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Synthesis and removal of different cholesterol esters by aortic smooth muscle cells in culture
483
Atherosclerosis, 26 (1977) 463-492 0 Elsevier/North-Holland Scientific
SYNTHESIS BY AORTIC
ALLAN
of Physiology,
(By invitation,
Ltd.
AND REMOVAL OF DIFFERENT CHOLESTEROL SMOOTH MUSCLE CELLS IN CULTURE *
J. DAY and MARION
Department
Publishers,
received
ESTERS
SHEERS
University of Melbourne,
November,
Parkuille, Victoria (Australia)
1976)
Summary
The incorporation of 3H-labelled oleic acid and of 14C-labelled linoleic acid into phospholipid, triglyceride and cholesterol ester in smooth muscle cells grown in incubation medium supplemented with either 5% normal or 5% hyperlipemic serum has been studied. Both fatty acids were incorporated into cholesterol esters to a greater extent when cells grown in incubation medium containing hyperlipemic serum. Oleic acid was incorporated into cholesterol esters in preference to linoleic acid. The addition of hyperlipemic serum to the incubation medium did not increase the incorporation of either 3H-labelled oleic acid or of “C-labelled linoleic acid into phospholipid or triglyceride. The removal of labelled lipid fractions has also been followed for four days in cells pulse labelled for 24 hours with 3H-labelled oleic acid and 14C-labelled linoleic acid. Both 3H- and 14C-labelled cholesterol esters were removed more rapidly when the smooth muscle cells were grown in medium containing normal serum than in medium containing hyperlipemic serum. The removal of both phospholipid and triglyceride was similar in normal and hyperlipemic serum. Comparison of the 3H/14C ratio indicated that the cholesterol oleate and cholesterol linoleate were removed at similar rates. Key words:
Atherogenesis - Cholesterol Triglyceride - Tissue culture
ester - Phospholipid
-Smooth
muscle
cells -
Introduction
Early atherogenesis is characterised by the proliferation smooth muscle cells and their conversion to lipid containing
of intimo-medial foam cells. Studies
This paper is dedicated to Professor F.G. Schettler in the occasion of his sixtieth birthday. * This work was supported by grants from the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia.
484
on arterial wall metabolism have demonstrated the presence of cholesterolesterifying enzymes in developing lesions [2--51 and this activity has been localised to the foam cells present [3,6]. Foam cells isolated from atherosclerotic lesions have been found to have marked cholesterol-esterifying activity [7] and homogenates of such cells shown to contain two distinct cholesterol-esterifying enzymes [8]. The foam cell appears therefore to play a significant role in the accumulation of cholesterol ester in the early lesion. Aortic smooth muscle cells from a variety of experimental animals have been grown in tissue culture [9,10] and it has been demonstrated that plasma low density lipoprotein promotes both cell growth and the intracellular accumulation of lipid [ 11,121. Smooth muscle cells grown from normal aortic explants in tissue culture in medium supplemented with normal serum do not demonstrate significant synthesis of cholesterol ester [13]. Where low density lipoprotein is added to the incubation medium however, cholesterol-esterifying activity is markedly increased [12]. These cells provide therefore a model system for studying atherogenesis at the cell level and for determining the mechanisms involved in the accumulation and removal of cholesterol ester from the early atherosclerotic lesion. In the present paper, the synthesis of cholesterol esters by smooth muscle cells in tissue culture has been further studied using 3H-labelled oleic acid and “C-labelled linoleic acid as precursors for cholesterol ester synthesis. In addition the removal of cholesterol oleate and of cholesterol lineolate has been investigated in order to determine whether the accumulated cholesterol ester can be removed from the cells by altering the lipoprotein content of the incubation medium. Materials and Methods Tissue culture methods Normal rabbits, 2-6 weeks of age, were killed by ether anaesthesia and their thoracic aortas carefully removed under sterile conditions. Adventitial connective tissue was removed and the aortas stripped into two layers. The inner layer (containing the intima and most of the media) was reserved for preparation of the tissue culture explants. The outer layer containing some media and the adventitia was discarded. Explants, approximately l-2 mm square, were plated into Falcon flasks. Three to four ml of incubation medium (Basal Eagle’s medium) containing 10% foetal calf serum was added and the flasks incubated at 37°C in a COZ incubator. Cell growth appeared within a week of primary culture and the cells were allowed to grow for a further period of approximately one week. At this stage the explants and new cells were removed by trypsinisation (3 ml of 0.25% trypsin - EDTA solution). The cells were centrifuged and resuspended in Basal Eagle’s medium containing 10% foetal calf serum and plated into Leighton tubes. After l-2 weeks when growth was confluent, the incubation medium was removed and replaced with incubation medium containing radioactively labelled fatty acids as set out below. Electron microscopy of the cells indicated differentiated smooth muscle cells with adequate evidence of myofibrils. Experimental procedure Two experimental procedures
were carried
out. In the first, duplicate
tubes
485
were incubated in Basal Eagle’s medium supplemented with 5% foetal calf serum and either 5% hyperlipemic rabbit serum (obtained from rabbits fed 1% cholesterol for 3-4 months) or 5% normal rabbit serum. A known amount of 14C-labelled linoleic acid (approximately 1.5 /Xi and 3H-labelled oleic acid (approximately 3 PCi) was added. After 24 h incubation, the medium was removed, the cells washed with 0.9% sodium chloride solution and their lipid extracted by chloroform-methanol (2 : 1, v/v) as described by Folch [ 141. In the second series of experiments cells were prelabelled in an incubation medium containing Basal Eagle’s medium supplemented with 5% foetal calf serum and 5% hyperlipemic rabbit serum, to which had been added a known amount of 3H-labelled oleic acid (approximately 3.0 FCi) and 14C-labelled linoleic acid (approximately 1.5 @i). After 24 h the cells were washed, a duplicate set of tubes taken down for immediate lipid extraction and the remainder divided into two groups. To one group was added incubation medium containing Basal Eagle’s medium, 5% foetal calf serum and 5% hyperlipemic rabbit serum and to the other Basal Eagle’s medium, 5% foetal calf serum and 5% normal rabbit serum. Duplicate flasks were taken down at daily intervals from each of the two groups for four days. The cells were washed with 0.9% sodium chloride solution and the lipid extracted with chloroform methanol as described above. Radioactive
