L. Bierman Division of Metabohsm, Endocrinology and Nutrition, Department of Medicrne, University of Washington, Seattle, WA (U.S.A.) (Received

Key words:

Acid cholesterol

esterase;

Cholesterol;

19 August

1987)

Fatty acid specificity;

(Human

arterial

smooth

muscle cell)

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of celfs with 0.2 mg/ml low-densi~ li~protefn and 50 PM c~oroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without cbloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with li~protein-deficient serum revealed that ~Iyunsa~rat~ cholesteryi esters (finoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA : cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We con&de that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydroIyzes polyunsaturated cholesteryl esters.

During the pathogenesis of atherosclerosis, the cholesteryl ester content of the arterial wall increases significantly. Chemical analyses have shown that cholesteryl oleate is the main subclass among the accumulated cholesteryl esters in fatty streaks [l]. This is in marked contrast to the fatty-acid composition of cholesteryl esters in circulating low-density lipoproteins (predominantly cholesteryl linoleate). It has therefore been suggested that the accumulated cholesteryl esters are synthesized endogenously by acyl-CoA : cholester-

Abbreviations: DMEM, Dulbecco’s LDL, low-density lipoprotein.

modified

Eagle’s medium;

Correspondence (present address): J.P. Slotte, Department of Biochemistry and Pharmacy, Abo Akademi, SF-20500 Turku, Finland. ~05-2760/88/~03.50

8 1988 Elsevier Science Publishers

01 acyltransferase, a reaction which preferentially uses oleic acid as the reaction co-substrate [2]. On the other hand, an enrichment of cholesteryl oleate in cholesteryl esters of arterial cells could also be due to a preferential lysosomal hydrolysis of cholesteryl linoleate in cholesteryl esters derived from internalized low-density lipoproteins. Indeed, it has been shown that partially purified cholesterol esterase from rabbit, rat and monkey arterial homogenates in vitro hydrolyzes polyunsaturated cholesteryl esters slightly faster than the more saturated cholesteryl-ester subclasses 13-51. A similar fatty-acid specificity has also been reported for partially purified lysosomal rat liver cholesterol esterase (61. The availability of a specific inhibitor of acylCoA : cholesterol acyltransferase (Sandoz Compound 58-035) makes it possible to determine the

B.V. (Biomedical

Division)

309

fatty-acid specificity of acid cholesterol esterase in intact cells. The acyl-CoA : cholesterol-acyltransferase reaction, which otherwise would produce cholesteryl oleate during a hydrolysis experiment, can now be inhibited. Compound 58-035 has been shown to be a specific inhibitor of acylCoA : cholesterol acyltransferase [7], and does not affect the activity of the lysosomal cholesterol esterase [S]. Hence, changes in the fatty-acid profile of cellular cholesteryl esters should be due only to the hydrolysis reaction, provided that the cytosomal concentration of cholesteryl esters is negligible compared to the pool of lysosomal cholesteryl esters. In this study, we used human arterial smooth muscle cells with a negligible mass of endogenously synthesized cholesteryl esters at the start of the experiment (less than 7.5 pmol cholesteryl ester/mg protein). We then enriched the lysosomal compartment of these cells with LDL-derived cholesteryl esters. A subsequent hydrolysis of cholesteryl esters was induced by incubating cells in medium containing lipoproteindeficient serum. The fatty-acid composition of the cellular cholesteryl esters in cells with or without acyl-CoA : cholesterol-acyltransferase activity (blocked by 58-035) was then determined before and during the incubation with lipoprotein-deficient serum. Cultured human arterial smooth muscle cells were derived from intima-medial explants of human aorta [9]. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum. Cells were plated in 35 mm diameter cell culture dishes (100 000 cells/dish) and grown for 9 days, with medium replaced every other day. The initial cholesterol mass in cells grown in 10% fetal calf serum was 65 and 7.5 pmol sterol/mg cell protein for free and esterified cholesterol, respectively. On day 1, 1.0 ml of serum-free DMEM with 0.2 mg/ml LDL and 50 PM chloroquine was added to each dish. After 24 h, the cells were rinsed five times with 2 ml phosphate-buffered saline (pH 7.4) and 1.0 ml DMEM with 5% lipoprotein-deficient serum was added. Some sets of cells also received 2 pg/ml of the acyl-CoA : cholesterol acyltransferase inhibitor, 58-035. The acyl-CoA : cholesterol acyltransferase reaction was completely blocked at this inhibitor concentration (not shown). The smooth

