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The effect of serum lipoproteins on cholesterol content and sterol exchange in cultured human endothelial cells
312
Biochimica et Biophysics Acta, 574 (1979) 312-320 0 Elsevier/Nortb-Holland Biomedical Press
BBA 57414
THE EFFECT OF SERUM LIPOPROTEINS ON CHOLESTEROL CONTENT AND STEROL EXCHANGE IN CULTURED HUMAN ENDOTHELIAL CELLS
T. HENRIKSEN, S.A. EVENSEN *, J.P. BLOMHOFF, H. TORSVIK and B. CARLANDER Institute
for Surgical Research
and Medicaf Department
A, Rikshospitalet,
Oslo 1 ~~orwa~~
(Received December 2lst, 1978)
Key words: Arteriosclerosis;
Lipoprotein;
~ndotheiial
cell; Cholesterol
Summary
Cultured human endothelial cells preincubated with the infranatant of human serum increased their content of cholesterol when subsequently exposed to low density lipoproteins (LDL) as compared to control cultures further incubated in the presence of infranatant only. Replacing LDL with high density lipoproteins (HDL) resulted in no change in the cellular cholesterol content compared to the control. The addition of HDL did not influence the increase in cellular cholesterol content mediated by LDL. HDL stimulated the efflux of endogenously synthesized 14C-labelled sterols compared to the infranatant fraction, whereas LDL had only a slight effect. Cells preincubated with whole serum did not change their cholesterol content when subsequently exposed to LDL, compared to cultures further incubated in presence of whole serum, Replacing whole serum (during the final incubation) with infranatant, resulted in a decrease of the cellular cholesterol content, which was not influenced by further addition of HDL.
Introduction
Previous studies from our laboratory have demonstrated that serum lipoproteins influence sterol synthesis in cultured human endotheli~ cells [ 11. In the course of this work we observed that high density lipoprotein (HDL) and low density lipoprotein (LDL) influenced the release of endogenously synthesized
* To whom correspondence should be addressed.
313
14C-labelled sterols from the endothelial cells. In cultures of other cell types the lipoproteins have been demonstrated to influence the cholesterol content of the cells [ 2 -41. In vivo, the endothelial cells form a barrier between the blood and the subendothelial layers. Therefore, the endothelial cells occupy a unique position with respect to the exchange of cholesterol between lipoproteins and the vessel wall. In the present study we have investigated further the effect of serum lipoproteins on the cholesterol content and sterol exchange in cultured endothelial cells. Materials and Methods Endothelial cell cultures. Endothelial cells were isolated from the vein of human umbilical cords as previously described [ 51. They were grown in a synthetic culture medium consisting of RPM1 1640 (Grand Island Biological Company, GIBCO cat. No. 240), supplemented with 20% v/v fetal calf serum (GIBCO cat. No. 519) and antibiotics. 3-8day-old cultures were used. The culture medium was changed every second day, and the cells were not subcultured. Lipoproteins. Blood samples drawn from 4-10 subjects were allowed to coagulate at room temperature for 2 h. Lipoprotein fractions were prepared by ultracentrifugation at 105 000 Xg at 4°C for 24 h in a Spinco L2 65B by a slight modification of the procedure described by Have1 et al, [6]. The following density intervals were used: very low density lipoproteins (VLDL), d < 1.006 g/ml; LDL, d = 1.019-1.063 g/ml; HDL, d = 1.090-1.21 g/ml; infranatant, d > 1.21 g/ml. The isolated fractions were dialyzed extensively against 0.15 mol/l NaCl at 4°C for 24 h. After dialysis the lipoproteins were sterilized by filtration through a Millipore filter (pore size 0.22 m). The purity of the fractions was checked by gel electrophoresis [ 71. The lipoproteins were stored at 4°C maximally for 12 days. [l-‘4C]Acetczte incorporation in to sterols. After washing of the cells in situ with Earle’s balanced salt solution 4 times, fresh culture medium (0.5 ml/well) consisting of RPM1 1640 with 20% v/v infranatant (12 mg protein/ml), antibiotics and 1 &i/ml (final concentration), [1-14C]acetate (The Radiochemical Centre, Amersham, U.K., specific activity 60 Ci/mol) was added to series of endothelial cell cultures derived from the same umbilical cord. The incubation was continued for 48 h at 37°C in an atmosphere of 5% CO? in humidified air. Then the cells were washed in situ 4 times. Fresh culture medium (0.5 ml/well) containing various amounts of the separated lipoprotein fractions was added and incubation was continued for 24 h. The compositions of these media are described in the legends to the figures and tables. Determination of “C-labelled sterol radioactivity in cells and culture medium. The culture media were removed and the cells were washed in situ 4 times with Earle’s balanced salt solution. The washing waters were added to the incubation media. The cells were scraped off with a rubber policeman. Lipids were extracted from the cells and freeze-dried media according to Folch et al. [8] as previously described [9]. The lipid extracts were hydrolyzed in methanolic KOH (1 mol/ml) for 1 h at 60°C. The sterols of the unsaponifiable fraction were precipitated by digitonin and the digitonide precipitate was collected on a glass fibre filter (Whatman GFIA, W&R. Balston Ltd., England) [ 91. Free
314
