HUVECs were obtained from Capsugel, Morristown, NJ, USA). The cells were cultured in Endothelial Growth Basal Medium (EBM-2; Lonza Group, Ltd., Basel, Switzerland) supplemented with growth factors according to the manufacturer's protocols. To analyze the changes in SIRT4 expression in response to oxLDL (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at various concentrations, cells were treated with oxLDL for 24 h at concentrations of 0, 10, 50 and 100 µM at 37°C. In order to analyze changes in SIRT4 expression in response to oxLDL treatment following different time periods, cells were treated with 50 µM oxLDL for 0, 12, 24 and 48 h at 37°C. Following treatment, cells were harvested and assessed for changes in SIRT4 expression using western blotting.

The complete open reading frame of SIRT4 was cloned into the pLVX–IRES-ZsGreen1 (Clontech Laboratories, Inc., Mountain View, CA, USA) plasmid (OV-SIRT4), using the empty vector as negative control (-NC). pLVX-SIRT4 was generated by transiently transfecting 293T cells (Beijing Zhongyuan Ltd., Beijing, China). Lentiviral production, concentration and titration were performed as follows: 293T cells (70–80% confluence) were seeded in 6-well plates, and 1.5 µg pLVX–IRES-ZsGreen1 was transfected using the Lipofectamine^® 2000 (40 µl; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Then, 293T cells were cultured for 48 h at 37°C. Lentivirus particles were directly collected and concentrated from the cell culture medium at 48 h following transduction by multi-steps of ultracentrifugation (50,000 × g at 4°C for 2 h). The titration (transduction units, TU) and multiplicity of infection of concentrated lentivirus particles were determined in 293T cells grown in 96-well plates by serial dilutions. For infection purposes, 2×10⁵ HUVECs were divided into 2 groups and subcultured in 6-well culture plates for 24 h prior to transduction. For infection, the cell culture medium was removed and cells were washed twice with phosphate-buffered saline (PBS). Next, 0.5 ml of lentiviral suspension (1×10⁸ IU/ml, multiplicity of infection=100) containing 8 µg/ml Polybrene was added to the cells. Cells were then incubated at 37°C overnight. Vector suspension was then aspirated from the cells and the transduced cells were added to 2 ml/flask of fresh growth medium. Cells were then incubated at 37°C in a humidified atmosphere containing 5% CO₂. The growth medium was replaced after 24 h. After allowing the cells to incubate for 72 h at 37°C, HUVECs cells were passaged twice per week with growth medium containing 5 µg/ml puromycin to select for cells expressing the transduced vector. Positively screened cell lines were sub-cloned three times using limiting dilution (diluent: EBM-2) and cultured in growth medium containing puromycin for 1 month to generate stable cell lines. HUVECs were infected by pLVX-SIRT4 and treated with 50 µM oxLDL. Following incubation for 48 h at 37°C and 5% CO₂, HUVECs overexpressing SIRT4 and NC cells were harvested for western blotting.

Following treatment or infection, cells were lysed using a cell lysis buffer containing protease inhibitors [Tris-HCl (pH 7.5) 50 mM, NaCl 250 mM, EDTA 10 mM, NP-40 0.5%, Leupeptin 10 µM, PMSF 1 mM and NaF 4 mM]. The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). Soluble lysate was mixed with loading buffer and boiled for 5 min. Equal amounts of the protein samples (50 ng/lane) were separated via 10% SDS-PAGE and transferred on to polyvinylidene difluoride membranes. Membranes were blocked with PBS, containing 10% non-fat dry milk, overnight at 4°C and incubated with anti-SIRT4 antibody (1:1,000; cat. no. ab124521), anti-GAPDH antibody (1:10,000; cat. no. 5174), anti-PI3K antibody (1:1,500; cat. no. 4249), anti-Akt antibody (1:1,500; cat. no. 4691), anti-phosphorylated (p)-Akt antibody (1:500; cat. no. 4060), anti-p-inhibitor of κBα (IκBα) antibody (1:500; cat. no. 2859), anti-total (t)-IκBα antibody (1:1,000; cat. no. 4812), anti-p65 NF-κB antibody (1:500; cat. no. 8242) and anti-p-p65 NF-κB antibody (1:500; cat. no. 3033) for 2 h at 25°C. Anti-SIRT4 was obtained from Abcam (Cambridge, MA, USA); all other primary antibodies were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA). Membranes were washed with TBS-0.05% Tween 20 and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G heavy and light chain secondary antibodies (1:10,000; cat. no. 7074; Cell Signaling Technology, Inc.) for 2 h at room temperature. This process was followed by the development of protein bands for visualization. Image-Pro Plus 6.0 software (Media Cybernetics, Silver Spring, MD, USA) was used to quantify relative protein densities. GAPDH was used as the loading control. Each experiment was replicated three times.

