339
Atherosclerosis, 28 (1977) 339-353 0 Elsevier/North-Holland Scientific Publishers, Ltd.
EFFECT OF HYPERLIPEMIC SERUM LIPOPROTEINS ON THE LIPID ACCUMULATION AND CHOLESTEROL FLUX OF RABBIT AORTIC MEDIAL CELLS
ROBERT M. CHEN and KATTI FISCHER-DZOGA Department of Pathology and Specialized Center for Research in Atherosclerosis, University of Chicago, Chicago, Ill. 60637 (U.S.A.) (Received 31 May, 1977) (Revised, received 5 August, 1977) (Accepted 15 August, 1977)
Summary The effects of hyperlipemic rabbit serum and its lipoproteins on the lipid accumulation in cultured rabbit aortic medial cells were evaluated in this study. Hyperlipemic serum stimulated the accumulation of cholesterol esters in these cells. Cells exposed to 540% normal serum had equivalent amounts of cholesterol ester, while cells exposed to only 2% hyperlipemic serum showed a 2-5 fold increase in cholesterol ester. This happened in spite of the fact that 2% hyperlipemic serum contained much less cholesterol than did 40% normal serum. Most of the cholesterol ester accumulation occurred in the initial 12 h and it could be reproduced by incubating these cells in a medium containing hyperlipemic low density lipoproteins. Cells exposed to low concentrations of hyperlipemic serum, as well as those in high concentrations of normal serum, also showed up to a 2-fold increase of free cholesterol and a smaller increase of triglycerides. This appeared to be a function of increased lipid level in the culture medium, not a specific effect of hyperlipemic serum. Furthermore, culture media containing a high lipid level, either from hyperlipemic serum or a high concentration of normal serum, showed a progressive increase in free cholesterol and a concomitant decline of cholesterol esters. These results indicated that the cells hydrolyzed a large amount of cholesterol ester. The present data do not permit a distinction to be drawn between hydrolysis in the cell and on the cell membrane. It is clear, however, that cells were required for hydrolysis and it is not due to enzymes excreted into the culture medium. This work is supported in part by USPHS Grants HL-15062 and GM-0093. Part of the data in this paper was incorporated into a paper which won the 1975 Young Investigators Award of American College of cardiologY.
340
Key words:
Hyperlipemia
-A therogenesis - Low
cells - Cholesterol ester accumulation
density lipoproteins - Aortic and hydrolysis
medial
Introduction Recently, attention has been focused on two major factors in the pathogenesis of atherosclerosis. First is the general recognition that the major cell involved in the pathological process is the smooth muscle cell [l-4]. This cell stores lipid [ 4,5], proliferates [ 6,7], synthesizes connective tissue elements and undergoes necrosis [S-lo] and thus contributes substantially to the lesions of atherosclerosis. Secondly, low density lipoproteins (LDL) appear to furnish most of the lipid in the atherosclerotic plaque [ 5,11-161. This is in agreement with the strong correlation between hyperlipemia and atherosclerosis, indicated both by epidemiological and experimental studies [4,5,17]. Lipid accumulation in artery wall cells has been considered one of the earliest events in atherogenesis [12--141. However, it is extremely difficult to study the kinetics of atherogenesis in the lesion itself. The in vitro experiments included in this report demonstrate some of the kinetics of lipid flux and accumulation in aortic medial cells as influenced by hyperlipemic serum and its LDL fraction. Studies concerning the effects of hyperlipemic serum on cell proliferation and increased cell death [18], cell lipid metabolism changes [19-211 and some of the mechanisms involved [22] are presented elsewhere. In this particular study, subcultured cells from the aortic media of normal adult rabbits, in a relatively stationary phase of growth, were exposed to various concentrations of either normal or hyperlipemic rabbit serum, as well as serum lipoprotein fractions. The accumulation of various lipids in cells exposed to hyperlipemic serum or its lipoproteins was compared to that of cells exposed to high concentrations of normal serum and its similarly prepared lipoproteins. The simultaneous increases and decreases in lipids in the culture media were also measured to provide an indication of the flux of lipids between the cells and the culture media.
Materials and methods Hyperlipemic serum Hyperlipemia was induced by feeding normal adult male rabbits ad libitum with an atherogenic diet for at least 1 month. The diet, which has been used for in vivo induction of atherosclerosis in rabbits, consisted of normal rabbit chow enriched with 10% sunflower oil and 0.4% cholesterol [23]. Blood was collected from rabbits, after overnight fasting, by heart puncture without anesthesia. Serum was obtained by centrifugation at 250 X g for 10 min after blood clot retraction, Routinely, serum was inactivated at 56°C for 30 min before adding it to the culture medium. Serum lipoproteins were fractionated and washed by sequential ultracentrifugation [24] at the following densities: VLDL p = 1.006; LDL p = 1.063, HDL p = 1.210. For chemical analysis the LDL was further fractionated into LDLl p = 1.006-1.019 and LDLz p =
341
1.019-1.063. Their purity was checked by agarose thin film electrophoresis [25], and the peptide pattern studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [26]. The lipid and protein contents of each lipoprotein fraction were determined as described below. Cells
To prepare the cultured aortic medial cells, the aortas were removed aseptically from normal adult male rabbits. The adventitia with the outer third of the media and the intima were carefully stripped off. The remaining inner two-thirds of the media of each aorta were cut into 100-150 pieces, approximately 1 mm in greatest cross-section and transferred to 250 ml plastic flasks (Falcon), with about 20 pieces in each flask. These explants were nourished with Eagle’s basal medium [27] supplemented with 5% normal rabbit serum. The culture medium was changed twice a week. Cell outgrowth was generally visible within 1 week and the cells grew almost to confluency by 1 month. They were then dislodged with 2 ml of 0.25% trypsin in a calcium and magnesium-free Hank’s basal salt solution. The dispersed cells were harvested by centrifugation at 250 X g for 10 min, resuspended in culture medium and transferred to new 30 ml Falcon flasks containing 3 ml of fresh culture medium. The number of cells seeded was approximately 10,000 cells per cm* of the flask bottom area. The initial cell doubling time varied from 40 to 48 h during the log phase of growth. These cells grew as a confluent monolayer on the bottom of the flasks and reached a relatively stationary phase of growth in 2 weeks. They exhibited bundles of myofilaments with fusiform