Bradykinin bk

HBMECs (10⁵ cells/mL) were incubated overnight for cell adhesion and, when achieved 60–70% confluence, cells were cultured with DENV-2 (16,681 strain), ZIKV_MR766 or ZIKV_PE243, at a multiplicity of infection (MOI) of 1. Supernatants of noninfected C6/36 cells (mock) or UV-inactivated DENV (iDENV) were used as controls. After 90 min of incubation at 37 °C, cells were washed with endotoxin-free PBS and cultured with M199 with 10% FCS. At 24 h post infection (h.p.i.), the cells (around 90% confluency) were treated or not with 10nM of bradykinin (BK) (Sigma-Aldrich, St. Louis, MO, USA) or Lys-Des-Arg⁹-bradykinin (DABK) (Sigma-Aldrich) after 30 min of preincubation with lisinopril (25 µM) (Sigma-Aldrich), an inhibitor of the angiotensin converting enzyme (ACE). At some experimental points, the cells were also treated with the antagonist of B2R-HOE-140; or with the antagonist of B1R Lys-[Leu⁸]-Des-Arg⁹-BK (LLDABK; 100 nM; (Sigma-Aldrich)); or with the inhibitor of nitric oxide synthase (NOS) L-NMMA (250 µM; Sigma-Aldrich); or with the nitric oxide donor SNAP (150 µM; Sigma-Aldrich). The antagonists and inhibitors were added 30 min before the addition of the BK or DABK, when indicated. The cells and supernatants were harvested at 48 h.p.i. and virus infection was analyzed by plaque-forming assay, or flow cytometry.

Peptidoglycan (from Staphylococcus aureus, lot number 77140), lipopolysaccharide (LPS), thrombin, and bradykinin (BK) were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). The COX-1 antibody was obtained from Santa Cruz Biotechnology Inc. (Dallas, TX, USA), whereas the COX-2 antibody was purchased from BD Biosciences (Franklin Lakes, NJ). The β-actin antibody was purchased from EMD Millipore (Billerica, MA, USA). U46619 (a TXA₂ analog), PGE₂, AH6809, and AH23848 were purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). α-SMA antibody was purchased from GeneTex Inc. (Hsinchu, Taiwan).

Linear peptides (> 95% pure) were commercially synthetized acetylated and amidated at the N- and C-termini, respectively (Supplementary Table S2). The cyclic-retro-inverse-vasoinhibin-(45–51)-peptide (CRIVi45–51) (> 98% pure) was synthetized with the sequence _DIle-_DPhe-Gly-_DArg-Gly-_DHis-_DThr and head-to-tail cyclization (GenScript, Piscataway, NJ). Recombinant vasoinhibin of 123 residues was produced as reported [27 (link)]. Recombinant human PRL was provided by Michael E. Hodsdon [28 (link)] (Yale University, New Haven, CT). Recombinant human vascular endothelial growth factor-165 (VEGF) was a gift from Genentech (South San Francisco, CA) and basic fibroblast growth factor (bFGF) was donated by Scios, Inc. (Mountain View, CA). Bradykinin (BK) and interleukin-1β were purchased from Sigma-Aldrich (St. Louis, MO) and R&D Systems (Minneapolis, MN), respectively. Anginex peptide was acquired from Phoenix Pharmaceuticals (Burlingame, CA), and cilengitide, human angiostatin K1-3, endostatin, pazopanib, sorafenib and sunitinib from Sigma-Aldrich (St. Louis, MO). Vasoinhibin and PRL cDNA were point-mutated by the two-step PCR technique, cloned in the pcDNA3 vector and produced by HEK293T/17 (ATCC, Manassas, VA) cells as reported [23 (link)].