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Hormones and Vasculature Laboratory (S.L., A.D., M.R.I.W., K.M., P.A.K., K.S.) and Morphology Laboratory (R.J.D.), Baker Medical Research Institute, Melbourne 3181, Australia
Address all correspondence and requests for reprints to: K. Sudhir, M.D., Ph.D., Pharmacyclics, 995 East Arques Avenue, Sunnyvale, California 94085-4521. E-mail: . ksudhir{at}pcyc.com
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Introduction |
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Materials and Methods |
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(20 ng/ml) as controls.
AR binding study
EA.hy926 cells in six-well plates (
4 x 106 cells/well) were washed (twice) with serum-free DMEM and incubated with serum-free DMEM (2 ml/well) for 30 min, then incubated at 37 C for 90 min in serum-free DMEM with [3H]R1881 (0.31–5.0 nmol/liter) and triamcinolone (1 µmol/liter), with or without nonradioactive DHT (1 µmol/liter). After washing with PBS (twice), cells were scraped into 0.1% Triton-acetic acid (1 ml/well), transferred to scintillation vials with 3 ml scintillation liquid Instagel (Bio-Rad Laboratories, Inc., Sydney, Australia), and counted for 5 min per vial by a ß-counter.
Measurement of DNA synthesis
EA.hy926 cells in 24-well plates were incubated with 1 µCi/well [3H]thymidine for 6 h. Cells were washed with ice-cold PBS (three times), incubated with ice-cold 0.2 N NaHClO4 (1 ml/well) on ice for 30 min, and washed (three times) with 0.2 N HClO4 (0.5 ml/well). Cells were incubated with 0.2 N NaOH (0.5 ml/well) at 37 C for 1 h, then neutralized with 6% acetic acid (0.2 ml/well), transferred to scintillation vials with 3 ml Instagel, and counted (2 min/vial) by a ß-counter.
Estimation of cell damage by counting floating cells
Between d 1–4 of serum deprivation, detached cells (floating cells) in the medium were collected and counted directly by an automatic cell counter (S.ST.II/ZM, Coulter Electronics Ltd., London, UK). Simultaneously, attached cells were harvested from culture plates by 0.05% trypsin digestion and counted. The extent of cell damage was estimated from the percentage of floating cells.
Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) stain
Annexin V is a calcium-dependent phospholipid-binding protein with high affinity for phosphatidylserine (PS) in the plasma membrane (14) and has been used for analysis of apoptotic damage in attached EC in this study. Cells were grown to confluence in 60-mm dishes with coverglasses and serum-deprived for 48 h in the presence of T (10 nmol/liter) or vehicle (control). Cells on coverglasses were stained by incubation (37 C, 10–15 min) with annexin V-Fluos label solution, prepared shortly before use by prediluting 20 µl annexin V-FITC (Roche Molecular Biochemicals, Castle Hill, Australia) and 20 µl PI (50 µg/ml; Sigma, St. Louis, MO) in 1 ml HEPES buffer [10 mmol/liter HEPES/NaOH (pH 7.4), 140 mmol/liter NaCl, and 5 mmol/liter CaCl2]. Stained cells in the same field were evaluated by fluorescence microscopy (x400) under transmitted light (showing all stained and unstained cells), FITC (showing annexin V-stained cells), and Texas Red (showing PI-stained cells), respectively. The numbers of total, annexin V-stained (FITC+/PI-), and annexin V/PI-stained (FITC+/PI+) cells were calculated from three random fields in each experiment. In experiments conducted in cells in serum-enriched medium, apoptosis-related damage was generally less than that in serum-free cultures; the degree of such damage was therefore analyzed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson and Co., Mountain View, CA).
