Scandinavian Journal of Clinical and Laboratory Investigation
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Toxicity of sitosterol to human umbilical vein endothelial cells in vitro K. M. Boberg, K. S. Pettersen & H. Prydz To cite this article: K. M. Boberg, K. S. Pettersen & H. Prydz (1991) Toxicity of sitosterol to human umbilical vein endothelial cells in vitro, Scandinavian Journal of Clinical and Laboratory Investigation, 51:6, 509-516 To link to this article: http://dx.doi.org/10.3109/00365519109104559
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Scand J Clin Lab Invest 1991: 51: 509-516
Toxicity of sitosterol to human umbilical vein endothelial cells in vitro K. M . BOBERG, K. S . PETTERSEN & H . PRYDZ"
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Institute of Clinical Biochemistry and *Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo, Norway.
Boberg KM, Pettersen KS, Prydz H. Toxicity of sitosterol to human umbilical vein endothelial cells in vitro. Scand J Clin Lab Invest 1991; 51: 509-516. The pathogenesis of the early development of atherosclerosis in sitosterolaernia is unknown. The effect of sitosterol on vascular endothelial cells in vitro was investigated by culturing human umbilical vein endothelial cells in the presence of up to 0.7 mmol I-' of sitosterol. Liposomes were used to supply the high sterol concentrations. Exposure to 0.7 mmol I-' of sitosterol for 72 h caused contraction of the endothelial cells and increased release of intracellular lactate dehydrogenase. After 96 h incubation the cells were partly detached from the substrate. At this time-point 0.35 mmol I-' of sitosterol also caused perturbation of the endothelial cells. However, we could not confirm previous reports that tissue plasminogen activator production was enhanced by sitosterol. Key-words: atherosclerosis; endothelial cells; lactate dehydrogenase release; sitosterolaemia; tissue plasminogen activator; von Willebrand factor Kirsten Muri Boberg, Institute of Clinical Biochemistry, Rikshospitalet, 0027 Oslo I , Norway.
Early development of atherosclerosis is a major clinical finding in the rare familial sterol storage disease sitosterolaemia [l-41. Some patients have died from coronary heart disease at an early age [I, 21. Plant sterols, mainly sitosterol and campesterol, are absorbed in excess and accumulate in serum, xanthomas, arteries, and other tissues in sitosterolaemia patients. In serum they may constitute 7-26% of the total sterols 11-41. The cholesterol level is normal or moderately elevated [ 1-41. The primary metabolic defect and the pathogenesis of the premature atherosclerosis in this disease are unexplained. Endothelial cell injury may be an important factor in atherogenesis 15-61. It is possible that minor disturbances in endothelial cell function
may initiate the atherogenic process [5, 61. Perturbation of endothelial cell function may initiate the atherogenic process [5, 61. Perturbation of endothelial cells by sitosterol has been suggested, since sitosterol has an ability to increase synthesis and secretion of plasminogen activator from cultured bovine carotid artery endothelial cells (7-91. In the present study we wanted to see if this finding is reproducible in human vascular endothelial cells and to investigate possible effects of sitosterol on other aspects of endothelial cell damage: release of tissue plasminogen activator (antigen) and activator inhibitor (activity and antigen), von Willebrand factor, lactate dehydrogenase, and 2-deo~y-D-[l-'~C]gIucose, and synthesis of thromboplastin. 509
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MATERIALS AND METHODS
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Muteriuls Sitosterol (24-ethyl-S-cholesten-3~3-ol)(98% pure) was obtained from Alltech Associates. Dearfield, IL, USA. The purity of this material was ascertained by high-performance liquid chromatography (HPLC) prior to use. Chotesterol, epicoprostanol (5[3-cholestan-3a-ol). oI.-a-phosphatidyl-1,-serine-dipalmitoyl, and t.-a-phosphatidylcholin P-oleoyl-palmitoyl were purchased from Sigma, St Louis, MO, USA. 2-Deoxy-r1-[l-"C] glucose (59 mCi mmol-') and [4-"C] sitosterol (56 mCi mmol-') were obtained from Amersham International, Amersham, UK.
cholesterol in the final incubation medium. epicoprostanol was added as internal standard before alkaline hydrolysis and lipid extraction with n-hexane. as described previously [ 111. Aliquots of the lipid extracts were analysed by gas chromatography, using a gas chromatograph (5880A Hewlett-Packard, Avondale, PA, USA) with a 25 m long capillary column (i.d. 0.32 mm) CP-WAX 52 CB. In one set of experiments sitosterol was dissolved in ethanol and added to the medium at final concentrations of 0.05 mmol I-' and 0.1 mmol I-' and 0.5'% ethanol. The suspensions were sonicated before use. Controls contained 0.5% ethanol only.
Uptuke of sitosterol by endothefial cells Cell cdrure Human umbilical vein endothelial cells were cultured according to Jaffe et ul. [ 101. The cells were plated in 12-well tissue culture clusters (Costar. Cambridge, MA, USA), using 1.0 ml of RPMI-1640 supplemented with 20% fetal bovine serum (FBS) (Sigma), penicillin (50 U ml I), streptomycin ( 5 0 pg ml-I), and Lglutamine (2 mmol I I) (Gibco, Paisley, UK). Medium was changed every second or third day. Primary cultures reached confluence after 3-7 days. Secondary cultures were established by treatment with trypsin-EDTA (Flow, Irvine, UK) and used in all experiments when confluent. All incubations and assays were performed in serum-containing media.
Preprution of liposornes Phosphatidylcholine, phosphatidylserine, and sitosterol (molar ratios 3.9: 1:5.9), or cholesterol (molar ratios 3.9: 1:6.3). were dissolved in chloroform. dried by rotary evaporation under nitrogen and resuspended in culture medium with 20'L FBS by shaking at 4 "C. followed by repeated sonication for periods of 10 s. Control liposomes were prepared a s above. without addition o f sitosterol o r cholesterol. The procedure was performed under sterile conditions. I n addition to incuhations with the three different kinds o f liposomecontaining media, control experiments were carried o u t with ordinary culture medium. To determine the concentration of sitosterol and
In some experiments the liposomes were prepared with '4C-sitosterol (0.56 mCi mmol I . ' ) . After incubation for 6, 12, or 24 h the culture medium was removed, and the cell layer was washed with ice-cold veronal-buffered saline (VBS). The cells were homogenized in 0.5 ml o f buffer, and a sample was assayed for radioactivity in a Tri-Carb liquid scintillation analyser, model 1500 (Packard Instrument, Downers Grove, IL, USA). Viability of the cells in culture during sitosterol o r cholesterol treatment was tested using tryphan blue by standard methods.
