Sulforaphane attenuates angiotensin II-induced human umbilical vein endothelial cell injury by modulating ROS-mediated mitochondrial signaling

Abstract

The study aimed to investigate whether sulforaphane (SFN) protects against angiotensin II (Ang II)-mediated human umbilical vein endothelial cell (HUVEC) injury. Ang II treatment decreased HUVEC viability, increased cell apoptosis, decreased mitochondria membrane potential (MMP), impaired cytochrome c release, activated caspase 3/9, and induced reactive oxygen species (ROS) production, and nicotinamide adenine dinucleotide phosphate oxidase activity. Moreover, SFN treatment blunted Ang II-stimulated oxidative stress and mitochondria-related apoptosis in HUVECs. The ROS scavenger N-acetyl-l-cysteine reduced Ang II-induced oxidative stress and apoptosis, indicating that ROS generation is involved in the Ang II-induced mitochondria-mediated apoptotic pathway. SFN induced nuclear factor erythroid 2 (Nrf2) activation and expression in Ang II-stimulated HUVECs. Downregulation of Nrf2 expression by a target-specific siRNA revealed an Nrf2-dependent effect on the SFN-mediated attenuation of Ang II-induced apoptosis in HUVECs. Pretreatment with brusatol, an Nrf2-specific inhibitor, reversed the protective effects of SFN on Ang II-induced HUVEC injury. SFN treatment protected HUVECs from Ang II-induced damage by decreasing oxidative stress and ameliorating mitochondrial injury.

Keywords

Sulforaphane angiotensin II human umbilical vein endothelial cells oxidative stress Nrf2

Introduction

Atherosclerosis (AS) is the leading cause of coronary heart disease and has high mortality and morbidity rates. Endothelial dysfunction predicts AS, cardiovascular events, and long-term clinical outcomes. Reactive oxygen species (ROS) play a key role in endothelial cell (EC) injury and in the development of cardiovascular disease (CVD).^1,2 Approaches designed to improve endothelial function may have additional therapeutic value in the prevention and treatment of atherosclerotic diseases.

Angiotensin II (Ang II) is a key independent risk factor for hypertension, inducing the development of AS and CVD.^3,4 Ang II has been shown to stimulate the generation of nicotinamide adenine dinucleotide phosphate (NADPH) in vascular cells. Small artery structures are then altered through the activation of signaling pathways such as the MEK1/ERK and PI3/Akt pathways.^5
–7 Consequently, intracellular ROS production increases and induces EC apoptosis and dysfunction.Previous studies have demonstrated that Ang II induces apoptosis in HUVECs. Amlodipine attenuates Ang II-induced endothelial apoptosis via the Bcl-2/Bax pathway.⁸ Safflor yellow B reverses Ang II-induced HUVEC injury by increasing Bcl-2 expression and suppressing ROS generation.⁹ Celastrol ameliorates Ang II-induced apoptosis in HUVECs via nuclear factor erythroid 2 (Nrf2)-related factor 2 activation.¹⁰ Ursodeoxycholic acid reverses acute aortic dissection formation in Ang II-treated apolipoprotein E deficient mice by reducing the generation of ROS and increasing Nrf2 activation. However, the molecular mechanisms of Ang II-induced HUVECs apoptotic signaling events are not yet fully understood.

Nrf2 is involved in protecting cells from oxidative stress. Low Nrf2 levels have been observed under normal conditions. Oxidative stress may lead to the activation of Nrf2 and its translocation to the cell nucleus. The accumulated Nrf2 in the nucleus upregulates the transcription of antioxidant enzymes.^11,12 Nrf2 cooperates with the antioxidant response element (ARE) to stimulate ARE-mediated gene expression. The deactivation of Nrf2 is binding to Kelch-like ECH-associated protein 1 (Keap1) under physiological conditions. Under oxidative stress, the activation of Nrf2 leads to its translocation into the cell nucleus to elicit the expression of heme oxygenase 1. Inhibition of Nrf2 by the specific inhibitor brusatol has been demonstrated to suppress the Nrf2-mediated antioxidant role.¹³ Here, we investigated the effect of Nrf2 on Ang II-mediated endothelial injury and detected its molecular mechanisms.

