17β-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells

Life Sciences 70 (2001) 403–413

17b-estradiol inhibits oxidized low density lipoprotein-induced generation of reactive oxygen species in endothelial cells C.H. Yen, C.C. Hsieh, S.Y. Chou, Y.T. Lau* Department of Physiology, Chang Gung University College of Medicine, Tao-Yuan, Taiwan, ROC Received 19 June 2000; accepted 29 December 2000

Abstract Increase of intracellular reactive oxygen species (ROS) has been proposed to cause endothelial injury, and oxidized LDL (oxLDL) actions are associated with an early increase of ROS. Estrogen protects vascular cells partly via its antioxidant effects and by preventing lipid peroxidation. However, whether it can inhibit oxLDL-induced stimulation of ROS generation in endothelial cells is unknown. We utilized the fluorescent dye (DCFH-DA) to measure ROS generation and compared the stimulant effect of tert-butylhydroperoxide (TBH) and oxLDL in human umbilical vein endothelial cells (HUVECs). We found that TBH, H2O2, and oxLDL rapidly stimulated ROS generation, and in a dose-dependent manner with TBH. A concentration of estrogen effective in preventing lipid peroxidation was employed either by pretreatment of cells 18h prior to or by direct co-incubation (30 min) with HUVEC and oxLDL. Estrogen (54 mM) pretreatment significantly suppressed both TBH- and oxLDL- induced stimulation of ROS generation. Both 1 and 54 mM concentration of estrogen could directly inhibit oxLDL-induced ROS production in HUVECs. Thus, either 18h pretreatment or 30 min co-incubation with estrogen reduced stimulated ROS generation, suggesting that both cellular and direct actions of estrogen may be involved. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Estrogen; Oxidized LDL; Dichlorofluorescin; Endothelial cells; Antioxidant

Introduction Oxidized low-density lipoprotein (oxLDL) has been implicated in the pathogenesis of atherosclerosis [1–4]. Several important steps during atherogenesis have been studied in cultured endothelial cells as a model for potential actions of oxLDL in vivo, including modulation of NO production [5,6], expression of adhesion molecules [7,8], potentiation of cytokine-stimulated expression of adhesion molecules [9,10], and cytotoxicity [11,12]. Reac* Corresponding author. Tel.: 011-886-3-328-3016, ext. 5095; fax: 011-886-3-328-3031. E-mail address: [email protected] (Y.T. Lau) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 4 8 6 -2

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tive oxygen species (ROS) are likely involved as an early response in some of those effects [11,13–16]. 17b-Estradiol (estrogen) exerts a protective effect on vascular endothelium [for review, see refs. 17,18] partly via its antioxidant actions [19–21], and recent results indicate that this may occur by estrogen acting as a ROS scavenger to inhibit lipid peroxidation [22,23] and the formation of oxLDL [24,25]. Furthermore, hormone replacement therapy in general improves cardiovascular functions in postmenopausal women [26,27]. However, whether estrogen inhibits the stimulated ROS generation induced by oxLDL in endothelial cells is unknown. With the successful application of 29, 79-dichlorofluorescin diacetate (DCFH-DA) in the measurement of intracellular ROS production in endothelial cells [28,29], it has become possible to evaluate directly the effect of estrogen on oxLDL-induced ROS production in cultured endothelial cells. We therefore investigated whether estrogen could prevent (by pre-incubation) or inhibit (by co-incubation) ROS generation upon stimulation by oxLDL or the chemical ROS generator (TBH) in cultured human umbilical vein endothelial cells (HUVECs). Materials and methods Isolation and culture of endothelial cells Human umbilical vein endothelial cells (HUVECs) were harvested as described previously and were grown in MCDB107 medium supplemented with 2% fetal calf serum (FCS) and 0.2% partially purified growth factor [30,31]. Near-confluent cell monolayer (2–5 passage) in 75T-flask were used. For experiments, HUVECs were pre-incubated in phenol red-free MCDB107 medium containing 2% charcoal-treated FCS and 0.2% growth factors (modified medium) overnight to avoid the interference of phenol red and steroid hormone. Determination of intracellular reactive oxygen species (ROS) generation The intracellular ROS measurement was performed in cell suspension by flow cytometry as described previously [28,32]. DCFH-DA is a lipid permeable, nonfluorescent compound and is oxidized by intracellular ROS to form the lipid impermeable and fluorescent compound DCF [28,29]. Organic tert-butylhydroperoxide (TBH), a model of a hydroperoxide compound, and H2O2 were also used [31] in addition to oxLDL to test whether estrogen’s effect against ROS generation was independent of how cells were activated. Two concentrations of estrogen were employed: 1 mM to mimic the maximum level of estrogen after hormone replacement therapy (0.2 – 1 mM) [23], and 54 mM to induce maximum vasorelaxation [34] and to prevent cell death [35]. This high concentration of estrogen was chosen because lipid peroxidation and cell death stimulated by oxLDL were also significantly reduced by 54 mM of estrogen [22,23]. To assess the direct effect of estrogen, the cells were preincubated in modified medium overnight. Cells were trypsinized (0.05% trypsin) and then resuspended in MCDB107 (1 ml, containing 2% FCS) to a final concentration of 106 cells/ml. DCFH-DA (10 mM) and stimulator (TBH or oxLDL) were added in cell suspension with or without estrogen followed by incubation at 378C for 30 min. The cells were then centrifuged and resuspended for immediate determination of ROS generation by flow cytometry (FACscan, Becton-Dickinson, CA) using 488 nm for excitation and 525 nm for emission.