Lactate dehydrogenuse ( L D H ) LDH was quantified by a photometric method [12]. The coefficient of variation in the actual range of measurements was 4.5'%. The cells were suspended in VBS and disrupted by sonication. LDH was assayed in the medium and in the cell lysate. Release of intracellular LDH to the medium is expressed as percentage of total L D H in medium and cells. Corrections were made for the content of LDH in fetal bovine serum, which was about 250 UI-'.
voii
Wilkhrund fuctor ( v WF)
vWF was measured by an enzyme-linked immunosorbent assay (ELISA) (F. Brostad. personal communication), coating the plates with a rabbit antibody against human vWF
Cytotoxicity of' sitosterol (Dakopatts, Denmark). After incubation with the medium samples, a goat anti-human von Willebrand factor (Atlantic Antibodies, Scarborough, ME, USA) was added as secondary antibody. The plates were then incubated with peroxidase-conjugated swine antigoat IgG (TAGO, Burlingame, C A , USA), and the peroxidase activity was determined. O n e unit of vWF antigen is defined as the quantity present in 1 ml of pooled normal platelet-poor citrated plasma.
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Thromboplustin activity (TPL) TPL of the endothelial cell homogenates was tested in a one-stage system [ 131, consisting of 0.1 ml normal human platelet-poor citrated plasma and 0.1 ml of the solution to be tested, both prewarmed separately for 3 min at 37 "C. Coagulation was started by the addition of 0.1 ml prewarmed 30 mmol I-' CaCI2. The results were compared with a standard preparation of human brain thromboplastin which clotted normal human plasma in 15-18 s. The curve (double logarithmic plot) was linear between 0.1 U ml-' (87 s) and 10 U ml-' (24 s). Protein was measured according to Markwell et ul. [14].
Tissue plusminogen uctivutor untigen (t-PA) t-PA was measured by an ELISA (Biopool lmulyse. t-PA, U m e i , Sweden). The values are given as ng t-PA mg-' cell protein or as ng t-PA mI supernatant.
'
Tissue plusminogen uctivutor inhibitor-] (PAI1)
PAI-1 activity was measured by the CoAS E T t-PA/PAI method (KabiVitrum, Molndal, Sweden), adjusted to assay PAI-1 in endothelial cell supernatants. A linear standard curve for PAI-1 was established by using the preparation supplied with the kits. PAI-1 antigen was measured by the Biopool Tintelize PAI-1 kit as described by the manufacturer. All tests were in duplicate (two wells from the same umbilical cord).
Release of 2 - d e o x y - ~ - l 1 - ' ~glucose. c/ This was used as a parameter ofendothelial cell damage and was assayed as described by
51 1
Andreoli et rzl. [ 1.51. Confluent layers of endothelial cells were radiolabelled by incubation with 3.0 x 10" d.p.m. of 2-deoxy-1>-(l-"C] glucose per well for I8 h. The cells were washed four times with VBS and subsequently incubated with control medium or medium containing liposomes for periods of 2, 4. 6, 12, and 24 h. Approximately 1.4% of the added "C-activity was retained in the cells after the radiolabelling procedure. A t the end of each experiment the monolayer was washed twice, and the cells were disrupted by homogenization. Aliquots of the supernatants, washes, and cell homogenates were counted. Release of radioactivity to the supernatants was calculated as per cent of total activity.
Stutisticx Student's t-test and Wilcoxon's signed-rank test were used for statistical analysis.
RESULTS By incorporating sitosterol into liposomes we obtained a final concentration of this sterol in the incubation medium of 0.6-0.7 mmol I - ' , which resembles the plasma sitosterol level in untreated sitosterolaemia patients [ 1-41, The cholesterol concentration in sitosterolenriched medium, medium with control liposomes, and ordinary medium was approximately 0.15 mmol I-', owing to the cholesterol content in fetal bovine serum. In incubations lasting 24, 48, 72, and 96 h medium containing only half the above sterol concentrations was also used. In medium prepared with cholesterol liposomes the total cholesterol concentration amounted to 1.18 mmol I-'. The cellular uptake of sitosterol was in the range 30-70 pg mg-' cell protein. There was no further significant increase in sterol incorporation into the cells after 6 h.
Morphology and L D H release Exposure to sitosterol or cholesterol-containing liposomes for up to 48 h caused no changes in the light microscopy appearance of the endothelial cells, and there was no uptake of Tryphan blue after the incubations. There was
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2'4
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Incubation time (h) FIG.1. Release o f LDH into the culture medium of endothelial cells. i n per cent of total LDH in medium and cells. The content of LDH in fetal bovine serum was corrected for. Ordinary medium (M), medium containing control liposomes without addcd sterols (L), liposomes with sitosterol, 0.7 mmol I~I (SI) and 0.35 mmol I ' (S2). and liposomes with cholesterol, I . 18 mmol I I ( C ) .Each value is mean k SEM of two independent experiments, each comprising three separate cultures (M, L, SI), o r of three separate cultures from the same umbilical cord (S2, C).
no significant difference between the experimental groups in the amount of L D H present in the conditioned media for incubations lasting up to 48 h. After incubation with 0.7 mmol I-' of sitosterol in liposomes for 72 h, however, contraction of the endothelial cells with formation of intercellular gaps was observed. After 96 h a small number of the sitosterolexposed cells were detached from the substrate and floating in the medium. At this time-point cell contraction and intercellular gaps were also visible at 0.35 mmol I-' o f sitosterol. The cell monolayer remained intact in incubations with cholesterol and in the control experiments. Concomitant with the morphological alterations release of LDH increased (Fig. 1). At 72 h the mean release in the presence of 0.7 mmol I-' of sitosterol was 63% (p=0.008) compared with 35% for the control cultures. At Y6 h the LDH release reached 83%)(p=O.OOl) at 0.7 mmol I-'. and 0.35 mmol I-' o f sitosterol led to 64% release. The release in the controls did not incrcase (36%).
t-PA utitigeti The amounts of t-PA antigen released varied greatly between the secondary endothelial cell
cultures originating from different umbilical cords. During the period up to 24 h the levels of t-PA antigen increased in the control cell supernatants as well as in the supernatants of cells incubated with sitosterol liposomes or control liposomes. There were no significant differences between these three sets of values (p>0.25). The kinetics of release of t-PA in the cholesterol-exposed cultures were slightly different; a rapid increase reaching a plateau at about 6 h. Also, in prolonged incubations up to 96 h the production of t-PA antigen in the control cultures increased essentially linearly with time (Fig. 2). The amounts of t-PA in the medium after incubations with 0.35 mmol I-' of sitosterol were equal to the controls, while a slight decrease was noted for 0.7 mmol I-' after Y6 h. In no case was the t-PA production increased in sitosterol-exposed cells compared with control cells.
