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Minimal effect of estrogens on endothelial cell growth and production of prostacyclin
THROMBOSIS RESEARCH 34; 303-310, 1984 0049-3848/84 $3.00 + .OO Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.
MINIMAL
EFFECT OF ESTROGENS ON ENDOTHELIAL PRODUCTION OF PhGSTACYCLIN
CELL GROWTH AND
E. CORVAZIER,E. DUPUY, A.M. DOSNE, J. MACLOUF. Lab. H&nostaseet U.156 INSERM, H^opitalLariboisisre, 6, Rue Guy Patin, 75010 PARIS, FRANCE. (Received 11.4.1983; Accepted in revised form 16.2.1984 by Editor M. Samama)
ABSTRACT The effeclnof natural and synthetic sex hormones (lo-*M) have been studied on human umbilical endothelial cell proliferation and prostacyclin production. 17 B estradiol and progesterone had no effect on cell multiplication. Ethinyl-estradiol increased pr;liferation when the initial plating density was 40,000 cells/ cm . However, the production of 6-keto PGFleinduced by the Catf ionophore A 23187 was not different between control, 17 B estradiol-or ethinyl estradiol-treated cultures. These results demonstrate an in vitro effect of synthetic estrogen, ethinyl estradiol on endothelial cell nroliferation. At the present time it is however difficult to correlate these results with the clinical observation of an increase in thromboembolic complicationsin women under oral contraceptives. INTRODUCTION The low incidence of atherosclerosis in premenopausal women compared to men of similar age (l), the increased risk of vascular complications in women under oral contraceptives (2-71, suggest that sex-linked factors may play a role in the development of vascular diseases. These clinical data suggest that there is a major difference between natural and synthetic estrogens in the development of atherosclerotic lesions. Endothelial cells synthesize collagen, basal membrane and elaborate the composition and structure of the subendothelium (8). Factors controlling endothelial cell proliferation are thought to be an important aspect in the comprehension of atherosclerosis which develops after endothelial injury (9) because they could accelerate or slowdown the regeneration of the normal endothelium barrier. Since estrogen binding sites have been demonstrated in endothelial cell cultures by Colburn and Buonassissi (lo), the effects of natural and synthetic estrogens and progesterone have been investigated on cell proliferation and on the production of 6-keto PG Flo by human endothelial cell cultures. KEY WORDS : Sex hormones, human endothelial cell, prostacyclin. 303
304
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
MATERIAL._. AND METHODS -..-_ .___ .._^ .___ Isolation and culture of endothelial cells. EndotheliaGells were-?solated from human umbilical veins by collagenase digestion (11). The endothelial cells were cultured on Linbro multiplates (2 cm2-24 wells) in the presence of Medium 199 supplemented with 10 % charcoal adsorbed fetal calf serum (FCS-A). The inital density of plating was either 20 000 or 40 000 cells per cm2. Preparation of estrogen depleted sera. Physiologicarconcentrationsof stad hormones are present in fetal calf serum (FCS) (12, 13). Therefore, the serum was depleted of endogenous steroids and prostaglandins by two adsorptions with dextran coated charcoal at 50°C for 45 min. (14). Charcoal was removed by centrifugation and the serum (FCS-A) was stored at -2OOC after sterilization by filtration. The remaining estradiol concentration of FCS-A was less than lo-1lM as measured by radioimmunoassay. Proliferation study. One day after plating (day 1); the