Cholesterol Cultured

Esterification and Cholesteryl Pigeon and Monkey Arterial

BETH P. SMITH, Arterio.derosis of

RICHARD W.

Research Center, Detrartnrent the Wake Forest Uniccrsitu,

ST. of

Ester Accumulation in Smooth Muscle Cells

CLAIR, AND Jos C. LEWIS

PathologrJ, Bowman Gray School of Medicine North Carolina 37103

Wimton-Salem,

Received Alay 19, 1978, and in recisctl

form

October

17, 1978

Aortic smooth muscle cells from atherosclerosis-susceptible White Cameau and resistant Show Racer pigeons were grown in cuiture utilizing conditions identical to those developed for the culture of rhesus monkey arterial smooth muscle cells and skin fibroblasts. Pigeon smooth muscle cells had ultrastructural and growth characteristics simdar to mammalian smooth muscle cells in culture including growth in multiple overlapping layers, numerous pinocytic vesicles, and abundant myofilaments. Cells were incubated for 21 to 52 hr with culture medium containing either lipoprotein deficient serum, fetal calf serum, normocholesterolemic pigeon serum or hypcrcholesterolemic pigeon serum, and difl’crences in cholesterol content and in the rate of cholesterol esterification were studied. Although there was snlxtantial variability in the absolute cholesterol content among different cell iines, cells of the same type behaved similarly in their rcsponsc to the various test sera. Generally, the higher the cholesterol content of the culture mcdiunr the greater the cholesterol content of the cells. There were, however, consideralrlc clillerences in the ma~nritucle and pattern of response among the dilferent ccl1 types. Incuhatron with 10% hypercholesterolemic serum resulted in an increase of similar magnitude in the free cholesterol content of all cell lines. This was not true, however, for the accumulation of cholesteryl esters. Inculx~tion with 10% hypercholeste~olc~nic serum produced a marked increase in the cholesteryl ester content of monkey skin fibrol~lasts and smooth muscle cells while prodrrcing only a slight incrcasc in the cholesteryl ester content of pigeon smooth muscle cells unless very high concentrations of hypercholesterolemic serum were used. Monkey skin filmrl:lnsts were the most responsive increase in cholesteryl to cholesteryl ester accumulation with a greater than 2%fold ester content occurring when inculx~tecl with hyp~rcllolcstcrolcn~ic serum. Incubation fur 21 hr with l~ypercl~olestrrc,lemic serum stimulated cholesterol estcrification 3 to g-fold in monkey celis in a manner that paralleled the time course of accumulation of choFestery1 esters, while no stimulation in cholesterol csterification occurred in pigeon cells, consistent with their lack of airility to accumulate large amounts of cholesteryl esters. No differences in the response to either normal or hypercholesterolemic serum were seen between smooth muscle cells from atherosclerosis-susceptible White Carneau and resistant Show Racer pigeons. There were, however, major differences between pigeon and monkey cells. Although results indicate that the mechanism of accumulation of cholesteryl esters by pigeon smooth muscle cells may be different than for mammalian cells, no differences were seen in any of the parameters measured that might help to explain the difference in susceptibility to atherosclerosis of the twa breeds of pigeons.

190 0014-4800,/79/020190-19$02.00/O Copyright All rights

0 1979 by Academic Press, of reproduction in any form

Inc. reserved.

CHOLESTEROL

METABOLISM

IN

ShlOOTii

MUSCLE

CELLS

191

INTRODUCTIOS The accumulation of cholesterol, particularly cholesteryl esters, and the stimulation of cholesterol esterification are among the earliest biochemical changes in the pathogenesis of atherosclerosis (St. Clair, 1976). In animals in which atherosclerosis can be rapidly induced experimentally, such as rabbits and pigeons, the changes in cholesteryl ester accumulation and cholesterol esterification can be demonstrated to occur after onI!- a few days of feeding of an atherogenie diet (St. Clair et al., 1970; Day and Proudlock, 1974). Hypercholesterolemia, in response to an atherogenic diet, clearly plays a major role in exacerbation of the esperimentnllp induced disease. Only a portion of the variability in the resulting atherosclerosis, however, can be explained by the hypercholesterolemia, since animals with similar plasma cholesterol concentrations may vary considerably in the estent of atherosclerosis. This is illustrated best; perhaps, in the pigeon where the White Carneau are susceptible to nortic atheroscler,osis while Show Racers arc resistant even though total plasma cholesterol concentrations are not significantly different ( Clarkson et al., 1959). Such results are consistent with the conclusion that other factors, probably genetically mediated, act to control susceptibility to atherosclerosis at the level of the arterial wall. The biochemical mechanism(s) that control susceptibility at the level of the arterial wall are unknown. It is reasonable to suggest, however, that genetic factors might determine the manner 1))~ which arterial smooth muscle cells respond to similar challenges, such as hypercholesterolemia. The pigeon is an ideal model in which to test such interactions since clear-cut differences have been demonstrated in susceptibility to atherosclerosis between the White Carnenu and Show Racer breeds. The advent of techniques of growing arterial smooth muscle cells in culture (Ross, 1971) has provided a mechanism by which to test the biochemical response of smooth muscle cells from atherosclerosis-resistant and susceptible strains of animals to identical factors such as hypercholesterolemic sera, lipoproteins, etc. In this report we describe a technique for growing smooth muscle cells from the aortas of atherosclerosis-susceptible White Carneau and atherosclerosisresistant Show Racer pigeons. These cells were used to study the influence of normo- and hypercholesterolemic pigeon serum on cholesterol accumulation and cholesterol esterification. We have compared the response of the pigeon smooth muscle cells with that of arterial smooth muscle cells and skin fibroblasts from rhesus monkeys. From these comparisons. we hoped to cletermine whether the increased susceptibility to atherosclerosis of the m’hite Carneau pigeon relative to the Show Racer or rhesus monkey ~‘1s reflected by increased cholesterol accumulation or esterification in cultured arterial smooth muscle cells in response to exposure to normo- or hypercholesterolemic pigeon sermix MATERIALS

AND

METHODS

Smooth Muscle Cell Cdtures Using sterile procedures, aortas were obtained from three White Carneau and three Show Racer pigeons, 4 to 8 weeks of age. The nortic segment removed