(−)-Sulforaphane (SFN), a key compound isolated from cruciferous vegetables, has anti-inflammatory, antioxidant, and antiangiogenic effects in vitro and in vivo.^14
–16 SFN has consistently been shown to increase apoptosis in cancer cells.^17,18 According to a few studies, SFN ameliorates the progression of atherosclerotic lesions and vascular injury.¹⁹ Accumulating evidence suggests that SFN inhibits endothelial inflammatory response.^20,21 SFN is regarded as a potential Nrf2 inducer, an antioxidant transcription factor that induces the expression of antioxidant enzymes, including glutathione transferase.^22,23 SFN is a potent activator of the Keap1-Nrf2-ARE pathway that can induce protective and preventive effects in oxidative stress-mediated cytotoxicity and genotoxicity.^24,25 Results from in vivo and in vitro reports indicate a protective role for SFN in vascular system injuries.²⁶ Most previous studies used a higher dose of SFN (>2 μM), thus most investigations have revealed the pharmacological effect of SFN in vitro and in vivo.²⁷ A past study showed that even lower concentrations (0.05–1 uM) of SFN are able to induce antioxidant mechanisms.²⁸ Consequently, the physiological effects of these SFNs remain unknown. In addition, researchers have not determined whether SFN ameliorates Ang II-induced damage to cells and mitochondrial dysfunction in human ECs through Nrf2 signaling.

In this study, we aimed to investigate the potential protective effects of SFN on Ang II-induced HUVEC injury, as well as the underlying mechanisms. Our study is the first to suggest that SFN attenuates Ang II-mediated HUVEC injury, at least in part, by activating Nrf2 signaling.

Materials and methods

Cell culture

HUVECs were obtained from All Cells Technology (Shanghai, China). Cells were seeded in complete medium (HUVEC-004, All Cells) and cultured in an incubator at 37°C with 5% CO₂. Different concentrations of [Val5]-Ang II acetate salt hydrate (Ang II, Sigma-Aldrich, St Louis, Missouri, USA), ranging from 0 nM to 400 nM, were added to the medium. SFN was also purchased from Sigma-Aldrich. The SFN stock solution was prepared in sterile double-distilled water. Brusatol was purchased from Tauto Biotech (Shanghai, China). HUVECs were pretreated with brusatol before Ang II and SFN were added.

Human umbilical vein EC viability assay

HUVEC viability was measured using an thiazolyl blue tetrazolium bromide (MTT) assay according to the manufacturer’s instructions (MTT, Sigma-Aldrich).²⁹ The absorbance was measured at 490 nm using a microplate reader (Bio-Rad, Berkeley, California, USA). The results are presented as percentages of the control.

LDH assay

Lactate dehydrogenase (LDH) release was analyzed using an LDH activity assay kit (Sigma-Aldrich) according to the manufacturer’s instructions.³⁰ Supernatants (50 μL) were incubated with NADH (reduced form) and pyruvate for 15 min at 37°C. Finally, the absorbance of the samples was measured at 440 nm in a microtiter plate reader (Bio-Rad).

Measurement of HUVEC apoptosis using Annexin V-FITC/PI staining

HUVECs were exposed to Ang II and SFN for 24 h, collected, washed with phosphate-buffered saline, and resuspended in binding buffer. The cell suspension was harvested and stained with Annexin V-FITC and propidium iodide (PI) apoptosis detection kit (Keygen Biotech, Nanjing, China) for 15 min at room temperature. The stained cells were analyzed using a flow cytometer.³¹ Flow cytometry data were analyzed using the CellQuest software (BD Biosciences, San Jose, California, USA). The images were measured by Annexin V-FITC/PI assay: necrotic cells were indicated in the first quadrant (Q1), cells in the later stage of apoptosis were indicated in the second quadrant (Q2), early apoptotic cells were indicated in the third quadrant (Q3), and the normal cells were indicated in the fourth quadrant (Q4).

Measurement of the mitochondrial membrane potential

The mitochondria membrane potential (ΔΨm, MMP) was measured using the lipophilic cationic probe JC-1 dye. The relative quantity of red and green fluorescence intensity was used to analyze the depolarization of MMP. A decrease in red/green ratio indicated cell apoptosis. Cells were incubated with JC-1 (10 µg/mL) for 15 min at 37°C in the dark. Subsequently, labeled cells were detected using a fluorescence microscope (Olympus, Tokyo, Japan).³²

Caspase activity assay

Cells were harvested and lysed, and caspase 3 and caspase 9 activities were measured using Colorimetric Activity Assay Kits (Beyotime Bio. Inc., Nantong, China), according to the manufacturer’s instructions.

Intracellular ROS production assay

Intracellular ROS levels were detected using the HDCF-DA Probe Assay Kit (Beyotime, Shanghai, China). HUVECs were pretreated with or without SFN, brusatol for 24 h, and incubated with 100 nM Ang II for another 4 h. HUVECs were incubated with HDCF-DA (10 μM) for 1 h at 37°C in the dark. Intracellular ROS generation was measured at 485 nm (excitation) and 535 nm (emission) using a microplate reader (Bio-Rad).

