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Department of Medicine and Experimental Oncology, Hypertension Unit, University of Torino, 10133 Torino, Italy
Address all correspondence and requests for reprints to: Dr. Tracy A. Williams, Department of Medicine and Experimental Oncology, Hypertension Unit, University of Torino, 10133 Torino, Italy. E-mail: tracy.williams{at}libero.it.
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Introduction |
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Spironolactone is a competitive antagonist of aldosterone that is widely employed in the treatment of hypertension and heart failure. The Randomized Aldactone Evaluation Study (RALES) has shown that spironolactone decreases mortality in patients with heart failure at dosages that are ineffective on blood pressure levels (2). It has been postulated that part of its protective effects may be mediated through an improvement of endothelial function (3); in fact, spironolactone has been shown to increase endothelium-dependent vasodilation in patients with heart failure and primary aldosteronism (4, 5). Interestingly, in essential hypertensives with normal aldosterone levels, spironolactone therapy resulted in an improvement in endothelial function (5). However, in addition to binding to the mineralocorticoid receptor (MR), the aldosterone receptor, spironolactone also binds as an agonist to the progesterone receptor (PR) and as an antagonist to the androgen receptor (AR), which causes some side effects of the drug such as development of gynecomastia, breast tenderness, and menstrual irregularities (6). Furthermore, spironolactone displays some antagonist activity toward the glucocorticoid receptor (GR), although its affinity for the GR is two orders of magnitude lower compared with that of the AR (7); as well as, under certain experimental conditions, significant mineralocorticoid agonist activity (8). Recently, a new aldosterone antagonist, eplerenone, has been developed that is selective for the MR in that it exhibits a far lower affinity for both the PR and AR (9, 10).
The strategic location of the endothelium as a protective barrier between circulating blood and all tissues, allows it to detect changes in hemodynamic forces and blood-borne signals and respond by releasing autocrine and paracrine factors to maintain vascular homeostasis. This balance is disrupted in endothelial dysfunction that predisposes the vessel wall to vasoconstriction, leukocyte adherence, platelet activation, thrombosis, vascular inflammation, and atherosclerosis (1).
The endothelium is a critical site for the control of apoptosis during processes such as inflammation, vascular remodeling, and angiogenesis. Although the suppression of endothelial cell apoptosis is required for the maintenance of blood vessel integrity and for angiogenesis, the activation of apoptosis has been suggested to contribute to pathological tissue destruction during vascular injury, inflammation, atherosclerosis, and allograft arteriopathy (11, 12). Endothelial cell perturbing agents, such as inflammatory cytokines, lipopolysaccharides, reactive oxygen species, and oxidized low-density lipoprotein, all induce endothelial cell apoptosis (13, 14). As in other cell types, endothelial cell apoptosis is executed by the activation of the cysteine protease family, the caspases (15). However, in contrast to other cell types, endothelial cells are more resistant to apoptosis being protected by the endothelial synthesis of nitric oxide (NO) which is the key endothelium-derived relaxing factor that plays a pivotal role in the maintenance of vascular tone and reactivity (15).
The aim of this study was to investigate whether spironolactone could exert a protective effect on the endothelium by inhibiting endothelial cell apoptosis; such an inhibitory effect could potentially be a contributing factor to the improvement in endothelial function observed in patients on spironolactone therapy. The presence of both MR mRNA and protein have been demonstrated in human vascular endothelial cells by RT-PCR and immunocytochemistry, respectively (16). We used primary cultures of human umbilical vein endothelial cells (HUVEC) that were induced to undergo apoptosis by serum deprivation, an in vitro simulation of a component of ischemia (17, 18), and we show that spironolactone inhibits caspase-3 activation, cytochrome c release from mitochondria, and poly (ADP-ribose) polymerase (PARP) cleavage in response to serum deprivation, while it increases cell viability. Progesterone induces the same antiapoptotic responses as spironolactone, whereas eplerenone does not; thus raising the possibility that the effects of spironolactone could be due to its agonistic binding to the PR. Furthermore, we show that this effect is independent of NO because the antiapoptotic effect of spironolactone is maintained even in the presence of the NOS inhibitor N-monomethyl-L-arginine (NMA).
