279
Biochimica et Biophysics Acta, 530 (1978) @ Elsevier/North-Holland Biomedical Press
279-291
BBA 57218
DIFFERENTIAL EFFECTS OF ISOLATED LIPOPROTEINS FROM NORMAL AND HYPERCHOLESTEROLEMIC RHESUS MONKEYS ON CHOLESTEROL ESTERIFICATION AND ACCUMULATION iN ARTERIAL SMOOTH MUSCLE CELLS IN CULTURE
R.W. St. CLAIR and M.A. LEIGHT The Arteriosclerosis Research Center, Department of Medicine, Winston-Salem, N.C. 27103 (U.S.A.) (Received
January
of Pathology, Bowman Gray School
9th, 1978)
Summary Whole serum obtained from hypercholesterolemic rhesus monkeys was found to stimulate cholesterol esterification and cholesteryl ester accumulation in rhesus monkey arterial smooth muscle cells in culture to a si~ific~tly greater extent than no~ocholesterolemic serum. This was true even when the cholesterol concentration of the culture medium was equalized. Isolation and characterization of the low density lipoproteins (LDL) from rhesus monkeys indicated that the LDL from hypercholesterolemic animals was 33% larger than LDL from normocholesterolemic animals due principally to an increase in the amount of cholesteryl ester per molecule. As a result, LDL from hypercholesterolemic animals transported over 50% more cholesterol per molecule than did normal LDL. The LDL of altered composition from hypercholesterolemic animals, when added to smooth muscle cells in culture, was nearly twice as effective in stimulating cholesterol esterification and cholesteryl ester accumulation than was LDL of normal composition. Results suggest that at least part of the exaggerated ability of whole hypercholes~rolemi~ serum to stimulate the esterification and accumulation of cholesterol in cells in culture is due to the presence of LDL of altered composition.
Introduction Although hypercholesterolemia is a major risk factor for development of atherosclerosis in human beings [ 1] and experiments animals [Z], the specific mechanism(s) by which it exerts this effect is unknown.
Abbreviation:
HEPES, N’-2-bydroxyethylpiperazine-N(-ethantlslfonic
acid.
280
Recent studies have indicated that whole serum from hypercholesterolemic animals will stimulate the accumulation of cholesteryl esters in organ cultures of arterial tissue [ 31, as well as in arterial smooth muscle cells [ 4-71 and other cells in culture [8,9]. Even when the total cholesterol concentration of the culture medium is equalized, hypercholesterolemic serum is more effective in stimulating accumulation of cholesteryl esters than is normocholesterolemic serum [ 4,5,83. The reason for this exaggerated response to hypercholesterolemit serum is unknown but could be due to differences in either the concentration or composition of the lipoproteins. Cholesterol feeding is known to increase the concentration of low density lipoproteins (LDL) in the plasma ] 10,11] ,and in some species decreases the concentration of high density lipoproteins (HDL) [11,12]. In certain animal species or individuals, cholesterol feeding also results in the production of LDL of altered composition. These LDL are larger, and of lower density, due principally to an increase in the amount of cholesteryl ester transported per molecule [ 10)) and they may also have an altered apolipoprotein composition [ 111. The purpose of this study was to determine whether lipoproteins of different composition when isolated from normal and hypercholesterolemic rhesus monkeys differed in their ability to stimulate cholesterol accumulation and cholesterol esterifieation in arterial smooth muscle cells in culture. Methods Tissue culture. Procedures for obtaining explants from the tunica media of the thoracic aorta of rhesus monkeys (Macaca mulatta) and for the growth of arterial smooth muscle cells in culture have been described previously [ 133. Cells were routinely grown in 75 cm* flasks until they appeared confluent, at which time they were removed by t~ps~ization with 0.05% trypsin and 0.02% ethylenediamine tetraacetic acid (EDTA)). For metabolic experiments 2-6 . lo5 cells were plated into lOO-mm dishes or 1 . lo5 cells were plated into 60-mm dishes. After the cells had grown to approximately 80% confluence they were washed with phosphate-buffered saline and culture medium containing lipoprotein-deficient calf serum (2.5 mg protein/ml) was added. After 24 h of incubation, fresh culture medium was added containing the same concentration of lipoprotein-deficient serum plus test serum or lipoprotein as indicated in the tables and figures. Cells were incubated with the test medium for 12 h after which [l-i4C]oleate (1 pCi,!ml, 0.17 mM) was added for an additional 4 h. Following incubation with [l-14C]oleate the cells were rinsed twice with phosphate-buffered saline, freed from the dishes with trypsin and EDTA, transferred to 12-ml centrifuge tubes and washed twice with phosphate-buffered saline. The cells were suspended in 1 ml of deionized water and disrupted by sonication. An aliquot was taken for determination of cell protein and another aliquot extracted for lipids. The lipids were separated by thin-layer chromatography and counted for radioactivity. The cholesterol and cholesteryl ester content of the cells were determined by gas-liquid chromatography. Isola tiorz and charac terim tion of Eipopro teins. Lipoproteins were isolated from the pooled plasma from 6-10 no~ocholesterolemic and 2-4 hypercholesterolemic rhesus monkeys. All animals were fed the following semi-
281
purified diet (quantities in g/100 g): lard 25.0, wheat flour 20.0, apple sauce 7.8, non-fat dry milk solids 30.0, casein, USP 13.0, USP XIV salts mixture 2.0, complete vitamin mixture (devoid of vitamin D) 2.2, vitamin D, in corn oil 25 000 I.U. The diet was formed into the consistency of bread dough by addition of 250 ml water and stored frozen. The cholesterol content of the basal diet was 0.004 mg/kcal derived from the lard. Crystalline cholesterol was added to this diet by dissolving it in the warm lard to a final cholesterol concentration of the diet of from 0.05 to 1.08 mg/kcal depending on the specific experiment. The salt and vitamin mixtures were obtained from ICN Biochemicals (Cleveland, Ohio). Hypercholesterolemic animals received this diet containing 1.08 mg cholesterol/kcal while normocholesterolemic animals received the same diet containing from 0.004 to 0.22 mg cholesterol/kcal. Normocholesterolemic animals were selected to have plasma cholesterol concentrations of less than 200 mg/dl, while the hypercholesterolemic animals had plasma cholesterol concentrations between 580-827 mg/dl. For isolation of lipoproteins, the animals were fasted overnight and the blood was collected in EDTA (1 mg/ml) and kept at 4°C during all subsequent procedures. The plasma lipoproteins were separated by the combined ultracentrifugal and agarose column procedure of Rude1 et al. [ 141. Following isolation, LDL and HDL were concentrated overnight by dialysis against approximately 50 ~01s. of a solution of 30% sucrose and 0.1% EDTA. The sucrose was removed by dialysis against three changes of 100 ~01s. each of 0.9% sodium chloride and 0.01% EDTA, pH 7.4, and an aliquot of each lipoprotein fraction was taken for analysis. The remainder of the lipoprotein preparations were dialyzed against 100 ~01s. of Eagles Minimum Essential Medium containing, as a buffer, 20 mM ~‘-2-hydroxyethylpiperazine-~‘-ethanesulfonic acid (HEPES), pH 7.4. Electrophoresis was carried out on the isolated lipoproteins by the method of Noble [15]. Apolipoproteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis as described by Rude1 et al. [ 161. Lipoprotein protein content was dete~ined by the method of Lowry et al. 1173 using bovine albumin as a standard. Lipids were extracted from the isolated lipoproteins using the method of Folch et al. [ 18 1. Phospholipid phosphorus [ 191 was determined after perchloric acid digestion and the results multiplied by 25 to obtain the amount of phospholipid. Total cholesterol and triglycerides were measured using the AutoAn~yzer II methodolo~ [20]. Free and esterified cholesterol were determined [20] following their separation by thin-layer chromatogrpahy on silica Gel G using Skellysolve B (Skelly Oil Co., Kansas City, MO.), ethyl ether, and acetic acid (146 : 50 : 4) as the developing solvent. The molecular weight of the isolated LDL was measured as described by Rude1 et al. [lo] using a r2’I-labeled LDL of known molecular weight as an internal standard. ResuI ts
Characterization of lipoproteins from normo- and hypercholesterolemic rhesus monkeys. Lipoproteins used for addition to cells in culture were isolated
from
pooled
plasma
from
normo-
and hypercholesterolemic
animals
I
OF PLASMA
LIPOPROTEIN
USED FOR ADDITION
TO CULTURES
OF RHESUS
MONKEY
ARTERIAL
SMOOTH
MUSCLE
CELLS
LDL HDL
LDL HDL Hyper~hole~erolemic
Normochole~erolemic
Preparation
4.5 .t 0.42 1.9 _r0.16 1.4 i: 0.39 1.0 i: 0.36
19.4 ?. 1.5 26.5 “- 3.3
16.2 rt 1.2 49.4 _C3.2
Triglyceride
20.2 i 1.8 30.0 + 1.7
Phospholipid
composition
20.2 t 1.6 42.4 + 0.67
Protein
Percentage
44.9 21.0
52.7 L 1.7 19.5 i 0.85
10.2 t 0.77 3.6 ? 0.24
t 2.2 + 1.6
Esterified cholesterol
10.2 + 1.0 4.6 + 0.13
Free cholesterol
4.4 c 0.25 -
3.3 ?: 0.43 -
Mol. wt. daltans X lo+’
2.76 -
t: 0.22
1.81 r 0.31 -
/rg Total cholesterol per pm01 LDL
Results are the mean ?: S.E. of lipoproteins isolated from four separate samples of pooled plasma from 6-10 normocholesterolemic and 2-4 hypercholsterolemic rhesus monkeys. Total plasma chofesterol concentration for the original pooled normo- and hypercholesterolemic plasma was 169 i 6.5 and 692 + 100 mg/dl, respectively, and triglycerides were 33 r 5.5 and 38 i 13.6 mg/dl, respectively. Esterified cholesterol was calculated based on the molecular weight of cholesteryl &ate. LDL molecular weight was estimated by agarose column chromatography against z2sI-labeled of known moieculw weight 1101. Total cholesterol (pg per Pmol LDL) was calculated from the molecutar weight of LDL and the percentage of LDL as free plus esterified choesterol.