192

SMITH,

ST. CLAIR,

AND LEWIS

from each laircl included the portion of the aorta from the level of l)ifurc:~ti()ll of the left brachiocephalic artery extending just proximal to the I)ifurc:~tion of the celiac artery. Smooth muscle cells were grown from explants oljtained from tlic proximal one-half of this aortic segment since we were uncertain of being able to completely remove the adventitia in the very thin distal portion of the aortic segment. The adventitia and the outer one-third of the media were removed from the proximal segment of aorta under a dissecting microscope and discarded. The remaining intima-media preparation was cut into approximately 1 mm explants and 40 to 50 of these explants were transferred to Falcon tissue culture flasks (75 cmZ) containing 5 ml of culture media. The culture medium consisted of Eagle’s minimunl essential medium ( MEM-Auto Pow) supplemented with Eagles MEM vitamins, 200 mM L-glutamine, n-glucose (1 mg/ml), and penicillin (100 IU/ml) : streptomycin (100 pg/ml) or gentamicin (50 ,g,/ml). For the routine growth and maintenance of cells in culture, this culture medium was further supplemented with 10% fetal calf serum. All tissue culture supplies were obtained from Flow Laboratories. Flasks of cells were maintained in an incubator at 100% humidity and 37°C with an atmosphere of 95~C air and 5% COs. The explants were allowed to adhere to the flasks by maintaining the flasks in ,an upright position for 2 to 3 hr. Following attachment of the explants to the plastic, the flasks were maintained in their normal horizontal position and were left undisturbed for 4 days at which time 10 ml of fresh culture media was added. The first outgrowth of cells from the explants occurred within 4 to 5 days. The culture mediunl was changed three times per week with 20 ml of media added at each change. After 2 to 3 weeks, the cells were trypsinized for the first time using a solution of O.O5c/c:trypsin and 0.02~$ ethylenediamine tetraacetic acid (EDTA). Confluent monolayers of smooth muscle cells from one 75 cm2 flask were trypsinized every 5 to 7 days and divided into five 75 cm? flasks, thus effecting a 1:5 split with each cell passage.For all experiments, the pigeon smooth muscle cells were used while in the seventh to ninth passSage. The monkey aortic smooth muscle cells and monkey skin fibroblasts were cultured as described previously (St. Clair et al., 1977: Guertler and St. Clair, 1977) . Experimental Design For individual experiments, cells from several confluent 75 cm? flasks were trypsinized, combined, and pl’ated into 100 mm dishes, 60 mm dishes, or 25 cm’ flasks at a cell density of from 5 to 7 x 10” cells per flask for pigeon cells and 2.5 to 5 x lo5 cells per flask for monkey cells. The higher plating density was used for experiments with 100 mm dishes. For all experiments, cells were grown to confluency prior to the addition of the various test media. Prior to the addition of the test medi’a, cell monolayers were washed with phosphate-buffered saline (PBS) at pH 7.4. Two types of experiments were carried out: [1-‘“Cloleate esterification studies and sterol content studies. For the [lJ’C]oleate esterification studies, the smooth muscle cells were incubated for various periods of time with the standard Eagles MEM culture medium containing different concentrations of hypercholesterolemic pigeon serum and 2.5 mg protein/ml of lipoprotein deficient serum (LPDS). For measurement of esterification of oleic acid, the cells were incubated with [1-14C]oleate for

CHOLESTEROL

METABOLISM

IN SMOOTH MUSCLE CELLS

193

4 hr prior to the termination of the experiment, essentially as described previously (St. Clair et al., 1977). For the sterol content studies, smooth muscle cells were incubated with normocholesterolemic or hypercholesterolemic pigeon serum at the concentrations and times indicated in the tables and figures. The cells were grown in the standard Eagle’s MEM culture medium containing 10% fetal calf serum until the initiation of the experiment. After the cell monolayers were washed with PBS, the cells were incubated for up to 72 hr with the standard Eagles MEM culture medium containing either LPDS (2.5 mg protein/ml), 10% fetal calf serum, 10yc normocholesterolemic pigeon serum, or lo%> hypercholesterolemic pigeon serum. At the termination of each experiment the cell monolayers were rinsed twice with PBS, removed from the dishes or flasks with trypsin-EDTA and transferred to 12 ml centrifuge tubes with several washes of PBS. The cells were pelleted by centrifugation and the cell pellets were washed twice with PBS. Deionized water (1 ml) w’as added, and the cells were disrupted by sonication for 5 set at 55 watts utilizing a Heat Systems Model W185 sonifier with a micro tip. A sample of this solution was taken for cell protein determination by the method of Lowry et al. (1951) using bovine serum albumin as the standard. For oleate esterification experiments, cholesterol 1,2-3H (0.02 &i) was added to each tube as a recovery standard. For sterol content studies, stigmasterol (24 pg) was used as the recovery standard. Lipids were extracted using the Bligh and Dyer method (1957) and for [1-“Cloleate studies individual lipids were separated by thin-layer chromatography and radioactivity determined as described previously (St. Clair and Harpold, 1975). The cholesterol and cholesteryl ester content of the cells was determined by gas-liquid chromatography by the method of Ishikawa et al. ( 1974) using a Tracer, hlodel 560, gas chromatograph equipped with a CSI-38 digital integrator (Columbia Scientific Industries). All results were corrected for recovery of the [1,2-3H]cholesterol or stigmasterol internal standards. Serum was obtained from noncholesterol-fed and cholesterol-fed adult male pigeons that had been fasted overnight, Noncholesterol-fed pigeons consumed a diet consisting exclusively of a mixture of four different grains: corn, Canadian peas, wheat, and milo. Cholesterol-fed pigeons consumed a diet consisting of pigeon pellets (Ralston Purina Co.) (89.5% ), lard (10% ), and cholesterol (0.5%) ). The cholesterol was dissolved in the melted lard, and this mixture added to the pigeon pellets. The pigeons ,consumed these diets for at least 2 months prior to blood collection. The individual blood samples were allowed to clot, and the serum was collected and pooled. Serum pools consisted of approximately equal volumes from White C’arneau and Show Racer pigeons. The serum pools were sterilized through a 0.45 pm Millipore filter and stored refrigerated. The total cholesterol and triglyceride content of the serum pools were determined using the AutoAnalyzer II methodology of Rush et d. (1971). Lipoprotein deficient serum was prepared by adjusting the density of calf serum to 1.215 g/ml with solid KBr and centrifugation at 15°C for 24 hr at 60,000 rpm in a Beckman 6OTi rotor using a Beckman L2-65B ultracentrifuge. The lipoprotein layer was removed and the lipoprotein-free infranatant solution was dialyzed exhaustively against PBS and Eagle’s MEM. For experiments in which LPDS was added to the culture medium, it was added at a final concen-