Electrophoretic analysis of genomic DNA fragments
EA.hy926 cells in 100-mm dishes were harvested by 0.05% trypsin digestion, and the genomic DNA was isolated with a DNA purification kit (Promega Corp., Madison, WI). Aliquots (10 µg) of DNA were run in 1% agarose gel with ethidium bromide at 70 V for 1.5 h, and after washing in water, DNA bands in the gel were visible under UV light and analyzed by comparison to DNA markers.
Morphological examination
EA.hy926 cells in six-well plates were washed with PBS and observed by phase contrast microscopy (x400, model 1X70-S8F2, Olympus Optical Co. Ltd., Tatsuno, Japan), or harvested by 0.05% trypsin digestion, smeared on glass slides, stained with 1:20 diluted Giemsa solution (Sigma), and examined under a light microscope (400x).
Western blot analysis
Total cell protein was isolated with lysis buffer [20 mmol/liter Tris-HCl (pH 7.7), 250 mmol/liter NaCl, 2 mmol/liter EDTA, 2 mmol/liter EGTA, 0.5% Nonidet P-40, 10% glycerol, 20 mmol/liter ß-glycerophosphate, 1 mmol/liter Na3VO4, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µmmol/liter pepstatin, 1 mmol/liter phenylmethylsulfonylfluoride, and 1 mmol/liter dithiothreitol], separated on 10% SDS-polyacrylamide gels, and transferred to nitrocellulose filters. After blocking in 5% nonfat milk overnight, the filters were hybridized by antihuman B-cell leukemia/lymphoma-2 protein (Bcl-2) or IL-1ß-converting enzyme/caspase-1 antibodies (Oncogene Research Products, Cambridge, MA) for 1 h, followed by horseradish peroxidase-conjugated secondary antibodies (DAKO Corp., Carpenteria, CA) for another hour. The filters were submersed in enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Arlington Heights, IL) for 1 min and exposed to x-ray film for 1–10 min to visualize the protein bands. For sample load control, nonspecific protein bands on the gel were visualized by staining with Coomassie blue (0.5 g in 500 ml methanol, 200 ml acetic acid, and 300 ml water). AR protein was also detected by reprobing with antihuman AR antibody (Oncogene Research Products).
Statistics
Data are presented as the mean ± SE. Comparisons between two means (effect of antagonists) were made by t test, and multiple comparisons (dose-response relationships) using two-way ANOVA and post-hoc testing using the Student-Newman-Keul test. Differences at P < 0.05 were considered significant.
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), with a density of 10,365 ± 829 sites/cell (Kd = 0.05 nmol/liter; binding capacity, 17.6 fmol). AR protein was also detected in these cells by Western blotting, with levels of expression remaining unchanged during the experimental period (see below).
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, DNA synthesis in EA.hy926 cells, assessed by [3H]thymidine incorporation, was decreased significantly by T treatment for 24 h in a dose-dependent manner. To verify whether such an effect was specific to T, two other steroid hormones, namely dehydroepiandrosterone (DHEA) and 17ß-E2 (E2) were examined simultaneously with T, and neither steroid influenced DNA synthesis at the same concentration as T (data not shown). The effect of T was abolished by the administration of the AR antagonist flutamide, indicating an AR-mediated effect.
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). Again, neither DHEA nor E2 increased the floating cells (data not shown). A similar trend was observed in experiments in human vascular endothelial cells (Clonetics, San Diego, CA; passages 2–3), at high concentrations of T in serum-enriched medium (data not shown); however, as these cells were poorly resistant to serum deprivation, the rest of the experiments in this study were carried out in EA.hy926 cells.
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). These fragments indicate the presence of apoptotic damage to these cells after serum deprivation, although such data are not quantitative and do not allow for comparison between experimental groups.
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). With Giemsa stain, apoptotic cells characterized by nuclear fragmentation and condensation at a relatively early stage or by nuclear disruption at a late stage were observed under light microscopy; their number significantly increased after serum withdrawal, and they were more frequent with T treatment (Fig. 5B).