COMPOSITION
TABLE
283
having plasma cholesterol concentrations averaging 169 and 692 mg/dl, respectively. Four separately isolated and characterized preparations of lipoproteins were used for these studies. Their average composition is shown in Table I. Relatively minor changes were seen in the composition of HDL from hypercholesterolemic animals. These changes consisted of an increase in the proportion of protein and a decrease in the proportion of phospholipid. The major compositions changes associated with hypercholesterolemia occurred in the LDL. Hypercholesterolemia resulted in a LDL composed of proportionally less protein and triglyceride, little change in phospholipid and free cholesterol, and a greater than 17% increase in the proportion of cholestery! esters, Largely as a consequence of this increased cholesteryl ester content there was a 33% increase in the molecular weight of the LDL and a 50% increase in the amount of cholesterol transported per molecule. The increase in the molecular weight of LDL from hypercholesterolemic animals can be readily seen in Fig. 1 which represents a typical agarose gel chromatography elution pattern of the isolated LDL from a representative pool of plasma from normo- and hypercholesterolemic animals. The LDL from hypercholesterolemit animals always eluted from the agarose gel before the LDL from normo-
Normocholrstrrolrmic 7000
-
6000
-
5000
-
4000
-
3000
-
2000
-
1000
-
LDL
-// Hypsrcholrsterolrmic 7000
-
6000
-
5000
-
4000
-
3000
-
2000
-
1000
-
LDL
-I/ 100
120
110 Elution
Volume
130
140
(ml)
Fig. 1. Typical agarose gel chromatography elution pattern of LDL from plasma of normo- and hypercholesterolemic rhesus monkeys (shaded area) compared with a lz51~abeled LDL internal standard with a molecular weight of 3.1 .106 daltons.
284
cholesterolemic animals, indicating that the LDL from hypercholesterolemic animals was larger than that from normocholesterolemic animals. The broader elution profile of the LDL from hypercholesterolemic animals also suggested greater size heterogeneity relative to the normal lipoprotein. Isolated lipoproteins used for addition to cells in culture were shown to be free of ~ont~ination by other lipoprote~s by agarose gel electrophoresis and immunoelectrophoresis against antisera to apolipoprotein B, HDL and LDL. Analysis of the apolipoproteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 2) indicated no major qualitative differences in the apolipoprotein patterns of LDL and HDL as a result of hypercholesterolemia. The identifiable apolipoproteins of HDL were apo-A-I and the apo-C peptides. There was no detectable apo-B in the HDL confirming the immunoelectrophoretic and agarose electrophoresis data. The principal apolipoprotein of LDL was apo-B which remained in the sample well or at or near the interface between the spacer gel and the running gel. A small amount of protein migrated in the position where the arginine-rich apolipoprotein is normally found. There appeared to be more of this arginine-rich apolipoprotein in the LDL from hypercholesterolemic animals but since we did not measure it directly any specific conclusion as to the influence of hypercholesterolemia on the concentration of the arginine-rich apolipoprotein will have to await such analyses.
~nf~~~~~e of lipoproteins on cholesterol content and metabolism in arterial smooth musca’e cells. Prior to the addition of isolated lipoproteins to smooth muscle cells in culture under our experimental
we wanted conditions,
to determine whether we could reproduce, the results of others indicating that hyper-
TABLE II INFLUENCE OF NORMOCHOLESTEROL CONTENT
AND HYPERCHOLESTEROLEMIC SERUM IN ARTERIAL SMOOTH MUSCLE CELLS
ON LIPID SYNTHEIS
AND
Cells were incubated with culture medium containing lipoprotein-deficient serum for 24 h prior to addition of the normo- or hypercholesterolemic serum-containing medium for 12 h. [l-1410bate was added for an additional 4 h. Each value represents the mean + S.E. of five replicate cultures. Only the x values are given for cholesteryl ester content since the cells from all five dishes were pooled for this analysis. Normocholesterolemic serum was pooled from five rhesus monkeys and had a cholesterol concentration of 167 mg/dl. Hypercholesterolemic serum was pooled from four rhesus monkeys and had a cholesterol concentration of 503 mg/dl. Treatment
Cholesterol content f&mg
f l-l41
Oleate esterified (nmolfmg protein)
Protein)
Free
Phospholipid
Triglyceride
Cholesteryl ester
Esterified
Lipoprotein-deficient serum Normocholesterolemic serum
28.2 + 5.4
5.0
58.5 !z 8.2
131 t 16.2
0.44 * 0.07
5 &ml 500 pg/ml Hyperehoiesterolemic serum
27.2 t 2.3 31.2 f 0.37
5.4 6.8
55.4 r 4.7 76.6 + 0.9
142 fr 13.6 120 i 5.0
0.45 + 0.05 4.3 f 3.6
5 f&ml 500 &ml
29.1 ?: 1.5 37.6 C 2.2
6.7 11.7
55.6 + 2.2 63.3 t 2.4
133 t 5.0 131 r 5.4
0.90 + 0.04 7.1 r 0.5
* Results are corrected for differences in the specific activity of the [l-14Cloleate tion with free fatty acids from the whole serum.