PAI-I uctivity In preliminary experiments there were no significant effects of sitosterol-containing lipow m e s on the release of either PAI-I activity (n=3) or antigen ( n = l ) up to Y6 h. The mean control levels at 24 h were 2.8 U ml-' and 0.8 pg mI-I, respectively.
Cytotoxicity of sitosterol 14
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-
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24
48 Incubation time (h)
FIG. 2. Production of t-PA antigen in confluent secondary cultures of human umbilical vein endothelial cells. Ordinary medium (M), medium containing control liposomes without added sterols (L), liposomes with sitosterol, 0.7 mmol I-' (Sl) and 0.35 mmol I-' (S2), and liposomes with cholesterol 1.18 mmol I-' (C). Results of one experiment with parallel cultures.
6
12
18
24
Incubation time (h)
FIG.3. Production of vWF antigen in confluent secondary cultures of human umbilical vein endothelial cells. Ordinary medium (A),medium containing control liposomes without added sterols (0).liposomes with and liposomes with cholesterol, 1.18 mmol I-' ( A ) . Each value is mean f SEM sitosterol, 0.7 mmol I-' (0). of two to five independent experiments, each comprising two separate cultures. Total number of cultures tested for each mean is given in brackets.
Thromboplastin activity
von Willebrand factor (v WF)
Untreated endothelial cells did not express thromboplastin activity. Neither the sitosteroland cholesterol-containing liposomes, nor the control liposomes, caused a detectable rise in thromboplastin activity. Previous control experiments have shown that induced thromboplastin is not degraded or inactivated during the isolation procedure prior to assay.
Endothelial cells isolated from different umbilical cords secreted various amounts of vWF to the culture medium, as observed by others [16]. The level of vWF antigen increased in a time-dependent manner during incubation with all four kinds of medium (Fig. 3 ) . Sitosterolcontaining medium did not provoke larger release of vWF than medium with control
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liposomes o r cholesterollliposomes. After 12 h and 24 h incubations cells treated with each of the three liposome-containing media had released less vWF antigen than had control cells (Fig. 3). The difference reached borderline significance (p=O.O5) at 24 h. Sonication of medium containing FBS did not cause a similar reduction.
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Release
of 2-deoxy-u-ll-'4Clgluc.ose
The release of intracellular 2-deoxy-~-[l-"C] glucose was linear up to about 6 h, whereafter it levelled off slightly to reach about 80% in all cultures at 12 h. There was no detectable effect of any kind of liposomes on the release. Using 0.5% ethanol as vehicle, only limited amounts (up to 0.1 mmol I I ) of sitosterol could be dissolved in the medium. Compared with control cultures, 0.05 mmol I-' and 0.1 mmol I-' of sitosterol did not perturb the endothelial cells. In particular, the production of t-PA was at no point increased in sitosterol-exposed cells.
DISCUSSION In the atherosclerotic lesions and xanthomas of sitosterolaemia patients plant sterols (sitosterol and campesterol), Sa-saturated stanols (Susitostanol, Su-campesterol. cholestanol). and cholesterol are deposited in approximately the same proportions as found in plasma [ 1-41, but the events promoting the excessive sterol uptake and atheroma development are unknown. Using liposomes as vehicle, we cultured the endothelial cells in the presence of sitosterol at concentrations similar t o those found in sitosterolaemia patients. From a biological point of view the optimal way of administering the sterol would be by incorporation into low-density lipoproteins (LDL). However, liposomes bind to the endothelial cell surface and exchange lipids or fuse with the cell membrane [17]. Sitosterol had n o effect on endothelial cell viability or morphology at the light microscopy level up to 48 h. The cells retained their ability to exclude Tryphan blue and did not release increased amounts of L D H . However, after prolonged incubations for 72 h and 06 h signs o f endothelial cell injury appeared. Contraction o f endothelial cells with simultaneously
increased LDH release has previously been reported after incubations with cholestane-3P. Sa. 6fLtriol [18]. This finding was associated with significant increase in albumin transfer across the endothelial cell monolayer, implying decreased barrier function. Exposure t o high sitosterol concentrations for 72-96 h was required to detect signs of endothelial cell perturbation in the present study. However, the plasma sitosterol levels in sitosterolaemia patients might be even higher. The mean value in a group of 14 patients investigated by Salen ef ul. [4] was 0.85 mmol I-'. In sitosterolaemia patients the endothelial lining of the blood vessels is continuously exposed to the enhanced levels of sitosterol. It seems possible that sitosterol contributes to the atherogenesis by interfering with the endothelial cell function. 7f3-Hydroxycholesterol, which has also been implicated in the pathogenesis ofatherosclerosis, caused similar changes o f endothelial cells. even though more pronounced. in studies in our laboratory [ 101. Other studies have indicated that sitosterol might interfere with cell viability. Sitosterol supplied with the diet retarded development of chemically induced colon carcinomas in rats [20]. Changes in cellular metabolism or kinetics secondary t o alterations in the epithelial cell membrane were suggested as possible mechanisms of this effect. I n experiments with Ehrlich ascites tumour cells in suspension culture sitosterol at the level of 0.1 mmol I-' in the culture medium reduced the cell survival to about 65% after 24 h (I. SmithKielland, personal communication). In the present experiments with human umbilical vein endothelial cells we could not confirm the previous reports that tissue plasminogen activator release was stimulated by sitosterol in cultured bovine carotid artery endothelial cells 17-91, despite using more than twice the highest concentrations of sterol previously used. These authors [7-91 administered the sterol dissolved in ethanol and found a marked increase in the activity of plasminogen activator in the conditioned media at sitosterol concentrations above 0.02 mmol 1 I , levelling off at 0.05 mmol I I. N o further increase was seen even at 0.25 mino1 I-'. The fihrinolytic activity was assayed by nephelometric measurement ofturbiditydecrease in a fibrin suspcnsion. Plasminogen activators are released from cultured human umbilical vein endothelial cells
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Cytotoxicity
of sitosterol
