Atorvastatin was purchased from Pfizer, Inc. (New York, NY, USA) and was diluted with anhydrous ethanol to 0.7, 7, 35 and 70 µM. Dulbecco's modified Eagle's medium (DMEM) was obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Fetal bovine serum (FBS) was from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Radioimmunoprecipitation assay (RIPA) lysis buffer was purchased from Beyotime Institute of Biotechnology (Haimen, China). The protease inhibitor cocktail was obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The lactate dehydrogenase (LDH) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Antibodies against neuron-specific enolase (NSE; #wl0278) and glial fibrillary acidic protein (GFAP; #wl0836) were purchased from Wanleibio (Shenyang, China). Antibodies against poly (ADP-ribose) polymerase-1 (PARP-1; #9542), Cleaved PARP-1 (#5625), caspase-3 (#9662), cleaved caspase-3 (#9664) β-actin (#3700), microtubule-associated protein 1A/1B-light chain 3 (LC3; #4108) and Beclin1 (#3738), and horseradish peroxidase-conjugated secondary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).

HUVECs were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were maintained in a humidified incubator at 37°C in a 5% CO₂ atmosphere.

HUVECs were seeded in 6-cm dishes. When the cells were 80% confluent, the medium was replaced with serum-free low-glucose DMEM and cells were treated with 0, 0.7, 7, 35 or 70 µM atorvastatin at 37°C for 24 h. Cells treated with an equal volume of anhydrous alcohol served as the control group and cells cultured with 10% serum-containing medium served as the normal group. Total RNA was extracted according to the manufacturer's protocol using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Then, 1 µg total RNA was reverse transcribed into cDNA according to the manufacturer's protocol, by the PrimeScript^™ RT reagent kit (RR037Q; Takara Biotechnology Co., Ltd., Beijing, China). The 20 µl RT reaction solution consisted of 4 µl 5X PrimeScript Buffer, 1 µl PrimeScript RT Enzyme Mix I, 1 µl Oligo(dT) Primer, 1 µl Random 6 mers, 1 µg Total RNA, and RNase Free dH₂O to 20 µl. The RT reaction procedure: 37°C for 15 min, followed by 5 sec at 85°C, and then 4°C for 10 min. PCR was performed using the following primers: NSE, forward, 5′-ACCTGACCTCTTGCTGTCTC-3′ and reverse, 5′-CTATGCACAGTTCACGGCTC-3′; neurofilament light polypeptide (NF-L), forward, 5′-TGGGTGTGGAGATTTGTTAGGA-3′ and reverse, 5′-TAGGACACCAACCTGCTGTG-3′; β-actin, forward, 5′-AAGATCAAGATCATTGCTCCTC-3′ and reverse, 5′-GGACTCATCGTACTCCTG-3′. PCR was performed using SYBR Premix Ex Taq II kit (#DR039A; Takara Biotechnology Co., Ltd., Dalian, China) and carried out in a ABI 7500 system (Applied Biosystems, Carlsbad, CA, USA). The PCR solution consisted of 12.5 µl 2X Premix, 1 µl Forward Primer (10 µM), 1 µl Reverse Primer (10 µm), 2 µl cDNA template and 8.5 µl RNase-free dH₂O. PCR procedure: 95°C for 5 min, followed by 40 cycles of 5 sec at 95°C, 45 sec at 60°C, and then 72°C for 1 min. The PCR products were resolved by 2.0% agarose gel electrophoresis and stained by GelRed (A616697; Sangon Biotech Co., Ltd., Shanghai, China). The ImageJ program (version 1.44p; National Institutes of Health, Bethesda, MD, USA) was used to densitometry analysis for semi-quantitation of the bands obtained by ChemiDoc™ XRS+ System. Relative target gene expression was normalized to β-actin expression.

A total of 2,000 HUVECs were seeded in 96-well plates. Following treatment with 0, 0.7, 7, 35 or 70 µM atorvastatin for 24 h, at 37°C in a 5% CO₂ atmosphere, and cells treated with an equal volume of anhydrous alcohol served as the control group, and cells cultured with 10% serum containing medium served as the normal group. Then cells were stained with 4% sulforhodamine B (SRB) at room temperature for 15 min. The absorbance of each sample was subsequently measured at 570 nm using a microplate reader.

