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Am J Physiol Endocrinol Metab 292: E585-E593, 2007. First published October 3, 2006; doi:10.1152/ajpendo.00413.2006
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Mitochondria play an important role in 17beta-estradiol attenuation of H2O2-induced rat endothelial cell apoptosis

Ailing Lu,* Michael Frink,* Mashkoor A. Choudhry, William J. Hubbard, Loring W. Rue, III, Kirby I. Bland, and Irshad H. Chaudry

Center for Surgical Research and Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama

Submitted 10 August 2006 ; accepted in final form 28 September 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Studies have shown salutary effects of 17beta-estradiol following trauma-hemorrhage on different cell types. 17beta-Estradiol also induces improved circulation via relaxation of the aorta and has an anti-apoptotic effect on endothelial cells. Because mitochondria play a pivotal role in apoptosis, we hypothesized that 17beta-estradiol will maintain mitochondrial function and will have protective effects against H2O2-induced apoptosis in endothelial cells. Endothelial cells were isolated from rats' aorta and cultured in the presence or absence of H2O2, a potent inducer of apoptosis. In additional studies, endothelial cells were pretreated with 17beta-estradiol. Flow cytometry analysis revealed H2O2-induced apoptosis in 80.9% of endothelial cells; however, prior treatment of endothelial cells with 17beta-estradiol resulted in an ~40% reduction in apoptosis. This protective effect of 17beta-estradiol was abrogated when endothelial cells were cultured in the presence ICI-182780, indicating the involvement of estrogen receptor (ER). Fluorescence microscopy revealed a 17beta-estradiol-mediated attenuation of H2O2-induced mitochondrial condensation. Western blot analysis demonstrated that H2O2-induced cytochrome c release from mitochondrion to cytosol and the activation of caspase-9 and -3 were decreased by 17beta-estradiol. These findings suggest that 17beta-estradiol attenuated H2O2-induced apoptosis via ER-dependent activation of caspase-9 and -3 in rat endothelial cells through mitochondria.

cytochrome c; estrogen receptors; mitochondria

17beta-ESTRADIOL, ONE OF THE ISOFORMS OF ESTROGEN, has been intensely studied for its protective effects on a number of nonreproductive tissues, including the cardiovascular system, immune cells, brain, bone, and others (2, 14, 27, 28, 36). Previous studies from our laboratory have shown that administration of 17beta-estradiol following trauma-hemorrhage restored the depressed cardiac, hepatocellular, and immune functions (2, 27). Studies have also demonstrated that the effects of estrogen are mediated via estrogen receptors (ER). The two subtypes of ER are estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor-beta (ERbeta). Several studies reported that, in human umbilical vein and coronary vessels, endothelial cells (EC) express functional ERs (8, 20, 38). We have recently found that 17beta-estradiol significantly enhanced the expression of ER{alpha} and ERbeta in rat aorta following trauma-hemorrhage (unpublished observation). Because vascular endothelium is critically interposed between circulating blood and other tissues, studying EC is likely to provide information about the interaction between the intra- and extravascular components.

Studies from our laboratory have shown that endothelium-dependent relaxation is depressed following sepsis (40). Studies have also shown that, during inflammation, vascular endothelium becomes the target of attack by the activated macrophages and neutrophils (30, 42, 43). One of the cytotoxic agents produced by these activated cells is H2O2 (3, 29). The local increase in H2O2 concentration causes apoptosis in EC and plays a key role in tissue injury under conditions such as trauma and sepsis (12).

