Interaction between macrophages and mesenchymal cells Effect of LDL- or HDL-containing media, added to cholesteryl ester-loaded macrophages, on cholesterol esterification in mesenchymal cells

Biochimica et Biophysics Acta, 7 12 (1982) 597-604 Elsevier Biomedical Press

597

BBA 51192

INTE~CTION

BE~EEN

~CROP~GES

AND MESENC~MAL

CELLS

EFFECT OF LDL- OR HDL-CONTAINING MEDIA, ADDED TO CHOLESTERYL ESTER-LOADED MACROPHAGES, ON CHOLESTEROL ESTERIFICATION IN MESENCHYMAL CELLS 0. STEIN,

Y. STEIN and G. HA~PERIN

Department Laboratory,

of Experimental Medicine und Cancer Research, Hebrew Unioersity~~adassah Department of Medicine B, Hadassah iJniversit_v Hospital, Jerusalem (Israel)

(Received

March

Key words: LDG

Medical

School

and Lipid

Research

1 lth, 1982)

HDL; Acefylation;

Hypercholesterolemia;

Choiesterol esterification;

(Smooth muscle ceil)

Mouse peritoneal macrophages were loaded with cholesteryl ester (200* 17 pg cholesteryl ester /mg cell protein) by incubation with acetylated LDL (50 pg protein/ml) for 72 h. Next, the loaded cells were incubated for 48 h in Dul~c~Vo~ medium containing 1% bovine serum ~bumin alone or together with low or high density lipoproteins, 15 pg protein/ml, or acetylated LDL, 25 gg protein/ml. Bovine aortic smooth muscle cells and human skin fibroblasts were labeled for 72 h with medium containing free [3H]cholesterol. The labeled cells were then incubated for an additional 48 h with media conditioned in the presence of macrophages. The cholesteryl ester content of the labeled smooth muscle cells incubated with macrophageconditioned media containing LDL or acetylated LDL increased !&fold and there was also a IO-fold increase in the amount of labeled cltoiesteryl ester over that recorded prior to exposure to the macrophage-conditioned medium. Similar results were obtained with macrophage-conditioned media containing HDL, but the effects were less prominent. No enhancement of cholesterol esterification was seen with macrophage-conditioned media containing albumin only. Increase in cellular cholesteryl ester content and in labeled cholesteryl ester after incubation with macrophage-conditioned media containing LDL or acetylated LDL was obtained also in human skin fibroblasts. Cells derived from contro1 donors and from a patient with familial homozygous hy~rcholesterolemia responded in a similar manner. The enhanced cholesterol esterification in human skin fibroblasts, incubated with macrophage-conditioned medium containing acetylated LDL, was counteracted by addition of HDL to the human skin fibroblast medium. The present results offer a mechanism for enrichment in cholesteryl ester of smooth muscle cells and fibroblasts which bypasses the receptor-mediated uptake of LDL and, thus, is not susceptible to autoregulation.

Introduction Even though cholesteryl aortic smooth muscle cells is of human and experimental mechanism of this process has Abbreviations: LDL, density lipoprotein(s).

low density

OOOS-2760/82/oooO-00/$02.75

Recently, we described a model system to test a possible role of macrophages in cholesteryl ester deposition in cultured smooth muscle cells. In that study [ 11, we used acetylated low density lipoprotein (LDL) to enrich macrophages with cholesteryl ester and showed that the macrophage-conditioned medium promotes cholesterol esterification in aortic smooth muscle cells. It appeared that fol-

ester deposition in a prominent feature atheroma, the exact not been elucidated.

lipoprotein(s);

