Estrogen Receptor Activation Protects Against TNF-α-Induced Endothelial Dysfunction

Abstract

Lack of estrogen is a cause of cardiovascular disease in men and postmenopausal women. We examined the effects of estrogen receptor (ERs) activation/inactivation on endothelial cells subjected to tumor necrosis factor (TNF) α, which is involved in vascular disease pathogenesis. Endothelial nitric oxide synthase (eNOS) and matrix metalloproteinases (MMP) 9 expression, as well as protein kinase B (PKB) activation were evaluated as markers of endothelial dysfunction. The TNF-α induces eNOS and MMP-9 expression and PKB activation. The ER activation by apigenin, a nonsteroidal compound with estrogen-like activity mediated through ER binding-dependent pathways, counteracts these effects. These effects are reversed by classic (ER-α and ER-β) and nonclassic (G-protein-coupled receptor) ER inhibitors (ICI182 780 and pertussis toxin, respectively). Our data suggest that ER activation counteracts endothelial dysfunction induced by TNF-α. The use of ER activators, such as apigenin, may represent a strategy to prevent vascular disease associated with endothelial dysfunction, while avoiding the feminizing effects of estrogens.

Keywords

apigenin endothelial dysfunction eNOS estrogen receptor MMP-9 TNF-α

Introduction

Epidemiological data suggest that premenopausal women are largely protected from cardiovascular disease (CVD) compared to men of similar age. This phenomenon, referred to as the “female advantage,” is gradually lost after menopause, so that by 60 years of age, women have the same incidence of CVD as men. The protection of premenopausal women from CVD has been attributed to the cardioprotective effect of female sex hormones^1,2 such as estrogens. Estrogens are involved in many physiological processes, which are important for cardiovascular health, and this effect may be mediated via estrogen receptors (ERs). In addition to the classic receptors, ER-α and ER-β, a novel G-protein-coupled receptor, GPR30, has been described as a nonclassic ER and appears to mediate the rapid nongenomic response to estrogens.^3,4

Reduced ER levels have been associated with the development of coronary artery disease (CAD) in females. In coronary artery samples from pre- and postmenopausal women, ERs were expressed in the majority of normal arteries, but only in a minority of arteries obtained from women with CAD.⁵ This is supported by reports indicating an increase in methylation (and an associated decrease in expression) of the gene encoding ER-α in human coronary atherosclerotic plaques compared to normal aortic tissue.⁶ Thus, reduced estrogenic action, either as a result of decreased estrogen levels or reduced numbers of functional ERs in blood vessels, likely constitutes a considerable CVD risk factor. Although the cardioprotective effects of estrogens can be attributed, in part, to indirect regulation of circulating lipid levels, vascular tone, and hemostasis, a direct effect on blood vessels has also been demonstrated. Several studies have reported the presence of functional ERs in vascular endothelial cells, suggesting that ERs can directly regulate endothelial function.^7,8 However, the precise mechanism of regulation is not fully understood.

The aim of this study was to investigate the effects of ER activation/inactivation on endothelial dysfunction induced by tumor necrosis factor (TNF) α, by evaluating endothelial function markers such as matrix metalloproteinases (MMPs) 9, endothelial nitric oxide synthase (eNOS), and protein kinase B (PKB). For this purpose, we used a pharmacological approach that included apigenin, a nonsteroidal compound to estrogen-like activity mediated through ER binding-dependent pathways, and ER inhibitors ICI182 780 and pertussis toxin (PTX).

Materials and Methods

Cell Culture Conditions and Treatments

EAhy926 endothelial cells were cultured in Dulbecco modified Eagle medium with 10% fetal calf serum. For treatment experiments, cells were cultured in serum-free medium (SFM) for 2 hours, and then left untreated or were treated with 20 or 50 µmol/L of the ER activator apigenin (A3145, Sigma-Aldrich, MO, USA) for 1 hour and then with 1 or 10 ng/mL of TNF-α (T6674, Sigma-Aldrich). In the experiments with ICI182 780 (I4409, 10 µmol/L; Sigma-Aldrich), PTX (P-7028, 4 µg/mL; Sigma-Aldrich), and PKB inhibitor (124 005, 40 µmol/L; Calbiochem, Germany), cells were pretreated with the inhibitors for 1 hour. After 24 hours, cells were lysed in radioimmunoprecipitation assay buffer (5 mmol/L Tris-HCl [pH 7.5], 150 mmol/L NaCl, 1% nonyl phenoxypolyethoxylethanol [NP-40], 0.5% deoxycholic acid, and 0.1% sodium dodecyl sulfate [SDS]), and conditioned media (CM) were collected, centrifuged to remove cells and debris, and immediately frozen at −80°C. For intracellular signaling experiments, confluent cells were preincubated for 6 hours in SFM and left untreated or were treated for short pulses (5-60 minutes) with TNF-α. The cells were lysed in ice-cold lysis buffer (1% NP-40, 150 mmol/L NaCl, 50 mmol/L Tris HCl [pH 8.0], 5 mmol/L EDTA, 10 mmol/L NaF, 10 mmol/L Na₄P₂O₇, and 0.4 mmol/L Na₃VO₄).