Apoptosis is essential for normal development of different organs in mammalians. However, abnormally high levels of apoptosis are linked to many degenerative diseases. Although the anti-apoptotic effect of 17beta-estradiol on apoptosis in EC has been described (1), the exact mechanism by which 17beta-estradiol prevents apoptosis is not yet clear. Because mitochondria play a central role in the regulation of apoptosis (4) and recent studies have shown that ER{alpha} and ERbeta are present in mitochondria of different cell types and tissues (5, 34), we hypothesized that treatment with 17beta-estradiol will maintain mitochondrial function and will have salutary effects against EC apoptosis.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Rat aortic EC isolation. Adult male (275–325 g) Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were used in this study. All animal experiments were carried out according to the guidelines of the National Institutes of Health, and our study was approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham. Aortic EC were isolated as previously described (7). In brief, after anesthesia with isoflurane (Attane; Minrad, Bethlehem, PA), a midline laparotomy incision was performed. The aorta was removed after branches were cauterized. Both ends were cannulated with Intramedic PE-90 tubing (BD Diagnostic System, Sparks, MD). The aorta was gently flushed with 1 ml PBS (pH 7.4) and then incubated three times with 0.5 ml of 0.25% Trypsin/EDTA (Sigma, St. Louis, MO) for 5 min. Afterward fluid was collected in a 15-ml tube containing 5 ml endothelial basal medium 2 (EBM-2; Clonetics, Walkersville, MD) with 10% FBS after charcoal-dextran stripping (Sigma). After centrifugation at 1,000 g for 10 min, the cell pellet was washed two times with 10 ml PBS and finally resuspended in 3 ml of EBM-2. Cells were plated in a 25-cm2 flask (Nalgene, Rochester, NY) and allowed to adhere for 7 days before being washed in EBM-2 to remove debris.

Rat aortic EC culture. EC were cultured in medium (EGM Complete Medium; Clonetics, San Diego, CA), until confluency was reached (37°C, 95% humidity and 5% CO2). EC were used between the 7th and 11th passages. EC showed the characteristic cobblestone morphology for >15 passages (data not shown).

EC marker anti-platelet/endothelial cell adhesion molecule-1 staining using immunocytochemistry and flow cytometry. To identify EC, immunocytochemistry staining was performed using a polyclonal goat antibody anti-platelet/endothelial cell adhesion molecule (PECAM)-1 (CD31) antibody (M-20; Santa Cruz Biotechnology, Santa Cruz, CA). EC suspensions were placed in chamber slides (Lab Tek 2; Nalgene) and cultured for 24 h to allow adherence. Cells were washed with PBS one time and fixed in 2% paraformaldehyde in PBS for 15 min. The cells were then permeabilized with 0.2% Triton X-100 (Sigma) before washing with PBS. Endogenous peroxidase was blocked by incubation with 1% hydrogen peroxidase (Sigma) for 30 min. Nonspecific protein binding was eliminated by incubation with 5% normal rabbit serum (Vector Laboratories, Burlingame, CA) for 30 min. Cells were incubated with the anti-PECAM-1 (M-20) antibody overnight at 4°C in a humid chamber at a concentration of 1 µg/ml. The next day, cells were incubated with a biotinylated rabbit anti-goat IgG antibody for 1 h at room temperature, followed by horseradish peroxidase-conjugated streptavidin for 30 min at room temperature (Vectastain kits; Vector Laboratories). Primary and secondary antibodies were diluted following the manufacturer's instructions. Peroxidase was visualized by 3,3'-diaminobenzidine hydrochloride (Vector Laboratories). Cells were counterstained with hematoxylin. In negative controls, primary antibodies were omitted and replaced by buffer.

For flow cytometry analysis, biotinylated rabbit anti-goat IgG secondary antibody was followed by streptavidin-allophycocyanin (APC) (BD Biosciences, Pharmingen, San Jose, CA).

Inducing and evaluating apoptosis. EC were cultured in 10% FBS (after charcoal-dextran stripping to remove estrogen; Sigma) EBM-2 for two passages before experiments. The appropriate concentrations of H2O2 for induction of apoptosis and 17beta-estradiol were determined by titration in preliminary experiments. When 80% confluency was reached, apoptosis was induced by exposing the cells to H2O2 (0.75 mM) for 20 min. This concentration was detected as adequate in preliminary experiments (data not shown). To evaluate the effect of 17beta-estradiol on H2O2-induced apoptosis, EC were incubated in the presence of 0.5 µM of 17beta-estradiol (Sigma) 30 min before incubation with H2O2. Furthermore, to determine the specificity of 17beta-estradiol, EC were preincubated with 17beta-estradiol and the ER antagonist ICI-182780 (1 µM; Tocris Bioscience, Ellisville, MO). EC were then incubated for 20 h (37°C, 95% humidity and 5% CO2).