cultures of each well were washed once with Medium 199 without FCS in order to eliminate floating cells. Exogenous hormones were added daily to the culture. Control cultures from the same cell preparation were carried out in Medium 199 with 10 % FCS-A but without adding hormone. Cell count of adherent cells was performed daily. Before cell counting, the culture was washed once with Medium 199 without serum and once with saline phosphate buffer. The cells were dissociated by collagenase (0.1 %) in EDTA (0.1 %) and counted in a hematocytometer. The steroid hormones were dissolved in ethanol and diluted in Medium 199 with 10 % of FCS-A at a final concentration of lo-*M which corresponds to the physiological concentration in human cord blood (15). Results were compared using Student's t test analysis. All culture reagents were from Gibco Laboratory, 17 B estradiol and ethinyl estradiol were obtained from Sigma Chemical Co, and progesterone from Besins Isovesco Laboratory. Dextran coated charcoal and TES buffer were purchased from Sigma. Production of 6-keto -PG Flo . For this investigation the cells were plated at 40 000 cells/cm2 in 35 mm Lux Petri dishes. At day 3 the cultures were washed four times with M 199 without serum in order to remove floating cells and serum. Cells were stimulated by calcium ionophore A 23187 (Calbiochem) at 0,25 ; 0,5 and 1 PM final concentration during 5 mins.,to induce the release of prostacyclin by the response was influenced hormone treated culture. In order to knowMhet&the by sex, matched sex cultures were also stimulated with 1 PM ionophore and analyzed. 6-keto PG F CL radioimmunoassay. Thestable'derivative of prostacyclin was measured by the method of Maclouf et al (16, 17) using 1251 -Histamide-6-keto PG Flc(-100 ul of the diluted sample were added to the medium containing 200 ~1 of Tris-HCl buffer pH 8, 100 ~1 of 1251 6-keto PGFlo (10.000 dpm), 200 pl of 0,5 % w/v bovine gammaglobulin and 100 ~1 of the diluted antiserum (final dilution : l/35,000). After 18 h incubation at 4OC, labelled immune complex was separated from the unbound by 700 ~1 of a 25 % w/v ice cold polyethylene glycol solution. After centrifugation the supernatant was discarded and the globulin precipitate counted in a gamma counter (Beckman). Standard curve was obtained using 1,25
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
305
to 80 pg per tube of 6-keto PG Fla . Assay detection limit was 1,2 pg and 50 % inhibition was obtained with 13 pg of 6-keto PG Fl~r . Cross reactivity was 3 % with PG Flu , 1 % with PG F2 a and less than 1 % for other prostaglandins. RESULTS Characterization of endothelial cell cultures. Electron microscopy demonstrates the presence of the Weibel-Palade bodies which are the characteristic of endothelial cells (18). Comparison between FCS-A and FCS on endothelial cell cultures. The proliferation amt
9 -0
',30. c u &I
t-t
Release of 6-keto PG Fla induced by Cat+ Ionophore A 23187(control culture). Mean of 4 experiments + SEM.
$ a .'Ct Y 9 y 0.25 0.5 Ca++ lonophorc
1 1 QM
FIG.3
306
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
FIG. Effect of sex hormones (lo-*M) -on the proliferation _of the endothelial cells --seeded at 20.000 cells/cm2. --Progesterone (a), 17 B estradiol (B)+or ethinyf estradiol (c) (-b-).Control (+). Mean of 5 experiments (mean Ir--S.D.).).
FIG. 2 of the endothelial -L__ cells seeded Effect of sex hormones -on the p roliferation ---at 40.000 cells/cm2. Meam experiments. Same symbols than fig. 1. Increase of 30 % (p <0,05) for ethinyl estradiol culture (mean I 2.S.D.).
