Vegf a165

Aortae were harvested from 8- to 12-week-old Insr^+/- mice under terminal isoflurane anesthesia and then processed according to the protocol of Baker et al (23 (link)). In brief, after dissection of perivascular fat and overnight storage in serum free OptiMEM media (Thermo Fisher Scientific), aortae were cut in to 1-mm-thick rings that were then embedded in rat type I collagen. Rings were incubated for 5 days at 37°C in 5% CO₂ in Opti-MEM media containing 2.5% fetal calf serum (FCS), 50 ng/mL VEGF-A₁₆₅ (R&D Systems, Abingdon, UK) and penicillin-streptomycin, with a media change on day 3. Rings were then fixed with 4% paraformaldehyde, stained with BS-1 lectin-fluorescein (Sigma Aldrich) to define endothelium, and then imaged with an inverted confocal microscope (LSM700, Carl Zeiss Microscopy Ltd.); tiled images were collected using a 10×/0.2NA objective and stitched using Zen software. Image analysis was performed with Image J (NIH), defining the number of fluorescein staining sprouts per ring and the mean length of these sprouts; mean data were then produced for each experimental animal from at least 4 rings.

Purified neutrophils (5×10⁶/mL, 500 μL) were incubated in RPMI-1640 solution (Gibco, Carlsbad, CA) supplemented with 5% fetal bovine serum (PAA Laboratories, Etobicoke, ON), 1% penicillin/streptomycin/GlutaMAX (P/S) (Gibco) and 25 mM HEPES (Sigma, Oakville, ON), and termed RPMI (for complete RPMI-1640 solution). Neutrophils were then stimulated for 2 and 24 hours with control vehicle (PBS), N-Formyl-Met-Leu-Phe (fMLP; 10^-7 M) (Sigma, Oakville, ON), bacterial lipopolysaccharide (LPS; Escherichia coli 0111:B4; 1 μg/mL) (Sigma, Oakville, ON) or tumor necrosis factor-α (TNF-α; 10 ng/mL) (Peprotech, Rocky Hill, NJ) at 37°C, 5% CO₂. Upon neutrophil stimulation, cells were centrifuged at 900 g for 7 minutes and supernatants stored at −80°C for future quantifications by ELISA of VEGF-A₁₆₅, IL-1RA and IL-8 (R&D Systems). The selected aforementioned agonists (i.e. fMLP, LPS or TNF-α) were used because of their capacity to promote VEGF-A₁₆₅, IL-1RA and IL-8 release by the neutrophils [28 (link)-31 (link)].

The in vitro permeability assay was performed as we previously described [34 (link)]. Briefly, hBMECs transfected with miR-24 mimic or miR scramble were grown on 0.4-mm fibronectin-coated (R&D Systems, Inc., Minneapolis, MN, USA) Transwell filters (Corning Inc., Corning, NY, USA). After 48 h, the medium in the upper well was replaced by FITC-dextran 70 kD (0.5 mg/mL in PB).
Cells were stimulated in the lower well with PBS alone or PBS containing 50 ng/mL VEGF-A₁₆₅ (R&D Systems). The entity of endothelial permeabilization was determined measuring at 520 nm the fluorescence of Dextran that passed in the bottom chamber through the cell monolayer.

Growth Factor Reduced Matrigel (BD Biosciences, Oxford UK) was utilized to provide extracellular matrix for cell culture. 65 µl of Matrigel were added to each well of a 96-well plate and incubated at 37°C for 30 minutes. 10,000 cells/well were then added in 100 µl of tube formation assay buffer (i.e. EBM-2 medium containing 2% FBS). Cells were cultured at 37°C/5%CO₂ in the presence of 50 µM PAR1- and/or PAR2-activating peptide and/or 25–100 ng/ml VEGFa₁₆₅ (R&D systems, Minneapolis US) or basic Fibroblast Growth Factor (FGF) (R&D systems, Minneapolis US) with or without pharmacological treatments (50 µM PD98059, Sigma-Aldrich, Poole UK) and phase contrast images were captured 4 hours after treatment using a EVOS FL microscope with an UPlan FL N 4X/0.13 objective. Experiments were repeated a minimum of three times and the vasculogenic response was measured as total number of tubes normalized to control (scrambled peptide) using the Angiogenesis Analyzer plugin of ImageJ (Gilles Carpentier, Faculté des Sciences et Technologie, Université Paris Est, Creteil Val de Marne, France). Validation experiments of this assay using human umbilical vein endothelial cells (HUVECs) are shown in Figure S2.

ESCs were plated at a density of 3 × 10³ cells/cm² on gelatinized plates in ESC medium with LIF and incubated overnight to attach. The next day the media was removed and cells were washed with PBS. Differentiation was induced by adding ESC medium without LIF and VEGF-A₁₆₅ (R&D Systems) at 20–60 ng/μl. VEGF-A₁₆₅ was supplemented to the culture every alternate day for 10 days to drive differentiation into endothelial lineage. Cell morphology was monitored using phase-contrast microscopy and pluripotency was monitored by alkaline phosphatase staining.

Collagen-coated 0.4μm pore size inserts were obtained from BD Biosciences. ECs were seeded at 40,000 cells per insert well in a total volume of 500μl of EGM-2 MV media and cultured for 8 hours to allow for cell attachment and adhesion. When confluence was reached, monolayers of ECs in the transwells were pre-treated with the five treatment groups (FFP, LP, LR and media control) for one hour at 10 and 30% total well volume. Permeability was then induced in monolayers with 50ng/ml VEGF-A165 from R&D (Minneapolis, MN), administered simultaneously with the addition of FITC-Dextran. EC monolayer permeability was tested by adding 50μl of 2mg/mL 40 kDa dextran conjugated to alexa fluor 480 (Sigma-Aldrich, St. Louis, MO) to the upper chamber of each well (final concentration). 75 μl samples were removed at timed intervals from the bottom well to determine the amount of fluorescent signal that had passed through the monolayer, which directly correlates to the degree of paracellular permeability in each sample. Measurements were determined with a fluorimeter (Biotek Synergy Biotek Winooski, VT) using excitation and emission wavelengths of 485nm and 530nm, respectively, from samples obtained 45 minutes after addition of FITC-Dextran. This is the same experiment that was run in Wataha et al. (2013) [16 (link)]

C57BL/6 8 to 10 week-old male mice (n = 3 animals per group) were subcutaneously injected with liquid containing Matrigel Gel (Thermo Fischer Scientific) mixed with heparin (0.1 mg/mL) (Ajinomoto Pharma, Tokyo, Japan) [24 ]. For the positive control recombinant human VEGF-A 165 (200 ng/mL) (R&D Systems) and fibroblast growth factor 2 (1 μg/mL) (R&D Systems) were added to the gel mix. The Matrigel (500 μL) was injected subcutaneously into flanks of mice. VCE-004.8 (20 mg/kg) was i.p. daily administered until experimental endpoint.