Flow cytometry analysis. EC monolayers were dispersed using trypsin (Sigma) to prepare EC suspension and washed with EBM-2. The percentage of apoptotic cells in each experimental condition as described above was determined by flow cytometry using a commercial kit (Vybrant Apoptosis Assay Kit no. 11; Molecular Probes, Eugene, OR). Approximately 5 x 105 cells/ml were stained according to the manufacturer's instructions, by incubation in culture medium containing 4 µl of 10 µM MitoTracker Red for 30 min in the dark (37°C, 95% humidity and 5% CO2). Cells were then washed with PBS and resuspended in 100 µl of annexin-binding buffer containing 5 µl of annexin V Alexa fluor 488. After incubation for 15 min at room temperature in the dark, cells were diluted in binding buffer and immediately analyzed by flow cytometry (LSR II; BD Biosciences).

Fluorescence microscopy and light microscopy. EC were grown on Lab-Tek CC2-treated chamber slides (Nalge Nunc International, Naperville, IL) and incubated overnight under the experimental conditions described above. After staining with MitoTracker Red and Alexa Fluor 488 annexin V, the cells were fixed in 2% paraformaldehyde/PBS for 20 min. Cells were washed and covered with a coverslip with antifade mounting medium. 4',6-Diamidino-2-phenylindole (Molecular Probes) was used as a nuclear counterstain. Cells were visualized using either an inverted epifluorescent microscope (model IX70; Olympus, Melville, NY) or a fluorescence microscope (Leica). Before labeling with MitoTracker Red and annexin V, Alexa fluor 488, EC's apoptotic morphology changes induced by H2O2 in culture were examined by inverted microscopy (ECLIPSE TS100; Nikon, Melville, NY) equipped with a digital camera.

Cytochrome c release. Cytochrome c release was measured in cells permeabilized with streptolysin O. EC were trypsinized, washed with PBS, and resuspended in 150 µl of PBS containing 1 U/µl streptolysin O (Sigma), 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), and 0.01% BSA. After incubation for 30 min at 37°C, cells were centrifuged at 14,000 g for 30 min at 4°C. Cytochrome c in the supernatant was determined by Western blot analysis (21).

Western blot analysis. EC were lysed in lysis buffer (50 mM Tris·HCl, 150 mM NaCl, 1 mM Na2-EDTA, 0.1 mM EGTA, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 1 mM PMSF, 10 µg/ml of aprotinin, and 10 µg/ml leupeptin; all from Sigma). After centrifugation at 10,000 g for 10 min, the supernatant was collected, and protein concentration was determined using a Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). Protein samples were mixed with an equal volume of 2x SDS gel loading buffer [100 mM Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, 200 mM dithiothreitol (DTT), 0.2% bromphenol blue], boiled for 5 min, and were resolved on 12% polyacrylamide gel and transferred to a nitrocellulose membrane. After incubation in blocking buffer (5% milk in 10 mM Tris, 100 mM NaCl, and 0.1% Tween 20, pH 7.5) for 120 min, membranes were probed with primary antibodies (Table 1). All antibodies were used in blocking buffer, and, for all antibodies, membranes were incubated overnight at 4°C. After the membranes were washed five times with Tris-buffered saline-Tween 20, they were incubated for 120 min with horseradish peroxidase-conjugated secondary antibodies (1:2,000; anti-rabbit or anti-mouse peroxidase-conjugated IgG; Bio-Rad, Hercules, CA). Protein content was detected by enhanced chemiluminescence reagent (NEN Life Science, Boston, MA).


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  		Table 1. Primary antibodies for Western blot analysis

 
Measurement of caspase-9 and -3 activities. Caspase-3 and -9 activity was determined by cleavage of the specific substrates Ac-DEVD-AMC for caspase-3 and Ac-LEHD-AMC for caspase-9 (both from Alexis Biochemicals, San Diego, CA; see Ref. 54). Cell extracts were prepared as described for Western blot analysis. Supernatants containing 100 µg of protein /100 µl were added to a 96-well plate, and 100 µl of 2x caspase reaction buffer (40 mM HEPES, pH 7.5, 20% glycerol, and 4 mM DTT) containing the substrate in a final concentration of 50 µM were added. After incubation for 2 h at 37°C, the increase in fluorescence resulting from its release was quantified in relative fluorescence units at an excitation wavelength of 380 nm and emission wavelength of 460 or 505 nm, using a microplate fluorescence reader (Packard Fusion Universal Microplate Analyzer; Packard Biosciences, Meriden, CT). Background fluorescence was determined by using 100 µl of lysate and 100 µl of reaction buffer containing 50 µM of substrate (35).

