Androgens inhibit tumor necrosis factor-α-induced cell adhesion and promote tube formation of human coronary artery endothelial cells

Steroids 77 (2012) 756–764

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Androgens inhibit tumor necrosis factor-a-induced cell adhesion and promote tube formation of human coronary artery endothelial cells Chun-Hou Liao a,b, Feng-Yen Lin c, Yi-No Wu b, Han-Sun Chiang a,b,⇑ a

Division of Urology, Department of Surgery, Cardinal Tien Hospital, Taipei, Taiwan College of Medicine, Nutrition and Food Sciences, Graduate Institute of Basic Medicine, Fu Jen Catholic University, Taipei, Taiwan c Department of Internal Medicine, School of Medicine, Taipei Medical University and Division of Cardiology, Taipei Medical University Hospital, Taipei, Taiwan b

a r t i c l e

i n f o

Article history: Received 16 February 2012 Received in revised form 25 March 2012 Accepted 27 March 2012 Available online 3 April 2012 Keywords: Androgen Atherosclerosis Endothelial cells HCAEC Testosterone

a b s t r a c t Endothelial cells contribute to the function and integrity of the vascular wall, and a functional aberration may lead to atherogenesis. There is increasing evidence on the atheroprotective role of androgens. Therefore, we studied the effect of the androgens—testosterone and dihydrotestosterone—and estradiol on human coronary artery endothelial cell (HCAEC) function. We found by MTT assay that testosterone is not cytotoxic and enhances HCAEC proliferation. The effect of testosterone (10–50 nM), dihydrotestosterone (5–50 nM), and estradiol (0.1–0.4 nM) on the adhesion of tumor necrosis factor-a (TNF-a)-stimulated HCAECs was determined at different time points (12–96 h) by assessing their binding with human monocytic THP-1 cells. In addition, the expression of adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), was determined by ELISA and Western blot analysis. Both testosterone and dihydrotestosterone attenuated cell adhesion and the expression of VCAM-1 and ICAM-1 in a dose- and time-dependent manner. Furthermore, androgen treatment for a longer duration inhibited cell migration, as demonstrated by wound-healing assay, and promoted tube formation on a Matrigel. Western blot analysis demonstrated that the expression of phosphorylated endothelial nitric oxide synthase (eNOS) increased, whereas that of inducible nitric oxide synthase (iNOS) decreased following the 96-h steroid treatment of TNF-a-stimulated HCAECs. Our findings suggest that androgens modulate endothelial cell functions by suppressing the inflammatory process and enhancing wound-healing and regenerative angiogenesis, possibly through an androgen receptor (AR)-dependent mechanism. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction Gender differences in the occurrence of cardiovascular diseases have been well established: males have a higher incidence of vascular disease than premenopausal women [1,2]. Consequently, estrogen has been considered atheroprotective [3]. However, another possibility is that androgen exposure in early life may predispose men to earlier atherosclerosis [2]. Serum testosterone levels are reported to decline with age [4,5] and low testosterone is considered to contribute to other cardiovascular risk factors [6,7], indicating the cardio-protective role of testosterone [5,8,9]. Another supporting evidence is that testosterone administration to men with coronary artery disease reduces myocardial ischemia [10] and improves endothelial function [11]. However, despite the

⇑ Corresponding author. Address: Graduate Institute of Basic Medicine, Fu Jen Catholic University, 510 Chung-Cheng Road, Hsinchuang, New Taipei City 24205, Taiwan. Tel.: +886 2 29052202; fax: +886 2 22193391. E-mail address: [email protected] (H.-S. Chiang). 0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2012.03.014

growing evidence on the protective effect of androgens on atherosclerosis, the picture is far from clear [12]. Endothelial cells of the vascular wall play an important role in the pathogenesis of atherosclerosis [13]. Normal vascular endothelium is essential for the maintenance of vascular wall function and integrity, and endothelial injury is one of the initiating events in atherosclerosis [14]. Endothelial cells express adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1); these molecules are the main constituents of the atherogenic process that regulate the recruitment, adhesion, and extravasation of leukocytes, thus sustaining atherogenesis [15,16]. On the other hand, endothelial cells are also known to secrete anti-inflammatory and anti-atherogenic factors such as nitric oxide (NO) that cause relaxation of the underlying smooth muscle cells and regulate vascular tone [17]. Endothelial cells also control the proliferation of the smooth muscle cells and maintain a non-thrombogenic surface [17]. The effect of testosterone on endothelial cells has been investigated in several in vitro studies, but the results are conflicting [9,18–22]. Testosterone action depends on its conversion to