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) are stained only by annexin V, appearing green-stained on their membranes under FITC microscope (Fig. 6A-ii), and are not stained by PI (Fig. 6A-iii), as the integrity of the cell membrane is not compromised. However, when cells enter into late stage apoptosis, and the membrane is destroyed, their DNA is also positively stained with PI, and they become indistinguishable from necrotic cells. Such cells (indicated, for example, by red arrows in Fig. 6A) stain positively with both annexin V (Fig. 6A-ii) and PI (Fig. 6A-iii), indicating either late stage apoptosis or necrosis. Most floating cells were FITC+/PI+ (data not shown), indicating that severe damage had occurred in cells by this stage. Of the attached cells, 10.8 ± 1.2% appeared damaged after serum deprivation for 48 h, with 6.8 ± 1.0% FITC+/PI- and 4.0 ± 0.2% FITC+/PI+; T (10 nmol/liter) significantly increased both FITC+/PI- and FITC+/PI+ cells to levels approximately twice those of controls (16.0 ± 1.1% and 8.3 ± 0.3%; P 
0.01 vs. control; Fig. 6B). The effect of T was also examined in serum-enriched (2.5–10%) culture conditions using flow cytometric analysis. In the presence of serum, 1.03 ± 0.08% of attached cells stained positively (0.56 ± 0.04% FITC+/PI- and 0.47 ± 0.03% FITC+/PI+). In the presence of T (10 nmol/liter), positively stained cells increased to 1.47 ± 0.13% (P = 0.05 vs. control), mainly in FITC+/PI+ cells (0.98 ± 0.05%; P < 0.01 vs. control; Fig. 7).
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). Consistent with the result of AR binding assay, the cells expressed AR protein, and this protein expression did not change during serum deprivation and hormonal treatment; thus, AR protein was used as an additional loading control for estimation of Bcl-2 (Fig. 8). Caspase-1 expression, with its main subunits (p45, p20, and p10) shown in Fig. 9, increased significantly after both 48-h serum deprivation (control 1) and serum deprivation plus TNF- (20 ng/ml; control 2). In contrast to bcl-2, however, no further changes in caspase-1 were observed in T-treated cells.
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Discussion |
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Vascular endothelial cells suffer apoptotic damage when growth factors are lacking. In cultured human EC, the absence of heparin, serum, and growth factors for 6–8 h results in significant apoptosis (15). The present study shows that human vascular EC under serum-free culture undergo apoptotic damage, confirmed by multiple apoptosis-associated determinations, including electrophoretic analysis of genomic DNA fragments, morphological observation of Giemsa-stained cells, annexin V-FITC/PI staining, and detection of apoptosis-associated gene expression. These changes in EC under serum-free culture in vitro appear very similar to the apoptotic damage occurring in vivo in the endothelium of arteries subject to ischemia (9).
To date there is still no single method that is very effective for the identification and quantification of apoptosis. We employed a new technique, annexin V-FITC/PI staining, for analysis of apoptosis in cultured EC. During early apoptosis, changes occur at the cell surface, such as the translocation of PS from the inner part of the membrane to the outer layer, by which PS becomes exposed at the external surface of the cell and can been bound by annexin V (14). Cells undergoing early apoptosis still have intact membranes and only stain positively with annexin V on their membranes; necrotic cells that also expose PS due to loss of membrane integrity can be discriminated from apoptotic annexin V-stained cells by their DNA staining with PI (14). In the present study within first 24 h of serum deprivation treatment most apoptotic EC stained only positively with annexin V (FITC+/PI-) and were very different in appearance from cells with necrotic damage (FITC+/PI+). With longer treatments, many apoptotic cells become FITC+/PI+ due to the loss of their membrane integrity and cannot be distinguished from other cells undergoing necrosis by annexin V-FITC/PI staining. Most floating EC in this study were FITC+/PI+, indicating the cells in this phase were undergoing severe damage, whereas attached apoptotic EC were mainly FITC+/PI-, reflecting a relatively early stage of cell damage. In combination with agarose gel analysis of DNA fragments and morphological studies, we obtained a reliable and quantifiable analysis of apoptosis in our study using this simple and sensitive technique.
