Endothelial basal medium (EBM), penicillin, streptomycin, fetal calf serum (FCS), human endothelial growth factor β (β-ECGF), TRIzol reagent and Lipofectamine 2000 were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Zoledronate (full name, 2-(imidazole-1-yl)-hydroxy-ethylidene-1, 1-bisphosphonic acid, disodium salt, 4.75 hydrate; molecular weight, 401.6) was purchased from Novartis International AG (Basel, Switzerland). A stock solution of Zoledronate (100 mM) was prepared in filter-sterilized phosphate-buffered saline (PBS). Chloroquine, 3-methyladenine (3-MA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33258 were obtained from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany).

Human umbilical cord veins were obtained from the Affiliated Nanjing University Medical School and human umbilical vein endothelial cells (HUVECs) were isolated as previously described (25). The study was approved by the Medical Research Ethics Committee of Medical School of Nanjing University and informed content was obtained from 20 pregnant women giving birth between 2012 and 2014. In brief, cells were harvested from umbilical cord by 0.125% trypsin, and then cultured in EBM supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 20% FCS and 100 µg/ml EGF in an incubator with 5% CO2 and 95% air at 37°C.

Total RNA from HUVECs treated with various concentrations of Zoledronate (25, 50, 75 and 100 µM) for 48 h was isolated using TRIzol reagent according to the manufacturer's protocols. Total RNA (1 µg) was reverse-transcribed using SuperScript III First-Strand Synthesis system (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Amplification was performed using Fast SYBR^® Green Master Mix Kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) in an Applied Biosystems 7300 Fast RT-PCR system and LC3B expression was calculated using the 2-^ΔΔCq method as previously described (26). A total of 35 cycles were conducted consisting of denaturation at 95°C for 15 sec, primer annealing at 60°C for 1 min and primer extension at 72°C for 30 sec. The specific primer sequences used in this study were as follows: LC3B, forward, 5′-AGCAGCATCCAACCAAAATC-3′, reverse, 5′-CTGTGTCCGTTCACCAACAG-3′; 18S, forward, 5′-CGGCTACCACATCCAAGGAA-3′, reverse, 5′-CTGGAATTACCGCGGCT-3′.

Proteins from HUVECs (2×105 cells) treated with various concentrations of Zoledronate (25, 50, 75 and 100 µM) for 48 h were isolated with radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Jiangsu, China) containing protease and phosphatase inhibitor cocktail (Merck Millipore, Darmstadt, Germany). The protein content of cell lysates was determined by Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). An equal amount of protein (50 µg) from HUVEC lysates was separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto Immobilon-P membranes (Merck Millipore, Volketswil, Switzerland), blocked with 5% non-fat milk with TBST (Tris-HCL 20 mM, NaCl 150 mM, 0.1% Tween 20) and then incubated with antibodies against LC3B (cat. no. 3868), sequestome 1 (SQSTM1) (cat. no. 8025), Beclin-1 (cat. no. 3738), cleaved caspase-9 (cat. no. 7237), cleaved caspase-3 (cat. no. 9579) and β-actin (cat. no. 3700) (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) overnight at 4°C. Following incubation with the horseradish peroxide-labeled goat anti-rabbit (cat. no. A0208) or goat anti-mouse (cat. no. A0216) antibody (Beyotime Institute of Biotechnology) at room temperature for 1 h, the signals were visualized on radiographic film using enhanced chemiluminescence reagents (Bio-Rad Laboratories, Inc.). The density of the appropriate sized bands was quantified using ImageJ software, version 1.41 (National Institutes of Health, Bethesda, MD, USA).

Adenovirus encoding GFP-LC3 (Ad-GFP-LC3) was obtained from Cyagen Biotechnology Co., Ltd. (Santa Clara, CA, USA). HUVECs (2×105 cells) were cultured in EBM containing 2% FCS and Ad-GFP-LC3 (multiplicity of infection, 100) for 3 h, following which, the medium was replaced with fresh complete medium. HUVECs were infected with Ad-GFP-LC3 for 24 h prior to treatment with various concentrations of Zoledronate for a further 48 h. Following treatment, cells were harvested and fixed in 4% paraformaldehyde and visualized and photographed in Zeiss LSM 5 Pascal laser scanning confocal fluorescent microscope (Zeiss AG, Oberkochen, Germany). The average number of GFP-LC3 punctae per cell were counted to quantify autophagy activities using ImageJ software, version 1.41 (National Institutes of Health).