dense body attachments, basal lamellae, other features which characterized them as smooth muscle cells [ 18,281. To test the effect of hyperlipemic serum and its lipoproteins on these cells, various concentrations of serum and lipoproteins were added to the basal medium. Cells were exposed to these enriched media for a specified period. At the end of each experiment, the culture media were saved for further chemical analysis. Cells were recovered by trypsinization and centrifugation at 250 X g for 10 min, twice washed with phosphate buffered saline (PBS), and resuspended in 0.6 ml of PBS by drawing into a tuberculin syringe through a No. 25 needle 5 times. From the 0.6 ml of cell suspension, 2 samples of 50 ~1 each were utilized for cell count and protein determination respectively, and the remainder was injected into lipid extraction solutions for further analysis. Chemical analysis The protein of cells, serum, and serum lipoprotein fractions was determined according to Lowry et al. [29]. Lipids were extracted with 20 vol of 2 : 1 mixtures of chloroform and methanol, and washed with 2 M potassium chloride solution and distilled water according to Tarlov and Kennedy [30]. Standard calorimetric procedures were used for the determination of total cholesterol [31], triglycerides [32] and lipid phosphorus [33]. Cholesterol ester and free cholesterol were separated by silica gel thin-layer chromatography as described below and quantitated by fluorometry according to Bondjers and Bjiirkerud [ 341, or by densitometry as described below. For the separation of neutral lipids, Silica Gel G thin-layer plates (250 pm
342
thick, 20 cm X 20 cm glass plates, Brinkmann or Analtech) were prewashed in chloroform overnight and preactivated in a 110” C oven for 30 min immediately before use. Lipid extracts from cells or culture media were dried in small tubes in a vacuum oven or under a stream of nitrogen. Two hundred ~1 of chloroform-methanol (2 : 1, v/v) were added to each tube containing dried lipids, which were then redissolved in a Vortex mixer. These tubes were capped with rubber tops and kept on ice to prevent evaporation of solvents. Eighty ~1 lipid extracts were spotted in duplicate on thin-layer plates, as 1 cm long horizontal streaks, 1 cm apart from one another and 2 cm above the bottom of the plate. Usually 4 samples were spotted on the left half of the plates and 4 reference standards were spotted on the right, consisting of lipid mixtures ranging in amount from 0.5 to 20 Ergof each lipid class. The developing solvent system for neutral lipids was modified from Skipski et al. [35] and Freeman and West [36]. The plates were first developed in a solvent containing di-isopropyl ether-benzene-lacial acetic acid (48 : 48 : 4, v/v/v) to within 8 cm of the top of the plate. After being dried in air for 10 min, these plates were developed in a second solvent containing petroleum ether-diethyl etheracetic acid (95 : 5 : 0.5, v/v/v) to within l-2 cm of the top of the plate. The neutral lipids were separated from top to bottom in the following sequence: hydrocarbons, cholesterol ester, fatty acid methyl ester, triglycerides, free fatty acids, diglycerides, free cholesterol, monoglycerides. The phospholipids remained at the origin. The lipid extracts from serum and from cultured cells contained recognizable amounts of cholesterol ester, triglycerides, free cholesterol, phospholipid and trace amounts of free fatty acids. The chromatographically separated lipids were visualized by spraying the plates evenly with 10% of phosphomolybdic acid in ethanol, and heated in a 120°C oven for 30 min. Lipid quantities as small as 0.5 pg may appear as purple blue spots on the plate and the optical density is proportional to the amount of lipid. These spots were scanned in a Zeiss PMQ II reflectance chromatoscan according to Stahl [37]. The results of densitometry are consistent with those obtained from standard calorimetric procedures for triglycerides and lipid phosphorus, and with fluorometric procedure for cholesterol, as described above. Results (1) Hyperlipemic serum Chemical analysis of the hyperlipemic rabbit serum revealed an elevated level of cholesterol, both in free and esterified form, that exceeded those of other lipid classes. The lipid content of the hyperlipemic serum was as follows: cholesterol esters, 982 f 224 mg% (normal 81 f 17 mg%); free cholesterol, 320 + 56 mg% (normal 26 + 4 mg%); triglyceride, 162 + 58 mg% (normal 98 + 26 mg%); free fatty acids, 26 + 5 mg% (normal 12 + 3 mg%); phospholipid 608 f 53 mg% (normal 104 f 16 mg%). Ultracentrifuge analysis of the lipoproteins in the hyperlipemic serum revealed a striking increase in VLDL and LDL especially the density fraction p = 1.006-1.019, and a slight decrease in HDL (Table 1). SDS-polyacrylamide electrophoresis revealed that in addition to usual apo B in LDL, and the usual small peptides in VLDL, a peptide of
343
TABLE LIPID
I AND PROTEIN
COMPOSITION
OF RABBIT
SERUM
LIPOPROTEINS
The amount is expressed as mg/lOO ml of serum. The data are the means of analysis of 3 batches of normal and hyperlipemic sera. The variation between batches was within 20%. Lipoproteins were fractionated by sequential ultracentrifugation at the following densities: VLDL, p = 1.006; LDLI, p = 1.019; LDL2. p = 1.063; HDL, P = 1.210. Lipoprotein fractions Normal VLDL LDLl LDL2 HDL HLP VLDL LDL, LDL2 HDL
TABLE
Cholesterol ester
Free cholesterol
2.0 10.2 11.8 54.2
Triglyceride
0.5 2.8 3.5 18.6
310 428 169 82
Phospholipid
5.2 20.2 12.4 58.2
98 104 72 34
2.1 8.1 7.4 83.5
42 38 34 24
Protein
0.5 4.1 6.8 136.5
160 208 88 45
42.4 72.2 50.2 98.5
2
LIPID CONTENT HYPERLIPEMIC
OF RABBIT SERUM
AORTIC
CELLS
AND CULTURE
MEDIA
CONTAINING
NORMAL
OR
The amount of lipid in the cells is expressed as pg of lipid/mg of ceII protein, and that of medium is expressed as mg%. Each data point Is the mean of 3 flasks in one extierhnent. The coefficient of variation for cholesterol ester and free cholesterol is approximately + lo%, while that of triglyceride and phospholipid is f l(r201. Cells were grown in control serum for 2 months, and were subcultured with trypsin twice during that period. These cells were then exposed to basal media enriched with various concentrations of normal and hyperlipemic serum for 40 h. Three similar experiments showed UP to 20% difference of the mean value of ceII lipid content, but the response to the change of serum and its concentration leads to the same conclusion. Concentrations of serum in culture media
Cholesterol
Control (5% normal)
13
10% normal
10
20% normal
8
16
40% normal
13
32
42 a
2% HyperIipemic + 5% normal
26 a
24
44 a
1.7
394 a
5% HyperIIpemic + 5% normal
21 a
53
44 a
7.1
380a
10% Hyperlipemic + 5% normal
28a
102
43 a
365b
CeII
a P < 0.01 as compared b P < 0.05 as compared
ester Medium
Free cholesterol CeII
4.1
22
8.2
to control. to control.