194

SMITH,

ST. CLAIR,

AND LEWIS

tration of 2.5 mg protein/ml. This amount of LPDS provided less than 0.004 mg cholesterol~‘ml. [l-“C]oleic acid (250 &i; 4.26 qol) (A mc,rsham/Searlc) and nonradioactive oleic acid (38.2 pmol) (Applied Science Laboratories) were purified by thin-layer chromatography (TLC) and dissolved in 5 ml of redistilled 95% ethanol. To form the sodium salt, the oleic acid solution was titrated to a phenolphthalein end point (approximately pH 10) with 1 N NaOH, and the ethanol was evaporated under a stream of N, at 45°C. The sodium oleate was complexed to albumin at 40°C by addition of 2.5 ml of a 17,0 (‘1 solution of bovine albumin (essentially fatty acid-free) (Sigma). The final clear solution was sterilized through a 0.45 pm Millipore filter and was stored refrigerated. This solution had a final specific activity of 11,000 dpm,/nmol, a molar ratio of fatty acid to albumin of 6: 1, and was used as the substrate for measurement of [ 1-‘“Cloleate esterification to cholesterol, triglyceride, and phospholipid.

Electron iVicroscopy Cells for electron microscopy were fixed in the culture dishes for 1 hr with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2. Post fixation in lcjr osmium tetroxide in the samebuffer was followed by dehydration through a graded series of ethanol and embedding in situ in Epom 812 (Luft, 1961). Thin sections obtaiued with a diamond knife in a LKB ultrotome 3 were double stained with lead citrate and uranyl acetate before viewing in a Philips 201C electron microscope, RESULTS

Growth Characteristics Muscle Cells

and Celkdar

hforphology

of

Pigeon Aortic Smooth

The cells that grew from the White Carneau and Show Racer pigeon aortic explants had a pattern of growth typical of mammalian smooth muscle cells in that they grew in patterns of multiple overlapping layers in a configuration resembling “hills and valleys” (Fig. 1). The pigeon cells also exhibited the ultrastructural characteristics typical of mammalian smooth muscle cells (Fig. 2). The cytoplasm contained numerous ribosomes, microtubules, and filaments (Fig. fascicles. There were numerous pinocytotic vesicles associated with the fascicles in 3a). The filaments were from 60 to 70 A in diameter and were arranged in the peripheral cytopkasm (Fig. 3b). The perinuclear cytoplasm contained multiple Golgi zones and an abundance of rough endoplasmic reticulum (Fig. 3~). A few lysosome-like structures were observed in most cells (Fig. 3d). Even though the pattern of growth was similar for smooth muscle cells from rhesus monkeys and pigeons, there were differences in their rate of growth and in the density of cells at confluency. The cell doubling time measured during the period of most rapid log growth averaged 36 to 48 hr for both White Carneau and Show Racer pigeon smooth muscle cells compared with an average doubling time of 77 hr for rhesus monkey smooth muscle cells and skin fibroblasts. Smooth muscle cells continued to divide after reaching confluency and developed multiple overlapping layers of cells and, as a result, the number of cells continued to

CHOLESTEROL

FIG. 1. Growth characteristics seen to grow in areas of high and hematosylin. ~300.

FK 2. Electron micrograph position of the nlwleus (S) c)toplasm. x:3300.

METABOLISM

IN

SMOOTH

hlUSCLE

CELLS

of Show Racer smooth muscle cells in culture. ant1 lo\v cell density reseml)ling “lrilk ant1 dleys.”

and

of a typical \Vhite Carnenu the pwsrnce of IIII~CKIIIS

smooth filaments

muscle (\iF)

190

Cells were Oil Red 0

cell showing the in the peripheral

196

SMITH,

ST. CLAIR,

AND

LEWIS

FIG. 3. Ultrastructural features of cultured White Carneau and Show Racer smooth muscle cells. (a) Cytoplasm contains many ribosomes (R ), microtubules (MT), and filaments (MF). ~30,000. (b) Peripheral cytoplasm exhibiting numerous pinocytotic vesicles ( PV) associated with filaments. ~22,500. (c) Perinuclear cytoplasm containing Golgi zones (G) and rough endoplasmic reticulum (RER). ~23,500. (d) A few lysosome-like structures were observed jn most cells. Some highly differentiated cells contained multiple residual bodies (RB) and multivesicular bodies (MVB) in the perinuclear cytoplasm. X35,000,

CHOLESTEROL

METABOLISM

IN SAIOOTH

MUSCLE

CELLS

197

increase with continued duration of incubation, although not as rapidly as during the log growth phase. All experiments in this study utilized cells that had completed their log phase of growth and, thus, will be referred to as confluent. At this time the density of rhesus monkey skin fibrohlasts and smooth muscle cells ranged from 2 to 8 x lo4 cells/cm?, while for the pigeon smooth muscle cells there was an average of 14 to 20 x 10’ cells/cm’. At least part of this difference in the number of cells per unit area was due to the smaller size of the pigeon smooth muscle cells. Cholesterol Contellt Experiments To determine the effect of different sera on the cellular free cholesterol and cholesteryl ester content of pigeon and monkey cells, we incubated the cells for 72 hr with culture medium cont’aining either LPDS (2.5 mg protein/ml), lo? fetal calf serum, 10% normal pigeon serum, or 109, hypercholesterolemic pigeon serum. Results are shown in Table 1. These data were analyzed by a two way analysis of variance, as indicated in Table I, and individual groups were compared at the P < 0.05 level of significance using Tltkey’s test. We have included in Table I the results from the 10 different cell lines studied in order to indicate the magnitude of the variability in the absolute cholesterol content among different cell lines, even from the same species. Nevertheless, the pattern of response to the various test media of individual cell lines was similar for cells of the same type from a given species. Because of considerable individual variability there was no significant difference in the free cholesterol content of cells from the four species studied. There was, however, considerable difference in the free cholesterol content of the individual cell lines, particularlJr among the pigeon smooth muscle cells. AS the analysis of variance table indicates, there were significant differences in the cholesteryl ester content of the different cell lines. This was due almost eselusively to the significantly greater response of the rhesus monkey skin f?brobl;tits to normal and hypercholesterolemic serum, as there was little difference in the cholesteryl ester content of the different cell lines when cultured with LPDS or fetal calf serum. The specific culture medium with which the cells were incubated had a similar qualitative effect on the free and esterified cholesterol content of all of the cell lines studied, although the magnitude of this response varied significantly among cell lines. Rhesus monkey smooth muscle cells and skin fibroblasts contained significantly more free cholesterol when incubated with fetal calf serum compared to LPDS. There was no further increase in the free cholesterol content of rhesus monkey smooth muscle cells upon incubation with either normal or hypercholesterolemic serum. Rhesus monkey skin fibroblasts, however, contained significantly more free cholesterol when incubated with hypercholesterolemic serum than with normal serum. The major change in all cell lines upon incubation with the various test media, occurred in the cholesteryl ester fraction. The rhesus monkey skin fibroblnsts were the most responsive to the test media with significant and progressive increasesoccurring in the cholesteryl ester content upon incubation with fetal calf serum, normal and hypercholesterolemic serum. With hypercholesterolemic