Statistical analysis. Results are shown as means ± SE. Statistical differences among groups were determined by one-way ANOVA followed by Tukey's test (version 2.0; SigmaStat). Data are expressed as significant at P values <0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of primary rat aortic EC. Isolated EC were evaluated via light microscopy (Fig. 1, A–F), immunhistochemistry (Fig. 1G) and fluorescence-activated cell sorter (FACS) analysis (Fig. 1H). Expression of CD31 (PECAM-1) was shown as the best marker for identification of EC (24). Immunocytochemical staining and flow cytometry analysis demonstrated positive expression of EC marker PECAM-1 (CD31) in the primary culture rat aortic ECs. Flow cytometry analysis showed 83% cells stained positive for PECAM-1 (Fig. 1, G and H).


Figure 1
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  		Fig. 1. Analysis of morphological changes and characterization of isolated endothelial cell (EC) using EC marker platelet/endothelial cell adhesion molecule (PECAM)-1 (CD31). A: untreated EC demonstrated normal cobblestone morphology. B: exposure to H2O2 (0.75 mM) led to cell shrinkage, a characteristic morphological sign of apoptosis. C: treatment with 17beta-estradiol (E2) attenuated cell shrinkage. D: ICI-182780 (ICI), an estrogen receptor (ER) antagonist, abrogated protective effects of E2. Cytotoxic effects of E2 (E) or ICI (F) were excluded. G: immunocytochemical staining for characterization of isolated EC using PECAM-1 (CD31) as an endothelia cell marker. H: in flow cytometry, a purity of 83% PECAM-1 positive EC was proved.

 
17beta-Estradiol treatment reduced H2O2-mediated apoptotic changes. Untreated EC demonstrated typical cobblestone morphology (Fig. 1A); however, upon exposure to H2O2, the cells shrank in size (Fig. 1B). Prior treatment of cells with 17beta-estradiol attenuated this sign of apoptosis (Fig. 1C). However, this protective effect of 17beta-estradiol was abrogated by using ICI-182780, a specific ER antagonist (Fig. 1D). Incubation of cells with 17beta-estradiol or ICI alone did not cause apoptosis (Fig. 1, E and F). Although shrinkage is a characteristic of cells undergoing apoptosis, we also confirmed apoptosis using FACS analysis and fluorescence microscopy.

To quantify apoptosis, the percentage of apoptotic cells was measured using FACS analysis. In untreated EC, 34.8% of cells were apoptotic (Fig. 2A); however, upon exposure to H2O2, apoptosis increased to 80.9% (Fig. 2B). Pretreatment of EC with 17beta-estradiol decreased apoptosis to 41.9% (Fig. 2C). This protective anti-apoptotic effect of 17beta-estradiol was blocked by an ER-antagonist (Fig. 2D). Incubation with 17beta-estradiol or ICI-182780 alone did not influence EC apoptosis (Fig. 2, E and F).


Figure 2
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  		Fig. 2. Quantification of apoptotic cells. A: analysis of apoptotic cells with flow cytometry showed 32.3 ± 1.8% apoptotic EC. B: incubation with H2O2 resulted in an increase of up to 82.2 ± 1.8%. C: coincubation with H2O2 and 17beta-estradiol (E2) decreased the rate of apoptosis to 50.7 ± 6.7%. D: ICI-182780 blocked E2's protective effect on EC (83.5 ± 2.5%). Incubation with E2 (E) or ICI-182780 (F) alone showed no significant influence on EC apoptosis. P < 0.05 vs. untreated (*), vs. H2O2 (#), and vs. H2O2/E2 ($).

