Synthesis of Phospholipid by Foam Cells
Isolated from Rabbit Atherosclerotic Lesions
By Allen J. Day, M.D., D.Phil., H. A. I. Newman, Ph.D.,
and D. B. Zilversmit, Ph.D.
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ABSTRACT
In a preliminary study, foam cells from atherosclerotic lesions have been
isolated by incubation of the intima with collagenase and elastase. The
composition and metabolism of homogeneous preparations of foam cells obtained in this way have been compared with other parts of the atherosclerotic
intima. About 1% of the cholesterol and phospholipid of the intima was present
in the cells obtained. The phospholipids of the foam cells contained a
lower percentage of lecithin and of sphingomyelin and a higher percentage
of "phosphatidyl ethanolamine" than the other parts of the intima. The isolated
foam cells incubated in vitro incorporated PJf-phosphate into phospholipid
at a rate of about 0.5 m/ijnoles/100 cells/hr. The P 31 was incorporated mainly
into lecithin and phosphatidyl inositol with smaller amounts incorporated into
phosphatidyl ethanolamine, sphingomyelin and lysolecithin.
The amount of P 3 t incorporated into phospholipid by the foam cells was
compared with that incorporated into phospholipid by other portions of
the intima when the aorta was incubated in vitro prior to disruption. Up to
6.8% of the phospholipid synthesis was brought about by isolated cells. The
significance of the findings in relation to phospholipid synthesis in the atherosclerotic lesion was discussed.
ADDITIONAL KEY WORDS
phospholipid inositol
aortic atheroma
cholesterol
lecithin
sphingomyelin
phosphatidyl ethanolamine
aortic intima
• The presence of large numbers of lipidcontaining foam cells in the early atherosclerotic lesion has led to the suggestion that
these cells contribute in part to the metabolic
changes in the early lesion.1'2 These cells have
been investigated in situ by histochemical and
ultramicroscopic methods and such studies1' s ' * have indicated that metabolism and
synthesis of lipid by foam cells may be associated with the development of the atherosclerotic lesion. These studies have obvious
From the Department of Physiology and Biophysics, University of Tennessee Medical Units, Memphis, Tennessee.
Supported by Grant HE-2181 from the U. S. Public
Health Service.
Dr. Day is on study leave from the University of
Adelaide, supported by U. S. Public Health Service
Fellowship Award FO5-TW-812-01.
Dr. Zilversmit is a Career Investigator of the
American Heart Association.
Dr. Newman's present address is Lipid Research
Laboratory, Akron City Hospital, Akron, Ohio.
Accepted for publication February 15, 1966.
122
limitations, however, in investigations of biochemical changes taking place in the cells.
The metabolism of macrophages obtained from
the peritoneal cavity or lung of rabbits has
been studied previously,2' B~8 but there are difficulties in extrapolating the findings to foam
cells in the arterial wall. The separation of
foam cells from the aorta for metabolic studies
in vitro, however, would provide a means of
investigating in detail their biochemical properties and of determining what part these
cells play in the lipid changes occurring in
the early atherosclerotic lesion. Recently Rodbell" has developed a technique in which fat
cells were isolated from adipose tissue with
the aid of collagenase.
In the present paper a similar method has
been described for isolating aortic intimal
foam cells from rabbit atherosclerotic lesions
and some data are presented comparing the
lipid composition and phospholipid synthesis
Circulation Research, Vol. XDC July 1966
PHOSPHOLIPID SYNTHESIS BY FOAM CELLS
of such foam cells with that of other fractions
of the arterial wall.
Methods
ISOLATION OF FOAM CELLS BY ENZYMIC
DISRUPTION OF THE AORTIC INTIMA
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New Zealand albino rabbits, fed daily 100 g
Purina chow, containing 1 g of cholesterol and
2.8 g of cottonseed oil for periods ranging from
80 to 123 days, were used in these studies. The
rabbits were exsanguinated from the abdominal
aorta under ether anesthesia and the thoracic
aorta was removed, washed with saline, and
bathed in normal rabbit serum. The superficial
fat was removed and the intima stripped for subsequent incubation as set out in the accompanying flow diagram (fig. 1). The intima was incubated with Krebs Ringer phosphate solution
(pH 7.4) containing 4% bovine albumin (fatty
acid poor, Pentex, Kankakee, Illinois) 10 mg
coUagenase (Worthington Biochemical Corp.,
Freehold, New Jersey), 4 mg elastase (Nutritional Biochemicals Corp., Cleveland, Ohio) and
15 pjnoles of glucose in a volume of 5 ml. Incubation was done in 20 ml plastic screw top
counting vials at 37°C, the digest being shaken
gently every 5 min. After 1 hr the partially disrupted intima was teased apart with forceps and
incubation allowed to proceed for an additional
1 hr at 37°C. The digest was then filtered
through gauze in a 10 ml plastic syringe, the
residue washed with 1 to 2 ml of 4% bovine albumin in Krebs Ringer phosphate and the washings expressed from the residue (fraction I, fig.