muscle cells were then incubated in this medium for 0, 4, 8 and 23 h at 37 o C. The incubation was terminated by decanting the medium containing the lipoprotein-deficient serum and by rinsing the cell monolayer extensively (5 x 2 ml) with phosphate-buffered saline. The cellular neutral lipids were extracted with hexane/isopropranol (3 : 2, v/v) [lo]). The extraction solvent also contained 0.5 pg/ml of butylated hydroxytoluene as antioxidant. Free and esterified cholesterol were separated on Silica Gel H thin-layer chromatography plates (Analtech, Newark, DE) eluted with hexane/diethyl ether/glacial acetic acid (135 : 30 : 1.5, v/v). The spotting of lipids onto plates and the subsequent elutions were performed under nitrogen atmosphere. A part of the cholesteryl-ester spot was taken for fatty-acid analysis by gas liquid chromatography [ll]. Cholesterol mass in extracts from the chromatograms was determined by a cholesterol-oxidase method [12]. Cellular proteins remaining on dishes after lipid extraction were digested into 1.0 ml 0.1 M NaOH (1 h at room temperature). An aliquot was taken for protein mass determination with bovine albumin as standard [13]. Accumulation of LDL-derived cholesteryl esters in lysosomes of cultured human arterial smooth muscle cells was achieved by incubating cells for 24 h with 0.2 mg/ml LDL in the presence of 50 PM chloroquine. Chloroquine is known to inhibit effectively the lysosomal degradation of internalized cholesteryl esters in cultured cells [14], thus leading to lysosomal accumulation of unhydrolyzed cholesteryl esters. To force removal of cholesterol from cholesteryl-ester-enriched smooth muscle cells, the cells were incubated in DMEM supplemented with 5% lipoprotein-deficient serum. With this treatment, the mass of cellular cholesteryl ester was significantly reduced during the 23 h incubation period (Fig. 1). When the re-esterification of hydrolyzed free cholesterol by acylCoA : cholesterol acyltransferase was blocked (with 2 pg/ml 58-035) an even greater reduction in the mass of cellular cholesteryl ester could be observed (Fig. 1). Since only free cholesterol can leave cells [10,15,16], the cellular cholesteryl ester mass reduction was mainly the result of a lysosomal hydrolysis of cholesteryl esters and a subsequent efflux of the free cholesterol so produced.

310

0 A Control 0. ACAT-lnhlbltor 68-035)

Al6 Al6

0 1

SE 18’ 020 cl22

s: 2 4 4

P 3 : 5

Q

50-

A--A

A

/

01

9

5

IO 15 lncubatlon time (h)

20

25

Fig. 1. Cholesterol mass in human smooth muscle cells exposed to Iipoprot~in-deficient serum. Confluent cells were enriched with cholesteryl esters by treatment with LDL and chloroquine, as described in the text. Cells with or without 2 pg/ml 58035 were then incubated in serum-free DMEM supplemented with 5% lipoprotein-deficient serum for up to 23 h at 37 o C. Cholesterol and cholesteryl ester mass values (given as pmof sterol/mg cell protein) are averages from two or three dishes of two experiments (k SD.).