isotopes
[9,10-3H]Oleic acid (specific activity 2.5 Ci/mmol) and [l-‘4C]linoleic acid (specific activity 56 mCi/mmol) were obtained from the Radiochemical centre Amersham, U.K. Prior to use they were taken up in 0.5 N NaOH and added to serum prior to inclusion of the latter in the incubation medium. Thin layer chromatography
After initial counting, aliquots of the lipid extracts were separated into phospholipid, cholesterol, free fatty acid, triglyceride and cholesterol ester by thin layer chromatography on silica gel G using the solvent system n-hexane : ether : acetic acid (112:38:3). Internal standards were added to visualise the separated lipid and the identified lipids were scraped directly into counting vials for counting of the 3H, 14C content using the dioxane : water scintillator described by Snyder [15]. Double labelled techniques using a Packard Tricarb Model 3375 were employed. Chemical
analyses
Lipid phosphorus was assayed in the lipid extracts by the micro method of Bartlett [16]. Cholesterol and cholesterol ester were assayed by densitometry using a Schoeffel SD 3000 spectrodensitometer. Details were described previously [17]. Results The incorporation of 3H-labelled oleic acid and 14C-labelled linoleic acid into various lipid fractions in smooth muscle cells grown for 24 h in the presence of either normal or hyperlipemic serum is shown in Table 1. Data is presented as
486 TABLE,1 EFFECT OF HYPERLIPEMIC SERUM ON THE INCORPORATION LINOLEIC ACID INTO VARIOUS LIPID FRACTIONS IN SMOOTH CULTURE
OF 3H-OLEIC MUSCLE CELLS
AND 14CIN TISSUE
Data is expressed as dpm X.10-3/106 dpm in the incubation medium except where figures in parenthesis are shown. These give the specific activity (dpm X 10-3/1~6 dpm in incubation medialpg Pi or /pg cholesterol ester--cholesterol). 14C-Linoleic __~
3 H- Oleic NOrmal
Hyperlipemic
18’7 151
145 101
Triglyceride Cholesterol-ester
(4.58) 28 3.2
(3.37) 30 10.0
392 313 (9.48) 63 4.8
CE/PL ratio
(0.42) 0.02
(1.82) 0.10
(0.62) 0.015
294 202 (6.73) 12 14.7 (2.67) 0.07
122 96 (4.40) 11 11.3 (2.13) 0.12
94 15 (3.83) 13 3.9 (0.54) 0.05
98 15 (3.44) 13 8.3 (1.57) 0.11
Kxperimen t II Total lipid Phospholipid
Experiment
Hyperlipemic
III
Total lipid Phospholipid Triglyceride Cholesterol ester CE/PL ratio Experiment
NOrmal
105 85 (3.83) 11 5.5 (0.76 ) 0.06
IV
Total lipid Phoshpolipid Triglyceride Cholesterol ester CE/PL ratio
216 148 52 6.1 0.04
186 112 49 16.2 0.145
315 197 98 6.2 0.03
264 149 88 16.3 0.110
dpm X 10m3 incorporated into the respective lipid fractions in relation to the fatty acid present in the incubation medium. In two of the three experiments shown, the specific activities of the phospholipid and cholesterol ester fractions were calculated and this data is given in parenthesis in Table 1. The presence of hyperlipemic serum in the incubation medium did not appreciably influence the incorporation of either oleic or linoleic acid into phospholipid or triglyceride in the cells. The incorporation of both oleic and linoleic acid into cholesterol ester however, was markedly increased when hyperlipemic serum was present in the incubation medium. The cholesterol ester/phospholipid ratio is also presented in Table 1. This fraction relates the incorporation of the respective fatty acid into cholesterol ester to that into phospholipid. The presence of hyperlipemic serum resulted in an increase in this cholesterol ester phospholipid ratio by 2-5 times that shown when the cells were grown in medium supplemented with normal serum. In order to compare the incorporation into the various lipid fractions of 3Hlabelled oleic acid with that of 14C-labelled linoleic acid, the 3H/‘4C ratio for each fraction was calculated and this data is shown in Table 2. For both normal and hyperlipemic serum and in all experiments the 3H/14C ratio for the triglyceride fraction is appreciably less than that for phospholipid and the 3H/‘4C
487
TABLE
2
3H-OLEIC/14C-LINOLEIC CELLS AFTER 24 HOURS
ACID RATIO INCUBATION
OF VARIOUS LIPID IN TISSUE CULTURE
3H/14C ratio is expressed each experiment.
as a percentage
of the ratio for the phospholipid
Experiment
Phospholipid Triglyceride Cholesterol ester
I
Experiment
II
FRACTIONS
Experiment -___
IN SMOOTH (hyperlipemic)
III
MUSCLE fraction
Experiment
for
IV
Hyperlipemic
Normal
hyperlipemic
Normal
Hyperlipemic
Normal
Hywrlipemic
100 87 132
97 88 136
100 84 138
88 64 109
100 68 105
100 71 141
100 74 131
ratio for cholesterol ester is appreciably more than that for phospholipid. It is apparent therefore that oleic acid is preferentially incorporated into cholesterol ester and linoleic acid preferentially incorporated into triglyceride compared with their incorporation into phospholipid in the cells. The removal of the labelled lipid fractions from smooth muscle cells in culture in the presence of either hyperlipemic or normal serum is shown in Fig. 1 and 2 for one representative experiment. There were differences in relative incorporation into lipid fractions between different experiments, but the pattern of removal for each was similar in all experiments and is represented by
o--oHYPERLIPEMIC x--xNOAMOLlPEMlC IM
dpm.lO-‘/lO’dpm
dpm x IO-‘/W
dpm I.M
100
0-o HYPERLIPEMIC x---xNORMOLlPEMlC
SERUM SERUM
I
I \a
L-.
1
2
3
4 TlMEtdays)
‘H-CHOLESTEROL
ESTER
G-T-T--
5
‘H-TRIGLYCERIDE
5
1
TIMEldays)
Fig. 1. Removal of phospholipid and triglyceride labelled with -‘H-oleic acid from smooth muscle cells grown in medium supplemented with either normal or hyperlipemic serum. Calculated linear regression lines based on semllogarithmic plots are presented. Fig. 2. Removal of cholesterol ester labelled with 3H-oleic acid from smooth muscle cells grown in tissue culture in medium supplemented with either normal or hyperlipemic serum. Semllogaritbmic transformation of data and calculation of linear regression lines has been carried out.