Oxidation product assay

Malondialdehyde (MDA) concentrations were analyzed using a commercial kit (Cayman Chemical Company, Ann Arbor, Michigan, USA). Protein carbonyl concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Cell Biolabs, San Diego, California, USA). All standards and samples were assessed in duplicate.

Antioxidant enzyme activity assays

HUVECs were pretreated with or without SFN and brusatol for 24 h and incubated with 100 nM Ang II for another 4 h. The enzymatic activities of catalase (CAT) and superoxide dismutase (SOD) were measured using kits according to the manufacturer’s protocols (Cayman Chemical Company). Glutathione and oxidized glutathione (GSH and GSSG) levels were measured using the GSH and GSSG Assay Kit (Beyotime Bio. Inc.). Enzymatic activities are presented as percentages of the control.

NADPH oxidase activity assay

NADPH oxidase activity was detected using a Colorimetric Assay Kit according to the manufacturer’s protocol (Abcam, Cambridge, Massachusetts, USA). The absorbance was determined using a microplate reader (Bio-Rad), and NADPH oxidase activity was expressed as pg/mg protein.

Measurement of cytochrome c release

Measurement of the cytochrome c release to the HUVECs was performed as previously described.³⁰ The levels of cytochrome c in the cytosolic and mitochondrial fractions were detected by the Quantikine M Cytochrome C Immunoassay Kit (R&D Systems, Minneapolis, Minnesota, USA) according to the manufacturer’s instructions. The cells were lysed and centrifuged, and the supernatants were collected. The supernatants were subjected to centrifugation at 15,000 × g for 15 min at 4°C, and the pellets containing the mitochondria were collected and the supernatant was used as the cytosolic fraction. Mitochondria were lysed by 0.5% Triton X-100 for 10 min, and centrifuged at 15,000 × g for 10 min, and the supernatant was used as the mitochondrial fraction. The absorbance was measured by a microplate reader (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

RNAi-mediated silencing of the Nrf2 gene

Cells were transfected with an Nrf2 siRNA (GenePharma, Shanghai, China) using Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer’s instructions. A nonspecific non-targeting siRNA (scrambled) was used as a negative control. Cells were examined by Western blotting 48 h after transfection.

Western blotting

Western blots were performed as previously described.³³ Proteins were extracted from cells using radio immunoprecipitation assay (RIPA) buffer supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific). Protein concentrations were determined using the bicinchoninic acid protein assay kit. Subsequently, 30 µg of protein was separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene fluoride membranes. Membranes were blocked with 5% nonfat milk for 1 h at room temperature. Membranes were then incubated with primary antibodies overnight at 4°C. Subsequently, membranes were exposed to the horseradish peroxidase-conjugated secondary antibodies for 1 h at 37°C. Finally, membranes were incubated with an enhanced chemiluminescence reagent (Thermo Fisher Scientific). The bands were quantified using ImageJ Software (NIH, Bethesda, Maryland, USA).

Quantification of Nrf2 activity using an ELISA

Nrf2 activity was measured using a TransAM NRF2 Assay Kit (Active Motif, Carlsbad, CA) according to the manufacturer’s instructions. The absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific).

Statistical analysis

All standards and samples were assessed in duplicate. All data were analyzed with SPSS 21.0 statistical software (SPSS, Inc., Chicago, Illinois, USA). The results are presented as means ± standard deviations. Statistical significance was determined using one-way analysis of variance, followed by Tukey’s or Dunnett’s tests. A p value ≤0.05 was considered statistically significant.

Results

SFN ameliorated Ang II-induced reduction in HUVEC viability

HUVECs were treated with different concentrations of SFN to test the safety of this compound in our in vitro model. The 2 μM SFN treatment did not affect the viability of HUVECs after 24 h (Figure 1(a)). Moreover, Ang II significantly decreased the viability of HUVECs in a dose-dependent manner (Figure 1(b)). Thus, 2 μM SFN and 400 nM Ang II were used in the subsequent experiments.

Figure 1.