1) by means of the syringe plunger. The filtrate
THORACIC AORTA
I
•tripped
WTIM
Incubation 1 hr with
collaganaa«, alait-ata.
Taaaa, furthar 1 hr
incubation.
f i l t e r through 9aux«
123
was centrifuged at 220 g for 3 min and the deposited cells were washed twice with 0.9% sodium
chloride solution and recentrifuged. The initial
supernatant (fraction II, fig. 1) was reserved for
study while the washings were discarded. The
washed cells were taken up in Hanks solution10
for counting in a hemocytometer chamber and
then suspended in Hanks solution containing 0.5%
bovine albumin, dispensed into glass containers
(Leighton tubes for metabolic studies or stoppered conical flasks for chemical assay) and incubated for 60 min at 37°C. Most of the foam
cells adhered to the glass while other cells and
particles remained in suspension. The adhering
foam cells were washed with warm saline and the
cells which remained in suspension or were
washed off were counted to determine loss of
cells. The combined solution containing the cell
washings was then centrifuged (1250 g for 10
min) and the deposit, washed twice with saline,
was reserved for subsequent study (fraction
IV, fig. 1). All procedures except the final stage
in which the cells were stuck to glass vessels were
done in plastic containers. Four fractions were
obtained by the above procedure, viz. residue
(I), supernatant (II), foam cells (III) and washings (IV). These fractions were used for subsequent chemical or isotope assay or for metabolic
studies.
The fractions were extracted with 2:1 chloroform methanol as described by Folch et al. 11
and fractionated into phospholipid and neutral
lipid on silicic acid columns.12 Lipid phosphorus
was determined by the method of Bartlett18 and
distribution of individual phospholipids by thin
layer chromatography and elution by the method
of Skipski et al.14 Total cholesterol was determined as described by Zlatkis et al.16 in the
neutral lipid fraction following saponification by
the method of Abell et al.10 Free and ester cholesterol were determined by the same method following separation of free from esterified cholesterol on silicic acid columns37 and subsequent
saponification. Purity of column fractions was
checked by thin layer chromatography.18
RES1PUE (Fraction I)
Cantrifuga
22O g - 3 sin
SYNTHESIS OF PHOSPHOLIPID BY ISOLATED FOAM CELLS
BUPEWATAJTF (fraction II)
Wath 2 tljMi with
•alina and centrlfuga
•cspand in O.W albu»In
in HaaXa and into glaaa
vaiaal. Incubat* 6O ain
at 3 ? ' . Hash adbarant
c a l l s with aalina.
I.'^3 (Fraction IV)
TOW CUXS (fraction III)
FIGURE 1
Flow diagram illustrating separation of foam cells
from the atherosclerotic aortic intima.
CircnUtion Roemrch. VoL XDC July 1966
Two series of metabolic experiments were performed. In the first series the incorporation of
P3'-labeled phosphate into phospholipid by isolated foam cells was investigated. Approximately
1.5 X 10° foam cells were dispensed into Leighton tubes, allowed to adhere to the glass, and
washed. The cells which did not adhere to the
glass were counted. One ml of medium (1:1 v/v
Hanks:normal rabbit serum with 0.1 mg/ml of
streptomycin and penicillin) containing up to 30
fie of P"-labeled phosphate (Abbott Laboratories) was added. Duplicate batches of cells were
124
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incubated for 1/2, 1, 2, 4 or 18 hours after addition of the labeled medium. After the appropriate
incubation time the medium was removed and
the adhering cells washed twice with 0.9% sodium
chloride solution. The medium was centrifuged
and the small amount of deposit washed twice
with 0.9% sodium chloride solution and extracted
together with the adhering cells. The centrifuged
medium was also extracted. Aliquots of the
Folch-washed extracts were assayed for P3* with
the scintillator described by Gordon and Wolfe19
and for lipid phosphorus.
In the second series of experiments the rabbit
atherosclerotic thoracic aorta was incubated in
vitro with P " as described elsewhere.20 After
incubation the aorta was washed with 0.9% sodium
chloride solution, the intima removed by stripping
and incubated with collagenase and elastase in
order to free and isolate the foam cells as set out
above. The four fractions were extracted and
assayed for P " and lipid phosphorus. In some of
these experiments the supernatant (fraction II)
was filtered through a 1.2 fi millipore filter in
order to separate the large numbers of particles
present. The particles (which varied up to 3 to
4 fi in diameter) were washed twice with 0.9%
sodium chloride solution and extracted.
