Ethical approval was obtained by the Medical Ethics Committee of The Second Xiangya Hospital (Changsha, China). Human LDLs (d=1.019–1.063 g/ml) were isolated by sequential ultracentrifugation (235,000 × g at 4°C for 24 h) of plasma from 20 normolipidemic subjects (10 males and 10 females from January to July 2017) following overnight fasting, as described previously (25). Informed consent was obtained. The purity of the LDLs was assessed by agarose gel electrophoresis and the protein concentration was determined by the modified Lowry method (26). The LDL particles were dialyzed by semi-permeable membrane (3500D) for 24 h with 0.01 M PBS (pH 7.4) at 4°C to remove EDTA, then oxidized by exposure to CuSO₄ (10 mM CuSO₄, 24 h at 37°C) (27). EDTA was added and the LDL particles were dialyzed by semi-permeable membrane (3500D) for 24 h with PBS to terminate the oxidization at 4°C. Thiobarbituric acid-reactive substances and agarose gel electrophoretic mobility were determined. oxLDLs were sterilized by passing through a 0.22-µm Millipore filter (SLGP033RB; Merck KGaA, Darmstadt, Germany).

EPCs were cultured as described previously (28,29). Briefly, 40 ml peripheral blood from healthy volunteers [aged 18 to 33 years old (21.0±4.5 years)] who provided informed consent were subjected to density gradient centrifugation (671 × g for 20 min at room temperature) with Histopaque-1077 (10771; Sigma-Aldrich; Merck KGaA) to isolate peripheral blood mononuclear cells (PBMCs). Following purification and 3 washing steps, 10×10⁶ PBMCs per well were plated on fibronectin-coated 6-well plates. The cells were cultured in endothelial basal medium-2 (EBM-2; cat. no. CC3156; Clonetics; Lonza Group Ltd., Walkersville, MD, USA) with single EGM-2MV aliquots containing 5% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), vascular endothelial growth factor (VEGF), fibroblast growth factor-2, epidermal growth factor, insulin-like growth factor and ascorbic acid. After 4 days, non-adherent cells were removed by washing with PBS. Fresh medium was added, and the culture was continued for 8 days. Non-adherent cells were removed again by washing with PBS and the adherent cells were considered as EPCs and harvested for subsequent experiments.

To confirm the endothelial phenotype, the expression of endothelial protein markers was measured by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). EPCs were detached with 1 mM EDTA in PBS and incubated for 15 min with human fluorescein isothiocyanate (FITC)-conjugated kinase insert domain receptor (KDR; cat. no. FAB357F-025; R&D Systems, Minneapolis, MN, USA), anti-vascular endothelium cadherin (cat. no. sc9989; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), phycoerythrin (PE)-conjugated cluster of differentiation 31 (CD31; cat. no. 553373; BD Biosciences), or rat anti-mouse FITC-conjugated cluster of differentiation 34 (CD34; DS-MB-03816; Raybiotech Life, Inc., Atlanta, GA, USA). For vascular endothelial cadherin (VE-cadherin) analysis, cells were first incubated with mouse anti-human VE-cadherin (cat. no. 555661; BD Biosciences; 1:1,000) for 30 min at 4°C. Following washing with PBS twice for 5 min each, cells were incubated with FITC-conjugated goat anti-mouse secondary antibody (1:200) for 30 min at 4°C (cat. no. F9384: Sigma-Aldrich; Merck KGaA). Mouse IgG1 isotype control antibody (cat. no. 555121; BD Biosciences; 1:1,000) served as controls. Following incubation, the cells were fixed with 1% paraformaldehyde for 15 min at 4°C and quantitative analysis was performed on a FACScan flow cytometer (BD Biosciences) and analyzed with CellQuest software (version 5.1; BD Biosciences) with 20,000 cells/sample.

EPCs were treated without or with 100 mg/ml oxLDLs for 24 h. EPCs in the IGF-1 group were pretreated with 0.1 or 0.5 µg/ml IGF-1 for 30 min prior to exposure to oxLDLs, as described previously (30,31). An additional group of cells was also pretreated with 100 µM nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of eNOS, for 60 min and then with 0.5 µg/ml IGF-1 for 30 min prior to exposure to oxLDLs.

Apoptosis was analyzed using an Annexin V/propidium iodide kit (556547; BD Biosciences). Briefly, 100 µl 1×10⁶/ml cells were incubated with 5 µl Annexin V-FITC and 5 µl propidium iodide (PI) for 15 min at room temperature. Following washing, the cells were diluted in 400 µl Annexin V-binding buffer and immediately detected using a flow cytometer.

Mitogenic activity was measured using a colorimetric MTS assay (Cell-Titer 96^® AQueous Non-radioactive Cell Proliferation assay; cat. no. G1111; Promega Corporation, Madison, WI, USA). EPCs were harvested and seeded on a 96-well plate (1×10⁴ cells per well) in 0.1 ml EBM-2 medium supplemented with 0.5% bovine serum albumin (BSA; Gibco; Thermo Fisher Scientific, Inc.) in the presence of human recombinant vascular endothelial growth factor (100 ng/ml; cat. no. 293-VE-010; R&D Systems). After 24 h the MTS/phenazine methosulfate solution was added to each well for 3 h and the absorbance at 570 nm was measured using an ELISA plate reader (S5 Versa Analyzer, Cellular Technology Ltd., Cleveland, OH, USA).