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estradiol by aromatase and to dihydrotestosterone by 5a-reductase, and both these enzymes have been identified in several vascular cell types. Both testosterone and dihydrotestosterone bind to and activate the androgen receptor (AR), whereas estradiol acts through the estrogen receptor (ER). Therefore, the effect of testosterone in the modulation of a number of endothelial responses should be investigated by considering both the AR and ER pathways. It should be noted that a majority of these studies were conducted in human umbilical vein endothelial cells (HUVECs). Although a widely used model to study endothelial cell function, HUVECs are different from arterial endothelial cells and may not be suitable to study cardiovascular disease [23]. Human coronary arterial endothelial cells (HCAECs) better represent the potentially important effects in vivo at arterial sites prone to atherosclerosis. Therefore, we aimed to investigate the anti-atherogenic effect of testosterone and its androgen and estrogen derivatives on the function of HCAECs. 2. Experimental 2.1. Cell culture Male HCAECs (Cascade Biologics, Portland, OR, USA) were grown in endothelial cell growth medium (Medium 200; Cascade Biologics, CA, USA) supplemented with 2% fetal bovine serum (FBS), 1 lg/mL hydrocortisone, 10 ng/mL human epidermal growth factor, 3 ng/mL human fibroblast growth factor, 10 lg/mL heparin, 100 U/mL penicillin, 100 pg/mL streptomycin, and 1.25 mg/mL Fungizone (Gibco, Grand Island, NY, USA), at 37 °C in a 5% CO2 incubator and used at passages 3–8. The growth medium was changed every other day until confluence. The purity of HCAEC cultures was established by immunostaining with a monoclonal antibody directed against endothelial-specific human von Willebrand factor (vWF; R&D Systems, Minneapolis, MN, USA). The human acute monocytic leukemia cell line THP-1 (American Type Culture Collection; Rockville, MD, USA) was cultured in RPMI 1640 medium (R&D Systems, Minneapolis, MN, USA) containing 5% FBS and subcultured at a 1:5 ratio 3 times per week. Testosterone, dihydrotestosterone, and estradiol (all from Sigma–Aldrich Co.; St. Louis, MO, USA) were individually dissolved in ethanol to prepare a 10 mM stock solution, and stored at 4 °C. 2.2. Measurement of cell proliferation by MTT assay The effect of testosterone, dihydrotestosterone, and estradiol on cell proliferation was analyzed by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HCAECs (2  104 cells) were grown in 96-well plates, and incubated with various concentrations of testosterone (5–50 nM), dihydrotestosterone (2.5–100 nM), and estradiol (0.05–0.4 nM) for 2–96 h. Untreated cells were used as internal control for each experiment. Subsequently, MTT (0.5 lg/mL) was applied to cells for 4 h to allow the conversion of MTT into formazan crystals. After washing with phosphate-buffered saline (PBS), the cells were lysed with dimethyl sulfoxide (DMSO), and absorbance was read at 530 nm by using a DIAS Microplate Reader (Dynex Technologies, Chantilly, VA, USA). Cell proliferation values were reported as a percentage of absorbance of treated cells to the control (untreated cells). 2.3. THP-1 and endothelial cell adhesion assay HCAECs (5  105 cells) were seeded in 24-well plates and allowed to reach confluence. The growth medium was supplemented with testosterone, dihydrotestosterone, or estradiol at the indicated concentrations for 12–96 h, followed by stimulation with TNF-a (10 ng/mL) for 6 h at 37 °C. Simultaneously, THP-1 cells