Medium
Triglycerides
Phospholipids
CeII
Cell
Medium
Medium
1.3
256
24
2.6
250
10
309
23
5.2
326b
20
205b
21
4908
39
349
42
260
18
13
223
36
21
315
66
10
33
4.9
8.1
306
5.2 10
344
approximate molecular weight of 36,000 was dominant and VLDL, but not evident in normal serum counterparts
in hyperlipemic [Ml.
LDL
(2) Effect of hyperlipemic serum on cell lipid content To test the effect of hyperlipemic serum on lipid accumulation by aortic medial cells, subcultured cells that were grown in 5% normal serum for 2 weeks were incubated in media containing 5-40% normal or 2-10% hyperlipemic serum plus 5% normal serum for 40 h. Cells exposed to 40% normal serum showed only small changes in cholesterol ester content (Table 2), even though the culture medium had 8 times more cholesterol per ml than the control medium containing 5% normal serum. In another group, cells exposed to 2% hyperlipemic serum plus 5% normal serum showed a more than 2-fold increase in cholesterol ester content, although the cholesterol level in this medium is much lower than that in 40% normal serum. Cells further exposed to 5% or 10% hyperlipemic serum under the same condition showed equally large increases of cholesterol esters. These results clearly indicate that the increment of cholesterol ester in these cells is not purely a function of cholesterol content in the culture medium, but is influenced by some other factors in hyperlipemic serum such as changes in its LDL peptide or an increase in the arginine rich peptide. Cells exposed to 40% normal serum showed an 80% increase of free cholesterol, which approaches that in cells exposed to 2-10% hyperlipemic serum. This might be a function of cholesterol content in the LDL as well as that in the whole culture medium. The triglyceride content of cells exposed to hyperlipemic serum was notably higher than that in the control group but less than that in cells exposed to 40% normal serum. This appears to be a function of triglyceride level in the culture medium. The lipid phosphorus of these cells was variable and exhibited no definite correlation to concentration of serum or type of serum in the culture media. (3) Effect of hyperlipemic lipoproteins on cell lipid content Hyperlipemic serum was fractionated and washed at the densities of 1.006 (VLDL), 1.063 (LDL) and 1.210 (HDL) sequentially. The lipoproteins were added to the basal medium in amounts equivalent to those present in 10% of hyperlipemic serum, and cells were incubated in these enriched media for 12 h, 36 h and 60 h, respectively (Table 3). Cells exposed to hyperlipemic lipoproteins showed an accumulation of all the major lipid classes, with the exception of triglycerides in cells incubated in hyperlipemic LDL. Cholesterol ester and free cholesterol accumulation was predominant in cells exposed to LDL, while triglycerides were predominant in cells exposed to VLDL and phospholipid in those exposed to HDL. In one experiment, normal rabbit serum lipoproteins were fractionated at the same densities as was hyperlipemic serum. The culture media containing 5% normal serum were enriched with these various lipoprotein fractions to a final cholesterol concentration of 300pg/ml. Cells exposed to hyperlipemic VLDL and LDL showed the highest increment of cholesterol ester and free cholesterol (Table 4). These increments notably exceeded those in cells exposed to normal lipoproteins of the same cholesterol content. This result indicated that some
345 TABLE
3
LIPI? COMPOSITION LIPOPROTEINS
OF
RABBIT
AORTIC
CELLS
AND
MEDIA
CONTAINING
HYPERLIPEMIC
The amount of lipid in the cells is expressed as pg of Iipid/mg of cell protein, and that of medium is expressed as mg%. Each data point is the mean of 3 flasks in one experiment. Cells were originally grown in control medium. consisting of Eagle’s basal medium and 5% normal serum and then exposed to media enriched with hyperlipemic VLDL (p = 1.006). LDL (p = 1.006-1.063). or HDL (p = 1.063-1.210) in amounts equivalent to that present in 10% hyperlipemic serum for 12-60 h as stated. Lipoproteins in culture media Control
Cholesterol CeU
ester Medium
8.8
Free cholesterol Medium
Cell
Phospholipids
Cell
CeII
Medium
Medium
4.2
21.7
203
4.8
196
VLDL
12h 36 h 60 h
24.4 26.1 27.3
a a a
34.0
33.5 38.6 39.3
b b b
10.5
335 b 361 a 375 a
9.2
233 b 256 b 265 b
22.0
LDL
12 h 36 h 60 h
46.7 50.4 47.9
a a a
62.0
52.4 54.9 55.4
a a a
19.0
217 193 195
12.2
290 a 306 a 306 a
34.2
HDL
12h 36 h 60 h
12.8 13.5 12.9
12.0
27.8 27.7 28.3
7.4
342 a 347 a 363 a
9.6
a P < 0.01 b P < 0.05
as compared as compared
1.2
Triglycerides
4.6
275b 275 b 276 b
5.0
to control. to control.
properties (peptide pattern? protein-lipid combination?) of the hyperlipemic LDL fraction, in addition to its high cholesterol content, account for the cholesterol accumulation in these cells. (4) Sequential measurement of lipid accumulation in cells incubated in hyperlipemic serum We studied the accumulation rate of each class of lipids in these cells. Cells were incubated in various concentrations of normal and hyperlipemic serum from 0 to 48 h. The content of cholesterol ester was not changed by growth in 20% serum, but cells grown in 40% normal serum showed a slight increase of cholesterol esters (Fig. la). When these cells were exposed to only 2% hyperlipemic serum, the cell cholesterol ester content increased within 4 h of incubation and reaching a 24 h level 5 times higher than the initial level. Cells growing in 5% and 10% hyperlipemic serum had a similar but slightly higher degree of cholesterol ester accumulation. The major effect of exposure to higher levels of hyperlipemic serum was the larger initial increment in this ester accumulation. Cells incubated in 5% and 20% normal rabbit serum had similar free cholesterol levels throughout. Cells grown in 40% normal serum had substantially more free cholesterol and cells grown in 2%, 5% and 10% of hyperlipemic serum had an almost 2-fold increase of free cholesterol (Fig. lb). The time course and level of free cholesterol accumulation was not influenced by the level of hyperlipemic serum. It is noteworthy that more than 60% of the increase was achieved within the initial 4 h of incubation. In this part of the study, the free cholesterol increased only 2-fold as contrasted to a 5-fold increase of cholesterol esters.