SFN reversed Ang II-induced increase in HUVEC cytotoxicity: (a) HUVECs were treated with different concentrations of SFN for 24 h and cell viability was detected; (b) HUVECs were treated with different concentrations of Ang II for 24 h and cell viability was detected; (c) HUVECs were divided into a normal group, Ang II (400 nM) group, SFN (2 μM) group, SFN (2 μM) + Ang II (400 nM) group, and brusatol (40 nM) + SFN (2 μM) + Ang II (400 nM) group, treated for 24 h, and cell viability was detected; and (d) LDH release from different groups. The results of the cell viability assay are presented as a percentage of the control, and the results of the LDH assay are presented as a fold change compared with the control. Experiments were repeated at least thrice. Data are presented as the means ± SD of three experiments. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; ^#p < 0.05 compared with the Ang II group; ^$p < 0.05 compared with the Ang II + SFN group. SFN: sulforaphane; Ang II: angiotensin II; HUVEC: human umbilical vein endothelial cell; SD: standard deviation.

HUVECs were treated with 2 μM SFN and 400 nM Ang II for 24 h to assess the potential protective effects of SFN. SFN clearly mitigated the Ang II-induced decrease in cell viability (Figure 1(c)) and increase in LDH release (Figure 1(d)). When Nrf2 activity was blocked by brusatol, the effect of SFN on increasing cell viability was suppressed to 56% of the level of the control group (Figure 1(c)). Moreover, when HUVECs were pretreated with brusatol, the effect of SFN on LDH release was also ameliorated. Based on these data, SFN reversed the Ang II-induced reduction inviability of HUVECs via the Nrf2 signal.

SFN ameliorated the Ang II-induced increase in HUVEC apoptosis

Flow cytometry was used to quantitatively evaluate HUVEC apoptosis. SFN did not affect HUVECs apoptosis after 24 h of treatment (Figure 2(a)). Moreover, 24 h of incubation with Ang II (400 nM) increased the number of apoptotic HUVECs (Figure 2(b)). Interestingly, the SFN treatment alleviated Ang II-mediated HUVEC apoptosis (53.1% ± 3.2% vs. 29.3% ± 2.6%, p < 0.05; Figure 2(c) and (d)). When HUVECs were pretreated with brusatol, the protective effect of SFN on HUVEC apoptosis was reversed (29.3% ± 2.6% vs. 36.1% ± 2.5%, p< 0.05; Figure 2(c) and (d)).

Figure 2.

SFN reversed Ang II-induced increase in HUVEC apoptosis: (a) HUVECs were treated with different concentrations of SFN for 24 h and cell apoptosis was detected; (b) HUVECs were treated with different concentrations of Ang II for 24 h and cell apoptosis was detected; (c) HUVECs were divided into a normal group, Ang II (400 nM) group, SFN (2 μM) group, SFN (2 μM) + Ang II (400 nM) group, and brusatol (40 nM) + SFN (2 μM) + Ang II (400 nM) group, treated for 24 h, and the apoptosis rates were compared with those of the control group by flow cytometry; and (d) quantitative analysis of cell apoptosis presented in (c), the percentage of apoptotic cells/total number of cells. The experiment was repeated at least thrice. Data are presented as the means ± SD of three experiments. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; ^#p < 0.05 compared with the Ang II group; ^$p < 0.05 compared with the Ang II + SFN group. SFN: sulforaphane; Ang II: angiotensin II; HUVEC: human umbilical vein endothelial cell; SD: standard deviation.

SFN inhibited mitochondrial dysfunction in Ang II-treated HUVECs

The MMP was reduced in the Ang II-treated group (Figure 3(a)), indicating that the mitochondrial membrane was damaged. SFN increased the MMP in Ang II-treated HUVECs (Figure 3(a)), but when HUVECs were pretreated with brusatol, the protective effect of SFN on the MMP was reversed (Figure 3(a)).

Figure 3.

SFN reversed the Ang II-induced increase in HUVEC apoptosis through a mitochondria-dependent pathway. HUVECs were divided into a normal group, Ang II (400 nM) group, SFN (2 μM) group, SFN (2 μM) + Ang II (400 nM) group, and brusatol (40 nM) + SFN (2 μM) + Ang II (400 nM) group and treated for 24 h: (a) changes in the MMP were monitored using JC-1 staining and are shown in the histogram; (b) the mitochondrial levels of the cytochrome c protein are presented as a ratio relative to the control group; (c) the cytosolic levels of the cytochrome c protein are presented as a ratio relative to the control group; and (d) changes in caspase 9 and caspase 3 activities in HUVECs exposed to different treatments. Experiments were repeated at least thrice. Data are presented as the means ± SD of three experiments. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; ^#p < 0.05 compared with the Ang II group; ^$p < 0.05 compared with the Ang II + SFN group. SFN: sulforaphane; Ang II: angiotensin II; HUVEC: human umbilical vein endothelial cell; SD: standard deviation; MMP: mitochondrial membrane potential.

