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(The FASEB Journal. 2007;21:265-273.)
© 2007 FASEB

Progestins overcome inhibition of platelet aggregation by endothelial cells by down-regulating endothelial NO synthase via glucocorticoid receptors

Murielle Zerr-Fouineau*, Marta Chataigneau*, Christian Blot{dagger} and Valérie B. Schini-Kerth*,1

* Département de Pharmacologie et Physico-Chimie, UMR 7175-LC1, Université Louis Pasteur de Strasbourg, France; and

{dagger} Théramex, Monaco

1Correspondence: UMR CNRS 7175-LC1, Université Louis Pasteur de Strasbourg, Faculté de Pharmacie, 74, route du Rhin, B.P. 60024, F-67401 Illkirch, France. E-mail: valerie.schini-kerth{at}pharma.u-strasbg.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hormone replacement therapy with estroprogestin preparations is associated with an increased risk of venous and arterial thromboembolic events in postmenopausal women. This study examined whether progestins affect the formation of NO in endothelial cells, and, if so, to determine the underlying mechanism. Experiments were performed with human umbilical vein endothelial cells. Endothelial nitric oxide synthase (eNOS) expression was assessed by real-time polymerase chain reaction (PCR) and Western blot analysis, NO formation by electron spin resonance spectroscopy, nuclear translocation of the glucocorticoid receptor by immunofluorescence microscopy, and platelet aggregation by an aggregometer. Medroxyprogesterone acetate (MPA) and progesterone markedly decreased the eNOS mRNA and protein levels, whereas levonorgestrel and nomegestrol acetate had only small effects. This effect was associated with a decreased NO formation leading to a reduced ability of endothelial cells to prevent platelet aggregation and was prevented by knockdown of the glucocorticoid receptor using siRNA. MPA and progesterone, but not levonorgestrel and nomegestrol acetate, caused nuclear translocation of the glucocorticoid receptor. The present findings indicate that certain progestins, including MPA, reduce the antiaggregatory effect of endothelial cells by decreasing the expression of eNOS and the formation of NO in endothelial cells, an effect that is mediated via activation of glucocorticoid receptors.—Zerr-Fouineau, M., Chataigneau, M., Blot, C., Schini-Kerth, V. B. Progestins overcome inhibition of platelet aggregation by endothelial cells by down-regulating endothelial NO synthase via glucocorticoid receptors.


Key Words: thrombosis • hormone replacement • therapy • contraception


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ESTROGENS HAVE BEEN SHOWN to have a beneficial effect on the cardiovascular system. Indeed, they can inhibit the vascular response to balloon injury (1) and the development of atherosclerosis (2) . Potential mechanisms involved in the beneficial effect include an improvement of the lipid profile by reducing the level of LDL and by increasing the level of HDL (3) , an acceleration of the functional endothelial recovery after balloon injury through an increased expression of vascular endothelial growth factor (VEGF) (1) , and the prevention of the expression of proatherosclerotic factors, such as vascular cell adhesion molecule (VCAM)-1 and MCP-1 (4 , 5) . Alternatively, the beneficial effect may also be due to the direct action of estrogens on endothelial cells (6 7 8) . Indeed, estrogens can rapidly increase the endothelial formation of NO as a consequence of the PI3 kinase/Akt-dependent phosphorylation of endothelial NO synthase (eNOS) at Ser-1177 (9 10 11) . In addition, estrogens can also stimulate the expression of eNOS by increasing gene transcription and mRNA stability (12) .