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For the preventative study, the cells were preincubated with estrogen in modified medium for 18 hours. The cells were then trypsinized, washed, and resuspended to 106 cells/ml. DCFH-DA (10 mM) and stimulators (TBH or oxLDL) were added in the absence of estrogen followed by incubation at 378C for 30 min. Intracellular ROS was then analyzed as described previously. LDL preparation and oxidative modification Healthy human serum was obtained from Chang Gung Memory Hospital and LDL (density: 1.02 – 1.06) was isolated by sequential ultracentrifugation as described previously [32]. Freshly prepared native LDL was treated with 50 mM FeSO4 (in 0.9% NaCl solution) for up to 24 hours at 378C. The degree of oxLDL oxidation was determinated by analyzing the level of thiobarbituric acid-reactive substances (TBARS) expressed as malondialdehyde (MDA) content and by gel electrophoresis [32]. The TBARS level of oxLDL used in this study was 22.4 nmole MDA/mg LDL protein, and it exhibited a greater negative charge than the native LDL in gel electrophoresis. The TBARS level of the non-modified native LDL preparation (nLDL) was less than 0.1 nmole MDA/mg LDL protein. In most experiments, a comparable concentration of nLDL was added as a control for ROS determination. In all preparations of nLDL tested, none exerted any stimulation of the flurorescence signal (Table 1). For the sake of clarity these data were not shown in other tables or figures. Results We first tested the effect of native LDL on flurorescence response (ROS production) and found that at concentrations of 100 or 300 mg/ml, nLDL slightly suppressed ROS production (Table 1). Both H2O2 (100 mM) and TBH ($ 50 mM) caused significant increases of ROS as summarized in Table 1. Production of ROS by HUVECs exhibited a dose-dependent response over a 10-fold range of TBH concentration. H2O2 (100 mM) caused a smaller stimulation than TBH at an equimolar concentration. These results are consistent with the report that DCFH-DA serves as a useful index for intracellular ROS in non-adherent endothelial cell preparations [28,29]. Table 1 ROS generation following treatment by nLDL, H2O2, or TBH Fold-increase in fluorescence nLDL (mg/ml) 100 0.87 6 0.03

H2O2 (mM) 300 0.85 6 0.02

100 11

2.7 6 0.1

TBH (mM) 50 11,

4.8 * 6 0.4

100 11,

5.1 * 6 0.5

500 7.2111 6 0.1

HUVECs were treated by nLDL, H2O2 or TBH and DCF fluorescence measured after 30 min. Control (DCFHDA dye only) was present in each experiment (exhibited fluorescence intensity of about 100–200 units) and at least duplicate determinations were performed. The results were mean stimulation (normalized to the control of each experiment) from 2–7 separate experiments. (Different from control: 11 P,0.001; Different from 500 mM TBH: * P,0.05).

