title: CTLA4-Ig/CD86 interactions in cultured human endothelial cells: effects on VEGFR-2 and ICAM1 expression.
abstract: OBJECTIVE
  Previous studies have reported the presence of CD86 (B7.2) costimulatory molecule on endothelial cells (ECs) and recent data have shown that CTLA4-Ig (abatacept), used as a biological agent in rheumatoid arthritis, interacts with CD86 expressed on different cells involved in synovitis. Therefore, the effects of CTLA4-Ig/CD86 interaction on VEGFR-2 (vascular endothelial growth factor receptor 2) and ICAM1 expression, were evaluated in cultured ECs.
  
  
  METHODS
  Activated ECs (γIFN 500 U/ml or IL-17 100 ng/ml), treated with CTLA4-Ig (10, 100, 500 μg/ml) were analysed for CD86, VEGFR-2 and ICAM1 expression, by flow cytometry (FACS), by western blot (WB) and quantitative real time PCR (qRT-PCR).
  
  
  RESULTS
  Following CTLA4-Ig treatment (10, 100, 500 μg/ml; 24 hrs), activated ECs decreased their CD86-positivity at FACS: 66, 59, 51%, respectively, versus 68% of untreated cells (cnt) (for γIFN-activated cells) and 42, 47, 46% versus 71% (cnt) (for IL-17-activated ECs). Gamma-IFN-activated ECs, treated with CTLA4-Ig, showed a dose-dependent decrease only for ICAM1 fluorescence. Whereas, WB showed a significant decrease (p<0.05) for both ICAM1 and VEGFR-2 after CTLA4-Ig 500 μg/ml (3 and 24 hrs) and for VEGFR-2 also after CTLA4-Ig 100 μg/ml (3 hrs). QRT-PCR showed a significant decrease (p<0.05) for VEGFR-2 after CTLA4-Ig 500 μg/ml (3 and 24 hrs) and after CTLA4-Ig 100 μg/ml (limited at 3 hrs). QRT-PCR for ICAM1 was negative at 3 and 24 hrs, possibly since it was to late to be detected.
  
  
  CONCLUSIONS
  Results support a CTLA4-Ig/CD86 interaction on γIFN and IL-17 activated ECs modulation, in the expression of VEGFR-2 and ICAM1, both relevant for inflammatory and angiogenetic processes, suggesting ECs as a further target for abatacept.
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cutolo
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Montagna
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Soldano
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Contini
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Paolino
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Carmen
authors.initials: C
authors.lastname: Pizzorni
authors.affiliation: Division of Clinical Immunology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Bruno
authors.initials: B
authors.lastname: Seriolo
authors.affiliation: Division of Clinical Immunology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Sulli
authors.affiliation: Research Laboratory and Academic Division of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy.
authors.forename: Renata
authors.initials: R
authors.lastname: Brizzolara
mesh_terms: D000069594:Abatacept; D051940:B7-2 Antigen / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D018796:Immunoconjugates / Q000494:pharmacology*; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D020381:Interleukin-17 / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism*
chemical_list: D051940:B7-2 Antigen; C497964:CD86 protein, human; C573420:ICAM1 protein, human; D018796:Immunoconjugates; D007166:Immunosuppressive Agents; D020381:Interleukin-17; D012333:RNA, Messenger; D018799:Intercellular Adhesion Molecule-1; D000069594:Abatacept; D007371:Interferon-gamma; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2