TABLE
4
LIPID
COMPOSITION
OF RABBIT
AORTIC
CELLS
EXPOSED
TO NORMAL
OR HYPERLIPEMIC
LIPOPROTEINS The amount of lipid is expressed as pg of lipid/mg of ceII protein. Each data point is the mean of 3 flasks in one experiment. CeIIs were originally grown in Eagle’s basal medium containing 5% normal rabbit serum. and then exposed to media enriched with normal or hyperlipemic VLDL (p = 1.006). LDL 1.006-1.063) and HDL (p = 1.063-1.210) for 40 h. The cholesterol content of aII the culture media were adjusted to be 300 pl/mI. Cholesterol ester
Lipoproteins added
Free cholesterol
Triglyceride
Phospholipid
Control
10.2
28.5
182
270
Normal VLDL LDL HDL
12.2 11.5 13.0
31.5 36.6 34.1
410 a 340 a 245
310 275 410 b
HIP
18.6 a 21.3 a 12.4
48.4 a 52.1 a 41.0 b
298 b 234 248
265 298 350
VLDL LDL HDL
a P < 0.01 as compared b P < 0.05 as compared
to control. to control.
Cholesterol
a.
esters
b.
60
Free
cholesterol
60 IO%
HlpS
50
5% HIPS 50 2% HlpS
40
40
30
30
HlpS HlpS Hl;S NRS
/-•
20 40% NRS 20% NRS 1
c.
d.
Triglycerides
300
40% NRS 10% HlpS 5% Hips 20% NM
_a_----
;-I:--.-~-.--.______~_.
:
200
IO
._*______ __-. ,.-__ -IF .
300
NRS
20
10
400
20%
2% HIpS
*__.--
Phospholipids
_-a.__
--..__
.--mm.
1’
!‘,L..._ _--...::z;,
, ,,,r--
200
10%
HlpS
5% HIPS 2% H,pS
--*=.-_::::
.‘.*
??
40%
NRS
??
20%
NRS
100 100
1
1
4 0
1
1
24
4
4; DURATION
OF
INCUBATION
I
,
a
24
I
48
(hours)
Fig. 1. Sequential accumulation of (a) cholesterol ester, (b) free cholesterol, (c) triglycerides, and (d) phospholipids in rabbit aortIc medial ceIIs exposed to various concentrations of normal or hyperIipemIc rabbit serum. These ceils were 0-y grown in Eagle’s basal medium containing 5% normal rabbit serum and exposed to media containing 2040% normal serum or 2-1046 hyperllpemic serum ~1~s 5% normal serum for up to 48 h. Each flask contained about 8 X 105 cells In 2.5 ml of culture medium.
341
The triglycerides showed less than a 2-fold increase (Fig. lc) in contrast to a 2-5-fold increase of cholesterol ester, and this increment took place at a slower rate with an initial lag phase. Furthermore, the triglyceride accumulation was noted at least as much in cells grown in normal serum as that in hyperlipemic serum, apparently reflecting the triglyceride content of the medium in which the cells were grown. The phospholipid content of cells exposed to hyperlipemic sera changed a little with increased time of incubation (Fig. Id). (5) Sequential change of lipids in culture medium The culture media containing 5-40% normal serum and 2-10’S hyperlipemic serum were analyzed 4-48 h after incubation with cells. The most
IO00
1 Cholesterol
a.
.
esters
500
800
___---
400
??
57 HlpS
,__--’
600
=
400
F 4 z a”
40"1 NRS
200
27
100
2 2
1
f E a. ;
500
s
400
G 2 5
c.
*. '.
300
t- =.
‘-,.
.--___
1 r
I
4
a
d.
Phospholipids _____----'
NRS
'-a*
6oo
.
400
--
200
-
I
_I'
:
_'
-'
??
---___
.--._ 10%
HlpS
--*--__m_
---.*
5% HIPS
--- -_..
I
48 DURATION
409
NRS
__c_.____-----*
HIPS
20% NRS 2% HlpS
2% HlpS I
24
..__e-----
HlpS
5%
___*_______~___---------~*
. --.___
10%
.*
*. ... .
1
I
I
a00 -
Triglycerides
--•
100
I
I
40% '\
200
HlpS
2O'x NRS
I
4 OF
INCUBATION
a
WITH
24 CELLS
48
(hours)
Fig. 2. Sequential change of (a) cholesterol esters, (b) free cholesterol, (c) triglycerides and (d) phospholipids in culture media containing 2040% normal rabbit serum or 2-10% hyperlipemic rabbit serum plus 6% normal rabbit serum, incubated with rabbit aortic medial cells for up to 48 h. Each flask contained 2.5 ml of culture medium incubated with about 8 X 105 cells.
348
pronounced changes were observed with time in the concentration of cholesterol ester, which decreased considerably in all media, but which was proportionally higher in media containing higher concentrations of serum cholesterol (Fig. 2a). Changes of the free cholesterol in the culture media (Fig. 2b) contrasted greatly to those of cholesterol ester. The content of free cholesterol did not decrease during the incubation with cells, but those media containing high concentrations of cholesterol showed a considerable increase of this lipid subclass. In all media containing normal or hyperlipemic serum, there was some decrease in triglycerides (Fig. 2~). There was no remarkable change of phospholipids, except a very small but consistent increase in phospholipids during the 48 h incubation, notably in medium containing 10% hyperlipemic serum (Fig. 2d). (6) Reversal of lipid accumulation Cells exposed to 2040% normal serum or 2-10% hyperlipemic serum plus 5% normal serum for 2 days were returned to control medium (5% normal serum) for another 48 h to see how much of the accumulated lipids could be removed. At the end of a 2day exposure to these variously enriched media, the cells showed increments of cholesterol ester, free cholesterol and triglyceride (Table 5), consistent with experiments described earlier. The elevated cellular triglycerides and free cholesterol declined toward control level after 48 h incubation in control medium. However, the return of cholesterol esters to control levels was less complete in 48 h. About 20-30% of the excess cholesterol esters remained in the cells.
TABLE 5 REVERSAL
OF LIPID ACCUMULATION
IN RABBIT AORTIC MEDIAL CELLS
The amount of lipid is expressed as pg lipid/mg of ceII protein. Each data point is the mean of 3 flasks. Cells were exposed to these variously enriched media for 48 h. Some of the cells were harvested and the remainder were returned to control medium consisting of EagIe’s basal medium and 5% normal serum for 2 days. Concentration of serum in media
Cholesterol ester
Free cholesterol
Start
Start
5% normal serum
8.5
20% normal serum
8.9
End
End
26.5
start
End
192
Phospholipids start
End
176 179
170
40% normal serum
13.9 b
10.4 d
46.5 a
29.5 c
342 a
190 c
194
182
2% Hip serum + 5% normal serum
48.3 a
16.2 c
48.3 a
30.6 c
278 b
210 c
208
168 d
5% Hlp serum + 5% normal serum
48.6 a
20.4 c
50.6 a
28.4 c
240
189 d
199
178
10% Hip serum + 6% normal serum
41.8 a
21.2 c
43.5 a
30.6 c
310 a
210 c
262 a
189 d
a b c d
P P P P
< < <
Atherosclerosis, 28 (1977) 339-353 0 Elsevier/North-Holland Scientific Publishers, Ltd.