488
the data given in these figures. The removal of 3H-labelled phospholipid and triglyceride over the five day period studied was similar whether normal or hyperlipemic serum was added to the incubation medium (Fig. 1). The removal of the 3H-fatty acid labelled cholesterol ester, however, was increased by changing the supplement in the incubation medium from hyperlipemic serum to normal serum (Fig. 2). In order to obtain comparable data for the various experiments the fractional half time (ti) was calculated for each of the labelled lipid fractions and this data is shown in Table 3. The t$ was obtained from the slope of the removal line following log transformation of the data at each of the five time intervals. The significance of fit was determined by analysis of variance and the slope of the line and its intercept obtained from the linear regression equation. Comparison of the slopes of the lines was then made‘ by Student’s t-test for each of the lipid fractions for the individual experiments. There was no significant difference between the slopes of the removal lines for either 3H-oleic or 14C-linoleic labelled phospholipid or triglyceride when normal serum was substituted for hyperlipemic serum. There was, however, a statistically significant increase in the slope of the removal line for both 3H-oleic and “C-linoleic labelled cholesterol ester when normal serum was substituted for hyperlipemic serum in two of the experiments reported. In the other two experiments a trend was apparent but was not statistically significant.
TABLE
3
EFFECT
OF NORMAL
LIPID FRACTIONS
AND HYPERLIPEMIC
FROM
SMOOTH
SERUM
MUSCLE
CELLS
ON THE REMOVAL IN TISSUE
TIME (t;)
OF LABELLED
CULTURE
k$ (days) has been calculated from fitted exponential decay curves based on incorporation (dpm X 10m3 / lo6 dpm -incubation medium) at each of the 6 time intervals. Calculated lines of best fit which were not statistically significant by analysis of variance are indicated by italicizing the relevant t$. 3 H-Oleic
Experiment
a-a
Hyperlipemic
1.80 1.88 1.57
b
1.81 1.39 4.62
b
1.83 2.05 1.78
a
1.90 1.33 5.47a
a
5.80 1.07 3.60
a
3.68 1.04 1.18
a
4.97 1.07 4.44
6.66 1.06 1.07 2.42 1.86 2.75
2.33 1.61 63.3
2.08 I .40 2.44 ’
2.47 0.99 1.81
1.48 0.86
2.22 0.91
1.94
1.99
1.94 1.24 21.6 ’
IV
Phospholipid Triglyceride Cholesterol ester P < b-b P < cP < All other
Normal
III
Phospholipid Triglyceride Cholesterol ester Experiment
Hyperlipemic
II
Phospholipid Triglyceride Cholesterol ester Experiment
NOrmal I
Phospholipid Triglyceride Cholesterol ester Experiment
14 C-Linoleic
0.01 0.02 by Student’s t-test. 0.05 1 comparisons of slope (normal
vs hyperlipemic)
were not statistically
1.60 0.83 2.34
significant,
a
489 TABLE 4 EFFECT OF NORMAL AND HYPERLIPEMIC SERUM ON THE REMOVAL TIME (t;) OF LABELLED LIPID FRACTIONS FROM SMOOTH MUSCLE CELLS IN TISSUE CULTURE t$ (days) has been calculated from fitted exponential decay curves based on specific activity data. Calculated lines of best fit which were not statistically significant by analysis of variance are indicated by it&eking the relevant th .
3H-Oleic
Exmrimen
--
i4 C-Linoleic
-
Hyperlipemic
Normal
Hyperlipemic
3.63 0.97 1.64 a
2.00 0.83 0.19 a
3.29 0.96
1.33
2.10 1.40 1.86
1.66 1.09 2.45
t II
Phospholipid Triglyceride Cholesterol ester Experiment III Phospholipid Triglyceride Cholesterol ester
. 2.64 0.89 0.14 a 2.44 1.83 2.03
1.41 2.64
--._ I-
1.80 a
a-a P < 0.02 by Student’s t-test. All other comparisons of slope (normal vs hyperlipemic) were not statistically significant. SH-Oleic/‘4C-Linolaic
ratio
M
Hyperlipemic N0Ullal
.---.
I
---l------_~__---__r______. +
100
Cholesterol Ester
50
100 ____r
---T
__--
-__ T Trtglyceride
50 -
Photpholipid
Time Idays)
Fig. 3. 3H-oleic/14C-linoleic acid ratios (X100) for cholesterol ester, phospholipid and triglyceride fractions in relation to time in smooth muscle cells grown in tissue culture in medium supplemented with normal or hyperlipemic serum. Cells have been pulse labelled with labelled fatty acids for 24 h prior to commencement of the removal study. - Mean of 4 experiments _+SEM is shown.
490
In two of the four experiments reported, chemical analyses were possible and the specific activity of the phospholipid, triglyceride and cholesterol ester obtained. This specific activity data was used to calculate the removal times (ti) for these two experiments and this data is shown in Table 4. These times have been obtained in a similar way to those described for Table 3. When the slopes of these lines were compared statistically by Students t-test, there was no significant difference between normal and hyperlipemic serum added to the incubation medium with regard to the removal of the labelled phospholipid or of the labelled triglyceride. For cholesterol ester, however, the removal was accelerated by the substitution of normal for hyperlipemic serum in the incubation medium. This shows statistical significance for experiment II 03 < 0.02), but does not attain statistical significance for experiment III (Table 4). The relative removal (over the five day period) of 3H-oleic and 14C-linoleic acid incorporated into phospholipid, triglyceride and cholesterol ester, is shown in Fig. 3. The 3H/‘4C ratio for the three lipid fractions in the presence of either normal or hyperlipemic serum has been plotted in relation to time. The 3H/14C ratio does not change appreciably with time over the five day period studied for any of the three lipid fractions, indicating that the removal rates of the two fatty acids incorporated into phospholipid, triglyceride and cholesterol ester are similar. Three way analysis of variance with time, treatment, experiment indicated that there was no significant difference either with treatment or with time for the 3H/‘4C ratio for any of the lipid fractions studied. Discussion The effect of hyperlipemic serum on cholesterol ester synthesis in smooth muscle cells in culture is in agreement with findings reported by other investigators [ 141. In the present work a similar increase in synthesis occurred whether 3H-labelled oleic acid or 14C-linoleic acid were used as precursors for cholesterol ester. In early atherosclerotic lesions in both man and experimental animals the major cholesterol ester which accumulates is cholesterol oleate [l&19] and in vitro metabolic work carried out using atherosclerotic arteries isolated from atherosclerotic lesions have indicated that labelled oleic acid is incorporated more readily than other fatty acids into cholesterol