When the cells were 80% confluent, the medium was replaced with serum-free low-glucose DMEM and HUVECs were treated with 0, 0.7, 7, 35 or 70 µM atorvastatin for 24 h at 37°C. Cells treated with an equal volume of anhydrous alcohol served as the control group and cells cultured with 10% serum-containing medium served as the normal group. Following treatment, the cell media were collected. The cell necrosis rate was evaluated using an LDH assay kit, according to the manufacturer's protocols. Subsequently, the absorbance of each sample was measured at 440 nm using a microplate reader.

Following treatment detailed above, HUVECs were washed twice with PBS and lysed with RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride on ice for 15 min. The lysates were collected and centrifuged at 12,000 × g at 4°C for 15 min. Equal quantities of extracted protein (20–40 µg) were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked in 5% (w/v) nonfat dry milk dissolved in Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature, and then incubated with primary antibodies (PARP-1, 1:500; cleaved PARP-1, 1:500; caspase-3, 1:200; cleaved caspase-3, 1:500; β-actin, 1:3,000; LC3, 1:400; and Beclin1, 1:200) at 4°C overnight. The membranes were subsequently washed three times in TBST, and incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (#7074, 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. TBST was used to wash the membranes three times, and the protein bands were visualized using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.). β-actin was used as the loading control. Blots were semi-quantified using ImageJ software (version 1.44p; National Institutes of Health, Bethesda, MD, USA).

The results are expressed as the mean ± standard error of the mean. Data were collected from at ≥3 independent experiments. Data were then analyzed with the GraphPad Prism 5 software (version 5.01; GraphPad Software, Inc., La Jolla, CA, USA). Differences among groups were assessed using one-way analysis of variance followed by a post hoc Holm-Šídák test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

Following treatment with increasing concentrations of atorvastatin for 24 h, HUVECs gradually exhibited a long and thin, neuron-like cell morphology, which was particularly pronounced at 70 µM atorvastatin (Fig. 1). Following treatment for 48 h, these morphological alterations were more pronounced, when compared with the same concentration at 24 h (Fig. 1).

Since treatment with atorvastatin induced a neuronal-like morphology in HUVECs, the potential of atorvastatin to induce HUVEC trans-differentiation into neuron-like cells was investigated. The neuronal markers, NSE and NF-L, were detected using semi-quantitative RT-PCR, and NSE and GFAP were detected using western blot analysis. As presented in Fig. 2, the mRNA and protein expression levels of these markers remained unaltered following treatment with all concentrations of atorvastatin. Therefore, these results indicate that atorvastatin does not induce the trans-differentiation of HUVECs into neuron-like cells.

The effects of atorvastatin on cell survival and necrosis were investigated in HUVECs using SRB staining and an LDH assay, respectively. The results revealed that, following treatment with 7, 35 and 70 µM atorvastatin, the cell survival rate significantly decreased in a dose-dependent manner (Fig. 3A). In addition, a significant increase in necrosis was observed following treatment of HUVECs with 70 µM atorvastatin when compared with the control cells (Fig. 3B). These results suggest that high doses of atorvastatin may exert cytotoxic effects on HUVECs.

As a high dose of atorvastatin was revealed to decrease the survival rate of HUVECs, the authors investigated the possibility that apoptosis may have been responsible for these observed effects. To explore this hypothesis, the expression levels of apoptosis-associated proteins were determined by western blot analysis. The protein expression levels of cleaved caspase-3, which is a widely-used marker of apoptosis, were significantly upregulated in HUVECs following treatment with 35 µM atorvastatin, in the presence and absence of serum (Fig. 4). Consistent with these observations, the protein expression levels of an additional marker of apoptosis, cleaved PARP-1, were significantly upregulated in HUVECs treated with 35 µM atorvastatin in the presence and absence of serum when compared with control cells (Fig. 4).

In order to investigate the effect of atorvastatin on cell autophagy, the protein expression levels of LC3 and Beclin1, which are common markers of autophagy, were analyzed. The results demonstrated that the expression levels of LC3 and Beclin1 were significantly upregulated following treatment with atorvastatin in the absence of serum, regardless of the concentration that was used (Fig. 5). By contrast, LC3 and Beclin1 protein expression levels were significantly reduced following treatment with 0.7 µM atorvastatin in the presence of serum (Fig. 5). These results suggested that under different conditions, such as with or without serum, atorvastatin may exert varying effects on HUVECs.