Deposition and mobilization of cholesterol ester in cultured human skin fibroblasts

367

Biochimica et Biophysics 0 Elsevier/North-Holland

Acta,

450

Biomedical

(1976)

367-378

Press

BBA 56895

DEPOSITION AND MOBILIZATION OF CHOLESTEROL CULTURED HUMAN SKIN FIBROBLASTS

0. STEIN,

J. VANDERHOEK,

G. FRIEDMAN

ESTER IN

and Y. STEIN

Lipid Research Laboratory, Department of Medicine B, Hadassah University Hospital and Department of Experimental Medicine and Cancer Research, Hebrew University-Hadassah Medical School, Jerusalem (Israel)

(Received

Jurle 18th, 1976)

Summary Human skin fibroblasts in culture served as a model system to study intracellular cholesterol ester deposition in mesenchymal cells. Confluent cultures were exposed to homologous low density lipoprotein alone and together with chloroquine. In the presence of low density lipoprotein alone, even at half circulating serum concentrations, cellular free cholesterol increased no more than 12%, while the increase in cholesterol ester ranged from 13-100% during 48 h of incubation. Addition of chloroquine to the culture medium containing low density lipoprotein resulted in a very marked increase in cholesterol ester and the ratio of cellular esterified cholesterol to free cholesterol rose up to 2.2. In the presence of chloroquine the sum of uptake and degradation of ““I-labeled low density lipoprotein was enhanced and at higher chloroquine concentrations the more pronounced inhibition of degradation resulted in the intracellular retention of undegraded protein. Upon removal of the chloroquine-containing medium, there was a slight fall in the cellular cholesterol after 24 h incubation in a medium containing 10% fetal calf serum. Replacement of the fetal calf serum by lipoprotein-deficient serum and a mixture of high density apolipoprotein and sphingomyelin increased very significantly the loss of total cholesterol from the cells. At the same time the rate of loss of the retained labeled low density lipoprotein was also increased. The present results provide an adequate and reproducible model system for the study of cholesterol accumulation in human mesenchymal cells, which is one of the basic changes in atheromatosis. The availability of cholesterol ester laden cells also provides a good system for the study of agents active in cholesterol removal. Introduction Accumulation of cholesterol ester in the cells of mesenchymal origin (fibroblasts and smooth muscle cells) and later on in the extracellular material, is the

hallmark of atherosclerosis. The aim of the present study was to establish optimal conditions in vitro which would promote accretion of cellular cholesterol, which under normal conditions remains fairly constant. In an attempt to change the cholesterol content of cultured fibroblasts, Bailey has shown that increase of cellular cholesterol was related to the origin of the serum used, rather than to its cholesterol content [ 11. Using mouse L-cells and human skin fibroblasts, Rothblat [ 2) was able to increase cholesterol ester content of these cells by incubation with hyperlipemic rabbit serum. However, even in the presence of 20% hyperlipemic rabbit serum for 3 days, esterified cholesterol did not exceed 20 pg/mg cell protein and the ratio of cholesterol ester to free cholesterol was less than 1.0. Under similar conditions rat hepatoma cells accumulated up to 70 pg cholesterol esterlmg protein [2]. In previous attempts with rat aortic smooth muscle cells, we have used cholesterol containing emulsions as well as sera of different cholesterol content or isolated human low density lipoprotein, but the increase in cellular cholesterol was rather modest [ 31. Low density lipoproteins of bovine [ 41, human [4,5] and pig [6] origin have been shown to restore the cholesterol content of skin fibroblasts and pig aortic smooth muscle cells which had been depleted of cholesterol by culture in serum free media [4] or by preincubation in lipoprotein-deficient serum [5,6]. However, addition of human low density lipoprotein to culture medium resulted in an increase of only 75% in the cholesterol content of human skin fibroblasts, which had not been exposed to lipoprotein-deficient serum [7]. Following interiorization, low density lipoprotein has been shown to be concentrated in secondary lysosomes [ $1, the enzymes of which participate in the degradation of proteins [9] and cholesterol esters [lo]. An interference with the degradative process could result in an accumulation of the ingested products; lately inhibition of intralysosomal degradation has been achieved with the help of chloroquine [ 11-131. More recently, this drug was shown to inhibit the hydrolysis of protein and cholesterol ester of low density lipoprotein in human skin fibroblasts [14,15]. This approach was studied presently and a lo-fold increase in the concentration of cellular cholesterol ester was achieved. We have then attempted to study the reversal. of this process and have utilized “acceptors” which had been shown to promote the release of cholesterol from various cell types [ 7,16-181. Materials and Methods Human skin fibrobhsts