substrate due to dj.lu-
285
cholesterolemic serum had a greater influence on lipid metabolism and cholesterol accumulation than did normocholesterolemic serum added at the same cholesterol concentrations. Results of such an experiment are shown in Table II. At equivalent cholesterol concentrations, hypercholesterolemic serum was more effective in promiting accumulation of cholesterol and cholesteryl esters and in stimulating cholesterol esterification. These differences in cellular response to normo- and hypercholesterolemic serum persisted even if the whole serum was dialyzed against several changes of tissue culture medium prior to addition to cells in culture. The influence of LDL and HDL from normo- and hypercholesterolemic rhesus monkeys on the accumulation of cholesterol by arterial smooth muscle cells is shown in Table III. LDL from both normo- and hypercholesterolemic animals produced a slight but consistent increase in the free cholesterol concentration of the cells. This increase generally was maximum at the lowest concentration used (100 pg/ml) and did not increase when higher concentrations of lipoprotein were added. HDL from both normo- and hypercholesterolemic animals also produced some increase in cellular free cholesterol content but not always to the extent of that seen with the LDL. LDL from hypercholesterolemic animals was appriximately twice as effective as LDL from normocholesterolemic animals in stimulating accumulation of cholesteryl esters. The accumulation of cholesteryl esters in cells exposed to TABLE III INFLUENCE OF LIPOPROTEINS OF DIFFERENT COMPOSITION TENT OF ARTERIAL SMOOTH MUSCLE CELLS IN CULTURE
ON THE CHOLESTEROL
CON-
Cells were incubated for 24 h with culture medium containing lipoprotein-deficient serum (LDS). The culture medium containing the test lipoprotein was added for an additional 12 h. Results are the mean ? S.E. of six replicate cultures at each concentration for Experiment 1 and four for Experiment 2 with the exception of the HDL values where there were six replicates. Low density (LDL) and high density (HDL) lipoproteins from normocholesterolemic (N-LDL and ‘N-HDL) and hypercholesterolemic (H-LDL and H-HDL) animals were compared with cells cultured with LDS. Lipoproteins from preparation B (Table I) were used for Experiment 1, preparation D for Experiment 2 and preparation A for the HDL of Experiment 2. Lipoprotein
Cholesterol concn. of
Cholesterol content (pg/mg cell protein, mean + S.E.)
culture medium
Experiment 1
(pg/mI)
Free
LDS N-LDL
H-LDL
N-HDL H-HD L
100 250 500 100 250 500 100 500 82 100
Esterified
22.3 + 0.89 29.3 ? 1.3 29.4 * 31.0 2 1.6 30.6 28.7 30.1 27.2
-
Experiment 2
fr 4.0 ? 0.28 + 1.4 + 3.3
2.1 f 0.37 3.2 + 1.3 3.9 ? 0.60 6.2 i 0.62 10.4 3.2 3.2 1.7 -
f 2.4 + 0.41 _+0.45 + 0.33
Free 31.4 33.3 30.3 33.1 31.2 33.0 34.0 32.4 30.5
Esterified * r + r + r t f
0.43 0.28 0.28 1.6 2.8 1.2 1.5 1.5 **
+ 1.1 **
1.6 4.0 3.9 4.9 3.5 6.3 8.1 2.4
-
* 0.22 + 0.52 * 0.56 k 0.55 f 0.39 ? 0.87 * 1.1 k 0.57 **
2.0 r 0.04 **
* Only an n of 2 was available at this concentration: thus, the S.E. was not calculated. ** The LDS control values for these cultures were 29.5 f 1.4 for free cholesterol and 1.4 f 0.2 pg esterified choIesterol/mg protein.
286
;&$&
N-LDL
H-LDL
N-HDL
H-HDL
Fig. 2. Typical plasma apolipoprotein patterns of LDL and HDL isolated from normocholesterolemic (N-LDL, N-HDL) and hypercholesterolemic (H-LDL, H-HDL) rhesus monkeys. Electrophoresis was carried out on 10% polyacrylamide slab gels containing 0.1% sodium dodecyl sulfate, 0.5 M Tris, pH 8.8, with a 5% polyacrylamide spacer gel. 40 pg of protein was applied to each gel. Arg-Rich, arginine-rich.
LDL, p~i~u~~ly from hyper~holesterolemi~ animals, appeared to increase progressively as the concentration of the lipoprotein was increased and did not show the same saturation effect with increasing concentrations as seen for free cholesterol. At similar cholesterol concentrations HDL produced only a
_I too tig Lipoprotein
200
300
Cholesterol
400
500
/ml culture medium
100
200
300
pmoles Lipoprotein
1400 /ml
culture
1500
7812
medium
Fig. 3. Influence of increased concentrations of lipoproteins from normo- and hypercholesterolemic rhesus monkeys on cholesterol esterification in arterial smooth muscle cells. Cells were incubated for 24 h with culture medium containing lipoprotein-deficient serum and then for an additional 12 h with the lipoprotein to be tested. Cl-1 4Cl Oleate was added for an additional 4 h in order to measure esterification.
287
30-50% increase in cellular cholesteryl ester content with little difference seen between HDL isolated from normo- or hypercholesterolemic animals. The influence of increasing concentrations of lipoproteins on cholesterol es~~fication in arterial smooth muscle cells is shown in Fig. 3. The concentration of the lipoproteins is expressed both on the basis of cholesterol content (panel A) and on the basis of the number of molecules (panel B) of lipoprotein per ml of culture medium. The number of pmol of LDL was calculated from the actual measured molecular weight of the isolated LDL. For high density lipoprotein we used an estimated molecular weight of 250000. HDL produced only an approximate 25% increase in the rate of cholesterol esterification over that of the lipoprotein-deficient serum controls. In the range of cholesterol concentrations. tested there was no difference in the ability of HDL from normo- or hypercholesterolemic animals to stimulate cholesterol esterification. The LDL from hypercholesterolemic animals was approximately twice as effective in stimulating cholesterol esterification as the same amount of normal LDL. This difference persisted, even when the results were expressed on the basis of the number of molecules of LDL added. In four separate experiments utilizing three different lipoprotein preparations added to the culture medium at a concentration of 100 pg cholesterol/ml, LDL stimulated cholesterol esterification an average of 3.9 times that of the lipoprotein-deficient serum controls, while normal LDL stimulated cholesterol esterification 2.6 fold. In these same experiments in~o~oration of [I-14C]oleate into phospholipids and triglycerides was not altered by any of the lipoproteins tested. When [l-‘4C]oleate and LDL from either normo- or hypercholesterolemic animals were added to the cells in culture at the same time, the greater stimulatory effect on cholesterol esterification of the LDL from hypercholester-
4
8
16
12
lncubatmn
time
20
24
(hc )
Fig. 4. Influence of incubation time on stimulation of cholesterol estcrification by LDL from normo(N-LDL) and hypercholesterolemic (H-LDL) rhesus monkeys. Cells were incubated for 24 h with cultuxe medium containing ~poprotein~eficient serum prior to addition of the lipoprotein. LDL was added to smooth muscle cells in culture at a final concentration of 100 pg cholesterol/ml culture medium. [1-*4CJOleate substrate was added at the time of addition of the test lipoprotein. Separate lipoprotein preparations were used in each experiment. Each point represents the mean of duplicate cultures. Lipoproteindeficient serum (LDS) was included as a control for the influence of incubation time on cholesterol esterification.
288
I N= 3 at each paint i
;;*
L
--m
l
-*-A-.-.
i 4
I
2
_
I 6 Cell
(Ag
Cell
.
110
--
1
I
8
IO
I 12
I
1
I4
Density
Protein/cm2
Surface
Area1
Fig. 5. Influence of cell density on rate of incorporation of [l- 14~loleate into phospholipids, trigiyceridcs and cholesteryl esters in rhesus monkey arterial smooth muscle cells in culture. Cells were incubated with culture medium containing ~poprotei~~e~cient serum for 48 h prior to addition of LDL from hypercholesterolemic animals at a final concentration of 100 pg cholesterol/ml. After 12 h, [l-i4Clok?ate was added for 4 h in order to measure
esterification.