Cell proliferation was assessed using a Cell Counting Kit-8 assay (CCK-8; Beyotime Institute of Biotechnology, Shanghai, China), according to the manufacturer's protocols. HUVECs stably overexpressing SIRT4 and control cells were cultured in 96-well plates and treated with 50 µM oxLDL for 48 h at 37°C and 5% CO₂. Then, 10 µl of CCK-8 reagent was added to each well and mixed gently; cells were then incubated at 37°C for 4 h. The absorbance was evaluated at 450 nm using a microplate reader. Survival rate=optical density (OD)450_OV-SIRT4 group/OD450_NC group. Each experiment was repeated three times.

Cell apoptosis was analyzed using flow cytometry. HUVECs stably overexpressing SIRT4 and NC cells were cultured and treated with 50 µM oxLDL for 48 h at 37°C and 5% CO₂. The cells were collected and the assay was performed using a Propidium Iodide (PI)/Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (Nanjing Keygen Biotech Co., Ltd., Nanjing, China). Annexin V-FITC (5 µl) and PI (5 µl) were added, and cells were incubated at 25°C in the dark for 15 min. Within 1 h, the apoptotic cells was then assessed using a flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo version 10.07 software (FlowJo LLC, Ashland, OR, USA). The apoptotic rate was defined as the percentage of cells in the upper and lower right quadrants. Each experiment was repeated three times.

HUVECs stably overexpressing SIRT4 and NC cells (4×10⁵ cells/ml) were seeded into 24-well plates and treated with 50 µM oxLDL for 48 h at 37°C and 5% CO₂. Following treatment, the cells were washed with PBS and then fixed with 4% paraformaldehyde for 15 min at 37°C. The cell membranes were then permeabilized using 0.3% Triton X-100 in PBS with 0.2% (V/V) Tween-20 (PBST) on ice for 15 min, followed by blocking with 5% bovine serum albumin (Gibco; Thermo Fisher Scientific, Inc.) and 2.5% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) in PBST and were then incubated for 2 h at 25°C with rabbit anti-NF-κB p65 antibody (1:100; cat. no. 8242; Cell Signaling Technology, Inc.). Next, cells were washed three times with PBST and incubated with the Alexa Fluor^® 594-conjugated secondary antibody (1:200; cat. no. ab150084; Abcam) for 1 h at room temperature. Following three washes with PBST, the cells were counterstained with DAPI (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 5 min at 25°C. The cells were then visualized and images captured via fluorescence microscopy (magnification, ×400; Leica Microsystems GmbH, Wetzlar, Germany).

HUVECs stably overexpressing SIRT4 and NC cells (4×10⁵ cells/ml) were seeded into 24-well plates, followed by treatment with 50 µM oxLDL for 48 h at 37°C and 5% CO₂. Cell-free supernatants were collected and used in an assay for the detection of cytokines. The concentrations of the cytokines IL-1β (E-EL-H0149c), IL-6 (E-EL-H0102c) and TNF-α (E-EL-H0109c) were determined using commercially available ELISA kits (eBioscience; Thermo Fisher Scientific, Inc.). The entire procedure was performed according to the manufacturer's protocols. Each experiment was replicated three times.