 
To obtain further evidence of 17beta-estradiol-mediated attenuation of H2O2-induced apoptosis in rat EC, cells were labeled with MitoTracker Red dye (red fluorescence) and Alexa Fluor 488 annexin V (green fluorescence). Mito Tracker Red positive staining indicates the adequate status of the mitochondrial transmembrane potential, and annexin V identifies the exposed phosphatidylserine on apoptotic cells. Untreated EC showed fewer apoptotic cells (less green color) and normal mitochondria, identified by bright red staining (Fig. 3, top). EC exposed to H2O2 caused apoptotic cell change, as identified by brighter green fluorescence (annexin V positive staining). Additionally, EC showed less intensity of MitoTracker Red labeling, indicating the loss of mitochondria transmembrane potential (Fig. 3, row 2). Both findings were attenuated by 17beta-estradiol treatment (Fig. 3, row 3) and abrogated by additional treatment with ICI-182780 (Fig. 3, bottom).


Figure 3
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  		Fig. 3. Morphological changes of mitochondria. Top: analysis of apoptotic EC with fluorescence microscopy showed normal mitochondrial transmembrane potential. Row 2: loss of mitochondrial transmembrane potential (decreased red fluorescence) and increased number of apoptotic cells (increased green fluorescence) was detected after incubation with H2O2. Row 3: E2 attenuated apoptosis change induced by H2O2 visualized by increased red and decreased green fluorescence. Bottom: ICI-182780 abrogated protective effects of E2 in EC exposed to H2O2.

 
The mechanism of 17beta-estradiol protective effect is mitochondria and caspase dependent. Studies have shown that cytochrome c released from mitochondria is an essential component of this apoptotic pathway. Cytochrome c is normally localized in the mitochondrial intermembrane space; during cell apoptosis, cytochrome c translocates to cytosol, where it is required for activation of caspase-9 and caspase-3. This pathway leads to mitochondria condensation and causes cell shrinkage. To demonstrate the involvement of this pathway, we performed Western blot analysis and caspase-9 and -3 activity assays. Western blot analysis indicated that H2O2 treatment increased cytosolic cytochrome c (Fig. 4) along with the expression of cleaved caspase-9 and caspase-3 (Fig. 5). Enhanced caspase-9 and -3 activity supported these observations (Figs. 6, A and B). Increases in expression of cytosolic cytochrome c, cleaved caspase-9 and caspase-3, and caspase-3 and -9 activity were reduced by 17beta-estradiol treatment. Additional treatment with the ER antagonist abrogated the beneficial effects of 17beta-estradiol.


Figure 4
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  		Fig. 4. Analysis of cytochrome c release and changes in endothelial nitric oxide synthase (eNOS) expression. Cytosolic cytochrome c release from mitochondria in the cytosol induced by H2O2 was prevented by 17beta-estradiol (E2) treatment. The beneficial effects of E2 were prevented by ICI. E2 and ICI alone did not show any difference compared with untreated cells. P < 0.05 vs. untreated (*), vs. H2O2 (#), and vs. H2O2/E2 ($). Western blot analysis demonstrated E2 normalized eNOS expression, which was downregulated in H2O2-induced apoptosis. Detection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a protein loading control. Bars represent mean values for 2 experiments ± SD. P < 0.05 vs. untreated (*), vs. H2O2 (#), and vs. H2O2/E2 ($).

 

Figure 5
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  		Fig. 5. Expression of caspase-9 and -3. E2 reduced expression of the apoptotic signals caspase-9 and -3 after coculture with H2O2. These protective effects were prevented after additional treatment with ICI-182780. Treatment with E2 or ICI alone revealed no changes compared with untreated EC. GAPDH served as a protein loading control. P < 0.05 vs. untreated (*), vs. H2O2 (#), and vs. H2O2/E2 ($).

 

Figure 6
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  		Fig. 6. Activity of caspase-9 and -3. Activity of caspase-9 and -3 was determined by cleavage of the substrates Ac-LEHD-AMC and Ac-DEVD-AMC using Western blot analysis. H2O2 increased caspase activity, which was restored to normal levels after treatment with E2. The ER antagonist prevented effects of estrogen. E2 and ICI did not change caspase activity. P < 0.05 vs. untreated (*), vs. H2O2 (#), and vs. H2O2/E2 ($).