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1211. Due to concomitant production of plasREFERENCES minogen activator inhibitor (PAI). the t-PA is Bjdrkhem I, Skrade S. Familial diseases with storage of sterols other than cholesterol: cerebroinactive and not detectable in fibrinolytic assays tendinous xanthomatosis and phytosterolemia. In: 122). Using a sensitive ELISA, we observed Scriver CR, Beaudet AL. Sly WS, Valle D, eds. release of t-PA antigen that increased linearly The Metabolic Basis of Inherited Disease. New with time. Sitosterol-exposed cells and control York: McGraw-Hill. 1989: 1283-302. cells released similar amounts of t-PA antigen Salen G , Horak I, Rothkopf M, Cohen JL, Speck J, Tint GS, Shore V, Doyal B, Chen T, Shefer S. when either liposomes (up to 0.7 mmol I-' of Lethal atherosclerosis associated with abnormal sitosterol) or ethanol (up to 0.1 mmol l-') was plasma and tissue sterol composition in sitosteroused as vehicle. Species differences with respect lemia with xanthomatosis. J Lipid Res 1985; 26: to release of plasminogen activators might 1126-33. Miettinen TA. Phytosterolaemia, xanthomatosis explain the different results. Sitosterol has been and premature atherosclerotic arterial disease: a proposed as an agent for prevention and case with high plant sterol absorption, impaired therapy of thrombosis [ 7 ] , but the results of sterol elimination and low cholesterol synthesis. the present study do not support this idea. Eur J Clin Invest 1980; 10: 27-35. Salen G , Kwiterovich PO Jr, Shefer S, Tint GS, Human umbilical vein endothelial cells are Horak J, Shore V, Doyal B, Horak E. Increased considered a representative model for human plasma cholesterol and 5a-saturated plant sterol endothelial cells of various other origins with derivatives in subjects with sitosterolemia and respect to synthesis and release of vWF 1231. xanthomatosis. J Lipid Res 1985; 26: 203-9. vWF may be secreted constitutively or remain 5 Ross R, Glomset JA. The pathogenesis of atherosclerosis. N Engl J Med 1976; 295: 369-77. stored in intracellular organelles (Weibel-Palade 6 Ross R. Harker L. Hvoerlioidemia and atherobodies), available for relzase in response to sclerosis. Chronic hyperlipidemia initiates and vascular injury [24]. Neither sitosterol nor maintains lesions by endothelial cell desquamation cholesterol stimulated secretion of vWF above and lipid accumulation. Science 1976; 193: 1094lo(). the basal secretion; on the contrary, in all 7 Hagiwara H, Shimonaka M, Morisaki M, liposome-containing cultures the secretion was lkekawa N , Inada Y . Sitosterol-stimulative reduced. The incorporation of liposomal production of plasminogen activator in cultured material into the cell plasma membrane, thereendothelial cells from bovine carotid artery. fore, seemed to reduce vWF secretion. Thromb Res 1984; 33: 363-70. 8 Shimonaka M, Hagiwara H, Kojima S, lnada Y. Measurement of release of radiolabelled 2Successive study on the production of plasminogen deoxy-D-glucose is considered more sensitive activator in cultured endothelial cells by phytothan the "Cr, "-adenine, 3H-leucine. or LDH sterol Thromb Res 1984; 36: 217-22. release assays for detection of endothelial cell 9 Kojima S, Soga W, Hagiwara H, Shimonaka M, damage [lS] and was used to study the initial Saito Y, lnada Y. Viable fibrinolysis by endothelial cells: effect of vitamins and sterols. Biosci period after sitosterol addition. However, we Rep 1986; 6: 1029-33. did not detect any sterol-induced injury to the 10 Jaffe EA. Nachman RL, Becker CG, Minick CR. endothelial cells during the first 6-8 h by this Culture of human endothelial cells derived from method. The release of 2-deo~y-o-[l-'~CC]glucose umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: in control cell cultures reached about 80% after 2745 - 56. 12 h. Beyond that point differences in the 11 Skrede S,Bjdrkhem I. Biosynthesis of cholesterol release would have been difficult to assay with J from intestinal 7a-hydroxy-4-cholesten-3-one. this technique. Biol Chem 1982; 257: 8363-7. We conclude that sitosterol at concentrations 12 The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical relevant to the in vivo situation in sitosteroPhysiology. Recommended methods for the laemia patients has a marked effect on cell determination of four enzymes in blood. Scand J morphology and LDH release after 72-96 h, Clin Lab Invest 1974; 33: 291-306. whereas no effect on t-PA production was seen. 13 Prydz H, Allison AC. Tissue thromboplastin >.
ACKNOWLEDGMENTS This study was supported by The Norwegian Council on Cardiovascular Diseases.
I
activity of isolated human monocytes. Thromb Haemost 1978; 39: 582-91. 14 Markwell MAK, Haas SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 1978; 87: 206- 10.
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15 Andreoli SP. Bachner RL, Bergstein JM. In vilro detection of cndothclial cell damage using 2-
deoxy-o-'H-glucose. Comparison with chromium 51 , 'H-leucine. .'H-adcnine, and lactate dehydrogcnase. J Lab Clin Mcd 1985; 106: 253-61. I6 Locsberg C, Gonsalves MD. Zandbergcn J , Willems C, van Aken W G , Stel HV. van Mourik J A , de Groot PG. The effect of calcium on the secretion of factor VI11-related antigen by cultured human endothelial cells. Biochim Biophys Acta 19x3; 763: 160-8. 17 Zipper J . Sarrnch D, Hallc W. Wcchselwirkung von Liposomen mit GcT:' 'isscndothclzellen. '. Riomed Biochim Acta 1988: 47: 713-9. 18 Hennig B, Boissonneault G A . Cholestan-3(3. Stx, h(3-triol decreases harrier function of cultured cndothelial cell monolaycrs. Atherosclerosis 1987: 68: 255-61. I0 Petterscn KS, Boberg KM, Stahursvik A. Prydz H. Toxicity of oxygenated cholesterol derivativca toward cultured human umbilical vein cndothclial cells. Artcriosclerosis 1991: I I : 423-8. 20 Raicht RF. Cohen H I , Fazzini EP, Sarwal AN, Takahashi M. Protective effect of plant stcrols
against chemically induced colon tumors in rats. Cancer Res 1980; 40: 403-5. 21 Levin EG. Latent tissue plasminogen activator produced by human endothelial cells in culture: Evidence for an enzyme-inhibitor complex. Proc Net1 Acad Sci USA 1983; 80: 6804-8. 22 Rijken DC, van Hinsbcrgh VWM, Scns Et1C. Ouantitation o f tissue-type plasminogen activator in hunian endothelial ccll cultures by use of an enzyme immunoassay. Thromb Res 1984; 33: 145-53. 23 van Wachcm PB, Reindcrs J H , viin BuulWortclboer MF, de Groot PG, van Akcn W G , van Mourik JA. Von Willebrand factor in cultured human vascular endothclial cells from ;idult and umbilical cord arteries and veins. Thromb Hacinost 1986; 56: 189-92. 24 Sporn L A , Mardcr VJ, Wagner DD. Inducible secretion of large, biologically potent von Willebrand factor multimers. Cell 1986; 46: 185-90. Received 25 October 1990 Accepted 15 April 1991
ISSN: 0036-5513 (Print) 1502-7686 (Online) Journal homepage: http://www.tandfonline.com/loi/iclb20
Toxicity of sitosterol to human umbilical vein endothelial cells in vitro K. M. Boberg, K. S. Pettersen & H. Prydz To cite this article: K. M. Boberg, K. S. Pettersen & H. Prydz (1991) Toxicity of sitosterol to human umbilical vein endothelial cells in vitro, Scandinavian Journal of Clinical and Laboratory Investigation, 51:6, 509-516 To link to this article: http://dx.doi.org/10.3109/00365519109104559
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 10
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iclb20 Download by: [University of Otago]
Date: 07 October 2015, At: 09:42
Scand J Clin Lab Invest 1991: 51: 509-516
Toxicity of sitosterol to human umbilical vein endothelial cells in vitro K. M . BOBERG, K. S . PETTERSEN & H . PRYDZ"
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Institute of Clinical Biochemistry and *Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo, Norway.