Progestins are often associated with estrogens in both oral contraception and hormonal replacement therapy, to counteract the hyperplasic effect of estrogens on the endometrium. Numerous clinical studies have indicated that estrogen-progestin combinations used for oral contraception and postmenopausal replacement therapy increase the relative risk of thromboembolic events (13 14 15 16) . The higher incidence of thromboembolic events was attributed initially to the estrogen component of oral contraception, because reduction from 50 to 30 µg of estrogen (low-dose estrogen in newer preparations) per tablet had a favorable outcome (17) . Several recent independent studies, however, have shown a two-fold increased relative risk of thromboembolic events with combined oral contraceptives containing a third generation progestin such as gestodene or desogestrel, in combination with low-dose estrogen compared with those containing levonorgestrel and similar low-dose estrogen (18 19 20) . Moreover, the Women’s Health Initiative study has indicated that the risk of thromboembolic events is higher among postmenopausal users of combined estro-progestin treatments than users of only estrogen replacement therapy (21) . These findings highlight a potential determinant role also of the progestin component of estro-progestin treatments for the development of thromboembolic events.

The vascular effect of progestins, a heterogeneous group of compounds has been only poorly investigated. All progestins have potent progestogenic and antiestrogenic effects on the endometrium, but depending on their chemical structure, they can also interact with other nuclear receptors, including the glucocorticoid receptor and the mineralocorticoid receptor to induce biological responses (22 , 23) . In particular, medroxyprogesterone acetate has a high relative binding affinity for the glucocorticoid receptor, and it is also able to induce glucocorticoid-like effects in targets cells, whereas no such effects were observed with levonorgestrel and nomegestrol acetate (22 , 23) . Glucocorticoids have been shown to down-regulate eNOS mRNA and protein expression in endothelial cells resulting in a decreased endothelium-dependent NO-mediated relaxation and hypertension (24) . Therefore, the aim of the present study was to examine whether progestins affect the expression of eNOS and the formation of NO, a potent vasoprotective and antithrombotic factor, in human endothelial cells and, if so, to determine the role of their partial glucocorticoid activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Endothelial cells (HUVECs) were isolated from human umbilical veins as described previously (25) . Cells were cultured in MCDB 131 containing 2 mmol/L ultraglutamine, 105 U/L penicillin, 100 mg/L streptomycin supplemented with 10% FCS. Cells were used at first passage.

Hormonal treatments
At confluence, HUVECs were serum deprived for a 6-h period before being exposed to either solvent (0.01% DMSO) or a test compound. Two categories of progestins have been studied, those with partial glucocorticoid activity, such as progesterone, medroxyprogesterone acetate (MPA) and megestrol acetate (MEG), and those without partial glucocorticoid activity such as levonorgestrel (LNG) and nomegestrol acetate (NOMAC). In addition, experiments were also performed with the glucocorticoid compounds hydrocortisone (HYDRO) and dexamethasone (DEXA). Most experiments were performed with progestins used at concentrations up to 0.1 µmol/L, which can be reached in the plasma of postmenopausal women using hormonal replacement therapy (26 , 27) .

Real-time polymerase chain reaction
Total RNA was isolated from HUVECs using RNeasy Micro kit (Qiagen, Courtaboeuf, France). cDNA was synthesized from total RNA using iScript cDNA Synthesis kit (Bio-Rad, Marnes-la-Coquette, France), and PCR amplification was performed using IQ SYBR Green Supermix (Bio-Rad). The specific primers were as follows: eNOS sense, 5'-GGC ATC ACC AGG AAG ACC-3'; eNOS antisense, 5'-TCA CTC GCT TCG CCA TCA C-3'. The control housekeeping gene was human GAPDH. Relative quantitation was determined by standard 2(–{Delta}{Delta}CT) calculations.

Western blot analysis
Total proteins (20 µg) were subjected to SDS-PAGE (10%) and blotted on PVDF membranes. Immunodetection was carried out using an antibody (Ab) directed against eNOS (BD Biosciences, Marseille, France) or the glucocorticoid receptor (Alexis Biochemicals, Paris, France) and enhanced chemiluminescence (ECL) (Amersham, Orsay, France).

Small inhibitory RNA (siRNA) studies
HUVECs were transfected with four pooled siRNA duplexes (50 nmol/L) directed against separate glucocorticoid receptor mRNA target sequences (SMART-Pool; Dharmacon, Evry, France) for 24 h before hormonal treatments. Transfections were conducted using DharmaFECT transfection reagent according to the manufacturer’s instructions.