SFN inhibited Ang II-induced cytochrome c release into the cytoplasm of HUVECs

Compared with the control group, the Ang II treatment reduced the levels of the cytochrome c protein in mitochondria (Figure 3(b)) and increased the levels of the cytochrome c protein in the cytoplasm of HUVECs (Figure 3(c)). Moreover, SFN treatment increased the mitochondrial levels of the cytochrome c protein (Figure 3(b)) and decreased the cytoplasmic levels of the cytochrome c protein (Figure 3(c)) in Ang-II stimulated HUVECs. However, when HUVECs were pretreated with brusatol, the effects of SFN on cytochrome c release were reversed (Figure 3(b) and (c)).

SFN inhibited Ang II-induced caspase activity in HUVECs

Moreover, caspase 3 and caspase 9 activities were also increased in the Ang II-treated group compared with the control group (Figure 3(d)), but SFN decreased caspase 3 and caspase 9 activities in the Ang II-treated group (Figure 3(d)). However, when HUVECs were pretreated with brusatol, the effects of SFN on caspase activity were reversed (Figure 3(d)). Based on these results, SFN ameliorated Ang II-induced mitochondrial dysfunction and cell apoptosis through a mitochondria-dependent pathway.

SFN ameliorated Ang II-induced increase in oxidative stress in HUVECs

A fluorescent probe (DCDHF-DA) was used to detect ROS levels. The Ang II treatment increased ROS generation in HUVECs, whereas SFN decreased ROS generation in the Ang II-treated group (Figure 4(a) and (b)). Ang II increased MDA levels in HUVECs, whereas SFN decreased MDA levels in the Ang II-treated group (Figure 4(c)). Furthermore, Ang II increased protein carbonyl levels in HUVECs, whereas SFN reduced the protein carbonyl levels in the Ang II-treated group (Figure 4(d)). As shown in Figure 4(e), Ang II increased NADPH oxidase activity compared with the control group (p < 0.01). SFN abolished the Ang II-induced increase in NADPH oxidase activity (p < 0.01). Moreover, brusatol and siNrf2 treatments significantly increased ROS generation, MDA levels, protein carbonyl levels, and NADPH oxidase activity in HUVECs compared with the SFN-treated group (p < 0.05).

Figure 4.

SFN reversed the Ang II-induced changes in oxidative stress and NADPH oxidase activity. HUVECs were divided into a normal group, Ang II (400 nM) group, SFN (2 μM) group, SFN (2 μM) + Ang II (400 nM) group, brusatol (40 nM) + SFN (2 μM) + Ang II (400 nM) group, and siNrf2+ SFN (2 μM) + Ang II (400 nM) group treated for 24 h: (a) representative images showing the detection of DCFH-DA in the indicated groups using a fluorescence microscope and (b) quantification of the ROS levels. The activities of endogenous antioxidant enzymes were measured by determining the (c) MDA levels, (d) protein carbonyl levels, and (e) NADPH oxidase activity. Experiments were repeated at least thrice. Data are presented as the means ± SD of three experiments. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; ^#p < 0.05 compared with the Ang II group; ^$p < 0.05 compared with the Ang II + SFN group. SFN: sulforaphane; Ang II: angiotensin II; HUVEC: human umbilical vein endothelial cell; SD: standard deviation; ROS: reactive oxygen species; MDA: malondialdehyde; NADPH: nicotinamide adenine dinucleotide phosphate.

The ROS scavenger NAC alleviated Ang II-induced HUVEC apoptosis

N-acetyl-l-cysteine (NAC) significantly inhibited Ang II-induced ROS production (p < 0.05, Figure 5(a)). Moreover, NAC alleviated mitochondrial membrane injury in the Ang II-treated group and the red fluorescent protein/green fluorescent protein ratio of stained cells increased (Figure 5(b)). An evaluation of the percentage of apoptotic cells indicated that NAC decreased the Ang II-induced increase in the percentage of apoptotic cells (Figure 5(c)). Thus, NAC alleviated Ang II-mediated mitochondrial membrane injury and apoptosis in HUVECs to a similar extent as SFN.

Figure 5.

NAC reversed the Ang II-induced changes in ROS generation, the MMP, and apoptosis in HUVECs. HUVECs were treated with NAC and Ang II. The (a) ROS levels, (b) MMP, and (c) apoptosis were measured. The experiments were repeated at least thrice. Data are presented as the means ± SD of three experiments. *p < 0.05 compared with the control group; ^#p < 0.05 compared with the Ang II group. NAC: N-acetyl-l-cysteine; MMP: mitochondrial membrane potential; HUVEC: human umbilical vein endothelial cell; ROS: reactive oxygen species.

