Interaction between macrophages and aortic smooth muscle cells Enhancement of cholesterol esterification in smooth muscle cells by media of macrophages incubated with acetylated LDL

Biochimica et Biophysics Acta, 665 (198 1) 477-490

Elsevier/North-Holland

477

Biomedical Press

BBA 57886

INTERACTION BETWEEN MACROPHAGES AND AORTIC SMOOTH MUSCLE CELLS ENHANCEMENT OF CHOLESTEROL ESTERIFICATION IN SMOOTH MUSCLE CELLS BY MEDIA OF MACROPHAGES INCUBATED WITH ACETYLATED LDL 0. STEIN, G. HALPERIN and Y. STEIN ~epart~~ei~t of E~perime~~ta~ ~ed~c~~e and Cancer Research, Hebrew U~iversit~Hadassah ~ed~cai School, and Lipid Research Laboratory, ~e~rtment of Medicine B, Ha~ssah University Hospital, Jerusalem (IsraeEj

(Received May 5th. 1981)

Key words: LDL; HDL; Atherosclerosis;

Cell culture

Mouse peritoneal macrophages were cultured for 24 h in Dulbecco-Vogt medium ~ontain~g 10% calf serum. This medium was replaced with Dulbe~~o-Vogt medium contai~g 1% bovine serum ~burn~ to which all subsequent additions were made. Medium changes, accompanied by appropriate additions, were made every 48 or 72 h and the media were used for incubation of aortic smooth muscle cells, prelabeled with [ 3H]cholesterol. The amount of labeled cholesteryl ester in the smooth muscle cells incubated for 48 h with macrophage media which had been collected 48-144 h after addition of acetylated LDL was increased 3-4 times above that present prior to postincubation. A marked increment in cholesteryl ester mass occurred also after incubation of smooth muscle cells with macrophage media conditioned with acetylated LDL and this effect was shared by maleylated LDL, but not by other negatively charged compounds. The increase in labeled cholesteryl ester in smooth muscle cells was more pronounced with media collected at later time intervals of incubation with macrophages and was evident 8 h after postincubation. Only the d < 1.063 fraction of the medium enhanced cholesterol este~fi~tion in smooth muscle cells. The acetylated LDL reisolated from macrophage media at d < 1.063 did not compete with native LDL for degradation by smooth muscle cells. No increase in degradation of r 251-labeled acetylated LDL preincubated with macrophages was observed above that of non-preincubated acetylated LDL. The macrophage medium conditioned with acetylated LDL depressed [ 14C]acetate incorporation into sterols in smooth muscle cells and this effect was abolished by extraction of the medium with diethyl ether. The ratio of free to total cholesterol in the macrophage media collected after incubation with acetylated LDL increased from 28-70%, and a decrease occurred after incubation with smooth muscle cells. The enhancement of cholesterol esterification could be abolished by addition of high density apo~poprotein~sphingomyelin mixture during incubation with macrophages, even though excretion of free cholesterol into the medium increased 3-fold. It is proposed that when smooth muscle cells are presented with a lipoprotein in which an increase in the free to esterified cholesterol ratio occurred, and which is not recognized by a specific receptor, the enhancement of cellular cholesterol esterification is due mostly to a surface transfer of lipoprotein-free cholesterol. The present results offer another view of the possible interactions between macrophages and smooth muscle cells. A modified lipoprotein, not recognized by smooth muscle cells, is ingested by macrophages, which leads to accumulation of esterifed cholesterol. Part of the esterified cholesterol undergoes hydrolysis and is excreted back into the medium, leading to enrichment of the lipoproteins in the medium with free cholesterol. This enrichment with free cholesterol promotes cholesterol esterification in smooth muscle cells. Abbre~ations: LDL, Iow density ~poprote~(s); low density lipoprote~(s). 00052760/81/0000-0000/$02.50

VLDL, very

0 1981 ElsevierjNorth-Holland

Biomedical Press

478

Introduction In previous research we have studied the effect of plasma components added to medium on the content of cytoplasmic cholesteryl ester of smooth muscle cells in culture [I 1. To mimic conditions in vivo the agents used were those which might be present in extraceIluIar fluid, such as lipoproteins, free fatty acids and apolipoprotein-phospholipid complexes. During development of atherosclerosis the smooth muscle cells come in contact not only with interstitial fluid but also with other cells, namely macrophages [2,3]. Therefore it became of interest to investigate possible interactions between smooth muscle cells and macrophages, especially with regard to the metabolism of cytoplasmic cholesteryl ester. In a preliminary study, mouse peritoneal ma~rophages were incubated for several days in serum containing medium to which acetylated low density lipoprotein was added. When medium thus conditioned was used for postincubation of smooth muscle cells (prelabeled with f3Hjcholesterol), a marked stimulation of esterification of the smooth muscle cells cholesterol was found [4]. The present study was designed to investigate further this observation, i.e., to determine whether the presence of serum in the incubation medium is needed, whether otherwise modified lipoproteins will have the same effect and, finally, to elucidate the qechanism responsible for this finding.

Materials and Methods Peritoneal macrophages were obtained from male albino mice following intraperitoneal injection of phosphate-buffered saline [S] . The cells were peheted and washed by resuspension in Dulbecco-Vogt medium containing 10% new-born calf serum. The cell suspension was plated to give (4-6) * IO6 cells/60 mm petri dish, assuming that the percent of macrophages in the peritoneal fluid was about 30% [6]. After 2 h of preplating the medium and nonattached cells were removed, the cell layer was washed three times with phosphate-buffered saline and the cells were incubated for 24-48 h in medium containing 10% newborn calf serum. 100% of attached cells phagocytosed latex particles; the protein content per dish was 300400 fig. On day 1 or 2 after plating of the macrophages, the culture medium was removed, the ceil layer