ester in these preparations [20]. In the smooth muscle cells studied in the present paper, oleic acid was preferred to linoleic acid for cholesterol ester synthesis. This was true whether cells were synthesising small amounts of cholesterol ester or stimulated by exposure to hyperlipemic serum. The removal of labelled lipid fractions from the cells occurred relatively rapidly over the four day period studied. There was some variation in the ti for the various lipid fractions in different experiments as indicated by the data given for these experiments in Tables 3 and 4. However, a consistent pattern for removal between different lipid fractions was apparent in all experiments. Triglyceride was removed rapidly from the cells with a t3 from one to two days. The substitution of normal serum for the hyperlipemic supplement in the incubation medium did not alter the removal rate of the labelled triglyceride. Phospholipid removal was somewhat more variable in the cells but in each experiment was removed more slowly than the triglyceride with a ti from 1.48 to
491
6.66 days. The latter high figures however, were recorded in only one experiment and in the other three experiments a G of 1.48 to 2.47 days were observed. Again no effect of changing the cholesterol content of the incubation medium was apparent. In contrast, cholesterol ester showed a relatively long half life when the hyperlipemic serum supplement was continued throughout the four day removal period. These values varied fairly widely between experiments but were higher than those found for triglyceride and phospholipid. In contrast however, where the hyperlipemic supplement was changed to a normal serum supplement the removal time for cholesterol ester was reduced to values similar to those shown for phospholipid and triglyceride. It seems therefore that the fractional turnover time of cholesterol ester, but not of phospholipid or triglyceride can be reduced by changing the cholesterol content of the incubation medium. Removal of cholesterol ester, as well as its deposition, might therefore be expected in cells grown in tissue culture if they are subjected to a suitable regression regimen. The fractional turnovers of cholesterol oleate and of cholesterol linoleate were similar with both showing a similar reduction in ti when t.he hyperlipemic medium was changed to a normal serum supplement. Examination of the 3H/ 14C ratio showed little change throughout the course of the experiment for any of the lipid fractions studied. It can be concluded therefore that the cholesterol oleate and cholesterol linoleate are being removed from the cells at similar rates, as has been observed for arterial wall incubated in vitro [20]. Any cholesterol oleate accumulation which occurs in stimulated smooth muscle cells appears therefore to result from its increased synthesis in the cells rather than its reduced removal in relation to other cholesterol esters formed. Acknowledgements We are indebted to Dr. John Hurley, Department of Pathology, University of Melbourne for assistance with electron microscopy. We are also indebted to Mr. Dennis Vickery, Mrs. Jill Duda and Miss Margaret Ackland for technical assistance. References 1 G&z, G.S., Vesselinovitch, D. and Wissler. R.W.. A dynamic pathology of atherosclerosis, Amer. J. Med., 46 (1969) 657. 2 Lofland, H.B. and Clarkson, T.B.. Certain metabolic patterns of atheromatous pigeon aortas, Arch. Path.. 80 (1965) 291. 3 Day, A.J. and Wahlqvlst. M.L., Uptake and metabolism of 14C-labeled oleic acid by atherosclerotic lesions in rabbit aorta, Circ. Res., 23 (1968) 779. 4 St. Clair, R.W.. Lofland, H.B. and Clarkson, T.B., Influence of atherosclerosis on the composition, synthesis and esterification of lipids in aortas of squirrel monkeys (Saimb’i sciureus). J. Atheroscler. Res.. 10 (1969) 193. 5 Proudlock. J.W. and Day, A.J., Cholesterol esterlfying enzymes of atherosclerotic rabbit intima, Biochim. Biophys. Acta, 260 (1972) 716. 6 Wahlqvist, M.L.. Day, A.J. and Tome, R.K.. Incorporation of oleic acid into lipid by foam cells in human atherosclerotic lesions, Circ. Res. 24 (1969) 123. 7 Day, A.J. and Tome. R.K., In vitro incorporation of 14C-labelled oleic acid into combined lipid by foam cells isolated from rabbit atheromatous lesions, J. Atheroscler. Res., 9 (1969) 141. 8 Proudlock, J.W.. Day. A.J. and Tume, R.K., Cholesterol-esterifying enzymes of foam cells isolated form atherosclerotic rabbit intima, Atherosclerosis, 18 (1973) 451.
492 9 Jarmolych, proliferation 10 11 12 13 14 15 16 17
18 19
20
J., Daoud, A.S., and production
Landau, J.. Fritz, K.E. of mucopolysaccharides,
and McElvene, R., Aortic media explants, cell collagen and elastic tissue, EXP. Mol. Path.,
9 (1968) 171. Ross, R., The smooth muscle cell, Part 2 (Growth of smooth muscle in culture and formation of elastic fibers). J. Cell Biol.. 50 (1971) 172. Dzoga, K.. Wissler, R.W. and Vesselinovitch, D., The effect of normal and hyperlipemic low density lipoprotein fractions on aortic tissue culture cells, Circulation, 43 and 44 (Suppl. II) (1971) 6. St. Cl&, R.W.. Metabolism of the arterial wall and atherosclerosis. In: R. Paoletti and A.M. Gotto, Jr. (Eds.), Atherosclerosis Reviews, Vol. 1, Raven Press, New York. 1976, p. 61. Day, A.J., Lipid metabolism by rabbit aortic intimal and medial cells in tissue culture. Virchows Arch. A. Path. Anat. and Histol., 362 (1974) 89. Folch. J., Lees, M. and Sloane-Stanley, G.H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. Snyder, F., Radioassay of thin-layer chromatograms - A high-resolution zonal scraper for quantitative Cl4 and H3 scanning of thin-layer chromatograms, Anal. Biochem.. 9 (1964) 183. Bartlett, G.R.. Phosphorus assay in column chromatography. J. Biol. Chem.. 234 (1959) 466. Bretherton, K.N., Day, A.J. and Skinner. S.L., The effect of renal hypertension on the regional deposition of cholesterol and phospholipid in the aorta of normally- and cholesterol-fed rabbits, Atherosclerosis, 22 (1975) 517. Smith, E.B., The influence of age and atherosclerosis on the chemistry of aortic intima. J. Atheroscler. Res.. 5 (1965) 224. Peterson, M., Day, A.J.. Tume. R.K. and Eisenberg. E.. Ultrastructure, fatty acid content, and metabolic activity of foam cells and other fractions separated from rabbit atherosclerotic lesions, Exp. Mol. Path., 15 (1971) 157. of different fatty acids into combined Day, A.J., Wahlqvist. M.L. and Tume, R.K., Incorporation lipids in rabbit atherosclerotic lesions, Atherosclerosis, 12 (1970) 253.