olemic animals was first evident after about 12 h of incubation (Fig. 4). This difference was further exaggerated after 24 h of incubation. As shown in Fig. 5, the density of cells in the tissue culture dish, as measured by pg cell protein/cm2 surface area, profoundly influenced the absolute rate of esterification of oleic acid. This was particularly true for esterification to cholesterol which decreased dramatically as the density of cells in the dish was increased. A similar effect of cell density on uptake of LDL by arterial smooth muscle cells in culture has been reported by Stein and Stein 2211. Consequently, we have been careful to compare data only within the same experiment in which the cell densities were constant for all treatments. Discussion The increase in the size of the LDL from the hypercholesterolemic rhesus monkeys of this study was similar to that reported by Rude1 et al. 1121. Consistent with previous studies by others f11,12], the major reason for the increased size of the LDL from hypercholesterolem~c animals was an increase in
289
the cholesteryl ester content. Each molecule of LDL from the hypercholesterolemic animals transported over 50% more cholesterol than normal LDL. Cells, particularly those maintained in tissue culture, appear to obtain the bulk of their cholesterol for maintenance of normal growth and metabolism from LDL of the culture medium. This occurs by means of binding and subsequent uptake of intact LDL by LDL receptors located on the plasma membrane [22]. Cholesterol internalized by this mechanism acts to suppress cholesterol synthesis and stimulate cholesterol esterification. These processes appear to be controlled by a pool of cholesterol within the cell, since cholesterol added to cells in a manner that circumvents the receptor will also regulate cellular cholesterol synthesis and esterification. Thus, in the present study, for an equivalent number of molecules of LDL from hypercholesterolemic animals taken into the (cell by the lipoprotein receptor there would be approximately 50% more cholesterol available to the cell. As a result of this increased amount of cholesterol internalized, the initial rate of cholesterol esterification and cholesteryl ester accumulation would be expected to be stimulated to a greater extent with the LDL from hypercholesterolemic animals. Another mechanism by which LDL from hypercholesterolemic animals could be more effective in stimulating cholesterol esterification and accumulation is secondary to an increased affintiy for binding to the LDL receptor. Such a mechanism could be mediated by changes in the concentration of specific apolipoproteins, such as the arginine-rich peptide. This apolipoprotein has been shown to bind to the high affinity LDL receptor [23] with an even higher affinity than apolipoprotein B [24]. Apolipoprotein B constituted the major apolipoprotein (93-97%) of LDL from normo- and hypercholesterolemic animals [lo]. In the LDL used in this study a small amount of protein migrated in the position where the arginine-rich and C-apolipoproteins are normally found. These apolipoproteins were not quantified although their increased intensity of staining (Fig. 2) suggest that they may be present in somewhat greater amounts in LDL from hypercholesterolemic animals. Nevertheless, individually they never account for more than approximately 3% of the total apolipoprotein of rhesus monkey LDL (Rudel, L.L., personal communication). Whether changes in apolipoproteins that constitute such a small percentage of the lipoprotein molecule can alter the binding and ultimate metabolism of the lipoprotein by the cell is unknown. Cholesterol from LDL of altered composition might also be transferred to cells through a process that does not require uptake of the intact lipoprotein molecule, By such a mechanism an increased rate of transfer of free cholesterol from an abnormal lipoprotein to the cells would result in the enrichment of cellular membranes with cholesterol. Such a mechanism has been suggested by Arbogast et al. [25] to explain the stimulation of cholesterol esterification and accumulation of cholesteryl esters in cells exposed to cholesterol-phosphatidylcholine dispersions. If such a mechanism occurred the excess membrane-free cholesterol could be kept within physiologically compatible limits by esterification and storage as cholesteryl ester droplets within the cell. Consistent with this possibility is the observation by Rothblat et al. [26] that incubation of microsomal membranes from rat hepatoma cells with hyperlipemic rabbit serum results in the enrichment of these membranes with cholesterol and this
290
in turn stimulates cholesterol esterifieation, The same amount of cholesterol as normoli~emic rabbit serum was without effect in increasing membrane free cholesterol content or in stimulating cholesterol esterification. Cholesterol esterification is markedly stimulated in atherosclerotic arteries [27,28] and the bulk of this stimulation in cholesterol esterification is localized in cholesteryl ester-rich foam cells f29], In cells in culture, however, even though the cholesteryl ester content of the cells can be increased subst~ti~ly by addition of LDL to the culture medium [8,13,30,31], the rate of cholesterol esterification is rapidly inhibited as the cholesterol content of the cells increases [ 133. This appears to be due to regulation of high affinity binding sites for LDL by intracellular pools of cholesterol 132,331. In the atherosclerotic artery, cholesteryl ester synthesis can be stimulated as much as lOO-fold in tissue contain~g cells enriched with large amounts of cholesterol and cholesteryl esters [ 2834). Thus, the pathologic processes leading to the development of atherosclerosis cannot be adequately explained simply by an exaggerated uptake of LDL cholesterol by cells of the arterial wall via high affinity receptors for LDL. Whether this “pathological accumulation” of cholesteryl esters results from the unregulated entry of lipoproteins into cells of the arterial wall, as suggested by Goldstein and Brown 1221, or by other, as yet unreco~ized, mechanisms is unclear. The present studies suggest, however, that differences in the composition of lipoproteins, resulting from genetic, dietary or other environmental influences may play an important role in determining the efficiency of delivery of cholesterol to cells and perhaps ultimately its “atherogenic potential.” Acknowledgements The authors gratefully acknowledge the excellent technical assistance of Ns. Grayce Greene and the aid of Mrs. Brenda Warner in the preparation of this manuscript. This work was supported by a grant from the National Heart, Lung and Blood Institute (S.C.O.R.) HL-14164. References 1 Kannel. W.B.. Castelii. W.P., Gordon, T. and McNamara, P.M. (1971) The Framingham Study. Ann. Int. Med. 74, l-12 2 Clarkson, T.B.. P&hard, R.W.. Bullock, B.C., St. Clair. R.W.. Lehner, N.D.M., Jones, D.C., Wagner, W.D. and Rudel, L.L. (1976) Expt. Mol. Path& 24,264-286 3 St. Clah, R.W. and Harpold, G.J. (1975) Expt. Mol. Path&. 22.207-219 4 Fisher-Dzoga, K., Chen, R. and WissIer, R.W. (1977). Adv. Expt. Med. Bioi. 43. 299-311 5 Bates, S.R. and Wissler, R.W. (1976) Biochim. Biophys. Acta 450. 78-88 6 Pearson, J.D. (1976) Atherosclerosis 24, 233-242 7 Nikkari, T., Pietila. K. and Sale, M. (1976) Med. Biol. 54, 264-271 8 Rothblat, G.H. (1974) Lipids 9,526-535 9 Rothblat, G.H., Arbogast, L., Kritchevsky, D. and Noftulin. M. (1976) Lipids 11,97-108 10 Rudel, L.L., Pitts, III, L.L. and Nelson, C.A. (1977) J. Lipid Res. l&211-222 21 Mahleu, R.W., Weisglaber, K.H. and Inneraritv, ‘I’. (1976) Biochemistry 15,2979-2985 12 Rudel, L.L. and Lofland, H.B. (1977) Am. J. Med. 62,707-714 13 St. Clair, R.W., Smith, B.P. and Wood, L.L. (1977) Ciyc. Res. 40.166-173 14 Rudel. L.L., Lee, J.A., Morris, M.D. and Felts. J.M. (1974) Biochem. J. 139.89-95 15 Noble, R.P. (1968) J. Lipid Res. 9, 693-700 16 Rudel, L.L., Greene, D.G. and Shah, R. (1977) J. Lipid Res. 18,734-744
291 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
