In the past decades observational and mechanistic studies1,2 have supported the concept that the administration of hormone therapy (HT) to postmenopausal women is associated with protection from heart disease. However, major randomized clinical trials have subsequently failed to confirm these alleged advantages.3 The reason for this discrepancy is one of the greatest conundrums in modern medicine, and studies are currently undergoing to understand whether differences in genetic background, timing of HT initiation after the menopause, dosages or ways of administration, or other variables may influence the cardiovascular effects of HT.4
Recently, the publication of the conjugated equine estrogens–only arm of the Women’s Health Initiative trial has highlighted another challenge in this complex arena. Indeed, although in younger women receiving conjugated equine estrogens a significant benefit for combined cardiovascular outcomes was found,5 in the age-matched women treated with conjugated equine estrogens plus medroxyprogesterone acetate no advantage in coronary heart disease incidence was seen,3 supporting the hypothesis that the use of medroxyprogesterone acetate may have some specific effects related to the development of cardiovascular disease.
Along with this finding, growing evidence suggests that each progesterone receptor ligand may have specific cellular effects, and this seems to be true also in vascular cells. For example, detrimental effects of medroxyprogesterone acetate on coronary vasomotion6 and on arterial remodeling7 have been described in female monkeys, where natural progesterone has neutral effects.8 In addition, medroxyprogesterone acetate does not share the positive actions of progesterone on exercise-induced myocardial ischemia in humans.9
Endothelial cells are key regulators of vessel function. The lack of correct endothelial function results in impaired vascular dilatation and enhanced atherogenesis.10,11 Although estrogen preserves endothelial function in vitro and in vivo,1,2 progesterone or other synthetic progestins show variable effects. For example, natural progesterone, unlike medroxyprogesterone acetate, has positive effects on endothelial cell NO production and leukocyte adhesion molecule expression when administered alone; moreover, it enhances the positive effects of estrogens on these measures.12–14 We have previously shown that when given to human endothelial cells, natural progesterone and medroxyprogesterone acetate result in the recruitment of different patterns of intracellular signaling mediators through the differential modulation of progesterone receptors or through the interference with other steroid receptors, such as the glucocorticoid receptor.14 In support of these observations, different effects of progesterone and medroxyprogesterone acetate on neurons have been described.15,16
Nomegestrol acetate is a progestin that does not bind to the androgen receptor and, hence, does not exert clinical androgenic side effects.17 Nomegestrol acetate stimulates angiogenesis at high doses18 and has less detrimental effects on cardiovascular risk factors and peripheral insulin sensitivity in comparison with medroxyprogesterone acetate.19 However, the vascular actions of nomegestrol acetate are not established and no comparison is available with the effects of other gestagens.
In this study we compared the effects of nomegestrol acetate with those of natural progesterone or medroxyprogesterone acetate on human endothelial cell function. In particular, we comparatively studied the effects of these progestins on nitric oxide (NO) synthesis and on endothelial nitric oxide synthase (eNOS) activity and expression, with a focus on signal transduction.
MATERIALS AND METHODS
The cells used in the study were primary human endothelial cells extracted as previously described20 from umbilical veins obtained from normal pregnancies delivered at term. The newborn were either male or females, and the umbilical veins were chosen randomly. The cells were used after two to three passages, and the different experiments were repeated for confirmation using cells derived from different individuals. Approximately 40–50 different umbilical veins were used to derive cells that were subsequently grown in culture and treated according to the experimental protocol. Before treatments, human endothelial cells were kept 48 hours in Dulbecco’s Modified Eagle Medium containing steroid-deprived fetal bovine serum. Before experiments investigating nontranscriptional effects, human endothelial cells were kept in Dulbecco’s Modified Eagle Medium containing no fetal bovine serum for 8 hours. Whenever an inhibitor was used, the compound was added 30 minutes before starting the treatments.
Endothelial nitric oxide synthase activity was determined as conversion of [3H]arginine to [3H]citrulline in endothelial cell lysates. [3H]Citrulline was separated using an acidic ion-exchange resin, as previously described.21 Extracts incubated with N-Nitro-L-Arginine Methyl Ester (1 mM), served as blank.
Nitric oxide production was determined by a nitrite assay using 2, 3 diaminonaphtalene.22 Fluorescence of 1-(H)-naphtotriazole was measured with excitation and emission wavelengths of 365 nm and 450 nm. Standard curves were constructed with sodium nitrite. Nonspecific fluorescence was determined in the presence of NG-mono-methyl-L-arginin (3 mM).
