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Introduction

Pulmonary arterial hypertension (PAH) is a disease of the small pulmonary arteries that is characterized by a progressive increase in pulmonary vascular resistance and obliterative pulmonary vascular remodeling, leading to uncompensated right heart failure, and ultimately, death (1). The histopathological features of PAH include intima and media thickness, muscularization of distal pulmonary arteries, vascular occlusion and complex plexiform lesions (2). Although the pathogenesis of PAH is not completely understood, it has been reported that the genetic change resulting in endothelial dysfunction is an important contributing factor (2,3). Previous studies have revealed that bone morphogenetic protein type II receptor (BMPR2) mutations serve an important role in PAH pathogenesis via the Smad and transforming growth factor-β signaling pathways (4-6). However, as PAH is not penetrant, BMPR2 mutations alone may not be sufficient to cause disease, therefore, other genetic and/or non-genetic factors may serve as a ‘second hit’ to trigger PAH pathology (7), including inflammation and chronic hypoxia. Chronic hypoxia may serve a key role in the pathogenesis of PAH via pathological alterations to endothelial cells in the vascular system (8,9). Chronic hypoxia can also induce the generation of reactive oxygen species (ROS) in tissues, which triggers epigenetic changes and induces dysregulation of various signaling pathways in endothelial cells (10).

As an epigenetic factor, Sirtuin 1 (SIRT1) may respond to ROS (11,12). SIRT1 is expressed in the nucleus and cytoplasm of multiple tissues, acting as a histone deacetylase that participates in cell apoptosis, survival and proliferation (13). In a previous study on prostate cancer, Ruan et al (12) suggested that the generation of ROS can increase the expression level of SIRT1. Moreover, other studies have reported that SIRT1 expression is increased in endothelial and smooth muscle cells in PAH (14,15), but SIRT1-induced pathogenic alterations have not been explored further. Therefore, the present study investigated SIRT1 expression in hypoxia-induced arterial endothelial cells. SIRT1 expression was significantly increased in response to ROS generation, and SIRT1 promoted human arterial endothelial cell proliferation, but inhibited apoptosis. Furthermore, the results indicated that the SIRT1 inhibitor could decrease the activity of the Akt signaling pathway, which facilitates PAH development.

Materials and methods

Cell culture

Human pulmonary arterial endothelial cells (HPAECs; cat. no. PCS-100-022; American Type Culture Collection) were cultured in endothelial cell medium (ScienCell Research Laboratories, Inc.) supplemented with 10% FBS (Hyclone; Cytiva), 100 U/ml penicillin (Invitrogen; Thermo Fisher Scientific, Inc.) and 100 U/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C with 5% CO2. Cells up to passage 4 were used for subsequent experiments.

Cells (5x103 cells/well in 96-well plates) were treated with 0.4 µM SIRT1 antagonist (EX-527; dissolved in DMSO; Selleck Chemicals) (16), 4 µM SIRT1 activator (SRT1720; dissolved in DMSO (12); Selleck Chemicals) and 10 µM Akt antagonist (mk2206; dissolved in DMSO; Selleck Chemicals) (17). In addition, cells were treated with 1 mM hydrogen peroxide (H2O2; cat. no. H1009; Sigma-Aldrich; Merck KGaA) and 5 mM N-acetylcysteine (NAC; cat. no. ST1546; Beyotime Institute of Biotechnology) (18). The control group was treated with the same volume of DMSO. During the treatment, cells were maintained at 37˚C for 72 h.

Cell hypoxia experiments in vitro

To induce in vitro hypoxic conditions, HPAECs were placed in a hypoxic chamber (10% O2 and 5% CO2) for 72 h at 37˚C. Control cells were maintained under standard culture conditions (37˚C; 21% O2 and 5% CO2) for 72 h (19,20).

SIRT1 activity assay

To evaluate SIRT1 activity, a fluorescent activity kit for SIRT1 (SIRT1 Activity Assay kit (Fluorometric); cat. no. ab156065; Abcam) was used according to the manufacturer's protocol. The results were read at a fluorescent emission of 460 nm using a multimode plate reader (Thermo Fisher Scientific, Inc.).