EFFECT OF HYPERLIPEMIC SERUM LIPOPROTEINS ON THE LIPID ACCUMULATION AND CHOLESTEROL FLUX OF RABBIT AORTIC MEDIAL CELLS
ROBERT M. CHEN and KATTI FISCHER-DZOGA Department of Pathology and Specialized Center for Research in Atherosclerosis, University of Chicago, Chicago, Ill. 60637 (U.S.A.) (Received 31 May, 1977) (Revised, received 5 August, 1977) (Accepted 15 August, 1977)
Summary The effects of hyperlipemic rabbit serum and its lipoproteins on the lipid accumulation in cultured rabbit aortic medial cells were evaluated in this study. Hyperlipemic serum stimulated the accumulation of cholesterol esters in these cells. Cells exposed to 540% normal serum had equivalent amounts of cholesterol ester, while cells exposed to only 2% hyperlipemic serum showed a 2-5 fold increase in cholesterol ester. This happened in spite of the fact that 2% hyperlipemic serum contained much less cholesterol than did 40% normal serum. Most of the cholesterol ester accumulation occurred in the initial 12 h and it could be reproduced by incubating these cells in a medium containing hyperlipemic low density lipoproteins. Cells exposed to low concentrations of hyperlipemic serum, as well as those in high concentrations of normal serum, also showed up to a 2-fold increase of free cholesterol and a smaller increase of triglycerides. This appeared to be a function of increased lipid level in the culture medium, not a specific effect of hyperlipemic serum. Furthermore, culture media containing a high lipid level, either from hyperlipemic serum or a high concentration of normal serum, showed a progressive increase in free cholesterol and a concomitant decline of cholesterol esters. These results indicated that the cells hydrolyzed a large amount of cholesterol ester. The present data do not permit a distinction to be drawn between hydrolysis in the cell and on the cell membrane. It is clear, however, that cells were required for hydrolysis and it is not due to enzymes excreted into the culture medium. This work is supported in part by USPHS Grants HL-15062 and GM-0093. Part of the data in this paper was incorporated into a paper which won the 1975 Young Investigators Award of American College of cardiologY.
340
Key words:
Hyperlipemia
-A therogenesis - Low
cells - Cholesterol ester accumulation
density lipoproteins - Aortic and hydrolysis
medial
Introduction Recently, attention has been focused on two major factors in the pathogenesis of atherosclerosis. First is the general recognition that the major cell involved in the pathological process is the smooth muscle cell [l-4]. This cell stores lipid [ 4,5], proliferates [ 6,7], synthesizes connective tissue elements and undergoes necrosis [S-lo] and thus contributes substantially to the lesions of atherosclerosis. Secondly, low density lipoproteins (LDL) appear to furnish most of the lipid in the atherosclerotic plaque [ 5,11-161. This is in agreement with the strong correlation between hyperlipemia and atherosclerosis, indicated both by epidemiological and experimental studies [4,5,17]. Lipid accumulation in artery wall cells has been considered one of the earliest events in atherogenesis [12--141. However, it is extremely difficult to study the kinetics of atherogenesis in the lesion itself. The in vitro experiments included in this report demonstrate some of the kinetics of lipid flux and accumulation in aortic medial cells as influenced by hyperlipemic serum and its LDL fraction. Studies concerning the effects of hyperlipemic serum on cell proliferation and increased cell death [18], cell lipid metabolism changes [19-211 and some of the mechanisms involved [22] are presented elsewhere. In this particular study, subcultured cells from the aortic media of normal adult rabbits, in a relatively stationary phase of growth, were exposed to various concentrations of either normal or hyperlipemic rabbit serum, as well as serum lipoprotein fractions. The accumulation of various lipids in cells exposed to hyperlipemic serum or its lipoproteins was compared to that of cells exposed to high concentrations of normal serum and its similarly prepared lipoproteins. The simultaneous increases and decreases in lipids in the culture media were also measured to provide an indication of the flux of lipids between the cells and the culture media.
Materials and methods Hyperlipemic serum Hyperlipemia was induced by feeding normal adult male rabbits ad libitum with an atherogenic diet for at least 1 month. The diet, which has been used for in vivo induction of atherosclerosis in rabbits, consisted of normal rabbit chow enriched with 10% sunflower oil and 0.4% cholesterol [23]. Blood was collected from rabbits, after overnight fasting, by heart puncture without anesthesia. Serum was obtained by centrifugation at 250 X g for 10 min after blood clot retraction, Routinely, serum was inactivated at 56°C for 30 min before adding it to the culture medium. Serum lipoproteins were fractionated and washed by sequential ultracentrifugation [24] at the following densities: VLDL p = 1.006; LDL p = 1.063, HDL p = 1.210. For chemical analysis the LDL was further fractionated into LDLl p = 1.006-1.019 and LDLz p =
341
1.019-1.063. Their purity was checked by agarose thin film electrophoresis [25], and the peptide pattern studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis [26]. The lipid and protein contents of each lipoprotein fraction were determined as described below. Cells
To prepare the cultured aortic medial cells, the aortas were removed aseptically from normal adult male rabbits. The adventitia with the outer third of the media and the intima were carefully stripped off. The remaining inner two-thirds of the media of each aorta were cut into 100-150 pieces, approximately 1 mm in greatest cross-section and transferred to 250 ml plastic flasks (Falcon), with about 20 pieces in each flask. These explants were nourished with Eagle’s basal medium [27] supplemented with 5% normal rabbit serum. The culture medium was changed twice a week. Cell outgrowth was generally visible within 1 week and the cells grew almost to confluency by 1 month. They were then dislodged with 2 ml of 0.25% trypsin in a calcium and magnesium-free Hank’s basal salt solution. The dispersed cells were harvested by centrifugation at 250 X g for 10 min, resuspended in culture medium and transferred to new 30 ml Falcon flasks containing 3 ml of fresh culture medium. The number of cells seeded was approximately 10,000 cells per cm* of the flask bottom area. The initial cell doubling time varied from 40 to 48 h during the log phase of growth. These cells grew as a confluent monolayer on the bottom of the flasks and reached a relatively stationary phase of growth in 2 weeks. They exhibited bundles of myofilaments with fusiform dense body attachments, basal lamellae, other