PARP-1 Involvement in Autophagy and Their Roles in Apoptosis of Vascular Smooth Muscle Cells under Oxidative Stress

Original Article

PARP-1 Involvement in Autophagy and Their Roles in Apoptosis of Vascular Smooth Muscle Cells under Oxidative Stress

(PARP-1 / autophagy / AMPK-mTOR / oxidative stress / apoptosis)
Y. Y. MENG1, C. W. WU1, B. YU2,3, H. LI4, M. CHEN5, G. X. QI5

1Department of Cardiology, 5Department of Geriatrics, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning Province, China
2Department of Cardiology, The Second Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province, China

3The Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Harbin, Heilongjiang Province, China
4Department of Cardiology, No.1 Central Hospital of Baoding, Baoding, Hebei Province, China

Abstract. Autophagy and poly(ADP-ribose) poly- merase 1 (PARP-1) are activated and involved in a series of cell processes under oxidative stress, which is associated with pathogenesis of atherosclerosis. Research on their relationship under oxidative stress has been limited. In this study, we aimed to investi- gate the activation, relationship, and role of au- tophagy and PARP-1 in vascular smooth muscle cell (VSMC) death under oxidative stress. This study ex- plored the signal molecule PARP-1 and autophagy in VSMCs using gene silencing and the hydrogen peroxide (H2O2)-stimulated oxidative stress model. We observed that H2O2 could induce autophagy in

VSMCs, and the inhibition of autophagy could pro- tect VSMCs against oxidative stress-mediated cell death. Meanwhile, PARP-1 could also be activated by H2O2. Additionally, we analysed the regulatory role of PARP-1 in oxidative stress-mediated au- tophagy and found that PARP-1 was a novel factor involved in the H2O2-induced autophagy via the AMPK-mTOR pathway. Finally, PARP-1 inhibition protected VSMCs against caspase-dependent apop- tosis. These data suggested that PARP-1 played a critical role in H2O2-mediated autophagy and both of them were involved in apoptosis of VSMCs.

Introduction

Autophagy is a catabolic pathway for the bulk destruc- tion of long-lived proteins and damaged organelles via lysosomes, which generates amino acids and substrates for cellular metabolism to maintain cellular homeostasis (Kroemer et al., 2010; Mizushima and Komatsu, 2011). Autophagy is activated under nutrient deprivation and/ or oxidative stress (Mizushima et al., 2004; Lee et al., 2012) and associated with atherosclerosis (Martinet and De Meyer, 2009). It is well known that oxidative stress, especially reactive oxygen species (ROS), is involved in the pathogenesis of atherosclerosis (AS), serving as an important promoting factor of AS, as well as an impor- tant inducer and mediator of autophagy (Stocker and Keaney, 2004; Scherz-Shouval et al., 2007).

At present, accumulating evidence suggests that au- tophagy is a double-edged sword in determining cell fate (Shintani and Klionsky, 2004). Autophagy is in- volved in the maintenance of normal cellular homeosta- sis and generally serves as a pro-survival mechanism, whereas excessive autophagy triggers cell death (Gomez- Santos et al., 2003; Liao et al., 2012). In some studies,

Received April 16, 2018. Accepted July 27, 2018.

This study was supported by the Key Laboratory of Myocardial Ischemia, Harbin Medical University, Chinese Ministry of Edu- cation (Grant No. KF201511).

Corresponding author: Guoxian Qi, Department of Geriatrics, The First Affiliated Hospital of China Medical University, No. 155, Nanjingbei Street, Heping District, Shenyang 110001, Liaon- ing Province, China. E-mail: tianya1711@sina.com; Phone: (+ 86) 24-83282226; Fax: (+86) 24-83282226

Abbreviations: 3-MA – 3-methyladenine, 4-HNE – 4-hydroxy- nonenal, 7-KC – 7-ketocholesterol, AMPK – adenosine 5’mo- nophosphate (AMP)-activated protein kinase, AS – atherosclero- sis, ATP – adenosine triphosphate, α-SMA – α-smooth muscle actin, DMSO – dimethyl sulphoxide, ERK – extracellular regu- lated protein kinases, H2O2 – hydrogen peroxide, HCQ – hydroxy- chloroquine, JNK – c-Jun N-terminal kinase, LC3 – light chain 3, mTOR –mammalian target of rapamycin, PAR – poly(ADP-ri- bose), PARP-1 – poly(ADP-ribose)polymerases 1, PBS – phos- phate-buffered saline, p-P70S6K – phospho-ribosomal protein s6 kinase, ROS – reactive oxygen species, RT-PCR – real-time poly- merase chain reaction, SD – standard deviation, VSMC(s) – vas- cular smooth muscle cell(s).