Biopsies were obtained from the medial part of the forearm of normal male adults, after informed consent. The cells were grown in plastic Falcon petri dishes (60 mm in diameter), in 4 ml medium (modified Dulbecco-Vogt [19] containing 10% fetal calf serum), which was changed every other day. For each experiment, about 2 _ 10” cells were seeded and all experiments were performed on cells which had reached a confluent stage. For the present experiments the cells used were between the second and 8th passage. Preparation

Human

of lipoproteins,

apoproteins

low and high density

and liposomes

lipoproteins

were isolated

by ultracentrifuga-

369

tion according to the method of Have1 et al. [20] in a Spinco ultracentrifuge, model L2 65B, from human serum containing 1 mg/ml of EDTA. Low density lipoprotein and high density lipoprotein were isolated at d = 1.019-1.063 and d = 1.063-1.21, respectively. The lipoproteins were washed twice at their respective densities and then dialyzed exhaustively against 0.9% NaCl, 0.01% EDTA solution, pH 7.4. All procedures were performed at 4°C. The low density lipoproteins were examined under the electron microscope and were found to consist of particles of uniform size, 200-220 K in diameter. Low density lipoprotein was iodinated following the ~~acFarla~le [21] iodine monochloride technique, modified as described before [ 221. Free iodine was removed by repeated dialysis against 0.9% NaCl, 0.01 EDTA, pH 7.4. More than 98% of the label were precipitable with 10% trichloroacetic acid and not more than 5% were extractable in chloroform/methanol. To prepare high density apolipoprotein, the high density lipoprotein was delipidated according to the method of Scanu and Edelstein [23] and 90-98% of protein was recovered after the delipidation. To prepare the lipoprotein deficient serum, the d > 1.21 fraction (after removal of high density lipoproteins) was adjusted to d = 1.25 with KBr and subjected to an additional 48 h of centrifugation at 165 000 X g,,. This final fraction (d > 1.25) was dialyzed against 0.9% NaCI, heated for 30 min at 56°C and sterilized by filtration through a 0.45 pm Millipore filter. The protein concentration ranged between 50-70 mg/ml, total cholesterol ranged between 6-12 pg/ml, of which about 70% was esterified and the phospholipid content ranged between 2.5-5.0 pg lipid phosphorus per ml. Rat liver sphingomyelin was isolated and fractionated by silicic acid column chromatography as described previously [‘24]. The sphingomyelin recovered in the chloroform/ methanol fraction (2 : 3, v/v) was further purified from traces of lecithin and lysolecithin by mild alkaline hydrolysis (0.4 M KOH in 70% methanol incubated overnight at 37°C). The sphingomyelin proved to be more than 98% pure on silica gel thin-layer chromatography in two different solvent systems, chloroform/methanol/water (70 : 25 : 4, by vol.) and chloroform/methanol/acetic acid/water (50 : 25 : 8 : 4, by vol.). Prior to sonication, 5 mg of sphingomyelin in chloroform were placed in a glass tube (internal diameter 1.1 cm, length 10 cm) and the solvent was removed under nitrogen. 0.5-2.0 ml of 0.1 M Tris (pH 8.0) in 0.9% NaCl were added to the glass tube containing the dry phospholipid, and subjected to ultrasonic irradiation in a Braun-Sonic 300 instrument (Braun, Melsungen, Germany), using a microtip of a diameter of 4 mm, at the maximal scale. The sonication was carried out under N2 for 2 X 2.5 min, during which the solution was kept in a water-ice bath. To each ml of the sonicated liposomes 5 mg of high density apolipoprotein were added and the solution was left at room temperature for l-2 h, in which time the turbidity of the sphingomyelin - lipoprotein mixture had cleared completely. Prior to use, the apolipoprotein - sphingomyelin was centrifuged at 1200 rev./min 10 min. Experimental Enrichment

procedure of cells with free and esterified

cholesterol

All experiments were carried out on human skin fibroblasts which had grown to confluency for 4-5 days after seeding in medium containing 10% fetal calf