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were labeled for 1 h at 37 °C with 20 lM, 20 70 -bis(2-carboxyethyl)-5(6)-carboxyflorescein acetoxymethyl ester (BCECF/AM; Boehringer-Mannheim, MD, USA) in serum-free RPMI 1640 medium, then washed with PBS to remove the free dye, and resuspended in the RPMI 1640 medium containing 2% FBS. Labeled THP-1 cells (1  106 cells) were added to each HCAEC-containing well and incubated for 1 h. Non-adherent cells were removed by two gentle washes with PBS. The degree of THP-1 cell adhesion to HCAECs was measured by a Multilabel Counter Victor 2 (Wallace Co., CA, USA) at an emission of 530 nm and absorption of 435 nm after the cells were lysed with DMSO. 2.4. Enzyme-linked immunosorbent assay In order to measure the cell-surface expression of adhesion molecules, HCAECs in 96-well plates were treated with testosterone (10, 25, and 50 nM), dihydrotestosterone (2.5, 5, and 50 nM), or estradiol (0.1, 0.2, and 0.4 nM) for 12–96 h, and subsequently stimulated with TNF-a (10 ng/mL) for 8 h at 37 °C. Expression of cell-surface VCAM-1 and ICAM-1 was measured by separate incubations of 30 min at room temperature with specific goat antibodies against human VCAM-1 or ICAM-1 (0.5 lg/mL; R&D, CA, USA), followed by horseradish peroxidase-conjugated rabbit anti-goat IgG (0.5 lg/mL) for 1 h at room temperature. After each incubation step, the cells were washed with Hank’s Balanced Salt Solution (HBSS) containing 2% skim milk. Binding of the secondary antibody was determined by incubating the plates in the dark for 15 min with 100 lL of 3% o-phenylenediamine and 0.03% H2O2 in 50 mM citrate buffer and 100 mM phosphate buffer. The reaction was terminated by addition of 50 mL of 2 M H2SO4. Surface expression of the adhesion molecules was quantified by reading the optical density (OD) at 490 nm in an enzyme-linked immunosorbent assay (ELISA) plate reader. 2.5. Western blot analysis HCAECs were treated for 12 or 96 h with 50 nM testosterone, 50 nM dihydrotestosterone, or 0.4 nM estradiol, followed by TNFa (10 ng/mL) treatment for 8 (VCAM and ICAM expression) or 4 (iNOS and eNOS) h. Total cell lysates were prepared, subjected to SDS–PAGE, and transferred to a PVDF membrane. The membrane was probed with goat anti-hVCAM-1, goat anti-hICAM-1, rabbit anti-phospho-eNOS, mouse anti-phospho-iNOS, mouse anti-total eNOS, mouse anti-total iNOS antibody (all antibodies purchased from R&D Co., CA, USA), or mouse anti-b-actin antibody (Labvision/NeoMarkers, CA, USA) and then incubated with horseradish peroxidase-conjugated secondary antibody. b-actin, total eNOS, and iNOS were used as loading controls. Bound antibodies were detected with the enzyme-linked chemiluminescence detection reagent and exposed to an X-ray film. 2.6. Wound-healing assay In order to assess the ability of testosterone, dihydrotestosterone, or estradiol-treated HCAECs to facilitate cell migration, monolayer denudation assays were performed as described previously [24]. In brief, HCAECs were cultured in a 12-well plate, pretreated with testosterone (50 nM), dihydrotestosterone (50 nM), or estradiol (0.4 nM) at the indicated concentrations for 12 or 96 h, and then stimulated with TNF-a (10 ng/mL) for 8 h at 37 °C. The confluent cells (2  105/well) were wounded by scraping with a 100 lL pipette tip, which denuded a strip of the monolayer that was 300 lm in diameter. The cultures were washed twice with PBS, the medium was supplemented with 5% FBS, and the rate of wound closure was observed after 24 h. The distance of the gap was measured under a 4 phase objective of a light microscope (Olympus

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IX71; Olympus, CA, USA), monitored with a CCD camera (Macro FIRE 2.3A; Olympus, CA, USA), and captured with a video graphic system (Picture Frame Application 2.3 software; Olympus, CA, USA).

inhibited THP-1 cell/HCAEC binding (Fig. 2C). However, estradiol treatment did not alter the adhesion between THP-1 cells and HCAECs (Fig. 2D).

