1Research Unit, Hospital Clínico Universitario of Valencia and Departments of 2 Physiology, 3 Pediatrics, Obstetrics and Gynecology and 4 Functional Biology and Physical Anthropology, University of Valencia, 46010 Spain
5 To whom correspondence should be addressed at: Department of Physiology, Faculty of Medicine and Dentistry, Av. Blasco Ibañez, 17, E 46010 Valencia, Spain. Email: carlos.hermenegildo{at}uv.es
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Abstract |
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Key words: cyclooxygenase/endothelial function/hormones/prostaglandins/vasoactive agents
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Introduction |
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Prostacyclin is a potent endogenous anticoagulant for platelets and a strong vasodilator. Prostacyclin is a prostaglandin produced from free arachidonic acid through the catalytic activity of two different cyclooxygenases (COX), termed COX-1 and COX-2. COX represent the main control mechanism for prostacyclin production. COX-1 is considered to be expressed in a constitutive manner, whereas COX-2 is inducible by mitogens, cytokine growth factors and endotoxins and is over-expressed in inflammatory processes (Smith et al., 2000; Parente and Perretti, 2003
).
Clinical and experimental data support the consideration of endothelium as a target for sexual hormones (Mendelsohn and Karas, 1999). Estrogen receptors have been found in endothelium and estradiol actions have been exhaustively studied (Mendelsohn and Karas, 1999
; White, 2002
). For instance, estradiol enhances endothelial prostacyclin production through both COX-1 (Jun et al., 1998
) and COX-2 (Akarasereenont et al., 2000
) and reduces production of vasoconstrictors, reducing blood pressure (Dubey et al., 2002
).
Much less is known, however, regarding progestogen actions on the vascular wall. Several types of endothelial cells express progesterone receptors (PR) (Vazquez et al., 1999). Progestogens have been shown to regulate many physiological processes that impact on the atherosclerotic progression. For instance, progestogens can inhibit vasorelaxation (White et al., 1995
) and decrease endothelial cell proliferation (Vazquez et al., 1999
). Also, PR can regulate the vascular injury response (Karas et al., 2001
).
Progestogens are a second component of hormone substitution in post-menopausal women, and are recommended in women with uterus to reduce the risk of endometrial cancer. The effects of exogenous administration of progestogens in combination with estradiol on endothelial function are also unclear, given that in some studies, progestogens counteract estradiol's beneficial vascular effects, while in other trials progestogens do not (see reviews Cano, 1999; Dubey et al., 2002
; Ganz, 2002
).
Cardiovascular effects of hormone therapy are presently under discussion (Herrington and Klein, 2003; Kuller, 2003
). A better knowledge of the endothelial effects of two clinically used progestogens, progesterone (the natural progestogen) and medroxyprogesterone acetate (MPA), could contribute to clarify the physiological and clinical effects. Moreover, all progestogens do not have similar effects on vascular tone (Sarrel, 1999
).
The objectives of the work were the following: (i) to study the effects of two progestogens, progesterone and MPA, on prostacyclin production by endothelial cells; (ii) to uncover whether these actions are mediated by PR; (iii) to illuminate whether the observed effects are due to regulation of one or both COX isozymes; and (iv) to test whether progestogens interact with estradiol effects.
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Materials and methods |
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Cells were identified as endothelial by their characteristic cobblestone morphology and the presence of von Willebrand factor by immunocytochemistry using a specific antibody (F-3520; Sigma, Spain).