Folia Biologica (Praha) 64, 103-111 (2018)

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autophagy promotes vascular smooth muscle cell (VSMC) survival in response to reactive species, such as 7-keto- cholesterol (7-KC) (He et al., 2013), 4-hydroxynonenal (4-HNE) (Hill et al., 2008), and free cholesterol (Xu et al., 2010). A recent study demonstrated that autophagy inhibition by gene deletion prevented oxidative stress- induced cell death in VSMCs (Grootaert et al., 2015). Therefore, the role of autophagy in the viability of VSMCs under oxidative stress is uncertain and remains to be elucidated.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nu- clear enzyme that is activated by DNA injury and impli- cated in a wide range of essential cellular processes, in- cluding DNA repair, gene transcription, metabolism, and cell death (Langelier et al., 2012; Xu et al., 2014). However, the hyperactivation of PARP-1 induced by DNA damage results in various biological consequences, including cellular dysfunction and cell death (Szabo et al., 1996; Radovits et al., 2007). The ROS-DNA injury- PARP pathway has recently been established as a major downstream intracellular pathway of oxidative stress and implicated in the pathogenesis of various cardiovas- cular diseases (Pacher and Szabo, 2007).

It has been demonstrated that PARP-1 participates in the regulation of autophagy. Some chemotherapeutic agents such as alkylating agents (Zhou et al., 2013), doxorubicin (Munoz-Gamez et al., 2009) and radiother- apy (Chen et al., 2015) could induce autophagy through PARP-1 activation, but these studies are more concen- trated on the field of cancer. A recent report found that PARP-1 was involved in autophagy induction in umbili- cal vein endothelial cells (Li et al., 2015). In addition, PARP-1 hyperactivation not only led to necrotic cell death (Ha and Snyder, 1999), but was also involved in apoptosis (Wang et al., 2011). Recent studies have re- vealed that PARP-1 may mediate a new non-caspase dependent cell death type (Wang et al., 2011).

PARP-1 may be involved in multiple signal path- ways, but the specific mechanism by which PARP-1 af- fects cell fate remains unclear. The activation of au- tophagy and PARP-1 is closely related to oxidative stress and plays an important role in atherosclerosis. However, their precise role and relationship in VSMCs under oxidative stress has yet to be elucidated. Therefore, we sought to explore the role of PARP-1 in autophagy and their effects on cell death in VSMCs through a H2O2-stimulated oxidative stress model. In this study, we showed that inhibition of autophagy and PARP-1 de- ficiency could decrease cell death in VSMCs, and PARP-1 might contribute to the induction of autophagy via the AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) pathway.

Material and Methods

Reagents

We used the following antibodies from rabbits: antibod- ies against Beclin1, LC3, GAPDH, p-AMPK, p-P70S6K

(Cell Signaling Inc, Beverly, MA), antibody against PARP-1 (Santa Cruz Biotechnology, Santa Cruz, CA), antibody against PAR (Enzo Life Sciences, Farmingdale, NY). Chemicals: H2O2 was obtained from Sigma-Aldrich (St. Louis, MO); 3-methyladenine(3-MA) and hydroxy- chloroquine sulphate (HCQ) were purchased from Sel- leck (Houston, TX).

Cell culture

VSMCs were isolated from the thoracic aorta of C57BL/6J mice by tissue explantation and cultured in DMEM supplemented with 10% FBS, 100 U/ml peni- cillin and 100 U/ml streptomycin in air supplemented with 5% CO2 at 37 °C. Cultures were confirmed to con- sist primarily (> 95%) of VSMCs both morphologically, by their classical “hill and valley” appearance, and immu- nohistochemically, by α-smooth muscle actin (α -SMA) immunoreactivity. VSMCs at passages four to six and grown to 70% to 80% confluence were used for the ex- periments.

MTT assay

The MTT assay procedure was used to determine cell viability of the drug-treated VSMCs and untreated cells. MTT assays were performed by adding 20 μl MTT (5 mg/ml) to wells in a 96-well plate according to manu- facturer’s instructions (Sigma-Aldrich). After incubat- ing 4 h in the dark, the medium was removed and the cells were dissolved in 150 μl dimethyl sulphoxide (DMSO). The absorbance value was measured for each well using a microplate reader (Bio-Tek, Winooski, VT) at 490 nm (A490).