Line

monkey

17.4 15.9 11.4 14.9 20.0 12.2 19.3 17.2

f + f f + & f f

FC

0.66 0.22 0.35 0.67 0.24 0.62 0.88 0.92

5 5

6 3

5 5 5

1.7 0.76 1.2

0.15 0.96 0.30

* 0.46 + 0.20 f 0.28

0.91 f 1.2 f 1.0 f

1.3 f 0.51 2.3 f 0.33 2.2 f 0.34 2.0 3.z 0.23 0.95 It 0.12 1.2 f 0.53 2.3 f 0.92 1.5 zt 0.36

CE

6 6

6 6

6 6 6

6 6 6

N

0.49 0.27 0.29

0.26 0.46 0.23 0.58 0.28 0.26 1.3 0.92

6 6

6 6

5 5 6

6 5 6

N

FCS

Cell line Culture medium Interaction

2-\Vay

3O.Ortl.O 26.0 f 0.60 28.0 f 0.83

f f f

f f f f f f f f

FC

10yfi

0.18 0.07 0.31 0.25 0.24 0.61 0.80 0.43

f f f 6 6

ti 5

6 6 6

6 6 6

P < 0.001

FC N.S. P < 0.001

of variance

0.91 0.26 0.86

z!z 0.24 f 0.51 f 0.30

analysis

6.8 2.0 4.4

2.9 3.7 3.3

f f f f f A f zt

cell protein

fig/nig 0.88 1.7 1.9 1.5 0.67 2.4 3.1 2.1

N

+ + f f f do ?t f

0.41 0.48 0.35 1.1 0.21 0.64 0.73 1.0

Cl5 I’ < 0.005 P < 0.001 P < 0.001

31.0 + 0.26 29.9 f 0.35 30.5 f 0.26

24.4 f 0.27 23.2 f 0.58 23.8 ItI 0.34

26.3 28.6 18.0 24.3 27.7 19.5 28.4 25.2

FC

6 6

6 5

6 5 5

6 6 6

h’

lo%< NCS

f f f f f f f f

CE

13.5 zk 0.92 8.4 f 0.51 11.0 f. 0.91

0.11 0.43 0.70

0.55 1.5 0.28 0.5’2 0.30 0.85 1.3 0.51

6 6

6 6

6 6 6

6 5 6

li

34.8 34.7 34.8

27.6 24.4 26.0

31.9 35.6 22.2 29.6 30.1 22.8 28.4 “7.1

f f f

f f f

f zt f f f zk f rk

FC

0.36 0.59 0.33

0.42 0.76 0.64

0.96 0.39 0.39 1.4 0.43 0.37 0.66 0.80

10yc

Culture Media Cont,aining Pigeon Serum~~

2.6 f 6.9 f 4.5 f

3.4 4.8 4.1 4.1 3.6 3.4 5.3 4.1

Cells Following Incubation with Normaand Hypercholesterolemic

CE

of Pigeon and Monkey Fetal Calf Serum, and

17.2 18.4 13.1 16.3 19.6 14.2 22.1 18.6 “5.9 25.1 25.5

Content Seriuu,

I

6 6

6 6

6 5 6

6 6 6

N

HCS

30.5 37.2 33.8

0.47 1.1 1.2

1.5

1.5 0.69 0.70 0.58 1.0 0.44

zt 1.6 f 1.9 f 0.55

9.2 f 16.3 f 12.7 f

8.9 f 7.7 f 7.8 * 8.1 f 6.8 f 6.1 f 16.4f2.4 30.0 +

CE

Cl\Vhite Carncau (WC) pigeon smooth muscle cells (SIIC), Show Racer (SR) pigcon SRIC, rhesus monkey SMC, and rhesus monkey skin fibroblasts (SF) were grown to confluency in culture medium containing 10 C;> fetal calf serunr (FCS) prior to the initiation of the experiment. The cells were washed twice wit,h PBS, and test media containing either lipoprotein deficient serum (LPDS) (2.5 mg protein/ml), 10cyO FCS (0.4 mg cholest,erol/ml), 1OC;;; normal pigeon serum (SC%) (0.32 mg cholesterol/ml), or 10 yc hypercholesterolemic pigeon serum (HCS) (1.84 mg cholesterol/ml) was incubated with the cells for 72 hours.

f 0.50 + 0.53 * 0.70

N

‘4 6 6

LPDR

and Esterified Cholesterol Lipoprotein Deficient

SRlC 6 22.9 f 0.80 6 19.3 + 0.72 21.1 zt 0.76

6 6 6

6 6 6

N

Rhesus monkey SF 31 6 24.2 458 6 20.1 .F 22.2

Rhesus 632 703 s

Pigeon SpttC \vc 891 I\% 600 \vc 100 s SB 977 SRI00 SR 510 f

Crll

Free

TABLE

CHOLESTEROL

10 %

hIETAROLISh1

FIG. 4. Influence of duration in cells incubated with either (NCS) pigeon strum. Cells fetal calf serum, then rinsed fibroblasts ( Rh 31) A-A

O----•

hlUSCLE

CELLS

199

NCS

lncubollon

(SR 977)

IN Sh~lOOTII

Time

with

Test

Media

(hrs

)

of incubation on accumulation of free and esterified cholsterol 10% hypercholesterolemic (HCS) or 105% normocholesterolemic were grown to confluency in culture medium containing 10% in PBS prior to addition of the test media. Rhesus monkey skin and smooth muscle cells ( Rh 703) A- - --A and Show Racer

and White

Carneau

incubated with the test media containing mg/ml) or 10% normal pigeon strum (0.30 the mean 2 SEM of three replicate cultures.

