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CARDIOVASCULAR
Research Unit, Hospital Clínico Universitario of Valencia, Valencia, Spain (C.H., M.A.G.-P.); and Departments of Physiology (C.H.), Pediatrics, Obstetrics, and Gynecology (P.J.O., A.C.), and Functional Biology and Physical Anthropology (J.J.T.), University of Valencia, Valencia, Spain
Received June 2, 2005; accepted July 21, 2005.
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Abstract |
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-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17
diol] completely abolished the isoflavone-induced increase in prostacyclin production, COX-2 mRNA expression, and COX-2 protein content. The results clearly support the hypothesis that genistein and daidzein increased HUVEC prostacyclin production through estrogen receptor-dependent mechanism, which involved the enhancement of COX-2 protein and activity.
; Ososki and Kennelly, 2003).
Phytoestrogens have been shown to exert favorable effects on some factors relevant to cardiovascular health, such as the lipid profile or other parameters related to atherosclerosis development and coronary heart disease risk (Clarkson and Anthony, 1998; Ososki and Kennelly, 2003). Less is known about the cardiovascular actions of phytoestrogens that are mediated through direct modification of endothelial physiology. The endothelium is crucial to the modulation of vessel tone and to the control of platelet adhesion and aggregation, two key factors in the initiation and development of atherosclerosis (Ross, 1999). Endothelium, including HUVECs, expresses both types of ER, and , and the actions of estrogens on endothelium have been exhaustively studied (Mendelsohn, 2000). Moreover, clinical and experimental data support the consideration of endothelium as a target for sexual hormones (Mendelsohn, 2002). Although phytoestrogens bind to both ER isoforms, higher affinity has been shown for the ER (Morito et al., 2001).
Endothelial actions are mediated through the release of such vasoactive compounds as prostacyclin, a potent endogenous inhibitor of platelet aggregation and a strong vasodilator. Prostacyclin is a prostaglandin produced from free arachidonic acid through the catalytic activity of two different cyclooxygenases (COXs), termed COX-1 and COX-2. Both COXs 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 it is overexpressed in inflammatory processes (Smith and O'Malley, 2000; Parente and Perretti, 2003).
It has been demonstrated that prostacyclin production by HUVECs is stimulated after exposure to serum from postmenopausal women treated with a mixture of phytoestrogens (Garcia-Martinez et al., 2003). Nevertheless, because the data on the activity of purified phytoestrogens are more convincing than obtained with a mixture, the present study is focused both on the effects of genistein and daidzein on HUVEC prostacyclin production, as well as the role of ER and of the two COXs on the observed effects. Genistein and daidzein were chosen because they are two of the most abundant substances in phytoestrogenic preparations (Ososki and Kennelly, 2003) and because of their different reported actions, since both are active through ER activation but genistein is also a nonselective inhibitor of tyrosine-kinase (Akiyama et al., 1987).
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Materials and Methods |
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). Briefly, HUVECs were grown in 25-cm2 flasks (Orange Scientific, Waterloo, Belgium) in human endothelial cell-specific medium EBM-2 (Cambrex Bio Science Walkersville, Walkersville, MD), supplemented with EGM-2 (Cambrex Bio Science Walkersville), in an incubator at 37°C with 5% CO2. 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-Aldrich, St. Louis, MO). Cells from passages 4 to 6 were seeded 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% confluence, culture medium was exchanged for a phenol red-free Medium 199 (Invitrogen, Carlsbad, CA) supplemented with 20% charcoal/dextran-treated fetal bovine serum (Invitrogen), EGM-2, pyruvic acid, and antibiotics (hormone-free medium) and maintained for 24 h. Then, different concentrations (0.01-100 µM) of genistein or daidzein and other compounds were added. The pure antiestrogen ICI 182780 (1 µM; Biogen, Madrid, Spain), also called Fulvestrant or Faslodex, was used to evaluate whether this production was mediated by ER modulation. The selective COX-1 (0.1 µM SC-560; Cayman Chemical, Ann Arbor, MI) or COX-2 (1 µM NS-398; Cayman Chemical) inhibitors, and the nonselective one, indomethacin (10 µM, Sigma-Aldrich), were used to discern which of two COX isoenzymes was implicated in prostacyclin production.
Assay of Prostacyclin. After different amounts of incubation (1-48 h in Fig. 1, 24 h in Figs. 2 and 3, and in Table 1) with the desired compounds, medium was collected and stored at -20°C until prostacyclin assay. Culture wells were then washed with phosphate-buffered saline, and adherent cells were collected in 0.5 N NaOH solution for protein determination by the modified Lowry method using bovine serum albumin as standard (Lowry et al., 1951).