HDL,

high

0 1982 Elsevier Biomedical

Press

598

lowing ingestion of the acetylated LDL, by the macrophage, the cholesteryl ester is hydrolyzed and , while a major portion is re-esterified, considerable amounts of unesterified cholesterol are excreted into the medium, when the latter contains acetylated LDL. The acetylated LDL in the medium enriched in unesterified cholesterol became a cholesterol donor to smooth muscle cells in which it promoted cholesterol esterification by activating the acyl-CoA: cholesterol O-acyltransferase reaction. At present we have extended this study to determine whether non-modified lipoproteins such as LDL and high density lipoprotein (HDL) can transfer free cholesterol from cholesteryl ester-enriched macrophages to smooth muscle cells and to human cells, such as skin fibroblasts. Since LDL, unlike acetylated LDL [2], is recognized by the specific apolipoprotein B-E receptor [3,4], use was made of human skin fibroblasts derived from a familial homozygous hypercholesterolemic patient in order to test whether the presence of the apolipoprotein B-E receptor is involved in the process of stimulation of cholesterol esterification by macrophage-conditioned media. We have also tested the possible role of HDL in the interaction between cholesterol-enriched lipoproteins and mesenchymal cells. Materials and Methods Cell cultures and experimental design Mouse peritoneal macrophages were isolated from normal albino mice of the Hebrew University strain after intraperitoneal injection of phosphate-buffered saline [5]. The cells were sedimented and washed by centrifugation, plated in 60-mm petri dishes (12. lo6 cells/dish) and following incubation for 2 h, the nonadherent cells were removed. The adherent cells (about 30%) were cultured in a medium containing 10% newborn calf serum for 24 or 48 h [l]. Subsequently, the serum-containing medium was removed, the cells were washed three times with phosphatebuffered saline and the macrophages were then incubated with culture medium containing 1% bovine serum albumin, supplemented with acetylated LDL, 30-50 pg protein/ml, and incubation was carried out for 72 h. This medium was collected, the cell layer was washed twice with

phosphate-buffered saline and serum-free medium containing 1% bovine serum albumin was added. Different lipoproteins were added to triplicate dishes of thus-treated macrophages: LDL, 15 pg protein/ml: acetylated LDL, 25 pg protein/ml; HDL, 5-75 pg protein/ml; and incubation was carried out for 48 h. A further set of macrophage dishes, preloaded with acetylated LDL, was incubated with medium containing 1% bovine serum albumin. The macrophage-conditioned media were centrifuged 400 X g X 10 min, and used for incubation with human skin fibroblasts or bovine aortic smooth muscle cells. Bovine aortic smooth muscle cells were cultured as described before [6]. Bovine aortae from the abattoir were dissected and medial explants were prepared [7]. The cells were cultured in modified Dulbecco-Vogt medium [6] containing 5% fetal bovine serum and 5% new-born calf serum. Control human skin fibroblasts were obtained from normal donors with informed consent, and cultured in the same medium. Fibroblasts from a patient with homozygous familial hypercholesterolemia (M.C.) were purchased from Human Genetic Mutant Cell Repository, designated cell line GM-2000. These cells were cultured in Ham’s F-12 medium supplemented with 20% uninactivated fetal bovine serum. Prior to each experiment, the cells were 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 bovine serum to which [7ty(n)-3H] cholesterol was added [S] to give 1 pCi/ml medium. After 72 h of incubation in the labeled medium, the medium was removed, the cells (smooth muscle cells or human skin fibroblast) were washed three times with phosphate-bufferd saline. At that time, zero-time controls were terminated (as described below) and triplicate dishes were incubated for 48 h with media derived from the macrophages, or with non-preincubated media supplemented with lipoproteins. The experiment was terminated after 48 h. The media were collected, the cells were washed three times with 0.2 albumin in phosphate-buffered saline and three times with phosphate-buffered saline, and scraped from the dish with 50% methanol using a teflon policeman. Following the addition of chloroform to give a chloroform:methanol ratio of 1: 1 (v/v) and of campesterol (an internal standard for