Cells were suspended in 20 µl PBS and incubated with 10 µg/ml 1,19-dioctadecyl-3,3,3939-tetramethylindocar-bocyanine perchlorate (Dil)-acetylated LDLs (ac-LDL) for 4 h at 37°C. Following washing with PBS, the cells were fixed with 2% paraformaldehyde for 10 min at room temperature and incubated with FITC-Ulex europaeus agglutinin 1 (UEA-1; 50 µg/ml) for 1 h at 4°C. The fluorescence signals were observed using an inverted fluorescence microscope (magnification, ×200; Nikon Corporation, Tokyo, Japan).

NO is an unstable product. Following metabolism, it transforms to nitrate and nitrite rapidly. In addition, it is difficult to measure NO directly. In the present study, NO production in EPCs were measured by a colorimetric assay kit (cat. no. A012; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) using a nitrate reductase method according to the manufacturer's protocol. Absorbance was measured at 550 nm by a spectrophotometer. The NO concentration was expressed as µmol/l.

EPCs were washed and incubated in 75 µl lysis buffer at 4°C for 40 min, as described previously (32). The nuclear and cytosolic fractions were separated by a commercially available kit (NE-PRE Nuclear and Cytoplasmic Extraction Reagents) according to the protocol of the manufacturer (cat. no. 78833; Pierce Chemical Co., Dallas, TX, USA), as described previously (33). Proteins (30–50 µg/lane) measured by a bicinchoninc acid Protein Assay kit (Beyotime Institute of Biotechnology, Nanjing, China) were loaded on 10% SDS-PAGE gels and blotted on polyvinylidene difluoride (PVDF) membranes. Then, PVDF membranes were incubated with 1% BSA at room temperature for 1 h. Western blot analysis was then performed using antibodies against eNOS (1:500; mouse monoclonal anti-eNOS antibody; cat. no. 612706; BD Biosciences) at 4°C overnight. Following washing with TBST (0.1% Tween-20) for 3 times (5 min each), the PVDF membranes were incubated with a horseradish peroxidase-conjugated donkey anti-mouse secondary antibody (cat. no. SA00001-8; ProteinTech Group, Inc., Chicago, IL, USA; 1:10,000) for 1 h at room temperature. Finally, following washing with TBST, the autoradiographs were scanned and semi-quantitatively analyzed to calculate the protein ratio.

SPSS 22.0 statistical software (IBM Corp., Armonk, NY, USA) was used for data analysis. All data are presented as mean ± standard deviation. Statistical analyses were performed using one-way analysis of variance followed by a Least Significant Difference test. P<0.05 was considered to indicate a statistically significant difference.

The levels of thiobarbituric acid-reactive substances were 2.13±1.59 and 24.4±8.31 nmol/mg protein in native LDLs and oxLDLs, respectively. Compared with native LDLs, oxLDLs indicated a 1.4±0.4 fold increase in electrophoretic mobility on agarose gels.

Flow cytometry was used to identify the endothelial phenotype of the EPCs. After 8 days of culture, the expression rates of KDR, VE-cadherin, CD34, and CD31 in the attached cells were 68.8±7.5, 73.9±6.3, 25.4±9.1 and 77.1±7.2%, respectively (Fig. 1). After 8 days in culture, the attached cells took up Dil-acLDL and bound FITC-UEA-1 (Fig. 2A). Cells that were positive for these 2 factors simultaneously were considered EPCs. They constituted up to 90% of all attached cells. These results indicate that EPCs were successfully isolated from PBMCs.

EPCs were characterized as adherent cells that were doubly-positive for lectin and Di-LDL. The toxic effects of oxLDLs were examined in EPCs; oxLDL significantly decreased the number of EPCs. IGF-1 (0.1 or 0.5 µg/ml) significantly prevented the decrease of EPCs caused by oxLDLs; the effect of 0.5 µg/ml IGF-1 was more marked. When EPCs were incubated with L-NAME (100 µM), 0.5 µg/ml IGF-1 and oxLDL for 24 h, L-NAMsE significantly decreased the protective effect of IGF-1 against oxLDL (Fig. 2B). These results suggest that IGF-1 may protect EPCs against the toxic effects of oxLDL.

The increase in the number of EPCs following IGF-1 treatment may be attributed to a combination of factors, including inhibition of apoptosis and stimulation of proliferation. Therefore, the levels of apoptosis and proliferation of EPCs were examined following oxLDL challenge and in response to IGF-1. The results of the MTS assay demonstrated that treatment of EPCs with IGF-1 significantly prevented EPC apoptosis and improved EPC proliferation. These effects were significantly attenuated by L-NAME (Fig. 3A and B).

As the eNOS/NO axis may serve a role in the effects of IGF-1 on EPCs, the effects of IGF-1 on the eNOS protein were examined, and the effects of L-NAME, an eNOS inhibitor. NO generation was decreased by treatment with 100 mg/ml oxLDLs. This inhibitory effect of oxLDLs was prevented by the presence of 0.5 µg/ml IGF-1. Treatment with L-NAME significantly decreased NO generation compared with the 0.5 µg/ml IGF-1 group (Fig. 3C). To verify the hypothesis that IGF-1 protects EPCs against oxLDL through the eNOS pathway, eNOS protein expression was assessed by western blot analysis. Incubation of EPCs with 100 mg/ml oxLDL significantly suppressed eNOS protein expression. Pretreatment with IGF-1 caused a partial restoration of the downregulation of eNOS protein expression induced by oxLDL (Fig. 3D).