Vascular endothelial growth factor (vegf)

The mice were divided into four groups: sham plus saline, UC plus saline, sham plus VEGF, UC plus VEGF. Four days before UC placement, saline or VEGF (0.02 mg/kg; Cat#: 583106; Bio Legend, San Diego, CA) was injected into the mouse for three days, via tail veins of the mouse, before the placement of UC.

HUVEC isolation was performed as previously described [Frangos et al., 1988 (link)]. Human umbilical cords were obtained from Sharp Memorial Hospital (San Diego, CA) under the auspices of Sharp Healthcare Institutional Review Board protocol no. 011081. Freshly harvested cells were cultured in flasks and then seeded onto glass microscope slides and grown to confluence within 4 days in M199 media (Irvine Scientific) supplemented with 20% fetal bovine serum. Prior to all experimental procedures, the HUVECs were serum-starved overnight in ATP-free M199 with 1% bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO) to establish quiescence in the monolayer. Cells used as positive control for Akt-1 activation were treated for 5 min with a combination 100 ng ml⁻¹ final of VEGF (Biolegend, San Diego, CA) and 10 nM EGF (Peprotech, Rocky Hill, NJ).
Silencing of Gα_q/11 was performed using a siRNA target sequence common to both human Gα_q and Gα₁₁ transcripts (sense: 5′-AAGATGTTCGTGGACCTGAA-3′). One hundred fifty nanomolars of final siRNA were combined to Lipofectamine siRNAmax (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol and added to HUVEC for 5 h before cells were reseeded at high density onto slides for 2 days to allow cells to reestablish confluency.

Purified Macrophages were cultured in the presence of Kynurenine (10 and 100 nM/ml) or lactic acid (0.1, 0.5 μM/ml) (R&D systems) or G-CSF (10ng/ml) or VEGF (10 ng/ml) (Biolegend) or a cocktail containing all of them for 24h. Supernatants collected were assayed for the cytokines, TGF-β, TNF-α and IL-10. Cells were stained for CD200R using flow cytometry.