Atherosclerosis, 26 (1977) 463-492 0 Elsevier/North-Holland Scientific
SYNTHESIS BY AORTIC
ALLAN
of Physiology,
(By invitation,
Ltd.
AND REMOVAL OF DIFFERENT CHOLESTEROL SMOOTH MUSCLE CELLS IN CULTURE *
J. DAY and MARION
Department
Publishers,
received
ESTERS
SHEERS
University of Melbourne,
November,
Parkuille, Victoria (Australia)
1976)
Summary
The incorporation of 3H-labelled oleic acid and of 14C-labelled linoleic acid into phospholipid, triglyceride and cholesterol ester in smooth muscle cells grown in incubation medium supplemented with either 5% normal or 5% hyperlipemic serum has been studied. Both fatty acids were incorporated into cholesterol esters to a greater extent when cells grown in incubation medium containing hyperlipemic serum. Oleic acid was incorporated into cholesterol esters in preference to linoleic acid. The addition of hyperlipemic serum to the incubation medium did not increase the incorporation of either 3H-labelled oleic acid or of “C-labelled linoleic acid into phospholipid or triglyceride. The removal of labelled lipid fractions has also been followed for four days in cells pulse labelled for 24 hours with 3H-labelled oleic acid and 14C-labelled linoleic acid. Both 3H- and 14C-labelled cholesterol esters were removed more rapidly when the smooth muscle cells were grown in medium containing normal serum than in medium containing hyperlipemic serum. The removal of both phospholipid and triglyceride was similar in normal and hyperlipemic serum. Comparison of the 3H/14C ratio indicated that the cholesterol oleate and cholesterol linoleate were removed at similar rates. Key words:
Atherogenesis - Cholesterol Triglyceride - Tissue culture
ester - Phospholipid
-Smooth
muscle
cells -
Introduction
Early atherogenesis is characterised by the proliferation smooth muscle cells and their conversion to lipid containing
of intimo-medial foam cells. Studies
This paper is dedicated to Professor F.G. Schettler in the occasion of his sixtieth birthday. * This work was supported by grants from the National Health and Medical Research Council of Australia and the National Heart Foundation of Australia.
484
on arterial wall metabolism have demonstrated the presence of cholesterolesterifying enzymes in developing lesions [2--51 and this activity has been localised to the foam cells present [3,6]. Foam cells isolated from atherosclerotic lesions have been found to have marked cholesterol-esterifying activity [7] and homogenates of such cells shown to contain two distinct cholesterol-esterifying enzymes [8]. The foam cell appears therefore to play a significant role in the accumulation of cholesterol ester in the early lesion. Aortic smooth muscle cells from a variety of experimental animals have been grown in tissue culture [9,10] and it has been demonstrated that plasma low density lipoprotein promotes both cell growth and the intracellular accumulation of lipid [ 11,121. Smooth muscle cells grown from normal aortic explants in tissue culture in medium supplemented with normal serum do not demonstrate significant synthesis of cholesterol ester [13]. Where low density lipoprotein is added to the incubation medium however, cholesterol-esterifying activity is markedly increased [12]. These cells provide therefore a model system for studying atherogenesis at the cell level and for determining the mechanisms involved in the accumulation and removal of cholesterol ester from the early atherosclerotic lesion. In the present paper, the synthesis of cholesterol esters by smooth muscle cells in tissue culture has been further studied using 3H-labelled oleic acid and “C-labelled linoleic acid as precursors for cholesterol ester synthesis. In addition the removal of cholesterol oleate and of cholesterol lineolate has been investigated in order to determine whether the accumulated cholesterol ester can be removed from the cells by altering the lipoprotein content of the incubation medium. Materials and Methods Tissue culture methods Normal rabbits, 2-6 weeks of age, were killed by ether anaesthesia and their thoracic aortas carefully removed under sterile conditions. Adventitial connective tissue was removed and the aortas stripped into two layers. The inner layer (containing the intima and most of the media) was reserved for preparation of the tissue culture explants. The outer layer containing some media and the adventitia was discarded. Explants, approximately l-2 mm square, were plated into Falcon flasks. Three to four ml of incubation medium (Basal Eagle’s medium) containing 10% foetal calf serum was added and the flasks incubated at 37°C in a COZ incubator. Cell growth appeared within a week of primary culture and the cells were allowed to grow for a further period of approximately one week. At this stage the explants and new cells were removed by trypsinisation (3 ml of 0.25% trypsin - EDTA solution). The cells were centrifuged and resuspended in Basal Eagle’s medium containing 10% foetal calf serum and plated into Leighton tubes. After l-2 weeks when growth was confluent, the incubation medium was removed and replaced with incubation medium containing radioactively labelled fatty acids as set out below. Electron microscopy of the cells indicated differentiated smooth muscle cells with adequate evidence of myofibrils. Experimental procedure Two experimental procedures
were carried
out. In the first, duplicate
tubes
485
were incubated in Basal Eagle’s medium supplemented with 5% foetal calf serum and either 5% hyperlipemic rabbit serum (obtained from rabbits fed 1% cholesterol for 3-4 months) or 5% normal rabbit serum. A known amount of 14C-labelled linoleic acid (approximately 1.5 /Xi and 3H-labelled oleic acid (approximately 3 PCi) was added. After 24 h incubation, the medium was removed, the cells washed with 0.9% sodium chloride solution and their lipid extracted by chloroform-methanol (2 : 1, v/v) as described by Folch [ 141. In the second series of experiments cells were prelabelled in an incubation medium containing Basal Eagle’s medium supplemented with 5% foetal calf serum and 5% hyperlipemic rabbit serum, to which had been added a known amount of 3H-labelled oleic acid (approximately 3.0 FCi) and 14C-labelled linoleic acid (approximately 1.5 @i). After 24 h the cells were washed, a duplicate set of tubes taken down for immediate lipid extraction and the remainder divided into two groups. To one group was added incubation medium containing Basal Eagle’s medium, 5% foetal calf serum and 5% hyperlipemic rabbit serum and to the other Basal Eagle’s medium, 5% foetal calf serum and 5% normal rabbit serum. Duplicate flasks were taken down at daily intervals from each of the two groups for four days. The cells were washed with 0.9% sodium chloride solution and the lipid extracted with chloroform methanol as described above. Radioactive