Cells from passages 46 were seeded onto 6-well plates with fibronectin-treated coverslips for immunocytochemistry, onto 24-well plates for prostacyclin measurements, onto 96-well plates for measurement of cell viability, and onto 25 cm2 flasks for western blot and mRNA isolation. When cells were at 75% of confluence, culture medium was exchanged for a phenol red-free medium 199 (Gibco-BRL, Life Technologies, UK) supplemented with 20% charcoal/dextran-treated fetal bovine serum (Gibco-BRL) and maintained for 24 h. The culture medium was then eliminated and immediately replaced with phenol red-free medium 199. The desired concentrations of progesterone, MPA and estradiol were obtained by successive dilutions of a stock solution (1 mmol/l in ethanol) with phenol red-free medium. The desired concentrations of RU-486, SC-560 and NS-398 were obtained by successive dilutions of a stock solution [10 mmol/l in dimethylsulphoxide (DMSO)] with phenol red-free medium. Therefore, cells were exposed to <0.1% ethanol and/or <0.1% DMSO. Control cells were exposed to the same concentrations of ethanol and/or DMSO as treated cells.
Immunoblotting
HUVEC were treated in 25 cm2 flasks for 24 h with the desired products. Flasks were then washed twice with pre-warmed medium 199. A volume of 150 µl of lysis buffer (0.1% Triton X-100, 0.5% sodium deoxicholate acid, 0.1% sodium dodecyl sulphate (SDS), in 100 ml of phosphate saline buffer containing protease inhibitors: 1 µg/ml leupeptin, 0.5 µg/ml pepstatin and 1 µg/ml bestatin) was added and incubation was maintained at 4 °C for 30 min. Cells were then collected using a cell scraper, boiled for 5 min and sonicated for 10 s. Protein content was measured (Lowry et al., 1951) and samples were frozen at 20 °C until assay.
Equal amounts of protein (ranging 40125 µg) were then separated by 10% of SDSpolyacrylamide gel electrophoresis, and the protein was transferred to PVDF sheets (PVDF Transfer Membrane Westran, Schleicher & Schuell, Germany). Immunostaining was achieved using specific antibodies anti-PR (sc-538; Santa Cruz Biotechnology, USA), anti-COX-1 (sc-1752; Santa Cruz Biotechnology) or anti-COX-2 (cat no. 160107; Cayman Chemical, USA). Development was performed with alkaline phosphatase-linked anti-goat antibody (for COX-1) or anti-rabbit antibody (for PR and COX-2) (both from Sigma), followed with nitroblue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate, p-toluidine salt (BCIP) colour development reaction. Blots were digitized using a Gelprinter Plus (TDI, Spain), and the densities of spots were analysed with the program 1-D Manager. Equivalent protein loading and transfer efficiency were verified by staining for -actin (from Sigma).
Assay of prostacyclin
After incubation with the desired products, medium was collected and stored at 20 °C until prostacyclin assay. Culture wells were then washed with PBS and adherent cells were collected in 0.5 mol/l NaOH solution for protein determination by the modified Lowry method using bovine serum albumin as standard (Lowry et al., 1951).
The amount of prostacyclin produced, calculated as the concentration of stable hydrolysis product, 6-keto-prostaglandin F1, was assessed in duplicate by a commercial enzyme immunoassay kit (Cayman Chemical). The production of prostacyclin was expressed as ng prostacyclin/mg protein.
With the aim of discerning which of two COX isoenzymes was implicated in prostacyclin production, selective COX-1 (0.1 µmol/l SC-560; Cayman Chemical) or COX-2 (1 µmol/l NS-398; Cayman Chemical) inhibitors were added to some wells.
RNA isolation and real-time RTPCR assay
Total cellular RNA was extracted by using the TRIzol® reagent (Invitrogen, USA) following the manufacturer's instructions. RT was carried out using SuperScriptTM First-Strand Synthesis System for RTPCR (Invitrogen) by using a personal Mastercycler Eppendorf Thermocycler (Eppendorf, Germany). One microgram of total RNA was reverse-transcribed to cDNA following the manufacturer's instructions. For each RT, a blank was prepared using all the reagents except the RNA sample (for which an equivalent volume of diethylpyrocarbonate-treated water was substituted) and was used as non-template control in real-time PCR experiments.