Vo1.34, No.4
307
ESTROGENS AND ENDOTHELIAL CELLS
TABLE I Effect of estrogens on the production of 6-keto PG Fla from endothelial cells stimulated by the Cat+ Ionophore A 23187. The results are expressed in % of the control. Mean of 4 experiments A SEM. Ionophore (PM)
17 B estradiol
Ethinyl estradiol 114 _+42
0,25
0,s
llO-+
11
138 f 48
1
119 f
22
136 2 46
At 1 PM Ionophore the production of 6-keto PG Fla was regardless of sex (data not shown). No difference was observed. DISCUSSION This study was undertaken in order to investigate the action Of Sexual on endothelial cell proliferation and modification of their prostacyclin production. The understanding of atherosclerosis developing after endothelial injury is of importance because factors controlling endothelial cell proliferation are thought to play an important role in the regeneration of normal endothelial barrier. Vascular abnormalities induced by synthetic estrogens could be related to a decreased regeneration of the endothelium. -8 In this study, 17 B Estradiol and Progesterone at 10 M had no effect on cell multiplication regardless of the initial density of cultured human endothelial cells. However, 10e8M Ethinyl-estradiol stimulated cell proliferation at the highest initial cell density (40.000 cells/cm2). This density could reflect an effect resulting from endothelial cells in confluency. Harrison and McKee (19) reported that 17 B Estradiol causes an increased rate of human endothelial cell multiplication, but synthetic estrogen was not tested in their study. Specific estrogen binding sites have been described on the surface of endothelial cells from animal species : e.g. rabbit aorta (lo), canine arteries and inferior vena cava (20). The proliferating effect induced by ethinyl-estradiol on human endothelial cells might be related to a direct action of this compound on this tissue although it is not clear wether these cells possess estrogen binding sites. The lack of effect of natural estrogen could also be due to a decrease of the estrogen receptors as a result of the high level of hormones during pregnancy and delivery ; such fact has been described for endcthelial cells (21). The effect induced by ethinyl estradiol might be related to the intimal proliferation of endometrial vessels observed in vivo in 80 % of women taking oral contraceptives (22, 23). In this regard the study of other vascular cells could be of interest although it remains difficult to extrapolate our data to human adult blood vessels. Prostacyclin is synthesized by endothelial cells (24) and its production may reflect an important part of the internal metabolism of these cells. In order to obtain a maximal response, we have used a non physiological inducer, the calcium ionophore A 23187 that stimulates prostaglandin synthesis (25). No difference could be shown in 17 B Estradiol and Ethinyl-estradiol treated cultures compared to control in spite of a noticeable scattering of the results. The influence of sexual steroids on prostacyclin production depends on the target tissue. It has been shown in vitro that 17 B Estradiol and hormones
308
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
Progesterone reduced the production of prostacyclin-like activity in the uterus from ovariectomized rats (26). Estradiol treatment of cultured rat aortic smooth muscle cells induced a stimulatory effect on prostacyclin production after the addition of arachidonic acid or prostaglandin H2 (27). The authors suggested that estradiol could stimulate both cyclooxygenase and prostacyclin synthetase activities by inducing new protein biosynthesis in smooth muscle cell cultures. In our study, prostacylin production was comparable in endothelial cell cultures supplemented with F.C.S. or F.C.S.-A. The much reduced concentration of arachidonic acid is not involved in prostacyclin synthesis. However it remains possible that the selection of another stimulus may have brought different results. There has been considerable epidemiological evidence to correlate sex and sexual hormones to the incidence of vascular disease. Our study does not support a correlation between the cell proliferation and synthesis of PG 12 The link between Ethinyl-estradiol induced endothelial cell proliferation and the vascular complications of oral contraceptives remains unclear. The nature of this cell proliferation must be further defined by the investigation of other endothelial cell functiorsin relation to thrombosis like collagen synthesis, fibrinolytic activities or factor VIII von Willebrand. ACKNOWLEDGEMENT We wish to thank the maternity of Lariboisiere for providing the umbilical cords and Dr Coussieu for the determining the level of estrogen in fetal calf serum. This work was supported by,Grant of the Ministere de la Sante.
REFERENCES 1.
Final Mortality Statistics no 23. National Centre for Health Statistics. Department of Health, Education and Welfare publication. Washington. D.C. 1971.
2.
VESSEY, M. MANN, J. Female sex hormones and thrombosis. Epidemiological Aspects. ---_ Brit. Med. Bull. 34, 157-162, 1978.
3.
ALMEN, T. HARTEL, M. NYLANDER, G. OLIVECRONA H. The effect of estrogen on the vascular endothelium and its possible relation to thrombosis. Surg. Gynecology and Obstetrics, 1110, 938-940, 1975.
4.