isotopes
[9,10-3H]Oleic acid (specific activity 2.5 Ci/mmol) and [l-‘4C]linoleic acid (specific activity 56 mCi/mmol) were obtained from the Radiochemical centre Amersham, U.K. Prior to use they were taken up in 0.5 N NaOH and added to serum prior to inclusion of the latter in the incubation medium. Thin layer chromatography
After initial counting, aliquots of the lipid extracts were separated into phospholipid, cholesterol, free fatty acid, triglyceride and cholesterol ester by thin layer chromatography on silica gel G using the solvent system n-hexane : ether : acetic acid (112:38:3). Internal standards were added to visualise the separated lipid and the identified lipids were scraped directly into counting vials for counting of the 3H, 14C content using the dioxane : water scintillator described by Snyder [15]. Double labelled techniques using a Packard Tricarb Model 3375 were employed. Chemical
analyses
Lipid phosphorus was assayed in the lipid extracts by the micro method of Bartlett [16]. Cholesterol and cholesterol ester were assayed by densitometry using a Schoeffel SD 3000 spectrodensitometer. Details were described previously [17]. Results The incorporation of 3H-labelled oleic acid and 14C-labelled linoleic acid into various lipid fractions in smooth muscle cells grown for 24 h in the presence of either normal or hyperlipemic serum is shown in Table 1. Data is presented as
486 TABLE,1 EFFECT OF HYPERLIPEMIC SERUM ON THE INCORPORATION LINOLEIC ACID INTO VARIOUS LIPID FRACTIONS IN SMOOTH CULTURE
OF 3H-OLEIC MUSCLE CELLS
AND 14CIN TISSUE
Data is expressed as dpm X.10-3/106 dpm in the incubation medium except where figures in parenthesis are shown. These give the specific activity (dpm X 10-3/1~6 dpm in incubation medialpg Pi or /pg cholesterol ester--cholesterol). 14C-Linoleic __~
3 H- Oleic NOrmal
Hyperlipemic
18’7 151
145 101
Triglyceride Cholesterol-ester
(4.58) 28 3.2
(3.37) 30 10.0
392 313 (9.48) 63 4.8
CE/PL ratio
(0.42) 0.02
(1.82) 0.10
(0.62) 0.015
294 202 (6.73) 12 14.7 (2.67) 0.07
122 96 (4.40) 11 11.3 (2.13) 0.12
94 15 (3.83) 13 3.9 (0.54) 0.05
98 15 (3.44) 13 8.3 (1.57) 0.11
Kxperimen t II Total lipid Phospholipid
Experiment
Hyperlipemic
III
Total lipid Phospholipid Triglyceride Cholesterol ester CE/PL ratio Experiment
NOrmal
105 85 (3.83) 11 5.5 (0.76 ) 0.06
IV
Total lipid Phoshpolipid Triglyceride Cholesterol ester CE/PL ratio
216 148 52 6.1 0.04
186 112 49 16.2 0.145
315 197 98 6.2 0.03
264 149 88 16.3 0.110
dpm X 10m3 incorporated into the respective lipid fractions in relation to the fatty acid present in the incubation medium. In two of the three experiments shown, the specific activities of the phospholipid and cholesterol ester fractions were calculated and this data is given in parenthesis in Table 1. The presence of hyperlipemic serum in the incubation medium did not appreciably influence the incorporation of either oleic or linoleic acid into phospholipid or triglyceride in the cells. The incorporation of both oleic and linoleic acid into cholesterol ester however, was markedly increased when hyperlipemic serum was present in the incubation medium. The cholesterol ester/phospholipid ratio is also presented in Table 1. This fraction relates the incorporation of the respective fatty acid into cholesterol ester to that into phospholipid. The presence of hyperlipemic serum resulted in an increase in this cholesterol ester phospholipid ratio by 2-5 times that shown when the cells were grown in medium supplemented with normal serum. In order to compare the incorporation into the various lipid fractions of 3Hlabelled oleic acid with that of 14C-labelled linoleic acid, the 3H/‘4C ratio for each fraction was calculated and this data is shown in Table 2. For both normal and hyperlipemic serum and in all experiments the 3H/14C ratio for the triglyceride fraction is appreciably less than that for phospholipid and the 3H/‘4C
487
TABLE
2
3H-OLEIC/14C-LINOLEIC CELLS AFTER 24 HOURS
ACID RATIO INCUBATION
OF VARIOUS LIPID IN TISSUE CULTURE
3H/14C ratio is expressed each experiment.
as a percentage
of the ratio for the phospholipid
Experiment
Phospholipid Triglyceride Cholesterol ester
I
Experiment
II
FRACTIONS
Experiment -___
IN SMOOTH (hyperlipemic)
III
MUSCLE fraction
Experiment
for
IV
Hyperlipemic
Normal
hyperlipemic
Normal
Hyperlipemic
Normal
Hywrlipemic
100 87 132
97 88 136
100 84 138
88 64 109
100 68 105
100 71 141
100 74 131
ratio for cholesterol ester is appreciably more than that for phospholipid. It is apparent therefore that oleic acid is preferentially incorporated into cholesterol ester and linoleic acid preferentially incorporated into triglyceride compared with their incorporation into phospholipid in the cells. The removal of the labelled lipid fractions from smooth muscle cells in culture in the presence of either hyperlipemic or normal serum is shown in Fig. 1 and 2 for one representative experiment. There were differences in relative incorporation into lipid fractions between different experiments, but the pattern of removal for each was similar in all experiments and is represented by
o--oHYPERLIPEMIC x--xNOAMOLlPEMlC IM
dpm.lO-‘/lO’dpm
dpm x IO-‘/W
dpm I.M
100
0-o HYPERLIPEMIC x---xNORMOLlPEMlC
SERUM SERUM
I
I \a
L-.
1
2
3
4 TlMEtdays)
‘H-CHOLESTEROL
ESTER
G-T-T--
5
‘H-TRIGLYCERIDE
5
1
TIMEldays)
Fig. 1. Removal of phospholipid and triglyceride labelled with -‘H-oleic acid from smooth muscle cells grown in medium supplemented with either normal or hyperlipemic serum. Calculated linear regression lines based on semllogarithmic plots are presented. Fig. 2. Removal of cholesterol ester labelled with 3H-oleic acid from smooth muscle cells grown in tissue culture in medium supplemented with either normal or hyperlipemic serum. Semllogaritbmic transformation of data and calculation of linear regression lines has been carried out.