599

separated by thin-layer chromatography on silicic acid plates, using petroleum ether/diethyl ether (70:3O,v/v) as solvent. The compounds were visualized with iodine vapors, identified with the help of reference standards. The regions were cut and counted. Total and unesterified cholesterol was determined by gas liquid chromatography [ 111. Isolation of lipoproteins. LDL and HDL were isolated by ultracentrifugation according to the method of Have1 et al. [ 121 at d 1.019-1.063 g/ml and d 1.063- 1.2 1 g/ml, respectively, from human plasma containing 1 mg/ml of EDTA. Acetylation of LDL was carried out with acetic anhydride [ 131. All radioactive materials were obtained from the Radiochemical Centre, Amersham, U.K.

cholesterol), the cellular lipids were extracted and , following centrifugation to remove the non-lipid residue, were purified according to the method of Folch et al. [9]. Protein was determined on the pellet using bovine serum albumin as standard [lo]. The chloroform extract was used for the determination of lipid radioactivity and cholesterol content. Radiochemical and chromatographic procedures Radioactivity was determined on samples of culture medium and lipid extracts of cells, using a Tricarb liquid scintillation spectrometer (Packard Instruments, La Grange, IL, U.S.A.). To determine the percent of [3H] cholesterol recovered in esterified cholesterol, the lipid extracts were

TABLE

I

COMPARISON ESTER-LOADED CELLS

OF MEDIA SUPPLEMENTED MACROPHAGES, TO INDUCE

VARIOUS LIPOPROTEINS, WITH CHOLESTEROL ESTERFICATION

ADDED TO CHOLESTERYL IN AORTIC SMOOTH MUSCLE

Conditions: Mouse peritoneal macrophages were cultured in Dulbecco-Vogt medium containing 10% calf serum for 24 h after plating; this medium was replaced by serum-free medium containing 1% bovine serum albumin and acetylated LDL, 50 pg protein/ml. After 72 h, this medium was collected and replaced by fresh medium containing 1% albumin and the indicated lipoproteins. 25 /.tg protein/ml of acetylated LDL had been used, as about 40-50% were shown to be taken up by the macrophages during subsequent 48 h; the concentration of LDL and HDL was 15 pg protein/ml. This medium was collected after 48 h and used for incubation of aortic smooth muscle cells. The smooth muscle cells were plated at d 7. 104/30-mm petri dish in medium containing 10% fetal calf serum and [3H] cholesterol and labelled for 72 h. The medium was removed, the cell layer washed thrice with phosphate-buffered saline and postincubated 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 chromatography and the amount of total and esterified cholesterol by gas liquid chromatography. Zero time, petri dishes terminated prior to postincubation. Values are means -t S.E. of triplicate dishes of four experiments. Smooth muscle cells incubated with

‘H label after 48 h postincubation (dpm) Medium (Total,. IO-‘)

Cellular

Smooth Total

Medium added to macrophages, AcetylatedLDL LDL Albumin Nonincubated medium AcetylatedLDL LDL HDL Zero time

preloaded with cholesteryl 103*23 13li 7 114* 6 55* 6

cholesterol

( u g/mg protein) muscle cells

(. 10p3)

ester, supplemented 125* 112% 132* 162*

6.4 6.4 5.0 2.6

Cholesteryl (. 10-s)

Total

Esterified

54.6k2.4 58.7k5.9 43’2.3 32.3 2 1.5

14.9* 1.9 15.3A3.8 8.1 t2.0 3.5 kO.8

37.2k2.5 40.7kO.8 26.7 * 2.0

4.9* 1.0 4.8kO.7 2.6”0.1

28.220.7

3.1 f0.7

ester

with 198* 2.0 220* 15 141*11 25*4

containing 1322 5 124’ 8 70? 13

109* 10 126* 8 143k 19

23k4.0 2356.0 9f2.5

255 * 14

192

2.0

600

Results The present experiments were planned to test the ability of native lipoproteins to serve as cholesterol acceptors when added to cholesteryl ester-loaded (200 2 17 p/mg protein) macrophages and as cholesterol donors in the presence of smooth muscle cells. The data of four experiments with bovine aortic smooth muscle cells are presented in TableI. In agreement with our previous findings [l], it can be seen that acetylated LDL added to macrophages which had been preloaded with cholesteryl ester confers to the medium ability to enhance cholesterol esterification in smooth muscle cells. This parameter was determined by two inde-

