Huvecs

Human umbilical vein endothelial cells (HUVECs) were purchased from iCell Bioscience Inc. (Shanghai, China) and cultured in endothelial cell medium (ScienCell Research Laboratories, Carlsbad, CA, USA) supplemented with 5% fetal bovine serum (FBS), endothelial cell growth supplement (ECGS), and antibiotic (penicillin/streptomycin) solution. HUVECs were passaged no more than 6 times prior to use in experiments. Human brain vascular pericytes (HBVPs) were purchased from Fenghbio Co., Ltd. (Changsha, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific Inc.). PLC/PRF/5 cells were cultured in minimum Eagle’s medium (MEM; Gibco, Thermo Fisher Scientific Inc.) containing 10% FBS and non-essential amino acids (NEAA, Gibco, Thermo Fisher Scientific Inc.). Hep1-6 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in DMEM containing 10% FBS.

Human umbilical vein endothelial cells (HUVECs) (iCell Bioscience Inc, Shanghai; 5 × 10⁵ cells/mL) were seeded in six‐well plates (10% serum medium for HUVECs culture and expansion, HUVEC‐90011 with the growth factor supplementation, OriCell®) and cultured in 37°C incubators at 5% CO₂ for 24 h. The six‐well plate was removed from the incubator after the cells grew to the logarithmic phase and the adhesion rate reached 80%–90%. The spent medium was aspirated and a 20 μL pipette tip was used to make a transverse scratch on the culture plate, with the tip remaining vertical during the procedure. Each well was manipulated in the same method. Subsequently, the cells were rinsed three times with phosphate buffered saline (PBS). Next, the scratched cells were aspirated and divided into four groups by adding 2% low serum medium (HUVEC‐90011 without the growth factor supplementation, OriCell®) with 1× PBS (Wuhan Servicebio Technology Co., Ltd.), 10 pM Recombinant human frizzled‐related protein 2 (rhsFRP2) (CSB‐MP021139HU, Cusabio Biotech),¹⁸ 10 nM Recombinant human Yes‐associated protein 1 (rhYAP1) (CSB‐YP026244HU, Cusabio Biotech) and 10 pM rhsFRP2⁺ 10 nM Peptide 17 (YAP‐TEAD Inhibitor 1, S8164, Selleck).¹⁹ The medium was stored in the incubator for 12 h before imaging (100×, the same area of the well imaged at both time points).

Human umbilical vein endothelial cells (HUVECs) (iCell Bioscience, CHN) were cultured in PriMed-iCELL-002 (iCell Bioscience, CHN) supplemented with 5% FBS. Subsequent experiments were conducted using cells at passage 2 to 4. Culture plates were pre-cooled to − 20 °C before the experiments. matrigel (Becton, Dickinson & Co., USA, 356230) was laid on the bottom of the lower transwell chamber, with the chamber then placed at 37 °C for 2 h for solidification of the matrigel. HUVECs (1 × 10⁵/well) were seeded into the lower chamber. hADSCs or HUVECs (as a control) (1 × 10⁴/well) were plated into the upper chamber of the transwell, followed by incubation for 24 h before the experiments. Angiogenesis (tube formation) was observed and photographed after 0, 2, 4, 6, 8, and 10 h. The number of tube structures was counted using an Image-Pro Plus 6 (Media Cybernetics, USA).

Human monocytic leukemia cells (THP-1) and human umbilical vascular endothelial cells (HUVECs) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. THP-1 cells were cultured in BPMI 1640 medium (TBD, Tianjin, China) with 10% fetal bovine serum (Procell, Wuhan, China), and HUVECs were cultured in a primary HUVEC cell culture system (iCell, Shanghai, China) with 10% fetal bovine serum. The cells were maintained at 37°C with 5% CO₂. HUVECs (5 × 10⁵/well) were seeded in a 6-well plate and incubated for 24 h for integration. The cells were then transfected with Opti-MEM (Sigma, USA) and Lipofectamine® RNAiMAX (Life Technologies, Shanghai, China) according to the manufacturer's instructions. The expression of miR-221-3p was detected at 48 h posttransfection.

Human umbilical vein endothelial cells (HUVECs) and HemECs were both obtained from iCell Bioscience Inc (Shanghai, China). HUVECs and HemECs were cultured in high-glucose DMEM medium (Procell, Wuhan, China) supplemented with 10% fetal bovine serum (Procell, Wuhan, China) and 1% penicillin–streptomycin-gentamicin solution (Solarbio, Beijing, China). All cells were cultivated in a humid environment with 5% CO₂ at a temperature of 37 °C.