washed twice with phosphate-buffered saline and 67 ml of fresh medium were added. In this medium the calf serum was replaced by 1% bovine serum albun~in and all additions were made to this medium. Unless otherwise stated the concentration of the lipoproteins added was 25 ,ug protein~ml. Following varying time intervals the medium was collected under sterile conditions, centrifuged at 400 Xg for 10 min and used for incubation with smooth muscle cells. The macrophages were either harvested or incubated for additional intervals of 48 or 72 h with fresh medium containing appropriate additions of lipoproteins. To harvest the macrophages, the dishes were washed three times with 0.2% albumin and three times with phosphate-buffered saline and scraped with a Teflon policeman into methanol [7]. The conditioned media were used for incubation with smooth muscle cells which had been prelabeled with [3H]cholestero1. Rat and bovine aortic smooth muscle cells were cultured as described before [I $1. Aortae were removed from 3-month-old male rats under diethyl ether anaesthesia, bovine aortae were obtained from the abbattoir. The aortae were dissected and medial explants were prepared [g]. The cells were cultured in modified Dulbecco-Vogt medium containing 5% fetal calf serum and 5% new-born calf serum. Prior to each experiment, the aortic smooth muscle cells were trypsinized and seeded in 30-mm petri dishes at a cell density of 7 . 104]dish. The medium used consisted of Dulbecco-Vogt medium supplemented with 10% fetal calf serum to which 7~-~-~3H~cholesterol was added [9] to give 1 i.tCi/ml medium. After 48-72 h of incubation in the presence of the labeled medium, the medium was removed. The smooth muscle cells layer was washed three times with phosphate-buffered saline and the cells were postincubated for 48 h with the following media: a, Dulbecco-Vogt I~lediuIn containing 1% bovine serum albumin incubated with macrophages for different time intervals with native or modified LDL (25 I.tg lipoprotein protein~~~). b, The same media collected from macrophages incubated in the absence of lipoproteins, to which LDL or modified LDL (25 I.rg protein~ml) were added at the onset of postincubation with smooth muscle cells. An additional control was the same medium not preincubated with macrophages and supplemented with LDL or modified LDL (25 hg protein/ml). The media which had been collected after incubation with

479

macrophages were designated either ‘preincubated’ or conditioned; the term ‘postincubation’ refers to subsequent incubation of the various media with smooth muscle cells. In some experiments the native or acetylated LDL used for preincubation with macrophages had been iodinated with lzsI. Fo~owing 48 h incubation of the macropllages~ the medium was collected and dialyzed extensively against several thousand-fold volumes of 0.15 M NaCl to remove “‘1 degradation products. When more than 98% trichloroacetic acid-soluble “‘1 had been removed, the medium was dialyzed against 10 vol. of Dulbecco-Vogt medium. Acetylated 12’1labeled LDL was added to Dulbecco-Vogt medium, dialyzed as above and used for postincubation of smooth muscle cells. The amount of radioactivity in the form of trichloroacetic acid-precipitable lipoprotein added to smooth muscle cell was the same when either medium obtained after incubation of 12’1labeled acetylated LDL with macrophages or non-preincubated medium to which ‘251-labeled acetylated LDL had been added prior to dialysis were used. The concentration of 1251-labeled LDL used for incubation with smooth muscle cells was the same as that of 12sI-labeled acetylated LDL. The smooth muscle cells were incubated for 24 h, the medium was collected and the non-iodine trichloroacetic acid-soluble radioactivity was determined as described before [S] . To reisolate the acetylated LDL from the medium after incubation with n~acrophages, the medium was adjusted with KBr to a density of 1.063 g/ml and centrifuged for 24 h at 50000 rev./min in a Ti60 rotor. The top 4.7 ml were cut, the subnatant was adjusted to density of 1.21 g/ml and centrifuged for 48 h at 50000 rev./min in a Ti60 rotor. The top 7 ml were designated d < 1.21, the rest d > 1.2 1. To measure the incorporation of acetate into lipids, sodium [ 1-14C]acetate (1 ~Ci~ml) was added to the medium at the start of postincubation of smooth muscle cells, prelabeled with [3H]cholesterol. To terminate the experiment, the medium was collected, the smooth muscle cell layer was washed 3 times with 0.2% albumin and 3 times with phosphate-buffered saline and the cells were scraped with a Teflon policeman into methanol 171. Following addition of chloroform and campesterol, the lipids were extracted and purified according to Folch et al. [lo] and aliquots of the chloroform phase were used

for determ~ation of radioactivity. The % of labeled cholesterol recovered as cholesteryl ester was determined by thin-layer chromatography [ 111. To determine the incorporation of [14C]acetate into nonsaponifiable lipids and fatty acids, the chloroform extract was subjected to methanolic alkaline hydrolysis 1121. The hydroly~te was acidified by addition of HCl and re-extracted with petroleum ether to determine the incorporation of [ 14C]acetate into fatty acids [ 121. Protein was determined on the delipidated cell pellet according to the method of Lowry et al. [13]. Free and total cholesterol were determined by gas-liquid chromatography as described before [ 141. Human low density (LDL) were isolated by dtracentrifugation according to the method of Have1 et al. [15] at d 1.019-l .063 g/ml from serum containing 1 mg/ml of EDTA. Acetylation of LDL and albumin was carried out with acetic anhydride [ 161. Maleylation of LDL was achieved by addition of maleic anhydride [ 17). Reductive methylation of LDL was carried out according to Weisgraber et al. [I 81, High density apolipoprotein was prepared from human high density lipoprotein (1.063-I .2 I g/ml) and sonicated apolipoprotein phospholipid dispersions (liposomes) were prepared using rat liver sphingomyelin, as described before [9]. To study incorporation of L-[4,5-3H]leucine (130 Ci/mmol), D-[6-3H]-glucosamine WC1 (20 Ci~mmol) and [6-3H]thymidine (5 Ci/ mmol), the radioactive precursors were added to the medium used for incubation of smooth muscle cells for 48 h. This medium was either macrophage medium conditioned with acetylated LDL or the same medium, not preincubated with macrophages but supplemented with acetylated LDL, 25 pg protein/ml. The cells were harvested, extracted with chloroform/ methanol (1 : 1) and treated with tric~oroacetic acid as described before [ 191. Bacto-Latex particles, size 0.81 pm, were obtained from Difco Laboratories, Detroit, MI. Trypsin, soy bean trypsin inhibitor and bovine serum albumin was obtained from Sigma. Poly(L-glutamic acid) M,. 30600, was obtained from Miles-Yeda Ltd., Rechovot, Israel. All radioactive materials were obtained from the Radiochemical Centre, Amersham, U.K. Radioactivity of 3H and 14C was determined with a fl and 12’1 with a y scintillation spectrometer (Packard Instruments, La Grange, IL, U.S.A.).