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Materials and Methods |
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-dihydrotestosterone, flutamide, or vehicle (all compounds purchased from Sigma-Aldrich, St. Louis, MO, unless otherwise stated) were added for 0–48 h before washing the cells with 2x PBS and incubating for a further 24 h in EBM-2 to induce apoptosis by growth factor and serum deprivation in the presence of the appropriate compound or vehicle used for the pretreatment period. Drugs or steroids were added to culture medium for pretreatment and apoptosis induction periods from 1000-fold concentrated stock solutions in ethanol to give final ethanol concentrations of 0.1%. An exception to this was eplerenone which we found to be soluble in ethanol to a maximum concentration of 2 mM; therefore, to avoid high ethanol concentrations, 1 mM acqueous stock solutions of eplerenone were prepared and ethanol was added to culture medium to a final concentration of 0.1%. In experiments with flutamide, the final ethanol concentration was 0.2%. Under cell culture conditions in the absence of drug or steroid, 0.1 or 0.2% ethanol as the vehicle was added as appropriate. Preliminary experiments of cells cultured in phenol red-free medium followed by induction of apoptosis by serum deprivation also in phenol red-free medium, resulted in the same response to both spironolactone (10 µM) and progesterone (1 µM) in terms of serum-deprivation-induced caspase-3 activity compared with cells cultured in the presence of phenol red (data not shown). Therefore, in all experiments described in this study, cells were grown in medium containing phenol red. Initial experiments showed that the apoptotic-protective effect of spironolactone was observed if the spironolactone was added at the same time as the serum was withdrawn, but only after 48 h (consistent with a long-term, time-dependent effect). The 48-h serum deprivation period resulted in such large amounts of cell death, particularly with vehicle-treated and eplerenone-treated cells, that the method was adapted to maintain at least 48 h total incubation with spironolactone while reducing the serum deprivation period to 24 h; therefore, we incorporated a preincubation period.
Measurement of caspase-3 activity
Caspase-3 activity was measured using a colorimetric assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. In brief, cell extracts (25 µg total protein) were incubated in the presence of the N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA, 200 µM) and the release of the chromophore p-nitroaniline was measured photometrically at 405 nm.
Measurement of cell viability
HUVECs were plated onto six-well plates at a density of 5000 cells/cm2 in EGM-2 medium containing 2% FBS. Cells were grown to confluence before being pretreated for 48 h with spironolactone (10 µM), progesterone (1 µM), eplerenone (10 µM), or vehicle (0.1% ethanol) in EGM-2 medium + 2% FBS. Cell monolayers were then washed with 2x PBS before induction of apoptosis in serum-free, basal medium (EBM-2) for 24 h. Cell viability was assessed by trypan blue exclusion, and counts were performed on triplicate wells in three independent experiments.
Preparation of cytosolic and mitochondrial extracts
Cytosolic and mitochondrial fractions were isolated using the ApoAlert cell fractionation kit (BD Biosciences, Palo Alto, CA) as described in the user’s manual.
Preparation of nuclear protein extracts
Nuclear protein extracts were prepared as described previously (19).