Boston collaborative drug surveillance Program. Oral contraceptives and venous thromboembolism diseases, surgically conformed gall-bladder disease, and breast tumors. Lancet, 1, 1399-1404, 1973.
5.
Collaborative group for study of stroke in young women. Oral contraception and increased risk of cerebral ischemia or thrombosis. New Engl. J. Med., 288, 871-878, 1973.
6.
VESSEY, M.P. DOLE, R. Investigations of relation between use of oral 2 199-205, contraceptives and thromboembolic disease. Brit. Med. J., _, 1968.
7.
VESSEY, M.P. PHERSON, M.C. JOHNSON, B. Mortality among women participating in the Oxford family planning association contraceptive study. Lancet, 2, 731-733, 1977.
Vo1.34, No.4
ESTROGENS AND ENDOTHELIAL CELLS
309
0.
JAFFE, E. MINICK, R. ADELMAN, B. BECKER, C. NACHMAN, R. Synthesis of basement membrane collagen by cultured human endothelial cells. J. Exp. Medicine, 144, 209-225, 1976.
9.
ROSS, R. GLOMSET, J. HARKER, L. Response to injury and atherogenesis. 86 664-675, 1977. Am. Journ. Path _-L'-)
10.
COLBURN, P. BUONASSISSI, V. Oestrogen-binding sites in endothelial cell cultures. Sciences, 201, 817-819, 1978.
11.
JAFFE, E.A. NACHMAN, R. BECKER, C.G. MINICK, R. Culture of human endothelial cells. Identification by morphologic and immunologic criteria. -J. Clin. Invest. 52, 2745-2755, 1973.
12.
ESBER, M. PAYNA, I. BOGDEN, A. Variability of hormon concentrations and ratios in commercial sera used for tissue culture. ---__ J. Natl. Cancer Inst. 50, 559-562, 1973.
13.
MILO, G.E. MALARKEY, W.B. POWELL, J.E. BLAKESLEE, J.R. YOHN, D.S. Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro. In vitro, 12, 23-30, 1976.
14.
ARMELIN, H. WISHIKAWA, K. SKATO, G. Control of mammalian cell growth in culture. The action of protein and steroid hormones as effector substances. In control of proliferation in animal cells, CLARDSON and BASERGA, R. (Ed.), Cold Spring Harbor NY, 1974, 97-104.
15.
MACCOBY, E. DOERING, C.H. JACKLIN, C. KRAEMER, H. Concentration of sex hormones in umbilical cord blood : their relation to sex and birth order of Infants. Child Development, 50, 532-642, 1979.
16.
MACLOUF, J. PRADEL, M. PRADELLES, P. DRAY, F. 1251 derivatives of prostaglandins. A novel approach in prostaglandin analysis bj radioimmunoassay. Biochim. Biophys. &, 431, 139146, 1976.
17.
MACLOUF J. Radioimmunoassay for 6 keto PG Flcl . Methods -in enzymology, 86, 273-286, 1982.
18.
WEIBEL, F. PALADE, G. New cytoplasmic components in arterial endothelia. J. -- Cell. Biol. , 23_, 101-112, 1964.
19.
HARRISON R.L., MCKEE P.A. The effect of estrogen on von Willebrand Factor production and endothelial cell number in culture. Thromb. Haemost., 2, 20 (A), 1981.
20.
HORWITZ K.B., HORWITZ L.D. Canine vascular tissues on target for androgens, estrogens, progestins and glucocorticoids. -J. Clin. Invest., -69 : 750-758, 1982.
21.
MURAI, J. LIEBERMAN, R. YANG, J. GERSCHENSON, L. Decrease of estrogen receptors induced by 17 B estradiol and progesterone in cultured rabbit endometrial cells. Endocrine. Res. Comm. 5, 235-247, 1979.
22.
OSTERHOLZER, H. GRILLO, D. KRUGER, P. DUNNIHOO, D. The effect of oral contraceptive steroids on branches of the uterine artery. Obtet. Gynecol. -29 : 227-232, 1977.
310
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
23.
