Rhvegf165

When specified, CM derived from either CC angiogenesis assays or monocultures, cultured for 72 h in ¼ ECGM-2, was added to both angiogenesis assays instead of the control medium. Furthermore, the following substances were used: recombinant human vascular endothelial growth factor (rhVEGF₁₆₅, 10 ng/ml; PeproTech, London, United Kingdom), angiopoietin-1 and angiopoietin-2 [rhAng-1 and rhAng-2, 400 ng/ml (Teichert et al., 2017 (link)); both PeproTech], suramin sodium (anti-angiogenic compound, 100 μM; Santa Cruz Biotechnology, Heidelberg, Germany), and ZM 323881 hydrochloride [selective VEGFR2 antagonist, 1 μM (Busceti et al., 2017 (link)); Santa Cruz Biotechnology].

Scaffolds with nanoclay and LAP-free were 3D printed, crosslinked following 10 min exposure to 100 mmol/L CaCl₂ and allowed to adsorb for 30 min with recombinant human vascular endothelial growth factor (rhVEGF 165, PeproTech, USA) at 100 μg/mL at 4 °C. 3D printed constructs were washed three times with 1× HBSS prior storage overnight at 4 °C.

Vascular endothelial cells proliferation has been validated as an initial step in angiogenesis, followed by migration, adhesion, and differentiation (Lamalice et al., 2007 (link)). Therefore, for the cell migration assay, we adopted HUVECs, which have been abundantly used (Lamalice et al., 2007 (link); Wang M. et al., 2019 (link)). These cells were seeded in a 6-well plate until they reached a density of 2 × 10⁵ cells per well. After a confluent monolayer was formed, a wound was generated in each well by a sterilized 200 μl peptide tip, and the cells were washed three times with PBS. The cells were then treated with 1 μg/ml recombinant human vascular endothelial growth factor-165 (rhVEGF₁₆₅, PeproTech Inc., United States) and combined with 10% PBS, XAV939 (10 μM) or XAV939 NPs (10 μM XAV939 equivalent) and incubated with a fresh DMEM medium containing 1% FBS. Cell migration and wound closure were observed at 0, 12, and 24 h with an Olympus inverted light microscope, and microphotographs were taken at 10× magnification with an Oplenic digital camera. The scratch widths and the percentage of wound closure were quantified using ImageJ software (National Institutes of Health, Bethesda, United States).

A male Heterodontus francisci shark was immunized iv with 1 μg rhVEGF₁₆₅ (Peprotech (Rocky Hill, NJ, USA), 300-01A) in 1× PBS every 15 days for 20 weeks. The dissection of the spleen, mRNA purification, and library generation were performed as described [48 (link)]. For the phage display, 3 rounds of panning were completed against 250 ng/well of rhVEGF₁₆₅ with 5, 10, and 20 wash cycles with 1× PBS/0.5% Tween (PBST-0.5). Positive colonies were selected by PCR with the ompseq and gback primers [49 ]. Positive PCR clones were grown in LB medium (Sigma (St. Louis, MO, USA), L3022), and plasmids were isolated using commercial kits (Qiagen (Hilden, Germany), 27104). Their sequences were obtained by capillary electrophoresis (Seqxcel, San Diego, CA, USA), analyzed on a CLC DNA Workbench (Qiagen, version 121 7.9.0, Redwood, CA, USA), and compared with internal and external databases using NCBI BLAST.