and esterified sterols were separated by thin-layer chromatography as previously described [9]. The radioactivity of the isolated sterols was assayed in a liquid scintillation counter (counting efficiency 75%). The external standard ratio model was used for quench correction. The recovery of sterols was controlled by an internal standard (1,2-3H]cholesterol, spec. act. 30 000 Ci/mol, The Radiochemical Centre, Amersham U.K.). The number of nmol 14C-labelled sterols synthesized was calculated assuming 12 mol [1-14C]acetate per mol 14C-labelled sterols. Determination of endothelial cell cholesterol con tent. Endothelial monolayers were established as described above, using the same batch of fetal calf serum. After washing with Earle’s balanced salt solution 4 times, the cells were incubated for 48 h with RPM1 1640 containing either 20% v/v infranatant (12 mg protein/ml final medium) or 20% v/v human whole serum. Thereafter the cells were washed 4 times with Earle’s balanced salt solution and then exposed for 24 h to fresh incubation medium containing the various lipoprotein fraction. Then the incubation medium was removed and the cells were washed 5 times in situ with Earle’s balanced salt solution. The cells were scraped off with a rubber policeman and the lipids were extracted and hydrolyzed as described above. The unsaponifiable fraction was evaporated to dryness under Nz at 60°C and redissolved in heptan and stored at -20°C. The cholesterol content was assayed by gas-liquid chromatography [lo]. [ l-3H]cholesterol was used as an internal standard and loss of cholesterol during the extraction procedures was corrected. The coefficient of variation in cellular cholesterol content per well incubated under identical conditions was less than *18%). Determination of the number of cells per culture well. After 24 h incubation with the various lipoprotein fractions the cells were washed 4 times in situ and then exposed to trypsin-EDTA (GIBCO cat. No. 530) for 20 min at 37°C in The number of cells per well was order to obtain a single cell suspension. assayed using a Coulter counter [ 51. In the experiments presented below, estimation of cell cholesterol per well has not been corrected for variations in cell numbers since the limited number of endothelial cells obtained from one umbilical vein did not allow us to perform cell counting in parallel to cell cholesterol quantitation. The actual number of cells per well averaged 1.3 . 105. Chemical determinations. Cell proteins were separated by filtering the CHClJCH30H solution obtained during the Folch extraction through a glass fibre filter. The collected proteins were solubilized by exposure to 1 mol/l NaOH for 24 h at room temperature. The amount of protein was determined according to Lowry et al. [ll]. Lipoprotein cholesterol was assayed by gasliquid chromatography [ 101 and triacylglycerols were measured fluorimetrically [ 121. Statistical analysis. The Wilcoxon test for paired observations was used for calculation of the statistical significance [ 131. Results Table I shows the effect of the different lipoproteins on the cell number per culture well after 24 h incubation. The maximal variation in the cell number was *14%.
315 TABLE
I
THE EFFECT
OF THE DIFFERENT
LIPOPROTEINS
ON THE NUMBER
OF CELLS
PER CULTURE
WELL The cells
were preincubated
for
48 h in presence
of 15% v/v mfranatant
(d > 1.21)
dissolved
in RPM1
1640. followed by exposure to a medium containing 80% v/v RPM1 1640 and 15% v/v of infranatant to which was added 5% of the indicated lipoprotein fractions, or 20% v/v whole serum. Then the cultures were washed, the cells dispersed by trypsin-EDTA and the cell number determined using a Coulter counter. The figures represent the mean f S.D. of three triplicate experiments (i.e. 9 wells in each group). 24 h exposure to RPM1 1640 + infranatant the indicated fraction or whole serum
supplemented
with
Saline LDL HDL LDL + HDL Whole serum * The actual number
of cells per well averaged
1.3
pig lipoprotein cholesterol per ml medium
Number of cells per well {arbitrary units) *
0 250 100 350 350
1OOi 117 * 110 f 119 f 121 f
8 13 10 13 14
* 105.
Cells preincubated with 1.21 g/ml infranatant increased their cholesterol content when exposed to LDL (Table II) whereas HDL no signific~t effect. A 5-fold increase of the LDL or HDL concentration (from 50 to 250 ,zg cholesterol/ml) did not further increase the cell cholesterol content. The amount of cholesterol per mg cell protein was approx. 50, 25 and 17 pg after exposure to LDL, HDL or saline, respectively. Furthermore, replacing the infranatantlipoprotein combination with whole serum (20% v/v) during the final 24 h incubation, resulted in a cellular cholesterol content of approx. 45 gg/mg cell protein. The presence of HDL in the medium did not significantly change the LDLinduced accumulation of cholesterol in the cultured cells (Table III).
TABLE
II
CHOLESTEROL CONTENT OF FOLLOWED BY 24 h EXPOSURE
ENDOTHELIAL TO DIFFERENT
CELLS PREINCUBATED LIPOPROTEIN CLASSES
WITH
INFRANATANT
Established monolayers of endothelial cells were preincubated for 48 h in RPM1 1640 supplemented with 20% v/v infranatant (12 mg protein/ml medium). After 4 times washing. the cdls were incubated for 24 h in RPM1 1640 supplemented with 15% v/v infranatant (8 mg p~tein/mi) and 5% v/v HDL or LDL dissolved in saline or saline alone. At the end of the experiment the cells were washed and the cholesterd content of each well was determined. Each figure represents the mean f SD. of triplicate experiments. Expt. No.
pg cholesterol/well after 48 h preincubation with infranatant
&I cholesterol/well containing
-
1.5 i: 0.2 0.8 r 0.1 0.8 * 0.1
* /.cg cholesterol/ml
2.4 * 0.2 2.4 f 0.3 incubation
medium.