IREY, N.S. HENRY, M.D. NORRIS, J. Intimal vascular lesions associated with female reproductive steroids. Arch. Pathol. -96 : 227-234, 1973.
24.
WEKSLER, B. LEY, C.N. JAFFE, E.A. Stimulation of endothelial cell prostaglandin production by thrombin, trypsin, and the ionophore A 23187. J. Clin. Invest. -62 : 923-930, 1978. --
25.
PICKETT, W. HESSE, R. COHEN, P. Initiation of phospholipase A2 hctivity in human platelets by the calcium ionophore A 23187. Biochim. Biophys. Acta. 486 : 209-213. 1977.
26.
GIMENO, M.F. BORDA, E.S. LAZZARI, M.A. GIMENO, A.L. Ovarian hormones inhibit the release of prostacyclin-like material from isolated rat uterus. Prostaglandins, -20 : 223-232, 1980.
27.
CHANG-CHANG, W. NAKAO, J. ORIMO, H. MUROTA, S.I. Stimulation prostaglandin cyclooxygenase and prostacyclin synthetase activities b, estradiol in rat aortic smooth muscle cells. Biochim., Biophys. e, 620 : 472482, 1980.
MINIMAL
EFFECT OF ESTROGENS ON ENDOTHELIAL PRODUCTION OF PhGSTACYCLIN
CELL GROWTH AND
E. CORVAZIER,E. DUPUY, A.M. DOSNE, J. MACLOUF. Lab. H&nostaseet U.156 INSERM, H^opitalLariboisisre, 6, Rue Guy Patin, 75010 PARIS, FRANCE. (Received 11.4.1983; Accepted in revised form 16.2.1984 by Editor M. Samama)
ABSTRACT The effeclnof natural and synthetic sex hormones (lo-*M) have been studied on human umbilical endothelial cell proliferation and prostacyclin production. 17 B estradiol and progesterone had no effect on cell multiplication. Ethinyl-estradiol increased pr;liferation when the initial plating density was 40,000 cells/ cm . However, the production of 6-keto PGFleinduced by the Catf ionophore A 23187 was not different between control, 17 B estradiol-or ethinyl estradiol-treated cultures. These results demonstrate an in vitro effect of synthetic estrogen, ethinyl estradiol on endothelial cell nroliferation. At the present time it is however difficult to correlate these results with the clinical observation of an increase in thromboembolic complicationsin women under oral contraceptives. INTRODUCTION The low incidence of atherosclerosis in premenopausal women compared to men of similar age (l), the increased risk of vascular complications in women under oral contraceptives (2-71, suggest that sex-linked factors may play a role in the development of vascular diseases. These clinical data suggest that there is a major difference between natural and synthetic estrogens in the development of atherosclerotic lesions. Endothelial cells synthesize collagen, basal membrane and elaborate the composition and structure of the subendothelium (8). Factors controlling endothelial cell proliferation are thought to be an important aspect in the comprehension of atherosclerosis which develops after endothelial injury (9) because they could accelerate or slowdown the regeneration of the normal endothelium barrier. Since estrogen binding sites have been demonstrated in endothelial cell cultures by Colburn and Buonassissi (lo), the effects of natural and synthetic estrogens and progesterone have been investigated on cell proliferation and on the production of 6-keto PG Flo by human endothelial cell cultures. KEY WORDS : Sex hormones, human endothelial cell, prostacyclin. 303
304
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
MATERIAL._. AND METHODS -..-_ .___ .._^ .___ Isolation and culture of endothelial cells. EndotheliaGells were-?solated from human umbilical veins by collagenase digestion (11). The endothelial cells were cultured on Linbro multiplates (2 cm2-24 wells) in the presence of Medium 199 supplemented with 10 % charcoal adsorbed fetal calf serum (FCS-A). The inital density of plating was either 20 000 or 40 000 cells per cm2. Preparation of estrogen depleted sera. Physiologicarconcentrationsof stad hormones are present in fetal calf serum (FCS) (12, 13). Therefore, the serum was depleted of endogenous steroids and prostaglandins by two adsorptions with