 
Fluorescence microscope examination of EC labeled with MitoTracker Red dye indicates the adequate status of the mitochondrial transmembrane potential and reveals mitochondria pathological morphology changes. In control cells, EC exhibit normal worm-like mitochondria (Fig. 7A), whereas in apoptotic EC, condensation of mitochondria is demonstrated as dot-like mitochondria (Fig. 7B). 17beta-Estradiol treatment attenuated mitochondria condensation, as demonstrated by almost worm-like mitochondria (Fig. 7C). Additional treatment with the ER antagonist prevented the beneficial effect of 17beta-estradiol (Fig. 7D).


Figure 7
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  		Fig. 7. Changes in mitochondrial transmembrane potential. A: evaluation of mitochondrial transmembrane potential using fluorescence microscope examination showed normal worm-like mitochondria in untreated EC. B: after incubation with H2O2, EC condensed dot-like mitochondria. E2 treatment attenuated mitochondria condensation. C: EC exhibit almost worm-like mitochondria. D: ICI-182780 blocked E2's protective effect to EC.

 
17beta-Estradiol normalizes endothelial nitric oxide synthase expression. Treatment of EC with H2O2 resulted in a decrease of endothelial nitric oxide synthase (eNOS) expression; pretreatment with 17beta-estradiol normalized this effect (P < 0.05). The sue of an ER antagonist abrogates the effects of 17beta-estradiol. However, incubation with 17beta-estradiol or ICI-182780 alone did not induce changes compared with untreated EC (Fig. 4).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
By measuring annexin V-conjugated aberrant surface exposure of phosphatidylserine, we have demonstrated that apoptosis is increased in EC exposed to H2O2. Consistent with this, we also found characteristics of apoptotic cell death, including shrinkage of cells and condensation of mitochondria. This was further confirmed by mitochondria release of cytochrome c and an increase in caspase-9 and -3 activation. During stress-induced apoptosis, the outer membrane of the mitochondria is permeabilized, leading to cytochrome c release from mitochondria to the cytosol (11). Cytosolic cytochrome c, apoptotic protease activating factor-1, and caspase-9 form a complex called "apoptosome," which activates downstream caspases such as caspase-3 (17).

Several studies have shown that 17beta-estradiol protected a number of nonreproductive tissues and cell types from apoptosis (16, 22, 27, 33, 36). The present study indicates that 17beta-estradiol attenuated all apoptotic changes that occurred in the presence of H2O2. Moreover, our additional studies using EC cultured with the ER antagonist ICI-182780 further confirmed that the salutary effects of 17beta-estradiol against apoptosis were mediated via ER. This finding supports the results of other studies showing protective effects of 17beta-estradiol against vascular injury (32).

The involvement of ER in mediating 17beta-estradiol's beneficial effects on vascular injury has been extensively investigated. Studies in ER{alpha} and ERbeta knockout mice indicate that both ER mediate the vasculoprotective effect of 17beta-estradiol (15, 18). Other investigators have also reported multiple abnormalities in ion channel function in vascular smooth muscle cells from ERbeta-deficient (ERbeta–/–) mice (47).

The strongest support for the protective effect of 17beta-estradiol on EC was the attenuation of the mitochondrial transmembrane potential. Incubation with H2O2 caused mitochondria condensation, indicating the loss of the mitochondrial transmembrane potential because of the opening of mitochondrial permeability transition pores (31). Treatment with 17beta-estradiol resulted in near-normal mitochondria morphology. This observation supports our hypothesis that the protective effects of 17beta-estradiol in EC apoptosis are mediated through mitochondria.

Studies have shown that the lack of the mitochondrial transcription factor A increased apoptosis in mouse heart, indicating the involvement of mitochondria during the apoptotic process (39). Studies have also shown that ER{alpha} and ERbeta are present in mitochondria membrane of different cell types and tissues. In the human lens, epithelial cells (HLE-B3) express ER{alpha} and ERbeta associated with the nucleus and ERbeta in mitochondria (5). ER{alpha} positive staining was observed in the cytoplasm, whereas ERbeta was specifically enriched at the site of the mitochondria in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells (34). Studies of Zhai et al. (44, 45) suggest a cardioprotective role of ER{alpha} following myocardial ischemia-reperfusion in males. Compared with wild-type mice, swollen and fragmented mitochondria with amorphous and granular bodies, loss of matrix, and rupture of cristae were found in hearts from ER{alpha}–/– mice (44). In ovariectomized female mice, mitochondrial respiratory function was found to be decreased in ovariectomized hearts (45). The abnormalities in mitochondrial structure and function in ovariectomized rats were restored following 17beta-estradiol treatment (45).

