Patients and samples
The current study was conducted on 20 formalin-fixed paraffin-embedded (FFPE) vascular anomalies tissues and 5 cases healthy control individuals from Xuzhou Central Hospital during the period from 2017 to 2018. All cases were re-evaluated and confirmed by experienced professionals according to the current clinical, radiographic, and histopathological guidelines for VM.
Prior to this study, we obtained the informed consent of all patients and passed the review of the ethics committee.
Mutation Analysis
DNA was extracted from paraffin-embedded tissues of the VM patients by a QIAamp DNA FFPE Tissue Kit (Qiagen, Duesseldorf, Hilden, Germany). An approximately 300 bp DNA sequence of TEK 17 exon (NCBI Gene ID 7010) was amplificated by allele-specific PCR. The PCR primer sequences were as follows: TEK-F: 5’-TCTCTTAAATGTCATAGCTGTTCAG-3’, TEK-R: 5’-AGGGAACTCCACAGGAAAGAT-3’. TaKaRa Taq Version 2.0 plus dye (TaKaRa, Shiga, Tokyo, Japan) was used for PCR amplification. Subsequently, the product was examined by agarose gel electrophoresis, and the sequencing was performed by Sangon (Shanghai, China).
Cell Culture And Treatment
Under a protocol approved by the Ethics and Research Committee of Nanjing Medical University, primary HUVECs and SMCs were obtained from fresh human umbilical vessels. Informed consent was obtained before volunteers were enrolled in this study. Primary HUVECs were cultured in medium dishes with ECM (Endothelial Cell Medium, Sciencell) containing 5% fetal bovine serum. SMCs were cultured in medium dishes with DMEM/F12 medium containing 10% fetal bovine serum. The medium was changed every 3 days until 80–90% confluence was achieved. To avoid genetic variation resulting from different individuals, all cells from three or more different donors were pooled together. Cells at no more than five passages were used for the following experiments.
Lentiviral Transfection
About 2 × 105 HUVECs were seeded on six-well plate overnight than transfected with lentiviral vectors expressing the GFP, TIE-WT, and TIE2-L914F in the presence of polybrene (8 mg/ml). After incubation for 48 h, cells were treated with DMSO or rapamycin (MCE, Shanghai, China) for another 48 h for subsequent experiments.
Apoptosis Analysis
HUVECs (3 × 105/well) were induced to undergo apoptosis via starvation in serum-free ECM medium overnight. After treated with DMSO or rapamycin for 48 h, cells were collected according to the protocol for fluorescein isothiocyanate (FITC) Annexin V Apoptosis Detection Kit (BioLegend, San Diego, CA, USA). Briefly, cells were resuspended in cold (4 °C) binding buffer and incubated for 15 minutes at room temperature following addition of 5 mL of Annexin V-FITC and 5 mL of 7-AAD solutions. Flow cytometry analysis was performed using FACSCalibur2 and analyzed with FlowJo 10.04 software. This experiment was independently repeated three times.
Migration Analysis
Migration assay was performed using a transwell chamber. Briefly, 8-µm pore size chambers with transparent polyester were placed into 24-well plates. 5 × 104 cells of each group were seeded on up chamber, with cells starved overnight, complete ECM was placed on bottom chamber. After 24 h of incubation, non-migrated HUVECs were removed by cotton swabs. Migrated cells were fixed and stained with crystal violet (0.1%). Experiments were performed in duplicate, and the number of cells present in 5 fields per well was counted at × 10 magnification in a blinded manner using an EVOS microscope (Invitrogen).
Bromodeoxyuridine (BrdU) Assay
Before BrdU experiment, the cells had been transfected with lentivirus and pretreated with rapamycin for 48 h. 4 × 104 ECs per well were seeded on 12-well plate. BrdU assay was performed as elsewhere described[28].
CCK-8
After cells starved overnight, HUVECs (3000/well) were placed on 96-well plate with 100 µl complete ECM. Cells (at day 1, 2, 3 and 4 after plating) were added with CCK-8 10 µl per well. 450 nm OD was tested after 3 h incubation.
Tube Formation Assay
Tube formation assay was performed as elsewhere described[29]. Briefly 48 h following lentiviral transfection, HUVECs (2 × 104 per well) were seeded in duplicate onto 96-well culture dishes coated with 50 µl of Matrigel (BD Biosciences). Tube formation was observed every 3 h post seeding with an inverted Olympus phase-contrast microscope, and five high-power fields at 100 magnifications were imaged randomly by using Olympus DP12 digital camera. The vessel length and branch points for each group was quantified by ImageJ. This experiment was independently repeated three times.