Lowry, O.J.. Rosebrough, N.J.. Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 Folch, J., Lees. M.. Sloane Stanley, G.H. (1957) J. Biol. Chem. 226. 497-509 Fiske, C.H. and SubbaRow, Y. (1925) J. Biol. Chem. 66.357400 Rush, R.L.. Leon, L. and Turrell. J. (1970) in Advances in Automated Analysis-Technicon International Congress (1970). Vol. 1, pp. 503-507, Futura Publishing Co., Mt. Kisco. N.Y. Stein, 0. and Stein, Y. (1975) Biochim. Biophys. Acta 398. 377-384 Goldstein, J.L. and Brown, M.S. (1977) Ann. Rev. Biochem. 46. 897-930 Bersot, T.P. and Mahley, R.W. (1976) J. Biol. Chem. 251, 2395-2398 Innerarity, T.L. and Mahley, R.W. (1977) Circulation 56. III-101 Arbogast. L.Y.. Rothblat, G.H.. Leslie, M.H. and Cooper, R.A. (1976) Proc. Natl. Acad. Sci. U.S. 73, 3680-3684 Rothblat. G.H., Naftulin, M. and Arbogast. L.Y. (1977) Proc. Sot. Expt. Biol. Med. 155, 501-506 Lofland. H.B.. Moury, D.M. and Hoffman. C.W. (1965) J. Lipid Res. 6,112-118 St. Clair, R.W.. Lofland. H.B. and Clarkson, T.B. (1970) Circ. Res. 27. 213-225 Day. A.J. and Wilkinson, G.K. (1967) Circ. Res. 21.593-600 Mahlev. R.W., Innsrarity. T.L., Weisgraber. K.H. and Fry, D.L. (1977) Am. J. Path. 87. 205-219 Brown, M.S., Faust, J.R. and Goldstein, J.L. (1975) J. Clin. Invest. 55. 783-793 Goldstein, J.L., Dana, SE., Brown, M.S. (1974) Proc. Natl. Acad. Sci. U.S. 71. 42884292 Goldstein, J.L.. Anderson, R.G.W., Buja, L.M., Basu, S.K. and Brown, M.S. (1977) J. Clin. Invest. 59.1196-1202 St. Clair, R.W. (1976) Atheroscler. Rev. 1. 61-117
Biochimica et Biophysics Acta, 530 (1978) @ Elsevier/North-Holland Biomedical Press
279-291
BBA 57218
DIFFERENTIAL EFFECTS OF ISOLATED LIPOPROTEINS FROM NORMAL AND HYPERCHOLESTEROLEMIC RHESUS MONKEYS ON CHOLESTEROL ESTERIFICATION AND ACCUMULATION iN ARTERIAL SMOOTH MUSCLE CELLS IN CULTURE
R.W. St. CLAIR and M.A. LEIGHT The Arteriosclerosis Research Center, Department of Medicine, Winston-Salem, N.C. 27103 (U.S.A.) (Received
January
of Pathology, Bowman Gray School
9th, 1978)
Summary Whole serum obtained from hypercholesterolemic rhesus monkeys was found to stimulate cholesterol esterification and cholesteryl ester accumulation in rhesus monkey arterial smooth muscle cells in culture to a si~ific~tly greater extent than no~ocholesterolemic serum. This was true even when the cholesterol concentration of the culture medium was equalized. Isolation and characterization of the low density lipoproteins (LDL) from rhesus monkeys indicated that the LDL from hypercholesterolemic animals was 33% larger than LDL from normocholesterolemic animals due principally to an increase in the amount of cholesteryl ester per molecule. As a result, LDL from hypercholesterolemic animals transported over 50% more cholesterol per molecule than did normal LDL. The LDL of altered composition from hypercholesterolemic animals, when added to smooth muscle cells in culture, was nearly twice as effective in stimulating cholesterol esterification and cholesteryl ester accumulation than was LDL of normal composition. Results suggest that at least part of the exaggerated ability of whole hypercholes~rolemi~ serum to stimulate the esterification and accumulation of cholesterol in cells in culture is due to the presence of LDL of altered composition.
Introduction Although hypercholesterolemia is a major risk factor for development of atherosclerosis in human beings [ 1] and experiments animals [Z], the specific mechanism(s) by which it exerts this effect is unknown.
Abbreviation:
HEPES, N’-2-bydroxyethylpiperazine-N(-ethantlslfonic
acid.
280
Recent studies have indicated that whole serum from hypercholesterolemic animals will stimulate the accumulation of cholesteryl esters in organ cultures of arterial tissue [ 31, as well as in arterial smooth muscle cells [ 4-71 and other cells in culture [8,9]. Even when the total cholesterol concentration of the culture medium is equalized, hypercholesterolemic serum is more effective in stimulating accumulation of cholesteryl esters than is normocholesterolemic serum [ 4,5,83. The reason for this exaggerated response to hypercholesterolemit serum is unknown but could be due to differences in either the concentration or composition of the lipoproteins. Cholesterol feeding is known to increase the concentration of low density lipoproteins (LDL) in the plasma ] 10,11] ,and in some species decreases the concentration of high density lipoproteins (HDL) [11,12]. In certain animal species or individuals, cholesterol feeding also results in the production of LDL of altered composition. These LDL are larger, and of lower density, due principally to an increase in the amount of cholesteryl ester transported per molecule [ 10)) and they may also have an altered apolipoprotein composition [ 111. The purpose of this study was to determine whether lipoproteins of different composition when isolated from normal and hypercholesterolemic rhesus monkeys differed in their ability to stimulate cholesterol accumulation and cholesterol esterifieation in arterial smooth muscle cells in culture. Methods Tissue culture. Procedures for obtaining explants from the tunica media of the thoracic aorta of rhesus monkeys (Macaca mulatta) and for the growth of arterial smooth muscle cells in culture have been described previously [ 133. Cells were routinely grown in 75 cm* flasks until they appeared confluent, at which time they were removed by t~ps~ization with 0.05% trypsin and 0.02% ethylenediamine tetraacetic acid (EDTA)). For metabolic experiments 2-6 . lo5 cells were plated into lOO-mm dishes or 1 . lo5 cells were plated into 60-mm dishes. After the cells had grown to approximately 80% confluence they were washed with phosphate-buffered saline and culture medium containing lipoprotein-deficient calf serum (2.5 mg protein/ml) was added. After 24 h of incubation, fresh culture medium was added containing the same concentration of lipoprotein-deficient serum plus test serum or lipoprotein as indicated in the tables and figures. Cells were incubated with the test medium for 12 h after which [l-i4C]oleate (1 pCi,!ml, 0.17 mM) was added for an additional 4 h. Following incubation with [l-14C]oleate the cells were rinsed twice with phosphate-buffered saline, freed from the dishes with trypsin and EDTA, transferred to 12-ml centrifuge tubes and washed twice with phosphate-buffered saline. The cells were suspended in 1 ml of deionized water and disrupted by sonication. An aliquot was taken for determination of cell protein and another aliquot extracted for lipids. The lipids were separated by thin-layer chromatography and counted for radioactivity. The cholesterol and cholesteryl ester content of the cells were determined by gas-liquid chromatography. Isola tiorz and charac terim tion of Eipopro teins. Lipoproteins were isolated from the pooled plasma from 6-10 no~ocholesterolemic and 2-4 hypercholesterolemic rhesus monkeys. All animals were fed the following semi-
281
purified diet (quantities in g/100 g): lard 25.0, wheat flour 20.0, apple sauce 7.8, non-fat dry milk solids 30.0, casein, USP 13.0, USP XIV salts mixture 2.0, complete vitamin mixture (devoid of vitamin D) 2.2, vitamin D, in corn oil 25 000 I.U. The diet was formed into the consistency of bread dough by addition of 250 ml water and stored frozen. The cholesterol content of the basal diet was 0.004 mg/kcal derived from the lard. Crystalline cholesterol was added to this diet by dissolving it in the warm lard to a final cholesterol concentration of the diet of from 0.05 to 1.08 mg/kcal depending on the specific experiment. The salt and vitamin mixtures were obtained from ICN Biochemicals (Cleveland, Ohio). Hypercholesterolemic animals received this diet containing 1.08 mg cholesterol/kcal while normocholesterolemic animals received the same diet containing from 0.004 to 0.22 mg cholesterol/kcal. Normocholesterolemic animals were selected to have plasma cholesterol concentrations of less than 200 mg/dl, while the hypercholesterolemic animals had plasma cholesterol concentrations between 580-827 mg/dl. For isolation of lipoproteins, the animals were fasted overnight and the blood was collected in EDTA (1 mg/ml) and kept at 4°C during all subsequent procedures. The plasma lipoproteins were separated by the combined ultracentrifugal and agarose column procedure of