2.7. Endothelial cell tube-formation assay

3.3. Testosterone and dihydrotestosterone reduce TNF–induced expression of VCAM-1 and ICAM-1

The in vitro tube-formation assay was performed to determine the ability of testosterone, dihydrotestosterone, or estradiol-treated HCAECs to form tubes (capillaries), which are required for angiogenesis, using the Angiogenesis Assay Kit (Chemicon, Brea, CA, USA) according to the manufacturer’s instructions. HCAECs were incubated in an M200 medium with testosterone (50 nM), dihydrotestosterone (50 nM), or estradiol (0.4 nM) for 12 or 96 h. ECMatrix gel solution was thawed at 4 °C overnight, mixed with ECMatrix diluent buffer, and placed in a 96-well plate at 37 °C for 1 h to allow the matrix solution to solidify. HCAECs were harvested with trypsin/EDTA, and 1  104 cells were placed on the matrix solution with M200 medium and incubated at 37 °C with TNF-a (10 ng/mL) for 12 h. Tubule formation was observed under an inverted light microscope. Four representative fields were evaluated, and the average of the total area of complete tubes formed by the cells was compared by Image-Pro Plus computer software. 2.8. Statistical analysis All study results were shown as mean ± standard error of the mean (SEM) of three experiments in triplicate. Student’s t-test was adapted for statistical analysis, with statistical significance of p-value < 0.05. 3. Results 3.1. Testosterone, dihydrotestosterone, and estradiol have different effects on cell proliferation Testosterone was not cytotoxic and did not alter cell proliferation of HCAECs when treated for 2–24 h. However, testosterone significantly increased cell proliferation in a dose-dependent manner between 48–96 h when compared with control cells (72 h: 135.2% ± 11.2% with 25 nM, 136.2% ± 10.8% with 50 nM; 96 h: 138.5% ± 9.9% with 10 nM, 136.0% ± 13.3% with 25 nM, and 139.6% ± 15.2% with 50 nM) (Fig. 1A). Treatment with dihydrotestosterone (2.5, 5, and 50 nM; Fig. 1B) for 2–72 h and with estradiol (0.05, 0.1, 0.2, and 0.4; Fig. 1C) for 96 h did not affect HCAEC proliferation. However, incubation with 100 nM dihydrotestosterone for 96 h caused a significant decrease in the number of total living cells present. (75.8% ± 9.0%; Fig. 1B). 3.2. Testosterone and dihydrotestosterone alter adhesion of monocytes to endothelial cells THP-1 cell/HCAEC adhesion assay was performed to assess the effect of testosterone, dihydrotestosterone, and estradiol on the binding of monocytes to endothelial cells. As expected, the binding of THP-1 cells to TNF-a-stimulated HCAECs was significantly increased as compared to the non-stimulated cells (control). Treatment with testosterone (10, 25, and 50 nM) for less than 72 h significantly increased the adhesion of THP-1 cells to the TNF-astimulated HCAECs (Fig. 2A). However, the effect was significantly reversed when the treatment with 50 nM testosterone continued for more than 72 h, resulting in a significant reduction of cell adhesion at 72 h (TNF-a treatment: 310.4% ± 28.6%; 50 nM testosterone treatment: 198.3% ± 41.1%) (Fig. 2B). Additionally, a further decrease at 96 h with 25 and 50 nM testosterone treatment (199.2% ± 29.6% and 135.2% ± 16.5%, respectively) was observed. Treatment with 5 and 50 nM dihydrotestosterone for 96 h also