Determination of NO formation by electron spin resonance spectroscopy
Determination of NO formation was assessed by electron spin resonance spectroscopy (ESR) after formation of [Fe(II)NO(DETC)2], a paramagnetic diethyldithiocarbamate iron complex with NO, in endothelial cells. The ESR methodology was used, as reported previously with minor modifications (28 , 29) . HUVECs were washed twice with HBSS buffered with 10 mmol/L HEPES, and then they were incubated in a HBSS-HEPES solution in the presence of bovine serum albumin (20.5 mg/mL), 1.5 mmol/L CaCl2, 0.3 mmol/L L-arginine for 15 min at 37°C. Spin trap chemicals FeSO4 (0.8 mmol/L) and DETC (1.6 mmol/L) were rapidly mixed to obtain a colloid form [Fe(II)(DETC)2], which was added to HUVECs at a final concentration of 0.2 mmol/L. After 5 min, the endothelial formation of NO was induced by the addition of thrombin (1 U/mL) for 30 min. Thereafter, dishes were placed on ice, and the incubation medium was removed before the addition of 0.2 µl of the HBSS-HEPES buffer. Cells were then scraped, and the cell suspension was collected in a calibrated tube. Tubes were rapidly frozen at 77K for ESR measurements. ESR measurements were performed on an MS100 spectrometer (Magnettech Ltd., Berlin, Germany) under the following conditions: temperature 77K, microwave frequency 9.34 GHz, microwave power 20 mW, modulation frequency 100 kHz, modulation amplitude 1 mT. The third component of the ESR signal was used for relative comparison of the concentration of NO trapped in each sample.

Immunofluorescence detection of intracellular glucocorticoid receptor distribution
HUVECs were seeded on Lab-Tek culture chambers (Nunk, Rochester, NY) coated with collagen. After treatment, cells were fixed in 4% formaldehyde in PBS at room temperature for 1 h. Cells were exposed to 5% goat serum for 1 h before exposure to an Ab directed against the human glucocorticoid receptor (dilution 1:200). Immunodetection was carried out with FITC-labeled secondary goat Ab (dilution 1:500). Nuclei were stained with 4'6-diamidino-2-phenylindole dihydrochloride (4',6'-diamidino-2-phenylidole (DAPI), Sigma, St. Louis, MO).

Platelet aggregation studies
Blood was obtained from healthy volunteers, who did not take any medication for at least 10 days. Washed platelet suspensions were kindly provided and prepared by Etablissement Français du Sang (Strasbourg, France), as described previously (30) . Suspensions of platelets (450 µl, 3x108 platelets/mL) were incubated for 2 min in a Chronolog 490 aggregometer (Havertown, PA) with continuous stirring at 1,000 rpm before addition of a submaximal concentration of thrombin (0.07 U/mL). HUVECs were cultured on cytodex-3 beads and exposed to either solvent or a compound for 24 h. In some experiments, HUVECs were treated with mifepristone (10 µmol/L) for 30 min before addition of a progestin for 24 h. Thereafter, they were added to platelet suspensions 1 min before addition of thrombin. In some experiments, HUVECs were preincubated with N{omega}-nitro-L-arginine (NLA, 300 µmol/L) for 30 min before addition to platelets.