Rude1 et al. [ 141. Following isolation, LDL and HDL were concentrated overnight by dialysis against approximately 50 ~01s. of a solution of 30% sucrose and 0.1% EDTA. The sucrose was removed by dialysis against three changes of 100 ~01s. each of 0.9% sodium chloride and 0.01% EDTA, pH 7.4, and an aliquot of each lipoprotein fraction was taken for analysis. The remainder of the lipoprotein preparations were dialyzed against 100 ~01s. of Eagles Minimum Essential Medium containing, as a buffer, 20 mM ~‘-2-hydroxyethylpiperazine-~‘-ethanesulfonic acid (HEPES), pH 7.4. Electrophoresis was carried out on the isolated lipoproteins by the method of Noble [15]. Apolipoproteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis as described by Rude1 et al. [ 161. Lipoprotein protein content was dete~ined by the method of Lowry et al. 1173 using bovine albumin as a standard. Lipids were extracted from the isolated lipoproteins using the method of Folch et al. [ 18 1. Phospholipid phosphorus [ 191 was determined after perchloric acid digestion and the results multiplied by 25 to obtain the amount of phospholipid. Total cholesterol and triglycerides were measured using the AutoAn~yzer II methodolo~ [20]. Free and esterified cholesterol were determined [20] following their separation by thin-layer chromatogrpahy on silica Gel G using Skellysolve B (Skelly Oil Co., Kansas City, MO.), ethyl ether, and acetic acid (146 : 50 : 4) as the developing solvent. The molecular weight of the isolated LDL was measured as described by Rude1 et al. [lo] using a r2’I-labeled LDL of known molecular weight as an internal standard. ResuI ts
Characterization of lipoproteins from normo- and hypercholesterolemic rhesus monkeys. Lipoproteins used for addition to cells in culture were isolated
from
pooled
plasma
from
normo-
and hypercholesterolemic
animals
I
OF PLASMA
LIPOPROTEIN
USED FOR ADDITION
TO CULTURES
OF RHESUS
MONKEY
ARTERIAL
SMOOTH
MUSCLE
CELLS
LDL HDL
LDL HDL Hyper~hole~erolemic
Normochole~erolemic
Preparation
4.5 .t 0.42 1.9 _r0.16 1.4 i: 0.39 1.0 i: 0.36
19.4 ?. 1.5 26.5 “- 3.3
16.2 rt 1.2 49.4 _C3.2
Triglyceride
20.2 i 1.8 30.0 + 1.7
Phospholipid
composition
20.2 t 1.6 42.4 + 0.67
Protein
Percentage
44.9 21.0
52.7 L 1.7 19.5 i 0.85
10.2 t 0.77 3.6 ? 0.24
t 2.2 + 1.6
Esterified cholesterol
10.2 + 1.0 4.6 + 0.13
Free cholesterol
4.4 c 0.25 -
3.3 ?: 0.43 -
Mol. wt. daltans X lo+’
2.76 -
t: 0.22
1.81 r 0.31 -
/rg Total cholesterol per pm01 LDL
Results are the mean ?: S.E. of lipoproteins isolated from four separate samples of pooled plasma from 6-10 normocholesterolemic and 2-4 hypercholsterolemic rhesus monkeys. Total plasma chofesterol concentration for the original pooled normo- and hypercholesterolemic plasma was 169 i 6.5 and 692 + 100 mg/dl, respectively, and triglycerides were 33 r 5.5 and 38 i 13.6 mg/dl, respectively. Esterified cholesterol was calculated based on the molecular weight of cholesteryl &ate. LDL molecular weight was estimated by agarose column chromatography against z2sI-labeled of known moieculw weight 1101. Total cholesterol (pg per Pmol LDL) was calculated from the molecutar weight of LDL and the percentage of LDL as free plus esterified choesterol.
COMPOSITION
TABLE
283
having plasma cholesterol concentrations averaging 169 and 692 mg/dl, respectively. Four separately isolated and characterized preparations of lipoproteins were used for these studies. Their average composition is shown in Table I. Relatively minor changes were seen in the composition of HDL from hypercholesterolemic animals. These changes consisted of an increase in the proportion of protein and a decrease in the proportion of phospholipid. The major compositions changes associated with hypercholesterolemia occurred in the LDL. Hypercholesterolemia resulted in a LDL composed of proportionally less protein and triglyceride, little change in phospholipid and free cholesterol, and a greater than 17% increase in the proportion of cholestery! esters, Largely as a consequence of this increased cholesteryl ester content there was a 33% increase in the molecular weight of the LDL and a 50% increase in the amount of cholesterol transported per molecule. The increase in the molecular weight of LDL from hypercholesterolemic animals can be readily seen in Fig. 1 which represents a typical agarose gel chromatography elution pattern of the isolated LDL from a representative pool of plasma from normo- and hypercholesterolemic animals. The LDL from hypercholesterolemit animals always eluted from the agarose gel before the LDL from normo-
Normocholrstrrolrmic 7000
-
6000
-
5000
-
4000
-
3000
-
2000
-
1000
-
LDL
-// Hypsrcholrsterolrmic 7000
-
6000
-
5000
-
4000
-
3000
-
2000
-
1000
-
LDL
-I/ 100
120
110 Elution
Volume
130
140
(ml)
Fig. 1. Typical agarose gel chromatography elution pattern of LDL from plasma of normo- and hypercholesterolemic rhesus monkeys (shaded area) compared with a lz51~abeled LDL internal standard with a molecular weight of 3.1 .106 daltons.
284
cholesterolemic animals, indicating that the LDL from hypercholesterolemic animals was larger than that from normocholesterolemic animals. The broader elution profile of the LDL from hypercholesterolemic animals also suggested greater size heterogeneity relative to the normal lipoprotein. Isolated lipoproteins used for addition to cells in culture were shown to be free of ~ont~ination by other lipoprote~s by agarose gel electrophoresis and immunoelectrophoresis against antisera to apolipoprotein B, HDL and LDL. Analysis of the apolipoproteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (Fig. 2) indicated no major qualitative differences in the apolipoprotein patterns of LDL and HDL as a result of hypercholesterolemia. The identifiable apolipoproteins of HDL were apo-A-I and the apo-C peptides. There was no detectable apo-B in the HDL confirming the immunoelectrophoretic and agarose electrophoresis data. The principal apolipoprotein of LDL was apo-B which remained in the sample well or at or near the interface between the spacer gel and the running gel. A small amount of protein migrated in the position where the arginine-rich apolipoprotein is normally found. There appeared to be more of this arginine-rich apolipoprotein in the LDL from hypercholesterolemic animals but since we did not measure it directly any specific conclusion as to the influence of hypercholesterolemia on the concentration of the arginine-rich apolipoprotein will have to await such analyses.
~nf~~~~~e of lipoproteins on cholesterol content and metabolism in arterial smooth musca’e cells. Prior to the addition of isolated lipoproteins to smooth muscle cells in culture under our experimental
we wanted conditions,
to determine whether we could reproduce, the results of others indicating that hyper-
TABLE II INFLUENCE OF NORMOCHOLESTEROL CONTENT
AND HYPERCHOLESTEROLEMIC SERUM IN ARTERIAL SMOOTH MUSCLE CELLS
ON LIPID SYNTHEIS
AND
Cells were incubated with culture medium containing lipoprotein-deficient serum for 24 h prior to addition of the normo- or hypercholesterolemic serum-containing medium for 12 h. [l-1410bate was added for an additional 4 h. Each value represents the mean + S.E. of five replicate cultures. Only the x values are given for cholesteryl ester content since the cells from all five dishes were pooled for this analysis. Normocholesterolemic serum was pooled from five rhesus monkeys and had a cholesterol concentration of 167 mg/dl. Hypercholesterolemic serum was pooled from four rhesus monkeys and had a cholesterol concentration of 503 mg/dl. Treatment
Cholesterol content f&mg
f l-l41
Oleate esterified (nmolfmg protein)
Protein)
Free
Phospholipid
Triglyceride
Cholesteryl ester
Esterified
Lipoprotein-deficient serum Normocholesterolemic serum
28.2 + 5.4
5.0
58.5 !z 8.2
131 t 16.2
0.44 * 0.07
5 &ml 500 pg/ml Hyperehoiesterolemic serum
27.2 t 2.3 31.2 f 0.37
5.4 6.8
55.4 r 4.7 76.6 + 0.9
142 fr 13.6 120 i 5.0
0.45 + 0.05 4.3 f 3.6
5 f&ml 500 &ml
29.1 ?: 1.5 37.6 C 2.2
6.7 11.7
55.6 + 2.2 63.3 t 2.4
133 t 5.0 131 r 5.4
0.90 + 0.04 7.1 r 0.5
* Results are corrected for differences in the specific activity of the [l-14Cloleate tion with free fatty acids from the whole serum.