SFN increased the activities of endogenous antioxidant enzymes in Ang II-treated HUVECs

Ang II reduced SOD activity in HUVECs to 52% of the control cells, whereas SFN increased SOD activity in the Ang II-treated group to 88% of the control cells (Figure 6(a)). Ang II decreased CAT activity in HUVECs to 58% of the control, whereas SFN increased CAT activity in the Ang II-treated group to 71% of the controls (Figure 6(b). Ang II increased the GSSG/GSH ratio in HUVECs to 246% of the control, whereas SFN decreased the GSSG/GSH ratio in the Ang II-treated group to 173% of the control (Figure 6(c)), indicating that SFN protects cellular redox homeostasis. Moreover, the brusatol pretreatment reversed the SFN-induced changes in SOD activity, CAT activity, and the GSSG/GSH ratio in HUVECs to a significant extent (p < 0.05).

Figure 6.

SFN reversed the Ang II-induced decreases in the activities of antioxidant enzymes. HUVECs were divided into a normal group, Ang II (400 nM) group, SFN (2 μM) group, SFN (2 μM) + Ang II (400 nM) group, and brusatol (40 nM) + SFN (2 μM) + Ang II (400 nM) group and treated for 24 h. The activities of (a) SOD, (b) intracellular CAT, and the (c) GSH/GSSG ratio were measured. Experiments were repeated at least thrice. Data are presented as the means ± SD of three experiments. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; ^#p < 0.05 compared with the Ang II group; ^$p < 0.05 compared with the Ang II + SFN group. SFN: sulforaphane; Ang II: angiotensin II; HUVEC: human umbilical vein endothelial cell; SD: standard deviation; SOD: superoxide dismutase; GSH: glutathione; GSSH: oxidized glutathione.

Knockout of Nrf2 with siRNA abrogated the protective effect of SFN on Ang II-induced ROS generation and apoptosis

As shown in Figure 7(a), the Ang II treatment reduced Nrf2 expression in HUVECs. In addition, the activation of Nrf2 in the Ang II-treated group was reduced compared with the control group (p < 0.05, Figure 7(b)). After treatment with SFN, both the expression and the activation of Nrf2 increased (p < 0.05, Figure 7(a) and (b). However, the brusatol pretreatment significantly reduced the level and activation of Nrf2 in HUVECs compared with the SFN-treated group (p < 0.05, Figure 7(a) and (b).

Figure 7.

SFN reversed the Ang II-induced decrease in Nrf2 activity. HUVECs were divided into a normal group, Ang II (400 nM) group, SFN (2 μM) group, SFN (2 μM) + Ang II (400 nM) group, and brusatol (40 nM) + SFN (2 μM) + Ang II (400 nM) group and treated for 24 h: (a) Nrf2 levels in HUVECs were determined using Western blotting; (b) Nrf2 activation in HUVECs was measured using the ELISA-based TransAM Nrf2 assay. The experiment was repeated at least thrice. Data are presented as the means ± SD of three experiments. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; ^#p < 0.05 compared with the Ang II group; ^$p < 0.05 compared with the Ang II + SFN group. SFN: sulforaphane; Ang II: angiotensin II; HUVEC: human umbilical vein endothelial cell; SD: standard deviation; ELISA: enzyme-linked immunosorbent assay.

We knocked out Nrf2 expression in HUVECs using siNrf2 to confirm that the protective effects of SFN on Ang II-induced HUVEC apoptosis were mediated by the Nrf2 pathway. Cells transfected with si control were used as the control. The efficacy of the Nrf2 knockout was confirmed by Western blotting, which revealed a significant reduction in Nrf2 expression in the siNrf2-transfected cells compared with the si control-transfected cells (Figure 8(a)). The protective effects of SFN on Ang II-induced apoptosis (Figure 8(b) and ROS levels (Figure 8(c)) were abolished in the siNrf2-transfected cells. These data confirmed that the protective effects of SFN on Ang II-induced apoptosis and ROS generation were mediated by the Nrf2 pathway.

Figure 8.

