Human umbilical vein endothelial cells (HUVECs) were purchased from Gibco; Thermo Fisher Scientific, Inc. (cat. no. C0155C). Cells were cultured in Medium 200 (cat. no. M200500) supplemented with LSGS (cat. no. S00310; both from Gibco; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. HUVECs were digested with Trypsin/EDTA at the appropriate confluency (~70–80%). Cells were cultured in an incubator under normal conditions or with sevoflurane treatment (1 and 3%). Treatment with sevoflurane was performed according to a previously reported method (18) and was achieved by connecting the incubator with the sevoflurane vaporizer (Abbott Laboratories) attached to the anesthetic machine (Dräger). The infrared gas analyzer (Puritan-Bennett) was used to monitor the sevoflurane concentration at the inflow and outflow connectors.

Cell viability was performed by MTT assay (cat. no. KA1606; Abnova). HUVECs were seeded in a 96-well plate at 2,000 cells/well under different conditions for 12, 24 48, and 72 h. Reagent medium (15/80 µl per well) was added followed by incubation for 4 h at 37°C. For the treatment with the VEGFA antibody, the cells were incubated with the antibody (20 µM; cat. no. AF-493-NA; R&D Systems) to chelate the effects of VEGFA in the culture medium and the corresponding control antibody (20 µM; cat. no. AB-108-C; R&D Systems) was applied as a control during the exposure of sevoflurane. Then 100 µl of the solubilizer was added to each well. OD570 nm was measured for each well on an absorbance plate reader. The cell viability was calculated by the ratio of OD570 at each time-point to OD570 at 0 h of each well and presented as the percentage of the ratio.

Total RNA was extracted from HUVECs using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). For mRNA analysis, 2 µg total RNA was reverse-transcribed to cDNA using the PrimeScript^® reagent kit (Takara Bio, Inc.). Quantitative PCR for mRNA was performed using SYBR-Green (Takara Bio, Inc.) with an ABI 7900 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 3 min; followed by 40 cycles of 95°C for 10 sec, 60°C for 5 sec and final extension at 72°C for 10 sec. Quantification of the mRNA expression was normalized to β-actin. The fold-change of the mRNA levels relative to the control cells was calculated using the 2^−ΔΔCq method (19). The sequences of the human-specific primers were as follows: VEGFa forward, 5′-CGAAGAGAAGAGACACATTG-3′ and reverse, 5′-GGATGGAGGAAGGTCAAC-3′; VEGFb forward, 5′-ACAGGACAGAGTTGGAAGA-3′ and reverse, 5′-GGAAGAGCCAGTTGTAAGAT-3′; VEGFc forward, 5′-TGTGTCCAGTGTAGATGAAC-3′ and reverse, 5′-TCTTCTGTCCTTGAGTTGAG-3′; VEGFR1 forward, 5′-ACTCGTGGCTACTCGTTA-3′ and reverse, 5′-ACCTTGCTTCGGAATGATT-3′; VEGFR2 forward, 5′-ACTGTCATCCTTACCAATCC-3′ and reverse, 5′-CCTCCAACTGCCAATACC-3′ and VEGFR3 forward, 5′-ATGCGAATACCTGTCCTAC-3′ and reverse: 5′-GTTGCCGATGTGAATGAG-3′.

Cells subjected to siRNA transfection were firstly cultured in half the volume of the culture medium and transfected with 3 ng/ml siRNA (sense, 5′-GCAGCGACAAGGCAGACUAUU-3′ and antisense, 5′-UAGUCUGCCUUGUCGCUGCGU-3′) or control siRNA (sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′) (Shanghai GenePharma Co., Ltd.) by RNAiMAX (Invitrogen; Thermo Fisher Scientific, Inc.) for 8 h according to the product protocol. After 8 h of incubation, the other half of the culture medium was added, and cells were incubated for another 40 h so that the total interference lasted for 48 h. After 48 h of incubation, the culture medium was changed to normal conditions without siRNA and subjected to further experiments.