Western blot analyses
For detection of PARP cleavage by Western blot analysis, nuclear protein extracts (20 µg total protein) were separated by 10% SDS-PAGE before transfer to nylon membranes (0.45 µm; Bio-Rad, Hercules, CA). Membranes were probed with a mouse monoclonal PARP antibody (diluted 1:500 in 5% nonfat milk powder dissolved in TBS/0.05% Tween 20) that reacts with both full-length (116 kDa) and cleaved (85 kDa) forms of PARP (clone F-2; Santa Cruz Biotechnology, Santa Cruz, CA). Cytochrome c release from mitochondria to the cytosol was visualized by Western blot analysis of the appropriate cell fraction after separation of 5 µg protein on 12% SDS-PAGE and detection using a rabbit polyclonal antibody to cytochrome c (diluted 1:100; BD Biosciences). Nylon membranes containing resolved cytosolic proteins were reprobed with a rabbit polyclonal actin antibody (Sigma-Aldrich), diluted 1:5000 in 5% BSA, TBS/0.05% Tween 20, whereas nylon membranes containing mitochondrial resolved proteins were stripped (Restore Western blot stripping buffer; Pierce, Rockford, IL), and reprobed with mouse monoclonal antibody to cytochrome oxidase subunit IV (1:500 dilution, BD Biosciences). The appropriate secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology) were diluted 1:5000 and visualized using LumiGLO chemiluminescent substrate (Upstate Cell Signaling Solutions, Lake Placid, NY).
Statistical analysis
SAS V8 software (SAS Institute Inc., Cary, NC) was used for statistical analyses. Data are expressed as mean ± SD and differences between two independent variables were evaluated using a t test or the Mann-Whitney test where appropriate. ANOVA between groups was performed by ANOVA, and the Bonferroni test was used to correct for multiple comparisons. A nonparametric ANOVA using a Kruskal-Wallis test was used to evaluate differences between variables where appropriate. A probability of less than 0.05 was considered statistically significant.
Other analyses
Protein concentrations were determined by the Bradford assay (Sigma-Aldrich). Data are presented as the mean ± SD of at least three separate experiments. For Western blots, a representative blot is shown with a histogram displaying mean band density of at least three blots from three independent experiments ± SD. Band densities on Western blots were quantified using a Gel Documentation system (Gel Doc XR; Bio-Rad) with Quantity One software. Comparisons between values were analyzed using the Student’s t test or by ANOVA. For multiple comparisons, we performed a post hoc Bonferroni test. Differences were considered significant when P < 0.05.
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); the presence of the pan-caspase inhibitor z-VAD-fmk during the 24-h induction of apoptosis period blocked the activation of caspase-3 under all conditions (data not shown). These results are consistent with spironolactone exerting a time-dependent protective effect from apoptosis in HUVECs induced by serum deprivation.
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; 85 ± 0.94%, n = 3, caspase-3 activity relative to controls incubated with vehicle alone) that is within the range of the plasma concentration of spironolactone and its multiple active metabolites in humans on spironolactone therapy [80–391 ng/ml (0.2–0.94 µM)] (10). Spironolactone is a nonselective MR antagonist with affinity for the AR, GR, and PR (10); in contrast, eplerenone is a significantly more selective MR antagonist compared with spironolactone, and at a concentration range of 0.5–10 µM, displayed no inhibitory effect on caspase-3 activation induced by serum deprivation (Fig. 1B). This demonstrates that 1) the apoptotic-protective effect of spironolactone in HUVECs results from its nonselectivity and 2) the effect of spironolactone was not due to antagonism of aldosterone binding to the MR.
Inhibition of caspase-3 by progesterone, hydrocortisone, and dexamethasone, but not by aldosterone and dihydrotestosterone
In addition to the data with eplerenone, a role for the MR in the antiapoptotic effect of spironolactone is further excluded by the observation that high doses of aldosterone have no effect on caspase-3 activity in response to serum withdrawal (Fig. 2A).
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). The stronger protective effect of dexamethasone compared with hydrocortisone was expected due to its higher relative potency in activating the GR. Therefore, under these conditions, the GR mediates an antiapoptotic effect. As spironolactone acts as a GR antagonist, this demonstrates that its effect on caspase-3 activity is not dependent on binding to the GR, although we cannot exclude the possibility of any putative agonist activity of spironolactone under our experimental conditions toward the GR.
Spironolactone binds strongly as an antagonist to the AR (7). Dihydrotestosterone had no effect at both concentrations tested (1 and 10 µM, Fig. 2B). In addition, flutamide, a nonsteroid antiandrogen, did not antagonize the antiapoptotic effect of spironolactone with respect to caspase-3 activity, thus ruling out the possibility of the AR mediating the effects of spironolactone.