substrate due to dj.lu-
285
cholesterolemic serum had a greater influence on lipid metabolism and cholesterol accumulation than did normocholesterolemic serum added at the same cholesterol concentrations. Results of such an experiment are shown in Table II. At equivalent cholesterol concentrations, hypercholesterolemic serum was more effective in promiting accumulation of cholesterol and cholesteryl esters and in stimulating cholesterol esterification. These differences in cellular response to normo- and hypercholesterolemic serum persisted even if the whole serum was dialyzed against several changes of tissue culture medium prior to addition to cells in culture. The influence of LDL and HDL from normo- and hypercholesterolemic rhesus monkeys on the accumulation of cholesterol by arterial smooth muscle cells is shown in Table III. LDL from both normo- and hypercholesterolemic animals produced a slight but consistent increase in the free cholesterol concentration of the cells. This increase generally was maximum at the lowest concentration used (100 pg/ml) and did not increase when higher concentrations of lipoprotein were added. HDL from both normo- and hypercholesterolemic animals also produced some increase in cellular free cholesterol content but not always to the extent of that seen with the LDL. LDL from hypercholesterolemic animals was appriximately twice as effective as LDL from normocholesterolemic animals in stimulating accumulation of cholesteryl esters. The accumulation of cholesteryl esters in cells exposed to TABLE III INFLUENCE OF LIPOPROTEINS OF DIFFERENT COMPOSITION TENT OF ARTERIAL SMOOTH MUSCLE CELLS IN CULTURE
ON THE CHOLESTEROL
CON-
Cells were incubated for 24 h with culture medium containing lipoprotein-deficient serum (LDS). The culture medium containing the test lipoprotein was added for an additional 12 h. Results are the mean ? S.E. of six replicate cultures at each concentration for Experiment 1 and four for Experiment 2 with the exception of the HDL values where there were six replicates. Low density (LDL) and high density (HDL) lipoproteins from normocholesterolemic (N-LDL and ‘N-HDL) and hypercholesterolemic (H-LDL and H-HDL) animals were compared with cells cultured with LDS. Lipoproteins from preparation B (Table I) were used for Experiment 1, preparation D for Experiment 2 and preparation A for the HDL of Experiment 2. Lipoprotein
Cholesterol concn. of
Cholesterol content (pg/mg cell protein, mean + S.E.)
culture medium
Experiment 1
(pg/mI)
Free
LDS N-LDL
H-LDL
N-HDL H-HD L
100 250 500 100 250 500 100 500 82 100
Esterified
22.3 + 0.89 29.3 ? 1.3 29.4 * 31.0 2 1.6 30.6 28.7 30.1 27.2
-
Experiment 2
fr 4.0 ? 0.28 + 1.4 + 3.3
2.1 f 0.37 3.2 + 1.3 3.9 ? 0.60 6.2 i 0.62 10.4 3.2 3.2 1.7 -
f 2.4 + 0.41 _+0.45 + 0.33
Free 31.4 33.3 30.3 33.1 31.2 33.0 34.0 32.4 30.5
Esterified * r + r + r t f
0.43 0.28 0.28 1.6 2.8 1.2 1.5 1.5 **
+ 1.1 **
1.6 4.0 3.9 4.9 3.5 6.3 8.1 2.4
-
* 0.22 + 0.52 * 0.56 k 0.55 f 0.39 ? 0.87 * 1.1 k 0.57 **
2.0 r 0.04 **
* Only an n of 2 was available at this concentration: thus, the S.E. was not calculated. ** The LDS control values for these cultures were 29.5 f 1.4 for free cholesterol and 1.4 f 0.2 pg esterified choIesterol/mg protein.
286
;&$&
N-LDL
H-LDL
N-HDL
H-HDL
Fig. 2. Typical plasma apolipoprotein patterns of LDL and HDL isolated from normocholesterolemic (N-LDL, N-HDL) and hypercholesterolemic (H-LDL, H-HDL) rhesus monkeys. Electrophoresis was carried out on 10% polyacrylamide slab gels containing 0.1% sodium dodecyl sulfate, 0.5 M Tris, pH 8.8, with a 5% polyacrylamide spacer gel. 40 pg of protein was applied to each gel. Arg-Rich, arginine-rich.
LDL, p~i~u~~ly from hyper~holesterolemi~ animals, appeared to increase progressively as the concentration of the lipoprotein was increased and did not show the same saturation effect with increasing concentrations as seen for free cholesterol. At similar cholesterol concentrations HDL produced only a
_I too tig Lipoprotein
200
300
Cholesterol
400
500
/ml culture medium
100
200
300
pmoles Lipoprotein
1400 /ml
culture
1500
7812
medium
Fig. 3. Influence of increased concentrations of lipoproteins from normo- and hypercholesterolemic rhesus monkeys on cholesterol esterification in arterial smooth muscle cells. Cells were incubated for 24 h with culture medium containing lipoprotein-deficient serum and then for an additional 12 h with the lipoprotein to be tested. Cl-1 4Cl Oleate was added for an additional 4 h in order to measure esterification.
287
30-50% increase in cellular cholesteryl ester content with little difference seen between HDL isolated from normo- or hypercholesterolemic animals. The influence of increasing concentrations of lipoproteins on cholesterol es~~fication in arterial smooth muscle cells is shown in Fig. 3. The concentration of the lipoproteins is expressed both on the basis of cholesterol content (panel A) and on the basis of the number of molecules (panel B) of lipoprotein per ml of culture medium. The number of pmol of LDL was calculated from the actual measured molecular weight of the isolated LDL. For high density lipoprotein we used an estimated molecular weight of 250000. HDL produced only an approximate 25% increase in the rate of cholesterol esterification over that of the lipoprotein-deficient serum controls. In the range of cholesterol concentrations. tested there was no difference in the ability of HDL from normo- or hypercholesterolemic animals to stimulate cholesterol esterification. The LDL from hypercholesterolemic animals was approximately twice as effective in stimulating cholesterol esterification as the same amount of normal LDL. This difference persisted, even when the results were expressed on the basis of the number of molecules of LDL added. In four separate experiments utilizing three different lipoprotein preparations added to the culture medium at a concentration of 100 pg cholesterol/ml, LDL stimulated cholesterol esterification an average of 3.9 times that of the lipoprotein-deficient serum controls, while normal LDL stimulated cholesterol esterification 2.6 fold. In these same experiments in~o~oration of [I-14C]oleate into phospholipids and triglycerides was not altered by any of the lipoproteins tested. When [l-‘4C]oleate and LDL from either normo- or hypercholesterolemic animals were added to the cells in culture at the same time, the greater stimulatory effect on cholesterol esterification of the LDL from hypercholester-
4
8
16
12
lncubatmn
time
20
24
(hc )
Fig. 4. Influence of incubation time on stimulation of cholesterol estcrification by LDL from normo(N-LDL) and hypercholesterolemic (H-LDL) rhesus monkeys. Cells were incubated for 24 h with cultuxe medium containing ~poprotein~eficient serum prior to addition of the lipoprotein. LDL was added to smooth muscle cells in culture at a final concentration of 100 pg cholesterol/ml culture medium. [1-*4CJOleate substrate was added at the time of addition of the test lipoprotein. Separate lipoprotein preparations were used in each experiment. Each point represents the mean of duplicate cultures. Lipoproteindeficient serum (LDS) was included as a control for the influence of incubation time on cholesterol esterification.
288
I N= 3 at each paint i
;;*
L
--m
l
-*-A-.-.
i 4
I
2
_
I 6 Cell
(Ag
Cell
.