features which characterized them as smooth muscle cells [ 18,281. To test the effect of hyperlipemic serum and its lipoproteins on these cells, various concentrations of serum and lipoproteins were added to the basal medium. Cells were exposed to these enriched media for a specified period. At the end of each experiment, the culture media were saved for further chemical analysis. Cells were recovered by trypsinization and centrifugation at 250 X g for 10 min, twice washed with phosphate buffered saline (PBS), and resuspended in 0.6 ml of PBS by drawing into a tuberculin syringe through a No. 25 needle 5 times. From the 0.6 ml of cell suspension, 2 samples of 50 ~1 each were utilized for cell count and protein determination respectively, and the remainder was injected into lipid extraction solutions for further analysis. Chemical analysis The protein of cells, serum, and serum lipoprotein fractions was determined according to Lowry et al. [29]. Lipids were extracted with 20 vol of 2 : 1 mixtures of chloroform and methanol, and washed with 2 M potassium chloride solution and distilled water according to Tarlov and Kennedy [30]. Standard calorimetric procedures were used for the determination of total cholesterol [31], triglycerides [32] and lipid phosphorus [33]. Cholesterol ester and free cholesterol were separated by silica gel thin-layer chromatography as described below and quantitated by fluorometry according to Bondjers and Bjiirkerud [ 341, or by densitometry as described below. For the separation of neutral lipids, Silica Gel G thin-layer plates (250 pm
342
thick, 20 cm X 20 cm glass plates, Brinkmann or Analtech) were prewashed in chloroform overnight and preactivated in a 110” C oven for 30 min immediately before use. Lipid extracts from cells or culture media were dried in small tubes in a vacuum oven or under a stream of nitrogen. Two hundred ~1 of chloroform-methanol (2 : 1, v/v) were added to each tube containing dried lipids, which were then redissolved in a Vortex mixer. These tubes were capped with rubber tops and kept on ice to prevent evaporation of solvents. Eighty ~1 lipid extracts were spotted in duplicate on thin-layer plates, as 1 cm long horizontal streaks, 1 cm apart from one another and 2 cm above the bottom of the plate. Usually 4 samples were spotted on the left half of the plates and 4 reference standards were spotted on the right, consisting of lipid mixtures ranging in amount from 0.5 to 20 Ergof each lipid class. The developing solvent system for neutral lipids was modified from Skipski et al. [35] and Freeman and West [36]. The plates were first developed in a solvent containing di-isopropyl ether-benzene-lacial acetic acid (48 : 48 : 4, v/v/v) to within 8 cm of the top of the plate. After being dried in air for 10 min, these plates were developed in a second solvent containing petroleum ether-diethyl etheracetic acid (95 : 5 : 0.5, v/v/v) to within l-2 cm of the top of the plate. The neutral lipids were separated from top to bottom in the following sequence: hydrocarbons, cholesterol ester, fatty acid methyl ester, triglycerides, free fatty acids, diglycerides, free cholesterol, monoglycerides. The phospholipids remained at the origin. The lipid extracts from serum and from cultured cells contained recognizable amounts of cholesterol ester, triglycerides, free cholesterol, phospholipid and trace amounts of free fatty acids. The chromatographically separated lipids were visualized by spraying the plates evenly with 10% of phosphomolybdic acid in ethanol, and heated in a 120°C oven for 30 min. Lipid quantities as small as 0.5 pg may appear as purple blue spots on the plate and the optical density is proportional to the amount of lipid. These spots were scanned in a Zeiss PMQ II reflectance chromatoscan according to Stahl [37]. The results of densitometry are consistent with those obtained from standard calorimetric procedures for triglycerides and lipid phosphorus, and with fluorometric procedure for cholesterol, as described above. Results (1) Hyperlipemic serum Chemical analysis of the hyperlipemic rabbit serum revealed an elevated level of cholesterol, both in free and esterified form, that exceeded those of other lipid classes. The lipid content of the hyperlipemic serum was as follows: cholesterol esters, 982 f 224 mg% (normal 81 f 17 mg%); free cholesterol, 320 + 56 mg% (normal 26 + 4 mg%); triglyceride, 162 + 58 mg% (normal 98 + 26 mg%); free fatty acids, 26 + 5 mg% (normal 12 + 3 mg%); phospholipid 608 f 53 mg% (normal 104 f 16 mg%). Ultracentrifuge analysis of the lipoproteins in the hyperlipemic serum revealed a striking increase in VLDL and LDL especially the density fraction p = 1.006-1.019, and a slight decrease in HDL (Table 1). SDS-polyacrylamide electrophoresis revealed that in addition to usual apo B in LDL, and the usual small peptides in VLDL, a peptide of
343
TABLE LIPID
I AND PROTEIN
COMPOSITION
OF RABBIT
SERUM
LIPOPROTEINS
The amount is expressed as mg/lOO ml of serum. The data are the means of analysis of 3 batches of normal and hyperlipemic sera. The variation between batches was within 20%. Lipoproteins were fractionated by sequential ultracentrifugation at the following densities: VLDL, p = 1.006; LDLI, p = 1.019; LDL2. p = 1.063; HDL, P = 1.210. Lipoprotein fractions Normal VLDL LDLl LDL2 HDL HLP VLDL LDL, LDL2 HDL
TABLE
Cholesterol ester
Free cholesterol
2.0 10.2 11.8 54.2
Triglyceride
0.5 2.8 3.5 18.6
310 428 169 82
Phospholipid
5.2 20.2 12.4 58.2
98 104 72 34
2.1 8.1 7.4 83.5
42 38 34 24
Protein
0.5 4.1 6.8 136.5
160 208 88 45
42.4 72.2 50.2 98.5
2
LIPID CONTENT HYPERLIPEMIC
OF RABBIT SERUM
AORTIC
CELLS
AND CULTURE
MEDIA
CONTAINING
NORMAL
OR
The amount of lipid in the cells is expressed as pg of lipid/mg of ceII protein, and that of medium is expressed as mg%. Each data point Is the mean of 3 flasks in one extierhnent. The coefficient of variation for cholesterol ester and free cholesterol is approximately + lo%, while that of triglyceride and phospholipid is f l(r201. Cells were grown in control serum for 2 months, and were subcultured with trypsin twice during that period. These cells were then exposed to basal media enriched with various concentrations of normal and hyperlipemic serum for 40 h. Three similar experiments showed UP to 20% difference of the mean value of ceII lipid content, but the response to the change of serum and its concentration leads to the same conclusion. Concentrations of serum in culture media
Cholesterol
Control (5% normal)
13
10% normal
10
20% normal
8
16
40% normal
13
32
42 a
2% HyperIipemic + 5% normal
26 a
24
44 a
1.7
394 a
5% HyperIIpemic + 5% normal
21 a
53
44 a
7.1
380a
10% Hyperlipemic + 5% normal
28a
102
43 a
365b
CeII
a P < 0.01 as compared b P < 0.05 as compared
ester Medium
Free cholesterol CeII
4.1
22
8.2
to control. to control.