The effect of testosterone, dihydrotestosterone, and estradiol on VCAM-1 and ICAM-1 expression of TNF-a-stimulated HCAECs was determined by cell ELISA and Western blot analysis. In ELISA, as expected, TNF-a was found to induce VCAM-1 (302.2% ± 25.1%) and ICAM-1 (287.5% ± 23.4%) expression in HCAECs. While treatment with the androgens for 12 h had no effect (Figs. 3A and C), testosterone (25 and 50 nM) and dihydrotestosterone (2.5, 5, or 50 nM) treatment for 96 h resulted in attenuation of TNF-a-induced VCAM-1 expression in HCAECs (25 nM testosterone: 196.5% ± 12.5%; 50 nM testosterone: 152.3% ± 23.6%; 2.5 nM dihydrotestosterone: 204.6% ± 19.9%; 5 nM dihydrotestosterone: 145.6% ± 16.3% of; and 50 nM dihydrotestosterone: 116.3% ± 29.6%) (Fig. 3B). In addition, treatment with 50 nM testosterone and dihydrotestosterone significantly downregulated ICAM-1 expression in TNF-a-stimulated HCAECs (165.7% ± 23.6% and 166.8% ± 29.7%, respectively) (Fig. 3D). Pretreatment with various concentrations of estradiol for 12 or 96 h had no effect on the expression of the cell adhesion molecules (Fig. 3A–D). The findings of cell ELISA were confirmed by Western blot analysis. Pretreatment with 50 nM testosterone or dihydrotestosterone for 96 h significantly inhibited VCAM-1 and ICAM-1 expression (Fig. 3E). 3.4. Testosterone and dihydrotestosterone inhibit TNF–induced HCAEC cell migration To explore the potential effects of testosterone, dihydrotestosterone, and estradiol on the ability of endothelial cells to migrate, the wound-healing/migration assays were performed on TNFa-stimulated HCAECs. The 8-h treatment with10 ng/mL TNF-a induced migration of HCAECs when compared with untreated cells (control) (256.3% ± 30.2%). While both androgens (50 nM) and estradiol (0.4 nM) did not affect TNF-a-induced cell migration after 12-h treatment, a longer exposure (96 h) to androgens caused a statistically significant reduction in HCAEC migration (124.6% ± 25.7% and 158.6% ± 30.2% for testosterone and dihydrotestosterone, respectively) (Figs. 4A and B). No difference was observed with the 96-h estradiol treatment (289.6% ± 31.2%). 3.5. Testosterone and dihydrotestosterone promote tube formation by HCAECs Next, we investigated the potential effects of androgens and estradiol on endothelial cell neovascularization, using tube-formation assays on HCAECs. TNF-a treatment resulted in inhibition of tube formation when compared with untreated cells. However, 96-h treatment with testosterone or dihydrotestosterone (50 nM) resulted in reversion of the tube formation ability of HCAECs to near-control levels (TNF-a: 20.5% ± 6.2%; testosterone: 79.6% ± 9.5%; and dihydrotestosterone: 64.4% ± 8.5%) (Fig. 5A and B). In contrast, 12-h treatment with testosterone or dihydrotestosterone, and 12-h and 96-h treatment with estradiol did not alter the tube formation ability of TNF-a-stimulated HCAECs. 3.6. Testosterone and dihydrotestosterone differentially activate endothelial and inducible nitric oxide synthase NO plays an important role in the maintenance of endothelial cell function and in inflammation; its production by iNOS is known to be higher than that by eNOS [25]. Therefore, we investigated the

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Fig. 1. Effect of testosterone, dihydrotestosterone, and estradiol on HCAEC proliferation. Cell proliferation was assessed by MTT assay. HCAECs were grown in 96-well plates and incubated with the indicated concentrations of testosterone, dihydrotestosterone, and estradiol. MTT was applied to the cells for 4 h. The cells were lysed with dimethyl sulfoxide (DMSO), and absorbance was read at 530 nm by a microplate reader. (A) Treatment of HCAECs with 5–50 nM testosterone for 2–12 h (left graph) or 24–96 h (right graph). (B) Treatment of HCAECs with 2.5–100 nM dihydrotestosterone for 2–12 h (left graph) or 24–96 h (right graph). (C) Treatment of HCAECs with 0.05–0.4 nM estradiol for 2–12 h (left graph) or 24–96 h (right graph). Data are expressed as the mean ± SEM of percentage of control (naïve HCAECs at the same time point) of 3 experiments performed in triplicate. ⁄p < 0.05 (Student’s t-test).

effects of testosterone, dihydrotestosterone, and estradiol on eNOS and iNOS levels in TNF-a-stimulated HCAECs by Western blot analysis. Phosphorylated iNOS levels (activated) were significantly increased in TNF-a-stimulated HCAECs (Fig. 6A). However, the 96h treatment with testosterone (50 nM), dihydrotestosterone (50 nM), or estradiol (0.4 nM) resulted in a decrease in the activated iNOS levels when compared with TNF-a treatment alone. In contrast, eNOS phosphorylation was significantly decreased in TNF-a-stimulated HCAECs, but the levels increased upon treatment with testosterone (50 nM), dihydrotestosterone (50 nM), or estradiol (0.4 nM) for 96 h (Fig. 6B). On the other hand, 12-h treatments with the steroids had no effect on the eNOS and iNOS levels when compared with the TNF-a treatment. 4. Discussion Despite the increasing evidence of the beneficial cardioprotective effects of androgens, their mechanism of action remains to

