HUVECs were purchased from the China Center for Type Culture Collection (Wuhan, China), and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 5 U/ml penicillin G, 5 µg/ml streptomycin sulfate (Invitrogen, Carlsbad, CA, USA). The present study was approved by the Ethical Committee of The General Hospital of Ningxia Medical University (Yinchuan, China; registration no. 2016-230).

HUVECs (1×10⁵/well) were seeded on 96-well plates, incubated for 24 h to allow for attachment to the surface of plate, then starved by incubating another 24–48 h in serum-free DMEM. The cell-free supernatants were harvested for measuring the secreted LOX using an ELISA kit according to the manufacturer's recommendations (SEC580Ra; Cloud-Clone Corp., Houston, TX, USA). Optical density values of the samples were used to calculate the concentrations of all of the samples.

A fibrin gel in vitro angiogenesis assay kit (ECM630; Chemicon, Temecula, CA, USA) was used to evaluate tube formation of HUVECs in 96-well plates. A fibrinogen mixture was dispensed into the well with thrombin. Finally, the plate was placed at 37°C for 30 min for polymerization. We added 100 µl HUVECs (with the cell concentration of 5×10⁴ cells/ml) in basal medium (DMEM supplemented with 1% BSA and antibiotics) with vascular endothelial growth factor (VEGF; Genentech, South San Francisco, CA, USA) 10 ng/ml, basic fibroblast growth factor (bFGF; Becton-Dickinson Labware, Bedford, MA, USA) 10 ng/ml, phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO, USA) 30 ng/ml with or without BAPN (0.1, 0.2, 0.4 mM) (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). They were grown at 37°C overnight in the tissue-culture incubator. After removing the culture medium we added 30 µl of fibrinogen per well in each medium of the noted wells, followed by 20 µl thrombin placed in every well. Then, the plate was placed in a 37°C incubator for 5 min to polymerize. Images were captured using a Zeiss microscope (magnification, ×100) after a week. Vessels were quantified by counting branch points in 6 random view-fields per well and the values were averaged.

Boyden Chamber assay (Corning Costar, New York, NY, USA) was used to assess cell migration. After cells were grown to 80% confluence, they were harvested. 600 µl of medium containing 10% FBS without or with BAPN (0.1, 0.2, 0.3 and 0.4 mM) was added into each well of the 24-well (?) plate. Then, we placed 1×10⁵ cells in 100 µl serum-free medium with or without BAPN into the insert wells of 8 µl pore membrane Boyden Chambers. After incubating for 24 h at 37°C, the unattached cells were removed. Then, cells were fixed by methanol for 5 min, washed once with PBS, and stained with 0.05% Giemsa stain. We quantified the cells that moved to the lower sides of the membrane by counting in 5 random visual fields (magnification, ×200) for each well under a light microscope.

HUVECs were cultured with or without BAPN (0.4 mM) for 24 h in serum-free medium. Total RNA was extracted using Tri Reagent (Molecular Research Center, Inc. Cincinnati, OH, USA) based on the manufacturer's instructions. The RNA concentration was evaluated by an optical density (OD) ratio of 260:280 and with gel electrophoresis. The cDNA was synthesized by reverse transcription of the RNA. The cDNA microarrays were generated with a SPBIO2000 robotic arrayer, and it had 1080 cDNA clones. Fluorescence intensities, produced by Cy5 or Cy3 impaired to the target sequences on the microarray slides, were quantified with a ScanArray 5000 laser confocal microscope equipped scanning system (General Scanning, Watertown, MA, USA) using the necessary excitation and emission filters. The signal from every impaired cDNA target on the microarray slide was found, and the expression ratio among the experimental and reference samples (Cy5/Cy3 ratio) was established with ImaGene version 3.0 software (Biodiscovery, Los Angeles, California, USA). Microarray analysis was conducted in triplicate, and the Cy5/Cy3 ratio values are the averages.

Total RNA was isolated using the TRI Reagent from the HUVECs treated with and without BAPN as described above. The purity of the RNA preparation was checked by the 260/280 nm ratio. Reverse transcription was conducted with 5 µg total RNA using random hexamer primers with the SuperScript^™ First-Strand Synthesis System for RT-PCR kit (Invitrogen Life Technologies, Carlsbad, CA, USA) as detailed by the company. PCR primers were designed with IDT OligoAnalyzer 3.0 (Integrated DNA Technologies, Coralville, Iowa, USA). The forward primer for myeloid progenitor inhibitory factor 1 (MPIF-1) gene (Ass no.: NM_005064) is: 5′-TGCCTCATGCTTGTTACTGC-3′, and the reverse primer is: 5′-TGATCCGTGTGTCCAGCTTC-3′. The RNA analysis was performed with DyNAmo™ HS SYBR-Green qPCR kit (MJ Bioworks, Inc., South San Francisco, CA, USA). All PCR reactions were performed in 20 µl reaction volumes. PCR conditions were 95°C for 30 sec for denaturation, and 45 cycles of amplification at 95°C for 5 sec and at 60°C for 20 sec, modified based on previously described (18). Amplification and detection were performed using the DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Bioworks, Inc.). Relative quantification of mRNA levels was conducted with the standard curve method, with amplification of first-strand cDNA in regular PCR. The standard curves were established with 4 dilutions (100,000, 10,000, 1,000 and 100) of cDNA, and the samples were then run in duplicate with the primers.