pendent methods, one involving incorporation of labeled cholesterol into cholesteryl ester, the other determination of cellular cholesterol content by gas liquid chromatography. There was fair agreement between the two determinations. As can be seen, macrophage medium supplemented with the two non-modified lipoproteins, i.e., LDL and HDL, promoted also cholesterol esterification in smooth muscle cells, but to a different extent. Macrophage medium supplemented only with bovine serum albumin did not affect cholesterol esterification. So far the experiments had been performed with bovine aortic smooth muscle cells and it seemed of interest to test the system with human cells also. As seen in Table II, human skin

TABLE II COMPARISON ESTER-LOADED TROL DONORS Conditions

OF MEDIA SUPPLEMENTED WITH VARIOUS LIPOPROTEINS, ADDED TO CHOLESTERYL MACROPHAGES, TO INDUCE CHOLESTEROL ESTERIFICATION IN SKIN FIBROBLASTS OF CONAND OF A FAMILIAL HOMOZYGOUS HYPERCHOLESTEROLEMIC PATIENT

as in Table I.

Human skin fibroblast incubated with:

‘H label after 48 h postincubation Medium (Total, 10m3)

Human

(dpm)

LDL HDL Albumin Nonincubated Acetylated LDL HDL Zero time

preloaded with cholesteryl 64k2.0

protein)

Cholesteryl (. 10-2)

Total

Esterified

49.0& 1.6

ester

ester, supplemented 52t 4.7

with: 129*

17k2.7

50% 3.5 522 3.6 80-c 10

137% 10 72) 3 10% 0.3

53.3e6.6 40.2 * 3.4 25.5k2.1

14.4% 1.5 12.3-t0.9 7.8’1.4 1.3kO.2

68k5.1 55t5.2 631-2.5 _

411 1.4 45* 1.8 30-c 0.3 110*10

5-t 0.6 16i: 1.2 4% 0.4 17% 1.2

36.2 * 2.5 34.520.9 22.6-c 1.0 32.9* 1.6

4.6-t0.2 4.4‘0.8 2.3 to.4 4.3 t 0.7

with 109-t_ 18 702 5.2 611 6.8 5

67.3’- 1.8 62.9k2.5 48.6kO.9 39.7

23.0t 1.3 19.4k2.2 10.2iO.8 5.1

37.22 1.3 40.722.9 25.6kO.5 30.5 f 0.9

5.7*0.8 6.92 1.2 3.6~0.6 4.9-to.s

61i6.2 58k8.5 medium LDL

cholesterol

skin fibroblast

Total (. 10m3)

Control human skin fibroblast Medium added to macrophages, Acetylated LDL

Cellular (ng/mg

9

containing:

Familial homozygous hypercholesterolemic human skin fibroblast Medium added to macrophages, preloaded with cholesteryl ester, Acetylated LDL 52 5 4.0 33t LDL 51’3.0 315 HDL 57k2.1 32-c Albumin 18 48 Nonincubated medium containing Acetylated LDL 71 k2.8 25t LDL 73 -f 7.8 28% HDL 45 * 2.6 34* Zero time _ 87f