Boberg KM, Pettersen KS, Prydz H. Toxicity of sitosterol to human umbilical vein endothelial cells in vitro. Scand J Clin Lab Invest 1991; 51: 509-516. The pathogenesis of the early development of atherosclerosis in sitosterolaernia is unknown. The effect of sitosterol on vascular endothelial cells in vitro was investigated by culturing human umbilical vein endothelial cells in the presence of up to 0.7 mmol I-' of sitosterol. Liposomes were used to supply the high sterol concentrations. Exposure to 0.7 mmol I-' of sitosterol for 72 h caused contraction of the endothelial cells and increased release of intracellular lactate dehydrogenase. After 96 h incubation the cells were partly detached from the substrate. At this time-point 0.35 mmol I-' of sitosterol also caused perturbation of the endothelial cells. However, we could not confirm previous reports that tissue plasminogen activator production was enhanced by sitosterol. Key-words: atherosclerosis; endothelial cells; lactate dehydrogenase release; sitosterolaemia; tissue plasminogen activator; von Willebrand factor Kirsten Muri Boberg, Institute of Clinical Biochemistry, Rikshospitalet, 0027 Oslo I , Norway.
Early development of atherosclerosis is a major clinical finding in the rare familial sterol storage disease sitosterolaemia [l-41. Some patients have died from coronary heart disease at an early age [I, 21. Plant sterols, mainly sitosterol and campesterol, are absorbed in excess and accumulate in serum, xanthomas, arteries, and other tissues in sitosterolaemia patients. In serum they may constitute 7-26% of the total sterols 11-41. The cholesterol level is normal or moderately elevated [ 1-41. The primary metabolic defect and the pathogenesis of the premature atherosclerosis in this disease are unexplained. Endothelial cell injury may be an important factor in atherogenesis 15-61. It is possible that minor disturbances in endothelial cell function
may initiate the atherogenic process [5, 61. Perturbation of endothelial cell function may initiate the atherogenic process [5, 61. Perturbation of endothelial cells by sitosterol has been suggested, since sitosterol has an ability to increase synthesis and secretion of plasminogen activator from cultured bovine carotid artery endothelial cells (7-91. In the present study we wanted to see if this finding is reproducible in human vascular endothelial cells and to investigate possible effects of sitosterol on other aspects of endothelial cell damage: release of tissue plasminogen activator (antigen) and activator inhibitor (activity and antigen), von Willebrand factor, lactate dehydrogenase, and 2-deo~y-D-[l-'~C]gIucose, and synthesis of thromboplastin. 509
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MATERIALS AND METHODS
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Muteriuls Sitosterol (24-ethyl-S-cholesten-3~3-ol)(98% pure) was obtained from Alltech Associates. Dearfield, IL, USA. The purity of this material was ascertained by high-performance liquid chromatography (HPLC) prior to use. Chotesterol, epicoprostanol (5[3-cholestan-3a-ol). oI.-a-phosphatidyl-1,-serine-dipalmitoyl, and t.-a-phosphatidylcholin P-oleoyl-palmitoyl were purchased from Sigma, St Louis, MO, USA. 2-Deoxy-r1-[l-"C] glucose (59 mCi mmol-') and [4-"C] sitosterol (56 mCi mmol-') were obtained from Amersham International, Amersham, UK.
cholesterol in the final incubation medium. epicoprostanol was added as internal standard before alkaline hydrolysis and lipid extraction with n-hexane. as described previously [ 111. Aliquots of the lipid extracts were analysed by gas chromatography, using a gas chromatograph (5880A Hewlett-Packard, Avondale, PA, USA) with a 25 m long capillary column (i.d. 0.32 mm) CP-WAX 52 CB. In one set of experiments sitosterol was dissolved in ethanol and added to the medium at final concentrations of 0.05 mmol I-' and 0.1 mmol I-' and 0.5'% ethanol. The suspensions were sonicated before use. Controls contained 0.5% ethanol only.
Uptuke of sitosterol by endothefial cells Cell cdrure Human umbilical vein endothelial cells were cultured according to Jaffe et ul. [ 101. The cells were plated in 12-well tissue culture clusters (Costar. Cambridge, MA, USA), using 1.0 ml of RPMI-1640 supplemented with 20% fetal bovine serum (FBS) (Sigma), penicillin (50 U ml I), streptomycin ( 5 0 pg ml-I), and Lglutamine (2 mmol I I) (Gibco, Paisley, UK). Medium was changed every second or third day. Primary cultures reached confluence after 3-7 days. Secondary cultures were established by treatment with trypsin-EDTA (Flow, Irvine, UK) and used in all experiments when confluent. All incubations and assays were performed in serum-containing media.
Preprution of liposornes Phosphatidylcholine, phosphatidylserine, and sitosterol (molar ratios 3.9: 1:5.9), or cholesterol (molar ratios 3.9: 1:6.3). were dissolved in chloroform. dried by rotary evaporation under nitrogen and resuspended in culture medium with 20'L FBS by shaking at 4 "C. followed by repeated sonication for periods of 10 s. Control liposomes were prepared a s above. without addition o f sitosterol o r cholesterol. The procedure was performed under sterile conditions. I n addition to incuhations with the three different kinds o f liposomecontaining media, control experiments were carried o u t with ordinary culture medium. To determine the concentration of sitosterol and
In some experiments the liposomes were prepared with '4C-sitosterol (0.56 mCi mmol I . ' ) . After incubation for 6, 12, or 24 h the culture medium was removed, and the cell layer was washed with ice-cold veronal-buffered saline (VBS). The cells were homogenized in 0.5 ml o f buffer, and a sample was assayed for radioactivity in a Tri-Carb liquid scintillation analyser, model 1500 (Packard Instrument, Downers Grove, IL, USA). Viability of the cells in culture during sitosterol o r cholesterol treatment was tested using tryphan blue by standard methods.