dextran coated charcoal at 50°C for 45 min. (14). Charcoal was removed by centrifugation and the serum (FCS-A) was stored at -2OOC after sterilization by filtration. The remaining estradiol concentration of FCS-A was less than lo-1lM as measured by radioimmunoassay. Proliferation study. One day after plating (day 1); the cultures of each well were washed once with Medium 199 without FCS in order to eliminate floating cells. Exogenous hormones were added daily to the culture. Control cultures from the same cell preparation were carried out in Medium 199 with 10 % FCS-A but without adding hormone. Cell count of adherent cells was performed daily. Before cell counting, the culture was washed once with Medium 199 without serum and once with saline phosphate buffer. The cells were dissociated by collagenase (0.1 %) in EDTA (0.1 %) and counted in a hematocytometer. The steroid hormones were dissolved in ethanol and diluted in Medium 199 with 10 % of FCS-A at a final concentration of lo-*M which corresponds to the physiological concentration in human cord blood (15). Results were compared using Student's t test analysis. All culture reagents were from Gibco Laboratory, 17 B estradiol and ethinyl estradiol were obtained from Sigma Chemical Co, and progesterone from Besins Isovesco Laboratory. Dextran coated charcoal and TES buffer were purchased from Sigma. Production of 6-keto -PG Flo . For this investigation the cells were plated at 40 000 cells/cm2 in 35 mm Lux Petri dishes. At day 3 the cultures were washed four times with M 199 without serum in order to remove floating cells and serum. Cells were stimulated by calcium ionophore A 23187 (Calbiochem) at 0,25 ; 0,5 and 1 PM final concentration during 5 mins.,to induce the release of prostacyclin by the response was influenced hormone treated culture. In order to knowMhet&the by sex, matched sex cultures were also stimulated with 1 PM ionophore and analyzed. 6-keto PG F CL radioimmunoassay. Thestable'derivative of prostacyclin was measured by the method of Maclouf et al (16, 17) using 1251 -Histamide-6-keto PG Flc(-100 ul of the diluted sample were added to the medium containing 200 ~1 of Tris-HCl buffer pH 8, 100 ~1 of 1251 6-keto PGFlo (10.000 dpm), 200 pl of 0,5 % w/v bovine gammaglobulin and 100 ~1 of the diluted antiserum (final dilution : l/35,000). After 18 h incubation at 4OC, labelled immune complex was separated from the unbound by 700 ~1 of a 25 % w/v ice cold polyethylene glycol solution. After centrifugation the supernatant was discarded and the globulin precipitate counted in a gamma counter (Beckman). Standard curve was obtained using 1,25
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
305
to 80 pg per tube of 6-keto PG Fla . Assay detection limit was 1,2 pg and 50 % inhibition was obtained with 13 pg of 6-keto PG Fl~r . Cross reactivity was 3 % with PG Flu , 1 % with PG F2 a and less than 1 % for other prostaglandins. RESULTS Characterization of endothelial cell cultures. Electron microscopy demonstrates the presence of the Weibel-Palade bodies which are the characteristic of endothelial cells (18). Comparison between FCS-A and FCS on endothelial cell cultures. The proliferation amt
9 -0
',30. c u &I
t-t
Release of 6-keto PG Fla induced by Cat+ Ionophore A 23187(control culture). Mean of 4 experiments + SEM.
$ a .'Ct Y 9 y 0.25 0.5 Ca++ lonophorc
1 1 QM
FIG.3
306
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
FIG. Effect of sex hormones (lo-*M) -on the proliferation _of the endothelial cells --seeded at 20.000 cells/cm2. --Progesterone (a), 17 B estradiol (B)+or ethinyf estradiol (c) (-b-).Control (+). Mean of 5 experiments (mean Ir--S.D.).).
FIG. 2 of the endothelial -L__ cells seeded Effect of sex hormones -on the p roliferation ---at 40.000 cells/cm2. Meam experiments. Same symbols than fig. 1. Increase of 30 % (p <0,05) for ethinyl estradiol culture (mean I 2.S.D.).