Cell viability was determined by MTT assay. HUVECs (2×103 cells/well) were plated in 96-well culture plates and rendered quiescent by culturing in serum-free medium overnight at 37°C. Following treatment with different concentrations of Zoledronate for 48 h, 10 µl MTT (5 mg/ml) was added to each well, and plates incubated for a further 4 h. The medium was removed and 100 µl of dimethyl sulfoxide was added to solubilize the formazan crystals. Absorbance of the medium was measured at 570 nm using the SPECTRAMax M5 reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

HUVEC were treated with various concentrations of Zoledronate (25, 50, 75 and 100 µM) for another 48 h, or pretreated with 3-MA (5 mM) for 30 min prior to Zoledronate (100 µM) incubation for another 48 h. Cell apoptosis was evaluated by fluorescein isothiocyanate (FITC)-conjugated Annexin-V and propidium iodide (PI) staining by using FITC-Annexin V and PI double staining kit (KeyGen Biotech Co., Ltd, Nanjing, China) according to the manufacturer's protocols. Treated or untreated cells (2×105) were digested and suspended in 500 µl binding buffer plus 5 µl FITC-labeled Annexin V and 5 µl PI solution. The mixtures were incubated on ice for 10 min, and then plotted for FITC-conjugated Annexin V and PI in a two-way dot plot to count the apoptotic cells with flow cytometry (BD Biosciences, San Jose, CA, USA).

Apoptotic morphology of HUVECs was observed by Hoechst 33258 dye. Cells were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 and incubated with 2.5 µg/µl Hoechst 33258 for 5 min at room temperature. Stained cells were washed, and nuclei observed by fluorescence microscopy using an IX83 inverted motorized microscope (Olympus Corporation, Tokyo, Japan).

Beclin-1 siRNA (5′-GGUCUAAGACGUCCAACAA-3′) and non-targeting negative control siRNA (5′-UGGUUUACAUGUCGACUAA-3′; Invitrogen; Thermo Fisher Scientific, Inc.) were transiently transfected with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Briefly, siRNA and Lipofectamine 2000 were mixed at ratio of 1:4, then incubated in serum- and antibiotic-free EBM for 15 min. The mixture was added to 70% confluence of HUVECs and swirled gently to ensure uniform distribution. Following 3 h incubation at room temperature, the transfection mixture was removed and fresh complete medium was added prior to further incubation. HUVECs were incubated for 24 h following siRNA transfection, followed by 48 h Zoledronate (100 µM) treatment.

All data are expressed as the mean ± standard error, and n values indicate the number of independent experiments performed. Comparisons between multiple groups were analyzed by one-way analysis of variance, followed by Tukey's multiple comparison post-hoc test. All statistical analyses were performed using SPSS software (version 17.0; SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

qPCR analysis of HUVECs revealed a significant dose-dependent increase in the mRNA expression levels of LC3B following Zoledronate treatment (Fig. 1A). To further confirm autophagy in HUVECs was mediated by Zoledronate, the conversion of LC3B-I to LC3B-II was analyzed, as this suggests an increase in the number of autophagosomes within cells (27). Although the protein level of LC3B-I was marginally increased, Zoledronate treatment significantly increased the levels of LC3B-II and the ratio of LC3B-II/I compared with untreated cells (P<0.05; Fig. 1B). Furthermore, infection with GFP-LC3 adenovirus revealed a dose-dependent increase in the number of GFP puncta in the cytoplasm following Zoledronate exposure, indicating increased transformation of LC3B-I to LC3B-II (P<0.05; Fig. 1C and D). By contrast, the levels of SQSTM1, which is degraded in the lysosome following autophagosome fusion (28), were significantly decreased in HUVECs that were treated with >50 µM Zoledronate in a dose-dependent manner (P<0.01; Fig. 1E). Western blot analysis of another autophagy marker, Beclin-1, which is required for the formation of autophagosomes (29), revealed that the protein expression level of Beclin-1 was increased dose-dependently following exposure to Zoledronate (P<0.05; Fig. 1F). Zoledronate was, therefore, demonstrated to induce autophagy in HUVECs.