Nucleoprotein Separation And Extraction
HUVECs were treated with rapamycin for an additional 48 h after they underwent serum-free starvation and then harvested. The nuclear proteins were isolated using the Invent Nuclear and Cytoplasmic Extraction Reagents Kit (Invent Biotechnologies, Plymouth, MN, USA) according to the manufacturer’s instructions.
Western Blot
The details of western blot were described elsewhere[30]. Briefly, cells were lysed by RIPA buffer (Beyotime, China), and the lysate was loaded onto 10% or 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked in 5% fat-free milk for 2 hours before incubated with the following primary antibodies: GAPDH (Proteintech, #10494-1-AP), AKT (CST, #2920), p-AKT (CST, #4060), FOXO1 (CST, #2880), p-FOXO1 (CST, #9461), mTOR (CST, #2983), p-mTOR (CST, #5536), p62 (Proteintech, #18420-1-AP), LC3B (Abcam, #ab51520), α-SMA (Proteintech, #23081-1-AP), OPN (Proteintech, #22952-1-AP), Histone-H3 (Proteintech, #17168-1-AP) at 4 °C overnight. Then the membranes were incubated for 30 minutes at 37 °C with secondary antibodies after washing. ImageJ software was used for quantitatively analyzing.
Real-time Quantitative PCR
Total RNA from cultured cells was extracted using Trizol reagent (Vazyme, Nanjing, Jiangsu, China) according to the manufacturer’s instructions. Quantitative real-time PCR analyses were performed in triplicate using SYBR Green PCR Master Mix (TaKaRa), and reactions were detected using an Applied Biosystems 7300 Real-time PCR system (Applied Biosystems, Gaithersburg, CA, USA). The primer sequences used for real-time PCR are listed as follows. PDGFB-F: 5’-TCTCTGCTGCTACCTGCGTCTG-3’, PDGFB-R: 5’-AGGTCCAACTCGGCTCTGTCTTC-3’; TIE2-F: 5’-GGGACTTTGCAGGAGAACTGG-3’, TIE2-R: 5’-AAATGCTGGGTCCGTCTCCA-3’.
Immunostaining
Immunohistochemical staining was performed using continuous paraffin sections of venous malformations. Details of immunohistochemical staining are described elsewhere [30]. PDFGB (Abclonal, #A1195) and α-SMA (Proteintech, #23081-1-AP) primary antibody were used to detect relative molecular expression.
Immunofluorescence
NC, TIE2-WT, TIE-L914F HUVECs 5000/well were seeded on 12-well plate coating with slides. Immunofluorescence staining was performed after cells were treated with DMSO or rapamycin for 48 h. Briefly, cell slides were fixed with 4% paraformaldehyde for 15 minutes and then washed with 0.1M Phosphate buffered saline (PBS) for three times. After 10 minutes of drilling with 0.1% Triton X-100 (Beyotime, Shanghai, China), the sections were treated with normal goat serum for 1 h at 37 °C, and then anti-mouse FOXO1 was used as primary antibody at dilutions of 1:200 overnight at 4 ℃.
ELISA
The concentration of PDGFB-B in the supernatants of the cultured HUVECs was detected by ELISA kits (Human PDGFB; CUSABIO, WuHan, China) according to the manufacturer’s procedures.
Co-culture Systems
We established the coculture system through 6-well plate and 3 µm pore size transwell inserts (Corning). The SMCs (5 × 105 per well) were seeded in 6-well plate and the same quantity of different treated ECs were seeded in the transwell inserts located in adjoining wells. The transwell inserts with ECs were moved to the wells containing SMCs when cells attached to the wall firmly (24 h). After another 48-hour culture, SMCs were harvest for western blot. Similarly, we set up another co-culture system for migration experiments, 24-well plate and 8 µm pore size transwell chambers (Corning) were used instead, 5 × 104 different treated ECs per well were seeded in 24-well plate and the same number of SMCs were seeded in up chambers adjacently. After cells attached firmly, the transwell chambers with ECs were moved to the wells containing ECs. 48 h later, chambers were taken out for migration experiments.
Statistical analysis
The results are represented as means ± SEM or as specified in the figure legends. The statistical analysis was performed using Prism 7 software (GraphPad Software, La Jolla, CA, USA). Comparisons between the 2 groups were analyzed using the 2-tailed, unpaired Student’s t test. Comparisons among ≥ 3 groups were performed using 1-way ANOVA followed by Tukey’s multiple comparisons. Adjusted values of P༜0.05 were considered significant.