serum. Chloroquine diphosphate dissolved in serum-free culture medium at pH 7.0 and low density lipoprotein were sterilized by Millipore filtration prior to the addition to the petri dish. The enrichment with free and esterified cholesterol was performed under two conditions: (1) 20-70 ,uM chloroquine diphosphate and low density lipoprotein (to give 10 or 360 1.18protein/ml of incubation medium) were added to medium containing 10% fetal calf serum and the cells were incubated for 48 h. (2) The fetal calf serum in the medium was replaced by the lipoprotein-deficient serum to give a final concentration of 2.5 mg/ml and the cells were incubated with the lipoprotein deficient serum for 24 h. At the end of this period, the medium was removed and chloroquine and low density lipoprotein were added to fresh medium of the same composition and the incubation was continued for 48 h. At the end of incubation, the medium was removed and saved for the determination of f251-labeled protein degradation products [253. The cell layer was washed 3 times with 0.2% albumin in phosphate buffered saline [26] and 3 times with phosphate buffered saline. The cell layer was scraped with two l-ml portions of 50% methanol and two 2-ml portions of 100% methanol. Following the addition of 5 ml chloroform, the cell suspension was gassed with N2 and lipids were extracted overnight. Following centrifugation at 2500 rev.fmin for 30 min, the pellet was removed and used for the determination of ‘*‘I radioactivity and for protein determination after hydrolysis in 1 M NaOH. To the chloroform/methanol (1 : 1) extract were added 62.5 pg campesterol as internal standard for gas chromatographic estimation of cholesterol. Following addition of a further 5 ml of chloroform, the lipids were washed according to Folch et al. [ 271. The purified chloroform phase was used for cholesterol determination.

Cholesterol

removal from enriChed cells

To study cholesterol removal, cells were enriched as described above. The medium was removed, the cell layer was washed 3 times with phosphate buffered saline and replaced by 2 ml of medium containing 10% fetal calf serum or lipoprotein-deficient serum to give a final protein concentration of about 30 mg/ml. The sphingomyelin - apoprotein mixture was added to 1.8 ml of the latter medium to give a final concentration of 0.5 mg/ml of each. The cells remained in contact with the “depleting” medium for 24 h and the experiment was terminated as described above.

A~alyt~~a~ and ~hrornato~ra~h~~ procedures Cholesterol determination. During these experiments,

we observed in various samples the presence of an unidentified component whose gas chromatographic retention time was nearly identical to that of 5a-cholestane, the internal standard that we [7] as well as others 1281 have used. In order to avoid this situation, we now routinely use campesterol as the internal standard. Free and total cholesterol determinations were carried out according to a modification of the procedure of Ishikawa et al. [28] and are reported in detail in a previous publication [ 71. Lipid phosphorus was determined according to Bartlett [ 291 and protein was determined according to Lowry et al. [30] using bovine serum albumin as stan-

371

dard. Radioactivity was determined using the Autogamma scintillation spectrometer (Packard, La Grange, Ill.). Radioactive iodine, as Nat*‘1 carrier free, was obtained from the Radiochemical Centre, Amersham, England. Chloroquine diphosphate salt was obtained from Sigma Chemical Company, St. Louis, MO. Campesterol was purchased from Supelco.Inc., Bellefonte, Pa., and the purity was found to be greater than 98% by gas liquid chromatography. Results The aim of the present study was to define conditions under which normal fibroblasts in culture will accumulate considerable amounts of cholesterol ester, and to determine the reversibility of such a process. To that end, use was made of ‘251-labeled low density lipoprotein, which is known to be ingested by the fibroblasts and of chloroquine, which interfered with its degradation. In order to determine optimal conditions for maximal accumulation of cholesterol ester, two experimental designs were used. In the first, the fibroblasts had been preincubated for 24 h in the presence of lipoprotein-deficient serum (2.5 mg protein/ml), and then chloroquine and 1251-labeled low density lipoprotein were added to fresh medium, containing lipoprotein-deficient serum. In the second design, the cells were maintained in culture medium containing 10% fetal calf serum throughout the experimental period. Results of three experiments, in which cells from different donors were used, are presented in Table I. When trace amounts or low density lipoprotein were added (10 pg protein/ml), the amount of lipoprotein retained with the cells plus the amount which had been degraded (total amount of lipoprotein metabolized) was 2.2-7.6 times higher when the cells had been preincubated with lipoprotein-deficient serum. However, when the concentration of low density lipoprotein was 360 I-18protein! ml, the total amount of lipoprotein metabolized by the cells preincubated with lipoprotein deficient serum was only up to 1.5 times higher and in some experiments the amount of lipoprotein recovered with these cells was even lower than in those maintained with 10% fetal calf serum (Table I). Next, the effect of chloroquine was compared under the two experimental conditions, i.e., in the presence of 10% fetal calf serum or lipoprotein-deficient serum in the medium. When the cells were exposed to trace amounts of ‘2SI-labeled low density lipoprotein, addition of 20 PM chloroquine resulted in a 20-50 fold increase in the amount of labeled protein recovered with the cells under both experimental conditions. In the presence of 360 pg/ml of low density lipoprotein in the medium, the increase was still 3-4.8 fold (Table I). However, while the addition of 20 PM chloroquine resulted in a 1.9-2.8 fold increase in the total amount of lipoprotein metabolized by cells maintained with 10% fetal calf serum, this increase was maximally 1.6 times in cells preincubated with lipoprotein deficient serum. When the concentration of chloroquine was raised to 70 I.IM and that of low density lipoprotein protein was 360 ,ug protein/ml, there was a twofold increase in the total amount of lipoprotein metabolized, but owing to a greater interference with degradation, up to 13 times more lipoprotein protein remained in the cells incubated in the presence of 10% fetal calf serum. This increase was only &fold in the cells which had been preincubated with lipoprotein deficient serum.