Medium
Triglycerides
Phospholipids
CeII
Cell
Medium
Medium
1.3
256
24
2.6
250
10
309
23
5.2
326b
20
205b
21
4908
39
349
42
260
18
13
223
36
21
315
66
10
33
4.9
8.1
306
5.2 10
344
approximate molecular weight of 36,000 was dominant and VLDL, but not evident in normal serum counterparts
in hyperlipemic [Ml.
LDL
(2) Effect of hyperlipemic serum on cell lipid content To test the effect of hyperlipemic serum on lipid accumulation by aortic medial cells, subcultured cells that were grown in 5% normal serum for 2 weeks were incubated in media containing 5-40% normal or 2-10% hyperlipemic serum plus 5% normal serum for 40 h. Cells exposed to 40% normal serum showed only small changes in cholesterol ester content (Table 2), even though the culture medium had 8 times more cholesterol per ml than the control medium containing 5% normal serum. In another group, cells exposed to 2% hyperlipemic serum plus 5% normal serum showed a more than 2-fold increase in cholesterol ester content, although the cholesterol level in this medium is much lower than that in 40% normal serum. Cells further exposed to 5% or 10% hyperlipemic serum under the same condition showed equally large increases of cholesterol esters. These results clearly indicate that the increment of cholesterol ester in these cells is not purely a function of cholesterol content in the culture medium, but is influenced by some other factors in hyperlipemic serum such as changes in its LDL peptide or an increase in the arginine rich peptide. Cells exposed to 40% normal serum showed an 80% increase of free cholesterol, which approaches that in cells exposed to 2-10% hyperlipemic serum. This might be a function of cholesterol content in the LDL as well as that in the whole culture medium. The triglyceride content of cells exposed to hyperlipemic serum was notably higher than that in the control group but less than that in cells exposed to 40% normal serum. This appears to be a function of triglyceride level in the culture medium. The lipid phosphorus of these cells was variable and exhibited no definite correlation to concentration of serum or type of serum in the culture media. (3) Effect of hyperlipemic lipoproteins on cell lipid content Hyperlipemic serum was fractionated and washed at the densities of 1.006 (VLDL), 1.063 (LDL) and 1.210 (HDL) sequentially. The lipoproteins were added to the basal medium in amounts equivalent to those present in 10% of hyperlipemic serum, and cells were incubated in these enriched media for 12 h, 36 h and 60 h, respectively (Table 3). Cells exposed to hyperlipemic lipoproteins showed an accumulation of all the major lipid classes, with the exception of triglycerides in cells incubated in hyperlipemic LDL. Cholesterol ester and free cholesterol accumulation was predominant in cells exposed to LDL, while triglycerides were predominant in cells exposed to VLDL and phospholipid in those exposed to HDL. In one experiment, normal rabbit serum lipoproteins were fractionated at the same densities as was hyperlipemic serum. The culture media containing 5% normal serum were enriched with these various lipoprotein fractions to a final cholesterol concentration of 300pg/ml. Cells exposed to hyperlipemic VLDL and LDL showed the highest increment of cholesterol ester and free cholesterol (Table 4). These increments notably exceeded those in cells exposed to normal lipoproteins of the same cholesterol content. This result indicated that some
345 TABLE
3
LIPI? COMPOSITION LIPOPROTEINS
OF
RABBIT
AORTIC
CELLS
AND
MEDIA
CONTAINING
HYPERLIPEMIC
The amount of lipid in the cells is expressed as pg of Iipid/mg of cell protein, and that of medium is expressed as mg%. Each data point is the mean of 3 flasks in one experiment. Cells were originally grown in control medium. consisting of Eagle’s basal medium and 5% normal serum and then exposed to media enriched with hyperlipemic VLDL (p = 1.006). LDL (p = 1.006-1.063). or HDL (p = 1.063-1.210) in amounts equivalent to that present in 10% hyperlipemic serum for 12-60 h as stated. Lipoproteins in culture media Control
Cholesterol CeU
ester Medium
8.8
Free cholesterol Medium
Cell
Phospholipids
Cell
CeII
Medium
Medium
4.2
21.7
203
4.8
196
VLDL
12h 36 h 60 h
24.4 26.1 27.3
a a a
34.0
33.5 38.6 39.3
b b b
10.5
335 b 361 a 375 a
9.2
233 b 256 b 265 b
22.0
LDL
12 h 36 h 60 h
46.7 50.4 47.9
a a a
62.0
52.4 54.9 55.4
a a a
19.0
217 193 195
12.2
290 a 306 a 306 a
34.2
HDL
12h 36 h 60 h
12.8 13.5 12.9
12.0
27.8 27.7 28.3
7.4
342 a 347 a 363 a
9.6
a P < 0.01 b P < 0.05
as compared as compared
1.2
Triglycerides
4.6
275b 275 b 276 b
5.0
to control. to control.
properties (peptide pattern? protein-lipid combination?) of the hyperlipemic LDL fraction, in addition to its high cholesterol content, account for the cholesterol accumulation in these cells. (4) Sequential measurement of lipid accumulation in cells incubated in hyperlipemic serum We studied the accumulation rate of each class of lipids in these cells. Cells were incubated in various concentrations of normal and hyperlipemic serum from 0 to 48 h. The content of cholesterol ester was not changed by growth in 20% serum, but cells grown in 40% normal serum showed a slight increase of cholesterol esters (Fig. la). When these cells were exposed to only 2% hyperlipemic serum, the cell cholesterol ester content increased within 4 h of incubation and reaching a 24 h level 5 times higher than the initial level. Cells growing in 5% and 10% hyperlipemic serum had a similar but slightly higher degree of cholesterol ester accumulation. The major effect of exposure to higher levels of hyperlipemic serum was the larger initial increment in this ester accumulation. Cells incubated in 5% and 20% normal rabbit serum had similar free cholesterol levels throughout. Cells grown in 40% normal serum had substantially more free cholesterol and cells grown in 2%, 5% and 10% of hyperlipemic serum had an almost 2-fold increase of free cholesterol (Fig. lb). The time course and level of free cholesterol accumulation was not influenced by the level of hyperlipemic serum. It is noteworthy that more than 60% of the increase was achieved within the initial 4 h of incubation. In this part of the study, the free cholesterol increased only 2-fold as contrasted to a 5-fold increase of cholesterol esters.