Detection of cell apoptosis

1) Hoechst 33258 staining: VSMCs were fixed and stained with Hoechst 33258. Subsequently, the cells were examined and photographed under a fluorescence mi- croscope (Olympus, Tokyo, Japan). Apoptotic cells were stained brighter due to chromatin condensation.

2) Flow cytometry: Apoptosis was analysed by using an Annexin V-FITC Apoptosis Detection Kit (Sigma- Aldrich, according to the instructions). VSMCs were collected together, washed twice with cold phosphate- buffered saline (PBS) and resuspended in 500 μl of binding buffer. Cells were then labelled for 15 min with annexin V-FITC (5 μl) and propidium iodide (5 μl) at room temperature in the dark. Cells were analysed with Accuri C6 flow cytometry (BD Biosciences, La Jolla, CA).

Western blot analysis

After the treatments, cells were collected and lysed in a lysis buffer. Cell samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes (PVDF) (Millipore, Bedford, MA). Membranes were subsequently blocked with 5% skimmed milk powder for 2 h and probed with primary antibodies overnight at 4 °C. Thereafter, membranes were incubated with HRP- conjugated secondary antibodies for 2 h with shaking.

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The optical density of bands was quantified by Quanti- tyOne software (Bio-Rad, Hercules, CA).

PARP1 gene silencing

The small interfering RNA (siRNA) targeting PARP-1 (5’-GAGCGACGCTTATTACTGT-3’) and the negative control siRNA were purchased from GeneChem Bio- technology (Shanghai City, China). VSMCs were trans- fected with PARP-1-specific siRNA or siRNA control according to the manufacturer’s instructions. The effi- ciency of siRNA-silenced genes was evaluated by real- time polymerase chain reaction (RT-PCR) and western blotting of the targeted proteins.

Quantitative RT-PCR analysis

Total mRNA was extracted from the cultured cells by the Trizol extraction method (BioTeke, Beijing, China). An equal amount of RNA was reverse-transcribed to cDNA by reverse transcription and amplified by PCR using primers. The expression of PARP-1 and housekeep- ing β-actin were measured by RT-PCR analysis using SYBR Green (Solarbio, Beijing, China) and the Exicycler 96 Real-Time PCR System (Bioneer, Daejeon, Korea) according to the manufacturer’s protocol. Data analysis was performed by the comparative method (2-ΔΔCT) using β-actin transcripts as an internal control.

Caspase-3 activity assay

The activity of caspase-3 like protease in the lysate was measured using a colorimetric caspase-3 assay kit (Sigma Aldrich) according to the manufacturer’s proto- col. VSMCs from differently treated groups were har- vested, lysed, and centrifuged. Then, aliquots of super- natants were collected and incubated with caspase-3 substrates at 37 °C overnight. The absorbance was read at 405 nm and the results were calculated using a cali- bration curve prepared using a stock solution of p-ni- troaniline (at the concentration range of 0–200 μM).

Statistical analysis

The results were expressed as mean ± standard devia- tion (SD). SPSS19.0 was used to analyse the differences between experimental groups. Statistical analyses were performed by using t-tests (2 groups) or one-way ANOVA with Bonferroni’s procedure for multiple com- parison tests (3 groups). P value < 0.05 was considered statistically significant.

Results

The effect of autophagy on VSMC survival under oxidative stress

In the present study, we first investigated VSMC death after the treatment with different concentrations of H2O2 and at different time points. H2O2 induced cell death in a dose-dependent manner (Fig. 1A-a) and gen- erally, a time-dependent manner (Fig. 1A-b). In addi- tion, we measured the autophagy response in VSMCs

treated with H2O2 at different time points. Our results showed that autophagy marker protein LC3-I to LC3-II conversion and beclin-1 expression significantly in- creased in VSMCs in a time-dependent manner after the treatment with H2O2 (Fig. 1B-a). Next, we examined the autophagy flux by lysosomal inhibitor HCQ. The pres- ence of HCQ further increased the LC3-II/LC3-I ratio (Fig. 1B-b), suggesting that H2O2 is capable of promot- ing the autophagy flux.