Cells were lysed on the plate with CelLytic mammalian tissue lysis/extraction reagent, supplemented with a proteinase inhibitor cocktail (Sigma, St. Louis, MO, USA). The protein concentration was established using the Bradford assay with bovine serum albumin as a standard. Total 30 µg proteins in each lane were isolated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The samples were then move to nitrocellulose paper. Following blockage with 5% skim milk, the blots were incubated with phospho-p44/42 MAPK (Thr202/Tyr204, ERK1/ERK2) monoclonal antibody (9106; Cell Signaling Technology, Inc., Danvers, MA, USA) at 1:2,000, phospho-Akt (Ser473) monoclonal antibody (587F11, 4051, Cell Signaling Technology, Inc.) at 1:1,000 in 5% milk in Tris-buffered saline-Tween overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:500; RPN2133; Amersham, Arlington Heights, IL, USA) for 1 h at room temperature. The blots were then incubated with the enhanced chemiluminescence (ECL) technique (Amersham). Chemiluminescence was evaluated through exposing the membranes to Kodak X-Omat films (Eastman Kodak, Rochester, NY, USA).

The data are displayed as means ± standard deviations (SD). Statistical analyses were performed with SPSS statistical software (version 20.0; IBM, Armonk, NY, USA). Continuous variables that were normally distributed were compared with one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls (SNK) post hoc test. A value of P<0.05 was considered significant.

We first examined the LOX expressions in HUVECs before we studied the role of BAPN on HUVECs. Supernatants of HUVECs were harvested after incubating for 24 and 48 h to measure LOX. The LOX concentrations were 53.12 pg/ml in the 24-h supernatant and 94.72 pg/ml in the 48-h supernatant.

HUVECs were induced to form anastomosing networks of capillary-like tubes in fibrin gel. Imaging was captured after a week, and the branch points in 6 random view-fields per well were counted and the values were averaged (Fig. 1). In the absence of growth factors, capillary sprout formation was minimal (control, 0.0 mM BAPN), and there were no significant changes after adding BAPN which inhibits LOX in the basal medium group (control with 0.1, 0.2 and 0.4 mM BAPN). VEGF, bFGF and PMA induced obvious sprouting of HUVECs (P<0.05). Importantly, BAPN (0.1, 0.2 and 0.4 mM) significantly inhibited angiogenesis induced by VEGF, bFGF and PMA compared with control (P<0.05).

Since angiogenesis greatly relies on endothelial cell motility, we evaluated the impact of BAPN on movement of HUVECs using an in vitro assay (Fig. 2). The results showed that when FBS significantly induced migration of HUVECs migration, BAPN at 0.1, 0.2 and 0.4 mM significantly inhibited cell migration of HUVECs (P<0.05).

We implemented a microarray assay to determine the changes of gene expression of HUVECs treated with BAPN. As shown in Tables I and II, BAPN upregulated 178, and downregulated 29 angiogenesis-related genes. The upregulated genes, including NADH dehydrogenase (ubiquinone) flavoprotein 1 and thymidine kinase, were involved in activities such as cell signal transduction and enzyme activity. The downregulated genes included AH-receptor mRNA and thrombin receptor mRNA. Noticeably, BAPN suppressed expression of MPIF-1 (hmrp-2a), a human chemokine, in HUVECs.

Since the microarray assay showed a significant change in MPIF-1 mRNA, we investigated the expression of MPIF-1 in HUVECs using RT-qPCR. The results showed that MPIF-1 mRNA reduced by 2-fold, from 1,250±35 copies (control) to 638±52 copies in BAPN-treated cells, and that there was significantly different between these two groups (P<0.05).

In order to understand the mechanism by which BAPN abrogated angiogenesis, we investigated whether BAPN would affect phosphorylation of MAPK and Akt in HUVECs because both of MAPK and Akt involve in angiogenesis. The results showed that after treatment with 0.2 mM BAPN for 48 h, both MAPK1/2 and Akt phosphorylation was reduced (Fig. 3). These results suggest that BAPN may be able to serve as alternative strategy to prevent angiogenesis via inhibiting LOX.