480

Results In the present experiments, mouse peritoneal macrophages were incubated for 2-6 days with medium in which the calf serum had been replaced by 1% bovine serum albumin. This medium had been supplemented with acetylated LDL 2.5 I.tg protein~ml and after exposure to macrophages was used for postincubation of bovine aortic smooth muscle cells, prelabeled with [3H]cholesterol. As seen in Table I, the labeled cholesterol which had been present in the cells prior to postincubation appeared also in the medium. While the medium cholesterol was only in unesterified form, cellular cholesterol was also esterified in part. When the medium used for postincubation had been preincubated with macrophages and acetylated LDL, the amount of

label recovered in smooth muscle cells in esterified cholesterol increased 3-4 times above that present at zero time. A concomitant increase in cellular total cholesterol and cholesteryl ester occurred in smooth muscle cells postincubated with macrophage medium conditioned in the presence of acetylated LDL (Table I). When acetylated LDL had been added to macropllage~onditioned medium or to nonpreincubated medium (used for postincubation), the amount of label present in esterified cholesterol in the smooth muscle cells was similar to that present at zero time. A slight increase in esterified cholesterol in the smooth muscle cells above that at zero time occurred when LDL had been added to macrophage medium during corldition~g, but it was similar to that encountered when the LDL had been added either to macrophage-conditioned medium or to non-preincu-

TABLE I EFFECT OF SERUM-FREE MEDIUM PREINCUBATED WITH MACROPHAGES AND ACETYLATED LDL ON CHOLESTEROL ESTERIFICATION IN AORTIC SMOOTH MUSCLE CELLS Conditions: Mouse peritoneal macrophages were cultured in Dulbecco-Vogt medium containing 10% calf serum. 24-48 h after plating, the medium was replaced by serum-free Dulbecco-Vogt medium, containing 1% bovine serum albumin. The concentration of acetylated LDL or LDL was 25 pg protein/ml and incubation was carried out for 48 or 72 h. The medium was collected, centrifuged for 10 min at 2 000 rev./min and used for postincubation of aortic smooth muscle cells. Media derived from macrophages incubated without lipoproteins, as well as non-preincubated Dulbecco-Vogt medium, containing 1% bovine serum albumin, were supplemented with acetylated LDL or LDL, 25 pg protein/ml, and were used as controls. The smooth muscle cells were plated at a density of 7 .,104/60 mm petri dish in medium containing 10% fetal calf serum and [3H]cholesterol and labeled for 48-72 h. The medium was removed, the cell layer washed thrice with phosphate-buffered saline and postin~ubated in the presence of the indicated media. After 48 h the medium was collected, the cell layer washed and extracted. Following lipid extraction the amount of label recovered in cholesteryl ester was determined by thin-layer chron~at~~raphy and the amount of total and esterified cholesterol by gas-liquid chromatogrpahy. Zero time = petri dishes terminated prior to post incubation. Values are iS.E. of triplicate dishes of six experiments. Postincubation of smooth muscle cells in medium preincubated in presence of:

Added to medium after preincubation

._ ll-___l_ Zero time Macrophages Macrophages+ acetylated LDL Macrophages acetylated LDL Macrophages + LDL Macrophages LDL Nonpreincubated medium acetylated LDL Nonpreincubated medium LDL Nonpreincubated medium -

3H label after 48 h postincubation

(dpm)

Medium Total

Smooth muscle cells

(10-3)

Total

_ 38 f 3.8 75 + 4.0 112t6.0 13Ok 6.0 110t7.0 95 ir 9.0 152 k9.0 50 * 7.0

Cellular cholesterol (fig rug-1 protein) Total

(10-3)

Cholesteryl ester (10-a)

161 If:4.7 116 27.5 92+ 1.9 7.5 + 1.7 69 + 3.5 73 i- 4.7 79 rt 2.4 80+ 1.8 105+2

69f 12 25k 3 235 t 14 7ort 6 875 7 74+ 6 55i13 8125 19+ 3

28.1 30.1 48.8 32.5 33.3 34.8 31.9 32.1

Esterified

* 1.4 ?I 0.9 t 2.6 t 2.5 .i 5.0 $2.2 t 2.0 -t-2.0

3.4 3.2 12.7 5.8 5.0 6.3 4.4

+ 0.6 t 0.3 + 1.4 IO.8 i 0.4 f 0.5 i 0.8

3.8 i 0.7 -.

481 TABLE II COMPARISON OF MEDIA OF MACROPHAGES CONDITIONED ESTERIFICATION IN AORTIC SMOOTH MUSCLE CELLS

WITH VARIOUS

REAGENTS ON CHOLESTEROL

Conditions: Mouse peritoneal macrophages were cultured in Dulbecco-Vogt medium containing 10% calf serum for 24 h. The medium was replaced by serum-free medium containing 1% bovine serum albumin and supplemented with the modified lipoproteins (25 gg protein/ml), poly(L-glutamic acid) (25 #g/ml) and with latex particles, and the macrophages were incubated for 48 h, the medium was removed and fresh medium containing the same additions was collected after 48 h and used for postincubation of smooth muscle cells. Methylated LDL = LDL which had undergone reductive methylation. The concentration of reagents added to non-preincubated medium was 25 pg protein/ml for the lipoproteins, 25 erg/ml for poly(L-glutamic acid). Values are means of 3-6 dishes in four experiments. Postincubation of smooth muscle cells in medium preincubated with:

[sH]cholesteryl (dpm)

Zero time Macrophages + acetylated LDL Macrophages + acetylated albumin Macrophages + maleylated LDL Macrophages + methylated LDL Macrophages + poiyglutamic acid Macrophages + latex Nonpreincubated medium + acetylated LDL Nonpreincubated medium + maleylated LDL Nonpreincubated medium + methylated LDL Nonpreincubated medium

Cholesterol (lug . mg-i protein)

ester

4 950 22 088 2 287 22 800 6 686 5 433 2 979 5 318 5 700 6100 3 243

Total

Esterified

28.3 58.1 30.0 59.1 28.7 25.9 30.6 34.5 35.5 29.4 30.1

4.6 21.7 2.5 19.8 4.1 4.9 3.9 6.8 5.5 4.7 5.0

TABLE III EFFECT OF CONCENTRATION OF ACETYLATED LDL IN MACROPHAGE MEDIUM ON THE ENHANCEMENT OF CHOLESTEROL ESTERIFICATION DURING POSTINCUBATION WITH AORTIC SMOOTH MUSCLE CELLS Conditions: The macrophages were incubated with various concentrations of acetylated LDL for 24 h; the medium was removed, fresh medium containing acetylated LDL was added, collected after 48 h and used for postincubation of smooth muscle cells. All other conditions as in Table I. Values are means * SE. of triplicate dishes. Zero time = petri dishes terminated prior to postincubation. Postincubation of smooth muscle cells in medium pre~cubated with macrophages and acetylated LDL @g/ml)

Zero time Macrophages Macrophages + acetylated LDL 5 10 20 50 Acetylated LDL, 50 &ml added to non-preincubated