Western blotting

Cells were collected and then centrifugated at 500 x g for 5 min at room temperature and media was aspirated. Subsequently, the cell pellets were washed with ice-cold PBS, centrifuged at 500 x g for 5 min at 4˚C and supernatant was aspirated. Cell pellets were lysed in RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) containing proteinase inhibitors (cat. no. P1006; Beyotime Institute of Biotechnology). Equal amounts of protein (20 µg/lane) were separated via 8-10% SDS-PAGE and electrotransferred onto PVDF membranes (EMD Millipore). The membranes were blocked with 5% BSA (cat. no. ST023-50 g; Beyotime Institute of Biotechnology) for 1 h at room temperature. Following blocking, membranes were incubated at 4˚C overnight with primary antibodies (1:3,000) targeted against: Anti-SIRT1 (rabbit; cat. no. ab189494; Abcam), anti-Akt (rabbit; cat. no. 9272; Cell Signaling Technology, Inc.), anti-phosphorylated (p)-Akt (rabbit; cat. no. P00024-5; Boster Biological Technology), anti-Bcl-2 (rabbit; cat. no. ab59348; Abcam), anti-hypoxia-inducible factor-1 (HIF-1; rabbit; cat. no. ab82832; Abcam) and anti-GAPDH (mouse; cat. no. sc-32233; Santa Cruz Biotechnology, Inc.). Subsequently, the membranes were incubated with HRP-conjugated anti-rabbit IgG (1:3,000; cat. no. ab205718; Abcam) or HRP-conjugated anti-mouse IgG secondary antibody (1:3,000; cat. no. ab205719; Abcam) at room temperature for 1 h. Protein bands were visualized using the Western Bright ECL kit (Bio-Rad Laboratories, Inc.). GAPDH was used as the loading control. The protein expression levels were quantified with ImageJ software (version 1.8.0; National Institutes of Health).

Reverse transcription-quantitative PCR

Total RNA was extracted from cells using TRIzol® (Beyotime Institute of Biotechnology). Total RNA (500 ng) was reverse transcribed into cDNA using a SuperScript III Reverse Transcriptase kit (cat. no. 18080093; Invitrogen; Thermo Fisher Scientific, Inc.) at 50˚C for 50 min and 85˚C for 5 min. Subsequently, qPCR was performed using SYBR Green (cat. no. 4309155; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol on the ABI PRISM 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following primers were used for qPCR: GAPDH forward, 5'-TGTGGGCATCAATGGATTTGG-3' and reverse, 5'-ACACCATGTATTCCGGGTCAAT-3'; and SIRT1 forward, 5'-TAGCCTTGTCAGATAAGGAAGGA-3' and reverse, 5'-ACAGCTTCACAGTCAACTTTGT-3'. The following thermocycling conditions were used for the qPCR: Initial denaturation for 10 min at 95˚C; 40 cycles of 15 sec at 95˚C for denaturation, 30 sec at an annealing temperature of 60˚C and a 30 sec extension at 72˚C. mRNA expression levels were quantified using the 2-ΔΔCq method and normalized to the internal reference gene GAPDH (21).

Nitroblue tetrazolium (NBT) assay

An NBT assay kit (Sigma-Aldrich; Merck KGaA) was used to assess ROS generation. Briefly, cells were seeded (5x103 cells/well) into 96-well plates and maintained under hypoxic or normal conditions for 72 h. Subsequently, the plates were washed with PBS to remove cell debris. Cells were counted and incubated with 0.1% NBT for 90 min at 37˚C. Cells were collected, sonicated (20 kHz/s, 30 sec, on ice) and dissolved in acetic acid. The absorbance of each well was measured at a wavelength of 560 nm using a multiple plate reader.