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The amount of prostacyclin produced, calculated as the concentration of its stable hydrolysis product, 6-keto-prostaglandin F1, was assessed in duplicate by a commercial EIA kit (Cayman Chemical). The production of prostacyclin was expressed as nanograms of prostacyclin per milligram of protein.
Immunoblotting. HUVECs were treated in 25-cm2 flasks for 24 h with the desired products. Then, flasks were washed twice with prewarmed Medium 199. A volume of 150 µl of lysis buffer (0.1% Triton X-100, 0.5% sodium deoxicolic acid, and 0.1% 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. Then, cells were 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 (range, 40-90 µg) were then separated by 10% SDS-polyacrylamide gel electrophoresis, and the protein was transferred to PVDF sheets (PVDF Transfer Membrane Westran; Whatman Schleicher and Schuell, Keene, NH). Immunostaining was achieved using specific antibodies anti-COX-1 (sc-1752; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-COX-2 (cat no. 160107; Cayman Chemical). Development was performed with alkaline-phosphatase-linked anti-goat antibody (for COX-1) or anti-rabbit antibody (for COX-2) (both from Sigma-Aldrich), followed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate, p-toluidine salt color development reaction. Blots were digitalized using a Gelprinter PLUS (TDI, Madrid, Spain), and the densities of spots were analyzed with the program 1-D Manager. Equivalent protein loading and transfer efficiency were verified by staining for -actin (Sigma-Aldrich).
RNA Isolation and Real-Time PCR Assay. Total cellular RNA was extracted using the TRIzol reagent (Invitrogen) following the manufacturer's instructions. Reverse transcription (RT) was carried out using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) with a personal Mastercycler Eppendorf Thermocycler (Eppendorf, Hamburg, 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 DEPC-treated water was substituted) and was used as nontemplate control in real-time PCR experiments.
Primers for quantitative RT-PCR were designed using the Primers Express Software (Applied Biosystems, Fosters City, CA) and synthesized by Custom Primers (Invitrogen). The sequence of the GAPDH sense primer was 5'-CTGCTCCTCCTGTTCGACAGT-3' and that of the antisense primer was 5'-CCGTTGACTCCGACCTTCAC-3' (NCBI no. NM_002046 [GenBank] ), giving rise to an expected PCR product of 100 bp. The COX-1 primers, 5'-TACTCACAGTGCGCTCCAAC-3' for the sense primer and 5'-GCAACTGCTTCTTCCCTTTG-3' for the antisense one (NCBI no. AF440204 [GenBank] ), were designed to amplify a 168-bp PCR product. For COX-2, the primers used were 5'-ATCATAAGCAGGGCCAGCT-3' for the sense primer and 5'-AAGGCGCAGTTTACGCTGTC-3' for the antisense one, and a 101-bp product was expected (NCBI no. D28235 [GenBank] ).
An RT-PCR 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 1/10. 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, TaqDNA polymerase, MgCl2, and reaction buffer with SYBR green; 1.5 µl of each 2.5 µM 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, 3-fold serial dilutions of well known cDNA concentrations were run as calibration curves. After the amplification process was ended, the melting curves program was used to assure that all the amplicons were obtained at the same temperature and to assure there was no amplification of other products. Data were analyzed with the ABI PRISM Sequence Detection version 1.7 analysis software (PerkinElmer Life and Analytical Sciences, Boston, MA). Duplicates showing more than a 5% variation were discarded. To validate an RT-PCR, standard curves with r > 0.95 and slope values between -3.1 and -3.4 were required. The amount 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 bands were purified using a MiniElute PCR Purification Kit (QIAGEN, Valencia, CA) and then sequenced to prove that the amplified products corresponded to previously published COX-1, COX-2, and GAPDH sequences. Agarose gel electrophoreses were also performed to demonstrate that RT-PCR 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 0.5 mg/ml MTT dissolved in phenol red-free Medium 199 for 3 h. Then, the medium was removed by aspiration, and formazan contained in cells was solubilized with 100 µl of dimethyl sulfoxide. 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, Hercules, CA). Results of different treatments were expressed as a relative percentage of formazan produced by cells maintained in phenol red-free Medium 199 without treatments.