Vo1.34, No.4
307
ESTROGENS AND ENDOTHELIAL CELLS
TABLE I Effect of estrogens on the production of 6-keto PG Fla from endothelial cells stimulated by the Cat+ Ionophore A 23187. The results are expressed in % of the control. Mean of 4 experiments A SEM. Ionophore (PM)
17 B estradiol
Ethinyl estradiol 114 _+42
0,25
0,s
llO-+
11
138 f 48
1
119 f
22
136 2 46
At 1 PM Ionophore the production of 6-keto PG Fla was regardless of sex (data not shown). No difference was observed. DISCUSSION This study was undertaken in order to investigate the action Of Sexual on endothelial cell proliferation and modification of their prostacyclin production. The understanding of atherosclerosis developing after endothelial injury is of importance because factors controlling endothelial cell proliferation are thought to play an important role in the regeneration of normal endothelial barrier. Vascular abnormalities induced by synthetic estrogens could be related to a decreased regeneration of the endothelium. -8 In this study, 17 B Estradiol and Progesterone at 10 M had no effect on cell multiplication regardless of the initial density of cultured human endothelial cells. However, 10e8M Ethinyl-estradiol stimulated cell proliferation at the highest initial cell density (40.000 cells/cm2). This density could reflect an effect resulting from endothelial cells in confluency. Harrison and McKee (19) reported that 17 B Estradiol causes an increased rate of human endothelial cell multiplication, but synthetic estrogen was not tested in their study. Specific estrogen binding sites have been described on the surface of endothelial cells from animal species : e.g. rabbit aorta (lo), canine arteries and inferior vena cava (20). The proliferating effect induced by ethinyl-estradiol on human endothelial cells might be related to a direct action of this compound on this tissue although it is not clear wether these cells possess estrogen binding sites. The lack of effect of natural estrogen could also be due to a decrease of the estrogen receptors as a result of the high level of hormones during pregnancy and delivery ; such fact has been described for endcthelial cells (21). The effect induced by ethinyl estradiol might be related to the intimal proliferation of endometrial vessels observed in vivo in 80 % of women taking oral contraceptives (22, 23). In this regard the study of other vascular cells could be of interest although it remains difficult to extrapolate our data to human adult blood vessels. Prostacyclin is synthesized by endothelial cells (24) and its production may reflect an important part of the internal metabolism of these cells. In order to obtain a maximal response, we have used a non physiological inducer, the calcium ionophore A 23187 that stimulates prostaglandin synthesis (25). No difference could be shown in 17 B Estradiol and Ethinyl-estradiol treated cultures compared to control in spite of a noticeable scattering of the results. The influence of sexual steroids on prostacyclin production depends on the target tissue. It has been shown in vitro that 17 B Estradiol and hormones
308
ESTROGENS AND ENDOTHELIAL CELLS
Vo1.34, No.4
Progesterone reduced the production of prostacyclin-like activity in the uterus from ovariectomized rats (26). Estradiol treatment of cultured rat aortic smooth muscle cells induced a stimulatory effect on prostacyclin production after the addition of arachidonic acid or prostaglandin H2 (27). The authors suggested that estradiol could stimulate both cyclooxygenase and prostacyclin synthetase activities by inducing new protein biosynthesis in smooth muscle cell cultures. In our study, prostacylin production was comparable in endothelial cell cultures supplemented with F.C.S. or F.C.S.-A. The much reduced concentration of arachidonic acid is not involved in prostacyclin synthesis. However it remains possible that the selection of another stimulus may have brought different results. There has been considerable epidemiological evidence to correlate sex and sexual hormones to the incidence of vascular disease. Our study does not support a correlation between the cell proliferation and synthesis of PG 12 The link between Ethinyl-estradiol induced endothelial cell proliferation and the vascular complications of oral contraceptives remains unclear. The nature of this cell proliferation must be further defined by the investigation of other endothelial cell functiorsin relation to thrombosis like collagen synthesis, fibrinolytic activities or factor VIII von Willebrand. ACKNOWLEDGEMENT We wish to thank the maternity of Lariboisiere for providing the umbilical cords and Dr Coussieu for the determining the level of estrogen in fetal calf serum. This work was supported by,Grant of the Ministere de la Sante.