372

TABLR

I

COMPARISON

OF

RUM

RETENTION

ON

TEIN The

BY cells

TIIE

MEDIA

CHLOROQUINE were

AND TRl?ATED

incubated

either

period

or lipoprotein-deficient

bation

with

dishes.

Retention

chloroquine

CONTAINING

LIPOPROTEIN-DEFICIHNT

DEGRADATION HUMAN

in medium serum

(2.5

I *sI.labeled

and

= l 2 s I-protein

wcovcrcd

SKIN

low

10%

fetal

protein/ml) density

OR

LOW

FKThL

DENSITY

CALF

for

24

lipoprott‘in.

calf

s~r‘um

h prior

to

Values

throughout and aw

the rxperimental

during

the

means

of

48 h of incu-

duplicate

Additions

to medium

..I?SI.f,ipoprotein

protein.

(ng/mg

cell protein

l)vr 48

With fetal .“__

protein

h)

I

donor Lipo-

petri

in the cells.

-___“. Cell

SF.-

LIPOPRO-

FIBROBLASTS

containing mg

SERUM

‘2sI-L,ABELED

OF

calf

serum . -

With

liitoprotein-dPficicnt

protein

lkten-

Degra-

Reten-

DC@?%-

(irglmlf

tion

nation

tion

dation

_-_-

serum

-_..-_ A.B.

LA.

M.M[.

A.B.

LA.

M,M. __

50

10

0

1453

45

4528

10

20

942

1882

987

6568

10

0

45

1265

10

20

SO3

2785

73

1700

10

0

10

70

1733 11135

250

1000

19633

5506

16939

4823

360

0

1044

7571

360

20

3881

13894

2000

I2300

26600

4333

14485

20

0

67

1814

360

70

21088

9239

0

360

9958

1989

3330

360

360

83

4633

797

1 5000

3800

15700

1200

6140

9600

4160 ._ _..

^...i.

I-_..-

The accumulation of fret?. cholesterol and cholesterol ester in the fibroblasts was examined in the presence of chloroquine and at two concentrations of low density lipoprotein, 10 and 360 pg/ml. The total amount of cholesterol in cells of donor M&I. maintained in 10% fetal calf serum was 44.9 pg/mg cell protein and 29.7 ,ug/mg in those exposed to lipoprotein deficient serum. When 20 PM chloroquine and low density lipoprotein (360 I.tg/ml) was added to medium containing 10% fetal calf serum, free chole&erol increased from 41.3 to 61.6 pg/mg cell protein and cholesterol ester from 3.6 to 50.2 pg/mg cell protein after 48 h of incubation. When 20 ,&V?chloroquine and low density lipoprotein (360 ,ug/ml) were added to medium containing Iipoprotei~l-deficient serum, free cholesterol rose from 25.0 to 35.2 pgjmg cell protein and esterified cholesterol from 4.7 to 32.6 pg/mg cell protein after 48 h of incubation. Hence, in most experiments the cells were incubated in medium containi~~~ 10% fetal calf serum throughout the experimental period. As the cholesterol ester content of human skin fibroblasts derived from different donors varied under standard culture conditions, and ranged between 5 and 15 ,ug/mg cell protein, it seemed preferable to treat the data derived from different donors ~nd~v~dua~ly. The response to chforoquine and low density lipoprotein with respect to cholesterol ester accretion was determilled in severai ceil lines and the results of representative experiments are shown in Table II. A 2 to 3.5fold increase in cholesterol ester content of the cells was observed following 48 h incubation wit.h trace amounts of Low density lipoprotein and

373

TABLE

11

EFFECT AND The

OF

CHLOROQUINK

ESTIXRIFIED cells

were

10%

fetal

calf

with

10 v~/ml

AND

CHOLESTEROL

exposed

to

serum

for

of low

density

Cell

Additions

donor

__

LOW IN

chloroquine

48

h.