Because spironolactone exhibits agonist activity exclusively to the PR, we considered the PR as the most likely candidate to mediate the observed effects. In accordance with this hypothesis, progesterone displayed a concentration-dependent inhibition of caspase-3 activity induced by serum-deprivation in HUVECs (Fig. 2C), achieving a level of inhibition at a concentration of 1 µM, similar to that observed with spironolactone at a concentration of 10 µM (Figs. 1B and 2C; 66.2 ± 2.6% and 65.1 ± 0.75% caspase-3 activity relative to vehicle-treated controls, 0.1% ethanol, in the presence of 1 µM progesterone and 10 µM spironolactone, respectively). This effect of progesterone was significant compared with vehicle-treated controls at the lowest concentration tested of 0.05 µM, that is within the range of peak progesterone levels during the luteal phase [0.006–0.06 µM 2–20 ng/ml)].
Spironolactone and progesterone inhibit mitochondrial cytochrome c release into the cytosol
In mammalian cells, the highly conserved 15 kDa cytochrome c protein is normally localized to the mitochondrial intermembrane space. During apoptosis, cytochrome c is translocated from the mitochondrial membrane to the cytosol where it serves to amplify caspase-3 activation via the formation of an apoptosome, that is, a complex of cytochrome c, Apaf-1, dATP, and procaspase-9. Consequently, caspase-9 is activated, which in turn, processes and activates other caspases, including caspase-3 (22). Because it has been demonstrated previously that cytochrome c is released from the mitochondria to the cytosol in HUVECs in response to serum deprivation (20), we investigated the effect of spironolactone and progesterone on this apoptotic signaling pathway. As shown in Fig. 3A, the withdrawal of 2% serum resulted in cytochrome c release into the cytosol, as demonstrated by Western blotting of cytosolic proteins. The observed that translocation of cytochrome c was unaffected by the presence of eplerenone, whereas it was dramatically reduced in the presence of either spironolactone or progesterone. Additionally, the prevention of cytochrome c release by spironolactone and progesterone was correlated with an increased concentration of cytochrome c in a mitochondrial fraction that contained cytochrome c oxidase subunit IV (COX IV), a marker enzyme for mitochondria localized to the inner mitochondrial membrane (Fig. 3B).
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; ratio of cleaved to uncleaved PARP = 0.50 ± 0.1 and 0.51 ± 0.12 for 10 µM spironolactone and 1 µM progesterone, respectively, compared with a ratio of 1.16 ± 0.14 and 1.07 ± 0.08 for cells incubated with vehicle (0.1% ethanol) or 10 µM eplerenone, respectively].
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), in contrast to the presence of the same concentration of eplerenone that did not significantly increase cell viability. Consistent with a role for the PR, progesterone (1 µM) also significantly increased cell viability compared with vehicle-treated control cells (16.1 ± 2.1%).
The antiapoptotic effect of spironolactone is partially abrogated by mifepristone
To further support the hypothesis that the effect of spironolactone is mediated by the PR, we tried to block its action using the progesterone antagonist mifepristone (RU 486); however, the pharmacology of mifepristone is complex and can act as either an agonist or antagonist (referred to as a type II antagonist) depending on the cell-type and the activation of other signaling pathways (23, 24). In fact, a concentration of 1 µM mifepristone displayed agonist activity to the PR and inhibited caspase-3 activation in response to serum deprivation in contrast to 0.1 µM mifepristone that had no significant effect alone (Fig. 5A). Therefore, because type II antagonists can inhibit progestin induction substoichiometrically (25), we used a lower concentration of mifepristone, that displayed no effect alone, compared with spironolactone and progesterone to antagonize their effects. In agreement with a role for the PR in mediating the protective effect of spironolactone on apoptosis, the lower concentration of mifepristone (0.1 µM) partially antagonized the inhibitory effect of spironolactone (1 µM) on caspase-3 activation in response to serum deprivation (79.8 ± 4.9% caspase-3 activity relative to vehicle-treated controls in the presence of 1 µM spironolactone alone and 91.2 ± 2.8% caspase-3 activity in the presence of 1 µM spironolactone and 0.1 µM mifepristone, n = 5), and similar results were observed with progesterone (66.9 ± 2.1% caspase-3 activity relative to vehicle-treated controls in the presence of 1 µM progesterone alone and 82.7 ± 2.5% caspase-3 activity in the presence of 1 µM progesterone and 0.1 µM mifepristone, n = 5) (Fig. 5A).