3H label after 48 h postincubation Medium Total (10-a)

127?

medium

(dpm)

Smooth muscle celIs cholesterol @g ’ mg-1 protein)

Smooth muscle cells Total Total (10-a)

9.6

738 + 5 1.0 545 t 13.3

1432 7.7 234k 4.2 353 f 12.9 518$ 4.5

545 rt 490 + 430 + 352k

5492

277 k 13.9

9.9

Esterified

Cholesteryl ester

36.6 17.5 14.6 5.7

39572411 1409rt 91

21.6 20.2

3.4 1.5

31232 95 11517rt397 12 3.55 zf 671 13312t637

26.4 32.4 43.2 42.1

5.7 10.4 15.2 15.5

3571 rt411

21.5

4.6

482

bated medium. To determine whether the stimulation of cholesterol esterification was limited to acetylated LDL, we have incubated macrophages with differently modified LDL, negatively charged macromolecules and inert particles. These conditioned media were tested as to their stimulating capacity of cholesterol ester formation in smooth muscle cells. Data of several experiments are summarized in Table II and it can be seen that only maleylated LDL reproduced the effect obtained with acetylated LDL. Medium supplemented with polyglutamic acid, acetylated albumin or latex particles, which had been phagocytized by all adherent cells, had no effect on the labeled or chemically determined cholesterol ester of smooth muscle cells during 48 h of postincubation. The stimulation of cholesteryl ester formation in the smooth muscle cells was related to the concentration of acetylated LDL used for the incubation with macrophages (Table III). Even a concentration of 5 pg acetylated LDL protein/ml of macrophage medium elicited an increase in total cholesterol and cholesteryl ester in smooth muscle cells and the effect was maximal between 20 and 50 pg acetylated LDL protein/ml. Therefore, in all subsequent experiments the concentration of acetylated LDL used was 2.5 pg protein/ml. So far, the time of conditioning of the macrophages with acetylated LDL was 48 or 72 h.

TABLE

Therefore, macrophages were incubated with acetylated LDL for 48 h intervals, the media were collected, fresh medium containing acetylated LDL was added and the macrophages were incubated for up to 144 h. As seen in Table IV, the effect on cholesterol esterification was evident with macrophage-conditioned media collected at all time intervals studied, but was higher with media collected at the second and third time interval. Next we have determined the time of contact between the conditioned medium and the smooth muscle cells needed to observe an increase in cholesterol esterification. As seen in Fig. 1. some stimulation of cholesterol esterification was evident already after 8 h, and progressed linearly between 8 and 24 h of postincubation; a further increase occurred between 24 and 48 h. To determine whether the property to stimulate cholesterol esterification in smooth muscle cells resides in a lipoprotein fraction, medium of macrophages which had been incubated with acetylated LDL was subjected to differential flotation at densities of 1.063 and 1.21 Following dialysis to remove the KBr the various density cuts were used for incubation with the smooth muscle cells. The amount of each fraction added was calculated on the basis of its contribution to the total cholesterol of the original medium determined at the end of incubation of

IV

EFFECT OF TIME OF INCUBATION AND MEDIUM CHANGES ON THE EFFECT OF MACROPHAGE TIONED WITH ACETYLATED LDL ON CHOLESTEROL ESTERIFICATION DURING POSTINCUBATION SMOOTH MUSCLE CELLS

MEDIA CONDIWITH AORTIC

Conditions: Macrophages were cultured in medium containing 10% calf serum for 24 h. The medium was replaced by serum-free medium containing 1% bovine serum albumin and acetylated LDL, 25 ng protein/ml. At 48 h intervals the medium was collected and replaced with fresh medium of the same composition. Postincubation of smooth muscle cells was carried out for 48 h. Zero time = petri dishes terminated prior to postincubation. Values are means f of triplicate dishes. Postincubation of smooth muscle cells in medium preincubated with macrophages and acetylated LDL, 25 @g/ml collected at intervals

3H label after 48 h postincubation Medium

Smooth

(dpm)

muscle

cells

Total ( 10m3)

Zero time 0- 48 h 48- 96 h 96-144 h Macrophage

medium

+ acetylated

LDL (2.5 pg/ml)

89.2 _+0.5 81.2k4.0 85.3 k 0.7 97.4 + 0.3

Total (10e3)

Cholestervl

242 12257 125 +3 126f4 12.5 +3

40 184 k 14 301 f 8.0 389 f 30 525 6

ester (lOma)

483

Time of tncubafmn

(hours)

1. Effect of duration of postincubation of smaoth muscle cells (SMC) on cholesterol esterification. Mouse peritoneal macrophages were cultured in Dulbe~co-Vogt medium containing 10% calf serum for 24 h. The medium was collected, and replaced with serum-free medium containing 1% bovine serum albumin and acetylated LDL, 25 pg protein/ml. After 72 h this medium was removed and fresh medium containing the same ingredients was added and incubation was continued for additional 48 h. The smooth muscle cells were prelabeled with [3H]cholesterol for 48 h and were postincubated with the macrophage medium conditioned with acetylated LDL (4) or with non-pre~cubated serum-free medium (0) containing 1% bovine serum albumin for the indicated time intervals. Values are means of triplicated dishes which varied less than 5%. Fig.

macrophages with acetylated LDL. As seen in Table V, the capacity to enhance cholesterol esterification resided mainly in the d < 1.063 fraction. Addition of d < 1.21 alone or together with d 3 1.21 fraction did not enhance further the activity present in the d < 1.063 fraction. The possib~ity was tested that during incubation with macrophages, the noningested acetylated LDL undergoes a change which permits its better recognition and uptake by smooth muscle cells. To that end the macrophages had been incubated with 12’1labeled acetylated LDL and following dialysis against saline (to remove the degradation products) and then against serum-free culture medium, the labeled medium was postincubated with smooth muscle cells. i2s1Iabeled acetylated LDL or LDL added to the medium immediately prior to incubation with smooth muscle cells served as control As seen in Table VI, the degradation by smooth muscle cells of ‘2sI-labeled acety-

lated LDL which had been preincubated with macrophages did not differ from that added directly to smooth muscle cells medium, and both were taken up and degraded much less extensively than 12SI-labeIed LDL. In another experiment, acetylated LDL had been reisolated from the ma~rophage~onditioned medium by flotation at d = 1.063. The ability of this ‘conditioned’ acetylated LDL, controi acetylated LDL and native LDL to compete for degradation of ‘2sI-labeled LDL was compared. As seen in Fig. 2, neither the ‘conditioned’ nor the control acetylated LDL competed with native LDL. These results indicated that the stimulation of cholesterol esterification in the smooth muscle ceils may not be due to increased uptake of ‘conditioned’ acetylated LDL. Further support for the possibiIity that stim~ation of cholesterol esterification was not dependent on increased uptake of the acetylated LDL particle was obtained in an experiment shown in Table VII. Acetylated LDL was incubated with macrophages in serum-free medium supplemented with 0.2% albumin. The medium was then incubated with trypsin (200 pug/ml) for 30 min at 37°C and the reaction was terminated by soybean trypsin i~ibitor -.-..-.-

oo~-“~-

..-.