Values

and

low

are means

lipoproteins

to medium

DENSITY

HUhtAN

LIPOPROTEIN

SKIN density of

ON

ACCUMULATION

OF

FREE

FIBROBLASTS lipoprotein

duplicate

petri

added dishes.

to

culture

Controls

medium were

cells

containing incubated

only.

Cellular

cholesterol

Lipo-

Chloro-

fig/mg

protein

quine,

--

protein,

&M

Free

10

70

30.4

35.9

113

205

360

0

28.7

19.8

107

113

360

70

45.2

75.3

168

430

10

70

37.2

52.3

120

344

360

0

34.7

17.2

112.

113

360

70

55.2

96.8

171

637

cell

protein

% of control ---_--

Ester

Free

Ester

/.&/ml A.B.

B.W.

S. G.

10

J.K.

--

70

31.4

42.4

111

260

360

0

31.5

23.2

112

142

360

70

44.0

96.6

156

593

10

70

41.4

50.7

137

354

360

0

33.7

29.1

112

203

360

70

55.7

102.0

184

713

^

70 I_IMchloroquine. Addition of low density lipoprotein at a concentration of 360 pg protein~ml to culture medium containing fetal calf serum did not change the cholesterol ester content of the cells in some cell lines (A.B.; B.W.) and resulted in a 1.5 to 2-fold increase in others (S.G.; J.K.). Quite a pronounced accumulation of cholesterol ester ranging from 4 to 7-fold occurred in all experiments, when the cells were exposed to both 70 FM chloroquine and to high concentrations of low density lipoprotein. The increase in free cholesterol was less than Z-fold, and was also more pronounced and reproducible when both chloroquine and high concentration of low density lipoprotein were present in the culture medium. The cells were examined every day with a phase microscope. Several hours after the addition of chloroquine to the culture medium, numerous droplets and granules were seen in the perinuclear area. Their number increased with the time of exposure and at the end of 48 h the entire cell cytoplasm was filled with these droplets. In ultrathin sections examined under the electron microscope, the droplets could be identified as secondary lysosomes. A full morphological description of the changes occurring during chloroquine treatment in the presence of high doses of low density lipoprotein, as well as their regression (see below) will form a part of a separate report. We next studied cholesterol removal from cells which had been enriched with free and esterified cholesterol by exposure to chloroquine and low density lipoprotein. In order to vary the amount of cholesterol which accumulates in the cells, 20 or 60 PM chloroquine was added to culture medium containing either lipoprotein deficient serum (Expts. 1 and 2) or 10% fetal calf serum (Expt. 3) and low density lipoprotein 360 ,ug protein/ml. After 48 h, the me-

374

dium was removed, the cell layer washed three times with phosphate buffered saline and fresh culture medium was added. The latter contained either 10% fetal calf serum or the d > 1.25 fraction of human serum at final concentration of 30 mg protein/ml and high density apolipoprotein - sphingomyelin mixture (“depleting medium”) (see Methods). The cells had been confluent prior to loading and no further increase in the protein content of the dished occurred. As seen in Table III, the free or esterified cholesterol content of the cells incubated for 24 h with 10% fetal calf serum decreased to 25%. In the presence of the “depleting medium”, the loss of free cholesterol was 24-44s while that of esterified cholesterol ranged between 37-49%. Omission of high density apolipoprotein-sphingomyelin mixture from the “depleting medium” reduced the loss of cellular cholesterol. It seems of interest that under similar conditions of incubation, the amount of cholesterol ester decreased by 46 pg/mg protein in cells which contained 126 pg cholesterol ester/mg cell protein and by 20 ,Ftgimg protein only, in those which contained 42 pg cholesterol ester/mg cell protein. Since during the loading period “‘I-labeled low density lipoprotein was included in the culture medium, it was possible to follow the fate of the protein moiety of the ingested lipoprotein under the above described conditions. When cells, which have accumulated labeled low density lipoprotein in the presence