IDENTIFICATION OF LABELED PHOSPHOLIPID
Separation of the lipid extract to identify and
quantitate the individual phospholipids labeled
by the foam cells was done by thin layer chromatography by the method of Skipski et al.14 In
view of the small amount of lipid phosphorus
present in the individual extracts in these metabolic experiments, the cell extract was taken
up in a lipid extract of aortic intima as carrier.
The phospholipid spots were located by spraying
with iodine vapor or by the method of Dittmer
and Lester21 and then scraped into counting vials
for counting by the method of Snyder.22 Identification of spots in aortic extracts by staining reactions has been described elsewhere.20
P"-labeled phospholipid was identified by
comparison of R values with standards and by
cochromatography using three systems as follows.
The labeled spots, located by radioautography,
were eluted and taken up with the appropriate
standard for rechromatography(i) by the method of Skipski et al., (ii) with silica gel G and
the solvent system chloroform :methanol: water
(140/50/9 v/v/v) and (iii) in the case of the
phosphatidyl inositol and phosphatidyl ethanolamine spots on an alkaline plate prepared as
described by Skipski et al., but using the solvent
system di-isobutyl ketone : acetic acid : water
( 4 0 : 2 5 : 5 v / v / v ) . Satisfactory separations of
phosphatidyl inositol and phosphatidyl serine
DAY, NEWMAN, ZILVERSMIT
were attained by this method. Labeled spots were
located by radioautography.
In the cochromatography experiments butylated hydroxytoluene was added to all solvents to
minimize oxidation of labile compounds.2'1 Standards used were phosphatidyl ethanolamine,
phosphatidyl serine and sphingomyelin obtained
from Applied Science Laboratories (State College, Pennsylvania), lecithin (containing lysolecithin) from Mann Research Laboratories (New
York, New York) and phosphatidyl inositol
kindly supplied by Dr. Faure (Pasteur Institute,
Paris). These standards were checked by staining
reactions as described elsewhere.20
Remits
The number of cells obtained from a single
intima varied in 18 experiments from 4.0 to
16.8 X 10° (mean 7.8 X 10° ± 0.68 SEM). Most
of these cells stuck to glass, but 10 to 15%
of cells did wash off and were counted in the
washings. Over the time period studied (80
to 123 days) there was no obvious correlation
between the duration of feeding and the yield
of foam cells although the lowest figure was
in fact from the 80 day rabbit.
The foam cells are shown, in figures 2 and
3, adhered to glass cover slips. All of the
cells observed were mononuclear, varying in
size from 15 to 50 fi, and morphologically
resembling macrophages. They contained a
large number of granules which stained positively for lipid with oil red O. In some prep-
FIGURE 2
Foam cells isolated from atherosclerotic intima.
Viewed unstained adhering to cover slips. Phase contrast.
Circulation Reran*, VoL XIX, July 1966
125
PHOSPHOLIPID SYNTHESIS BY FOAM CELLS
arations the granules were absent from parts
of the cell. In the final cell preparation shown
in figures 2 and 3 there were practically no
contaminating particles or other cells. Figure
4 is a free suspension of washed cells [Cells
(2) in fig. 1] obtained prior to plating in
glass vessels. Contaminating cells were present
together with relatively large numbers of particles ranging in size up to 3 to 4 //,. These
particles resembled morphologically the granules in the foam cells and, in some areas,
clusters of them resembling parts of cells can
be seen. Cells present in the supernatant
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FIGURE 3
Foam cells isolated from atherosclerotic intima.
Viewed unstained adhering to cover dips. Phase contrast.
(fraction II) were counted in some experiments and accounted for about 10% of the
cells present in the foam cell fraction (fraction III). However, the supernatant contained
large numbers of particles both singly and in
clusters (fig. 5). These are removed by filtration of the supernatant through millipore
filters most being removed by a 1.2 fi filter.
They are not appreciably sedimented however, at 1800 g for 15 min.
Some cells could be obtained with collagenase or with elastase alone, but better
yields were found when both enzymes were
used. When serum was used as incubation
medium, the yield was lower than with 4%
albumin in Krebs Ringer phosphate.