50

Competing. Compound

in Medrum

(,ng I ml )

Fig. 2. Ability of LDL (*), acetylated LDL (0) and d < 1.063 of serum-free medium preincubated with macrophages and acetylatcd LDL (A) to inhibit degradation of ‘2SI-labeled LDL (1251-LDL) by smooth muscle ceils during 6 h incubation. The d < 1.063 fraction was prepared as described in legend to Table IV. Values are means of triplicate dishes.

484 TABLE V ISOLATION OF THE FRACTION RESPONSIBLE FOR ENHANCEMENT OF CHOLESTEROL ESTERIFICATION DIFFERENTIAL FLOTATION OF MACROPHAGE MEDIUM CONDITIONED WITH ACETYLATED LDL

BY

Conditions: Medium was collected from several dishes of macrophages which had been incubated with acetylated LDL, 50 ).rg protein/ml, for 72 h. A portion of the medium waskept and about 70 ml was brought to d = 1.063 with KBr and centrifuged for 24 h at 50000 rev./mm in a Ti60 rotor. The tubes were sliced, the top fraction, 4.7 ml per tube, was designated d < 1.063. The bottom fraction was brought to d = 1.21, and centrifuged under the same conditions for 48 h. The top fraction, 7 ml/tube, was designated d < 1.21, the rest d > 1.21. Total cholesterol content of the original medium and the fractions d < 1.063 and d < I .21 was 33, 2.50, 17.4 fig/ml, respectively. The percent contribution of each fraction to the total cholesterol of the original medium was calculated and served as a basis for the amount of each fraction to be added to the medium used for postincubation. The numbers in parentheses represent the pg of total cholesterol present in 2 ml medium. Values are means + SE. of triplicate dishes. Zero time = petri dishes terminated prior to postincubation. Postincubation of smooth muscle cells in medium preincubated with

Cholesterol in smooth muscle cells after 48 h postincubation ___..-.-Total (pg mg+ protein) --

Esterified (c(g I mg-l protein)

1400 11 3661214

28.9 52.3 f 0.3

2.8 11.1

13 200 * 970 15932 129 1105 i 92 113502321 12617 2126f 39 1221

47 38.3 29.3 44.8 44.3 31.9 30.5

8.1 3.8 t 0.7 1.1 io.2 11.0 9.9 2.9 2.4

[3H]Cholesteryl fdpm) Zero time Macrophages + acetylated LDL (66) Macrophages + acetylated LDL, separated by differential flotation d < 1.063 (55) d < 1.21 (9) d > 1.21 (3) d < 1.063 id < 1.21 (64) d < l.O63+d < 1.21 +d > 1.21 (67) Non-preincubated medium acetylated LDL (66) Non-preincubated medium

ester

C 0.8 f 0.5 i 1.7 Il.3

TABLE VI EFFECT OF PREINCUBATION WITH MACROPHAGES OF r2sI-LABELED OF THE PROTEIN MOIETY BY AORTIC SMOOTH MUSCLE CELLS

ACETYLATED LDL ON THE DEGRADATION

Conditions: Mouse peritoneal macrophages were cultured in Dulbccco-Vogt medium containing lo%, calf serum for 24 II. The medium was removed and replaced with serum-free medium, containing 1% bovine serum albumin and *2sI-labeled acetylated LDL, 25 pg protein/ml. After 48 h, the medium was collected, dialyzed against saline to remove r *sI degradation products and then against 10 vol. Dulbecco-Vogt.medium. 12sI-labeled acetylated LDL and rzsi-labeled LDL not preincubatcd with macrophages were dialyzed under the same conditions. The same amount of trichloroacetic acid-precipitablc label was used for incubation of smooth muscle cells with r251-labeled acetylated LDL preincubated with macrophages or non-preincubated r2sI-labeled acetylated LDL or rzsI-labeled LDL. Additions to smooth muscle cells

12sI-labeled protein degraded (ng/mg cell protein per 24 h) Experiment:

Medium pre~ncubated with 1251~labeled acetylated LDL and macrophages 12sI-labeledacetylated LDL added to non-preincubated medium 1*sI-labeled LDL added to non-preincubated medium

1

2

3

290 185 1844

199 209 1182

286 212 4 748

485 TABLE VII EFFECT OF EXPOSURE TO TRYPSIN AND EXTRACTION WITH DIETHYL ETHER OF MEDIUM PREINCUBATED WITH MACROPHAGES AND ACETYLATED LDL ON CHOLESTEROL ESTERIFICATION IN AORTIC SMOOTH MUSCLE CELLS Conditions: Mouse peritoneal macrophages were cultured in Dulbecco-Vogt medium containing 10% calf serum for 24 h. The medium was replaced with medium containing 0.2% albumin and acetylated LDL, 25 pg protein/ml. After 24 h this medium was collected and replaced with the same medium and incubation with macrophages was continued for 48 h. The medium was then incubated with trypsin (200 &ml) for 30 min at 37°C and the reaction was terminated by soy bean trypsin inhibitor (600 pg/ ml). To determine the % degradation by trypsin, r asI-labeled acetylated LDL was incubated under the same conditions and 14% of the label was recovered as noniodide trichloroacetic acid-soluble radioactivity. Another aliquot of acetylated LDL conditioned medium was extracted 3 times with 5 vol. of diethyl ether and the diethyl ether was blown off under Na. Values are means of triplicate dishes. Treatment of macrophage medium prior to postincubation with smooth muscle cells