incubation
with infranatant
HDL *
LDL * 50
1 2 3
after 24 h subsequent
Saline
250
50
100
250
3.6 * 0.6 2.1 f 0.4 2.9 i 0.4
0.8 * 0.1
1.8 f 0.3 1.7 i 0.3 -
1.1 f 0.1
1.2 f 0.2 1.3 f 0.2 1.3 f 0.2
316 TABLE
III
CHOLESTEROL CONTENT OF ENDOTHELIAL FOLLOWED BY 24 h EXPOSURE TO DIFFERENT LIPOPROTEINS
CELLS PREINCUBATED WITH INFRANATANT COMBINATIONS OF HIGH AND LOW DENSITY
Established monolayers of endothelial cells were preincubated for 48 h in RPM1 1640 supplemented with 20% v/v infranatant (12 mg/ml medium). After 4 times washing, the cells were incubated for 24 h in RPM1 1640 containing 15% v/v infranatant (8 mg protein/ml medium) and 5% v/v of the indicated lipoprotein combinations dissolved in saline or saline alone. At the end of the experiment the cells were washed and the cholesterol content of each well was determined. Each figure represents the mean * S.D. of triplicate experiments. Cells from 3 different donors and HDL from 3 different preparations were used. Expt. No.
1 2 3
pg cholesterol/ well after 48 h weincubation with infrantant
0.8 * 0.1 -
* @g cholesterol/ml
pg cholesterol/well
after 48 h subsequent
LDL *
HDL *
250
90
1.9 f 0.3 3.0 * 0.4 3.2iO.4
1.1 * 0.1 -
incubation
incubation
with infranatant
containing
LDL * + HDL *
LDL
* + HDL * Saline
200
250
250
+ 200
I.7 f 0.2 1.8 * 0.2
2.1 * 0.2 3.2 + 0.4 3.5 f 0.5
+ 90
3.4 * 0.5 3.8 * 0.6
0.8 t 0.1 1.0 * 0.1 1.1 * 0.1
medium.
The effect of LDL and HDL on the cholesterol content of endothelial cells after preincubation with whole serum instead of infranatant was also investigated. Exposure to LDL in a concentration of 250 pug cholesterol/ml did not change the cholesterol content compared to values at the end of the preincubation. Replacing LDL with saline or HDL (100 pg cholesterol/ml) resulted in a similar reduction of the cellular cholesterol content (50 and 5276, respectively). Table IV shows that the presence of lipoproteins in the culture medium enhanced the efflux of 14C-labelled sterols from the cells. Heat inactivation of the serum did not change the efflux of 14C-labelled sterols, indicating that 1ecithin:cholesterol acyltransferase activity [ 141 and complement-dependent immunological factors were not involved. These findings made us investigate the effect of the different lipoproteins on the efflux of endogenously syn-
TABLE
IV
THE EFFECT OF HUMAN WHOLE SERUM AND 1.21 g/ml INFRANATANT ON THE EFFLUX 14C-STEROLS IN CULTURED ENDOTHELIAL CELLS ENDOGENOUSLY SYNTHESIZED
OF
[l-l 4 C] Acetate was incorporated into sterols of endothelial cells during 48 h incubation (in 24 cm2 flasks). After thorough washing the cells were exposed for 24 h to RPM1 1640 containing either 20% v/v whole serum (native or heat inactivated at 56’C for 30 mln). 20% v/v infranatant or saline 20% v/v. Thereafter the amount of 14C-labelled sterols in the cells and the medium was determined. Figures represent the mean of duplicate incubations (range less than 5%). Supplement
to RPM1 1640
Saline Infranatant Whole serum Heat-inactivated
whole serum
mol 14 Clabelled
sterols
Cells
Medium
Total
35.2 32.0 15.0 17.0
0.8 8.0 26.0 24.0
36.0 40.0 41.0 41.0
317
24
6 Incubotfon(h)
Fig. 1. Effect of whole serum, VLDL, LDL, HDL and infranatant on the efflux of endogenously synthesized 14C-labelled sterols. [l- 14C)Acetate was incorporated into sterols of endothelial cells during 48 h incubation as described in Methods. After thorough washing, the cells were exposed for 24 h to RPM1 1640 containing either 20% v/v isotonic saline (X); 20% v/v whole serum, n------n; or 17% vfv infranatant, the latter supplemented with 3% vjv of either VLDL; o----o, 160 pg triacylglycerol/mI medium; LDL, lA, 250 ng choiesterol/ml medium; HDL: A------a* 90 &g cholesterol/ml medium; or isotonic saline, b0. The efflux of 14C-~abelled sterols was calculated using the following formula: 96 efflux = dpm medium/dpm medium + cells X 100. Each point represents the mean of 3 separate experiments each run with duplicate determinations (S.D. = *4%).
I
8
I
50
100
155
pg LOL oi- WDi. cholesterol
i ml
medium
Fig. 2. Effect of various LDL and HDL cholesterol concentrations on the efflux of endogenously synthesixed 14C-labehed sterols. [I-14C1Acetate was incorporated into sterols of endothelial cells during a 48 h incubation. After thorough washing, the cells were exposed for 24 h to RPM1 1640 (80% v/v) containing either saline supplemented with HDL (a), or infranatant (17% v/v, 9.5 mg protein/ml medium) containing LDL () or HDL (a-a). The efflux of 14C-labelled sterols was calculated as described in the legend to Fig. 1. Each point represents the mean of duplicate incubations.