Primers for quantitative RTPCR were designed using the Primers Express Software (Applied Biosystems, USA) and synthesized by Custom Primers (Life Technologies, Spain). The sequence of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense primer was 5'-CTGCTCCTCCTGTTCGACAGT-3' and that of the antisense primer was 5'-CCGTTGACTCCGACCTTCAC-3' (NCBI#: NM_002046) giving rise to an expected PCR product of 100 bp. The COX-1 primers were designed to amplify a 168 bp PCR product and these were the following: 5'-TACTCACAGTGCGCTCCAAC-3' for the sense primer and 5'-GCAACTGCTTCTTCCCTTTG-3' for the antisense primer (NCBI#: AF440204). Related to COX-2, primers used were the following: 5'-ATCATAAGGAGGGCCAGCT-3' for the sense primer and 5'-AAGGCGCAGTTTACGCTGTC-3' for the antisense primer, and a 101 bp product was expected (NCBI#: D28235).
RTPCR was performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems) with a heated lid (105 °C), an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. To amplify cDNA, the RT samples were diluted 1/100 and 1/200. In each reaction a total of 1 µl from each RT tube was mixed with 12.5 µl of SYBR Green PCR master mix (Applied Biosystems) containing nucleotides, Taq DNA polymerase, MgCl2 and reaction buffer with SYBR green; 1.5 µl of each 2.5 µmol/l specific primers and double-distilled water were added to a final volume of 25 µl. Each sample was amplified in duplicate for COX-1, COX-2 and GAPDH. In parallel, 5-fold serial dilutions of well-known cDNA concentrations were run as calibration curves. Data were analysed with the ABI Prism 1.7 analysis software (Applied Biosystems). Duplicates showing >5% variation were discarded. To validate a RTPCR, standard curves with r>0.95 and slope values between 3.1 and 3.4 were required. The amounts of COX-1 and COX-2 were normalized to the corresponding values of the housekeeping gene GAPDH to estimate and compare the relative COX-1 and COX-2 expression among samples. Experiments were performed four times.
In some samples, PCR products were purified by using MiniElute PCR Purification Kit (Qiagen, USA) and were sequenced to prove that the amplified bands corresponded to previously published COX-1, COX-2 and GAPDH corresponding sequences. Agarose gel electrophoreses were also performed to demonstrate that RTPCR yielded a unique band.
Cell viability measurement
Cell respiration, an indicator of cell viability, was assessed by mitochondrial-dependent reduction of 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan. Experiments were performed in parallel and with the same protocol described for prostacyclin production, but were performed in 96-well plates. At the end of each experiment, medium was discarded and cells were incubated with 0.1 mg/ml MTT dissolved in phenol red-free medium 199 for 3 h. Medium was then removed by aspiration and formazan contained in cells was solubilized with 100 µl DMSO. The extent of reduction of MTT to formazan was quantified through the measurement of optical density at 540 nm by using a microplate reader (Bio-Rad, USA). Results were expressed as relative percentage of formazan produced by cells maintained in phenol red-free medium 199 without treatments.
Statistical analysis
Values are reported as means ± SEM. Repeated-measures analysis of variance test was applied for comparisons of means, and then Student's t-test was performed. P<0.05 was considered significant.
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Results |
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The physiological interaction between estradiol and progestogens on prostacyclin production was tested by exposing cells to a physiological concentration of estradiol and either progesterone or MPA (Figure 8). Treatment of cells with 1 nmol/l estradiol alone increased prostacyclin production by 25% (P<0.005 versus control values). When used in combination with progestogens, the amount of prostacyclin released was the addition of the effects of estradiol (25%) and progestogens (56 and 55% for progesterone and MPA alone respectively). In both cases, prostacyclin production induced by co-exposure to estradiol and progestogens was significantly higher than by exposure to estradiol alone (P<0.05).
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Discussion |
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Our results confirm previous reports regarding the presence of both forms of PR on cultured endothelial cells (Vazquez et al., 1999). Nevertheless, PRB expression is much less detectable than PRA expression, which is in contrast with data from previous studies. For instance, some authors have obtained similar levels of protein expression for PRA and PRB in other types of human endothelial cells (Vazquez et al., 1999
). Also, mRNA expression studies demonstrated a high expression of the PRB isoform in HUVEC (Tatsumi et al., 2002
). The relative roles of PRA and PRB tend to support the view that PRB is the active PR, whereas PRA is either inactive or acts as an inhibitor of PRB activity. However, both isoforms are often co-expressed in normal physiology and exert both cooperative actions and distinct activities (Graham and Clarke, 2002
).