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, B and C, respectively). Consistent with the above data obtained with caspase-3, when mifepristone (0.1 µM) was added in the presence of spironolactone (1 µM), the release of cytochrome c to the cytosol was increased significantly compared with the presence of spironolactone alone in response to serum withdrawal; that is, the antiapoptotic effect of spironolactone was partially antagonized by mifepristone (Fig. 5B; spironolactone alone, 0.62 ± 0.03-fold difference in cytochrome c release relative to vehicle medium containing 0.2% ethanol, n = 3, compared with a combination of spironolactone and mifepristone, 0.74 ± 0.02-fold difference, n = 3). Similarly, mifepristone antagonized the inhibition of cytochrome c release by progesterone, thereby causing an increase in cytochrome c release to the cytosol from 0.53 ± 0.02-fold in the presence of progesterone (1 µM) to 0.73 ± 0.04-fold with a combination of both progesterone (1 µM) and mifepristone (0.1 µM).
Further validating these results, mifepristone antagonized the inhibition of nuclear PARP cleavage by spironolactone. With spironolactone alone (1 µM), the ratio of cleaved to uncleaved PARP was 0.71 ± 0.15; whereas in the presence of both spironolactone (1 µM) and mifepristone (0.1 µM), an increased relative amount of the cleaved form was observed (ratio cleaved to uncleaved PARP = 1.44 ± 0.9, n = 4, Fig. 5C); and similarly with progesterone alone (1 µM), the ratio of cleaved to uncleaved PARP was 0.36 ± 0.10 compared with a ratio of 1.34 ± 0.21 in the presence of both progesterone (1 µM) and mifepristone (0.1 µM).
The above data are consistent with a role for the PR in mediating the antiapoptotic effect of spironolactone. However, because mifepristone is also an antagonist of the GR and due to the complex agonist-antagonist behavior of steroids reported under certain experimental conditions, we cannot exclude the possibility that the antiapoptotic effect of spironolactone could be due at least in part to agonist activity to the GR. Nonetheless, agonist behavior of spironolactone to the GR has never been previously described (7).
The protective effect of spironolactone on serum-deprivation-induced apoptosis is independent of NO
Many studies have reported the protective effect of NO on endothelial cell apoptosis (20, 26). Therefore, we hypothesized that spironolactone could increase endothelial NO synthase (eNOS) protein expression thereby increasing NO bioavailability and protecting HUVEC from apoptosis. However, by Western blot analysis of total cell extracts using a specific antibody to eNOS, we found no evidence for increased eNOS expression (data not shown). Furthermore, the NO synthase inhibitor NMA at a concentration of 1 mM did not block the protective effect of spironolactone on HUVEC apoptosis induced by serum deprivation when analyzed by both caspase-3 activation (Fig. 6A) and by PARP cleavage (Fig. 6B). This is consistent with the antiapoptotic properties of spironolactone in HUVECs being independent of NO.