Lactate dehydrogenuse ( L D H ) LDH was quantified by a photometric method [12]. The coefficient of variation in the actual range of measurements was 4.5'%. The cells were suspended in VBS and disrupted by sonication. LDH was assayed in the medium and in the cell lysate. Release of intracellular LDH to the medium is expressed as percentage of total L D H in medium and cells. Corrections were made for the content of LDH in fetal bovine serum, which was about 250 UI-'.
voii
Wilkhrund fuctor ( v WF)
vWF was measured by an enzyme-linked immunosorbent assay (ELISA) (F. Brostad. personal communication), coating the plates with a rabbit antibody against human vWF
Cytotoxicity of' sitosterol (Dakopatts, Denmark). After incubation with the medium samples, a goat anti-human von Willebrand factor (Atlantic Antibodies, Scarborough, ME, USA) was added as secondary antibody. The plates were then incubated with peroxidase-conjugated swine antigoat IgG (TAGO, Burlingame, C A , USA), and the peroxidase activity was determined. O n e unit of vWF antigen is defined as the quantity present in 1 ml of pooled normal platelet-poor citrated plasma.
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Thromboplustin activity (TPL) TPL of the endothelial cell homogenates was tested in a one-stage system [ 131, consisting of 0.1 ml normal human platelet-poor citrated plasma and 0.1 ml of the solution to be tested, both prewarmed separately for 3 min at 37 "C. Coagulation was started by the addition of 0.1 ml prewarmed 30 mmol I-' CaCI2. The results were compared with a standard preparation of human brain thromboplastin which clotted normal human plasma in 15-18 s. The curve (double logarithmic plot) was linear between 0.1 U ml-' (87 s) and 10 U ml-' (24 s). Protein was measured according to Markwell et ul. [14].
Tissue plusminogen uctivutor untigen (t-PA) t-PA was measured by an ELISA (Biopool lmulyse. t-PA, U m e i , Sweden). The values are given as ng t-PA mg-' cell protein or as ng t-PA mI supernatant.
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Tissue plusminogen uctivutor inhibitor-] (PAI1)
PAI-1 activity was measured by the CoAS E T t-PA/PAI method (KabiVitrum, Molndal, Sweden), adjusted to assay PAI-1 in endothelial cell supernatants. A linear standard curve for PAI-1 was established by using the preparation supplied with the kits. PAI-1 antigen was measured by the Biopool Tintelize PAI-1 kit as described by the manufacturer. All tests were in duplicate (two wells from the same umbilical cord).
Release of 2 - d e o x y - ~ - l 1 - ' ~glucose. c/ This was used as a parameter ofendothelial cell damage and was assayed as described by
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Andreoli et rzl. [ 1.51. Confluent layers of endothelial cells were radiolabelled by incubation with 3.0 x 10" d.p.m. of 2-deoxy-1>-(l-"C] glucose per well for I8 h. The cells were washed four times with VBS and subsequently incubated with control medium or medium containing liposomes for periods of 2, 4. 6, 12, and 24 h. Approximately 1.4% of the added "C-activity was retained in the cells after the radiolabelling procedure. A t the end of each experiment the monolayer was washed twice, and the cells were disrupted by homogenization. Aliquots of the supernatants, washes, and cell homogenates were counted. Release of radioactivity to the supernatants was calculated as per cent of total activity.
Stutisticx Student's t-test and Wilcoxon's signed-rank test were used for statistical analysis.
RESULTS By incorporating sitosterol into liposomes we obtained a final concentration of this sterol in the incubation medium of 0.6-0.7 mmol I - ' , which resembles the plasma sitosterol level in untreated sitosterolaemia patients [ 1-41, The cholesterol concentration in sitosterolenriched medium, medium with control liposomes, and ordinary medium was approximately 0.15 mmol I-', owing to the cholesterol content in fetal bovine serum. In incubations lasting 24, 48, 72, and 96 h medium containing only half the above sterol concentrations was also used. In medium prepared with cholesterol liposomes the total cholesterol concentration amounted to 1.18 mmol I-'. The cellular uptake of sitosterol was in the range 30-70 pg mg-' cell protein. There was no further significant increase in sterol incorporation into the cells after 6 h.
Morphology and L D H release Exposure to sitosterol or cholesterol-containing liposomes for up to 48 h caused no changes in the light microscopy appearance of the endothelial cells, and there was no uptake of Tryphan blue after the incubations. There was
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Incubation time (h) FIG.1. Release o f LDH into the culture medium of endothelial cells. i n per cent of total LDH in medium and cells. The content of LDH in fetal bovine serum was corrected for. Ordinary medium (M), medium containing control liposomes without addcd sterols (L), liposomes with sitosterol, 0.7 mmol I~I (SI) and 0.35 mmol I ' (S2). and liposomes with cholesterol, I . 18 mmol I I ( C ) .Each value is mean k SEM of two independent experiments, each comprising three separate cultures (M, L, SI), o r of three separate cultures from the same umbilical cord (S2, C).
no significant difference between the experimental groups in the amount of L D H present in the conditioned media for incubations lasting up to 48 h. After incubation with 0.7 mmol I-' of sitosterol in liposomes for 72 h, however, contraction of the endothelial cells with formation of intercellular gaps was observed. After 96 h a small number of the sitosterolexposed cells were detached from the substrate and floating in the medium. At this time-point cell contraction and intercellular gaps were also visible at 0.35 mmol I-' o f sitosterol. The cell monolayer remained intact in incubations with cholesterol and in the control experiments. Concomitant with the morphological alterations release of LDH increased (Fig. 1). At 72 h the mean release in the presence of 0.7 mmol I-' of sitosterol was 63% (p=0.008) compared with 35% for the control cultures. At Y6 h the LDH release reached 83%)(p=O.OOl) at 0.7 mmol I-'. and 0.35 mmol I-' o f sitosterol led to 64% release. The release in the controls did not incrcase (36%).
t-PA utitigeti The amounts of t-PA antigen released varied greatly between the secondary endothelial cell
cultures originating from different umbilical cords. During the period up to 24 h the levels of t-PA antigen increased in the control cell supernatants as well as in the supernatants of cells incubated with sitosterol liposomes or control liposomes. There were no significant differences between these three sets of values (p>0.25). The kinetics of release of t-PA in the cholesterol-exposed cultures were slightly different; a rapid increase reaching a plateau at about 6 h. Also, in prolonged incubations up to 96 h the production of t-PA antigen in the control cultures increased essentially linearly with time (Fig. 2). The amounts of t-PA in the medium after incubations with 0.35 mmol I-' of sitosterol were equal to the controls, while a slight decrease was noted for 0.7 mmol I-' after Y6 h. In no case was the t-PA production increased in sitosterol-exposed cells compared with control cells.