All statistical analyses were performed using SPSS version 19.0 (IBM Corp., Armonk, NY, USA). Results are presented as the mean ± standard deviation, and experiments were repeated in triplicate. Student's t-test was performed for comparison between two groups. Multiple groups were compared using one-way analysis of variance, followed by a least significant difference post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

In order to investigate changes in SIRT4 expression in response to oxLDL, HUVECs were treated with oxLDL at various concentrations. Western blotting results demonstrated that oxLDL treatment significantly reduced SIRT4 expression in HUVECs, compared with in control cells treated with 0 µM oxLDL. Higher oxLDL concentrations resulted in lower SIRT4 expression levels indicating a dose dependent association between oxLDL concentrations and SIRT4 expression (Fig. 1A). Next, HUVECs were treated with the same oxLDL concentration (50 µM) for different periods of time. Western blotting results demonstrated that oxLDL treatment significantly reduced SIRT4 expression in HUVECs compared with control cells treated with 0 µM oxLDL, in a time dependent manner; longer durations of treatment resulted in lower SIRT4 expression levels (Fig. 1B).

Cells overexpressing SIRT4 were selected by screening and treated with 50 µM oxLDL for 48 h. SIRT4 expression was determined via western blotting. SIRT4 expression levels were significantly higher in cells overexpressing SIRT4, compared with the NC group following oxLDL treatment for 48 h (P<0.001; Fig. 2). The results indicated successful overexpression of SIRT4 in HUVECs.

To investigate the effect of SIRT4 overexpression in response to oxLDL on HUVEC proliferation, HUVECs overexpressing SIRT4 and NC HUVECs were treated with 50 µM oxLDL for 48 h. Cell proliferation was assessed using a CCK-8 assay. The survival rate was significantly higher in HUVECs overexpressing SIRT4, compared with NC HUVECs following oxLDL treatment (Fig. 3A). The results indicated that SIRT4 overexpression may reverse oxLDL-induced inhibition of cell proliferation. In order to explore the effects of SIRT4 overexpression in response to oxLDL on HUVEC apoptosis, HUVECs overexpressing SIRT4 and NC HUVECs were treated with 50 µM oxLDL for 48 h. Cell apoptosis was examined via flow cytometry. Following oxLDL treatment, the apoptotic rate significantly decreased in HUVECs overexpressing SIRT4 compared with in NC HUVECs (Fig. 3B and C). These results indicated that overexpression of SIRT4 may inhibit oxLDL-induced apoptosis.

In order to elucidate the role of SIRT4 in regulating the PI3K/Akt/NF-κB signaling pathway, HUVECs overexpressing SIRT4 and NC HUVECs were treated with 50 µM oxLDL for 48 h. Western blotting analysis was conducted to quantify protein expression associated with the PI3K/Akt/NF-κB signaling pathway. Compared with the NC, SIRT4 overexpression suppressed the PI3K/Akt/NF-κB pathway in HUVECs by inhibiting PI3K expression (Fig. 4A and B). It also significantly inhibited p-Akt, p-IκBα and p-P65 NF-κB expression compared with the control. P65 NF-κB nuclear translocation was analyzed via immunofluorescence. Immunofluorescence analysis revealed that P65 NF-κB was largely expressed in the nucleus, while a small quantity was expressed in the cytoplasm of cells in the oxLDL + NC treatment group. In contrast, in the oxLDL + OV-SIRT4 treatment group, P65 NF-κB was largely expressed in the cytoplasm, while a small quantity was expressed in the nucleus. These results suggest that OV-SIRT4 may inhibit P65 NF-κB nuclear translocation (Fig. 4C).

In order to further confirm the effect of SIRT4 overexpression on endothelial activation in response to inflammatory cytokines, HUVECs overexpressing SIRT4 and NC HUVECs were treated with 50 µM oxLDL for 48 h. The expression of IL-1β, IL-6 and TNF-α were assessed using ELISA. Compared with NC HUVECs, the expression levels of IL-1β, IL-6 and TNF-α were significantly decreased in HUVECs overexpressing SIRT4 (Fig. 5). The results indicated that SIRT4 overexpression attenuated the expression levels of IL-1β, IL-6 and TNF-α induced at the protein level by oxLDL.