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Discussion |
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In this study, we observed that the nonselective aldosterone antagonist, spironolactone, strikingly protected the cells from caspase-3 activation upon cell starvation by serum deprivation, thereby suggesting that spironolactone was exerting an antiapoptotic effect. In accordance with this hypothesis, inhibition of caspase-3 activation was associated with an increase in cell viability, an inhibition of cytochrome c release from mitochondria to the cytosol, and an inhibition of the cleavage of nuclear PARP. These effects were a result of the nonspecific nature of the action of spironolactone because eplerenone, a selective aldosterone antagonist, did not exhibit such antiapoptotic properties, and aldosterone itself had no effect. Spironolactone can also act as an antagonist to the AR, and to a far lesser degree, to the GR; however, it exhibits agonist activity exclusively to the PR (7). We investigated the effects of androgens, glucocorticoids, and progesterone under our experimental conditions; because dihydrotestosterone did not protect HUVEC from caspase-3 activation in response to serum deprivation, and the nonsteroid antiandrogen flutamide did not antagonize the effect of spironolactone, this ruled-out a role for the AR in mediating an antiapoptotic response. As others have reported in endothelial cells (21), we found that hydrocortisone and dexamethasone exhibited antiapoptotic properties, and therefore, GR antagonism by spironolactone should stimulate apoptosis rather than having a protective effect as we observed. The only mechanism by which the GR could mediate the antiapoptotic effect of spironolactone would be if spironolactone displayed agonist activity to the GR; however, to our knowledge, such an activity has never been described.
This led us to propose that the PR, the expression of which has been demonstrated in endothelial cells (30, 31), could mediate the antiapoptotic effects of spironolactone. In addition to our observation that eplerenone exhibits no such antiapoptotic effect in HUVECs, we provide two further lines of evidence in support of this hypothesis: 1) progesterone, as spironolactone, inhibits caspase-3 activation and increases cell survival in response to serum deprivation, and furthermore, inhibits PARP cleavage and cytochrome c release to the cytosol; 2) mifepristone partially abrogates the antiapoptotic effect of spironolactone with respect to both caspase-3 activity and the release of cytochrome c to the cytosol, and furthermore, mifepristone antagonizes the inhibition of nuclear PARP cleavage by spironolactone. Moreover, previous studies have reported that progesterone can protect against apoptosis in various cell types and tissues, for example, in the human endometrium in the late luteal phase (32), and has also been shown to increase survival of monoblastoid cells undergoing TNF--induced apoptosis (33). Additionally, progesterone inhibits apoptosis in spontaneously immortalized granulosa cells (34) and has been demonstrated to up-regulate expression of the antiapoptotic mitochondrial protein Bcl-2 in human uterine leiomyoma (35) and delay spontaneous neutrophil apoptosis by inhibition of the release of mitochondrial cytochrome c (36).
Spironolactone is rapidly eliminated from plasma; however, several long-lived active metabolites are produced from spironolactone that contribute to its pharmacological activity in man. Canrenone and 6ß-OH-7--thiomethylspirolactone are the major circulating metabolites in human plasma, and both have been shown to bind to MR in animals (10). The plasma concentration of spironolactone and its multiple active metabolites in humans on spironolactone therapy is usually within the range 80–391 ng/ml or 0.2–0.94 µM (10). In this study, the apoptotic-protective effect of spironolactone was most evident at a concentration of 10 µM; however, we also demonstrated that 1 µM spironolactone had a significant antiapoptotic effect on the inhibition of cytochrome c release, nuclear PARP cleavage, and caspase-3 activity; and furthermore, 0.5 µM spironolactone displayed a significant inhibitory effect on caspase-3 activity.
NO has been shown to regulate either the induction of apoptosis in some cell types or the prevention of apoptosis in others. However, in endothelial cells, NO reportedly protects from apoptosis (26, 37) and has been demonstrated to mediate the protective effect of sphingosine 1-phosphate in the prevention of serum-deprived HUVEC apoptotic cell death (20). However, the antiapoptotic effect of spironolactone we have reported in this study appeared to be independent of NO levels because serum deprivation in the presence of the NO synthase inhibitor NMA did not ablate the protective effect of spironolactone in terms of inhibition of caspase-3 activity and PARP cleavage.