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The effects of estrogen on ROS generation in HUVECs were tested by either directly adding estrogen together with stimuli (oxLDL or TBH) or pretreating cells (18 h) with estrogen before oxLDL exposure. The results of a typical experiment of the direct effect is shown in Fig. 1. OxLDL (200 mg/ml) produced about a 2-fold increase in ROS generation (Fig. 1B, dark trace) as compared with controls (Fig. 1A, light trace). Cominacini et al. [36] also reported that Cu21-modified LDL (oxLDL) dose-dependently increased ROS production in HUVECs over a 20-fold range of oxLDL concentration. Estrogen (1 mM) inhibited (left shift) the oxLDL-induced ROS significantly (Fig. 1C) while 54 mM almost completely inhibited ROS production (Fig. 1D). Fig. 2 summarizes the results from 3–7 such experiments with different stimuli. Estrogen produced a dose- dependent inhibition of oxLDL-induced ROS production in HUVECs (Fig. 2A) as well as of TBH-induced ROS (Fig. 2B). The preventative effect of estrogen on oxLDL-induced ROS production is shown in Fig. 3. We found that oxLDL stimulated ROS level by more than 2-fold (Fig. 3B dark trace vs. light trace). Estrogen (1 mM) slightly reduced ROS (Fig. 3C), while 54 mM of estrogen pretreatment completely eliminated oxLDL-induced ROS production (Fig. 3D). Similarly, estrogen pretreatment also protected against TBH-induced ROS generation (the results of 3 experiments are shown in Table 2). While 1mM estrogen exhibited borderline inhibition, the high concentration of estrogen produced almost the same degree of inhibition (near 60%) of oxLDL-induced ROS generation as that produced by direct application of estrogen to the cells (Fig. 2A). We next tested the involvement of estrogen receptors (ER) in these actions. The ER antagonist ICI 182,780 (1 mM) did not significantly alter ROS generation produced by various stimuli and in the presence of estrogen (data not shown). Tamoxifen (1 mM), on the other hand, enhanced estrogen’s protective action by 23%. Because only a limited range of concentrations of ER antagonist was employed, further investigations are required to evaluate the role of ER in cellular signaling. Discussion We have demonstrated that estrogen inhibited stimulant-generated ROS in endothelial cells under two different conditions: (1) direct exposure of estrogen to cells with stimulant (Fig. 2) and (2) pretreatment of cells with estrogen prior to stimulation (Table 2). The nature of the direct effect is not clear. In the absence of stimuli, estrogen exerted no effect on (basal) ROS production of HUVECs (data not shown). In the presence of oxLDL, co-incubation with estrogen reduced the stimulated ROS by more than one-half (Fig. 2A). A similar concentration of estrogen inhibited LDL oxidation by 70% in vitro [22,23], but whether such an antioxidative effect accounts for our observations is not known. A recent report also indicated that 30 min preincubation of HUVECs with radical scavengers (trolox, vitamin C, and troglitazone) significantly reduced the oxLDL-induced ROS production [36]. However, oxLDL stimulates ROS generation rapidly in HUVECs with a receptor mediated effect [36]. Our finding that estrogen acted rapidly does not exclude the possibility that the antioxidant effect of estrogen was mediated by indirect mechanism(s) as well. In fact, rapid non-genomic increase of endothelial NO has been reported with estrogen treatment [37,38,39], which may exert a protective effect. We further investigated whether pretreatment with estrogen prevented oxLDL-induced

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Fig. 1. Direct effect of estrogen on oxLDL-induced ROS production in HUVECs. A. control, dye only (light trace); B. oxLDL (200 mg/ml) alone (dark trace); C. oxLDL with 1mM estrogen (dark trace); D. oxLDL with 54mM estrogen (dark trace).

ROS in HUVECs. A direct effect of extracellular estrogen was excluded by washing and replacing solution to remove estrogen before oxLDL addition. We found that pretreatment with estrogen was able to inhibit the oxLDL-induced ROS generation (Fig. 3 and Table 2). The precise mechanisms have yet to be determined but several possible cellular effects could be

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Fig. 2. Direct effect of estrogen on oxLDL (200mg/ml) (A) and TBH (100 mM) -induced (B) ROS production in HUVECs. Results were obtained from 3–7 experiments (stimulants only; open column).

involved. Firstly, estrogen is known to up-regulate endothelial NO synthase and enhances NO production in cultured endothelial cells, thus acting as an antioxidant [40,41]. However in pretreatment experiments, neither an inhibitor of NO synthase, e.g., Nv-nitro-L-arginine methylester (L-NAME, 0.1 mM), nor a NO donor (SNAP, 0.1 mM) significantly altered estrogen’s protective role during oxLDL challenge (data not shown). Secondly, estrogen and estradiol benzoate stimulate the activity of detoxifying enzymes such as catalase [42] or glutathione peroxidase [37,43] and inhibits superoxide anion formation at a micromolar concen-

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Fig. 3. Preventative effects of estrogen on oxLDL-induced ROS production in HUVECs. A. control (fluorescent dye only, light trace); B. oxLDL (200 mg/ml) only (dark trace); C. estrogen (1 mM) -pretreated (18h); D. estrogen (54 mM) -pretreated.