Statistical Analysis. Values shown in the text, tables, and figures are means ± S.E.M. ANOVA 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 effects of different concentrations of genistein and daidzein on prostacyclin production are presented in Fig. 2. Both compounds exhibited a similar, dual effect. Prostacyclin production began to increase with concentrations of 0.1 and 1 µM, but higher concentrations did not modify (10 µM daidzein) and even decreased (10 µM genistein and 100 µM of both compounds) prostacyclin production, compared with control values. Moreover, the higher concentrations of 10 and 100 µM decreased prostacyclin production compared with the effects of 0.1 and 1 µM for both phytoestrogens. Experiments were performed to discard the interference of higher concentrations (10 and 100 µM) of genistein and daidzein with the EIA measurement of prostacyclin (data not shown).
To test whether the increased prostacyclin production induced by genistein and daidzein was mediated through ER activation, HUVECs were exposed to the ER antagonist ICI 182780 with or without different concentrations of phytoestrogens (Table 1). Treatment of HUVECs with 1 µM ICI 182780 alone reduced prostacyclin production by 37%. The interference, 1 µM ICI 182780, with the EIA measurement of prostacyclin standards was discarded (data not shown). When cells were exposed to different concentrations of genistein or daidzein, ICI 182780 reduced both genistein- and daidzein-increased prostacyclin production to the same values of ICI 182780 alone.
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Experiments were performed to clarify whether increased prostacyclin production was due to increased enzyme activity or expression. Phytoestrogens did not modify COX-1 protein (Fig. 4A) or mRNA (Fig. 4B) expressions at the tested concentrations. The ICI 182780 alone slightly reduced both the mRNA, as well as the protein content, without altering them in simultaneous incubation with phytoestrogens. Conversely, genistein and daidzein increased HUVEC expression of COX-2 protein (P < 0.05) (Fig. 5A). Those effects were dependent on ER activity since, in experiments with ICI 182780 alone, COX-2 protein content was significantly reduced up to 59% of control values (P < 0.001). Concomitant treatment with ICI 182780 and phytoestrogens reduced COX-2 protein (P < 0.05 versus phytoestrogen-treated cells) to the same levels of ICI 182780 alone. The study of COX-2 mRNA expression revealed a pattern quite similar to that of protein content (Fig. 5B). Phytoestrogens slightly increased COX-2 mRNA expression, but there were no statistical differences with control values. The P values were, respectively, 0.208, 0.068, 0.072, and 0.110 for 0.1 and 1 µM genistein and for 0.1 and 1 µM daidzein. Nevertheless, ICI 182780 alone reduced COX-2 mRNA expression (P < 0.05 versus control values), and coexposure of cells to ICI 182780 and phytoestrogens significantly decreased mRNA expression (P < 0.05 versus treatments with phytoestrogens alone) to the same values of ICI 182780 alone.
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Discussion |
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). Moreover, the stimulated HUVEC prostacyclin production is comparable with that obtained after HUVEC exposure to serum from postmenopausal women treated with a mixture of phytoestrogens (Garcia-Martinez et al., 2003).
Exposure of HUVECs to 10 µM and higher concentrations resulted in unchanged or even reduced prostacyclin production. At that range of concentration, genistein is both a nonselective tyrosine kinase inhibitor, as well as an inhibitor of epidermal growth factor receptor kinase and cAMP-dependent phosphodiesterase (Akiyama et al., 1987; Nichols and Morimoto, 1999). Although we have not tested the hypothesis, it is possible that decreased prostacyclin production with higher genistein doses could be attributed to decreased tyrosine kinase activity. In fact, exposure to high concentrations of genistein, within the range of tyrosine kinase inhibition, has already been demonstrated to inhibit endothelial cell production of prostacyclin. For example, genistein (20-370 µM) dose-dependent inhibited prostacyclin production (EC50 = 50 µM, maximal inhibition 50-60%) in cerebral endothelial cells (Parfenova et al., 1998). In another study, an even more dramatic inhibition (80-85%) was observed in HUVECs exposed to genistein (100 µM), with an undetectable effect for daidzein (Wheeler-Jones et al., 1996). These results were comparable with those obtained in our study (Fig. 2) and were attributed to post-translational modifications of tyrosine residues of COX (Parfenova et al., 1998).
Data obtained with high concentrations of daidzein are more difficult to explain since daidzein is inactive as a tyrosine-specific inhibitor of protein kinase activity, epidermal growth factor receptor kinase, and cAMP-dependent phosphodiesterase (Akiyama et al., 1987; Nichols and Morimoto, 1999), and it can therefore be discarded that the effects observed were due to such enzyme inhibition.