be elucidated. In this study, we demonstrated that testosterone induced proliferation of TNF-a-stimulated HCAECs in a timeand dose-dependent manner, as evident by an increase in number of living cells after testosterone treatment as compared to the TNF-a treatment alone. This result indicates that testosterone, at the concentrations used in the experiments in this study, is not cytotoxic to HCAECs and induces cell proliferation, which may be beneficial for the repair of endothelial injury/damage in the cardiovascular system. Our results are consistent with a previous report on HAECs, which showed that androgens but not 17b-estradiol stimulate cell proliferation through the upregulation of VEGF-A, cyclin A, and cyclin D [26]. Further, testosterone is reported to induce the proliferation of lung endothelial cells in male but not female rats [27]. However, in the same study estradiol promoted the proliferation of vascular endothelial cells from both sexes. Although estradiol did not show any cell proliferation effect at the time points and doses tested, they were not cytotoxic at the doses used in the assays reported in this study.

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Fig. 2. Effect of testosterone, dihydrotestosterone, and estradiol on monocyte adhesion to endothelial cells. HCAECs were treated for (A) 12 or 24 h or (B) 72 or 96 h with 10, 25, or 50 nM testosterone; (C) 12 or 96 h with 5 or 50 nM testosterone; or (D) 0.1 or 0.4 nM dihydrotestosterone; all of these treatments were followed by 10 ng/mL TNF-a stimulation for 8 h. Adherence was represented as relative fluorescent units. Following the addition of BCECF/AM-labeled THP-1 cells, adherence was measured using Multilabel Counter Victor 2 at an emission of 530 nm and absorption of 435 nm for 3 independent experiments and represented as relative fluorescent units. ⁄p < 0.05 (Student’s t-test).

Although dihydrotestosterone has greater potency at the AR than testosterone, only testosterone induces cell proliferation of HCAECs in our study. In addition, high dose dihydrotestosterone may have some anti-proliferative effect. This fact implies that cell proliferation induced by testosterone may be through an AR-independent pathway. Our reports are also consistent with previous report that testosterone induced endothelial cell growth via an AR-independent manner with activation of ERK1/2 activity while dihydrotestosterone inhibited endothelial cell growth in an AR-dependent manner [20]. The supplementation of testosterone at physiological levels is likely to benefit to endothelial repair and enhance resistance to atherosclerosis. Adhesion of leukocytes to endothelial cells, which is facilitated by adhesion molecules such as VCAM-1 expressed on the cell surface, is one of the early events in atherosclerosis [28]. Our study showed that treatment of HCAECs with testosterone and dihydrotestosterone for 72–96 h decreases cell adhesion, suggesting an atheroprotective role of androgens. This result was supported by our observation that TNF-a-induced expression of VCAM-1 and ICAM-1 was significantly attenuated following exposure to these androgens for a longer duration. Since testosterone is converted to estradiol by aromatase or to dihydrotestosterone, a non-aromatisable androgen, by 5a reductase, it can exert its effect through the ER and/or the AR pathways. The enzymes aromatase and 5a reductase are expressed in HUVECs [9] but are undetectable in human aortic endothelial cells (HAECs) [5]. To the best of our knowledge, there is no information regarding the expression of these enzymes in HCAECs. In this study, it is possible that the effect of testosterone on HCAECs was not via its conversion to estradiol. However, presence of ER on HCAECs has been demonstrated [29]. The inability of estradiol to inhibit cell adhesion and VCAM-1 and ICAM-1 expression in HCAEC cultures despite the presence of ER in contrast to the ability of dihydrotestosterone, a potent androgen, to do so suggests

that the effect of testosterone on these cells is mediated through the AR. In support of this possibility, dihydrotestosterone was shown to inhibit TNF-a and lipopolysaccharide (LPS)-induced expression of VCAM-1 and ICAM-1 mRNA partly through the AR [18]. Interestingly, in a separate study in HUVECs, 5a-androstane-3b, 17b-diol (3b-Adiol), a dihydrotestosterone metabolite, decreased TNF-a- and LPS-induced expression of adhesion molecules, including ICAM-1 and VCAM-1, both in HUVECs and mice aorta [30]. In HAECs, testosterone attenuated VCAM-1 expression by inhibiting TNF-a-induced activation of its transcription factor nuclear factor-kappa B (NFjB) through its interaction with the AR [5]. However, contrasting results have also been reported where dihydrotestosterone enhances VCAM-1 expression and adhesion to monocytes in HUVECs [19,21,22]. Zhang et al. [19] reported that both testosterone and 17b-estradiol increases the VCAM-1 expression in HUVECs, although the ICAM-1 expression does not change. A different mechanism of action for testosterone in HUVECs has been reported, where testosterone downregulates TNF-a-induced expression of VCAM-1 by converting to estradiol [9]. The same study also reported that estradiol attenuates VCAM-1 expression, whereas dihydrotestosterone has no affect, suggesting that the effect of testosterone is AR-independent. In addition to regulating leukocyte adhesion, endothelial cells also promote migration and tube formation, processes that contribute to angiogenesis [31]. Angiogenesis is a prerequisite for embryonic development and plays a critical role in adult physiological processes such as wound repair and tissue responses to ischemia. Stable attachment to the extracellular matrix is essential for endothelial cells to maintain the integrity of the endothelium [32]. Our findings demonstrate that at 96 h, androgens attenuate TNF-a-induced HCAEC migration, but have no effect at a shorter time point of 12 h. These results indicate that testosterone treatment helps maintain stability of HCAECs on the extracel-