To determine the role of autophagy in VSMC survival under oxidative stress, we studied the role of autophagy in H2O2-mediated cell death by inhibition of autophagy. The results showed that autophagy inhibition by 3-MA significantly decreased the expression of LC3-II (Fig. 1B-b). Compared to the H2O2-treated group, the inhibi- tion of autophagy markedly increased cell viability as determined by MTT (Fig. 1C-a). To clarify the features of the decreased cell viability induced by H2O2, we fur- ther examined morphologic changes of VSMCs. As shown in Fig. 1C-b, apoptotic cells were observed to have condensed or segmented nuclei accompanied by bright blue fluorescence in H2O2-treated cells. The apop- totic cells were reduced in H2O2+3-MA-treated cells. In addition, apoptosis was further evaluated by flow cy- tometry.

H2O2 treatment led to a dramatic increase in the per- centages of early apoptotic (annexin V+/PI) cells, end- stage apoptosis and dead cells (annexin V+/PI+) (P < 0.05), and to a lesser extent, in the percentage of dam- aged cells (annexin V-/PI+) (P > 0.05). Inhibition of au- tophagy attenuated H2O2-induced cell death mainly through reducing early apoptosis (P < 0.05) (Fig. 1C-c). Taken together, these data indicated that autophagy sup- pression protected VSMCs against oxidative stress-me- diated cell death.

The activation of PARP-1 and its modulation of VSMC autophagy under oxidative stress

PARP-1 is activated in response to oxidative stress and implicated in the cell death and autophagy process under certain conditions. In this study, we first examined the activation of PARP-1 under oxidative stress by de- tecting PAR formation at different time points. H2O2 rapidly induced massive PAR polymer formation and peaked after 0.5 h (Fig. 2A).

Based on the previous research, we hypothesize that PARP-1 activation, AMPK activation, and subsequent mTOR inhibition might contribute to the induction of autophagy under oxidative stress. To determine whether PARP-1 activation contributes to the cell autophagy in- duced by H2O2 and its potential downstream signalling molecules, we used the gene silencing technology to block PARP-1 function in order to detect changes of PARP-1, AMPK and mTOR activity of VSMCs treated with H2O2.

Compared with the untreated group, H2O2 treatment markedly increased the PAR formation, the expression of p-AMPK, decreased the expression of p-P70S6K,

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Fig. 1. H2O2 induces cell death and autophagy, and the effect of autophagy on VSMC survival
(
A) VSMCs were treated with different concentrations of H2O2 or for different time periods. (A-a) Treatment of VSMCs with H2O2 at various concentrations for 16 h. (A-b) Treatment of VSMCs with H2O2 (0.1 mmol/l) for different time peri- ods. Cell viability was assessed using the MTT assay. *P < 0.05 versus control. (B) Detection of autophagy and autophagy flux. (B-a) Treatment of VSMCs (0.1 mmol/l) with H2O2 for different time periods. (B-b) VSMCs were pre-treated with HCQ (10 μM) or 3-MA (5 mM) for 1 h and then incubated with H2O2 (0.1 mmol/l) for 16 h. The protein expression of LC3 and beclin-1 was determined by western blot. GAPDH served as loading controls. (C) The effect of autophagy on VSMC survival under oxidative stress. (C-a) Cell viability was measured using the MTT assay after the same treatment as in (B-b). (C-b) Hoechst 33258 staining was used to visualize cell death. Bars, 50 μm. (C-c) Cell death was further analysed using annexin V/propidium iodide (PI) staining and flow cytometry. Data were expressed as mean ± S.D. of three independent experiments. *P < 0.05

Vol. 64 PARP-1 and Autophagy in VSMC Apoptosis 107

Fig. 2. PARP-1 activation contributed to H2O2-induced autophagy.
(
A) H2O2 induces PARP-1 activation. Treatment of VSMCs with H2O2 (0.1 mmol/l) for different time periods. PAR ex- pression was determined by western blot. *P < 0.05 versus control. (B) VSMCs were pre-treated with PARP-1 siRNA and then treated with or without H2O2 (0.1 mmol/l), PAR was measured after 2 h, and LC3 was measured after 16 h by western blot. (C) VSMCs were treated as (B) and the expression of p-AMPK, p-p70S6K proteins was analysed by western blot. Data were expressed as mean ± S.D. of three independent experiments. *P < 0.05

and finally enhanced LC3-I to LC3-II conversion (Fig. 2B, Fig. 2C). The silencing of PARP-1 significantly in- hibited H2O2-induced PAR formation, the expression of p-AMPK, restored the expression of p-P70S6K and sub- sequently attenuated LC3-I to LC3-II conversion (Fig. 2B, Fig. 2C). These observations suggest that the PARP- 1-AMPK-mTOR axis seems to be involved in the induc- tion of autophagy in response to oxidative stress.