TABLE
4
LIPID
COMPOSITION
OF RABBIT
AORTIC
CELLS
EXPOSED
TO NORMAL
OR HYPERLIPEMIC
LIPOPROTEINS The amount of lipid is expressed as pg of lipid/mg of ceII protein. Each data point is the mean of 3 flasks in one experiment. CeIIs were originally grown in Eagle’s basal medium containing 5% normal rabbit serum. and then exposed to media enriched with normal or hyperlipemic VLDL (p = 1.006). LDL 1.006-1.063) and HDL (p = 1.063-1.210) for 40 h. The cholesterol content of aII the culture media were adjusted to be 300 pl/mI. Cholesterol ester
Lipoproteins added
Free cholesterol
Triglyceride
Phospholipid
Control
10.2
28.5
182
270
Normal VLDL LDL HDL
12.2 11.5 13.0
31.5 36.6 34.1
410 a 340 a 245
310 275 410 b
HIP
18.6 a 21.3 a 12.4
48.4 a 52.1 a 41.0 b
298 b 234 248
265 298 350
VLDL LDL HDL
a P < 0.01 as compared b P < 0.05 as compared
to control. to control.
Cholesterol
a.
esters
b.
60
Free
cholesterol
60 IO%
HlpS
50
5% HIPS 50 2% HlpS
40
40
30
30
HlpS HlpS Hl;S NRS
/-•
20 40% NRS 20% NRS 1
c.
d.
Triglycerides
300
40% NRS 10% HlpS 5% Hips 20% NM
_a_----
;-I:--.-~-.--.______~_.
:
200
IO
._*______ __-. ,.-__ -IF .
300
NRS
20
10
400
20%
2% HIpS
*__.--
Phospholipids
_-a.__
--..__
.--mm.
1’
!‘,L..._ _--...::z;,
, ,,,r--
200
10%
HlpS
5% HIPS 2% H,pS
--*=.-_::::
.‘.*
??
40%
NRS
??
20%
NRS
100 100
1
1
4 0
1
1
24
4
4; DURATION
OF
INCUBATION
I
,
a
24
I
48
(hours)
Fig. 1. Sequential accumulation of (a) cholesterol ester, (b) free cholesterol, (c) triglycerides, and (d) phospholipids in rabbit aortIc medial ceIIs exposed to various concentrations of normal or hyperIipemIc rabbit serum. These ceils were 0-y grown in Eagle’s basal medium containing 5% normal rabbit serum and exposed to media containing 2040% normal serum or 2-1046 hyperllpemic serum ~1~s 5% normal serum for up to 48 h. Each flask contained about 8 X 105 cells In 2.5 ml of culture medium.
341
The triglycerides showed less than a 2-fold increase (Fig. lc) in contrast to a 2-5-fold increase of cholesterol ester, and this increment took place at a slower rate with an initial lag phase. Furthermore, the triglyceride accumulation was noted at least as much in cells grown in normal serum as that in hyperlipemic serum, apparently reflecting the triglyceride content of the medium in which the cells were grown. The phospholipid content of cells exposed to hyperlipemic sera changed a little with increased time of incubation (Fig. Id). (5) Sequential change of lipids in culture medium The culture media containing 5-40% normal serum and 2-10’S hyperlipemic serum were analyzed 4-48 h after incubation with cells. The most
IO00
1 Cholesterol
a.
.
esters
500
800
___---
400
??
57 HlpS
,__--’
600
=
400
F 4 z a”
40"1 NRS
200
27
100
2 2
1
f E a. ;
500
s
400
G 2 5
c.
*. '.
300
t- =.
‘-,.
.--___
1 r
I
4
a
d.
Phospholipids _____----'
NRS
'-a*
6oo
.
400
--
200
-
I
_I'
:
_'
-'
??
---___
.--._ 10%
HlpS
--*--__m_
---.*
5% HIPS
--- -_..
I
48 DURATION
409
NRS
__c_.____-----*
HIPS
20% NRS 2% HlpS
2% HlpS I
24
..__e-----
HlpS
5%
___*_______~___---------~*
. --.___
10%
.*
*. ... .
1
I
I
a00 -
Triglycerides
--•
100
I
I
40% '\
200
HlpS
2O'x NRS
I
4 OF
INCUBATION
a
WITH
24 CELLS
48
(hours)
Fig. 2. Sequential change of (a) cholesterol esters, (b) free cholesterol, (c) triglycerides and (d) phospholipids in culture media containing 2040% normal rabbit serum or 2-10% hyperlipemic rabbit serum plus 6% normal rabbit serum, incubated with rabbit aortic medial cells for up to 48 h. Each flask contained 2.5 ml of culture medium incubated with about 8 X 105 cells.
348
pronounced changes were observed with time in the concentration of cholesterol ester, which decreased considerably in all media, but which was proportionally higher in media containing higher concentrations of serum cholesterol (Fig. 2a). Changes of the free cholesterol in the culture media (Fig. 2b) contrasted greatly to those of cholesterol ester. The content of free cholesterol did not decrease during the incubation with cells, but those media containing high concentrations of cholesterol showed a considerable increase of this lipid subclass. In all media containing normal or hyperlipemic serum, there was some decrease in triglycerides (Fig. 2~). There was no remarkable change of phospholipids, except a very small but consistent increase in phospholipids during the 48 h incubation, notably in medium containing 10% hyperlipemic serum (Fig. 2d). (6) Reversal of lipid accumulation Cells exposed to 2040% normal serum or 2-10% hyperlipemic serum plus 5% normal serum for 2 days were returned to control medium (5% normal serum) for another 48 h to see how much of the accumulated lipids could be removed. At the end of a 2day exposure to these variously enriched media, the cells showed increments of cholesterol ester, free cholesterol and triglyceride (Table 5), consistent with experiments described earlier. The elevated cellular triglycerides and free cholesterol declined toward control level after 48 h incubation in control medium. However, the return of cholesterol esters to control levels was less complete in 48 h. About 20-30% of the excess cholesterol esters remained in the cells.
TABLE 5 REVERSAL
OF LIPID ACCUMULATION
IN RABBIT AORTIC MEDIAL CELLS
The amount of lipid is expressed as pg lipid/mg of ceII protein. Each data point is the mean of 3 flasks. Cells were exposed to these variously enriched media for 48 h. Some of the cells were harvested and the remainder were returned to control medium consisting of EagIe’s basal medium and 5% normal serum for 2 days. Concentration of serum in media
Cholesterol ester
Free cholesterol
Start
Start
5% normal serum
8.5
20% normal serum
8.9
End
End
26.5
start
End
192
Phospholipids start
End
176 179
170
40% normal serum
13.9 b
10.4 d
46.5 a
29.5 c
342 a
190 c
194
182
2% Hip serum + 5% normal serum
48.3 a
16.2 c
48.3 a
30.6 c
278 b
210 c
208
168 d
5% Hlp serum + 5% normal serum
48.6 a
20.4 c
50.6 a
28.4 c
240
189 d
199
178
10% Hip serum + 6% normal serum
41.8 a
21.2 c
43.5 a
30.6 c
310 a
210 c
262 a
189 d
a b c d
P P P P
< < <