The distribution of lipid P and of cholesterol between the four fractions obtained is
shown in table 1. Most of the lipid P and of
the cholesterol was present in the supernatant and residue fractions; only about US of
the total lipid P and cholesterol was found
in the-isolated foam cells. The-cholesterol:
lipid P ratios of the various fractions were
similar with the possible exception of the
residue in which the cholesterol:lipid P ratio
was somewhat higher. The percentages of
cholesterol ester present in the foam cells and
washings were significantly greater (P < 0.01)
than the percentages in the supernatant or the
residue.
FIGURE 4
FIGURE 5
Suspension of cells obtained from atherosclerotic
intima following incubation with coUagenase and
elastase. Phase contrast.
Supernatant solution obtained from atherosclerotic
intima following incubation with coUagenase and
elastase and centrifugation to remove foam cells.
Phase contrast.
OicuUrion Reward], VoL XIX, July 1966
DAY, NEWMAN, ZILVERSMIT
126
The per cent distribution of individual phospholipids in the four fractions is shown in
table 2. In the foam cells and the washings
the percentages of the phospholipid, present
as "phosphatidyl ethanolamine" and as the
fraction which travels at the solvent front,
were greater than the percentages in the
supernatant and residue. The percentages of
sphingomyelin and lecithin present in the cells
and washings were lower than those of the
supernatant and residue.
The incorporation of phosphate-P" into
phospholipid by isolated foam cells is shown
in table 3. P"-phosphoh'pid increased in the
cells fairly uniformly over the time period
studied so that after 4 hr incubation almost
0.2$ and after 18 hr almost 0.7$ of the P"
added to the medium has been incorporated
TABLE 1
Distribution of Lipids Between Fractions of Aortic Intima (means of 4 ± SEM)
Llpid (% of
total in whole
intima)
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0.92 ±0.17
1.33 ±0.14
48.3 ± 4.28
54.4 ± 5.22
0.795 ±0.108
Foam cells (III)
Washings (IV)
Supernatant (II)
Residue (I)
Total in intima,
mg
Cholesterol (•/,
of total in
whole intima)
Cholesterol (%
presentt as
ester in eeacht)
Cholesterol/
lipid P
ratio*
•/.
0.86 ± 0.23
1.29 ±0.11
43.9 ±4.21
54.0 ±4.10
54.93 ± 7.94
§74.3 ± 0.99
72.0 ± 1.51
+57.6 ± 1.49
61.1 ± 2.02
§61.5±
67.3 ±
§60.9 ±
*78.2 ±
5.06
4.73
2.16
3.25
•Analysis of variance shows significant differences at the 5% level.
tAnalysis of variance shows significant differences at the X% level,
tmean of 5.
§mean of 6.
TABLE 2
Per Cent Distribution of PhosphoUpid Fractions of Aortic Intima (means of 4 for cells and supernatant and of 2
for washings and residue)
Origin
Foam cells (IU)
Washings (IV)
Supernatant (II)
Residue (I)
4.0 ±
2.3 ±
0.5 ±
0.2 ±
1.9
0.20
0.09
0.0
Lysolecithln Sphingomyelint
5.7
3.3
9.8
5.2
±
±
±
±
1.5
17.2 ±
0.26 17.0 ±
1.40 25.7 ±
0.16 27.8 ±
Lecithin*
1.8
39.3 ± 2.5
1.4
38.2 ± 3.1
0.44 48.1 ± 1.1
0.35 47.2 ± 0.25
Phosph.
inositol
"Phosph.
ethanolamine"tf
9.0 ± 1.4
13.5 ± 4.7
5.2 ± 0.61
7.2 ± 0 . 1 6
15.5 ±
14.7 ±
6.7 ±
8.2 ±
1.2
0.85
0.42
0.35
Solvent
frontt
9.4 ±
11.0 ±
4.0 ±
4.6 ±
1.1
0.20
0.60
0.28
•Analysis of variance shows significant differences at the 5% level.
tAnalysis of variance shows significant differences at the \% level.
TABLE 3
Incorporation of P"-labeled Phosphate into Phospholipid by Isolated Foam Cells
Incubation
time
Eip. 1
Nci. of cells, 1.38 X 10s
Phosphate-P" added, 41 :X 10« cpm
p°-phospholipholipid
(•/. of addled P")
Lipid P
Cell*
Cells
Medium
Exp. 2
No. of cells, 1.11 X 10«
«
Phosphate-P added, 49 |X 10° cpm
P"-phol[)holipid
Lipid P
(% of miIded P")
Cells
Cells
Medium
hr
I
2
4
18
3^6
3.39
3.25
2.92
—
0.0170
0.0430
0.0890
0.1770
—
0.0004
0.0009
0.0009
0.0048
—
2%
3.00
2.87
3.04
2.40
0.0130
0.0280
0.0740
0.1960
0.6860
0.0025
0.0016
0.0037
0.0080
0.0800
Grcolation Research, Vol. XIX, July 1966
127
PHOSPHOLIPID SYNTHESIS BY FOAM CELLS
into cellular phospholipid. Table 3 shows that
the medium contained very little P " labeled
phospholipid at the earlier time intervals.