It has been proposed that part of the protective effect of spironolactone in the RALES could be due to antagonism of the adverse effects of aldosterone on the endothelium (2, 3), in addition to the antagonism of other deleterious effects of aldosterone on the cardiovascular system such as the profibrotic and proinflammatory effects on heart and vessels (38, 39). We hypothesize that protection against endothelial dysfunction by spironolactone may be partially mediated by PR agonism in addition to MR antagonism, and that inhibition of endothelial apoptosis could be a contributing factor. This effect could act in addition to a reduction of plasminogen activator inhibitor-1 and proinflammatory cytokines and to a reduction of the expression of adhesion molecules (40). In our experimental setting, the concentrations of spironolactone are higher than those used in the RALES; however, our study investigates an acute effect of the drug; whereas in the RALES, the administration was continued for 2 yr. Furthermore, spironolactone is converted in vivo into active compounds (canrenone, 6ß-hydroxy-7-thiomethyl-spirolactone and 7-thiomethyl-spirolactone), which can mediate part of the effect of the drug. Therefore, a comparison between the protective effect observed in vivo and in our in vitro study should be done cautiously. Finally, it should be highlighted that many of the protective effects observed with spironolactone are also observed with eplerenone, both in vitro (7) and in vivo, as reported by the EPHESUS study (Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) (7, 41), thereby demonstrating that the main protective effect of spironolactone is mediated via antagonism of the MR.
In conclusion, we report that spironolactone exerts a protective effect against endothelial cell apoptosis via a mechanism of suppressed cytochrome c release and caspase-mediated signaling, events that appear to be mediated by the agonist activity of spironolactone to the PR.
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Footnotes |
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Author disclosure summary: T.A.W., A.V., A.M., F.V., and P.M. have nothing to declare.
First Published Online February 23, 2006
Abbreviations: AR, Androgen receptor; COX IV, cytochrome c oxidase subunit IV; EBM-2, endothelial basal medium; EBM-2, endothelial growth medium; eNOS, endothelial NO synthase; FBS, fetal bovine serum; GR, glucocorticoid receptor; HUVEC, human umbilical vein endothelial cell; MR, mineralocorticoid receptor; NMA, N-monomethyl-L-arginine; NO, nitric oxide; PARP, poly (ADP-ribose) polymerase; PR, progesterone receptor.
Received October 17, 2005.
Accepted for publication February 13, 2006.
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in endothelial programmed cell death mediated by ionizing radiation and bacterial endotoxin. Blood 86:4184–4193- and lipopolysaccharide-induced apoptotic cell death in bovine glomerular endothelial cells upstream of caspase-3 activation. Br J Pharmacol 127:1633–1640[CrossRef][Medline](q) transgenic mice. Circulation 108:3036–3041-induced apoptosis. FASEB J 13:793–803. Circulation 100:1983–1991This article has been cited by other articles:
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  Y. Wei, A. T. Whaley-Connell, J. Habibi, J. Rehmer, N. Rehmer, K. Patel, M. Hayden, V. DeMarco, C. M. Ferrario, J. A. Ibdah, et al. Mineralocorticoid Receptor Antagonism Attenuates Vascular Apoptosis and Injury via Rescuing Protein Kinase B Activation Hypertension, February 1, 2009; 53(2): 158 - 165. [Abstract] [Full Text] [PDF]
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, P < 0.05 vs. + serum and – serum at 24 and 48 h. B, Cells were pretreated for 48 h with vehicle or either spironolactone (0–10 µM) or eplerenone (0–10 µM) in EGM-2 + 2% FBS, as indicated. Vehicle cells were incubated a further 24 h in the presence or absence of 2% FBS (EGM-2 or EBM-2, respectively); all other groups of cells were incubated with the same pretreatment concentrations of spironolactone or eplerenone in serum-free medium (EBM-2), as indicated. Data are shown relative to caspase-3 activity under conditions of serum deprivation set to 100%. Data represent mean ± SD (n = 3–5). Concentrations are shown in micromolar. *, P < 0.05 vs. 0.5 µM, 1 µM and 5 µM spironolactone; 