The estrogen receptor antagonist ICI 182780 is an antagonist of both estrogen receptors, with a similar capacity to antagonize the ER and ER (Smith and O'Malley, 2004). ICI 182780 decreased prostacyclin production (Table 1), which can be attributed to an incomplete deprivation of estrogenic compounds in the culture medium. In the same experimental conditions; however, ICI 182780 did not decrease prostacyclin production (Oviedo et al., 2005). ICI 182780 completely abolished the effects of genistein and daidzein on prostacyclin production (Table 1), which strongly supports the hypothesis that phytoestrogens would increase it through an ER-mediated activity. The implication of ER on the observed effects does not necessary mean there was a genomic action since estrogenic compounds also exert non-genomic vascular actions (Simoncini et al., 2004; Klinge et al., 2005). Nevertheless, time course analysis, in which phytoestrogen effects are evident only after 24 h, suggests a genomic effect. The experiments designed to analyze the effect of phytoestrogens on COX expression revealed an increased expression of COX-2 protein without significant changes in mRNA levels (Fig. 5), which could be attributed to the instability of the COX-2 mRNA (Gou et al., 1998). That increased expression can be prevented by an ER blockade, thus confirming that genistein and daidzein increase prostacyclin production via genomic pathways. The ER-dependent genomic stimulation of prostaglandin production by genistein and daidzein has already been demonstrated in other tissues, such as bovine endometrium (Woclawek-Potocka et al., 2005). Nevertheless, genistein concentrations similar to those used in the present study did not modify prostacyclin production in cultured myometrial cells (Korita et al., 2002).
The use of specific inhibitors of COX-1 and COX-2 confirmed previous studies describing the contribution of both enzymes to the basal production of prostacyclin in endothelium (McAdam et al., 1999; Hermenegildo et al., 2005). Therefore, both enzymes act not only as constitutive but also inducible enzymes in that tissue. The use of the nonselective inhibitor indomethacin left a residual production of prostacyclin, which could reflect the release of intracellular prostacyclin produced before the exposure to inhibitors or the existence of other sources of prostacyclin not inhibited by indomethacin. Both concentration of ICI 182780, SC-560, NS-398, and indomethacin, as well as the incubation time were optimized and were suitable to ensure that the drugs effect on endothelial cell prostacyclin release was optimal, as reported earlier (Mikkola et al., 1996; Hermenegildo et al., 2005).
The stimulation of prostacyclin obtained with phytoestrogens is of a similar level to that obtained after exposure of HUVECs to estradiol or progestogens (Mikkola et al., 1995; Hermenegildo et al., 2005), although the concentrations of phytoestrogens needed to reach the same levels are approximately 100 times higher than those for the former steroids.
Data obtained in the present study could contribute to explain part of the already described vascular actions of phytoestrogens. Among these actions, genistein and daidzein have demonstrated an induction of vascular dilation in vivo. For instance, genistein relaxed porcine coronary artery at relatively high concentrations and at a physiologically relevant concentration (3 µM) enhanced nitroprusside-induced relaxation (Lee and Man, 2003). In addition, genistein and daidzein caused endothelium- and concentration-dependent relaxation of preconstricted rat aorta rings. In that case, however, relaxation brought about by genistein and daidzein were not significantly affected by the genomic estrogen receptor antagonist ICI 182780 (10 M) (Mishra et al., 2000).
In conclusion, our results demonstrate that genistein and daidzein stimulate prostacyclin production by HUVECs through ER-mediated mechanisms, mainly involving increased protein content and activity of COX-2.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ER, estrogen receptor; HUVEC, human umbilical vein endothelial cell; PVDF, polyvinylidene difluoride; COX, cyclooxygenase; EIA, enzyme-linked immunoassay; ICI 182780, 7-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17 diol; SC-560, 5-(4-chlorophenyl)-1-(4-methoxypjenyl)-3-(trifluoromethyl)-1H-pyrazole; NS-398, N-[2-(cyclohexyloxy)-4 nitrophenyl]-methanesulfonamide; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NCBI, National Center for Biotechnology Information; MTT, 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Address correspondence to: Dr. Carlos Hermenegildo, Research Unit, Faculty of Medicine and Dentistry, Av. Blasco Ibañez, 17, E 46010 Valencia, Spain. E-mail: carlos.hermenegildo{at}uv.es
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, P < 0.001 versus control values and P < 0.05 versus 0.01 µM daidzein values. 
, P < 0.01 versus control values and P < 0.001 versus 0.1 and 1 µM genistein values. 
, P < 0.01 versus 0.1 µM daidzein values and P < 0.05 versus 1 µM daidzein values. ||, P < 0.001 versus control values and 0.1 µM genistein values and P < 0.05 versus 0.01 and 1 µM genistein values. #, P < 0.001 versus control values and all other daidzein concentration values.