The MMP-9 Zymography

Equivalent protein amounts (by bicinchoninic acid assay methods) from CM were loaded onto a 0.28% w/v gelatin (type A) SDS-acrylamide gel; electrophoresed at 6°C to 8°C; rinsed twice for 30 minutes in 2.5% Triton X-100; incubated for 16 to 18 hours at 37°C in 40 mmol/L Tris-HCl, 0.2 mmol/L NaCl, and 10 mmol/L CaCl₂; stained with 0.2% Coomassie Blue in 50% methanol and 10% acetic acid; and destained in 50% methanol and 10% acetic acid.

Western Blotting

Fifty micrograms of protein from cell lysates (100 μg for intracellular signaling) were electrophoresed on reducing SDS-acrylamide gels and then electrotransferred to nitrocellulose membranes. The membranes were saturated at room temperature for 1 hour in Tween-Tris-buffered saline (TTBS; 20 mmol/L Tris-HCl [pH 7.5], 500 mmol/L NaCl, 0.01% Tween 20, and 5% nonfat milk). The membranes were incubated with primary antibody for 16 hours in TTBS and then with horseradish peroxidase-secondary antibody (Cell Signaling Technology, MA, USA) for 1 hour in TTBS. The bands were visualized by ECL chemiluminescence (Millipore, Germany). The membranes were stripped with a denaturing solution (62.5 mmol/L Tris-HCl (pH 6.8), 2% SDS, and 100 mmol/L β-mercaptoethanol) for 20 minutes at 50°C, and then reblotted with polyclonal actin. Bands were quantified by optical densitometry, using the gel analysis system GeneGenius (Syngene, Cambridge, UK). The primary antibodies used were polyclonal anti-eNOS (07-520, 1:1000 dilution; Millipore), polyclonal anti-phospho-PKB (sc-7985, 1:200 dilution; Santa Cruz, TX, USA), and polyclonal anti-actin (sc-1615, 1:1000 dilution; Santa Cruz, TX, USA).

Statistical Evaluation

Each experiment was repeated 3 to 5 times. Data shown are mean ± standard error of the mean. The statistical significance of the results was determined using analysis of variance followed by Fisher test. P < .05 was considered significant.

Results

The TNF-α-Induced Endothelial Dysfunction in EAhy926 Cells

We first examined whether TNF-α modulated the expression of eNOS and MMP-9, 2 key molecules associated with endothelial dysfunction. As shown in Table 1, TNF-α upregulated eNOS and MMP-9 expression in a dose-dependent manner. Similarly, treatment with TNF-α induced a time-dependent phosphorylation of PKB (Figure 1). To determine whether PKB could be involved in the induction of eNOS and MMP-9 by TNF-α, we treated cells with a PKB inhibitor prior to TNF-α treatment. Inhibition of PKB by its inhibitor led to a reduction in TNF-α-induced expression of eNOS, while only a slight, but significant, effect on MMP-9 expression was detected (Table 2). These data suggest that the induction of eNOS and, partially, of MMP-9, by TNF-α is mediated by PKB activation.

Table 1.

Effects of TNF-α on the Expression of eNOS and MMP-9 in EAhy926 Endothelial Cells.^a

	eNOS (% of Control)	MMP-9 (% of Control)
Control	100.0 ± 5.1	100.0 ± 12.2
TNF-α (1 ng/mL)	120.1 ± 10.0^b	230.3 ± 18.3^c
TNF-α (10 ng/mL)	171.5 ± 9.5^c	316.2 ± 19.1^c

Abbreviations: eNOS, endothelial nitric oxide synthase; MMP, matrix metalloproteinase; SD, standard deviation; TNF, tumor necrosis factor.