488
the data given in these figures. The removal of 3H-labelled phospholipid and triglyceride over the five day period studied was similar whether normal or hyperlipemic serum was added to the incubation medium (Fig. 1). The removal of the 3H-fatty acid labelled cholesterol ester, however, was increased by changing the supplement in the incubation medium from hyperlipemic serum to normal serum (Fig. 2). In order to obtain comparable data for the various experiments the fractional half time (ti) was calculated for each of the labelled lipid fractions and this data is shown in Table 3. The t$ was obtained from the slope of the removal line following log transformation of the data at each of the five time intervals. The significance of fit was determined by analysis of variance and the slope of the line and its intercept obtained from the linear regression equation. Comparison of the slopes of the lines was then made‘ by Student’s t-test for each of the lipid fractions for the individual experiments. There was no significant difference between the slopes of the removal lines for either 3H-oleic or 14C-linoleic labelled phospholipid or triglyceride when normal serum was substituted for hyperlipemic serum. There was, however, a statistically significant increase in the slope of the removal line for both 3H-oleic and “C-linoleic labelled cholesterol ester when normal serum was substituted for hyperlipemic serum in two of the experiments reported. In the other two experiments a trend was apparent but was not statistically significant.
TABLE
3
EFFECT
OF NORMAL
LIPID FRACTIONS
AND HYPERLIPEMIC
FROM
SMOOTH
SERUM
MUSCLE
CELLS
ON THE REMOVAL IN TISSUE
TIME (t;)
OF LABELLED
CULTURE
k$ (days) has been calculated from fitted exponential decay curves based on incorporation (dpm X 10m3 / lo6 dpm -incubation medium) at each of the 6 time intervals. Calculated lines of best fit which were not statistically significant by analysis of variance are indicated by italicizing the relevant t$. 3 H-Oleic
Experiment
a-a
Hyperlipemic
1.80 1.88 1.57
b
1.81 1.39 4.62
b
1.83 2.05 1.78
a
1.90 1.33 5.47a
a
5.80 1.07 3.60
a
3.68 1.04 1.18
a
4.97 1.07 4.44
6.66 1.06 1.07 2.42 1.86 2.75
2.33 1.61 63.3
2.08 I .40 2.44 ’
2.47 0.99 1.81
1.48 0.86
2.22 0.91
1.94
1.99
1.94 1.24 21.6 ’
IV
Phospholipid Triglyceride Cholesterol ester P < b-b P < cP < All other
Normal
III
Phospholipid Triglyceride Cholesterol ester Experiment
Hyperlipemic
II
Phospholipid Triglyceride Cholesterol ester Experiment
NOrmal I
Phospholipid Triglyceride Cholesterol ester Experiment
14 C-Linoleic
0.01 0.02 by Student’s t-test. 0.05 1 comparisons of slope (normal
vs hyperlipemic)
were not statistically
1.60 0.83 2.34
significant,
a
489 TABLE 4 EFFECT OF NORMAL AND HYPERLIPEMIC SERUM ON THE REMOVAL TIME (t;) OF LABELLED LIPID FRACTIONS FROM SMOOTH MUSCLE CELLS IN TISSUE CULTURE t$ (days) has been calculated from fitted exponential decay curves based on specific activity data. Calculated lines of best fit which were not statistically significant by analysis of variance are indicated by it&eking the relevant th .
3H-Oleic
Exmrimen
--
i4 C-Linoleic
-
Hyperlipemic
Normal
Hyperlipemic
3.63 0.97 1.64 a
2.00 0.83 0.19 a
3.29 0.96
1.33
2.10 1.40 1.86
1.66 1.09 2.45
t II
Phospholipid Triglyceride Cholesterol ester Experiment III Phospholipid Triglyceride Cholesterol ester
. 2.64 0.89 0.14 a 2.44 1.83 2.03
1.41 2.64
--._ I-
1.80 a
a-a P < 0.02 by Student’s t-test. All other comparisons of slope (normal vs hyperlipemic) were not statistically significant. SH-Oleic/‘4C-Linolaic
ratio
M
Hyperlipemic N0Ullal
.---.
I
---l------_~__---__r______. +
100
Cholesterol Ester
50
100 ____r
---T
__--
-__ T Trtglyceride
50 -
Photpholipid
Time Idays)
Fig. 3. 3H-oleic/14C-linoleic acid ratios (X100) for cholesterol ester, phospholipid and triglyceride fractions in relation to time in smooth muscle cells grown in tissue culture in medium supplemented with normal or hyperlipemic serum. Cells have been pulse labelled with labelled fatty acids for 24 h prior to commencement of the removal study. - Mean of 4 experiments _+SEM is shown.
490
In two of the four experiments reported, chemical analyses were possible and the specific activity of the phospholipid, triglyceride and cholesterol ester obtained. This specific activity data was used to calculate the removal times (ti) for these two experiments and this data is shown in Table 4. These times have been obtained in a similar way to those described for Table 3. When the slopes of these lines were compared statistically by Students t-test, there was no significant difference between normal and hyperlipemic serum added to the incubation medium with regard to the removal of the labelled phospholipid or of the labelled triglyceride. For cholesterol ester, however, the removal was accelerated by the substitution of normal for hyperlipemic serum in the incubation medium. This shows statistical significance for experiment II 03 < 0.02), but does not attain statistical significance for experiment III (Table 4). The relative removal (over the five day period) of 3H-oleic and 14C-linoleic acid incorporated into phospholipid, triglyceride and cholesterol ester, is shown in Fig. 3. The 3H/‘4C ratio for the three lipid fractions in the presence of either normal or hyperlipemic serum has been plotted in relation to time. The 3H/14C ratio does not change appreciably with time over the five day period studied for any of the three lipid fractions, indicating that the removal rates of the two fatty acids incorporated into phospholipid, triglyceride and cholesterol ester are similar. Three way analysis of variance with time, treatment, experiment indicated that there was no significant difference either with treatment or with time for the 3H/‘4C ratio for any of the lipid fractions studied. Discussion The effect of hyperlipemic serum on cholesterol ester synthesis in smooth muscle cells in culture is in agreement with findings reported by other investigators [ 141. In the present work a similar increase in synthesis occurred whether 3H-labelled oleic acid or 14C-linoleic acid were used as precursors for cholesterol ester. In early atherosclerotic lesions in both man and experimental animals the major cholesterol ester which accumulates is cholesterol oleate [l&19] and in vitro metabolic work carried out using atherosclerotic arteries isolated from atherosclerotic lesions have indicated that labelled oleic acid is incorporated more readily than other fatty acids into cholesterol ester in these preparations [20]. In