Zero time None Incubation with trypsin Extraction with diethyl ether Acetylated LDL added to non-preincubated

medium

(600 pg/ml). Another aliquot of acetylated LDL-conditioned medium was extracted three times with 5 vol. of diethyl ether and the diethyl ether was blown off under Na. As seen in Table VII partial degradation of the acetylated LDL (14% as judged by degradation of ‘251-labeled acetylated LDL subjected to the same treatment) did not affect the stimulation of cholesteryl ester synthesis in the smooth muscle cells, as measured by the formation of labeled cholesteryl ester. In the presence of untreated macrophage medium, the increase in cholesteryl ester content in smooth muscle cells was from 5 pg/mg cell protein in the zero time control to 25 I.cg/mg cell protein and with trypsin-treated macrophage medium the increase was up to 19 pg/mg cell protein. However, the effect was abolished by diethyl ether extraction which reduced the cholesterol content of the medium from 18 to 2.5 I.cg/ml and of free cholesterol from 9 to 1.6

ELglml . Since the lipid moiety in the medium played a crucial role in the stimulation of cholesterol esterification, the effect of macrophage medium conditioned with acetylated LDL on cholesterol synthesis in the smooth muscle cells was determined. As seen in Table VIII, concomitantly with the stimulation of cholesterol esterification the macrophage medium condi-

Esterified cholesterol in smooth muscle cells after 48 h postincubation dpm

fig mg-1 protein

5 793 17 300 19 200 3 396 4 800

5.0 25.2 18.8 6.5 7.4

tioned with acetylated LDL reduced markedly incorporation of [14C]acetate into nonsaponifiable lipids, when compared with macrophage medium conditioned without acetylated LDL or to medium to which acetylated LDL had been added prior to the incubation with smooth muscle cells. Extraction of the acetylated LDLconditioned medium with diethyl ether relieved completely the inhibition of cholesterol synthesis. Since no increase in the uptake of conditioned acetylated ‘251-labeled LDL protein was seen it seemed that a change in the composition of the lipid moiety of the acetylated LDL might occur during incubation with macrophages, which could then promote cholesterol esterification in the smooth muscle cells. To test this possibility, macrophages were incubated for specified time intervals (Table IX) with medium containing acetylated LDL, 25 I.rg/ml, and cells from several dishes were harvested after one period of incubation while in other dishes the medium was collected, replaced with fresh medium containing acetylated LDL and the macrophages were incubated for an additional period (Table IX). The amount of cholesteryl ester that accumulated in the macrophages increased with longer times of incubation and repeated medium changes. At the same time, there was excretion of free cholesterol into the medi-

486 TABLE VIII EFFECT OF MEDIUM PREINCUBATED WITH ACETYLATED LDL AND MACROPHAGES ON CHOLESTEROL SYNTHESIS AND ESTERIFICATION DURING POSTINCUBATION WITH SMOOTH MUSCLE CELLS Conditions: Mouse peritoneal macrophages were cultured in Dulbecco-Vogt medium containing 10% calf serum for 24 h. The medium was removed, the macrophages were incubated with serum-free medium containing 1% bovine serum albumin and acetyiated LDL 2.5 pg protein/ml. The medium used for the experiment was from a collection period of 72-120 h. The extraction with diethyl ether was performed as in Table VII. The concentration of acetyiated LDL added to non-preincubated medium was 25 p,g protein~ml. The smooth muscle cells had been prelabeled with [3H]cholesterol as in Table I. Sodium [ l-r4Clacetate (1 pCi/ ml) was added to the medium at the onset of postincubat~on, and the cells were harvested 48 h thereafter. Following extraction of the lipids, the amount of label recovered in nonsaponifiable lipids and fatty acids was determined as detailed in Materials and Methods. Values are means or means i: S.E. of triplicate dishes. Treatment of medium used for postincubation with smooth muscle cells

Preincubation with macrophages + acetylated LDL Preincubation with macrophages + acetylated LDL and extraction with diethyl ether Preincubation with macrophages Addition of acetylated LDL to nonpreincubated medium

Incorporation of [ r‘Q]acetate into lipids of smooth muscle cells

Smooth muscle cells cholesterol ester

Nonsaponifiable

Fatty acids

3H

@pm)

@pm)

Wmf

pg . mirl protein

28

3 276 f 257

I6 866 + 668

23.4

3106fl76 32665152 2588 r 140

4300f251 2131k 52 3366i-176

3523C364 4 748 t 144

3.1 8.3

792 i

TABLE IX UPTAKE OF ACETYLATED LDL BY PERITONEAL MACROPHAGES AND EXCRETION OF FREE CHOLESTEROL INTO MEDIUM Conditions: Mouse peritoneal macrophages were cultured in Dulbecco-Vogt medium containing lo%* calf serum for 24 h. The medium was removed and replaced by 7 ml serum-free medium containing 1% bovine serum albumin and acetylated LDL 25 wg protein/ml (total cholesterol 32.5 pg/ml, free cholesterol 28%). The medium was collected at time intervals indicated in the fourth column and the cells were either harvested or postincubated with fresh medium of the same composition for the indicated time interval. The cholesterol content of the medium at the end of each incubation period was determined and the amount of free cholesterol excreted by the macrophages into the medium was calculated by the estimation of the residual cholesteryl ester. If no change had occurred in the noningested acetylated LDL, free cholesterol should have been 28% of total; the excess of free cholesterol over that expected = free cholesterol excreted. Values are means of duplicate petri dishes and of 2-3 samples of the medium. Incubation (h)

48 48+ 24 48 + 48 48+48+24 48 + 48 + 48

Macrophage cholesterol Total (rg w-l protein)

Esterified

201 273 243 291 438

153 239 211 259 392

Medium collected at intervals (h)

pig/ml free cholesterol excreted into medium

Free cholesterol (% of total)

0- 48 48- 72 48- 96 96-120 96-144

4.7 6.0 6.5 5.0 7.0

66.9 58.5 59.5 54.7 70.4

(a . mP protein)

487

urn which amounted to 4.6-7.0 I.cg/ml and the ratio of free cholesterol to total cholesterol increased from 25-27% in the original acetylated LDL up to 70% after 144 h of incubation and three medium changes. When the free cholesterol to total cholesterol ratio was compared before and after incubation with smooth muscle cells this ratio decreased from 50.8 to 42.7, 55 to 40 and 45.5 to 38% in three different experiments. At the same time the free to total cholesterol ratio of acetylated LDL added to smooth muscle cells at the onset of postincubation changed from 27 to 25,29 to 28 and 27 to 26%. The excretion of free cholesterol into the medium increased from 5.3 pg/ml when macrophages were incubated with acetylated LDL to 13-I 5 pg/ml after addition of high density apolipoprotein sphingomyelin liposomes to the medium during incubation of the macrophages with acetylated LDL (Table X). The ratio of free: total cholesterol in the medium was 59 and 67% in the presence of 25 or 100 pg high density apolipoprotein/sphingomyelin per ml, but when this medium was used for postincubation with smooth muscle cells it enhanced the loss of labeled cholesteryl ester from the cells. When the macrophage medium conditioned with acetylated LDL and high denTABLE