PAI-I uctivity In preliminary experiments there were no significant effects of sitosterol-containing lipow m e s on the release of either PAI-I activity (n=3) or antigen ( n = l ) up to Y6 h. The mean control levels at 24 h were 2.8 U ml-' and 0.8 pg mI-I, respectively.
Cytotoxicity of sitosterol 14
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FIG. 2. Production of t-PA antigen in confluent secondary cultures of human umbilical vein endothelial cells. Ordinary medium (M), medium containing control liposomes without added sterols (L), liposomes with sitosterol, 0.7 mmol I-' (Sl) and 0.35 mmol I-' (S2), and liposomes with cholesterol 1.18 mmol I-' (C). Results of one experiment with parallel cultures.
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FIG.3. Production of vWF antigen in confluent secondary cultures of human umbilical vein endothelial cells. Ordinary medium (A),medium containing control liposomes without added sterols (0).liposomes with and liposomes with cholesterol, 1.18 mmol I-' ( A ) . Each value is mean f SEM sitosterol, 0.7 mmol I-' (0). of two to five independent experiments, each comprising two separate cultures. Total number of cultures tested for each mean is given in brackets.
Thromboplastin activity
von Willebrand factor (v WF)
Untreated endothelial cells did not express thromboplastin activity. Neither the sitosteroland cholesterol-containing liposomes, nor the control liposomes, caused a detectable rise in thromboplastin activity. Previous control experiments have shown that induced thromboplastin is not degraded or inactivated during the isolation procedure prior to assay.
Endothelial cells isolated from different umbilical cords secreted various amounts of vWF to the culture medium, as observed by others [16]. The level of vWF antigen increased in a time-dependent manner during incubation with all four kinds of medium (Fig. 3 ) . Sitosterolcontaining medium did not provoke larger release of vWF than medium with control
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liposomes o r cholesterollliposomes. After 12 h and 24 h incubations cells treated with each of the three liposome-containing media had released less vWF antigen than had control cells (Fig. 3). The difference reached borderline significance (p=O.O5) at 24 h. Sonication of medium containing FBS did not cause a similar reduction.
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Release
of 2-deoxy-u-ll-'4Clgluc.ose
The release of intracellular 2-deoxy-~-[l-"C] glucose was linear up to about 6 h, whereafter it levelled off slightly to reach about 80% in all cultures at 12 h. There was no detectable effect of any kind of liposomes on the release. Using 0.5% ethanol as vehicle, only limited amounts (up to 0.1 mmol I I ) of sitosterol could be dissolved in the medium. Compared with control cultures, 0.05 mmol I-' and 0.1 mmol I-' of sitosterol did not perturb the endothelial cells. In particular, the production of t-PA was at no point increased in sitosterol-exposed cells.
DISCUSSION In the atherosclerotic lesions and xanthomas of sitosterolaemia patients plant sterols (sitosterol and campesterol), Sa-saturated stanols (Susitostanol, Su-campesterol. cholestanol). and cholesterol are deposited in approximately the same proportions as found in plasma [ 1-41, but the events promoting the excessive sterol uptake and atheroma development are unknown. Using liposomes as vehicle, we cultured the endothelial cells in the presence of sitosterol at concentrations similar t o those found in sitosterolaemia patients. From a biological point of view the optimal way of administering the sterol would be by incorporation into low-density lipoproteins (LDL). However, liposomes bind to the endothelial cell surface and exchange lipids or fuse with the cell membrane [17]. Sitosterol had n o effect on endothelial cell viability or morphology at the light microscopy level up to 48 h. The cells retained their ability to exclude Tryphan blue and did not release increased amounts of L D H . However, after prolonged incubations for 72 h and 06 h signs o f endothelial cell injury appeared. Contraction o f endothelial cells with simultaneously
increased LDH release has previously been reported after incubations with cholestane-3P. Sa. 6fLtriol [18]. This finding was associated with significant increase in albumin transfer across the endothelial cell monolayer, implying decreased barrier function. Exposure t o high sitosterol concentrations for 72-96 h was required to detect signs of endothelial cell perturbation in the present study. However, the plasma sitosterol levels in sitosterolaemia patients might be even higher. The mean value in a group of 14 patients investigated by Salen ef ul. [4] was 0.85 mmol I-'. In sitosterolaemia patients the endothelial lining of the blood vessels is continuously exposed to the enhanced levels of sitosterol. It seems possible that sitosterol contributes to the atherogenesis by interfering with the endothelial cell function. 7f3-Hydroxycholesterol, which has also been implicated in the pathogenesis ofatherosclerosis, caused similar changes o f endothelial cells. even though more pronounced. in studies in our laboratory [ 101. Other studies have indicated that sitosterol might interfere with cell viability. Sitosterol supplied with the diet retarded development of chemically induced colon carcinomas in rats [20]. Changes in cellular metabolism or kinetics secondary t o alterations in the epithelial cell membrane were suggested as possible mechanisms of this effect. I n experiments with Ehrlich ascites tumour cells in suspension culture sitosterol at the level of 0.1 mmol I-' in the culture medium reduced the cell survival to about 65% after 24 h (I. SmithKielland, personal communication). In the present experiments with human umbilical vein endothelial cells we could not confirm the previous reports that tissue plasminogen activator release was stimulated by sitosterol in cultured bovine carotid artery endothelial cells 17-91, despite using more than twice the highest concentrations of sterol previously used. These authors [7-91 administered the sterol dissolved in ethanol and found a marked increase in the activity of plasminogen activator in the conditioned media at sitosterol concentrations above 0.02 mmol 1 I , levelling off at 0.05 mmol I I. N o further increase was seen even at 0.25 mino1 I-'. The fihrinolytic activity was assayed by nephelometric measurement ofturbiditydecrease in a fibrin suspcnsion. Plasminogen activators are released from cultured human umbilical vein endothelial cells
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Cytotoxicity
of sitosterol