Increased autophagy can be attributed either to enhanced autophagosome formation or reduction of lysosomal activity (30). Since the increased autophagosome formation or the decreased lysosomal degradation may result in LC3B-II accumulation, the protein expression levels of LC3B-II were measured in HUVECs treated with 100 µM Zoledronate in the presence or absence of the lysosome inhibitor, chloroquine (1 µM). Western blot analysis revealed that pretreatment with chloroquine increased basal LC3B-II protein expression levels compared with vehicle-treated cells (P<0.01) and further enhanced the Zoledronate-induced increase in LC3B-II levels compared with cells treated with Zoledronate only (P<0.01; Fig. 2A). Furthermore, accumulation of SQSTM1 was also enhanced in HUVECs following inhibition of lysosomal activity by chloroquine compared with vehicle-treated cells (P<0.01), and chloroquine treatment abolished the inhibitory effect of Zoledronate on SQSTM1 levels (P<0.01; Fig. 2B). Zoledronate was therefore demonstrated to induce autophagy through increased autophagic activity rather than inhibition of lysosome degradation.

The effect of Zoledronate on the viability or apoptosis of HUVECs was investigated, due to the intricate crosstalk between autophagy and apoptosis (24). Zoledronate was demonstrated to significantly decrease the viability of HUVECs in a dose-dependent manner compared with untreated control (P<0.01), as demonstrated in Fig. 3A. Cell viability following treatment with 100 µM Zoledronate was decreased to 68.4±5.8% compared with untreated control cells (P<0.01; Fig 3A).

To further determine whether Zoledronate-induced cell death was due to apoptosis, molecular and cellular changes associated with apoptosis were analyzed. Increased activation of caspase signaling was observed, as demonstrated by a significant increase in the levels of cleaved caspase-9 and caspase-3 following Zoledronate treatment, compared with untreated cells (P<0.05; Fig. 3B and C). HUVECs were subsequently stained with FITC-Annexin-V to detect early apoptotic cells, and PI to detect late apoptotic/necrotic cells (31). Dose-dependently increased PI and Annexin-V cell staining was observed in Zoledronate-treated cells compared with untreated control cells (Fig. 3D), resulting in a significant dose-dependent increase in the total apoptosis rate (P<0.01; Fig. 3E). However, the increased apoptosis induced by 100 µM Zoledronate was significantly inhibited by 59.2±6.7% following pretreatment with 5 mM 3-MA, an autophagy inhibitor (P<0.01; Fig. 3D and E). 3-MA treatment alone exerted no effect on cell apoptosis (data not shown). Increased nuclear fragmentation in Zoledronate-treated HUVECs compared with untreated control cells was also observed by Hoechst 33258 staining (P<0.01; Fig. 3F and G), and was attenuated by inhibition of autophagy with 3-MA (P<0.01; Fig. 3F and G). Increased autophagy is, therefore, indicated to be involved in Zoledronate-induced apoptosis in HUVECs.

As Zoledronate was demonstrated to induce an increase in Beclin-1 expression, it was speculated that the autophagy induced by Zoledronate was dependent on the Beclin-1 pathway. To examine this hypothesis, Beclin-1 expression was genetically knocked down in HUVECs prior to Zoledronate exposure, and changes in cell apoptosis were measured. Transfection efficiency was confirmed by a significant decrease in Beclin-1 protein expression in siRNA-treated cells (P<0.01; Fig. 4A). Inhibition of Beclin-1 expression decreased the basal LC3B-II protein level compared with untransfected cells (P<0.05; Fig. 4B), and also abrogated the Zoledronate-induced increase in LC3B-II protein expression levels when compared with untransfected cells treated with 100 µM Zoledronate (P<0.01; Fig. 4B). By contrast, inhibition of Beclin-1 expression increased basal SQSTM1 protein expression levels compared with untransfected cells (P<0.01; Fig. 4C), and the decreased SQSTM1 protein expression levels induced by Zoledronate treatment (P<0.01; Fig. 4C), were partially restored in Beclin-1 siRNA-transfected cells (P<0.01; Fig. 4C).

The involvement of Beclin-1-mediated autophagy in HUVEC apoptosis following Zoledronate exposure was subsequently investigated. Beclin-1 knockdown was revealed to significantly increase cell viability in HUVECs following 48 h exposure to 100 µM Zoledronate by MTT assay (P<0.01; Fig. 4D) and Hoechst 33258 staining (P<0.01; Fig. 4E and F) compared with untransfected/Zoledronate-treated control cells. However, knockdown of Beclin-1 did not significantly affect HUVEC cell viability or Hoechst staining in the absence of Zoledronate compared with untransfected control cells (Fig. 4D-F). Beclin-1 is, therefore, suggested to be important for Zoledronate-induced HUVEC apoptosis.