(WC 600)

O- ---0

smooth muscle cells were

10% hypercholesterolemic pigeon serum (2.05 mg/ml) for up to ITA9 hr. Each point represents

there was a greater than 28-fold increase in the cholesteryl ester conteut relative to the same cells incubated with LPDS. The change in cholesterol ester content of the other cell lines was similar qualitatively to the rhesus monkey skin fibroblasts but of a reduced magnitude, In the rhesus monkey, White Carneau, and Show Racer pigeon smooth muscle cells, there was no significant increase in the cellular cholesteryl ester content among cells incubated with LPDS, fetal calf serum, or normocholesterolemic serum, but there was a significant increase in cholesteryl ester content when incubated with hypercholesterolemic serum. The time course of cellular accumul~ation of free cholesterol and cholesteryl ester was studied by incubating monkey and pigeon cells with either hypercholesterolemic or normal pigeon serum for periods of time up to 72 hr (Fig. 4). Consistent with the trends shown in Table I, the pigcon smooth muscle cells serum

200

SMITH,

ST.

CLAIR,

AND

LEWIS

maintained on culture medium containing fetal calf serum (zero time) were found to contain less free and esterified cholesterol per mg cell protein th,an the monkey cells. Incubation with 10% normal pigeon serum produced little change in the free cholesterol content of the monkey smooth muscle cells and skin fibroblasts. In contrast, the free cholesterol content of both pigeon cell lines increased progressively for 24 and 48 hr where it plateaued. All four cell lines accumulated relatively small amounts of cholesteryl ester during the 72 h r incubation with normal serum. The monkey skin fibroblasts accumulated the largest amount of cholesteryl ester, gradually increasing their cholesteryl ester content at 72 hr to concentrations 3.5 times greater than baseline levels. In the monkey smooth muscle cells, cholesteryl ester accumulation increased for up to 12 hr where it reached a plateau that was maintained until the end of the incubation period. Both pigeon cell lines accumulated much less total cholesteryl ester than the monkey cells throughout the entire incubation period. Incubation with hypercholesterolemic serum resulted in accumulation of free cholesterol in all four cell lines. Both monkey cell lines reached a maximum cellular free cholesterol content by 8 hr. In the monkey smooth muscle cells the cellular free cholesterol content returned to baseline by 48 hr, while in the monkey skin fibroblasts the cellular free cholesterol content remained slightly elevated for the entire 72 hr. The free cholesterol content of the Show Racer cells steadily increased until reaching a peak level at 24 hr. At this time the free cholesterol content had increased 1.6-fold over the baseline level. The White Carneau cells exhibited a similar time course of free cholesterol accumulation except for an initial lag of 4 hr. After the 48 hr of incubation the White Carneau cells had accumulated 1.7 times more free cholesterol than baseline levels. All four cell lines accumulated subsbantially more cholesteryl ester following incubation with 10% hypercholesterolemic serum than when incubated with 10% normal serum. Consistent with the data in Table I, the most dramatic accumulation of cholesteryl ester occurred in the monkey skin fibroblasts. The cholesteryl ester content increased steadily throughout the 72 hr incubation. At 72 hr the cholesteryl ester content had increased to a concentration of nearly 32 pg/mg cell protein. The monkey smooth muscle cells exhibited a two-phase pattern of accumulation of cellular cholesteryl ester. Cholesteryl ester content peaked at 12 hr but had decreased by 24 hr. A seconcl phase of accumulation began at 48 hr and continued until 72 hr. The two pigeon cell lines accumulated less cholesteryl ester than the two monkey cell lines. In the Show Racer cells, cholesteryl ester content increased for 24 hr and remained elevated for the entire 72 hr. The White Carneau cells accumulated less cellular cholesteryl ester than the Show Racer cells. Cholesterol Esterificatiolt Studies In order to compare the changes in the rate of cholesterol esterification with changes in cholesterol content, four cell lines were incubated with culture medi’a containing increasing amounts of hypercholesterolemic pigeon serum, The rate of estcrifioation of [1-‘4C]oleate to cholesterol was compared with the accumulation of free and esterified cholesterol in the same cells (Fig. 5). The pattern of response of the monkey skin fibroblasts and monkey smooth muscle cells to increasing concentrations of hypercholesterolemic serum was similar except for

CHOLESTEROL

I

Rh

SMC

METABOLISM

IN

SMOOTH

MUSCLE

CELLS

703

WC

SMC

600

I

mg cholesterol

/ml

test

medlo

FIG. 5. Influence of increasing concentrations of hypercholesterolenlic serum in the culture medium on cholesterol esterification and cholesterol accumulation. Cells were grown to confluency in culture medium containing IO’$ fetal calf serum, then rinsed in PBS prior to addition of the test media. The test media contained LPDS (2.5 mg protein/ml) and from 0 to 5.0 mg/mI of cholesterol’ as hypercholesterolemic pigeon serum (total serum cholesterol concentration = 2035 m&d]). After 21 hr of incubation with the test media, the cells were pulse labeled with [I-“Cloleate for 4 hr in order to measure cholesterol esterification. Each point represents the mean ) SEM of five replicate cultures of rhesus monkey (Rh) smooth muscle cells (SMC) and skin fibroblasts (SF) and of Show Racer (SR) and White Carneau (WC) smooth muscle cells.

the much greater magnitude of response of the fibroblasts. There was a progressive increase in the free cholesterol content of the cells as the percentage of hypercholesterolemic strum in the culture medium was increased. The increase in the cholesteryl ester content was much more dramatic, however; increasing from less than 1 pg,‘mg protein to concentrations of nearly 58 pg/mg protein in fibroblasts exposed to culture medium containing 5 mg/ml cholesterol as hypercholesterolemic serum. This represented an 8%fold increase over the baseline level. The rate of esterification of [l-‘4C]oleate to cholesterol paralleled cholesteryl ester accumulation up to a media cholesterol concentration of 0.5 mgjml. At the peak of the response, cholesterol esterification had increased S-fold over control level. At culture mediwl cholesterol concentrations greater than 0.5 mg/ml

SMITH,

202

ST. CLAIR,

AND

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there was no further increase in the rate of cholesterol esterification. At concentrations of 5 mg/ml, some of the skin fibroblasts began to detach from the dish suggesting that the massive increase in the cholesterol content of the cells may have become toxic to the cells and was responsible for the drop in the rate of cholesterol esterification at this concentration. Actual detachment of cells occurred only for the skin fibroblast line at this high cholesterol concentration. Therefore, the approximately 20% hypercholesterolemic serum needed to achieve this high medium cholesterol concentration did not in itself appear toxic to the cells. The responses of the two pigeon cell lines to increasing concentrations of cholesterol as hypercholesterolemic serum were similar to each other but were different from the two monkey cell lines. The cellular free cholesterol content of both the Show Racer and White Carneau cells increased as the media cholesterol increased. Coincident with the increase in free cholesterol content was an increase in the cellular cholesteryl ester content that appeared to change in parallel with the increase in the free cholesterol content of the cells. This is in contrast to the rhesus monkey cells in which the rate of increase in the cellular cholesteryl ester content was much greater than for free cholesterol. -4lthough the cholesteryl ester content of the pigeon cells increased, the rate of incorporation of

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FIG. 6. Influence of duration of incubation on stimulation of cholesterol esterification in pigeon smooth muscle cells incubated with culture medium containing hypercholesterolemic serum following 48 hr of preincubation with LPDS. Cells were grown to confluency in 60 mm dishes in culture medium containing 10% fetal calf serum, rinsed with PBS, and incubated for 48 hr with culture medium containing LPDS (2.5 mg protein/ml). Cells were rinsed with PBS and incubated with culture medium containing LPDS (2.5 mg protein/ml) and hypercholesterolemic serimr at a concentration of 1 mg cholesterol/ml. After incubation for periods of from 4 to 8 hr the cells were pulse labeled with [l-“Cloleate for 4 hr in order to measure cholesterol esterification. Each point is the mean of duplicate cultures.