To elucidate which, if any, of the cholesteryl ester subclasses were preferentially hydrolyzed during the incubation, the fatty acid composition of the cholesteryl esters remaining in cells after 4, 8 and 24 h of incubation in DMEM containing lipoprotein-deficient serum was determined. At the beginning of the incubation, the fatty-acid profile of cellular cholesteryl esters (Fig. 2A and TABLE

I

FATTY ACID COMPOSITION OF CHOLESTERYL TERS IN LOW-DENSITY LIPOPROTEINS

ES-

The fatty acid composition of cholesteryl esters in LDL used for loading the smooth muscle cells with cholesteryl ester mass was determined as described in the text. Values were obtained from a representative batch of LDL. Cholesteryl subclass

ester

14:o 16:O 16:l 18:0 18:l 18:2 20:4 18: l/18:2

Composition (% of total) 1.7 12.5 1.5 1.9 17.9 57.3 7.2

ratio = 0.31

0

5

IO 15 Incubation time (h)

20

i 5

Fig. 2. Fatty-acid profile of cholesteryl esters remaining in cells after exposure to lipoprotein-deficient serum. The values were obtained from the ceils shown in Fig. 1. (A) Fatty-acid composition of cholesteryl esters in cells with an active acyl CoA : cholesterol-acyltransferase reaction without (58-035) during the incubation period. (B) Fatty-acid profile in cells with an inhibited acyl-CoA : cholesterol-acyltransferase reaction (2 ng/ml 58-035). Different cholesteryl esters are designated according to their fatty-acid composition (i.e., number of carbons: number of unsaturations (e.g., 16: 0) in the fatty acid chain). Values are expressed as pmol sterol/mg cell protein.

B, zero time) was very similar to that found for pure LDL (Table I). Cholesteryl linoleate was the dominant subclass (more than 50% of the total). This similarity of the cellular cholesteryl ester fatty-acid composition with that of pure LDL indicates that no change (i.e., hydrolysis) in composition had occurred during the 24 h pretreatment of cells with LDL and chloroquine. During the 23 h treatment of cells with lipoprotein-deficient serum, about 50% of the original cholesteryl linoleate (18 : 2) mass was lost (Fig. 2A and B). An examination of the absolute values and percent

311

TABLE

II

FATTY ACID COMPOSITION OF CELLULAR LIPOPROTEIN-DEFICIENT SERUM Confluent described cells were groups of d% is the the range Cholesteryl ester subclass 16:O 16:l 18:O 18: 1 18:2 20:4 2214

human

arterial

smooth

CHOLESTERYL

ESTERS

BEFORE

AND

AFTER

A 23 h EXPOSURE

TO

muscle

cells were loaded with cholesteryl esters by treatment with LDL and chloroquine. as among cellular cholesteryl esters was determined from the neutral lipid extract. Some also exposed to 2 pg/ml of an acyl CoA: cholesterol acyltransferase inhibitor (58-035). Values are averages from two cells with triplicate dishes in each group. The cholesteryl ester mass values are expressed as pmol sterol/mg cell protein. percent change observed in cholesteryl ester content at 23 h compared to the 0 h value. Numbers in parentheses represent of averages.

in the text. The fatty acid distribution

Fatty acid composition

(pmol sterol/mg

protein) compound

control Oh

23 h

19.3 1.3 5.6 28.8 88.0 11.1 12.3

13.9 1.9 5.1 26.8 47.4 7.5 7.5

58-035

(A%)

Oh

23 h

(A%)

-31 (*3) +50(+50) -lO(ill) -7 (k5) -46 (k2) -33 (+5) -39 (k4)