318 TABLE
V
EFFECT OF LIPOPROTEINS LY SYNTHESIZED STEROLS
AND WHOLE
SERUM
ON THE ESTERIFICATION
OF ENDOGENOUS-
The cells were preincubated for 48 h in the presence of [l-I4 Clacetate dissolved in RPM1 1640 (80% v/v) containing 20% v/v infranatant. Then the cells were washed and exposed to a medium consisting of 80% v/v of RPM1 1640 and 15% v/v of infranatant to which was added 5% of the indicated lipoprotein fractions. or 20% v/v whole serum. Then the cells were washed and the lipids extracted and separated by TLC. The radioactivity in the free and esterified fractions of sterols were assayed as described in Methods. 24 h exposure to RPM1 1640 + infranatant supplemented with the indicated fractions or whole serum
pg lipoprotein cholesterol/ml
medium
Esterified sterols
*
(%b) Saline LDL HDL LDL + HDL Whole serum
*
6 250 90 250 + 90 380
cpm esterified cpm esterified
+ free
1.2 23.2 1.0 31.8 38.2
x 100.
thesized sterols. Compared to a medium containing only RPM1 1640 and saline the addition of 17% v/v infranatant markedly increased the release of i4Clabelled sterols (Fig. 1). Addition of HDL further increased the efflux of 14Clabelled sterols (P < 0.02) whereas LDL had only a slight effect. In Fig. 2 the effect of different concentrations of LDL and HDL on the efflux of 14Clabelled sterols is shown. HDL increased efflux at concentrations above 50 yg HDL cholesterol/ml medium, whereas no similar effect of LDL was observed. In the absence of infranatant proteins, HDL mediated an efflux of nearly 15% of the 14C-labelled sterols (Fig. 2). Table V shows that LDL and whole serum in contrast to HDL, increased the esterified fraction of cellular 14C-labelled sterols. Furthermore, the presence of HDL did not interfere with LDL-induced esterification. Discussion Incubation of cultured endothelial cells with whole serum resulted in 2-3fold increase of the cholesterol content of the cultures as compared to infranatant. This effect of whole serum was probably mainly due to LDL. First, LDL dissolved in infranatant induced approximately the same net increment in the cellular cholesterol content as observed after incubation with whole serum. Second, the cholesterol level of cells preincubated with whole serum did not change when subsequently exposed to LDL (Table III). Under the same conditions, infranatant with or without HDL mediated a net loss of cellular cholesterol. The ability of LDL to increase the cholesterol content of cells has also been observed in smooth muscle cells and skin fibroblasts [3,4], and is attributed to the binding and uptake of LDL by specific receptors [2]. There is evidence that LDL receptors also exist in endothelial cells [9,15,16]. It is therefore probable that LDL increase the cholesterol content of endothelial cells by a similar mechanism. This would fit the observation that endothelial cells pre-
319
incubated with whole serum, and therefore supposedly having a minimum number of receptors, did not increase their cholesterol content after exposure to LDL [X,17]. The only serum fraction which mediated net loss of cholesterol was the infranatant. This marked effect of the infranatant has been observed in cultures of other cell types [2,18,19,20], and is assumed to be mainly due to its content of apolipoprotein A-I [ 20,211, However, further studies are necessary to characterize the nature of the marked cell cholesterol depleting effect of the infranatant. The observation that addition of HDL to infranat~t did not further promote net loss of cellular cholesterol, is consistent with observations in fibroblasts and smooth muscle cells [ 2,4,19]. It has been claimed that HDL can inhibit the LDL-induced increase of the cell cholesterol content [3,4]. A similar effect of HDL could not be demonstrated in endothelial cell cultures (Table III). This discrepancy may reflect different properties of endotlleli~ cells in culture, the preincubation time with the infranatant was different (48 h versus 18 h) and the described effect of HDL on cultured fibroblasts was only demonstrated at HDL/LDL ratios far above those found in vivo. Thus, it remains to be convincingly demonstrated that HDL can inhibit LDL induced increase of cellular cholesterol within physiological HDL/LDL ratios. The cholesterol content of cultured cells reflects the net effect of complex biochemical mechanisms. In the present study we also labelled cells with endogenously synthesized 14C-labelled sterols and studied how lipoproteins influence the efflux of these sterols. 48 h preincubation with [ l-14C]acetate was used in order to allow the 14C-labelled sterol to equilibrate with the sterol pools of the cultured cells. Preliminary experiments had showed that synthesis of 14C-labelled sterols in the first 24 h after removing the labelled precursor was minimal (less than 5% of the total amount of 14C-labelled sterols found at the end of the preincubation). HDL, but not LDL and VLDL, further increased the release of 14C-labelled sterols from cells when compared to infranatant. Therefore, HDL seemed to mediate increased exchange of cellbound sterols to the medium without net loss of cellular cholesterol. A similar ability of HDL to increase the exchange of sterols with the surroundings has been observed in L cells [ 221. It was demonstrated that LDL mediated increased esterification of sterols in endothelial cells when preincubated with infranatant (Table V). The less potent effect of LDL compared to HDL on the efflux of *4C-labelled sterols in cells preincubated with infranatant, could therefore be due to a decreased amount of free exchangeable sterols. However, whole serum also induced marked esterification, but simultaneously mediated a high degree of release of “C-labelled sterols. This result indicated that sterol esterification and sterol exchange with the medium may operate independently. The present study shows that the content of cholesterol in cultures of endothelial cells is markedly in~uenced both by lipoproteins and the infranatant. Whether alterations in the cholesterol content or metabolism of endothelial cells may influence the normal integrity of the endothelium is not known.