supplemented 2.2 1.2 0.9

1.2 1.3 1.4 3.5

62 614% 7%

0.5 0.5 0.4 0.7

601 TABLE III

fibroblasts responded to the macrophage-contioned media supplemented with the three lipoproteins in a manner analogous to the smooth muscle cells. This finding permitted to investigate whether the apolipoprotein B-E receptor is involved in the enhancement of cholesterol esterification by the macrophage-conditioned media. To that end, use was made of human skin fibroblasts from a patient with familial homozygous hypercholesterolemia. Results of three experiments are presented in TableII, lower part. It can be seen that the macrophage media supplemented with LDL enhanced cholesterol esterification in the familial homozygous hypercholesterolemic fibroblasts to the same extent as acetylated LDL; in analogy to results with normal fibroblasts, media containing HDL were less effective. Incubation of the homozygous cells with macrophage-conditioned media resulted in an enhancement of cholesteryl ester accumulation even more pronounced than that encountered with normal fibroblasts. Analysis of the cholesterol content of the macrophage media used for incubation with smooth muscle cell or fibroblasts showed enrichment of the media with free cholesterol and, thus, a prominent change in the ratio of free cholesterol to total cholesterol when compared to the ratio seen in the original

INCREASE IN FREE CHOLESTEROL IN MEDIA DURING POSTINCUBATION OF CHOLESTERYL ESTER-LOADED MACROPHAGES IN THE PRESENCE OF VARIOUS LIPOPROTEINS Conditions: Mouse peritoneal macrophages were cultured for 24 h in Dulbecco-Vogt medium containing 10% calf serum. Thereafter, the medium was changed to serum-free medium containing 1% bovine serum albumin and acetylated LDL, 50 pg protein/ml. The cells were exposed to this medium for 72 h. This medium was collected and postincubation with the various lipoproteins at concentrations indicated in Table I was carried out for 48 h. The cholesterol content of the media was determined by gas liquid chromatography. Values are means 5S.E. of 5-8 media obtained from different preparations of macrophages. Medium added to macrophages preloaded with acetylated LDL, supplemented with

Free cholesterol

Free cholesterol

(pg/ml medium)

(% of Total)

Acetylated LDL HDL

8.7’1.4 11.1*0.6 6.4kO.8

56.7k4.2 48.1 f 1.2 50.954.3

LDL

Nonincubated Acetylated LDL HDL

TABLE

lipoprotein LDL

26.1 kO.8 24.1-‘2.0 21.3* 1.6

IV

EFFECT OF CONCENTRATION OF HIGH DENSITY LIPOPROTEIN IN MEDIA, ADDED TO CHOLESTERYL ESTER-LOADED MACROPHAGES, ON THE INDUCTION OF CHOLESTEROL ESTERIFICATION DURING POSTINCUBATION WITH AORTIC SMOOTH MUSCLE CELLS Conditions:

As in Table I.

Smooth muscle cells incubated with:

3H label after 48 h postincubation Medium (Total, . 10K3)

(dpm)

Cellular

cholesterol

(gg/mg

protein)

Smooth muscle cells Total (. 10e3)

Cholesteryl

Total

Esterified

15 -co.1 8 -cl.0 10 22.0 8.5 kO.3 10 k1.5 3 to.5

ester

(. 10-l) Medium added to macrophages Acetylated LDL (25) HDL (5) HDL (15) HDL (25) HDL (50) Zero time

loaded with cholesteryl 103* 5.2 104k 1.8 8Ok3.6 105~4.8 125k3.3

ester supplemented

with (pg protein/ml)

90-t 92193* 752 63*

135* 31k 99fll 96* 98%

3 3 6 8

67* 1.2 41 t 1.4 53k2.3 57* 1.4 55* 1.9

36*

5

36* 1.4

5 3 5 4 3

180-c 12

602

TABLE V EFFECT DURING

OF HIGH DENSITY LIPOPROTEIN ON CHOLESTEROL INCUBATION WITH HUMAN SKIN FIBROBLASTS

Conditions: As in Table I: HDL means -C SE. of triplicate dishes. Cells incubated

was added

with

to the medium

at the onset of incubation

75 /.tg protein/ ml HDL added to medium of human skin

fibroblast Medium added to macrophages loaded with cholesteryl ester supplemented with acetylated LDL Nonincubated containing

medium acetylated

LDL

Zero time

ESTERIFICATION

‘H label in human skin fibroblast 48 h of postincubation

WHEN

ADDED

with the human

after (dpm)

TO THE MEDIA

skin fibroblast.