18.8 1.2 5.1 27.1 82.5 10.4 11.5

9.6 1.4 3.4 19.4 38.6 4.3 3.2

-49(k3) + 22 ( * 20) -33 (k5) -28 (k7) -53 (k2) -58 (k3) -70(*30)

changes of individual cholesteryl ester subclasses found in cells at the beginning of the experiment (zero time) and at the completion of the incubation (23 h) shows that the cholesteryl oleate reduction was only about 7% in cells with an active acyl-CoA : cholesterol-acyltransferase reaction (Table II). This small reduction can be compared with the 28% decrease found in cells with inhibited acyl-CoA : cholesterol-acyltransferase activity (Table II). This discrepancy in values shows that acyl-CoA : cholesterol acyltransferase produces cholesteryl oleate during the hydrolysis reaction, and thus leads to an incorrect estimation of cholesteryl oleate hydrolysis in cells with an active acyl-CoA : cholesterol acyltransferase. When the hydrolysis of the major cholesteryl ester subclasses was considered, it was found that cells with inhibited acyl-CoA : cholesterolacyltransferase activity displayed a preferential hydrolysis of cholesteryl linoleate (18 : 2, 53% reduction in 23 h) and cholesteryl arachidonate (20: 4, 58% reduction in 23 h), whereas the reduction in cholesteryl oleate (18 : 1) was only 28% in 23 h. Since the contribution of the cytosomally localized cholesteryl ester mass (the initial 7.5 pmol sterol/mg cell protein) to the observable rates of cholesteryl ester hydrolysis is negligible (less than 5% of the total load of cholesteryl ester

mass), the differences in concentrations of cellular cholesteryl ester subclasses before and after incubation in lipoprotein-deficient serum can be taken to represent selective lysosomal hydrolysis of the different cholesteryl ester subclasses. The ratio of cholesteryl oleate to cholesteryl linoleate in cellular cholesteryl esters changed continuously over the entire incubation period (Fig. 3). This ratio was 0.33 (Fig. 3) at the beginning of the experiment. At the end of the 23 h incubation with 5% lipoprotein-deficient serum, the ratio of oleic to linoleic acid in cholesteryl esters had increased to about 0.6 in cells with an active acyl-CoA : cholesterol acyltransferase enzyme and to about 0.5 in cells with blocked acylCoA : cholesterol-acyltransferase activity. In a similar study with cultured human skin fibroblasts, Stein and co-workers [17] obtained results indicating that lysosomal hydrolysis of LDL-derived cholesteryl esters resulted in an increase in the cholesteryl oleate/cholesteryl linoleate ratio in the remaining cholesteryl esters. They concluded that most of the changes in the cholesteryl oleate/ cholesteryl linoleate ratio was due to an ongoing acyl-CoA : cholesterol-acyltransferase reaction, and that the results did not reflect a preferential hydrolysis of polyunsaturated cholesteryl esters. Results in the present study

312

0

Control

A &CAT-lnhibrtor 06

(58-035)

,O

0 e

of cholesteryl oleate over other cholesteryl ester subclasses. We acknowledge the skilled assistance of Maria Culala, Daniela Hairabedian, and Karin Sundquist. Part of this study was supported by NIH Grant HL 18645. J.P.S. was a Research Fellow of the American Heart Association (Washington Affiliate). References 1 Smith,

Fig. 3. Ratio of cholesteryl oleate to cholesteryl linoleate in cellular cholesteryl esters during treatment with Iipoprotein-deficient serum. The values are derived from numbers given in Table II. The cells were treated as described for Fig. 1.

2 3 4

have shown that even when the acyl-CoA : cholesterol-acyltransferase reaction is completely inhibited, one still can observe an increase in the cholesteryl oleate/cholesteryl linoleate ratio, suggesting a preferential hydrolysis of cholesteryl linoleate over cholesteryl oleate. The results with respect to the substrate-specificity of lysosomal cholesterol esterase in intact cells are consistent with previously reported results on the substrate-specificity of the rat liver lysosomal cholesterol esterase, as measured with isolated lysosomes in vitro (61. In conclusion, it appears that the observed enrichment of cholesteryl oleate in accumulated cholesteryl esters (at least in this cell model system) is at least in part due to preferential hydrolysis of cholesteryl linoleate by the acid cholesterol esterase. It is clear, however, that the intracellular esterification of cholesterol by acyl-CoA : cholesterol acyltransferase also results in an enrichment

5 6 I 8 9 10 11 12 13 14 15 16 17

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