Statistical analysis
All data are expressed as means ± SEM. Statistical analysis of the data was performed using Student’s t test or a multiway ANOVA followed by Fisher’s protected least significant different test where appropriate. A value of P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sex steroids down-regulate eNOS expression
Exposure of HUVECs to MPA (0.1 µmol/L) markedly decreased the eNOS mRNA level in a time-dependent manner and this effect reached 48% after a 6-hour treatment period (Fig. 1 A). In contrast, the solvent (0.01% DMSO) was without effect (eNOS mRNA level was 110±13.7%, n=3). Similarly to MPA, progesterone and the glucocorticoid dexamethasone also significantly decreased eNOS mRNA level by 43 and 51%, respectively whereas levonorgestrel caused only a slight but significant reduction (23%) and nomegestrol acetate did not have such an effect (Fig. 1B ). MPA and progesterone also significantly decreased eNOS protein level in a concentration-dependent manner after a 24-hour treatment period with a significant effect observed at concentrations as low as 1 nmol/L and 10 nmol/L, respectively (Figs. 2 A and 3 ). The inhibitory effect was mimicked by the glucocorticoid compounds hydrocortisone and dexamethasone but not by levonorgestrel, nomegestrol acetate, and megestrol acetate (Fig. 2) .


Figure 1
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  		Figure 1. Effect of progestins on eNOS mRNA level in HUVECs. A) Time-dependent down-regulation of eNOS mRNA expression by medroxyprogesterone acetate (MPA). B) Cells were exposed to either solvent (0.01% DMSO), a progestin (LNG, levonorgestrel; NOMAC, nomegestrol acetate; PROG, progesterone; MPA) or the synthetic glucocorticoid dexamethasone (DEXA) for 6 h before eNOS mRNA level was determined. Data are shown as mean ± SEM of 3–5 (A) and 7 (B) different experiments. *P ≤ 0.05 vs. control.


Figure 2
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  		Figure 2. Effect of different progestins (A) and glucocorticoid compounds (B) on eNOS protein level in HUVECs. HUVECs were exposed to either solvent (0.01% DMSO) or a compound before determination of the level of eNOS protein. A and B) top: representative Western blot of eNOS protein level and corresponding ß-tubulin protein level; bottom: corresponding cumulative data. Data are shown as means ± SEM of 7 or 8 different experiments. *P ≤ 0.05 vs. control.


Figure 3
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  		Figure 3. Effect of different concentrations of progestins on eNOS protein level in HUVECs. Cells were exposed to either solvent (0.01% DMSO) or a progestin for 24 h before determination of the level of eNOS protein. Top) representative Western blot of eNOS protein level and corresponding ß-tubulin protein level; bottom) corresponding cumulative data. Data are shown as means ± SEM of 7 or 8 different experiments. *P ≤ 0.05 vs. control.

Effect of progestins on thrombin-induced NO formation
Treatment of HUVECs with thrombin caused about a 170% increase in the formation of NO as assessed by ESR (Fig. 4 ). The stimulatory effect of thrombin was abolished by N{omega}-nitro-L-arginine (100 µmol/L; from 166±4% to 78±8%, respectively, n=3), indicating that it is due to an increased eNOS activity. Pretreatment of HUVECs with MPA, progesterone, or dexamethasone for 24 h markedly reduced the stimulatory effect of thrombin, whereas nomegestrol acetate did not have such an effect (Fig. 4) .


Figure 4
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  		Figure 4. Effect of progestins on thrombin-induced formation of NO in HUVECs. HUVECs were exposed to either solvent (0.01% DMSO) or a compound (0.1 µmol/L) for 24 h before addition of thrombin. NO formation in HUVECs was assessed by ESR spectroscopy. Results are shown as means ± SEM of 5 to 7 different experiments. * and #P ≤ 0.05 vs. control and thrombin, respectively.