We have recently found that 17beta-estradiol administration following trauma-hemorrhage significantly enhanced the expression of estrogen receptor ER{alpha} and ERbeta in rat aortas' EC, which was associated with vascular relaxation (unpublished data). However, we did not examine whether or not apoptosis was involved in vascular relaxation. The present study shows that the protective effect of 17beta-estradiol on EC apoptosis was abrogated by the 17beta-estradiol antagonist ICI-182780, indicating the involvement of ER. Studies from other investigators have also shown that 17beta-estradiol-mediated inhibition of apoptosis appears to be mediated via mitochondrial ER. These findings support our hypothesis that 17beta-estradiol's protective effects in attenuating EC apoptosis are mediated through mitochondria via ERs.

eNOS generates nitric oxide (NO) in EC, which appears to have a vascular protective effect (23). Because it is well known that 17beta-estradiol is a potent stimulus for eNOS activation and NO release (19), this could be one pathway by which 17beta-estradiol mediates its protective effects in the vascular bed (41). However, the precise mechanism by which 17beta-estradiol produces its salutary effect is not entirely clear. Nonetheless, endothelial-dependent vascular relaxation is mediated by the production of NO (9, 25). Zhu et al. (47) reported an ERbeta-mediated increase in inducible nitric oxide synthase (iNOS) expression in wild-type mice, leading to an attenuation of vasoconstriction. In contrast, 17beta-estradiol augmented vasoconstriction in blood vessels from ERbeta-deficient mice (47). Other studies have shown less eNOS expression/nitrite production in ER{alpha}–/– mice (13, 44). Previous studies from our laboratory indicated that the decrease of eNOS in vascular EC is at least partly responsible for EC dysfunction that is observed during the early hyperdynamic stage of polymicrobial sepsis (46). This observation is further supported by other studies which showed that endotoxemia led to increased iNOS expression (37) and inhibition of NO synthesis was found to be detrimental during endotoxemia (6, 26). Eckhoff et al. (10) reported that 17beta-estradiol-mediated beneficial effects were associated with increased serum NO following warm ischemia-reperfusion to the liver. In our study, eNOS expression was downregulated in H2O2-induced apoptotic EC but was normalized with 17beta-estradiol treatment. Our results and those of others thus suggest that the beneficial effect of 17beta-estradiol may be the result of maintenance of NO production via the ER in mitochondria. However, further studies are needed to determine the precise mechanism of the salutary effect of 17beta-estradiol.

In summary, this study describes the effect of 17beta-estradiol on EC apoptosis. We have demonstrated that the protective effects of 17beta-estradiol against apoptosis are through mitochondria via ER. 17beta-Estradiol attenuated H2O2-induced cytochrome c translocation from mitochondria into the cytosol and prevented further activation of caspase-9 and -3. Moreover, 17beta-estradiol normalized eNOS expression, which may result in increased NO production. These findings may have important implications in clinical conditions in which endothelial dysfunction occurs (inflammation, trauma-hemorrhage, tumor onset and progression, transplant rejection, sepsis, ischemic heart disease, and atherosclerosis). However, additional studies are needed to clarify the exact mechanism by which estrogen maintains EC functions.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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 GRANTS
REFERENCES
 
This work was supported by National Institute on General Medical Sciences Grants R37 GM-39519 and RO1 GM-37127.


   ACKNOWLEDGMENTS
 
We thank Bobbi Smith for editing, Albert Tousson and Shawn Williams for support in fluorescence microscopy, and Zheng Feng Ba and Dr. Jianguo Chen for providing rats for EC isolation.


   FOOTNOTES
 

Address for reprint requests and other correspondence: I. H. Chaudry, Center for Surgical Research, The Univ. of Alabama at Birmingham, 1670 Univ. Boulevard, Volker Hall, Ste. G094 (e-mail: Irshad.chaudry{at}ccc.uab.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* A. Lu and M. Frink contributed equally to this study. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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