Amounts less than 0.001% probably represented traces of P"-labeled phosphate present in
the lipid extracts. However, the amounts above
this were phospholipid P " which appeared
in the medium in significant, although small,
amounts relative to those in the cell extract.
The individual phospholipids labeled by the
isolated foam cells are shown in figure 6.
Five spots were labeled corresponding in RF
value to lysolecithin, sphingomyelin, lecithin,
phosphatidyl inositol and phosphatidyl
ethanolamine. The latter four spots were
eluted and cochromatographed with individual standards as shown in figure 7A, B, C.
Sphingomyelin, lecithin and phosphatidyl inositol traveled as single spots together with the
standard preparations on both the alkaline
plate (fig. 7A) and the silica gel G plate (fig.
7B). In order to determine whether phosphatidyl serine contributed to spot 5, this spot was
rechromatographed on an alkaline plate
(fig. 7C) with the solvent system di-isobutyl
ketone: acetic acid: water (40:25:5) mixed
with either phosphatidyl serine or phosphatidyl inositol or with both standards. Spot 5
traveled with, and can therefore be tentatively identified as, phosphatidyl inositol. About
4.0$ of the label traveled with phosphatidyl
serine, however. Spot 6 which ran with phosphatidyl ethanolamine in the Skipski system
rnun i
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P/ETHANOLAMINE
O
P/INOSITOL
O
LECITHIN
0
SPHINGOMYELIN
LYSOLECITHIN
0
SPOT 6
t
•
SPOT 5
SPOT 4
SPOT 3
o
*
SPOT 2
ORIGIN
FIGURE 6
Identification of P3S-ldbeled phospholipids synthesized
by isolated foam ceUs. Separation of the labeled foam
cell extract by thin layer chromatography followed by
autoradiography. Alkaline plate with solvent system
chloroform:methanohacetic acid:water (23/15/4/1.9).
FRONT
A
•
o
0
O
o
o
0
0
ORIGIN
*
•
S/M LKithin
/Ly«o
o
*
*
P.I.
RE.
SPOT SPOT SPOT SPOT
3
4
5
6
FRONT
I
o
o
o
*
RS.
Rl.
„„,
PE
Standard SPOT SPOT
Mixture
5
5
SPOT SPOT
5
6
B
FIGURE 7
0
O
ORIGIN
S/M LKilhln PI
/Ly«o
PE
Grculitioii Research. Vol. XIX, July 1966
SPOT SPOT SPOT SPOT
3
4
5
6
Identification by cochromatography of Pst-labeled
phospholipids synthesized by isolated foam cells.
Badioautography of P3t-labeled spots and chemical location of standards are shown. A: Alkaline plate, solvent system chloroform:methanol:acetic ackLwater
(25/15/4/1.9). B: Neutral plate, solvent system chloroform-.methanoh water (140/50/9). C: Alkaline plate,
solvent system di-isobutyl ketone-.acetic acULwater
(40/25/5). Details in text.
DAY, NEWMAN, ZILVERSMIT
128
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did not rechromatograph as a single spot. In
both fig. 7B and 7C this spot contained, in
addition to a spot running with the standard
phosphatidyl ethanolamine, other unidentified spots.
The percentage distributions of individual
P'Mabeled phospholipids for the cell extracts
at different times of incubation are given in
table 4. Most of the P " has been incorporated
into cellular lecithin, phosphatidyl inositol
and the composite spot "phosphatidyl ethanolamine." The distribution was essentially
similar throughout the four-hour time period
although in the one experiment at 18 hours
the sphingomyelin and "phosphatidyl ethanolamine" accounted for a larger amount and
the phosphatidyl inositol for a smaller amount
of the P " than at the earlier incubation times.
The P " incorporated into phospholipid by
each of the four fractions of the aortic intima
after in vitro incubation of the intact aorta
with P"-phosphate is shown in table 5. In
the first three experiments [table 5 (a)] the
aorta was incubated with P " for four hours.