Cells were washed with cold phosphate buffered saline and incubated in ice-cold lysis buffer consisting of 0.1% sodium dodecyl sulfate, 1% Igepal CA-630 (nonionic, nondenaturing detergent), 0.2mM phenylmethylsulfonylfluoride, and 0.01‰ protease inhibitor mixture (Sigma) for 30 minutes at 4°C. Cell lysates were centrifuged at 12,000g for 10 minutes, and the concentration of protein in the supernatant was determined by the bicinchoninic acid protein assay (Sigma). Twenty micrograms of total protein from whole-cell lysates were separated under reducing and denaturing conditions by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane (Millipore). Nonspecific binding sites were blocked with 5% skim milk in phosphate buffered saline containing 0.05% Tween 20 (PBS-Tween) for 30′ at room temperature. Membranes were then incubated with anti eNOS (Transduction Laboratories, Lexington, KY), wild type or Tyr204-P-ERK 1/2 (Calbiochem, San Diego, CA), wild type or Thr308-P-Akt (Upstate Biotechnology, Lake Placid, NY) overnight at 4°C. Membranes were then incubated in horseradish peroxidase-conjugated secondary Ab for 1 hour at room temperature, and results were visualized by enhanced chemiluminescence. Institutional review board approval is not required by the University of Pisa for studies on isolated cells.
All reported values are expressed as mean plus or minus standard deviation. Differences between mean values were determined by analysis of variance, followed by Fisher’s protected least significance difference using the SPSS 11.5 Software (SPSS Inc., Chicago, IL).
Nomegestrol acetate administration for 48 hours increased NO synthesis (Fig. 1A) and eNOS activity (Fig. 1B) in human endothelial cells in a concentration-dependent manner. Increased NO synthesis and eNOS activity were coupled to enhanced eNOS expression that increased with increasing nomegestrol acetate concentrations (Fig. 1C). Furthermore, nomegestrol acetate increased NO synthesis (Fig. 2A) and eNOS activity time-dependently, starting as early as after 1 hour (Fig. 2B). Endothelial nitric oxide synthase expression was also induced in a time-dependent manner (Fig. 2C); however, a significant increase in eNOS protein levels was seen only after 8 hours of treatment. Thus, the earlier enzymatic eNOS activation induced by nomegestrol acetate may be related to rapid functional modulation of the enzyme through nontranscriptional pathways.13
Human endothelial cells were treated for 48 hours with progesterone (progesterone 10 nM), medroxyprogesterone acetate (medroxyprogesterone acetate 10 nM) or nomegestrol acetate (10 nM), either alone or in the presence of the mixed progesterone receptor/glucocorticoid receptor antagonist RU-486 (RU-486 10 μM). Nomegestrol acetate and progesterone administration to human endothelial cells was associated with comparable increases of NO synthesis (Fig. 3A) and eNOS activity/expression (Fig. 3B-C). On the opposite, medroxyprogesterone acetate had no effect on NO synthesis (Fig. 3A) and eNOS activity/expression (Fig. 3B-C). RU-486 prevented the effects of nomegestrol acetate and progesterone, indicating that nomegestrol acetate, like progesterone, signals to eNOS through progesterone receptor recruitment.
Additionally, human endothelial cells were treated with 17β-estradiol (E2, 1 nM) for 48 hours, either alone or in the presence of nomegestrol acetate 10 nM, progesterone (10 nM), or medroxyprogesterone acetate (10 nM). The administration of E2 resulted in a strong increase in NO release by human endothelial cells (Fig. 4A), due to enhanced eNOS activity (Fig. 4B) and expression (Fig. 4C). The addition of progesterone or nomegestrol acetate to E2 did not lead to significant changes in any of the parameters (Fig. 4A-C), whereas coadministration of medroxyprogesterone acetate significantly interfered with E2 effects, reducing the amount of NO synthesized by human endothelial cells due to reduced eNOS activation and expression (Fig. 4A-C). Addition of RU-486 to E2 plus nomegestrol acetate did not impair NO induction or eNOS activation or expression (Fig. 4A-C). Overall, these findings suggest that the administration of selected progestins might differently alter endothelial cell nitric oxide synthesis.