Role of the glucocorticoid receptor
Progestins, which inhibit eNOS expression and activity, such as MPA and progesterone, have been shown to bind not only to the progesterone receptor but also to the glucocorticoid receptor with relatively high affinity and to induce glucocorticoid-like effects in several experimental cell systems (22 , 23) . In contrast, the inactive progestins, such as levonorgestrel and nomegestrol acetate, bind almost exclusively to the progesterone receptor, and they do not bind to the glucocorticoid receptor (22 , 23) . Therefore, experiments were planned to determine whether progestins can activate the glucocorticoid receptor resulting in its nuclear translocation and also whether the inhibitory effect of progestins on eNOS expression is prevented by knockdown of the glucocorticoid receptor and by the progesterone and glucocorticoid receptor antagonist mifepristone. In control HUVECs, the glucocorticoid receptor is localized predominantly in the cytosol and also to some extent in the nucleus (Fig. 5 ). Levonorgestrel and nomegestrol acetate did not affect glucocorticoid receptor localization, whereas MPA, progesterone, and dexamethasone caused its translocation from the cytosol into the nucleus (Fig. 5) . We next examined the effect of inhibition of the glucocorticoid receptor expression on the MPA- and progesterone-induced down-regulation of eNOS expression. To address this, HUVECs were transfected with siRNA (50 nmol/L) specific to the glucocorticoid receptor resulting in a 54.3 ± 8.9% reduction of the glucocorticoid receptor protein level (Fig. 6 A, n=3). Knockdown of the glucocorticoid receptor expression prevented the MPA- and progesterone-induced down-regulation of eNOS mRNA expression (Fig. 6B ). In addition, exposure of endothelial cells to mifepristone also prevented the down-regulation of eNOS protein levels by MPA, progesterone, and dexamethasone, whereas the antagonist alone had only minor effects (Fig. 7 ).


Figure 5
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  		Figure 5. Effect of progestins on nuclear translocation of the glucocorticoid receptor in HUVECs. HUVECs were exposed to either solvent (0.01% DMSO) or a compound for 30 min before the localization of the glucocorticoid receptor (GR) was assessed by immunocytochemistry. Top photos represent GR staining and bottom photos show the corresponding nuclear staining with DAPI. Similar observations were made in 3 additional experiments.


Figure 6
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  		Figure 6. Glucocorticoid receptor (GR) activation mediates the progestin-induced down-regulation of eNOS mRNA expression in endothelial cells. HUVECs were transfected with siRNA (50 nmol/L, 24 h) specific for the GR before exposure to MPA or PROG for 6 h. A) GR expression was assessed by Western blot analysis. Top) representative Western blot of GR and corresponding ß-tubulin protein level. Similar observations were made in two additional experiments. B) eNOS mRNA expression was assessed by RT-polymerase chain reaction (RT-PCR). Data are shown as means ± SEM of 4 different experiments. *P ≤ 0.05 vs. control.


Figure 7
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  		Figure 7. Glucocorticoid receptor activation mediates the progestin-induced down-regulation of eNOS protein expression in HUVECs. Cells were exposed either to solvent or mifepristone, a glucocorticoid and progesterone receptor antagonist for 30 min before addition of either MPA, PROG, or DEXA for 24 h. Thereafter, eNOS protein level was assessed by Western blot analysis. Top) representative Western blot analysis of eNOS and corresponding ß-tubulin protein level; bottom) cumulative data. Data are shown as means ± SEM of 3 or 4 different experiments. *P ≤ 0.05 vs. control.

Progestins decrease the HUVECs-induced inhibition of platelet aggregation
Thrombin (0.07 U/mL) caused submaximal and irreversible aggregation of washed human platelet suspensions within 4 to 5 min. The stimulatory effect of thrombin was markedly reduced by addition of HUVECs (103 cells) to platelet suspensions one minute before their activation with thrombin whereas only a small inhibitory effect was observed with conditioned culture medium without HUVECs (Fig. 8 ). In contrast, addition of HUVECs, which have been treated with N{omega}-nitro-L-arginine (300 µmol/L) for 30 min, to platelet suspensions did not affect thrombin-induced platelet aggregation (Fig. 8A ). Similarly, treatment of HUVECs with either MPA, progesterone, or dexamethasone for 24 h decreased the inhibitory effect of HUVECs on platelet aggregation, whereas levonorgestrel and nomegestrol acetate were without effect (Figs. 8B –F). Pretreatment of HUVECs with mifepristone prevented the MPA-, progesterone- and dexamethasone-induced reduction of the inhibitory effect of HUVECs on platelet aggregation (Figs. 8B, E, F) . Addition of conditioned medium alone from either N{omega}-nitro-L-arginine-, MPA-, progesterone- or dexamethasone-treated HUVECs did not affect platelet aggregation (data not shown).