TABLE

III

REMUVAI, OF CHOLESTEROL FROM HUMAN SKIN FIBROBLASTS ENRICHED WITH CHOLESTEROL F:STI?R BY PRETREATMENT WITH CHLOROQUlNl+: AND LOW DENSITY LIPOPROTI~IN Conditions: enrichment with cholesterol ester was achieved by incubation of thv &Is with human low density lipoprotein (360 prg protein/ml) and either 20 !.&I (Expt.1) or 60 PM chloroquine (Expts. 2 and 3) for 48 11, added to either medium containing liltopfotein-deficient srrum to give 2.5 mg protein/ml (Expts. 1 and 2) or medium containing 10% fetal calf serum (Expt. 3). At the rnd of the 48 h loading IXriod the medium was removed, thta cell layer washed three times with phosphate buffrred saline and the petri dishes wwe post-incubated for 24 h with either medium containing 10% fetal calf swum or “drpleting” medium, in which the lattw was replaced by lipoprotein-deficient serum to givr 30 mg protein/ ml and the high density apolipoprotein - sphingomyelin mixture (see Methods). Cellular protein content Per petri dish was similar and did not changca at the end of 24 h post incubation. Values art* means + S.F. of triplicate dishes, except for “Now” (Expt. 2), in which frw choirstwo is from duplicate dishes. (a) vs. (b) P < 0.05; (c) vs. (d) and (e) vs. (f), I’ < 0.01.

.

EXPL.

no.

Post incubation for 24 h

Cellular cholesterol

% decrease

wg/mg cell protein

1

2

3

-.

Free

Total

10% fetal calf serum “Depleting” medium

65.8 i 0.8 58.5 i 6.3 49.8 + 4.2

106.9 99.3 70.6

None 10% fetal calf swum “Depleting” medium

49.9 37.4 ! 0.7 28.2 ’ 1 .I

None 10% fetal calf serum “Depleting” medium Lipoprotrin deficient serum (30 mg protrin/

73.4 f 1.5 72.4 t 3.5 42.1 + 2.2

199.4 180.4 122.1

ml)

63.5

+ 7.5

162.5

NOIIC

Free

Ester

i 8.6 i- 4.3 a + 8.8 b

11 24

19 49

92.3 -i 5.0 86.9 + 5.2 c 51.0 T 4.0 d

25

-

44

46

i 7.8 + 8.9 ’ i 10.0 f

2 43

14 37

+ 16.8

14

22

375

TABLE

IV

EFFECT

OF

INGESTED

CHOLESTEROL

REMOVAL

1251-LABELED

LOW

FROM

DENSITY

HUMAN

SKIN

LIPOPROTEIN

FlBROBLASTS

PROTEIN

ON

DURING

THE

RECOVERY

LOSS

OF

FROM

CHLOROQUINE Conditions: covered further -__.__

same

in cells.

as in Table from

three

Ill.

Values

are meaus

petri

dishes,

which

post-incubated, served _ _~_ .____--~~~~~-

Exp.

Percent

as 100%. -.____.

1251-labeled

low

I S.E.

had

of triplicate

ingested

the labeled

serum

The

lipoprotein

.~. _ _..~. density

lipoprotein

10%

Fetal

1

37.6

f 3.5

24.2

2

51.8

t 2.0

39.9

!: 3.4

3

62.9

+ 1.6

41.1

+ 1.9

“Depleting”

protein for

radioactivity 48

h and

__._ -. __.__-.__ in celis

no. calf

dishes.

post incubated _____-.---.-..

were

renot

__

for 24 h with .--.-..-...