110
--
1
I
8
IO
I 12
I
1
I4
Density
Protein/cm2
Surface
Area1
Fig. 5. Influence of cell density on rate of incorporation of [l- 14~loleate into phospholipids, trigiyceridcs and cholesteryl esters in rhesus monkey arterial smooth muscle cells in culture. Cells were incubated with culture medium containing ~poprotei~~e~cient serum for 48 h prior to addition of LDL from hypercholesterolemic animals at a final concentration of 100 pg cholesterol/ml. After 12 h, [l-i4Clok?ate was added for 4 h in order to measure
esterification.
olemic animals was first evident after about 12 h of incubation (Fig. 4). This difference was further exaggerated after 24 h of incubation. As shown in Fig. 5, the density of cells in the tissue culture dish, as measured by pg cell protein/cm2 surface area, profoundly influenced the absolute rate of esterification of oleic acid. This was particularly true for esterification to cholesterol which decreased dramatically as the density of cells in the dish was increased. A similar effect of cell density on uptake of LDL by arterial smooth muscle cells in culture has been reported by Stein and Stein 2211. Consequently, we have been careful to compare data only within the same experiment in which the cell densities were constant for all treatments. Discussion The increase in the size of the LDL from the hypercholesterolemic rhesus monkeys of this study was similar to that reported by Rude1 et al. 1121. Consistent with previous studies by others f11,12], the major reason for the increased size of the LDL from hypercholesterolem~c animals was an increase in
289
the cholesteryl ester content. Each molecule of LDL from the hypercholesterolemic animals transported over 50% more cholesterol than normal LDL. Cells, particularly those maintained in tissue culture, appear to obtain the bulk of their cholesterol for maintenance of normal growth and metabolism from LDL of the culture medium. This occurs by means of binding and subsequent uptake of intact LDL by LDL receptors located on the plasma membrane [22]. Cholesterol internalized by this mechanism acts to suppress cholesterol synthesis and stimulate cholesterol esterification. These processes appear to be controlled by a pool of cholesterol within the cell, since cholesterol added to cells in a manner that circumvents the receptor will also regulate cellular cholesterol synthesis and esterification. Thus, in the present study, for an equivalent number of molecules of LDL from hypercholesterolemic animals taken into the (cell by the lipoprotein receptor there would be approximately 50% more cholesterol available to the cell. As a result of this increased amount of cholesterol internalized, the initial rate of cholesterol esterification and cholesteryl ester accumulation would be expected to be stimulated to a greater extent with the LDL from hypercholesterolemic animals. Another mechanism by which LDL from hypercholesterolemic animals could be more effective in stimulating cholesterol esterification and accumulation is secondary to an increased affintiy for binding to the LDL receptor. Such a mechanism could be mediated by changes in the concentration of specific apolipoproteins, such as the arginine-rich peptide. This apolipoprotein has been shown to bind to the high affinity LDL receptor [23] with an even higher affinity than apolipoprotein B [24]. Apolipoprotein B constituted the major apolipoprotein (93-97%) of LDL from normo- and hypercholesterolemic animals [lo]. In the LDL used in this study a small amount of protein migrated in the position where the arginine-rich and C-apolipoproteins are normally found. These apolipoproteins were not quantified although their increased intensity of staining (Fig. 2) suggest that they may be present in somewhat greater amounts in LDL from hypercholesterolemic animals. Nevertheless, individually they never account for more than approximately 3% of the total apolipoprotein of rhesus monkey LDL (Rudel, L.L., personal communication). Whether changes in apolipoproteins that constitute such a small percentage of the lipoprotein molecule can alter the binding and ultimate metabolism of the lipoprotein by the cell is unknown. Cholesterol from LDL of altered composition might also be transferred to cells through a process that does not require uptake of the intact lipoprotein molecule, By such a mechanism an increased rate of transfer of free cholesterol from an abnormal lipoprotein to the cells would result in the enrichment of cellular membranes with cholesterol. Such a mechanism has been suggested by Arbogast et al. [25] to explain the stimulation of cholesterol esterification and accumulation of cholesteryl esters in cells exposed to cholesterol-phosphatidylcholine dispersions. If such a mechanism occurred the excess membrane-free cholesterol could be kept within physiologically compatible limits by esterification and storage as cholesteryl ester droplets within the cell. Consistent with this possibility is the observation by Rothblat et al. [26] that incubation of microsomal membranes from rat hepatoma cells with hyperlipemic rabbit serum results in the enrichment of these membranes with cholesterol and this
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in turn stimulates cholesterol esterifieation, The same amount of cholesterol as normoli~emic rabbit serum was without effect in increasing membrane free cholesterol content or in stimulating cholesterol esterification. Cholesterol esterification is markedly stimulated in atherosclerotic arteries [27,28] and the bulk of this stimulation in cholesterol esterification is localized in cholesteryl ester-rich foam cells f29], In cells in culture, however, even though the cholesteryl ester content of the cells can be increased subst~ti~ly by addition of LDL to the culture medium [8,13,30,31], the rate of cholesterol esterification is rapidly inhibited as the cholesterol content of the cells increases [ 133. This appears to be due to regulation of high affinity binding sites for LDL by intracellular pools of cholesterol 132,331. In the atherosclerotic artery, cholesteryl ester synthesis can be stimulated as much as lOO-fold in tissue contain~g cells enriched with large amounts of cholesterol and cholesteryl esters [ 2834). Thus, the pathologic processes leading to the development of atherosclerosis cannot be adequately explained simply by an exaggerated uptake of LDL cholesterol by cells of the arterial wall via high affinity receptors for LDL. Whether this “pathological accumulation” of cholesteryl esters results from the unregulated entry of lipoproteins into cells of the arterial wall, as suggested by Goldstein and Brown 1221, or by other, as yet unreco~ized, mechanisms is unclear. The present studies suggest, however, that differences in the composition of lipoproteins, resulting from genetic, dietary or other environmental influences may play an important role in determining the efficiency of delivery of cholesterol to cells and perhaps ultimately its “atherogenic potential.” Acknowledgements The authors gratefully acknowledge the excellent technical assistance of Ns. Grayce Greene and the aid of Mrs. Brenda Warner in the preparation of this manuscript. This work was supported by a grant from the National Heart, Lung and Blood Institute (S.C.O.R.) HL-14164. References 1 Kannel. W.B.. Castelii. W.P., Gordon, T. and McNamara, P.M. (1971) The Framingham Study. Ann. Int. Med. 74, l-12 2 Clarkson, T.B.. P&hard, R.W.. Bullock, B.C., St. Clair. R.W.. Lehner, N.D.M., Jones, D.C., Wagner, W.D. and Rudel, L.L. (1976) Expt. Mol. Path& 24,264-286 3 St. Clah, R.W. and Harpold, G.J. (1975) Expt. Mol. Path&. 22.207-219 4 Fisher-Dzoga, K., Chen, R. and WissIer, R.W. (1977). Adv. Expt. Med. Bioi. 43. 299-311 5 Bates, S.R. and Wissler, R.W. (1976) Biochim. Biophys. Acta 450. 78-88 6 Pearson, J.D. (1976) Atherosclerosis 24, 233-242 7 Nikkari, T., Pietila. K. and Sale, M. (1976) Med. Biol. 54, 264-271 8 Rothblat, G.H. (1974) Lipids 9,526-535 9 Rothblat, G.H., Arbogast, L., Kritchevsky, D. and Noftulin. M. (1976) Lipids 11,97-108 10 Rudel, L.L., Pitts, III, L.L. and Nelson, C.A. (1977) J. Lipid Res. l&211-222 21 Mahleu, R.W., Weisglaber, K.H. and Inneraritv, ‘I’. (1976) Biochemistry 15,2979-2985 12 Rudel, L.L. and Lofland, H.B. (1977) Am. J. Med. 62,707-714 13 St. Clair, R.W., Smith, B.P. and Wood, L.L. (1977) Ciyc. Res. 40.166-173 14 Rudel. L.L., Lee, J.A., Morris, M.D. and Felts. J.M. (1974) Biochem. J. 139.89-95 15 Noble, R.P. (1968) J. Lipid Res. 9, 693-700 16 Rudel, L.L., Greene, D.G. and Shah, R. (1977) J. Lipid Res. 18,734-744
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