tration [22] in cultured endothelial cells. Recent findings also indicate that estrogen, but not progesterone or testosterone, mediates the induction (24h) of the protein thiol/disulfide oxidoreductases which may be involved in antioxidant protection [44]. An estrogen receptorindependent synergistic interaction between estrogen and glutathione for neuroprotection against amyloid-induced toxicity has also been reported [45]. Estrogen also exerts antioxidant effects in myocardial ischemia/reperfusion by increasing glutathione levels and glucose6-phosphate dehydrogenase enzyme activity [46]. At the tissue level, estrogen activates SOD- and catalase- sensitive endothelium-dependent relaxation of blood vessels [47]. Thirdly, estrogen may directly or indirectly (e.g., via NO production) modulate endothelial Table 2 Inhibition of ROS generation by estrogen pretreatment Estrogen protection (% inhibition) Stimulation oxLDL TBH

1 mM

54 mM

42.5 6 16.5 17.9 6 4.5

57.8 6 20.8* 39.7 6 4.8*

HUVECs were stimulated by oxLDL (200 mg/ml) or TBH (0.1 mM) and fluorescence measured after 30 min, subtracting control, was taken as 100%. Cells pretreated by 1 or 54 mM estrogen (18h) exhibited less fluorescence and the mean differences (inhibition) were calculated from 4 independent determinations. The results were analyzed by one-way ANOVA with Tukey-Kramer multiple comparison test and * represents p , 0.05 when compared with control.

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oxidative enzymes such as xanthine oxidase [48] and cyclooxygenase [49]. Our results thus are consistent with the notion that pre-treatment (18h) of estrogen on HUVECs appeared to blunt the later challenge by TBH (Fig. 2) or oxLDL (Fig. 3), possibly by altering antioxidant/ oxidant balance to reduce ROS generation. The reduction of ROS production by supra-micromolar concentrations of estrogen employed here could potentially alleviate oxidative stress-induced cytotoxicity. Both lipid peroxidation and cytotoxicity are significantly reduced by estrogen (.10 mM) in endothelial cells [12] and hepatocytes [35] induced by oxLDL and TBH, respectively. We have recently demonstrated that catalase and deferoxamine reduced both ROS generation and extent of apoptosis in vascular smooth muscle cells [50]. Such evidence suggests that partial inhibition of ROS production or oxidative stress could provide cytoprotective effects. However, a quantitative relationship may be difficult to establish because ROS may exert both direct action and indirect (signaling) cascade to cells. Endogenous estradiol could be elevated more than 10-fold following hormone replacement therapy (HRT) in postmenopausal women (51,52). This increase of estradiol is associated with significant increases of serum total antioxidant status as well as of sulfhydryl groups, and with a decrease of lipoperoxides in women (52). Considering the antioxidant role of NO, these effects are consistent with the observations that HRT also increases serum NO (53) while ovariectomy reduced acetylcholine-induced vasodilation (54). Thus, it is conceivable that endogenous estradiol provides antioxidative effects. The effect of the putative active component of oxLDL, e.g. lysophosphatidylcholine, was recently examined by direct determination of ROS production in HUVECs [55]. Our approach could therefore be extended to investigate the sites/mechanisms/active component of oxLDL action and the role of estrogen protection. In summary, we found that estrogen, at a concentration effective in protecting against lipid peroxidation and DNA damage [22], significantly inhibited oxLDL-induced ROS production in endothelial cells, suggesting that it may be associated with its cardioprotective effect seen after estrogen therapy. Acknowledgments Work described in this study was supported by the National Science Council, ROC (NSC 89-2320-B-182-027) and Chang Gung University (CMRP 736) to Y.T.Lau. References 1. Avogaro P, Bon GB, Cazzolato G. Presence of a modified low density lipoprotein in humans. Arteriosclerosis 1988;8(1):79–87. 2. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol. Modification of lowdensity lipoprotein that increase its atherogenicity. New England Journal of Medicine 1989;320(14):915–24. 3. Rosenfeld ME. Oxidized LDL affects multiple atherogenic cellular responses. Circulation 1991;83(6):2012–20. 4. Wiklund O, Mattsson L, Bjornheden T, Camejo G, Bondjers G. Uptake and degradation of low density lipoproteins in atherosclerotic rabbit aorta: role of local LDL modification. Journal of Lipid Research 1991;32(1):55–62. 5. Cohen RA. The role of nitric oxide and other endothelium-derived vasoactive substances in vascular disease. Progress in Cardiovascular Diseases 1995;38(2):105–28.

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