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[ l-llC]oleate into cholesterol did not parallel cholcsteryl ester accumulation. In fact, there was very little increase in the rate of cholesterol esterification in the pigeon smooth muscle cells even :tt the highest concentration of cholesterol in the culture medium. For the cholesterol esterification studies, cells were transferred directly from their normal growth medium containing 1Ojl. fetal calf serum to the test medium containing 10% normal or hypercholesterolemic sermI1 and not preincubated with LPDS. In human skin fibroblasts preincubated with LPDS there is a stimulation in the synthesis of LDL receptors so that upm the adclition of LDL there is a maximum rate ‘of receptor mediated uptake of LDL cholesterol and resulting stimulation in cholesterol esterification. Upon continued incubation with LDL there occurs an inhibition of LDL receptor synthesis that results in a decreased rate of receptor mediated uptake of LDL and a corresponding reduction in the rate of cholesterol estcrificntion (Brown and Goldstein, 1975). A similar phenoneman occurs in rhesus monkey cells in culture in that cholesterol esterification is stimulated masimumly 8 to 12 hr after addition of whole serum then declines by 24 hr to a rate less than one-half of the maximum, but still several-fold higher than the rate while incubated with LPDS (St. Clair et al., 1977). The influence of hypercholesterolemic serum on cholesterol esterification in three pigeon

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smooth muscle cell lines preincubated with LPDS is shown in Fig. 6. Addition of hypercholesterolemic serum produced an initial decline in the rate of esterification at 4 hr followed by a stimulation in cholesterol esterification that reached a maximum rate by 12 hr. Unlike mammalian cells (St. Clair et al., 1977), the pigeon smooth muscle cells did not show an inhibition of cholesterol esterification upon continued incubation with hypercholesterolemic serum. As a result, we did not preincubate cells with LPDS prior to testing the influence of different concentrations of hypercholesterolemic serum on cholesterol esterification and accumulation (Fig. 5). Also, since LDL receptor activity is thought to be suppressed in cells in situ (Ho et al., 1976), by not preincubating the cells with LPDS the influence of the hypercholesterolemic serum on cholesterol estcrification should reflect more closely the actual response of these cells in situ. In all four cell lines the amount of [I-“Cloleate incorporated into triglyceride and phospholipid decreased as the media cholesterol concentration increased (Fig. 7). All four cell lines incorporated similar amounts of [I-‘“Cloleate into phospholipid. The Show Racer and White Carneau cells, however, incorporated more [lJC]oleate into triglyceride than either of the two monkey cell lines. Figure 7 also emphasizes the difference in cholesterol esterification rates among the four cell lines. The White Carneau and Show Racer cells esterified more [1-‘“Cloleate to cholesterol than the monkey cell lines when the media contained no cholesterol. Following addition of cholesterol to the culture medium, cholesterol esterification increased dramatically in the monkey cells, hut ch’anged very little in the pigeon cells. DISCUSSION The present study describes a method for the initiation and maintenance of aortic smooth muscle cells from young pigeons utilizing culture conditions identical to those developed for culture of monkey aortic smooth muscle cells and skin fibroblasts. The cultured pigeon smooth muscle cells exhibited the ultrastructural and growth characteristics generally used to characterize mammalian smooth muscle cells in culture (Ross et al., 1971). In addition, the ultrastructural characteristics were similar to those previously described for smooth muscle cells grown from expIants of embryonic pigeon aorta ( Wight et al., 1977). Wight described the cells initially growing from pigeon aortic explants as being undifferentiated and “fibroblast-like.” However, after the cells remained in culture for several ‘days, they became more differentiated and eventually acquired the typical characteristics of smooth muscle cells, By 10 days in culture, the majority of cells were identified as smooth muscle cells. Undifferentiated cells were seldom seen after 16 to 21 days. It is unclear whether these undifferentiated cells represent the occasional “fibroblast-like” interlaminar cells that have dedifferentiated in the process of migration and growth from the explant. Nevertheless, our findings are consistent with those of Wight et al. ( 1977), and indicate that under the conditions ‘of this study the predominant cell type exhibited the ultrastructural and growth characteristics of smooth muscle cells. Previous studies with smooth muscle cells from embryonic pigeon aorta have described the occurrence of large lipid vacuoles and osmophilic inclusions in the cells from White Carneau pigeons but not in those from Show Racer pigeons (Smith et al., 1966, Wight et al., 1977). In addition, cultured White Carneau