320
Acknowledgements We thank Dr. P. Fylling and his staff at Department of Obstetrics at Ulleva City Hospital for supplying us with umbilical cords. We are indebted to Miss Hilde Letnes, who performed the cholesterol determination by GLC. Dr. T. Henriksen is a Research Fellow of the Norwegian Research Council for Science and the Humanities. This work was supported by grants from the Norwegian Research Council for Science and the Humanities, the Norwegian Council on Cardiovascular Diseases and legacies from the University of Oslo. References Henriksen,
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Biochimica et Biophysics Acta, 574 (1979) 312-320 0 Elsevier/Nortb-Holland Biomedical Press
BBA 57414
THE EFFECT OF SERUM LIPOPROTEINS ON CHOLESTEROL CONTENT AND STEROL EXCHANGE IN CULTURED HUMAN ENDOTHELIAL CELLS
T. HENRIKSEN, S.A. EVENSEN *, J.P. BLOMHOFF, H. TORSVIK and B. CARLANDER Institute
for Surgical Research
and Medicaf Department
A, Rikshospitalet,
Oslo 1 ~~orwa~~
(Received December 2lst, 1978)
Key words: Arteriosclerosis;
Lipoprotein;
~ndotheiial
cell; Cholesterol
Summary
Cultured human endothelial cells preincubated with the infranatant of human serum increased their content of cholesterol when subsequently exposed to low density lipoproteins (LDL) as compared to control cultures further incubated in the presence of infranatant only. Replacing LDL with high density lipoproteins (HDL) resulted in no change in the cellular cholesterol content compared to the control. The addition of HDL did not influence the increase in cellular cholesterol content mediated by LDL. HDL stimulated the efflux of endogenously synthesized 14C-labelled sterols compared to the infranatant fraction, whereas LDL had only a slight effect. Cells preincubated with whole serum did not change their cholesterol content when subsequently exposed to LDL, compared to cultures further incubated in presence of whole serum, Replacing whole serum (during the final incubation) with infranatant, resulted in a decrease of the cellular cholesterol content, which was not influenced by further addition of HDL.
Introduction
Previous studies from our laboratory have demonstrated that serum lipoproteins influence sterol synthesis in cultured human endotheli~ cells [ 11. In the course of this work we observed that high density lipoprotein (HDL) and low density lipoprotein (LDL) influenced the release of endogenously synthesized
* To whom correspondence should be addressed.
313
14C-labelled sterols from the endothelial cells. In cultures of other cell types the lipoproteins have been demonstrated to influence the cholesterol content of the cells [ 2 -41. In vivo, the endothelial cells form a barrier between the blood and the subendothelial layers. Therefore, the endothelial cells occupy a unique position with respect to the exchange of cholesterol between lipoproteins and the vessel wall. In the present study we have investigated further the effect of serum lipoproteins on the cholesterol content and sterol exchange in cultured endothelial cells. Materials and Methods Endothelial cell cultures. Endothelial cells were isolated from the vein of human umbilical cords as previously described [ 51. They were grown in a synthetic culture medium consisting of RPM1 1640 (Grand Island Biological Company, GIBCO cat. No. 240), supplemented with 20% v/v fetal calf serum (GIBCO cat. No. 519) and antibiotics. 3-8day-old cultures were used. The culture medium was changed every second day, and the cells were not subcultured. Lipoproteins. Blood samples drawn from 4-10 subjects were allowed to coagulate at room temperature for 2 h. Lipoprotein fractions were prepared by ultracentrifugation at 105 000 Xg at 4°C for 24 h in a Spinco L2 65B by a slight modification of the procedure described by Have1 et al, [6]. The following density intervals were used: very low density lipoproteins (VLDL), d < 1.006 g/ml; LDL, d = 1.019-1.063 g/ml; HDL, d = 1.090-1.21 g/ml; infranatant, d > 1.21 g/ml. The isolated fractions were dialyzed extensively against 0.15 mol/l NaCl at 4°C for 24 h. After dialysis the lipoproteins were sterilized by filtration through a Millipore filter (pore size 0.22 m). The purity of the fractions was checked by gel electrophoresis [ 71. The lipoproteins were stored at 4°C maximally for 12 days. [l-‘4C]Acetczte incorporation in to sterols. After washing of the cells in situ with Earle’s balanced salt solution 4 times, fresh culture medium (0.5 ml/well) consisting of RPM1 1640 with 20% v/v infranatant (12 mg protein/ml), antibiotics and 1 &i/ml (final concentration), [1-14C]acetate (The Radiochemical Centre, Amersham, U.K., specific activity 60 Ci/mol) was added to series of endothelial cell cultures derived from the same umbilical cord. The incubation was continued for 48 h at 37°C in an atmosphere of 5% CO? in humidified air. Then the cells were washed in situ 4 times. Fresh culture medium (0.5 ml/well) containing various amounts of the separated lipoprotein fractions was added and incubation was continued for 24 h. The compositions of these media are described in the legends to the figures and tables. Determination of “C-labelled sterol radioactivity in cells and culture medium. The culture media were removed and the cells were washed in situ 4 times with Earle’s balanced salt solution. The washing waters were added to the incubation media. The cells were scraped off with a rubber policeman. Lipids were extracted from the cells and freeze-dried media according to Folch et al. [8] as previously described [9]. The lipid extracts were hydrolyzed in methanolic KOH (1 mol/ml) for 1 h at 60°C. The sterols of the unsaponifiable fraction were precipitated by digitonin and the digitonide precipitate was collected on a glass fibre filter (Whatman GFIA, W&R. Balston Ltd., England) [ 91. Free