medium

?- 0.9

of 20 ,uM chloroquine (Table I, Expt. l), were post incubated for 24 h in medium containing 10% fetal calf serum, they retained about 37% of the originally present labeled lipoprotein. When the chloroquine concentration had been 60 PM (Expts. 2 and 3) the percent lipoprotein that remained after 24 h post incubation ranged from 51 to 62%. In all experiments, the cells post-incubated for 24 h with the “depleting” medium retained significantly less labeled lipoprotein than those post-incubated in the presence of 10% fetal calf serum (Table IV). Discussion In the present study, human low density lipoprotein and chloroquine were used in order to promote maximal cholesterol accumulation in cells. Initially the lower concentration of chloroquine was used since this concentration was shown to cause accumulation of membrane-bound cytoplasmic inclusions and to interfere with the degradation of intracellular polysaccharides [13]. However, it appeared that even though at 20 FM chloroquine more labeled protein was recovered with the cells, the inhibition of the degradative process was only minimal. Thus, even when the concentration of low density lipoprotein in the medium was raised, only about 25% of the ingested protein was retained by the cells in 48 h. Pretreatment of the cells with lipoprotein-deficient serum did enhance low density lipoprotein uptake and degradation, but the stimulation was apparent for both the specific receptor mediated uptake [ 311 (studied at 10 pg protein/ml) and bulk uptake (at 360 pg protein/ml). However, net retention of undegraded protein was not increased under those conditions. Further inhibjtion of protein degradation and enhancement of protein accumulation was achieved by raising the concentration of chloroquine to 70 I.~Mbut this process was not stimulated by the presence of lipoprotein deficient serum. Higher concentrations of chloroquine (100 FM) were not used, as the latter were shown to detach cells from their support [ 121 and have other deleterious effects [ 131. The labeled protein served as an indicator for the retention of the lipoprotein particle, and the cholesterol content of the cells was measured independently in order to determine how it was affected by the composition of the medium

376

used. In cells treated with either medium (fetal calf serum or with lipoproteindeficient serum) some increase in cellular cholesterol ester was observed even at low lipoprotein concentrations in the medium at 20 /AM chloroquine. Raising the lipoprotein concentration in the medium at 20 and 70 PM chloroquine resulted in a further increase of both free and esterified cholesterol and these effects were much more pronounced when the cells had not been exposed to lipoprotein-deficient serum. Under these experimental conditions, enrichment of human skin fibroblasts derived from four different donors with free and esterified cholesterol was quite reproducible and the increment in total cholesterol ranged between 80-110 pg cholesterol/mg cell protein. The product which accumulated was mainly esterified cholesterol and the increment due to the action of chloroquine in the presence of high concentrations of lipoproteins ranged between 72-100 pg cholesterol ester/mg cell protein. The origin of the esterified cholesterol could be attributed to the medium lipoprotein, which in the face of cholesterol ester hydrolase inhibition by chloroquine [ 14,151 had accumulated in secondary lysosomes. Both lipoprotein uptake and the lack of suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in the presence of chloroquine [14] might have also contributed towards the slight rise of free cholesterol. Previously, we have studied cholesterol removal from Ehrlich ascites cells, rat aortic smooth muscle cells and normal human skin fibroblasts, in the presence of different acceptors in the medium [7,16--181. Maximal cholesterol loss without cell injury was achieved by addition of a mixture of sphingomyelin or phosphatidylcholine and human high density apolipoprotein to medium which contained lipoprotein-deficient serum at a concentration of 30 mg protein/ml [7]. Presently this “depleting” medium was tested for its capacity to promote cholesterol removal from cholesterol enriched cells. Under the current experimental conditions, two processes were operating during the 24 h period of post incubation; one was concerned with the restoration of activity of lysosomal enzymes, while the other was involved with the removal of the accumulated cholesterol from the cells. Studies on macrophages have shown that chloroquine loss from the cells is almost complete in 24 h [ll]. In human skin fibroblasts, restoration of protein degradation was shown to start 7 h after chloroquine removal [ 141. The amount of total cholesterol lost, from the cells during 24 h incubation in the depleting medium was higher than that removed in the presence of fetal calf serum in the medium. The enhanced loss of cellular cholesterol in the presence of depleting medium could be due to several factors, one of them being the stimulation of cholesterol ester hydrolysis. This possibility is supported by the increased loss of intracellular labeled protein (Table IV) during 24 h incubation in the depleting medium, a finding which suggests that labeled protein degradation was stimulated under these conditions. Furthermore, the cholesterol released during hydrolysis of cholesterol ester could be more readily lost into a medium containing cholesterol acceptors, rather than fetal calf serum. Even if a comparable restitution of lysosomal cholesterol ester hydrolase did occur in the presence of fetal calf serum, the cholesterol derived from hydrolysis of the ester might be c hanneled back into a reesterification pathway in the cytoplasm. Currently, insufficiency of lysosomal cholesterol esterase has been proposed