The effect of PARP-1 on VSMC survival under oxidative stress

To investigate the role of PARP-1 in the cell death induced by H2O2, we used a genetic approach to silence PARP-1 with siRNA and measured cell viability by

MTT. We observed that PARP-1 knockdown signifi- cantly attenuated H2O2-induced cell death (Fig. 3A).

We further examined the nature of death by Hoechst 33258 staining, as seen in Fig. 3B-a. Condensed bright apoptotic nuclei were readily observed in H2O2-treated cells, and PARP-1 inhibition reduced cell apoptosis. The presence of apoptotic cells was further confirmed by flow cytometry. H2O2 treatment of VSMCs mostly re- sulted in a dramatic increase in the percentages of early apoptotic (annexin V+/PI) cells, end-stage apoptosis and dead cells (annexin V+/PI+) (P < 0.05). PARP-1 inhi- bition significantly decreased early apoptosis, as well as end-stage apoptosis and dead cells (P < 0.05). There was no significant difference between damaged cells (Fig. 3B). Taken together, these results suggested that PARP-1

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Fig. 3. PARP-1 effect on apoptosis in VSMCs through a caspase-dependent way
(
A) VSMCs were pre-treated with PARP-1 siRNA and then treated with or without H2O2 (0.1 mmol/l) for 16 h, and then cell viability was measured using MTT. (B) The effect of PARP-1 on VSMC survival under oxidative stress. (B-a) Cells were treated with vehicle control or H2O2 (0.1 mmol/l) for indicated time periods, and the nuclear morphology was de- tected using fluorescent microscopy. Bar = 50 μm. (B-b) Cell death was analysed using flow cytometry after the same treatment as in A. (C) VSMCs were pre-treated with PARP-1 siRNA and then treated with or without H2O2 (0.1 mmol/l) for 6 h, and caspase-3 activity was analysed by the colorimetric method. Data were expressed as mean ± S.D. of three independent experiments. *P < 0.05

inhibition protected VSMCs from cell death in response to H2O2.

Parthanatos, also known as PARP-1-dependent cell death, is a new form of programmed cell death based on DNA damage and PARP-1 activation, but independent of caspase (David et al., 2009). To examine whether the effect of PARP-1 was associated with the activation of caspase – a key factor of apoptosis, we measured the activity of caspase-3. We observed that PARP1 gene si- lencing significantly reduced caspase-3 activation in VSMCs treated with H2O2 (Fig. 3C). Thus, the effect of PARP-1 on VSMC apoptosis was associated with cas- pase activation.

Discussion

Autophagy, which is a fundamental cellular process and links to several cellular pathways, impacts VSMC survival and function. Emerging evidence demonstrates that autophagy in VSMCs would contribute to athero- sclerosis. Some stimuli can directly or indirectly acti- vate autophagy in VSMCs through ROS, such as H2O2, 7-KC, 4-HNE, ox-LDL (Hill et al., 2008; Huang et al., 2009; Ding et al., 2013; He et al., 2013). The role of autophagy has been investigated in many studies, sug- gesting that autophagy may exert different roles depend- ing on the cell type and the autophagic trigger. Some

Vol. 64 PARP-1 and Autophagy in VSMC Apoptosis 109

studies have found that autophagy induced by hypoxia, 7-KC, 4-HNE protected VSMCs against cell death (Hill et al., 2008; He et al., 2013; Ibe et al., 2013), while in some cases, autophagy played an opposite effect, such as the autophagy triggered by osteopontin leading to VSMC death (Zheng et al., 2012). Moreover, a recent study demonstrated that the inhibition of autophagy by gene deletion reduced oxidative stress-induced cell death (Grootaert et al., 2015).

Whether induction of autophagy in VSMCs exposed to H2O2 is the cause of death or actually an attempt to support survival in response to cellular stress remains to be determined. In this study, we found that autophagy was activated by H2O2 in VSMCs, and autophagy sup- pression by chemical inhibitor protected VSMCs against oxidative stress-mediated cell death. Grootaert et al. (2015) found that the effect of autophagy gene deletion, which protected VSMCs against oxidative stress-medi- ated cell death, was attributed to nuclear translocation of nuclear factor erythroid 2-like 2 (NFE2L2) resulting in up-regulation of several anti-oxidative enzymes. Another explanation is that autophagy might lead to autophagic cell death. Due to the lack of methods and accurate mo- lecular hallmarks for measuring autophagic cell death specifically and quantitatively, the mechanistic study of autophagic cell death has been difficult. However, we failed to explore this point in the current study, and fur- ther investigation should be conducted.