Cell apoptosis assay

Flow cytometry was performed to evaluate cell apoptosis in vitro. After treatment, cells were collected, washed with PBS and resuspended in 500 µl binding buffer containing 5 µl annexin V-fluorescein isothiocyanate and 10 µl propidium iodide (cat. no. ANNEX300F; Bio-Rad Laboratories, Inc.). Following incubation for 30 min in the dark at room temperature, cell apoptosis was assessed using a FACScan flow cytometer (BD Biosciences). Data were analyzed using FlowJo software (version 7.6.5; FlowJo LLC).

Cell proliferation assay

Cells were seeded (5x103 cells/well) into 96-well plates and cultured overnight at 37˚C to attach. Cells were maintained under hypoxic or normal conditions for 72 h. Subsequently, cell proliferation was assessed using a WST-1 assay (Roche Diagnostics) according to the manufacturer's protocol. The absorbance of each well was measured at a wavelength of 440 nm using a multimode plate reader.

siRNA transfection

Cells were seeded (1x105 cells/well) into 12-well culture plates and transfected with 40 nM SIRT1 siRNA (sense, 5'-ACUUUGCUGUAACCCUGUA(dTdT)-3' and antisense, 5'-UACAGGGUUACAGCAAAGU(dTdT)-3'.) or scrambled siRNA (sense, 5'-UUCUCCGAACGUGUCACGU(dTdT)-3' or antisense, 5'-AGGUGACACGUUGGGAGAA(dTdT)-3') (Santa Cruz Biotechnology, Inc.) using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. At 24 h post-transfection, western blotting was performed to assess transfection efficiency and cells were used for subsequent experiments.

Statistical analysis

All experiments were performed in triplicate. Data were analyzed and illustrated using R (version 3.6.2; https://www.r-project.org/). Data are presented as the mean ± SEM. Comparisons between two groups were analyzed using the Student's t-test. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Results

SIRT1 expression and the PI3K/Akt/mTOR signaling pathway are upregulated in pulmonary artery endothelial cells under hypoxic conditions

The results indicated that hypoxia significantly increased the expression of SIRT1 in HPAECs in vitro compared with the control group (Fig. 1A). In addition, HIF-1α is involved in the pathogenesis of PAH and is also a downstream factor of the PI3K/Akt/mTOR signaling pathway (22). The results suggested that hypoxia activated the PI3K/Akt/mTOR signaling pathway, as indicated by significantly increased levels of phosphorylated Akt in the hypoxia group compared with the control group, and significantly increased HIF-1α expression levels in pulmonary vascular endothelial cells compared with the control group (Fig. 1B). Moreover, under hypoxic conditions, treatment with NAC significantly decreased SIRT1 expression levels compared with the hypoxia group (Fig. 1A and B), which indicated that hypoxia-induced activation of the Akt signaling pathway could be blocked by NAC.

Figure 1 Hypoxia increases SIRT1 expression and activates the Akt signaling pathway, which can be alleviated by anti-reactive oxygen species reagent (NAC). (A) Compared with the control group, SIRT1 mRNA expression levels were increased after hypoxia treatment, which was reversed by NAC. (B) SIRT1 protein expression increased after hypoxia treatment compared with the control group. Moreover, compared with the control group, Akt phosphorylation was increased in cells under hypoxic conditions, but inhibited by NAC. Bcl-2 and HIF-1α expression levels were increased by hypoxic conditions compared with the control group, but hypoxia-induced expression was inhibited by NAC. *P<0.05; #P<0.01. SIRT1, sirtuin 1; NAC, N-acetylcysteine; HIF-1α, hypoxia-inducible factor-1α; p, phosphorylated.

Hypoxia-induced SIRT1 activates the Akt signaling pathway

The present study investigated whether SIRT1 upregulation was associated with activation of the Akt signaling pathway in HPAECs. The results indicated that SIRT1 activator activated the Akt signaling pathway, as indicated by increased phosphorylation levels of Akt compared with the control group, which was reversed by SIRT1 inhibitor (Fig. 2). Moreover, Bcl-2(23) and HIF-1(24) are downstream molecules of the Akt/mTOR signaling pathway. The results also suggested that Bcl-2 and HIF-1α expression levels were significantly increased in the SIRT1 activator group compared with the control group. By contrast, SIRT1 inhibitor significantly decreased the expression levels of Bcl-2 and HIF-1α compared with the control group (Fig. 2).

