Interleukin 1β

In this study human and rat retinal Müller cells were used. Immortalized rat Muller cells (rMC-1, T0576) were purchased from ABM Good (Richmond, BC, Canada) and grown in Prigrow medium 1% antibiotics. For the human model, a spontaneously immortalized human Muller cell line MIO-M1 (Moorfields/Institute of Ophthalmology-Muller 1) was obtained from the UCL Institute of Ophtalmology, London, UK [28 (link)]. According to the manufacture’s instructions, MIO-M1 were cultured in DMEM L-Glutamax (Gibco, Thermofisher Scientific). FBS or CBS were added to growth medium at 5%. Hydrogen peroxide (H₂O₂) at 30% (Sigma Aldrich) was used to induce oxidative stress and was diluted in sterilized water at 9.88 mM, before preparation of working solutions in serum-free medium for cell treatments. Interleukin-1β (Sigma Aldrich) was chosen for the inflammatory stimulation and was diluted in sterilized water at 10 μg/ml, before addition to serum-free cultures.

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)].

HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). This immortalized, stable cell line can be repeatedly frozen, thawed and propagated. HepG2 cells were seeded (2×10⁶ cells/well) in vented T-75 flasks and grown to confluence in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin and 10% of fetal calf serum (FCS). Thereafter, cells were switched to 1% FCS and incubated (37°C) under normoxic (21% O₂, 5% CO₂) or hypoxic conditions (5% O₂, 5% CO₂) in a controlled O₂ water-jacketed CO₂ incubator (Forma Scientific Series II, 3131, Marietta, OH) or treated with TNF-α (10 ng/ml, Sigma, St Louis, MO), lipopolysaccharide (LPS, 10 ng/ml, Sigma), AII (80 pM, Sigma), Endothelin-1 (2 nM, Sigma), Apelin (100 nM, Phoenix Pharmaceuticals, Burlingane, Ca), Fibronectin (10 ng/ml, Sigma), Interleukin-1β (20 ng/ml, Sigma) and TGF-β (10 ng/ml, R&D Systems, Minneapolis, Mn). All experiments carried out in cell lines were reproduced three times in at least 2 independent assays. Conditioned media were harvested, concentrated (80∶1) using 3000 MW Amicon Ultra centrifugal filters (Millipore Corp) and the presence of the fibrinogen α C-chain was assessed by SELDI-TOF-MS as described above.