PARP-1 is a group of nuclear enzymes that partici- pate in DNA damage repair by poly(ADP-ribosyl)ation, which is activated by oxidative/nitrosative stress and critically involved in the pathogenesis of atherosclerosis through numerous cellular processes. This study explored the activation of PARP-1 and its regulatory function in VSMCs under oxidative stress. So far, the autophagy signalling pathway under oxidative stress has not been fully understood. Studies suggest that autophagy might be activated by up-regulation of NADPH oxidase 4 (Nox4) leading to inhibition of Atg4B activity, or be triggered by a JNK-dependent mechanism (Scherz- Shouval et al., 2007; Haberzettl and Hill, 2013). Recent studies have found that PARP-1 may be involved in the autophagy, but were mainly focused on the field of on- cology, such as effects of alkylating agents, radiation therapy, and doxorubicin (Huang et al., 2009; Munoz- Gamez et al., 2009; Zhou et al., 2013; Chen et al., 2015). These reports revealed a novel signalling pathway link- ing PARP-1 to autophagy through AMPK-mTOR.

In this study, we found that PARP-1 was activated by H2O2 and PARP-1 suppression drastically inhibited PARP-1 activation, AMPK activation, mTOR phospho- rylation and subsequently autophagy activation. Our data are found to be consistent with a very recent report that ox-Lp(a) induced human umbilical vein endothe- lial cells autophagy via PAPR-1-LKB1-AMPK-mTOR pathway (Li et al., 2015). Our observations suggest that the PARP-1-AMPK-mTOR axis appears to be an alter- native signalling pathway of autophagy during oxida- tive stress. We speculate the molecular mechanism to be

that ATP depletion caused by PARP-1 activation follow- ing oxidative DNA damage leads to AMPK activation. AMPK, an AMP-dependent protein kinase, is a sensor of the cellular energy level, which is activated during energy stress, particularly reduction in ATP levels (Jeon, 2016). There is now accumulating evidence indicating that AMPK is a key upstream regulator of autophagy via mTOR. AMPK negatively regulates mTOR activity ex- ecuted on downstream effectors, such as ribosomal S6 kinase, thereby regulating autophagy (Alers et al., 2012).

Besides autophagy, PARP-1 also plays an important role in determining cell fate. We further explored the role of PARP-1 in the death of VSMCs under oxidative stress. This study found that H2O2 mainly induced VSMC apoptosis, and apoptosis decreased after the PARP1 gene was silenced, indicating that PARP-1 inhibition has a protective effect. These results are consistent with the report that PARP-1 deletion conferred protection to VSMCs by blocking apoptosis in response to H2O2 and 7-KC (Hans et al., 2008). Parthanatos is a new form of programmed cell death, also known as PARP-1-dependent cell death. PARP-1 mediates parthanatos when it is over-activated in response to extreme genomic stress and synthesizes PAR, which causes nuclear transloca- tion of AIF from mitochondria to the nucleus, where it induces DNA fragmentation and ultimately cell death (David et al., 2009). Based on the fact that apoptosis is dependent on the caspase pathway activated by cy- tochrome c release, while the parthanatos pathway can act independently of caspase (Fatokun et al., 2014), we could distinguish them through caspase activation. To further clarify the mechanism of PARP-1-mediated ap- optosis, we examined the activity of caspase-3, a key apoptotic enzyme. The result showed that caspase-3 ac- tivity in the PARP-1 silencing group was significantly reduced compared to the control group after H2O2 expo- sure, indicating that PARP-1-mediated apoptosis is cas- pase-dependent. A previous study showed that the in- volvement of PARP-1 in apoptosis might be associated with extracellular regulated protein kinases (ERK) and c-Jun N-terminal kinase (JNK) signalling pathway (Hans et al., 2008).

Overall, our results indicated that PARP-1 contribut- ed to H2O2-induced autophagy through the AMPK- mTOR signalling pathway. Moreover, the inhibition of PARP-1 and autophagy conferred protection to VSMCs against cell apoptosis under oxidative stress. These find- ings provided novel insights into the function of PARP-1, which might be a promising therapeutic target for car- diovascular diseases.

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