Progesterone increased prostacyclin production in a dose-dependent manner. Physiological concentrations of progesterone were used in the present study, since in the follicular phase, plasma progesterone levels are 0.150.70 ng/ml (corresponding to 0.52.2 nmol/l) and in the luteal phase are 2.0025.0 ng/ml (corresponding to 6.479.5 nmol/l). Nevertheless, the use of the same concentrations of MPA resulted in an increased, although not dose-dependent, prostacyclin production. The explanation for that difference remains elusive, although the wide range of values could be the main reason, since there is a tendency to increase prostacyclin production with increased MPA concentrations.
Increased prostacyclin production induced by progestogens supports an active role for these hormones on endothelial function, thus discarding previous reports in which it has been denied (Lewis et al., 1986; Mueck et al., 2002
). Furthermore, the production of prostacyclin by HUVEC after exposure to progesterone or MPA is comparable to that afforded by physiological concentrations of other steroid hormones such as estradiol (Mikkola et al., 1995
, 1996b
; Akarasereenont et al., 2000
).
Time-course analysis reveals a considerable latency required for the increased prostacyclin production, as progesterone and MPA effects are evident only after 24 h of incubation, thus suggesting a genomic effect. The use of the antiprogestogen RU-486 at a dose within the adequate range to ensure PR antagonism (Xu et al., 2002) permits the ascription of the observed effects to PR. RU-486 alone had no effect on basal prostacyclin synthesis by HUVEC, as has already been demonstrated in pulmonary artery endothelial cells (Jun et al., 1999
). When used in combination with progesterone or MPA, RU-486 completely abolished their effects on prostacyclin production. Taken together, data from Figures 2 and 4 indicate that progesterone and MPA enhanced prostacyclin release which is, at least in part, a genomic action mediated through PR activation.
Nevertheless, other mechanisms cannot be completely refuted. For instance, a rapid (within 5 min) stimulation of prostacyclin production by physiological concentrations of progesterone (1100 nmol/l) in rat aorta strips has recently been reported, suggesting a non-genomic mechanism. The possible contribution of PR, however, has not been studied (Selles et al., 2002).
It is generally believed that COX-1 is ubiquitously expressed (termed as constitutive isoform), whereas COX-2 is induced by mitogens, growth factors, bacterial endotoxin, and cytokines (inducible COX) (Parente and Perretti, 2003). Nevertheless, in endothelium [and other tissues, such as uterus (Kim et al., 1999
)], things may not be so simple, and both COX shared characteristics of constitutive and inducible enzymes.
Data from Figures 5 and 6 demonstrated a basal expression of COX-1 and COX-2, suggesting a constitutive component of COX-dependent prostacyclin production. A careful consideration should be done, however, since other factors, including cytokines, growth factors, etc., may be regulating this level and have not been identified in the present work.
Our results demonstrated a PR-mediated induction of both enzymes after exposure to progesterone or MPA, measured as protein content and as mRNA expression (Figures 5 and 6). These results suggest that COX mRNA in endothelium after exposure to progesterone is highly correlated with protein expression levels, indicating a regulation at the mRNA level (Habermehl et al., 2000). Moreover, the use of COX-1 and COX-2 inhibitors (Figure 7) revealed not only that basal production of prostacyclin by HUVEC was equally mediated through both enzymes, but also that both inhibitors similarly abolished progestogen-increased prostacyclin production. Taken together, results from Figures 5
7 support a role for COX-1 and COX-2 in both basal- and progestogen-induced prostacyclin production.