sity apolipoprotein/sphingomyelin liposomes was used no increase in esterified cellular cholesterol in the smooth muscle cells was seen. As seen in Table X, less label was recovered in esterified cholesterol in smooth muscle cells incubated with acetylated LDL and the high density apolipoprotein/sphingomyelin liposomes added to nonpreincubated medium, and there was a very pronounced decrease in cellular total cholesterol when high density apolipoprotein/sphingomyelin was present in the incubation medium. To determine the effect of the acetylated LDLconditioned medium on the incorporation of labeled leucine or glucosamine into proteins and glycoproteins respectively, and incorporation of labeled thymidine into DNA, smooth muscle cells were incubated with labeled precursors for 48 h. No stimulation of incorporation of either precursor was observed at a time when enhancement of cholesterol esterification was found (data not shown). Discussion During the last years more attention has been given to the contention of older literature that macrophages form a considerable proportion of the cellular

X

EFFECT OF HIGH DENSITY APOLIPOPROTEIN/SPHINGOMYELIN MACROPHAGES WITH ACETYLATED LDL ON THE CAPACITY ESTERIFICATION IN AORTIC SMOOTH MUSCLE CELLS

MIXTURES ADDED DURING INCUBATION OF OF THE MEDIUM TO ENHANCE CHOLESTEROL

Conditions: Mouse peritoneal macrophages were cultured in Dulbecco-Vogt medium containing 10% calf serum for 24 h. The medium was removed, replaced with medium containing 1% bovine serum albumin and acetylated LDL, 25 pg protein/ml. After 72 h this medium was collected. Fresh medium containing acetylated LDL alone or with high density apolipoprotein/sphingomyelin (1: 1) liposomes (ApoSp) was added and collected after an additional 48 h of incubation. This medium was used for postincubation of smooth muscle cells prelabeled with [3H]cholesterol. Values are means and means f S.E. of duplicate or triplicate dishes. Postincubation of smooth muscle medium preincubated with

cells in

Cholesterol

in smooth

[3H]Cholesteryl

Zero time Macrophages + acetylated LDL (25 pg/ml) Macrophdges + acetylated LDL (25 fig/ml) + ApoSp (25 pg/ml) + ApoSp (100 fig/ml) Nonpreincubated medium + acetylated LDL (25 pg/ml) + acetylated LDL + ApoSp (100 pg/ml) + ApoSp (100 pg/ml) Non-preincubated medium

ester

muscle cells after 48 h postincubation

(dpm)

Total (rg mg-t

8 158 20192k290

23.5 58.9

protein)

Esterified (pg mg-1 protein) 2.5 22.5

2788f162 1397 + 140

27.0 f 0.5 21.0

2.3 ?r 0.13 2.0

4057 1815 1240f 58 2 725 f 155

30.5 15.8 14.4 ?r 0.3 24.2

4.4 1.1 1.2 + 0.2 1.9

488

population of atheroma [2,20]. In studies on rabbits, these cells had been isolated, identified by virtue of their Fc receptors and were shown to be filled with lipid [3 1. However, macrophages isolated from experimental animals or human monocytes accumulate only little cholesterol when exposed to the atherogenic plasma lipoprotein, i.e., LDL [6,21]. Exposure to LDL which had undergone certain chemical modification such as acetylation [6] or treatment with malondialdehyde 1221 or to hypercholesterolemic canine @-VLDL [23] results in an intracellular accumulation of cholesteryl ester. Still, smooth muscle cells are the main cellular representatives of human atheroma and the mechanism by which they accumulate cholesteryl ester remains to be elucidated. In the present study we have tested the possibility whether macrophages highly enriched with choiesteryl ester might promote cholesterol esterification in smooth muscle cells. In the first experiments the enrichment of macrophages with cholesteryl ester was accomplished by addition of acetylated LDL to serum-containing medium [4]. Accumulation of cholesteryl ester occurred in the macrophages and when the medium was removed after 2-3 days of incubation and used for incubation with smooth muscle cells, stimulation of cholesterol esterification was observed [4]. In these experiments we have also shown that medium derived from macrophages ~cubated with acetylated LDL promoted incorporation of [ 1-14Cloleate into cholesteryl ester to the same extent as formation of labeled choiesteryl ester from [3H]cholesterol 141. In order to eiucidate the mode of this interaction between the macrophages and the smooth muscle cells, which was obviously mediated by the medium, the present experiments were performed in serum-free medium, containing 1% albumin. When macrophage medium which had been conditioned in the presence of acetylated LDL was added to smooth muscle cells, 3-4 times more labeled cholesteryl ester was recovered in the smooth muscle cells when compared to cells incubated with either acetylated LDL or LDL added directly to the smooth muscle cells medium. This increase in labeled cholesteryl ester was accompanied also by an increase in cellular cholesterol mass. This effect was related to the concentration of acetylated LDL used for the incubation of macrophages and could be detected already after 8 h of contact between the medium and the smooth muscle cells. In

later experiments, the duration of exposure of the macrophages to acetylated LDL and the number of medium changes was increased and it appeared that the cholesterol~sterifying activity was more pronounced with media collected at later time intervals. Among the various reagents tested only maleylated LDL shared this property with acetylated LDL; other negatively charged molecules, latex particles or reductively methylated LDL did not render the macrophage medium more active with respect to cholesterol esterification in smooth muscle cells. The stimulating activity did not deteriorate upon storage at 4°C for several weeks, it was resistant to partial degradation with trypsin, but could be abolished by partial delipidation of the medium with diethyl ether. The property to enhance cholesterol esterification was recovered in a d < 1.063 fraction of the medium, suggesting that it was due to the presence of a lipoprotein. Since acetylated LDL is taken up much less extensively than LDL by fibroblasts [6] and by smooth muscle cells 1241, the possibility was tested that the negatively charged lipoprotein might have been changed by incubation with macrophages. However, no significant increase in the uptake of macrophage ‘conditioned’ 125I-labeled acetylated LDL protein by smooth muscle cells could be demonstrated. Furthermore, when the d < 1.063 fraction was isolated from the inacrophage medium conditioned with acetylated LDL, it did not compete with native LDL for uptake and degradation by smooth muscle cells. These data have suggested that the protein portion of the lipoprotein reisolated from macrophage conditioned medium had not been rendered more LDL-like, i.e., recognizable by the classical LDL receptor 1251 which could explain the stinlulation of cholesterol esterification. Since depression of cholesterol synthesis [26] and stimulation of cholesterol esterification [I,271 had been induced by media in which the ratio of free cholesterol to esterified cholesteryl ester was raised, this mechanisIn was considered further to explain the activity of the acetylated LDL-conditioned medium. Werb and Cohn [28] have provided evidence that macrophages which have accumulated cholesteryl ester excrete only free cholesterol, derived from intracelluinto serum-containing medium. lar hydrolysis, Indeed, a very marked rise in free to esterified cholesterol was encountered presently in serum-free media