314
and esterified sterols were separated by thin-layer chromatography as previously described [9]. The radioactivity of the isolated sterols was assayed in a liquid scintillation counter (counting efficiency 75%). The external standard ratio model was used for quench correction. The recovery of sterols was controlled by an internal standard (1,2-3H]cholesterol, spec. act. 30 000 Ci/mol, The Radiochemical Centre, Amersham U.K.). The number of nmol 14C-labelled sterols synthesized was calculated assuming 12 mol [1-14C]acetate per mol 14C-labelled sterols. Determination of endothelial cell cholesterol con tent. Endothelial monolayers were established as described above, using the same batch of fetal calf serum. After washing with Earle’s balanced salt solution 4 times, the cells were incubated for 48 h with RPM1 1640 containing either 20% v/v infranatant (12 mg protein/ml final medium) or 20% v/v human whole serum. Thereafter the cells were washed 4 times with Earle’s balanced salt solution and then exposed for 24 h to fresh incubation medium containing the various lipoprotein fraction. Then the incubation medium was removed and the cells were washed 5 times in situ with Earle’s balanced salt solution. The cells were scraped off with a rubber policeman and the lipids were extracted and hydrolyzed as described above. The unsaponifiable fraction was evaporated to dryness under Nz at 60°C and redissolved in heptan and stored at -20°C. The cholesterol content was assayed by gas-liquid chromatography [lo]. [ l-3H]cholesterol was used as an internal standard and loss of cholesterol during the extraction procedures was corrected. The coefficient of variation in cellular cholesterol content per well incubated under identical conditions was less than *18%). Determination of the number of cells per culture well. After 24 h incubation with the various lipoprotein fractions the cells were washed 4 times in situ and then exposed to trypsin-EDTA (GIBCO cat. No. 530) for 20 min at 37°C in The number of cells per well was order to obtain a single cell suspension. assayed using a Coulter counter [ 51. In the experiments presented below, estimation of cell cholesterol per well has not been corrected for variations in cell numbers since the limited number of endothelial cells obtained from one umbilical vein did not allow us to perform cell counting in parallel to cell cholesterol quantitation. The actual number of cells per well averaged 1.3 . 105. Chemical determinations. Cell proteins were separated by filtering the CHClJCH30H solution obtained during the Folch extraction through a glass fibre filter. The collected proteins were solubilized by exposure to 1 mol/l NaOH for 24 h at room temperature. The amount of protein was determined according to Lowry et al. [ll]. Lipoprotein cholesterol was assayed by gasliquid chromatography [ 101 and triacylglycerols were measured fluorimetrically [ 121. Statistical analysis. The Wilcoxon test for paired observations was used for calculation of the statistical significance [ 131. Results Table I shows the effect of the different lipoproteins on the cell number per culture well after 24 h incubation. The maximal variation in the cell number was *14%.
315 TABLE
I
THE EFFECT
OF THE DIFFERENT
LIPOPROTEINS
ON THE NUMBER
OF CELLS
PER CULTURE
WELL The cells
were preincubated
for
48 h in presence
of 15% v/v mfranatant
(d > 1.21)
dissolved
in RPM1
1640. followed by exposure to a medium containing 80% v/v RPM1 1640 and 15% v/v of infranatant to which was added 5% of the indicated lipoprotein fractions, or 20% v/v whole serum. Then the cultures were washed, the cells dispersed by trypsin-EDTA and the cell number determined using a Coulter counter. The figures represent the mean f S.D. of three triplicate experiments (i.e. 9 wells in each group). 24 h exposure to RPM1 1640 + infranatant the indicated fraction or whole serum
supplemented
with
Saline LDL HDL LDL + HDL Whole serum * The actual number
of cells per well averaged
1.3
pig lipoprotein cholesterol per ml medium
Number of cells per well {arbitrary units) *
0 250 100 350 350
1OOi 117 * 110 f 119 f 121 f
8 13 10 13 14
* 105.
Cells preincubated with 1.21 g/ml infranatant increased their cholesterol content when exposed to LDL (Table II) whereas HDL no signific~t effect. A 5-fold increase of the LDL or HDL concentration (from 50 to 250 ,zg cholesterol/ml) did not further increase the cell cholesterol content. The amount of cholesterol per mg cell protein was approx. 50, 25 and 17 pg after exposure to LDL, HDL or saline, respectively. Furthermore, replacing the infranatantlipoprotein combination with whole serum (20% v/v) during the final 24 h incubation, resulted in a cellular cholesterol content of approx. 45 gg/mg cell protein. The presence of HDL in the medium did not significantly change the LDLinduced accumulation of cholesterol in the cultured cells (Table III).
TABLE
II
CHOLESTEROL CONTENT OF FOLLOWED BY 24 h EXPOSURE
ENDOTHELIAL TO DIFFERENT
CELLS PREINCUBATED LIPOPROTEIN CLASSES
WITH
INFRANATANT
Established monolayers of endothelial cells were preincubated for 48 h in RPM1 1640 supplemented with 20% v/v infranatant (12 mg protein/ml medium). After 4 times washing. the cdls were incubated for 24 h in RPM1 1640 supplemented with 15% v/v infranatant (8 mg p~tein/mi) and 5% v/v HDL or LDL dissolved in saline or saline alone. At the end of the experiment the cells were washed and the cholesterd content of each well was determined. Each figure represents the mean f SD. of triplicate experiments. Expt. No.
pg cholesterol/well after 48 h preincubation with infranatant
&I cholesterol/well containing
-
1.5 i: 0.2 0.8 r 0.1 0.8 * 0.1
* /.cg cholesterol/ml
2.4 * 0.2 2.4 f 0.3 incubation
medium.