In the last three experiments [table 5 (b)]
the incubation of the aorta with P " was
limited to two hours. In the four-hour experiments the cells may have deteriorated
since many of the separated cells did not
adhere to glass and a relatively large amount
of lipid-P3* was present in the washings. In
both series of experiments the PSf-phospholipid was distributed mainly in the supernatant and residue fractions, the cells accounting for at best 6.8? of the label. The specific
activity of the P**-labeled phospholipid in
TABLE 4
Incorporation of P" into Individual Phospholipids by Isolated Foam Cells (% distribution at
different time intervals)
Incubation
time
Origin
Ly»olecithin
Sphlngomyel
3.3
0.6
0.4
0.2
0.1
1.3
0.4
1.0
0.7
0.5
1.5
1.0
0.9
2.3
8.3
hr
%
1
2
4
18
Lecithin
56.2
60.3
61.0
65.4
54.3
Phoapba.
inooitol
"Phoepha.
ethanolamine"
Solvent
front
24.5
25.7
26.5
20.7
16.4
11.4
8.9
7.5
7.5
17.1
2.2
3.2
2.9
2.4
3.2
TABLE 5
Contribution of Different Fractions of Aortic Intima to Phospholipid Synthesis in Vitro
(a) Incubation of aorta with Pst in vitro for 4 hr followed by separation into fractions. Mean of
3 ± SEM*
Cells (HI)
Washings (IV)
Residue (I)
Supernatant (II)
PM-pho»phollpid
Lipid P
% attribution
AC
2.0 ± 0.72
9.3 ± 0.35
40.4 ±3.14
48.2 ± 2.18
2.19 ± 0.47
10.13 ± 0.76
341 ± 38.2
420 ±55.3
Specific activity
2560 ±147
2760 ± 67.7
360 ± 9.14
350 ± 24.2
(b) Incubation of aorta with P" in vitro for 2 hr followed by separation. Mean of 3 ±
Cells
Washings (IV)
Residue (I)
Supernatant
Total (II)
Particles
P"-pho»phollpid
% distribution
Lipid P
6.8 ±1.45
6.8 ± 2.09
40.1 ± 1.50
5.70 ± 1.87
4.85 ± 1.18
252 ± 96.0
2260 ± 62.0
2460 ±170
330 ± 50.8
46.3 ± 0.81
18.0 ± 2.00
294 ± 93.5
63.3 ±15.0
315 ± 53.2
538 ± 83.7
AC
Specific activity
cpm/fLf
•34.8 X 106 cpm P"-phosphate added.
t59.0 X 10« cpm P"-phosphate added.
Circulation Research. VoL XIX, July 1966
PHOSPHOLIPID SYNTHESIS BY FOAM CELLS
the cells, however, was six to seven times that
in the supernatant and residue. In the three
experiments shown in table 5 (b) the distribution of P " in, and the specific activity
of, the particles of the supernatant were also
determined. This fraction accounted for 37%
of the Pf in the supernatant with a specific
activity almost twice that of the supernatant
and residue but considerably less than that of
the cells.
Discussion
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The cells obtained were free of contaminating cells and represented a microscopically
homogeneous preparation of lipid-containing
foam cells. It is known, that in the rabbit
atherosclerotic lesion some of the lipid-containing cells can be identified ultramicroscopically as smooth muscle cells.25 It is possible
that some of the cells obtained by the method
described may be smooth muscle cells, but
morphologically the preparation consisted of
rounded mononuclear cells, indistinguishable
from macrophages. Further, while the ability
to adhere to a glass surface is by no means
specific, this property is one well developed
in macrophages from other situations and
used for their separation from other cell
types.28'27
The lipid content of foam cells was high
compared with that of macrophages obtained
from the peritoneal cavity of normal rabbits.
It can be calculated from the data reported
in table 1, knowing the cell count in each
preparation, that the total cholesterol content
amounted to 72 fig/106 cells, and the lipid
phosphorus to 1.1 /tg/106 cells. This compared with about 1 /ig/106 cells for cholesterol (Day and Fidge, unpublished data) and
0.7 /Ag/10e cells for lipid phosphorus for peritoneal cavity macrophages derived from normal rabbits.7
The cells obtained were metabolically active
as indicated by their ready incorporation of
P"-phosphate into phospholipid and can
therefore be used to investigate the part played
by the foam cell in the development of the
early lesion. The incorporation of P ^ -phosphate into phospholipid was high when compared with that of peritoneal cavity macroCircuUtion Roemrch. Vol. XIX July 1966
129
phages. It can be calculated that foam cells
incubated with P3?-phosphate incorporated into phospholipid approximately 0.5 /imoles of
the medium phosphate per hr/106 cells. Peritoneal macrophages from normal rabbits incorporated approximately 0.05 /imoles/ hr/108
cells which is increased 44% in the presence
of a 0.06% cholesterol suspension.7
A further comparison with peritoneal macrophages can be made with respect to the individual phospholipids labeled following incubation. Lecithin represented the major
phospholipid labeled in both cell types, but
in the case of the foam cells, considerably
more phosphatidyl inositol and less sphingomyelin were labeled than was the case with
the peritoneal macrophages incubated in the
presence or absence of cholesterol.7
The amount of phospholipid present in the
foam cells obtained was only about 1% of
that in the intima as a whole. If some disruption of cells was occurring, it is possible that
the amount of phospholipid present in the
foam cells in the intima may account for more
than this. However, even if disruption of cells
had occurred during preparation, it seems unlikely that the foam cells could contain intracellularly more than a small amount of the
phospholipid present in the atherosclerotic
intima. In the metabolic experiments for
example, if one considers that all the P"phospholipid present in the supernatant arose
from disrupted cells, and this cannot of
course be asserted on the data, it can be calculated that the actively metabolizing foam
cells could account for 10 to 20% of the phospholipid in the intima. This is in contrast
to histochemical data presented previously,1
in which it was shown that much of the
phospholipid present in the rabbit atherosclerotic aorta was present intracellularly in
foam cells. The data, therefore, with regard
to phospholipid distribution in the aorta would
support the conclusion of Adams et al. who
have reported histochemical work demonstrating that phospholipid is distributed predominantly extracellularly in the rabbit lesion.