Androgens induce a variety of effects on blood vessels. T induces endothelium-independent vasodilation (16, 17); conversely, it can act through endothelium to oppose relaxation and produce vasoconstriction (8, 18, 19). The latter action varies according to gender, being more marked in men, a fact that may contribute to the increased risk of vascular events in men (19). More recently, androgens have been shown to increase VSMC proliferation (6) and increase monocyte adhesion to endothelial cells (7), both actions consistent with an atherogenic effect.
Apoptotic damage of vascular EC is an important cellular mechanism in atherogenesis. During apoptosis, there is an increase in the adhesiveness of EC for platelets and in the tendency to thrombus formation, reflecting a loss of membrane components with anticoagulant properties and an increased expression of adhesion molecules (15, 20, 21). Subsequently, migration of smooth muscle into the endothelium also occurs, with promotion of atherosclerotic lesions (9, 22). Estrogens inhibit expression of the adhesion molecule (23) and protect EC from apoptotic damage via ER (24, 25). By contrast, the androgen DHT increases EC expression of vascular cell adhesion molecule-1 adhesion and monocyte adhesion to vascular endothelium (7); however, apoptosis-associated changes were not determined in that study. Our study shows that T enhances apoptotic damage in human vascular EC cultured in serum-free conditions. In serum-enriched cultures in which the extent of apoptotic damage is generally much lower, T also enhanced EC damage, as shown by an increase in annexin V/PI-stained cells, but the increase was mainly in FITC+/PI+ cells, reflecting effects on both necrosis as well as apoptosis. Taken together, our studies suggest that androgens, as opposed to estrogens, may aggravate vascular EC dysfunction in men in situations such as ischemia, and thus contribute to gender-based differences in cardiovascular disease.
Apoptosis is a form of programmed cell death and occurs through activation of cell death defective (ced) gene expression (26). Recently, the protein Bcl-2 and caspase families have been recognized as important regulators of this process (27, 28). Bcl-2 is a 24- to 26-kDa protein produced by a 230-kb protooncogene, and its major function appears to be inhibition of apoptosis or programmed cell death, possibly via inhibition of cytochrome c release from mitochondria (27). Caspase-1 is a cysteine protease activated during apoptosis and causes the apoptotic morphological changes observed in cells and nuclei as well as chromosomal DNA degradation (28). Although at present most information concerning the actions of Bcl-2 and caspase on apoptosis has come from nonhuman species, it has been shown that in cultured human EC, apoptosis induced by TNF is associated with altered levels of expression of Bcl-2 and caspase-1 (25, 29). Our study has shown that apoptosis in human vascular EC induced by serum deprivation or TNF is also associated with a decrease in Bcl-2 and an increase in caspase-1 expression, and administration of T further decreases Bcl-2 expression, without altering caspase-1 expression.
We conclude that T at physiological levels enhances apoptotic damage in human vascular EC cultured in serum-deprived conditions, possibly related to decreased expression of Bcl-2 protein. This is consistent with a role for T in atherosclerosis and may contribute to the higher incidence of atherosclerotic vascular disease in men.
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Acknowledgments |
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Footnotes |
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2 P.A.K. and K.S. are equal contributors.
3 Senior Research Fellow of the National Health and Medical Research Council of Australia.
Abbreviations: DHEA, Dehydroepiandrosterone; EC, endothelial cells; FITC, fluorescein isothiocyanate; PI, propidium iodide; PS, phosphatidylserine.
Received May 29, 2001.
Accepted for publication November 5, 2001.
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-reductase activity and androgen-dependent proliferation of vascular smooth muscle cells. J Steroid Biochem Mol Biol 50:169–174[CrossRef][Medline]
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