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Fig. 3. Effect of testosterone, dihydrotestosterone, and estradiol on the expression of VCAM-1 and ICAM-1 in HCAECs. The expression of VCAM-1 and ICAM-1 in TNF-astimulated HCAECs was determined by cell ELISA and Western blot analysis. HCAECs were treated with testosterone (T), dihydrotestosterone (DHT), or estradiol (E2) for 12 h (A and C) or 96 h (B and D), followed by stimulation with TNF-a (10 ng/mL for 8 h). Expression of cell-surface VCAM-1 and ICAM-1 was measured by separate incubations with goat antibodies against human VCAM-1 or ICAM-1, followed by horseradish peroxidase-conjugated rabbit anti-goat IgG. Binding of the secondary antibody was determined by incubating the plates with o-phenylenediamine and H2O2 in citrate and phosphate buffer, and terminated by the addition of H2SO4. Surface expression of adhesion molecules was quantified by reading the optical density at 490 nm using an ELISA plate reader. (E) Expression of VCAM-1 and ICAM-1 was analyzed by Western blot analysis by probing with goat anti-hVCAM-1 and goat anti-hICAM-1 primary antibodies and horseradish peroxidase-conjugated secondary antibody. b-actin was used as a loading control. Bound antibodies were detected with the enzyme-linked chemiluminescence detection reagent and exposed to an X-ray film. Data are expressed as the mean ± SEM of 3 experiments in triplicate. ⁄p < 0.05 (Student’s t-test).

lular matrix in a TNF-a-stimulated situation. Tube formation by HCAECs is inhibited by TNF-a; however, this effect is reversed when the cells are treated for 96 h with androgens, but not with estradiol. Another study, supporting our results, showed that dihydrotestosterone induced a dose-dependent increase in cell migration and tubulogenesis in HUVECs [33]; however, the cells were not treated with TNF-a. A previous study showed the dose-dependent effect of TNF-a on tube formation [31], whereas

we showed that the effect of androgens in angiogenesis is timedependent. In the healthy vessel, the endothelium plays a cardioprotective role by releasing eNOS catalyzed NO, which prevents processes that contribute to atherogenesis, such as abnormal constriction of the coronary arteries, aggregation of platelets, adhesion of leukocytes, and expression of adhesion molecules [13]. On the other hand, iNOS is expressed in response to immunological stimuli,

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Fig. 4. Effect of testosterone, dihydrotestosterone, and estradiol on wound healing/migration of TNF-a stimulated HCAECs HCAECs were cultured with 50 nM testosterone (T), 50 nM dihydrotestosterone (DHT), or 0.4 nM estradiol (E2) for 12 or 96 h, followed by stimulation with 10 ng/mL TNF-a. Cells were wounded by scraping with a 100 lL pipette tip, which denuded a strip of the monolayer. The rate of wound closure was observed after 24 h. (A) HCAECs migrating to the denuded area were counted using the black baseline as the reference. Photographs were taken after wound scraping for 8 h. (B) Migration of HCAECs was evaluated by counting the migrated cells in six random fields under high magnification (100). Data are expressed as the mean ± SEM of three independent experiments and expressed as the percentage of control. ⁄p < 0.05 (Student’s t-test).