Cellular

cholesterol

(pg/mg

protein)

Total Total (. 10-3)

_ +

6Ot- 1.2 31-rO.8

_ +

42t 1.3 35’0.5

_

1502 1.7

lipoprotein (Table III). Even though the change in the free to total cholesterol ratio was quite similar with the three lipoproteins studied, the macrophage medium supplemented with HDL was the least active with respect to enhancement of cholesterol esterification in all lines examined (Tables I and II). In the next experiment, the concentration of HDL added to the macrophage medium during the conditioning period was varied

Cholesteryl (, 10-Z)

Values are

Esterified

ester

150*0.9 16*0.1 5t0.1 350.05 1410.2

63.62 36.9*

1.3 1.2

37.7t0.5 32.4 32.32

22.5 * 1.7 3.11-0.7 5.020.4 2.5

14

4.520.2

and it can be seen that maximal stimulation of esterification could be demonstrated with 15 pg HDL protein/ml and was much lower in the presence of only 5 pg HDL protein/ml (Table IV). It seemed of interest to examine whether HDL will affect the enhancement of cholesterol esterification by macrophage-conditioned medium. To that end, human skin fibroblasts were incubated with macrophage-conditioned medium containing

TABLE VI EFFECT ADDED Conditions:

OF CONCENTRATION DURING INCUBATION As in Tables

Cells incubated

OF HIGH DENSITY LIPOPROTEIN WITH HUMAN SKIN FIBROBLASTS

CHOLESTEROL

ESTERIFICATION

WHEN

I and VI.

with

HDL added to medium of human skin fibroblast (ug/ml)

Medium added to macrophages loaded with cholesteryl ester supplemented with acetylated LDL

Zero time

ON

0 5 25 75

3H-labeled cholesteryl ester (dpm. lo-*)

118k8.0 44-t 1.0 820.6 5-0.3 22) 1.0

Cellular

cholesterol

(pg/mg

protein)

Total

Esterified

59.320.2 45.8kO.5 36.9* 1.7 32.7t0.6 35.2kO.8

13.3-t 1.0 8.52 1.5 5.920.1 33-to.7 3.0-t0.8

603

acetylated LDL. When HDL, 75 pg protein/ml, was added to the dishes, no enhancement of cholesterol esterification was observed (Table V). The marked counteracting effect of HDL on the promotion of cholesterol esterification by the macrophage-conditioned medium was seen at 75, 50 and 25 pg protein/ml, but to a lesser extent only at 5 pg protein/ml (Table VI). Discussion

Enrichment of aortic smooth muscle cells with cholesteryl ester is the prominent feature of atheromatosis, yet it is quite difficult to reproduce such a process under conditions of culture. When LDL or /3 low density lipoproteins [ 141 are used as cholesterol donors, accumulation of cholesterol is prevented because the r~eptor-mediated uptake of these lipoproteins is subject to autoregulation. The other cells which are present in an atheroma are macrophages which accumulate cholesteryl ester when exposed to modified lipoproteins [2, IS]. In a previous study we described a model system in which macrophages loaded with cholesteryl ester were incubated with acetylated LDL and the macrophage medium, containing acetylated LDL enriched with free cholesterol, was used to stimulate cholesterol esterification in aortic smooth muscle cells [I]. At present, we have extended these observations to non-modified human plasma lipoproteins and to cultured human skin fibroblasts, so as to reduce interspecies effects. Thus, when LDL is added to ma~rophages preloaded with cholesteryl ester, the medium becomes enriched with free cholesterol and, in turn, promotes cholesteryl ester accumulation in bovine smooth muscle cells as well as in human fibroblasts. Recently, secretion of apolipoprotein E by peritoneal mouse macrophages was reported, especially after enrichment of the cells with cholesteryl ester [16]. One could argue that the presence of apolipoprotein E-cholesterol complex was operative in the presently described stimulation of cholesterol esterification in smooth muscle cells. However, our results do not support such a conclusion because of the finding that stimulation of cholesterol esterification by macrophage media was as prominent in familial homozygous hyperchoiesterolemic cells, which lack the apolipoprotein B-E receptor,