Figure 2 SIRT1 activates Akt and its downstream signaling pathway. Compared with the control group, SIRT1 activator promoted Akt phosphorylation and increased the expression of Bcl-2 and HIF-1α, whereas SIRT1 inhibitor displayed the opposite effect. *P<0.05; #P<0.01. SIRT1, sirtuin 1; HIF-1α, hypoxia-inducible factor-1α; p, phosphorylated.

Moreover, the SIRT1 inhibitor and activator did not alter SIRT-1 expression levels, because the activator and inhibitor blocked the deacetylase activity of SIRT1 rather than altering SIRT1 expression levels.

ROS production promotes SIRT1 expression

Following culture under hypoxic conditions for 72 h, the generation of cellular ROS by HPAECs was assessed. The results indicated that culture for 72 h in hypoxic conditions significantly increased ROS levels compared with the control group (0 h; Fig. 3A). Therefore, ROS production may serve as an explanation for increased SIRT1 expression under hypoxic conditions.

Figure 3 Hypoxia increases SIRT1 expression by inducing ROS generation in cells. (A) ROS levels in cells were increased under hypoxic conditions for 72 h compared with 0 h. (B) SIRT1 enzyme activity assay. (C) SIRT1 protein expression in cells following treatment with H2O2 (1 mM) for 72 h. H2O2-induced SIRT1 activity was blocked by NAC (5 mM). *P<0.05 SIRT1, sirtuin 1; ROS, reactive oxygen species; NAC, N-acetylcysteine.

Cells treated with H2O2 (a type of ROS) displayed significantly increased SIRT1 activity and expression levels compared with the control group (Fig. 3B and C). H2O2-mediated upregulation of SIRT1 activity and expression was inhibited by the anti-ROS reagent NAC (Fig. 3B and C). Therefore, the results suggested that hypoxic conditions could induce ROS generation, which increases SIRT1 expression and its downstream signaling pathway.

SIRT1 promotes cell proliferation and inhibits cell apoptosis via the Akt signaling pathway

To investigate the role of SIRT1 in apoptosis, cells were cultured under hypoxic conditions for 72 h in the presence of SIRT1 activator, SIRT1 inhibitor and Akt antagonist. Compared with the control group, SIRT1 inhibitor significantly increased the level of apoptosis, whereas SIRT1 activator significantly decreased the level of apoptosis (Fig. 4). The Akt antagonist inhibited the anti-apoptotic effect of SIRT1, which indicated that Akt antagonist alleviated the anti-apoptotic effect of the SIRT1 activator.

Figure 4 SIRT1 prevents endothelial cell apoptosis under hypoxic conditions in vitro. Compared with the control group, hypoxia treatment promoted cell apoptosis. The proapoptotic effect of hypoxia was attenuated by SIRT1 activator but enhanced by SIRT1 inhibitor. *P<0.05; #P<0.01. SIRT1, sirtuin 1; PI, propidium iodide.

Subsequently, the effect of SIRT1 on cell proliferation was investigated. SIRT1 activator significantly increased cell proliferation compared with the control group, an effect that was reversed by the Akt antagonist. SIRT1 inhibitor significantly decreased cell proliferation compared with the control group (Fig. 5A). SIRT1 was knocked down using an siRNA, and the results indicated that cell proliferation was significantly decreased in the siRNA group compared with the scramble control group (Fig. 5B).

Figure 5 SIRT1 promotes endothelial cell proliferation in vitro. (A) Compared with the control group, SIRT1 activator increased cell proliferation, whereas SIRT1 inhibitor inhibited cell proliferation. In addition, Akt antagonist alleviated SIRT1 activator-induced proliferation. (B) SIRT1 knockdown decreased cell proliferation compared with the scramble control group. *P<0.05; #P<0.01. SIRT1, sirtuin 1; siRNA, small interfering RNA.