REFERENCES 1.
Final Mortality Statistics no 23. National Centre for Health Statistics. Department of Health, Education and Welfare publication. Washington. D.C. 1971.
2.
VESSEY, M. MANN, J. Female sex hormones and thrombosis. Epidemiological Aspects. ---_ Brit. Med. Bull. 34, 157-162, 1978.
3.
ALMEN, T. HARTEL, M. NYLANDER, G. OLIVECRONA H. The effect of estrogen on the vascular endothelium and its possible relation to thrombosis. Surg. Gynecology and Obstetrics, 1110, 938-940, 1975.
4.
Boston collaborative drug surveillance Program. Oral contraceptives and venous thromboembolism diseases, surgically conformed gall-bladder disease, and breast tumors. Lancet, 1, 1399-1404, 1973.
5.
Collaborative group for study of stroke in young women. Oral contraception and increased risk of cerebral ischemia or thrombosis. New Engl. J. Med., 288, 871-878, 1973.
6.
VESSEY, M.P. DOLE, R. Investigations of relation between use of oral 2 199-205, contraceptives and thromboembolic disease. Brit. Med. J., _, 1968.
7.
VESSEY, M.P. PHERSON, M.C. JOHNSON, B. Mortality among women participating in the Oxford family planning association contraceptive study. Lancet, 2, 731-733, 1977.
Vo1.34, No.4
ESTROGENS AND ENDOTHELIAL CELLS
309
0.
JAFFE, E. MINICK, R. ADELMAN, B. BECKER, C. NACHMAN, R. Synthesis of basement membrane collagen by cultured human endothelial cells. J. Exp. Medicine, 144, 209-225, 1976.
9.
ROSS, R. GLOMSET, J. HARKER, L. Response to injury and atherogenesis. 86 664-675, 1977. Am. Journ. Path _-L'-)
10.
COLBURN, P. BUONASSISSI, V. Oestrogen-binding sites in endothelial cell cultures. Sciences, 201, 817-819, 1978.
11.
JAFFE, E.A. NACHMAN, R. BECKER, C.G. MINICK, R. Culture of human endothelial cells. Identification by morphologic and immunologic criteria. -J. Clin. Invest. 52, 2745-2755, 1973.
12.
ESBER, M. PAYNA, I. BOGDEN, A. Variability of hormon concentrations and ratios in commercial sera used for tissue culture. ---__ J. Natl. Cancer Inst. 50, 559-562, 1973.
13.
MILO, G.E. MALARKEY, W.B. POWELL, J.E. BLAKESLEE, J.R. YOHN, D.S. Effects of steroid hormones in fetal bovine serum on plating and cloning of human cells in vitro. In vitro, 12, 23-30, 1976.
14.
ARMELIN, H. WISHIKAWA, K. SKATO, G. Control of mammalian cell growth in culture. The action of protein and steroid hormones as effector substances. In control of proliferation in animal cells, CLARDSON and BASERGA, R. (Ed.), Cold Spring Harbor NY, 1974, 97-104.
15.
MACCOBY, E. DOERING, C.H. JACKLIN, C. KRAEMER, H. Concentration of sex hormones in umbilical cord blood : their relation to sex and birth order of Infants. Child Development, 50, 532-642, 1979.
16.
MACLOUF, J. PRADEL, M. PRADELLES, P. DRAY, F. 1251 derivatives of prostaglandins. A novel approach in prostaglandin analysis bj radioimmunoassay. Biochim. Biophys. &, 431, 139146, 1976.
17.
MACLOUF J. Radioimmunoassay for 6 keto PG Flcl . Methods -in enzymology, 86, 273-286, 1982.
18.
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