Nitric oxide production is modulated by steroids through nongenomic activation of eNOS.13,23–25 Because nomegestrol acetate-induced increases in NO release and eNOS activity were visible after 1 hour of treatment, when eNOS expression was not yet increased, we characterized this rapid action exposing endothelial cells for 30 minutes to increasing nomegestrol acetate concentrations. Nomegestrol acetate rapidly induced increases of NO synthesis (Fig. 5A) and eNOS activity (Fig. 5B) in a concentration-dependent manner. This indicates that this progestin does not simply regulate endothelial cells through the modulation of gene expression, but also through inducing rapid changes of endothelial enzymes function.
When rapid treatments (30 minutes) of human endothelial cells were performed with progesterone (10 nM), medroxyprogesterone acetate (10 nM), or nomegestrol acetate (10 nM), alone or in the presence of the mixed progesterone receptor/glucocorticoid receptor antagonist RU-486 (10 μM), divergent effects were observed. Indeed, nomegestrol acetate and progesterone were associated with similar inductions of NO synthesis (Fig. 6A) and eNOS activity (Fig. 6B) whereas medroxyprogesterone acetate was ineffective (Fig. 6A and B). The RU-486 prevented the effects of nomegestrol acetate and progesterone, indicating that these rapid, nongenomic actions depend on progesterone receptor recruitment. In support of the hypothesis that short-term administration of progesterone and nomegestrol acetate activates eNOS independently of modulations of gene expression, no changes in eNOS expressions were observed at this time-point (Fig. 6C).
To determine the effects of the administration of nomegestrol acetate on the rapid signaling of estrogen to eNOS, we also treated for 30 minutes human endothelial cells with E2 (1 nM) alone or in the presence of progesterone (10 nM), medroxyprogesterone acetate (10 nM), or nomegestrol acetate (10 nM), in the presence or absence of RU-486 (10 μM). Progesterone and nomegestrol acetate enhanced the synthesis of NO induced by E2 (Fig. 7A). On the contrary, the coadministration of medroxyprogesterone acetate with E2 resulted in a significant reduction of the effect of E2 (Fig. 7A). These modifications were due to changes in eNOS activity (Fig. 7B). The effects of the addition of nomegestrol acetate to E2 were prevented by RU-486 (Fig. 7A and B), indicating that activation of progesterone receptor results in an activation of eNOS that is synergic with the action of ER. Overall, these findings suggest that the rapid actions of progestins in endothelial cells are significantly diverse depending on the compound, which is of interest in the clinical setting.
To check for the molecular basis for the different effects on eNOS activation of the progestins we treated human endothelial cells with progesterone (10 nM), medroxyprogesterone acetate (10 nM) or nomegestrol acetate (10 nM), in the presence or absence of RU-486 (10 μM) and checked for the intracellular activation of the mitogen-activated protein kinases (MAPK) ERK 1/2 and of the serine or threonine kinase Akt. After 5 minutes of exposure nomegestrol acetate and progesterone significantly induced the phosphorylation of ERK 1/2 (Fig. 8A) whereas on the opposite medroxyprogesterone acetate did not (Fig. 8A). In parallel, upon 30 minutes of treatment nomegestrol acetate and progesterone induced the phosphorylation of the PI3K downstream effector, Akt (Fig. 8B). Again, medroxyprogesterone acetate had no effect on this target (Fig. 8B). RU-486 completely blocked the activation of both ERK 1/2 and Akt phosphorylation by nomegestrol acetate, confirming that progesterone receptor recruitment is required for these processes.
To assess the effects of the addition of the different progestins on estrogen-induced nongenomic activation of MAPK-dependent pathways, we treated human endothelial cells with 17β-E2 (E2 10−8M), alone or in the presence of progesterone (10 nM), medroxyprogesterone acetate (10 nM), or nomegestrol acetate (10 nM). The coadministration of E2 and progesterone or nomegestrol acetate resulted in enhanced ERK 1/2 phosphorylation with respect to E2 only (Fig. 9). Medroxyprogesterone acetate, on the contrary, significantly interfered with the effect of E2, resulting in reduced ERK 1/2 activation (Fig. 9). Overall, these findings indicate that the different functional regulation of endothelial cells observed with each progestin can be explained by differences in the recruitment of important intracellular signaling pathways.