It is possible that foam cells contribute to
the phospholipid accumulation in the atherom-
DAY, NEWMAN, ZILVERSMIT
130
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atous arterial wall29'80 in two ways. Phospholipid synthesized by such active metabolic
cells as have been obtained in the present
experiments may be transferred to the extracellular space. However, very little P"-labeled
phospholipid appeared in the medium in the
in vitro experiments, but conditions in the
intima may differ from those in the medium.
The second possibility is that foam cells degenerate and become necrotic following thenactive metabolic phase thus leading to the
accumulation of extracellular phospholipid. On
the other hand, degenerated cells may contain much phospholipid, demonstrable histochemically, but fall apart when the aorta is
disrupted with collagenase, etc.
The incorporation of P"-phosphate into individual phospholipids by the isolated foam
cells was almost identical with the incorporation of Pst-phosphate into individual phospholipids by the intact aortic intima.20 This
could be because the foam cells were responsible for most of the incorporation of
PJ* into phospholipid that occurs in the aorta,
but this conclusion cannot be supported on
the basis of the data in table 5, unless one
considers that the yield of cells obtained in
these experiments was relatively small.
In the present paper it can be concluded
that phospholipid can be synthesized by the
foam cells present in the atheromatous lesion.
The relative contribution to the synthesis of
phospholipid by the intima as a whole cannot
be assessed satisfactorily, however, until the
questions of yield of cells, possible disruption during preparation, and the synthesis of
phospholipid by other components of the intima are determined.
Acknowledgment
We thank Mrs. P. Thompson and Miss R. Ward
for technical assistance.
atheromatous plaques. J. Atherosclerosis Res.
4: 144, 1964.
4. GEEB, J. C.: Fine structure of canine experimental atherosclerosis. Am. J. Pathol. 47:
241, 1965.
5. DAY, A. J., AND FIDCE, N. H.: The uptake and
metabolism of ^C-labeled fatty acids by
macrophages in vitro. J. Lipid Res. 3: 333,
1962.
6. DAY, A. J., AND FIDCE, N. H.: The incorpora-
tion of ^C-labeled acetate into lipids by
macrophages in vitro. J. Lipid Res. 5: 163,
1964.
7. DAY, A. J., FmcE, N. H.,
11.
3. DUNNICAN, M. G.:
pholipid
within
The distribution
macrophages
in
12. NEWMAN, H. A. I., Liu, C. T., AND ZTLVERSMTT, D. B.: Evidence for the physiological
occurrence of lysolecithin in rat plasma. J.
Lipid Res. 2: 403, 1961.
13. BAHTLETT, G.
R.:
Phosphorus
assay
in
col-
umn chromatography. J. Biol. Chem. 234:
466, 1959.
14. SKTPSKL, V. P., PETERSON, R. F., AND BARCLAY,
M.: Quantitative analysis of phospholipids by
thin layer chromatography. Biochem. J. 90:
374, 1964.
15. ZLATKIS, A., ZAK, B., AND BOYLE, A. J.: A new
method for the direct determination of serum
cholesterol. J. Lab. Clin. Med. 4 1 : 486, 1953.
16. ABELL, L. L., LEVY, B. B., BRODIE, B. B., AND
KENDALL, F. E.: A simplified method for the
estimation of total cholesterol in serum and
demonstration of its specificity. J. Biol. Chem.
195: 357, 1952.