incubation
with infranatant
HDL *
LDL * 50
1 2 3
after 24 h subsequent
Saline
250
50
100
250
3.6 * 0.6 2.1 f 0.4 2.9 i 0.4
0.8 * 0.1
1.8 f 0.3 1.7 i 0.3 -
1.1 f 0.1
1.2 f 0.2 1.3 f 0.2 1.3 f 0.2
316 TABLE
III
CHOLESTEROL CONTENT OF ENDOTHELIAL FOLLOWED BY 24 h EXPOSURE TO DIFFERENT LIPOPROTEINS
CELLS PREINCUBATED WITH INFRANATANT COMBINATIONS OF HIGH AND LOW DENSITY
Established monolayers of endothelial cells were preincubated for 48 h in RPM1 1640 supplemented with 20% v/v infranatant (12 mg/ml medium). After 4 times washing, the cells were incubated for 24 h in RPM1 1640 containing 15% v/v infranatant (8 mg protein/ml medium) and 5% v/v of the indicated lipoprotein combinations dissolved in saline or saline alone. At the end of the experiment the cells were washed and the cholesterol content of each well was determined. Each figure represents the mean * S.D. of triplicate experiments. Cells from 3 different donors and HDL from 3 different preparations were used. Expt. No.
1 2 3
pg cholesterol/ well after 48 h weincubation with infrantant
0.8 * 0.1 -
* @g cholesterol/ml
pg cholesterol/well
after 48 h subsequent
LDL *
HDL *
250
90
1.9 f 0.3 3.0 * 0.4 3.2iO.4
1.1 * 0.1 -
incubation
incubation
with infranatant
containing
LDL * + HDL *
LDL
* + HDL * Saline
200
250
250
+ 200
I.7 f 0.2 1.8 * 0.2
2.1 * 0.2 3.2 + 0.4 3.5 f 0.5
+ 90
3.4 * 0.5 3.8 * 0.6
0.8 t 0.1 1.0 * 0.1 1.1 * 0.1
medium.
The effect of LDL and HDL on the cholesterol content of endothelial cells after preincubation with whole serum instead of infranatant was also investigated. Exposure to LDL in a concentration of 250 pug cholesterol/ml did not change the cholesterol content compared to values at the end of the preincubation. Replacing LDL with saline or HDL (100 pg cholesterol/ml) resulted in a similar reduction of the cellular cholesterol content (50 and 5276, respectively). Table IV shows that the presence of lipoproteins in the culture medium enhanced the efflux of 14C-labelled sterols from the cells. Heat inactivation of the serum did not change the efflux of 14C-labelled sterols, indicating that 1ecithin:cholesterol acyltransferase activity [ 141 and complement-dependent immunological factors were not involved. These findings made us investigate the effect of the different lipoproteins on the efflux of endogenously syn-
TABLE
IV
THE EFFECT OF HUMAN WHOLE SERUM AND 1.21 g/ml INFRANATANT ON THE EFFLUX 14C-STEROLS IN CULTURED ENDOTHELIAL CELLS ENDOGENOUSLY SYNTHESIZED
OF
[l-l 4 C] Acetate was incorporated into sterols of endothelial cells during 48 h incubation (in 24 cm2 flasks). After thorough washing the cells were exposed for 24 h to RPM1 1640 containing either 20% v/v whole serum (native or heat inactivated at 56’C for 30 mln). 20% v/v infranatant or saline 20% v/v. Thereafter the amount of 14C-labelled sterols in the cells and the medium was determined. Figures represent the mean of duplicate incubations (range less than 5%). Supplement
to RPM1 1640
Saline Infranatant Whole serum Heat-inactivated
whole serum
mol 14 Clabelled
sterols
Cells
Medium
Total
35.2 32.0 15.0 17.0
0.8 8.0 26.0 24.0
36.0 40.0 41.0 41.0
317
24
6 Incubotfon(h)
Fig. 1. Effect of whole serum, VLDL, LDL, HDL and infranatant on the efflux of endogenously synthesized 14C-labelled sterols. [l- 14C)Acetate was incorporated into sterols of endothelial cells during 48 h incubation as described in Methods. After thorough washing, the cells were exposed for 24 h to RPM1 1640 containing either 20% v/v isotonic saline (X); 20% v/v whole serum, n------n; or 17% vfv infranatant, the latter supplemented with 3% vjv of either VLDL; o----o, 160 pg triacylglycerol/mI medium; LDL, lA, 250 ng choiesterol/ml medium; HDL: A------a* 90 &g cholesterol/ml medium; or isotonic saline, b0. The efflux of 14C-~abelled sterols was calculated using the following formula: 96 efflux = dpm medium/dpm medium + cells X 100. Each point represents the mean of 3 separate experiments each run with duplicate determinations (S.D. = *4%).
I
8
I
50
100
155
pg LOL oi- WDi. cholesterol
i ml
medium
Fig. 2. Effect of various LDL and HDL cholesterol concentrations on the efflux of endogenously synthesixed 14C-labehed sterols. [I-14C1Acetate was incorporated into sterols of endothelial cells during a 48 h incubation. After thorough washing, the cells were exposed for 24 h to RPM1 1640 (80% v/v) containing either saline supplemented with HDL (a), or infranatant (17% v/v, 9.5 mg protein/ml medium) containing LDL () or HDL (a-a). The efflux of 14C-labelled sterols was calculated as described in the legend to Fig. 1. Each point represents the mean of duplicate incubations.