^a Data are represented as means ± SD after densitometric analysis of Western blot for eNOS expression and zymography for MMP-9 activity.

^b P < .05.

^c P < .01.

Table 2.

Effects of PKB Inhibitor on the Expression of eNOS and MMP-9 in EAhy926 Endothelial Cells Treated With TNF-α.^a

	eNOS (% of Control)	MMP-9 (% of Control)
Control	100.0 ± 5.1	100.0 ± 12.2
TNF-α (10 ng/mL)	171.5 ± 9.5	316.2 ± 19.1
TNF-α + PKB inhibitor	115.9 ± 6.2^b	262.5 ± 13.0^c

Abbreviations: eNOS, endothelial nitric oxide synthase; MMP, matrix metalloproteinase; PKB, protein kinase B; SD, standard deviation; TNF, tumor necrosis factor.

^a Data are represented as means ± SD after densitometric analysis of Western blot for eNOS expression and zymography for MMP-9 activity.

^b P < .05.

^c P < .01.

The ER Activation by Apigenin Counteracted TNF-α-Induced Endothelial Dysfunction in EAhy926 Cells

We used apigenin to activate ERs in endothelial cells subjected to TNF-α treatment. We assessed whether ER activation modulated TNF-α-induced eNOS and MMP-9 expression. As shown in Table 3, apigenin decreased the TNF-α-induced expression of eNOS in a dose-dependent manner. Similarly, MMP-9 expression, strictly associated with TNF-α treatment, was decreased to basal levels by apigenin (Table 3). Finally, apigenin significantly inhibited TNF-α-induced phosphorylation of PBK (Figure 2). These data suggest that ER activation counteracts TNF-α-induced endothelial dysfunction.

Table 3.

Effects of Apigenin on the Expression of eNOS and MMP-9 in EAhy926 Endothelial Cells Treated With TNF-α.^a

	eNOS (% of Control)	MMP-9 (% of Control)
Control	100.0 ± 5.1	100.0 ± 12.2
TNF-α (10 ng/mL)	171.5 ± 9.5	316.2 ± 19.1
TNF-α + apigenin (20 μmol/L)	120.3 ± 10.2^b	230.0 ± 18.5^c
TNF-α + apigenin (50 μmol/L)	91.4 ± 11.1^c	103.3 ± 17.6^c

Abbreviations: eNOS, endothelial nitric oxide synthase; MMP, matrix metalloproteinase; SD, standard deviation; TNF, tumor necrosis factor.

^a Data are represented as means ± SD after densitometric analysis of Western blot for eNOS expression and zymography for MMP-9 activity.

^b P < .05.

^c P < .01.

The ER Inactivation by ICI182 780, and PTX Reversed the Inhibitory Effects of ER Activation on Endothelial Dysfunction in EAhy926 Cells

We next investigated whether inhibition of the classic (ER-α and ER-β) and/or the nonclassic (GPR30) ERs could reverse the inhibitory effects of apigenin on TNF-α-induced eNOS and MMP-9 expression. For this purpose, we used the ER-α and ER-β antagonist ICI182 780 and PTX, a potent inhibitor of the GPR30. As shown in Table 4, ICI182 780 and PTX treatment significantly counteracted the effect of ER activation on TNF-α-induced eNOS expression. Moreover, the inhibitory effect of ER activation on MMP-9 expression was slightly reversed by both ICI182 780 and PTX pretreatment (Table 4). Finally, treatment with ICI182 780 and PTX reversed the effect of apigenin on the TNF-α-triggered activation of PKB (Figure 3). Our data indicate that both classic (ER-α and ER-β) and nonclassic (GPR30) ER activation counteract TNF-α-induced endothelial dysfunction.

Table 4.

Effects of ER Inhibitors on the Expression of eNOS and MMP-9 in EAhy926 Endothelial Cells Treated With TNF-α and Apigenin.^a

	eNOS (% of Control)	MMP-9 (% of Control)
TNF-α (10 ng/mL)	100.0 ± 9.5	100.0 ± 19.1
TNF-α + apigenin (50 μmol/L)	53.2 ± 5.2	32.9 ± 18.5
TNF-α + apigenin (50μmol/L) + ICI187 780	98.7 ± 8.0b	54.5 ± 6.5b
TNF-α + apigenin (50 μmol/L) + PTX	73.0 ± 3.2c	49.4 ± 4.5b

Abbreviations: eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; MMP, matrix metalloproteinase; PTX, pertussis toxin; SD, standard deviation; TNF, tumor necrosis factor.