In addition, the results indicated that SIRT1 inhibitor significantly decreased cell proliferation and increased cell apoptosis in a dose-dependent manner compared with the control group (Fig. S1).

Discussion

The present study suggested that, compared with control cells, SIRT1 expression was increased in HPAECs under hypoxic conditions as a prosurvival gene expression profile triggered by chronic hypoxia. SIRT1 promoted cell proliferation and prevented cell apoptosis via regulation of the Akt signaling pathway. In addition, the present study indicated that SIRT1 expression was induced by ROS generation under hypoxic conditions. Collectively, the results of the present study furthered the current understanding of the role of SIRT1 in endothelial cells of the microvascular system under hypoxic conditions.

Hypoxic conditions can induce pathological alterations in endothelial cells of the vascular system. Under hypoxic conditions, ROS generation increases, overwhelming the antioxidant systems, which consist of stable oxidants, such as H2O2, and unstable free radicals, such as superoxide anion, nitric oxide, hydroxyl moiety and hypochlorite (25). Demarco et al (26) suggested that ROS are continuously generated in both endothelial and smooth muscle cells of the pulmonary vascular system under normal physiological conditions, which then participate in cellular events and regulate cell bio-behavior (27). However, excess ROS generation can induce DNA, lipid and peptides oxidation, which damages cells and induces tissue dysfunction, contributing to the pathogenesis of a number of diseases, such as pulmonary and systemic hypertension (28), heart failure (29) and ventricular hypertrophy (30). Furthermore, ROS may promote smooth muscle and endothelial cell vasoconstriction and proliferation in the microvascular system, which induces vascular remodeling and contributes to the pathogenesis of PAH (31). For instance, in mouse models of hypoxia-induced PAH, intrapulmonary artery ROS levels are elevated (32,33). Moreover, exposure to hypoxia increases ROS generation, xanthine oxidase levels and enzyme activities of pulmonary artery endothelial cells in mice (34). Therefore, the generation of ROS may induce expression alterations to genes and signaling pathways that contribute to the pathogenesis of PAH, including SIRT1.

Ruan et al (12) demonstrated that ROS generation induced by androgen deprivation therapy promoted SIRT1 expression in prostate cancer. In another study, Hasegawa et al (35) suggested that H2O2 can promote the expression of SIRT1 in renal tubular epithelial cells. In the present study, the results indicated that increased SIRT1 expression may be induced by cellular ROS generation, whereas NAC treatment alleviated SIRT1 upregulation in cells. It was hypothesized that the regulatory effect of NAC on SIRT1 expression may be attributed to its anti-ROS role in cells. However, NAC has multiple roles in cells, such as participation in cell apoptosis (36) and antibody-dependent cellular cytotoxicity (37). Therefore, the relationship between ROS and SIRT1 expression requires further investigation.

SIRT1 is a nicotinamide adenine dinucleotide+-dependent histone deacetylase (38) that can regulate cell apoptosis, migration and proliferation (39). The present study suggested that SIRT1 activated the Akt signaling pathway in vascular endothelial cells. Akt/mTOR is a classic signaling pathway that is regulated by SIRT1(40). Under basal conditions, Akt is acetylated in various tissues, including the heart, liver, brain and skeletal muscle, which suppresses Akt activity (41). Deacetylation of lysines by SIRT1 is necessary for the binding of Akt to phosphatidylinositol (3-5)-trisphosphate (PIP3), and for its membrane localization and activation (42). Activated Akt then phosphorylates mTOR and activates its downstream signaling pathway. Sundaresan et al (42) suggested that SIRT1 enhances the binding of Akt to PIP3 and promotes their activation via deacetylation in cancer and cardiac hypertrophy, whereas SIRT1 knockout in mice reduces cardiac hypertrophy. Furthermore, the SIRT1 and Akt signaling pathways serve a cardinal role in the process of angiogenesis, and SIRT1-deficient mice display an impaired ability to develop new blood vessels in response to angiogenic signals (43). In addition, decreased SIRT1 expression is observed in aging hearts (44), where it downregulates the activity of the Akt signaling pathway and induces cell apoptosis (45).

