Vegfr1

Endothelial progenitor cells were washed with cold PBS and were resuspended in PBS with FcR blocking reagent (MACS), 0.2% FBS, and 2 mmol/L EDTA at 4°C for 30 min. They were stained with monoclonal antibodies specific for the following surface antigens: CD34 (Becton Dickinson, San Jose, CA), CD51/61 (integrin αv/β3; Becton Dickinson), CD54 (ICAM1; Becton Dickinson), CD106 (VCAM1; Becton Dickinson), CD117 (c‐Kit; MACS), CD133 (MACS), CD144 (VE‐cadherin; Beckman Coulter, Brea, CA), CD202b (Tie2; R&D), CD309 (VEGF‐R2; R&D), and VEGF‐R1 (R&D). After incubation at 4°C for 30 min they were washed twice and were analyzed using two‐color flow cytometry.

The recombinant extracellular domains of the receptors DR5 (100 ng/well), VEGFR1 or VEGFR2 (50 ng/well) (R&D Systems, Minneapolis, MN, USA) were immobilized on ELISA plates overnight at 4 °C in 0.1 M carbonate–bicarbonate buffer (pH 9.4). The plates were washed three times with PBST (phosphate-buffered saline + 0.05% Tween), and wells were blocked by 2% BSA in PBST for 1 h at 37 °C. After blocking, dilutions of DR5-B, HRH–DR5-B or SRH–DR5-B (in 3 replicates) at concentrations from 0.032 to 2500 nM were added, and the plates were incubated for 1 h at 37 °C. Captured ligands were detected by subsequent incubation with monoclonal antibodies to TRAIL (MAB375, R&D Systems, Minneapolis, MN, USA) and anti-mouse polyclonal goat IgG conjugated with horseradish peroxidase (HAF007, R&D Systems). Unbound antibodies were washed 3 times with a PBST buffer, and color was developed by OPD (o-phenylenediamine dihydrochloride) colorimetric substrate. After 15 min of incubation with the substrate at 37 °C, the reaction was stopped by a 1 N H₂SO₄ solution. The optical density was determined at 450 nm by iMark reader (Bio-Rad, Hercules, CA, USA). Dissociation constant (K_D) values were calculated by GraphPad Prism 8 software (GraphPad Software Inc., San Diego, CA, USA), using the nonlinear regression option in the XY analysis section.

Cultured media were collected and centrifuged at 1,200 g for 5 min. Media were supplemented with 17.5 μg mL⁻¹ phenyl-methylsulfonylfluoride, 1 μg mL⁻¹ pepstatin A, 2 μg mL⁻¹ aprotinin and 50 μg mL⁻¹ bacitracin, and stored at −80 °C. Human specific ELISA kits were used to measure angiopoietin-1, MCP-1, FGF-2, EGF, IGF-1, VEGF, VEGFR-1, PICP and IL-6 (all from R&D Systems), OPG (Bender MedSystems GmbH), RANKL (Biomedica Gruppe) and M-CSF (Abcam). For TGF-β1 detection, cell layers were washed with PBS and extracted with 5 × 10⁻² M Tris-HCl pH 8.0, 5 × 10⁻¹ M NaCl and 1% Triton X-100, supplemented with protease inhibitors as above described. The amount of TGF-β1 in the extracts was determined using a human specific ELISA (RayBiotech) and the data were normalized to the total protein amount in cell layers, as determined by a Bradford-based protein assay (Bio-Rad Laboratories Inc.).

A MSD standard bind 96-well plate was coated with 0.25 μg/ml VEGFR1 or VEGFR2 (R&D Systems) in bicarbonate buffer, and sealed plates were incubated overnight at 4 °C. The following day plates were washed with Tris wash buffer and blocked with 3% BSA in PBS for 1 h at room temperature. Plates were washed as described above before the addition of anti-VEGF molecule and incubated at room temperature for 10 min before the addition of VEGF-165 (R&D Systems). The anti-VEGF molecules and VEGF were diluted in 0.1% BSA in PBS. The final VEGF concentration used was 10 ng/ml. No VEGF and VEGF-only wells were also included as controls on all plates. Plates were incubated at room temperature for 2 h before being washed again before the addition of the detection reagent (goat anti-human VEGF biotinylated antibody; R&D Systems) at 0.25 μg/ml in 1% BSA in PBS and incubated at room temperature for 1 h. Plates were washed again before the addition of the streptavidin sulfo-TAG (MSD) at 2 μg/ml in 1% BSA in PBS and incubated at room temperature for 30 min. Plates were washed, and 2× Read Buffer T was added before being read on a SECTOR 6000 MSD imager. GraphPad Prism was used to fit curves using a sigmoidal dose response (variable slope) model and to calculate the IC₅₀ values (with S.D. calculated over the mean) from n = 2–11 experiments.

A four-color FC analysis was performed on FC500 analyzer (Beckman Coulter) to characterize MSCs and S-MSCs at day 28: FITC-conjugated CD105 (endoglin), CD31 (PECAM1) antibodies and PE-conjugated CD90 (Thy-1), CD140a (Platelet-derived growth factor receptor alpha, PDGF RA), CD140b (Platelet-derived growth factor receptor beta, PDGF RB), CD144 (Ve-Cadherin, CDH5), VEGF R1 (Vascular endothelial growth factor receptor 1, FLT1) antibodies and PE-Cy5-conjugated HLA-DR (human leukocyte antigen-D related), CD34, CD45, CD11b (integrin alpha M), CD146 (Melanoma adhesion molecule, MCAM), CD184 (CXCR4), CD106 (VCAM1) antibodies and PE-Cy7 conjugated CD73 (5′ nucleotidase) and VEGF R2 (KDR, CD309) antibodies. Mouse anti-Human CD105, CD90, CD73, HLA-DR, CD140a, CD31, CD184, CD106 were obtained from BD Biosciences (Le Pont de Claix, France), CD34, CD45, CD11b, CD146, VEGFR2 from Beckman Coulter, CD140a, VEGFR1 from R&D Systems Inc (Minneapolis, USA) and CD144 from Santa-Cruz Biotechnology inc SCBT (Dallas, USA). The isotype-matched mouse IgG1-FITC, IgG1-PE, IgG1-PCy5 and IgG1-PCy7 were used as negative controls. Acquisition and processing data from 7,000 events were analysed using Kaluza software.