Fig. 5. Effect of testosterone, dihydrotestosterone, and estradiol on the tubeformation ability of HCAECs. Tube formation was determined using an in vitro assay with an ECMatrix gel. HCAECs were incubated in M200 medium with 50 nM testosterone (T), 50 nM dihydrotestosterone (DHT), or 0.4 nM estradiol (E2) for 12 or 96 h. After harvesting, cells were placed on a matrix solution with the M200 medium and incubated at 37 °C with TNF-a (10 ng/mL) for 12 h. (A) Representative photos for in vitro angiogenesis. (B) The cells were stained with crystal violet, and the averages of the total area of the complete tubes formed by the cells were compared by Image-Pro Plus computer software. Data are expressed as mean ± SEM; n = 3. ⁄p < 0.05 (Student’s t-test).

and once activated, generates large amounts of NO that contribute to pathological conditions. Disruption of the endothelial layer and initial loss of eNOS is a hallmark of the development of atherosclerosis. The upregulation of iNOS is considered to compensate for the loss of a functional endothelium and eNOS during injury and atherosclerosis, although the presence of excess NO may lead to additional tissue damage. While iNOS levels are undetectable in normal cardiomyocytes, increased iNOS expression has been observed in heart failure patients [34]. Estradiol had been reported to inhibit ovariectomy or lipopolysaccharide induced iNOS expression [35–36]. In our study, we demonstrated that not only estradiol but also androgens attenuate the levels of TNF-a-induced expression of activated iNOS in HCAECs, a finding which is in agreement with that of an earlier study in murine macrophages in which testosterone treatment inhibited iNOS [37]. On the other hand, we also found that activated eNOS levels increase in the presence of androgens and estradiol when compared with the presence of TNF-a alone. In a previous study, testosterone was shown to induce NO production in HAECs via AR-dependent activation of eNOS [38]. Another study showed that in HUVECs, both testosterone and dihydrotestosterone induced NO synthesis via the activation of eNOS, along with increases in the level of eNOS [39]. A contrasting result was presented by Hishikawa et al. in their study in HAECs: while 17b-estradiol increased the expression levels of eNOS, testosterone had no effect [40]; this study, however, did not measure the levels of activated eNOS. Taken together, our study shows that the effect of steroids may differ considerably across various endothelial cell types and therefore, it is important that in vitro results are interpreted with caution when extending them to in vivo studies. Nonetheless, our study also has some limitations, and further investigations using other cytokines such as IL-1B may help to validate our results. In addition, the use of neutralizing antibodies to VCAM-1 and ICAM-1, and AR inhibitors in a future study will help further strengthen our results.

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Fig. 6. Effect of testosterone, dihydrotestosterone, and estradiol on the phosphorylation of iNOS and eNOS in HCAECs. HCAECs were treated for 12 or 96 h with 50 nM testosterone (T), 50 nM dihydrotestosterone (DHT), or 0.4 nM estradiol (E2); all of these treatments were followed by TNF-a (10 ng/mL) treatment for 4 h. Expression of activated (phosphorylated) and total (A) iNOS and (B) eNOS was determined by Western blot analysis using mouse anti-phospho-iNOS, mouse anti-total iNOS, rabbit antiphospho-eNOS, and mouse anti-total eNOS primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Total iNOS and eNOS were used as loading controls. Bound antibodies were detected with the enzyme-linked chemiluminescence detection reagent and exposed to an X-ray film. ⁄Significant relative to control. + Significant relative to TNF-a treatment. ⁄,+p < 0.05 (Student’s t-test).

5. Conclusion We demonstrated that testosterone and dihydrotestosterone play a beneficial role in coronary artery HCAECs by inhibiting TNF-a-induced THP-1/HCAEC adhesion, decreasing the expression of VCAM-1 and ICAM-1, increasing stability, and inducing tubulogenesis. All these effects are dose- and time-dependent. Since these results were not observed with estradiol treatment, it is reasonable to suggest that the effects are AR-dependent. However, further investigations are required to elucidate the mechanism of androgen action in HCAECs. While our study suggests that testosterone is atheroprotective, the overall effect of testosterone on the other components of the vasculature such as the smooth muscle cells, myocardial fibers, macrophages, and platelets will determine its role in the vascular wall and subsequently, in atherosclerosis. Institutional Approval The study is approved by the institutional review board of Cardinal Tien Hospital (CTH-97-3-5-018). Acknowledgments We thank Feng-Pin Hsiao, Tze-Liang Yang, and Min-Yu Lo for provided technical assistance. This work was supported by

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