Akt/mTOR is an important intracellular signaling pathway for regulating multiple events in cells (46). Increasing evidence has demonstrated that the Akt/mTOR signaling pathway is activated and involved in PAH via vasoconstriction and remodeling (47). Furthermore, blocking the Akt/mTOR signaling pathway reverses PAH in animal models (48,49). The present study indicated that SIRT1 upregulated Akt/mTOR in endothelial cells under hypoxic conditions. Moreover, the results suggested that SIRT1 expression regulated HIF-1α protein levels as a downstream molecule of the Akt/mTOR signaling pathway. HIF-1 contains two subunits, HIF-1α (oxygen-sensitive) and HIF-1β (constitutive subunit), and its expression is activated by the restriction of oxygen supply (50). HIF-1 is another critical molecule in PAH. Abnormalities in HIF-1 that underlie PAH are observed in the rat model and blocking the expression of HIF-1 increases the level of voltage-gated potassium channel expression, thereby reducing pathologic alterations in PAH (22).

The present study had a number of limitations. In addition to endothelial cells, smooth muscle and adventitial cells may contribute to the pathogenesis of PAH. Zurlo et al (15) suggested that SIRT1 can regulate human pulmonary artery smooth muscle cell proliferation, serving a role in PAH development. Adventitia serves as another key regulator of pulmonary vascular wall function and structure (51). Burke et al (51) suggested that hypoxia induced vascular inflammation, which increased the thickness and inflammatory cells in the adventitia of the rat pulmonary artery. Therefore, the role of SIRT1 in smooth muscle and adventitial cells under hypoxic conditions requires further investigation. Furthermore, the present study did not use an animal model to investigate alterations in SIRT1 expression in PAH. The mouse model of PAH has been widely used in research, and the role of SIRT1 in vascular endothelial cells in vivo needs to be explored. For example, application of hypoxia and Sugen is a method commonly used to trigger pulmonary hypertension (52). Further research on SIRT1 needs to be performed in a mouse model to prove its translational value in the clinical setting. The administration of SIRT1 inhibitor in vivo has been reported by previous studies. Huang et al (53) suggested that a treatment-selective SIRT1 inhibitor with EX-527 attenuated lipopolysaccharide-induced histological abnormalities in lung tissue in mice. Furthermore, certain studies have used SIRT1 inhibitor to treat human Huntington disease. For example, the SIRT1 inhibitor selisistat has been used to treat Huntington's disease (54,55) in clinical trials, proving the safety and tolerance of SIRT1 inhibitor.

Collectively, the present study indicated that ROS generation promoted SIRT1 expression under hypoxic conditions, which activated the Akt signaling pathway to promote cell proliferation and inhibit cell apoptosis in microvascular endothelial cells. Therefore, inhibition of SIRT1 may serve as a potential therapeutic strategy against hypoxia in vascular endothelial cells.

Supplementary Material

SIRT1 inhibitor decreases cell proliferation and promotes cell apoptosis in a dose-dependent manner. (A) Compared with the control group, SIRT1 inhibitor decreased cell proliferation in a dose-dependent manner. (B) Compared with the control group, SIRT1 inhibitor increased cell apoptosis in a dose-dependent manner. *P<0.05. SIRT1, sirtuin 1; PI, propidium iodide.

Acknowledgements

Not applicable.

Funding

This study was supported by the Colleges and Universities in Hebei Province Science and Technology Research Project (grant no. QN2019189).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Authors' contributions

LX and LR designed and performed experiments, analyzed the data and wrote the manuscript. XY, DZ and HY performed experiments. QL and CY contributed to study design and wrote the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References