the smooth muscle cells studied in the present paper, oleic acid was preferred to linoleic acid for cholesterol ester synthesis. This was true whether cells were synthesising small amounts of cholesterol ester or stimulated by exposure to hyperlipemic serum. The removal of labelled lipid fractions from the cells occurred relatively rapidly over the four day period studied. There was some variation in the ti for the various lipid fractions in different experiments as indicated by the data given for these experiments in Tables 3 and 4. However, a consistent pattern for removal between different lipid fractions was apparent in all experiments. Triglyceride was removed rapidly from the cells with a t3 from one to two days. The substitution of normal serum for the hyperlipemic supplement in the incubation medium did not alter the removal rate of the labelled triglyceride. Phospholipid removal was somewhat more variable in the cells but in each experiment was removed more slowly than the triglyceride with a ti from 1.48 to
491
6.66 days. The latter high figures however, were recorded in only one experiment and in the other three experiments a G of 1.48 to 2.47 days were observed. Again no effect of changing the cholesterol content of the incubation medium was apparent. In contrast, cholesterol ester showed a relatively long half life when the hyperlipemic serum supplement was continued throughout the four day removal period. These values varied fairly widely between experiments but were higher than those found for triglyceride and phospholipid. In contrast however, where the hyperlipemic supplement was changed to a normal serum supplement the removal time for cholesterol ester was reduced to values similar to those shown for phospholipid and triglyceride. It seems therefore that the fractional turnover time of cholesterol ester, but not of phospholipid or triglyceride can be reduced by changing the cholesterol content of the incubation medium. Removal of cholesterol ester, as well as its deposition, might therefore be expected in cells grown in tissue culture if they are subjected to a suitable regression regimen. The fractional turnovers of cholesterol oleate and of cholesterol linoleate were similar with both showing a similar reduction in ti when t.he hyperlipemic medium was changed to a normal serum supplement. Examination of the 3H/ 14C ratio showed little change throughout the course of the experiment for any of the lipid fractions studied. It can be concluded therefore that the cholesterol oleate and cholesterol linoleate are being removed from the cells at similar rates, as has been observed for arterial wall incubated in vitro [20]. Any cholesterol oleate accumulation which occurs in stimulated smooth muscle cells appears therefore to result from its increased synthesis in the cells rather than its reduced removal in relation to other cholesterol esters formed. Acknowledgements We are indebted to Dr. John Hurley, Department of Pathology, University of Melbourne for assistance with electron microscopy. We are also indebted to Mr. Dennis Vickery, Mrs. Jill Duda and Miss Margaret Ackland for technical assistance. References 1 G&z, G.S., Vesselinovitch, D. and Wissler. R.W.. A dynamic pathology of atherosclerosis, Amer. J. Med., 46 (1969) 657. 2 Lofland, H.B. and Clarkson, T.B.. Certain metabolic patterns of atheromatous pigeon aortas, Arch. Path.. 80 (1965) 291. 3 Day, A.J. and Wahlqvlst. M.L., Uptake and metabolism of 14C-labeled oleic acid by atherosclerotic lesions in rabbit aorta, Circ. Res., 23 (1968) 779. 4 St. Clair, R.W.. Lofland, H.B. and Clarkson, T.B., Influence of atherosclerosis on the composition, synthesis and esterification of lipids in aortas of squirrel monkeys (Saimb’i sciureus). J. Atheroscler. Res.. 10 (1969) 193. 5 Proudlock. J.W. and Day, A.J., Cholesterol esterlfying enzymes of atherosclerotic rabbit intima, Biochim. Biophys. Acta, 260 (1972) 716. 6 Wahlqvist, M.L.. Day, A.J. and Tome, R.K.. Incorporation of oleic acid into lipid by foam cells in human atherosclerotic lesions, Circ. Res. 24 (1969) 123. 7 Day, A.J. and Tome. R.K., In vitro incorporation of 14C-labelled oleic acid into combined lipid by foam cells isolated from rabbit atheromatous lesions, J. Atheroscler. Res., 9 (1969) 141. 8 Proudlock, J.W.. Day. A.J. and Tume, R.K., Cholesterol-esterifying enzymes of foam cells isolated form atherosclerotic rabbit intima, Atherosclerosis, 18 (1973) 451.
492 9 Jarmolych, proliferation 10 11 12 13 14 15 16 17
18 19
20
J., Daoud, A.S., and production
Landau, J.. Fritz, K.E. of mucopolysaccharides,
and McElvene, R., Aortic media explants, cell collagen and elastic tissue, EXP. Mol. Path.,
9 (1968) 171. Ross, R., The smooth muscle cell, Part 2 (Growth of smooth muscle in culture and formation of elastic fibers). J. Cell Biol.. 50 (1971) 172. Dzoga, K.. Wissler, R.W. and Vesselinovitch, D., The effect of normal and hyperlipemic low density lipoprotein fractions on aortic tissue culture cells, Circulation, 43 and 44 (Suppl. II) (1971) 6. St. Cl&, R.W.. Metabolism of the arterial wall and atherosclerosis. In: R. Paoletti and A.M. Gotto, Jr. (Eds.), Atherosclerosis Reviews, Vol. 1, Raven Press, New York. 1976, p. 61. Day, A.J., Lipid metabolism by rabbit aortic intimal and medial cells in tissue culture. Virchows Arch. A. Path. Anat. and Histol., 362 (1974) 89. Folch. J., Lees, M. and Sloane-Stanley, G.H., A simple method for the isolation and purification of total lipids from animal tissues, J. Biol. Chem., 226 (1957) 497. Snyder, F., Radioassay of thin-layer chromatograms - A high-resolution zonal scraper for quantitative Cl4 and H3 scanning of thin-layer chromatograms, Anal. Biochem.. 9 (1964) 183. Bartlett, G.R.. Phosphorus assay in column chromatography. J. Biol. Chem.. 234 (1959) 466. Bretherton, K.N., Day, A.J. and Skinner. S.L., The effect of renal hypertension on the regional deposition of cholesterol and phospholipid in the aorta of normally- and cholesterol-fed rabbits, Atherosclerosis, 22 (1975) 517. Smith, E.B., The influence of age and atherosclerosis on the chemistry of aortic intima. J. Atheroscler. Res.. 5 (1965) 224. Peterson, M., Day, A.J.. Tume. R.K. and Eisenberg. E.. Ultrastructure, fatty acid content, and metabolic activity of foam cells and other fractions separated from rabbit atherosclerotic lesions, Exp. Mol. Path., 15 (1971) 157. of different fatty acids into combined Day, A.J., Wahlqvist. M.L. and Tume, R.K., Incorporation lipids in rabbit atherosclerotic lesions, Atherosclerosis, 12 (1970) 253.