318 TABLE
V
EFFECT OF LIPOPROTEINS LY SYNTHESIZED STEROLS
AND WHOLE
SERUM
ON THE ESTERIFICATION
OF ENDOGENOUS-
The cells were preincubated for 48 h in the presence of [l-I4 Clacetate dissolved in RPM1 1640 (80% v/v) containing 20% v/v infranatant. Then the cells were washed and exposed to a medium consisting of 80% v/v of RPM1 1640 and 15% v/v of infranatant to which was added 5% of the indicated lipoprotein fractions. or 20% v/v whole serum. Then the cells were washed and the lipids extracted and separated by TLC. The radioactivity in the free and esterified fractions of sterols were assayed as described in Methods. 24 h exposure to RPM1 1640 + infranatant supplemented with the indicated fractions or whole serum
pg lipoprotein cholesterol/ml
medium
Esterified sterols
*
(%b) Saline LDL HDL LDL + HDL Whole serum
*
6 250 90 250 + 90 380
cpm esterified cpm esterified
+ free
1.2 23.2 1.0 31.8 38.2
x 100.
thesized sterols. Compared to a medium containing only RPM1 1640 and saline the addition of 17% v/v infranatant markedly increased the release of i4Clabelled sterols (Fig. 1). Addition of HDL further increased the efflux of 14Clabelled sterols (P < 0.02) whereas LDL had only a slight effect. In Fig. 2 the effect of different concentrations of LDL and HDL on the efflux of 14Clabelled sterols is shown. HDL increased efflux at concentrations above 50 yg HDL cholesterol/ml medium, whereas no similar effect of LDL was observed. In the absence of infranatant proteins, HDL mediated an efflux of nearly 15% of the 14C-labelled sterols (Fig. 2). Table V shows that LDL and whole serum in contrast to HDL, increased the esterified fraction of cellular 14C-labelled sterols. Furthermore, the presence of HDL did not interfere with LDL-induced esterification. Discussion Incubation of cultured endothelial cells with whole serum resulted in 2-3fold increase of the cholesterol content of the cultures as compared to infranatant. This effect of whole serum was probably mainly due to LDL. First, LDL dissolved in infranatant induced approximately the same net increment in the cellular cholesterol content as observed after incubation with whole serum. Second, the cholesterol level of cells preincubated with whole serum did not change when subsequently exposed to LDL (Table III). Under the same conditions, infranatant with or without HDL mediated a net loss of cellular cholesterol. The ability of LDL to increase the cholesterol content of cells has also been observed in smooth muscle cells and skin fibroblasts [3,4], and is attributed to the binding and uptake of LDL by specific receptors [2]. There is evidence that LDL receptors also exist in endothelial cells [9,15,16]. It is therefore probable that LDL increase the cholesterol content of endothelial cells by a similar mechanism. This would fit the observation that endothelial cells pre-
319
incubated with whole serum, and therefore supposedly having a minimum number of receptors, did not increase their cholesterol content after exposure to LDL [X,17]. The only serum fraction which mediated net loss of cholesterol was the infranatant. This marked effect of the infranatant has been observed in cultures of other cell types [2,18,19,20], and is assumed to be mainly due to its content of apolipoprotein A-I [ 20,211, However, further studies are necessary to characterize the nature of the marked cell cholesterol depleting effect of the infranatant. The observation that addition of HDL to infranat~t did not further promote net loss of cellular cholesterol, is consistent with observations in fibroblasts and smooth muscle cells [ 2,4,19]. It has been claimed that HDL can inhibit the LDL-induced increase of the cell cholesterol content [3,4]. A similar effect of HDL could not be demonstrated in endothelial cell cultures (Table III). This discrepancy may reflect different properties of endotlleli~ cells in culture, the preincubation time with the infranatant was different (48 h versus 18 h) and the described effect of HDL on cultured fibroblasts was only demonstrated at HDL/LDL ratios far above those found in vivo. Thus, it remains to be convincingly demonstrated that HDL can inhibit LDL induced increase of cellular cholesterol within physiological HDL/LDL ratios. The cholesterol content of cultured cells reflects the net effect of complex biochemical mechanisms. In the present study we also labelled cells with endogenously synthesized 14C-labelled sterols and studied how lipoproteins influence the efflux of these sterols. 48 h preincubation with [ l-14C]acetate was used in order to allow the 14C-labelled sterol to equilibrate with the sterol pools of the cultured cells. Preliminary experiments had showed that synthesis of 14C-labelled sterols in the first 24 h after removing the labelled precursor was minimal (less than 5% of the total amount of 14C-labelled sterols found at the end of the preincubation). HDL, but not LDL and VLDL, further increased the release of 14C-labelled sterols from cells when compared to infranatant. Therefore, HDL seemed to mediate increased exchange of cellbound sterols to the medium without net loss of cellular cholesterol. A similar ability of HDL to increase the exchange of sterols with the surroundings has been observed in L cells [ 221. It was demonstrated that LDL mediated increased esterification of sterols in endothelial cells when preincubated with infranatant (Table V). The less potent effect of LDL compared to HDL on the efflux of *4C-labelled sterols in cells preincubated with infranatant, could therefore be due to a decreased amount of free exchangeable sterols. However, whole serum also induced marked esterification, but simultaneously mediated a high degree of release of “C-labelled sterols. This result indicated that sterol esterification and sterol exchange with the medium may operate independently. The present study shows that the content of cholesterol in cultures of endothelial cells is markedly in~uenced both by lipoproteins and the infranatant. Whether alterations in the cholesterol content or metabolism of endothelial cells may influence the normal integrity of the endothelium is not known.
320
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