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1211. Due to concomitant production of plasREFERENCES minogen activator inhibitor (PAI). the t-PA is Bjdrkhem I, Skrade S. Familial diseases with storage of sterols other than cholesterol: cerebroinactive and not detectable in fibrinolytic assays tendinous xanthomatosis and phytosterolemia. In: 122). Using a sensitive ELISA, we observed Scriver CR, Beaudet AL. Sly WS, Valle D, eds. release of t-PA antigen that increased linearly The Metabolic Basis of Inherited Disease. New with time. Sitosterol-exposed cells and control York: McGraw-Hill. 1989: 1283-302. cells released similar amounts of t-PA antigen Salen G , Horak I, Rothkopf M, Cohen JL, Speck J, Tint GS, Shore V, Doyal B, Chen T, Shefer S. when either liposomes (up to 0.7 mmol I-' of Lethal atherosclerosis associated with abnormal sitosterol) or ethanol (up to 0.1 mmol l-') was plasma and tissue sterol composition in sitosteroused as vehicle. Species differences with respect lemia with xanthomatosis. J Lipid Res 1985; 26: to release of plasminogen activators might 1126-33. Miettinen TA. Phytosterolaemia, xanthomatosis explain the different results. Sitosterol has been and premature atherosclerotic arterial disease: a proposed as an agent for prevention and case with high plant sterol absorption, impaired therapy of thrombosis [ 7 ] , but the results of sterol elimination and low cholesterol synthesis. the present study do not support this idea. Eur J Clin Invest 1980; 10: 27-35. Salen G , Kwiterovich PO Jr, Shefer S, Tint GS, Human umbilical vein endothelial cells are Horak J, Shore V, Doyal B, Horak E. Increased considered a representative model for human plasma cholesterol and 5a-saturated plant sterol endothelial cells of various other origins with derivatives in subjects with sitosterolemia and respect to synthesis and release of vWF 1231. xanthomatosis. J Lipid Res 1985; 26: 203-9. vWF may be secreted constitutively or remain 5 Ross R, Glomset JA. The pathogenesis of atherosclerosis. N Engl J Med 1976; 295: 369-77. stored in intracellular organelles (Weibel-Palade 6 Ross R. Harker L. Hvoerlioidemia and atherobodies), available for relzase in response to sclerosis. Chronic hyperlipidemia initiates and vascular injury [24]. Neither sitosterol nor maintains lesions by endothelial cell desquamation cholesterol stimulated secretion of vWF above and lipid accumulation. Science 1976; 193: 1094lo(). the basal secretion; on the contrary, in all 7 Hagiwara H, Shimonaka M, Morisaki M, liposome-containing cultures the secretion was lkekawa N , Inada Y . Sitosterol-stimulative reduced. The incorporation of liposomal production of plasminogen activator in cultured material into the cell plasma membrane, thereendothelial cells from bovine carotid artery. fore, seemed to reduce vWF secretion. Thromb Res 1984; 33: 363-70. 8 Shimonaka M, Hagiwara H, Kojima S, lnada Y. Measurement of release of radiolabelled 2Successive study on the production of plasminogen deoxy-D-glucose is considered more sensitive activator in cultured endothelial cells by phytothan the "Cr, "-adenine, 3H-leucine. or LDH sterol Thromb Res 1984; 36: 217-22. release assays for detection of endothelial cell 9 Kojima S, Soga W, Hagiwara H, Shimonaka M, damage [lS] and was used to study the initial Saito Y, lnada Y. Viable fibrinolysis by endothelial cells: effect of vitamins and sterols. Biosci period after sitosterol addition. However, we Rep 1986; 6: 1029-33. did not detect any sterol-induced injury to the 10 Jaffe EA. Nachman RL, Becker CG, Minick CR. endothelial cells during the first 6-8 h by this Culture of human endothelial cells derived from method. The release of 2-deo~y-o-[l-'~CC]glucose umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: in control cell cultures reached about 80% after 2745 - 56. 12 h. Beyond that point differences in the 11 Skrede S,Bjdrkhem I. Biosynthesis of cholesterol release would have been difficult to assay with J from intestinal 7a-hydroxy-4-cholesten-3-one. this technique. Biol Chem 1982; 257: 8363-7. We conclude that sitosterol at concentrations 12 The Committee on Enzymes of the Scandinavian Society for Clinical Chemistry and Clinical relevant to the in vivo situation in sitosteroPhysiology. Recommended methods for the laemia patients has a marked effect on cell determination of four enzymes in blood. Scand J morphology and LDH release after 72-96 h, Clin Lab Invest 1974; 33: 291-306. whereas no effect on t-PA production was seen. 13 Prydz H, Allison AC. Tissue thromboplastin >.
ACKNOWLEDGMENTS This study was supported by The Norwegian Council on Cardiovascular Diseases.
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activity of isolated human monocytes. Thromb Haemost 1978; 39: 582-91. 14 Markwell MAK, Haas SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 1978; 87: 206- 10.
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15 Andreoli SP. Bachner RL, Bergstein JM. In vilro detection of cndothclial cell damage using 2-
deoxy-o-'H-glucose. Comparison with chromium 51 , 'H-leucine. .'H-adcnine, and lactate dehydrogcnase. J Lab Clin Mcd 1985; 106: 253-61. I6 Locsberg C, Gonsalves MD. Zandbergcn J , Willems C, van Aken W G , Stel HV. van Mourik J A , de Groot PG. The effect of calcium on the secretion of factor VI11-related antigen by cultured human endothelial cells. Biochim Biophys Acta 19x3; 763: 160-8. 17 Zipper J . Sarrnch D, Hallc W. Wcchselwirkung von Liposomen mit GcT:' 'isscndothclzellen. '. Riomed Biochim Acta 1988: 47: 713-9. 18 Hennig B, Boissonneault G A . Cholestan-3(3. Stx, h(3-triol decreases harrier function of cultured cndothelial cell monolaycrs. Atherosclerosis 1987: 68: 255-61. I0 Petterscn KS, Boberg KM, Stahursvik A. Prydz H. Toxicity of oxygenated cholesterol derivativca toward cultured human umbilical vein cndothclial cells. Artcriosclerosis 1991: I I : 423-8. 20 Raicht RF. Cohen H I , Fazzini EP, Sarwal AN, Takahashi M. Protective effect of plant stcrols
against chemically induced colon tumors in rats. Cancer Res 1980; 40: 403-5. 21 Levin EG. Latent tissue plasminogen activator produced by human endothelial cells in culture: Evidence for an enzyme-inhibitor complex. Proc Net1 Acad Sci USA 1983; 80: 6804-8. 22 Rijken DC, van Hinsbcrgh VWM, Scns Et1C. Ouantitation o f tissue-type plasminogen activator in hunian endothelial ccll cultures by use of an enzyme immunoassay. Thromb Res 1984; 33: 145-53. 23 van Wachcm PB, Reindcrs J H , viin BuulWortclboer MF, de Groot PG, van Akcn W G , van Mourik JA. Von Willebrand factor in cultured human vascular endothclial cells from ;idult and umbilical cord arteries and veins. Thromb Hacinost 1986; 56: 189-92. 24 Sporn L A , Mardcr VJ, Wagner DD. Inducible secretion of large, biologically potent von Willebrand factor multimers. Cell 1986; 46: 185-90. Received 25 October 1990 Accepted 15 April 1991