Nrf2 knockdown prevented SFN decreases in the inflammatory response: (a) the siRNA-induced reduction in Nrf2 expression was determined using western blot analysis; (b) quantification of the immunoreactive bands; (c) Nrf2 siRNA reversed the SFN-induced decrease in cell apoptosis; and (d) Nrf2 siRNA reversed the SFN-induced decrease in the ROS level. Experiments were repeated at least thrice. Data are presented as the means ± SD of three experiments. *p < 0.05 compared with the control group; **p < 0.01 compared with the control group; ^#p < 0.05 compared with the Ang II group; ^$p < 0.05 compared with the Ang II + SFN group. SFN: sulforaphane; Ang II: angiotensin II; HUVEC: human umbilical vein endothelial cell; SD: standard deviation; ROS: reactive oxygen species.

Discussion

Ang II is a crucial factor responsible for cardiovascular pathologies, including hypertension and AS.^4,34 Recently, some studies have focused on exploring the effect of Ang II on ECs.^35

–38 However, the detailed mechanism by which Ang II mediates HUVEC injury has not yet been investigated. This study shows that SFN inhibited Ang II-modulated ROS-mediated mitochondrial signaling in HUVECs and thus abrogated apoptosis. In particular, 2-μM SFN inhibited Ang II-induced HUVEC apoptosis through a ROS-mediated mitochondrial apoptotic pathway, including the decrease in the MMP, impaired cytochrome c release, and caspase 3/9 activation. In addition, SFN abolished Ang II-induced ROS generation, NADPH oxidase activity, and antioxidant enzyme activity. Interestingly, the SFN treatment prevented all of these detrimental effects of Ang II. We then explored the potential protective mechanism by which SFN ameliorated Ang II-mediated vascular injury. SFN increased the expression ofNrf2. Importantly, downregulation of Nrf2 expression with a target-specific siRNA illustrated anNrf2-independent effect of SFN that diminished ROS generation and inhibited apoptosis in HUVECs.

A high concentration of SFN (50 μM) inhibited mitochondrial fission dynamics by an independent mechanism of the Keap1-Nrf2-ARE pathway.²² Moreover, high concentrations of SFN (50–200 μM) suppressed the inflammatory response by an independent mechanism of Nrf2 signaling in vitro and in vivo. These results suggest that SFN may be a potential agent for the treatment of inflammatory response-mediated diseases, including AS.³⁹ Ang II treatment reduced the MMP, an important characteristic indicator of aggravated mitochondrial damage, in HUVECs. Subsequently, cytochrome c was released into the cytoplasm, and caspase 9 and caspase 3 were activated to trigger cell apoptosis through the mitochondrial signaling pathway. Mitochondria are a key source of intracellular ROS generation. Conversely, mitochondria are also the main target of ROS, and ROS generation may decrease the MMP and initiate apoptosis. Apoptosis and necrosis have been implicated as the major causes of cell death induced by oxidative stress. Thus, we compare the direct cytotoxicity of Ang II toward HUVECs, and our results showed that Ang II induced apoptosis and necrosis in HUVECs. We next detected whether SFN could protect against Ang II-mediated apoptosis and necrosis using Annexin V-FITC assay. The results indicated that SFN inhibited the Ang II-mediated HUVEC injury by decreasing apoptosis and necrosis. Although many studies have focused on how SFN attenuates Ang II-mediated apoptosis, stress via ROS is known to play a key role in the regulation of apoptosis and necrosis. Our data support these previous findings, and our results showed that SFN may act as a protective agent against Ang II-induced oxidative stress by alleviating apoptosis and necrosis. In addition to increased antioxidant activity, SFN treatment restored the MMP in HUVECs and inhibited mitochondrial cytochrome c release of caspase 9/3. Therefore, SFN treatment participated in scavenging oxygen free radicals and maintaining cell function. From our data presented in this study, it is shown that the Nrf2-independent pathway plays an important role in the protective effects of SFN, especially in terms of oxidative stress.

Based on accumulating evidence, high ROS levels play a key role in vascular injury and dysfunction.^40,41 Mitochondrial ROS generation enhances the accumulation of proapoptotic molecules, prompting the activation of the apoptotic signaling pathway and ultimately leading to cell apoptosis.^42,43 According to the results of the fluorescent probe assay performed in this study, Ang II increases ROS levels and damages the intracellular redox homeostasis in HUVECs. MDA and protein carbonyl levels reflect the severity of oxidative stress in cells, and NADPH oxidase activity indirectly reflects the ability of cells to scavenge oxygen free radicals.⁴⁴ SFN reversed Ang II-induced increases in MDA and protein carbonyl levels as well as NADPH oxidase activity, while it induced the activity of antioxidant enzymes, including SOD, CAT, and the GSSG/GSH ratio. Based on these data, SFN protects Ang II-injured HUVECs by preventing oxidative stress. Therefore, we hypothesized that oxidative stress plays a fundamental role in Ang II-mediated vascular endothelial injury and SFN protects against Ang II-mediated injury in HUVECs by inhibiting oxidative stress.