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smooth muscle cells have been reported to contain greater aniolmts of squalene, cholesteryl ester, triglyceride and total lipid thau Show Racer cells ( Nicolosi et cd., 1972). In the present study wc tailed to observe morphoIogi~;~l or chemical differences between smooth muscle cells from White Carneau and Show Racer pigeons. Regardless of the serum to which they were exposed, smooth muscle cells from both breeds of pigeons contained similar numbers of lipid inclusions as seen by staining of the cells with Oil Red 0. This microscopic impression was supported by chemical analysis which indicated no major differences in the sterol content between the smooth muscle cells from the two breeds of pigeons. We have no explanation for our failure to observe differences in morphology and chemical composition between smooth muscle cells from White Carneau and Show Racer pigeons other than to point out that we utilized cells derived from 4 to 6-week-old animals rather than embryos ancl that the culture conditions and the duration of tissue culture differed between this and previous studies. Pigeon and monkey cells incubated with 10% hypercholesterolemic pigeon sermn accumulated more free cholesterol and cholesteryl ester than cells incubated with 10% normal pigeon serulii. Other investigators have also reported that hypercholesterolemic serum causes greater sterol accumulation in cultured cells than comparable amounts of normal serum (Bailey and Keller, 1971; Nikkari et al., 1976; Fisher-Dzoga et al., 1974). This difference is not clue solely to the higher cholesterol content of the hypercholesterolemic serum since even at equivalent cholesterol concentrations hypercholesterolemic serum is more effective than normal serum in promoting accumulation of cholesterol in cells in culture (Rates and Wissler, 1976; St. Clair and Leight, 197s; Arbogast and Rothblat, 1976). Part of this effect of hypercholesterolemic serum can be explained by the presence of low density lipoproteins of abnormal composition (St. Clair and Leight, 1978 ) . In the present study we made no attempt to determine whether there were differences in the response of pigeon smooth muscle cells to sera or lipoproteins frorn White Carneau and Show Racer pigeons, rather we were interested exclusively in possible differences between smooth muscle cells from the two breeds of pigeons in response to the same stimulus. Thus, we utilized a pool of serum derived from both breeds of pigeons. There are major differences in lipoprotein content and composition between normal and hypercholesterolemic pigeons. Noncholesterol-fed pigeons transport approximately 75’;: of their cholesterol as high density lipoprotein (HDL). Upon cholesterol feeding the absolute amount of HDL cholesterol remains relatively unchanged while the excess of the increase in plasma cholesterol concentration occurs in the lipoproteins of density < 1.063 gm/ml. These lipoproteins are composed of both LDL of abnormal size as well as cholesteryl ester-rich beta migrating VLDL (L. L. Rudel, personal commm~ication). Whether there are differences in the composition or content of these lipoproteins in White Cnrneau and Show Racer pigeons that could explain the differences in susceptibility to atherosclerosis is unknown, although total plasma cholesterol concentrations are not different between the two breeds (Clarkson et al., 1959). The monkey smooth muscle cells and skin fibroblasts and the pigeon smooth muscle cells differed in the total amount of free cholesterol and cholesteryl ester accumulated and in the time course of this accumulation. The monkey skin

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fibroblasts consistently accumulated larger quantities of free and esterified cholesterol and showed greater stimulation of cholesterol esterification than either the monkey or pigeon smooth muscle cells. This observation of a greater response by skin fibroblasts relative to smooth muscle cells to normal and hyperhpemic serum is generally consistent with reports by others (Stein et al., 1975; Bierman and Albers, 1977). Because of the inherent variability in response among cell lines, even of the same type, it is difficult to know whether fibroblasts are consistently more responsive to hyperlipemic serum than are smooth muscle cells or whether the observed differences in response simply reflect the normal variability among cell lines, To answer this question conclusively will require the comparison of several lines of skin fibroblasts and smooth muscle cells obtained from the same individuals which are subsequently exposed to identical test conditions. A consistent finding of this study was the greater magnitude of response to normal and hypercholesterolemic serum of the monkey smooth muscle cells relative to the pigeon smooth muscle cells. In all cases the monkey cells accumulated more cholesteryl esters than did the pigeon cells and, based on the characteristics of the ‘accumulation, the monkey cells appeared to accumulate cholesteryl esters by a different mechanism. In response to both normal and hypercholesterolemic serum, there was a greater increase in the free cholesterol coutent of the pigeon smooth muscle cells relative to the monkey cells. On the other hand, the monkey cells were able to increase their cholesteryl ester content in response to much lower concentrations of normal and hypercholesterolemic serum, while relatively minor increases in the cholesteryl ester content were observed in the pigeon smooth muscle cells, unless exposed to very high concentrations of hypercholesterolemic serum. This increase in cholesteryl ester content in the monkey cells was associated with a stimulation in cholesterol esterification. No such stimulation ‘occurred in the pigeon cells. This suggests that cholesterol esterification may play a major role in regulating the cholesteryl ester content of cells. This conclusion is consistent with studies by other investigators (Rothblat et al., 1976; Bates and Wissler, 1976). The relatively small amount of cholesteryl ester accumulation in pigeon smooth muscle cells appeared to be the result of a different mechanism than that which accounted for the cholesteryl ester accumulation in monkey cells. Since there was no stimulation of cholesterol esterification in the pigeon cells and the increase in the cellular concentration of free and esterified cholesterol paralleled one another, it is possible that simply bulk phase pinocytosis may have been responsible for the increase in the cholesterol content of the pigeon cells. This is in contrast to the monkey cells in which the increase in the cholesteryl ester occurred at a much greater rate than for free cholesterol. Therefore, it appears likely that while some process such as esterification of cholesterol was responsible for actively increasing the cholesteryl ester content of the monkey cells, no such process was involved in increasing the cholesteryl ester content of the pigeon cells. Although studies comparing the extent of atherosclerosis in pigeons have shown that White Carneau pigeons develop more severe atherosclerosis than Show Racer pigeons, the present study revealed no consistent differences between cultured aortic smooth muscle cells from Show Racer and White Carneau pigeons in their metabolic response to normal or hypercholesterolemic serum. The ab-

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sence of a stimulation in cholesterol esterification in the pigeon smooth muscle cells incubated with hypercholesterolemic serum was unespected due to the substantial increase in cholesterol esterification (up to lOO-fold) previously shown to occur in atherosclerotic pigeon aortas (St. Clair et al., 1970). The discrepancy between these two observations emphasizes the fact that the spectrum of biochemical changes seen in the atherosclerotic artery cannot be reproduced simply by exposure of cultured arterial smooth muscle cells to hyperlipemic serum. Unlike smooth muscle cells in culture, cholesterol esterification and cholesteryl ester accumulation were stimulated utilizing organ cultures of normal pigeon aorta exposed to pigeon serum for up to 9 days (St. Clair and Harpold, 1975). It is interesting to speculate that, as a result of the passage of serum through the extracellular matrix of the arterial wall, serum lipoproteins may be altered so that they are taken up by the smooth muscle cells in higher concentrations, perhaps analogous to cationized low density lipoproteins (Goldstein et a?., 1977). Thus, smooth muscle cells iu culture having a much less developed extracellular matrix than the whole artery may not respond to normal or hyperlipemic serum in a manner similar to the whole artery. Such an hypothesis could also provide an explanation for our failure to show any differences in the accumulation of cholester)ll esters or stimulation of cholesterol esterification between smooth muscle cells from the atherosclerosis susceptible White Carneau and atherosclerosis resistant Show Racer pigeons. ACKNOWLEDGMENT The authors gratefully acknowledge the excellent technical assistance of Patricia Hester, Grayce Greene, Molly Leight and Richard Taylor. This work was supported by a National Heart, Lung, and Blood Institute grant (SCOR) HL-14164 and by an Institutional National Research Service Award, HL 07115.