Therefore, it seems that COX-2 is, at least in part, a constitutive enzyme in HUVEC, and COX-1 an inducible one. Consistent with this role of COX-2, clinical studies with celecoxib, a selective inhibitor of COX-2, have shown that this enzyme exerts control of most systemic prostacylin production in healthy humans (McAdam et al., 1999).
In addition, COX-1 induction in cultured endothelial cells has previously been reported. As stated in the Introduction, estradiol augments the protein content of both COX-1 (Jun et al., 1998) and COX-2 (Akarasereenont et al., 2000
). Agreeing with our data, 10 day treatment with progesterone increases COX-1 expression in ovine renal artery endothelium (Rupnow et al., 2002
). Also, pregnancy (a state of both high estrogen and progesterone) increases both COX-1 protein and mRNA expression in endothelial cells from sheep (Janowiak et al., 1998
). The dependence of COX-1 on progesterone and PR activity has been shown in baboon endometrium, where COX-1 expression was completely inhibited by the use of an antiprogestogen (Kim et al., 1999
).
Our data could have important clinical and therapeutic implications, taking into account that progestogens are currently and frequently used as a second component of hormonal substitution in post-menopausal women. On the one hand, progestogens are thought to have a neutral effect or even to counteract the estradiol effects on several cardiovascular and haemostatic parameters, such as blood pressure, vascular tone, lipid profile, fibrinogen plasma concentration or the fibrinolytic system (Winkler, 1999; Kawano et al., 2001
; Dubey et al., 2002
; Ganz, 2002
). Related to prostacyclin, progestogens counteract estrogen-induced prostacyclin release in endothelial cultured cells exposed to serum or plasma from post-menopausal women in some studies (Mikkola et al., 2000
), but not others (Mikkola et al., 1996a
). Results of the present study support an active role for progestogens per se on endothelium-dependent vasodilator production. Moreover, progestogens do not counteract estradiol-induced prostacyclin production and in fact both hormones have an additional effect (Figure 8). Reported differences in the progestogen used or in the design of the experiments seem crucial to an adequate understanding of those results.
On the other hand, and related to the foregoing, our study reveals an equal action for progesterone and MPA. Despite the differences between them on lipid metabolism (Writing Group for the PEPI Trial, 1995), blood pressure (Dubey et al., 2002
), vascular tone (Minshall et al., 1998
), experimental atherosclerosis (Adams et al., 1997
), or HUVEC expression of vascular cell adhesion molecule-1 (VCAM-1) (Otsuki et al., 2001
), with a more prejudicial profile for MPA, both progestogens behaved similarly in every parameter assayed in our study.
Moreover, results of the present work cannot explain the differences observed in the Women's Health Initiative (WHI) clinical trial, which showed that there were no cardioprotective effects of estradiol + MPA, as judged by comparing the number of adverse cardiovascular events in the estradiol + MPA group with that in the placebo group (Rossouw et al., 2002). As stated before, there were no differences between MPA and MPA + estradiol-induced prostacyclin production (Figure 8).
Nevertheless, the possible benefits of increased prostacyclin production by progestogens should be carefully considered. It seems clear that the primary effects of prostacyclin should be to induce vasodilatation, but enhanced expression and activity of both COX could also increase the production of other prostanoids with proaggregatory and vasoconstrictor activity, such as thromboxane A2 or prostaglandin F2 (Ospina et al., 2003
). In that case, vasodilator and vasoconstrictor effects should be unbalanced, and possible pathological mechanisms promoted.
The reported COX stimulation by progestogens could have other consequences. For instance, it could have an important role on embryo implantation, since estradiol promotes uterine vascular permeability whereas progesterone stimulates uterine angiogenesis during pregnancy (Ma et al., 2001), probably through COX-2-derived prostaglandins (Matsumoto et al., 2002
).
In conclusion, our results demonstrate that progesterone and MPA stimulate prostacyclin production by HUVEC through PR-mediated mechanisms, probably involving both COX-1 and COX-2 enzymes, without counteracting the effects of estradiol.
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Acknowledgements |
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Submitted on October 13, 2004; resubmitted on January 14, 2005; accepted on January 19, 2005.