as in normal skin fibroblasts. In addition to LDL, HDL enriched in free cholesterol was shown to serve as cholesterol donor to the presently studied mesenchymal cells and, thus, enhance cholesterol esterification. HDL enriched in free cholesterol was shown also to donate cholesterol to hepatoma cells [ 17). Even though the increase in the ratio of free to total cholesterol in HDL was the same as in acetylated LDL, macrophage-conditioned media supplemented with HDL enhanced cholesterol esterification to a lesser extent. This is probably due to the greater capacity of HDL to bind free cholesterol and act as cholesterol acceptor rather than cholesterol donor [ 11,I 81, It seems plausible that when the cholesterol-enriched HDL is the only lipoprotein in the medium it will serve as cholesterol donor, while in the presence of other lipoproteins, HDL will act as an acceptor of free cholesterol. Thus, addition of HDL to medium containing acetylated LDL enriched in free cholesterol counteracted the enhancement of cholesterol esterifi~ation in the fibroblasts. This effect of HDL was concentration-dependent and was hardly apparent at low HDL concentration. This finding could provide yet another explanation for the inverse correlation reported recently [19,20] between plasma levels of HDL and incidence of myocardial infarction in the human. The present results offer a mechanism for enrichment in cholesteryl ester of smooth muscle cells and fibroblasts which bypasses the receptor-mediated uptake of LDL and, thus, is not susceptible to autoregulation. Acknowledgements

The excellent technical help of Mrs. A. Mendeles, Mrs. M. Ben-Naim, Mrs. Y. Dabach and Mr. G. Hollander is gratefully acknowledged. References 1 Stein, O., Halperin, G. and Stein, Y. (1981) Biochim. Biophys. Acta. 665, 447-490 2 Goldstein, J-L., Ho, Y.K., Basti, S.K. and Brown, M.S. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 333-337 3 Goldstein, J.L. and Brown, M. S. (1977) Annu. Rev. Bio&em. 46, 897-930 4 Innerarity, T.L., Kempner, ES., Hui, D.Y. and Mahley, R.B. (1981) Proc. Nat]. Acad. Sci. U.S.A. 78, 43784382

604

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13 Fraenkel-Conrat, H. (1957) Methods Enzymol. 4. 247-269 14 Mahley, R.W., Innerarity, T.L., Weisgraber, K.H. and Fry. D.L. (1977) Am. J. Pathol. 87, 205-226 15 Goldstein, J.L., Ho, Y.K., Brown, M.S., Innerarity, T.L. and Mahley, R.W. (1980) J. Biol. Chem. 255, 1839- 1848 I6 Basu, S.K., Brown, M.S.. Ho,Y.K., Have], R.J. and Goldstein, J.L. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 75457549 17 Rothblat, G.H., Arbogast, L.Y. and Ray, E.K. (1978) J. Lipid Res. 19, 350-358 18 Stein, 0.. Vanderhoek. J. and Stein,Y. (1976) Biochim. Biophys. Acta 43 1, 3477358 19 Gordon, T., Castelli, W., Hjortland, M., Hames, C.G. and Dawber, T.R. (1977) Am. J. Med. 62. 707-714 20 Miller, N.E., Thelle, D.S., F&de, O.H. and Mjefs, O.D. (1977) Lancet i, 965-968