as a key factor in the development of atherosclerosis [10,32,33,34]. Thus the presently described model system, in which inhibition of lysosomal cholesterol esterase was combined with a high lipoprotein load to induce cholesterol ester deposition in mesenchymal cells, seems to be quite relevant. However, since in most atheromatous lesions cholesterol ester accumulates also in extralysosomal, cytoplasmic lipid droplets 1331, studies are in progress to extend further the similarity between the presently described cholesterol ester laden cells and xanthoma or atheroma cells encountered in vivo. If the cholesterol esterase insufficiency in primate or human atherosclerosis could be relieved by enhanced removal of cellular cholesterol then perhaps the “depleting” medium studied here might have wider applications. Acknowledgements The exceIlent technical help of Mrs. A. Mandeles, Mrs. Y. Dabach, Mr. G. Hollander and Miss H. Rose is gratefully acknowledged. This investigation was supported in part by grants from the United States-Israel Binational Foundation, by grant No. 409-MH of the Israeli Ministry of Health to 0. Stein, and by grant No. 22 of the Israeli Ministry of Health to Y. Stein (Established Investigator of the Minist~ of Health). References 1 Bailev, J.M. (1961) Proc. Sot. Exp. Biol. Med. 107, 30-35 2 Rothblat., G.H. (1974) Lipids 9, 626-535 3 Stein, Y., Stein, 0. and Vandwhoek, J. (1976) in Swnposiurn on Lipoprotein Metabolism (Greten, H., ed.), pp. 99-105, Springer Verlag, H&d&berg 4 Maea. R.D. and Connor, W.F:. (1971) Proc. Sot. Esp. Biol. Mrd. 138.913-919 5 Brown, M.S., Faust, J.R. and Goldstein. J.L. (1975) J. Clin. Invest. 55, 783-793 6 Weinstrin. D.B., Carrw. T.E. and Steinberg, D. (1976) Biochim. Biophvs. Acta 424. 404-421 7 Stein, 0.. Vanderhoek, .J. and Stein, Y. (1976) Biochim. Biophvs. Acta 431, 347-358. 8 Stein. 0. and Stein, Y. (1975) Circulation Res. 36, 436-443 9 Coffv, J.W. and dr Duwz, C. (1968) J. Biol. Chcm. 243, 3255-3263 10 Peters, T.J. and de Duve, C. (1974) Exp. Mol. Path& 20, 228-256 11 Izedorko, M.E., Hirsch, J.G. and Cohn, Z.A. (1968) d. Cell Binl. 38. 392-402 12 Wibo, M. and Poolr:, B. (1974) J. Cell Biol. 63, 430.--440 13 Lie, S.O. and Schofield, B. (1973) Biochrm. Pharmacol. 22. 3109-~3114 14 Goldstein, ,J.L., Brunschede, G.Y. and Brown, M.S. (1975) J. Biol. Chem. 250, 7854-7862 15 Brown, M.S., Dana. S.E. and Goldstein, J.L. (1975) Proc. Natl. Acad. Sci. U.S. 72. 2925-2929 16 Stein, 0. and Stein, Y. (1973) Biochim. Biophys. Acta 326, 232-244 17 Stein, Y., Glangeaud, ME., Izainaru, M. and Stein, 0. (1975) Biochim. Biophys. Acta 380, 106-118 18 Jackson, R.L.. Stein. O., Gotto, A.M. and Stein, Y. (1975) 1. Rio]. Cht-m. 250, 7204-7209 19 Ross. R. (1971) J. Cell Bioi. 50, 172-186 20 Havrl, R.J., Edrr, H.A. and Bragdon. J.A. (1955) J. Clin. Inwst. 34. 1345-1353 21 MacFarlanc, A.S. (1958) Nature 182, 53 22 Bilhcimer, D.W., Eisenberg, S. and Levy, R.I. (1972) Biochim. Biophys. Acta 260, 212-221 23 Scanu, A.M. and Edelsttsin, C. (1971) Anal. Biochem. 44, 576-588 24 Ra~hmil~wi~~, D., Eisenberg, S., Stein, Y. and Stein, 0. (1967) Biochim. Biophss. Acta 144, 624632 25 Bitwnan. E.L., Stein, 0. and Stein, Y. (1974) Circulation Res. 35, 136--350 26 Dulbecco. R. and Vogt, M. (1954) J. EXP. Med. 99, 167-187 G.H. (1957) J. Biol. Chem. 226. 497-509 27 Folch, .J., Lrcs, M. and Sloane-Stanlrv, 28 Ishikawa. T.T., MacGre, J., Morrison, J.A. and Gluwk. C..J. (1974) J. Lip. R~s. 15. 286.--291 29 Bartlett, G.R. (1959) J. Biol. Chem. 234, 466-468 30 Lowry, O.H., Roscbrough, N.J., Farr, A.L. and Randall, R-d. (1951) J. Biol. Chem. 193, 265-275

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12 (Porter,

K. and