NAC, a ROS scavenger, inhibited the Ang II-increase in ROS levels and apoptosis in HUVECs. Moreover, NAC pretreatment attenuated Ang II-mediated mitochondrial injury. Therefore, Ang II induces mitochondrial injury and promotes ROS formation, and ROS accumulation impairs the mitochondrial membrane, which further triggers apoptosis.

In addition, Nrf2 is an important transcription factor that regulates the antioxidant response by enhancing the expression of cytoprotectivegenes.⁴⁵ Increased Nrf2 expression enhances cell survival under stress conditions.^46
–48 Nrf2 activation has been regarded as a central regulatory mechanism underlying the upregulation of antioxidant enzyme expression.⁴⁹ Here, Ang II reduced both the expression and the activation of Nrf2 compared with the control group. Cells may not be able to survive in such extreme conditions (400 nM Ang II treatment), and eventually decrease Nrf2 activation. Moreover, Nrf2 expression and activation were increased in cells treated with SFN.

Many previous reports have shown that brusatol was used to inhibit Nrf2 in vivo and in vitro.^10,50–52 Brusatol aggravates a rapid reduction of Nrf2 expression and subsequently regulates cell processes, thereby triggering cell responses.⁵³ In a past study, brusatol was regarded as a general translation inhibitor, which reduced the levels of many short-lived proteins, including Nrf2.⁵⁴ The author also highlighted that the data do not negate previous findings that show that the inhibition of Nrf2 via brusatol is a useful strategy. Brusatol suppresses the protective effect of SFN by attenuating Ang II-mediated cellular injury. Therefore, we hypothesized that SFN might attenuate Ang II-mediated cellular injury, at least in part, via the Nrf2 pathway. The SFN treatment induced Nrf2 activation in Ang II-treated HUVECs, as revealed by the increased expression and nuclear translocation of Nrf2. Our findings were consistent with the results reported in a recent study showing that SFN efficiently suppresses oxidative stress-induced injury.^55,56 This inhibitory effect is mediated by the antioxidant activity, which might be partially activated by the Nrf2 signaling pathway, because SFN increases nuclear expression of Nrf2 and reduces HUVEC apoptosis. After treatment with Nrf2 siRNA, the SFN-mediated inhibition of oxidative stress and apoptosis in HUVECs decreased. Therefore, the Nrf2 signaling pathway is required for SFN treatment to ameliorate Ang II-mediated HUVECs apoptosis. Thus, the Nrf2 pathway, at least in part, is involved in protecting against Ang II-induced endothelial damage. Moreover, there is a need to perform Nrf2 gain-of-function and loss-of-function studies in vivo using mice to confirm the critical role of Nrf2. Moreover, SFN markedly decreases the ICAM-1 and VCAM-1expression on the vascular wall via suppressing STAT3 phosphorylation signaling.²¹ SFN attenuates HepG2-induced EC angiogenesis via inhibition on the STAT3/HIF-1α/VEGF pathway.⁵⁷ SFN protects the neurovascular system against a systemic inflammatory response by both Nrf2-dependent and independent signal pathways.⁵⁸ Thus, further efforts are underway to study how SFN attenuates Ang II-induced endothelial apoptosis.

The underlying mechanism of how SFN can prevent Ang II-induced EC injury remains unclear; however, based on recent reports, epigenetic regulation of Nrf2 by SFN may be involved. Nrf2 expression can be regulated by modifying the methylation status of the Nrf2 promoterin prostate cancer. SFN directly enhances Nrf2 transcription and expression by decreasing methylation and elevating histone 3 acetylation in the Nrf2 promoter in prostate cancer cells. Therefore, we assume that Nrf2 transcription and expression are upregulated and retained at elevated levels by SFN via epigenetic regulation, which results in indirect Nrf2 translocation into nuclei and activation, which provides sustained prevention of Ang II-induced oxidative damage and subsequent ECs apoptosis. Nevertheless, the molecular mechanism responsible for the protective action of SFN on angiogenesis requires further study.

Conclusion

In conclusion, our study suggests that SFN ameliorated Ang II-induced cell damage by inhibiting ROS-mediated mitochondrial injury and, at least in part, by activating the Nrf2 signaling pathway.

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) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by a grant from the research fund of Shanghai Tongren Hospital, Shanghai Jiao Tong University School of Medicine (TRYJ201614).

ORCID iD

L Jiang

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