Cells from each group were scraped in RIPA lysis buffer (Thermo Fisher Scientific, Inc.) with serine protease inhibitor. Proteins (50 µg) were separated on 10% SDS-polyacrylamide gels and electroblotted onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore). After incubating with blocking buffer, the membranes were incubated overnight at 4°C with the primary antibodies: Anti-VEGFR2 (dilution, 1:2,000; product code ab2349), anti-phosphorylated (p)-VEGFR2 (phosphorylated at Tyr 1175; dilution, 1:2,000; product code ab194806) and anti-beta Actin (dilution, 1:2,000; product code ab8226; all obtained from Abcam). Next, the membranes were washed and incubated with appropriate anti-rabbit HRP-conjugated secondary antibodies (dilution, 1:3,000; product code ab6728, Abcam) at 37°C for 30 min. Protein bands were detected using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.). The integrated optical density of the detected protein band was normalized to the control band for quantitative comparison by ImageJ (v. d1.47; National Institutes of Health).

Axitinib, a VEGFR inhibitor, was purchased from Selleck Chemicals and was dissolved in DMSO at a stock concentration of 10 mM. The inhibitor was diluted to an appropriate final concentration in the culture medium.

All experiments were performed at least three times with data expressed as the mean ± SEM or as otherwise stated. Statistical analyses were performed with GraphPad Prism software (version 7; GraphPad Software, Inc.). Group comparisons were performed using one-way ANOVA with post hoc test using Dunnett's test or Sidak's multiple comparisons test. The two-column comparison was performed using Student's t-test. P<0.05 or P<0.01 were considered to indicate a statistically significant difference (*P<0.05 and **P<0.01, as indicated in the figures and legends).

To investigate whether sevoflurane was able to stimulate proliferation of HUVECs, the HUVECs were cultured in different concentrations of sevoflurane. The MTT colorimetric assay was used to determine HUVEC activity at different concentrations (1 and 3%, respectively) of sevoflurane at different time-points (12, 24, 48 and 72 h, respectively). The results revealed that sevoflurane increased cell viability in a dose- and time-dependent manner (Fig. 1).

The VEGF pathway has been demonstrated to serve essential roles in promoting HUVEC proliferation (5). The most well-known VEGFs and their receptors (VEGFRs) include VEGFa, b, and VEGFR-1, 2, 3. Thus qPCR was used to examine the expression levels of VEGFs and VEGFRs of HUVECs treated with 1, and 3% sevoflurane, respectively. As revealed in Fig. 2A, compared with the control group, only the expression level of VEGFa was increased after exposure to sevoflurane for 48 h, and the increase was in a concentration-dependent manner (P<0.05; Fig. 2A). Similarly, VEGFR2 expression was significantly increased after sevoflurane exposure (P<0.05; Fig. 2B). These data indicated that sevoflurane induced the expression of VEGFa, which may regulate VEGFR2 and activation of the following signaling pathway.

To explore whether VEGFa secreted by HUVECs is involved in regulating the cell proliferation under sevoflurane exposure, HUVECs in the presence of 1 and 3% sevoflurane were transfected with VEGFa siRNA. The interference efficiency is presented in Fig. S1. The data revealed that silenced expression of VEGFa resulted in decreased Ki67-positive HUVECs (Fig. 3A and B). Moreover, the decreased expression of VEGFa exhibited a similar effect on the viability of cells (Fig. 3C and D). To further assess whether VEGFa was sufficient to target sevoflurane stimuli, a VEGFa chelation experiment was performed in cell culture using anti-VEGFa antibodies. As revealed in Fig. 3E and F the proliferation of HUVECs under either 1 or 3% sevoflurane medium was significantly blocked by the addition of anti-VEGFa while the group receiving control IgG antibodies exhibited high proliferation. These data indicated that the enhanced proliferation of HUVECs due to the effect of sevoflurane was regulated by the expression level VEGFa.

To investigate whether VEGF signaling was the underlying mechanism of sevoflurane in enhancing HUVEC proliferation, a small molecular VEGFR inhibitor, axitinib, was used to block the VEGFR signaling pathway. Firstly, western blotting was used to assess the levels of VEGFR and p-VEGFR after exposure of HUVECs to different concentrations of Sevoflurane with VEGFR inhibitor for 48 h. As revealed in Fig. 4A and B, the significant decrease of the p-VEGFR level was observed in HUVECs exposed to axitinib and sevoflurane when compared with cells treated with sevoflurane alone. Notably, following the inhibition of the VEGFR pathway, there was significantly decreased cell proliferation in the cells treated with axitinib plus sevoflurane than with sevoflurane alone (Fig. 4C and D), implying that sevoflurane promotes the proliferation of HUVECs via activation of the VEGF/VEGFR pathway.