Figure 8
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  		Figure 8. Effect of progestins on the NO-mediated inhibitory effect of HUVECs on platelet aggregation. HUVECs (ECs) were exposed to either solvent, a progestin (LNG, levonorgestrel; NOMAC, nomegestrol acetate; PROG, progesterone; MPA), all tested at 0.1 µmol/L, or dexamethasone (DEXA, 0.1 µmol/L) for 24 h before they were added to washed platelets. In some experiments, HUVECs were pretreated with mifepristone (10 µmol/L) for 30 min before addition of a progestin. After 1 min, platelet aggregation was initiated by thrombin (0.07 U/mL). As indicated, the effect of conditioned culture medium alone and HUVECs, which were incubated with N{omega}-nitro-L-arginine (NLA, 300 µmol/L) for 30 min, on platelet aggregation is also shown. Similar findings were made in 2–4 additional experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major novel finding of the present study indicates that certain progestins substantially down-regulate the expression of eNOS, resulting in a subsequent depressed agonist-induced formation of NO in human endothelial cells. As an important consequence of these progestin-induced changes, the ability of endothelial cells to prevent platelet aggregation is reduced leading to an amplification of prothrombotic responses.

Interestingly, the down-regulation of eNOS expression is not induced by all members of the progestin family but occurs specifically with MPA and progesterone but not with levonorgestrel and nomegestrol acetate. The inhibitory effect of MPA on eNOS mRNA level occurred after a delay of 1 hour and reached ~50% after 6 h. This effect is associated with a decreased eNOS protein level by ~70% at 24 h, and results in a dramatic reduction of the endothelial formation of NO. Moreover, the progestin-induced down-regulation of eNOS expression is observed at concentrations as low as 1 nmol/L MPA and 10 nmol/L progesterone. In contrast, both levonorgestrel and nomegestrol acetate did not affect eNOS expression even at concentrations up to 1 µmol/L. During hormonal replacement therapy, peak serum concentrations of MPA are ~5 nmol/L after intake of 2 mg 17ß-estradiol and 5 mg MPA (26) , and mean serum concentrations of progesterone are ~10 nmol/L after use of a vaginal ring delivering estradiol 160 µg/day and progesterone 20 mg/day (27) . Thus, the inhibitory effect of progestins on eNOS expression is observed at concentrations that are likely to be of clinical significance. In addition, such an effect might also explain the impaired endothelium-dependent flow-mediated brachial artery dilatation in chronic users of contraceptive depot MPA (31) .

Progestins are a heterogeneous family of steroids that, in addition to their interaction with the cytosolic progesterone receptor, might also bind strongly to other steroid receptors, such as those for androgens, mineralocorticoids, and glucocorticoids to induce biological responses (22 , 23) . The down-regulation of eNOS expression and NO release occurred selectively with MPA and progesterone, whereas levonorgestrel and nomegestrol acetate, despite their similar progestin potency, had no effect. Such a differential effect is not consistent with a major role for the progesterone receptor in the down-regulation of eNOS. Interestingly, all stimulatory progestins have distinct intrinsic glucocorticoid activity, as opposed to the inactive ones (22 , 23) . Moreover, dexamethasone is a potent inhibitor of eNOS expression both at the mRNA and protein levels in endothelial cells (24 , 32) . In addition, chronic intake of dexamethasone caused down-regulation of eNOS in the aorta and several other tissues of glucocorticoid-treated rats and was associated with a pronounced attenuation of acetylcholine (Ach)-induced vasodilatation in resistance arteries (24) . Therefore, the role of the glucocorticoid receptor in the progestin-induced down-regulation of eNOS was investigated. The present findings indicate that, similar to dexamethasone, MPA and progesterone caused major translocation of the glucocorticoid receptor from the cytosol to the nucleus, whereas no such effect was observed with nomegestrol acetate and levonorgestrel. Moreover, the progestin-induced down-regulation of eNOS was abolished by knockdown of the glucocorticoid receptor using siRNA and also by mifepristone, a potent progesterone and glucocorticoid receptor antagonist. Taken altogether, the present findings indicate that activation of the glucocorticoid receptor rather than the progesterone receptor appears to be the key event in the progestin-induced down-regulation of eNOS. Activation of the glucocorticoid receptor might decrease eNOS expression by reducing both the stability of eNOS mRNA and the activity of the endothelial promoter by decreasing the binding activity of the essential transcription factor GATA (24) . In addition, glucocorticoid receptor activation might also decrease the bioavailability of NO by causing an overproduction of reactive oxygen species (ROS) in endothelial cells, which subsequently scavenge NO (32) .