REFERENCES ARBOCAST, L. Y., ROTHBLAT, G. H.,

LESLIE, M. H., and CO~PEH, R. A. (1976). Cellular cholesterol ester accumulation induced 1,~ free cholesterol-rich lipid dispersions. ~roc. eat/. Acad. Sci. 73, 36803684. BAILEY, P. J., and KELLER, D. ( 1971). The deposition of lipids from serum into cells cultured in vitro. Atherosclerosis 13, 333-343. BATES, S. R., and WISSLEH, R. W. (1976). Effect of hyperlipemic serum on cholesterol accumulation in monkey aortic media cells. Biochim. Biophys. Acta 450, ‘i&-88. BIERMAN, E. L., and ALUERS, J. (1977). Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells. Biochim. Biophys. Acta 488, 152-160. BLIGH, E. G., and DYER, W. J. (1957). A rapid method of total lipid extraction and purification of total lipids from animal tissues. J. Biol. Chern. 226, 497-509. BHOWN, M. S., and GOLDSTEIN, J. L. (1975). Regulation of the activity of the low density lipoprotein receptor in human fibroblasts. Ccl1 6, 307316. CLARKSON, T. B., PRICHARD, R. W., NETYKY, M. G., and LOFLAND, H. B. (1959). Atherosclerosis in pigeons. Its spontaneous occurrence and resemblance to human atherosclerosis. Arch. Path. 68, 143-147. DAY, A. J., and PROUDLOCK, J. W. (1974). Changes in aortic cholesterol-esterifying activity in rabbits fed cholesterol for three days. Atlwro.dero.ris 19, 2Fj3-21jtL

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FISIIER-DZOGA, K., CHEN, R., and WUSLEI~, R. W. (1974). Effects of serum lipoproteins on the morphology, growth, and metal)olism of arterial smooth muscle cells. 111 Arterial hlcsenchyme and Atherosclerosis (W. I>. Wa~ncr and T. B. Clarkson, cds.), pp. 299-31 1, I’lenun~ Press, New York. GOLDSTEIN, J. L., ANDERSON, 1~. G. W., BuJ,~, L. M., BASU, S. K. and BROWN, M. S. ( 1977). Overloading hm~~an aortic smooth muscle cells with low density lipoprotein-cholesteryl esters reproduces features of atherosclerosis in vitro. 1. Clin. Inoest. 59, 1196-1202. GUERTLER, L., and ST. CLAIR, R. W. (1977). In zjitro regulation of cholesterol metabolism by low density lipoproteins in skin fibroblasts from hypoand hyperresponding squirrel monkeys. Biochim. Biophys. Acta 487, 458-471. Ho, Y. K., BROWN, M. S., BILHEIMER, D. W., and GOLDSTEIN, J. L. (1976). Regulation of low density lipoprotein receptor activity in freshly isolated human lymphocytes. 1. Clin. Inoest. 58, 1465-1474. ISHIKA~A, T. T., MACGEE, J., hlomusonr, J. A., and GLUECX, C. J. (1974). Quantitative analysis of cholesterol in 5 to 20 pl of plasma. 1. Lipid Res. 15, 286-291. LOWRY, 0. H., ROSEBHOUGH, N. J., FAHR, A. L., and RANDALL, R. J. ( 1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. LUFT, J. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. cytoz. 9, 409414. NICOLOSI, R. J., SANTEHRE, R. F., and ShlrTH, S. C. (1972). Lipid accumulation in muscular foci in White Carncau and Show Racer pigeon aortas. Exp. Mokc. Puthol. 17, 2937. NIKKARI, T., PIETIL;~, K., and SALO, M. (1976). Increased cholesterol content and esterification in rabbit aortic medial cells cultured in hyperlipidemic serum. Med. Biol. 54, 264-271. Ross, R. (1971). The smooth muscle cell. II. Growth of smooth muscle in culture and formation of elastic fibers. J. Cell. Biol. 50, 172-186. ROTHBLAT, G. H., ARBOGAST, L., KMTCHEVSKY, D., and NAFTULIN, M. (1976). Cholesteryl ester metabolism in tissne culture cells: II. Source of accumulated esterified cholesterol in Fu 5AH rat hepatoma cells. Lipids 11, 97-108. RUSH, R. L., LEON, L., and TUMSELL, J. (1971). Automated simultaneous cholesterol and triglyc-ride determination on the AutoAnalyzer II. In Advances in Automated AnalysisTechnicon International Congress, 1970 (E. C. Barton et al., eds.), Vol. 1, pp. 503-507. Thurman Associates, Miami, Fla. SMITH, S. C., STROUT, R. G., DUNLOP, W. R., and SMITH, E. C. (1966). Mitochondrial involvement in lipid vacuole formation in cultured aomrtic cells from White Cameau pigeons. J. Athero. Res. 6, 489-496. ST. CLAIR, R. W. (1976). Metabolism of the arterial wall and atherosclerosis. In Atherosclerosis Reviews (R. Paoletti and A. M. Gotto, Jr., eds.), Vol. 1, pp. 61-117, Raven Press, New York. ST. CLAIR, R. W., and HAI~POLD, G. J. (1975). Stimulatioa of cholesterol esterification in vitro in organ cultures of normal pigeon aorta. Exp. Molec. Pothol. 22, 207-219. ST. CLAIR, R. W., LOFLAND, H. B., and CLARKSON, T. B. (1970). Influence of duration of cholesterol feeding on esterification of fatty acids by cell-free preparation of pigeon aorta. Studies on the mechanism of cholesterol esterification. Circ. Rcs. 27, 213-225. ST. CLAIR, R. W., SMITII, B. P., and WOOD, L. (1977). Stimulation of cholesterol esterification in rhesus monkey arterial smooth muscle cells. Circ. Res. 40, 166-173. effects of isolated lipoproteins from ST. CLAIR, R. W., and LEI~HT, M. A. (1978). D’ff1 erential normal and hypercholesterolcmic rhesus monkeys on cholesterol esterification and accumulation in arterial smooth muscle cells in culture. Biochim. Biophys. Acta 530, 279-291. STEIN, O., and STEIN, Y. ( 1975). Comparative uptake of rat and human serum low-density and high-density lipoproteins by rat aortic smooth muscle cells in culture. Circ. Res. 36, 436-443. WIGHT, T. N., Coo=, P. H., and S~~ITH, S. C. (1977). A n electron microscopic study of pigeon aorta cell cultures. Cytodifferentiation and intracellular lipid accumulation. Exp. Molec. Pathol. 27, 1-18.