^a Data are represented as means ± SD after densitometric analysis of Western blot for eNOS expression and zymography for MMP-9 activity.

^b P < 0.

^c P < .05.

Discussion

The initiation and progression of CVD are associated with dysregulated expression of molecules involved in vascular tone, inflammation, and remodeling that results in endothelial dysfunction.⁹ It is well-known that estrogens protect individuals from CVD by playing a role in lipid metabolism, vascular tone, and hemostasis.¹⁰ The presence of functional ERs in vascular endothelial cells suggests that ERs can directly regulate endothelial function. However, the precise mechanism by which this occurs is not fully understood. To further elucidate this mechanism, we examined the effects of ER activation/inactivation on endothelial cells rendered dysfunctional by TNF-α treatment. We chose TNF-α because many risk factors, such as aging, smoking, inflammation, hyperlipidemia, and hyperglycemia, contribute to the pathogenesis of vascular disease, at least in part, through modulation of TNF-α signaling.¹⁰ These data suggest that TNF-α may play a pivotal role in the initiation and progression of vascular disorders. To focus on the role of ERs in regulating endothelial function, we used a pharmacological approach that included the ER-α and ER-β antagonist ICI182 780, a potent inhibitor of the GPR30, PTX, and the flavone ER activator apigenin.

The major finding of this study is that ER activation exerts a protective effect on endothelial cells by inhibiting TNF-α-induced expression of MMP-9 and eNOS, as well as activation of PKB signaling, all markers of endothelial dysfunction. The MMP-9 is responsible for many complications in vascular disease such as vessel wall degradation and angiogenesis.^11

–14 Furthermore, eNOS is implicated in vascular homeostasis regulation and plays a protective role in the vascular system. However, in the presence of TNF-α, this positive effect of eNOS on the endothelium is reversed. The TNF-α dramatically increased the level of reactive oxygen species (ROS) and transformed eNOS to an O₂-generating enzyme.^15,16 The dual activation of the superoxide- and NO-generating systems provided a favorable environment for protein nitration, which is strongly associated with connective tissue destruction, cytotoxic effects in endothelial cells, and vascular disease.^17,18,19 Therefore, the induction of eNOS expression after exposure to TNF-α represents a deleterious compensatory mechanism due to a significant decrease in NO bioavailability, coupled with dramatic increases in ROS levels and protein nitration. Thus, our results indicate that ERs play a protective role against TNF-α-induced endothelial dysfunction by preventing the deleterious activation of the prooxidant and proteolytic mechanisms responsible for vascular disease progression. In particular, eNOS expression is strongly regulated by classic ERs and, to a lesser extent, by GPR30, as indicated by our experiments with ICI187 780 and PTX. Moreover, a slight, but significant, involvement of all ERs in the regulation of MMP-9 was detected. Our results also indicate that activation of both classic ERs and GPR30 leads to a rapid nongenomic response that provides efficient inhibition of the PKB signaling pathway activated by TNF-α. The PKB activation by endothelial cell stimuli, including TNF-α, is involved in angiogenesis and vascular tone, suggesting that PKB plays a pivotal role in vascular disease progression.^20,21 Since our data indicate that eNOS and MMP-9 induction by TNF-α depends, to a different extent in each case, on PKB activation, the vasoprotective role of activated ERs described here may be mediated by their ability to inhibit PKB signaling. In this context, the use of ER activators, such as apigenin, may represent a new strategy for the prevention of vascular disease associated with endothelial dysfunction. Apigenin is a naturally occurring plant flavone that is abundantly present in common fruits and vegetables.²² It is a nonsteroidal compound with estrogen-like activity on the vascular endothelium, but with an antiestrogenic effect on the mammary glands and endometrium.^23,24 Apigenin’s role in counteracting the inflammatory response in endothelial cells has been investigated.²⁵ Our study suggests that ER activation by apigenin counteracts 2 key markers of endothelial dysfunction, MMP-9 and eNOS. Therefore, apigenin may be useful in both men and women to improve endothelial function, while avoiding the feminizing side effects of estrogens that have emerged with the use of estrogenic therapies against CVDs.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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