The data presented herein indicate that nomegestrol acetate stimulates nitric oxide synthesis, eNOS activity, and eNOS expression in human endothelial cells by transcriptional and nontranscriptional pathways and that these effects occur through progesterone receptor. The endothelial effects of nomegestrol acetate are similar to those of natural progesterone and they significantly differ from those induced by medroxyprogesterone acetate. In addition, nomegestrol acetate does not impair the estrogen-dependent induction of eNOS expression, and actually enhances the effects of estrogens on eNOS through nongenomic signaling.
The discrepancy of biologic effects of different progesterone receptor ligands in human cells is in agreement with previous evidence coming from our group14 as well as from other laboratories15,16 and strengthens the concept that synthetic progestins have very different actions in nonreproductive tissues. These differences stem from the peculiar ability of progestins to bind other steroid receptors and therefore to act through activation or interference with these receptors’ signaling.26 Our findings also indicate that progestins result in divergent signaling of progesterone receptors, therefore adding another degree of complexity to the already complicated set of genetic, biochemical, and molecular aspects of progesterone receptor function in human cells.27
The variable regulation of eNOS expression and activity in endothelial cells provides a good example of the composite effects of progestins. The identification of the molecular events that determine the differential regulation of eNOS expression by nomegestrol acetate or medroxyprogesterone acetate are beyond the scope of this manuscript. However, it may be hypothesized that progesterone receptors, depending on the specific ligand engaging the hormone binding pocket, could be differently able to interfere with transcription factors regulating eNOS expression, such as Sp1 and GATA,28 thus leading to divergent effects on eNOS expression. Interference with this important transcription factor could also explain the differential effects of medroxyprogesterone acetate, progesterone, or nomegestrol acetate on estrogen-dependent eNOS induction, because estrogen activates eNOS expression through regulation of Sp1.29,30
On the same line, our findings indicating differences among progesterone, nomegestrol acetate, and medroxyprogesterone acetate on rapid progesterone receptor signaling to MAPK and PI3K pathways may be explained by specific conformations of progesterone receptor. Indeed, most of the nontranscriptional actions of steroid receptors depend on protein–protein interaction at the cell membrane or inside the cytoplasm.13 As happens for estrogen receptor ligands,31 it is likely that the structural conformation of progesterone receptor induced by distinct progestins may differ, and that only selected conformations may activate the Src/ERK cascade or PI3K.13
On the other side, the clinical effects of the different synthetic progestins on the cardiovascular system are not yet established. A growing literature suggests the existence of significant differences on the modification of measurable cardiovascular risk markers, such as lipids26 and C-reactive protein32 as well as on in vivo experimental systems, such as on angiogenesis18 and atherogenesis in animals6,8,33–35 and humans.9,36
These findings have been corroborated recently by the difference in coronary heart disease found in the two arms of the Women’s Health Initiative trial, indicating a protection from heart disease in younger women receiving only conjugated equine estrogens,5 but not in those receiving conjugated equine estrogens plus medroxyprogesterone acetate.3
Although these results obtained in vitro in cells derived from umbilical veins may not represent what happens in coronary artery–derived endothelial cells, nor can they depict properly what happens in a complex living organism, our results on endothelial nitric oxide synthesis regulation are in agreement with previous data indicating that nomegestrol acetate does not antagonize the vasodilatory effects of estrogens in monkeys.34,37
Nomegestrol acetate also differs from other progestins in its effects on relevant metabolic mediators, such as insulin. Indeed, nomegestrol acetate administration to ovariectomized monkeys results in a partial opposition to the improvement in insulin sensitivity associated with estrogen treatment,19 but this effect is less marked than that of medroxyprogesterone acetate.35 However, when nomegestrol acetate is administered to fertile women, it does not alter glucose or insulin levels during oral glucose tolerance tests.38 Finally, treatment with hormonal preparations containing nomegestrol acetate is associated with reduced concentrations of total and low-density lipoprotein-cholesterol and lipoprotein (a)39 and with reduced circulating C-reactive protein.32
In conclusion, these data support the concept that progesterone and the different synthetic progestins are characterized by unique actions on vascular cells that depend on specific signaling events being recruited by the various steroids. The effects of nomegestrol acetate on human endothelial cells resembles that of natural progesterone, being associated with enhanced synthesis of nitric oxide through both transcriptional and nontranscriptional actions that partially add to the effects of estrogens. These results add to the current understanding of how progestins act on the cardiovascular system and help to interpret the results coming from clinical trials.
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