Endothelial cells play a major role in the maintenance of blood fluidity and in the prevention of prothrombotic responses, in part, by releasing NO. The antithrombotic properties of NO include its ability to inhibit platelet adhesion and aggregation, and to prevent the recruitment of platelets and the endothelial expression of tissue factor, the physiological activator of the coagulation cascade (33 34 35 36 37 38 39) . The present findings indicate that although endothelial cells effectively inhibit platelet aggregation, they were no longer able to do so after their treatment with either MPA, progesterone, or dexamethasone. Similarly, the inhibitory effect of endothelial cells was also not observed after treatment of endothelial cells with the competitive eNOS inhibitor, N{omega}-nitro L-arginine, indicating a major role of NO. In contrast, nomegestrol acetate and levonorgestrel did not impair the antiaggregatory effect of endothelial cells. Altogether, the present findings indicate that progestins can reduce the protective effect of endothelial cells on platelet aggregation and that this effect is most likely due to their ability to activate the glucocorticoid receptor leading to the subsequent down-regulation of the endothelial formation of NO. They also suggest that the reduced ability of endothelial cells to appropriately oppose platelet activation may be a key event in promoting initiation and progression of venous and arterial thromboembolic events after hormonal replacement therapy or oral contraception, including a progestin with partial glucocorticoid activity. In addition, progestins with partial glucocorticoid activity but not those without have also been shown to markedly potentiate the tissue factor-dependent vascular procoagulant effects of thrombin by increasing the availability of thrombin receptors at the smooth muscle (40) . The potential role of glucocorticoid receptor activation in promoting thromboembolic events is also supported by the fact that a twofold increased relative risk of venous thromboembolism and fatal pulmonary embolism is observed in women taking combined low-dose oral contraceptives containing a progestin with partial glucocorticoid activity, such as gestodene and desogestrel, compared to those taking levonorgestrel and estrogen at a similar dose (18 19 20) . It is also consistent with the increased risk of thromboembolism observed in patients under long-term glucocorticoid therapy or with an endogenous hypercortisolism (Cushing’s syndrome) (41) .

In conclusion, the present findings indicate that certain progestins currently used in hormonal replacement therapy, such as MPA, reduce the antiaggregatory effect of endothelial cells by down-regulating the expression of eNOS. They further suggest that activation of the glucocorticoid receptor is responsible for this action and thus might be a key determinant in the induction of a thrombotic state.


   ACKNOWLEDGMENTS
 
This study was supported, in part, by Fondation de France (France) and Théramex (Monaco). The authors thank Drs. C. Gachet, B. Hechler and D. Cassel (Etablissement Français du Sang, Strasbourg, France) for kindly providing washed human platelets.

Received for publication July 11, 2006. Accepted for publication August 14, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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I. C Villar, A. J Hobbs, and A. Ahluwalia
Sex differences in vascular function: implication of endothelium-derived hyperpolarizing factor
J. Endocrinol., June 1, 2008; 197(3): 447 - 462.
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