489

containing acetylated LDL, which were collected from macrophages after various periods of incubation with acetylated LDL. The ratio of free to esterified cholesterol in the medium after 24 h of incubation of macrophages was changed only slightly, and the ability to promote cholesterol esterification in smooth muscle cells as well as the increase in the free to esterified cholesterol ratio increased in media collected after longer periods of incubation. Following incubation with smooth muscle cells there was a decrease in the free: esterified cholesterol in the medium, concomitant with an increase in smooth muscle cells cholesteryl ester. Thus, it seems that when smooth muscle cells are presented with a lipoprotein in which an increased free to esterified cholesterol ratio occurred and which is not recognized by a specific receptor, the enhancement of cholesterol esterification is due mostly to a surface transfer of lipoprotein-free cholesterol, as described by Rothblat et al. [29]. Addition of high density apolipoprotein/sphingomyelin mixtures to the medium during incubation of macrophages with acetylated LDL resulted in even more pronounced increase in the free to esterified cholesterol in the medium. However, the presence of high density apolipoprotein/sphingomyelin mixtures abolished completely the stimulation of cholesterol esterification in smooth muscle cells, most probably by virtue of acting as a competing sink for the excess of free cholesterol excreted from the macrophages into the medium. Ho et al. 1301 have shown that macrophages which had been loaded with cholesteryl ester, following incubation with acetylated LDL, excreted free cholesterol in the presence of high density lipoprotein. Further studies are in progress to elucidate the role of high density lipoprotein in the presently described model system and in more complex systems containing other serum lipoproteins. The present results offer another view of the possible interactions between macrophages and smooth muscle cells. A modified lipoprotein not recognized by smooth muscle cells is ingested by macrophages, which leads to accumulation of esterified cholesterol. Part of the esterified cholesterol undergoes hydrolysis and is excreted back into the medium leading to enrichment of the lipoproteins in the medium with free cholesterol. This enrichment with free cholesterol promotes cholesterol esterification in smooth muscle cells.

Acknowledgements The excellent assistance of Mrs. M. Ben-Naim, Mrs. A Mendeles, Mrs. Y. Dabach, Miss L. Glickstein and Mr. G. Hollander is acknowledged gratefully. References 1 Stein, O., Coetzee, G.A. and Stein, Y. (1980) Biochim. Biophys. Acta. 620,538-549 Gerrity, R.G., Naito, H.K., Richardson, M. and Schwartz, C.J. (1979) Am. J. Pathol. 95,775-792 Fowler, S., Shio, H. and Haley, N.J. (1979) Lab. Invest. 41,372-378 Stein, O., Halperin, G. and Stein, Y. (1981) FEBS Lett. 123,303-306 5 Cohn, Z.A. and Benson, B. (1965) J. Clin. Invest. 34, 1345-l 353 6 Goldstein, J.L., Ho, Y.K., Basu, S.K. and Brown, M.S. (1979) Proc. Natl. Acad. Sci. U.S.A. 76,333-337 I Stein, O., Fainaru, M. and Stein, Y. (1979) Biochim Biophys. Acta 574,495 -504 8 Bierman, E.L., Stem, 0. and Stein, Y. (1974) Circ. Res. 35,136-150 9 Stein, Y., Glangeaud, M.C., Fainaru, M. and Stein, 0. (1975) Biochim. Biophys. Acta 380,106-118 10 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226,497-509 11 Stein, O., Goren, R. and Stein, Y. (1978) Biochim. Biophys. Acta 529,309-318 12 Stein, O., Vanderhoek, J. and Stem, Y. (1976) Biochim. Biophys. Acta 431,347-358 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 14 Stem, O., Vanderhoek, J., Friedman, G. and Stein, Y. (1976) Biochim. Biophys. Acta 450,367-378 15 Havel, R.J., Eder, H.A. and Bragdon, H.J. (1955) J. Clin. Invest. 34, 1345-l 353 16 FraenkelConrat, H. (1957) Methods Enzymol. 4, 247269 17 Johnson, W.D., Mei, B. and Cohn, Z.A. (1977) J. Exp. Med. 146,1613-l 626 18 Weisgraber, K.H., Innerarity, T.L. and Mahley, R.W. (1978) J. Biol. Chem. 253,9 053-9 062 19 Friedman, G., Stein, 0. and Stein, Y. (1980) Biochim. Biophys. Acta 619,650-659 20 Schaftner, T., Vesselinovitch, D. and Wiisler, R.W. (1979) Fed. Proc. Fed. Am. Sot. Exp. Biol. 38,1076 21 Basu, S.K., Brown, M.S., Ho, Y.K. and Goldstein, J.L. (1979) J. Biol. Chem. 254,7 141-7 146 22 Fogelman, A.M., Shechter, I., Seager, J., Hokom, M., Child, J.S. and Edwards, P.A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77,2 214-2 218 23 Goldstein, J.L., Ho, Y.K., Brown, M.S., Innerarity, T.L. and Mahley, R.W. (1980) J. Biol. Chem. 255, 18391 848

490 24 Stein, 0. and Stein, Y. (1980) Biochim. Biophys. Acta. 620,631-635 25 Goldstein, J.L. and Brown, MS. (1977) Annu. Rev. Biothem. 46,897-930 26 Ray, E., Bellini, F., Stoudt, G., Hemperly, S. and Rothblat, G. (1980) Biochim. Biophys. Acta 617,318-334 27 Stein, O., Coetzee, G.A. and Stein, Y. (1980) in Atherosclerosis V (Gotto, A.M., Smith, L.C. and Allen, B. eds.),

pp. 795-799, Springer Verlag, New York 28 Werb, Z. and Cohn, Z.A. (1972) J. Exp. Med. 135, 2144 29 Rothblat, G.H., Arbogast, L.Y. and Ray, E.K. (1978) J. Lipid Res. 19, 350-358 30 Ho, Y.K., Brown, M.S. and Goldstein, J.L. (1980) J. Lipid Res. 21, 391-398