title: Response to anti-VEGF-A treatment of endothelial cells in vitro.
abstract: This study was conducted to compare the effects of two anti-VEGF-A drugs, Ranibizumab and Aflibercept, on the expression and secretion of VEGFs family members, and on their influence in proliferation and migration of endothelial cells (HECV) in vitro. HECV cells were exposed 24 h (T1), 4 days (T2) and 6 days (T3) to Ranibizumab or Aflibercept at pharmacodynamically relevant concentrations (Ranibizumab: 12.5 μg/ml and 125 μg/ml; Aflibercept: 50 μg/ml and 500 μg/ml). Cell viability and then expression and secretion of VEGF-A, VEGF-B, VEGF-C and PlGF were evaluated respectively by Real Time-PCR and ELISA. Intracellular signaling activated by VEGF-A and VEGF-C was investigated evaluating phosphorylation of VEGFR2. Influence in would healing was evaluated through scratch assay. In general no differences were observed among the tested concentrations of anti-vegf drugs. Ranibizumab and Aflibercept did not affect HECV cell viability in all experimental times. At T1, Ranibizumab decreased mRNA levels of VEGF-A, induced VEGF-C secretion, abrogated phosphorylation of VEGFR2 stimulated by VEGF-A, and impaired ability of HECV cells to repair wound healing. Aflibercept decreased mRNA levels of VEGF-A, -B and PlGF; slightly increased basal level of phVEGFR2, and completely abrogated phosphorylation stimulated by VEGF-A and VEGF-C. No effects on secretion of VEGF-B and on would healing were observed after exposure to Aflibercept. Prolonged exposure to anti-VEGFs decreased expression and secretion of VEGF-A and VEGF-B, up-regulated VEGF-C mRNA levels and its secretion, and increased basal phosphorylation of VEGFR2. Acute treatment with Ranibizumab or Aflibercept evoked different responses on endothelial cells, however these differences were lost after prolonged exposure. Scratch test results suggest that treatment with Ranibizumab may be more effective than Aflibercept in reducing angiogenic potential of endothelial cells in vitro.
authors.affiliation: Department of Internal Medicine and Medical Specialties, Viale Benedetto XV, Genova, Italy. Electronic address: alep100@hotmail.com.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Puddu
authors.affiliation: Department of Internal Medicine and Medical Specialties, Viale Benedetto XV, Genova, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Sanguineti
authors.affiliation: Department of Neuroscience, Ophthalmology and Genetics, Viale Benedetto XV, Genova, Italy.
authors.forename: Carlo Enrico
authors.initials: CE
authors.lastname: Traverso
authors.affiliation: Department of Internal Medicine and Medical Specialties, Viale Benedetto XV, Genova, Italy.
authors.forename: Giorgio L
authors.initials: GL
authors.lastname: Viviani
authors.affiliation: Department of Neuroscience, Ophthalmology and Genetics, Viale Benedetto XV, Genova, Italy; Fondazione per la Macula onlus - Genova, Piazza della Vittoria, Genova, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Nicolò
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D002470:Cell Survival; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008268:Macular Degeneration / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D012313:RNA / Q000235:genetics*; D000069579:Ranibizumab / Q000494:pharmacology*; D060888:Real-Time Polymerase Chain Reaction; D040262:Receptors, Vascular Endothelial Growth Factor; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics
chemical_list: D020533:Angiogenesis Inhibitors; D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; C533178:aflibercept; D012313:RNA; D040262:Receptors, Vascular Endothelial Growth Factor; D000069579:Ranibizumab
keywords: Aflibercept; Endothelial cells; Neovascularization; Ranibizumab; VEGF