17. VAN HANDEL,
E.:
Separation
and
chemical
assay of lipid classes. J. Am. Oil Chemists'
Soc. 36: 294, 1959.
18.
MANGOLD, H. K., AND MALINS, D. C : Fractiona-
tion of fats, oils and waxes on thin layers of
silicic acid. J. Am. Oil Chemists' Soc. 37:
383, 1960.
of phos-
human
FOLCH, J., LEES, M., AND SLOANE STANLEY,
G. H.: A simple method for the isolation and
purification of total lipides from animal tissue. J. Biol. Chem. 226: 497, 1957.
References
1. DAY, A. J.: The relationship of arterial macrophages to the phospholipid content in rabbit
atheroma. J. Atherosclerosis Res. 2: 350, 1982.
2. DAY, A. J.: The macrophage system, lipid
metabolism and atherosclerosis. J. Athersclerosis Res. 4: 117, 1964.
AND WELKTNSON,
G. N.: Effect of cholesterol in suspension on
the incorporation of phosphate into phospholipid by macrophages in vitro. J, Lipid Res.
7: 132, 1966.
8. ELSBACH, P.: Uptake of fat by phagocytic cells.
An examination of the role of phagocytosis.
II. Rabbit alveolar macTophages. Biochim.
Biophys. Acta 98: 420, 1965.
9. RODBELL, M.: Metabolism of isolated fat cells.
I. Effects of hormones of glucose metabolism
and lipolysis. J. Biol. Chem. 239: 375, 1964.
10. HANKS, J. H.: The longevity of chick tissue
cultures without renewal of media. J. Cellular
Comp. Physiol. 31: 235, 1948.
19.
GORDON, C. F., AND WOLFE, A. L.:
Liquid
Circulinon Raeucb, Vol. XTX, July 1966
131
PHOSPHOLIPID SYNTHESIS BY FOAM CELLS
scintillation counting of aqueous
Analyt. Chem. 32: 574, 1960.
20.
samples.
NEWMAN, H. A. I., DAY, A. J., AND ZILVER-
25. STILL, W. J. S.: An electron microscope study
of cholesterol atherosclerosis in the rabbit.
Exptl. Mol. Pathol., suppl. 2: 491, 1963.
SMTT, D. B.: In vitro phospholipid synthesis
in normal and atheromatous rabbit aortas. Circulation Res. 19: 132, 1966.
26.
21. DITTMEB, J. C , AND LESTER, R. L.: A simple,
27.
specific spray for the detection of phospholipids on thin layer chromatograms. J. Lipid
Res. 5: 126, 1964
22. SNYDER, F.: Radio-assay of thin layer chromatograms: A high-resolution zonal scraper for
quantitative C1* and H 3 scanning of thin
layer chromatograms. Analyt. Biochem. 9:
183, 1964.
Downloaded from http://circres.ahajournals.org/ by guest on June 16, 2017
Circulation Raewch. VoL XIX, July 1966
on
RABINOWITZ, Y.:
Separation
of
lymphocytes,
polymorphonuclear leucocytes and monocytes
on glass columns including tissue culture observations. Blood 23: 811, 1964.
28. ADAMS, C. W. M., BAYLJSS, O. B., AND IBABHTM,
M. Z.: The distribution of lipids and enzymes
in the aortic wall in dietary rabbit atheroma
and human atherosclerosis. J. Pathol. Bacteriol.
86: 421, 1963.
29.
ZELVERSMIT, D. B., SHORE, M. L., AND ACKER-
MAN, R. F.: The origin of aortic phospholipid
in rabbit atheromatosis. Circulation 9: 581,
1954.
identification and analysis of phosphatides. J.
Lipid Res. 3: 1, 1962.
matography of lipids in the presence of an
antioxidant, 4-methyl-2, 6 ditert.-butylphenol.
J. Chromatog. 14: 405, 1964.
drugs
intracellular tubercle bacilli. J. Pathol. Bacteriol. 64: 429, 1952.
23. MARINETTI, G. V.: Chromatographic separation,
24. WHEN, J. J., AND SZCZEPANOWSKA, A. D.: Chro-
MACKANESS, G. B.: The action of
30.
ZLLVERSMTT, D. B., AND MCCANDLESS, E. L.:
Independence of arterial phospholipid synthesis from alterations in blood lipids. J. Lipid
Res. 1: 118, 1959.
Synthesis of Phospholipid by Foam Cells Isolated from Rabbit Atherosclerotic Lesions
ALLEN J. DAY, H. A. I. NEWMAN and D. B. ZILVERSMIT
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Circ Res. 1966;19:122-131
doi: 10.1161/01.RES.19.1.122
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