title: Endothelin receptor blockade ameliorates vascular fragility in endothelial cell-specific Fli-1-knockout mice by increasing Fli-1 DNA binding ability.
abstract: OBJECTIVE
  It is generally accepted that blockade of endothelin receptors has potentially beneficial effects on vasculopathy associated with systemic sclerosis (SSc). The aim of this study was to clarify the molecular mechanism underlying these effects using endothelial cell-specific Fli-1-knockout (Fli-1 ECKO) mice, an animal model of SSc vasculopathy.
  
  
  METHODS
  Levels of messenger RNA for target genes and the expression and phosphorylation levels of target proteins were determined in human and murine dermal microvascular endothelial cells by real-time quantitative reverse transcription-polymerase chain reaction and immunoblotting, respectively. The binding of Fli-1 to the target gene promoters was evaluated using chromatin immunoprecipitation. Expression levels of Fli-1 and α-smooth muscle actin in murine skin were evaluated using immunohistochemistry. Vascular structure and permeability were evaluated in mice injected with fluorescein isothiocyanate-dextran and Evans blue dye, respectively.
  
  
  RESULTS
  In human dermal microvascular endothelial cells, endothelin 1 induced phosphorylation of Fli-1 at Thr(312) through the sequential activation of c-Abl and protein kinase Cδ, leading to a decrease in Fli-1 protein levels as well as a decrease in binding of Fli-1 to the target gene promoters, whereas bosentan treatment reversed those effects. In Fli-1 ECKO mice, 4 weeks of treatment with bosentan increased endothelial Fli-1 expression, resulting in vascular stabilization and the restoration of impaired leaky vessels.
  
  
  CONCLUSIONS
  The vascular fragility of Fli-1 ECKO mice was improved by bosentan through the normalization of Fli-1 protein levels and activity in endothelial cells, which may explain, in part, the mechanism underlying the beneficial effects of endothelin receptor blockade on SSc vasculopathy.
authors.affiliation: University of Tokyo Graduate School of Medicine, Tokyo, Japan.
authors.forename: Kaname
authors.initials: K
authors.lastname: Akamata
authors.forename: Yoshihide
authors.initials: Y
authors.lastname: Asano
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamashita
authors.forename: Shinji
authors.initials: S
authors.lastname: Noda
authors.forename: Takashi
authors.initials: T
authors.lastname: Taniguchi
authors.forename: Takehiro
authors.initials: T
authors.lastname: Takahashi
authors.forename: Yohei
authors.initials: Y
authors.lastname: Ichimura
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Toyama
authors.forename: Maria
authors.initials: M
authors.lastname: Trojanowska
authors.forename: Shinichi
authors.initials: S
authors.lastname: Sato
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000077300:Bosentan; D002199:Capillary Permeability; D002460:Cell Line; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D042783:Endothelial Cells / Q000378:metabolism*; D065128:Endothelin Receptor Antagonists / Q000494:pharmacology; D019332:Endothelin-1 / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D018345:Mice, Knockout; D055806:Microvessels / Q000166:cytology; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D051745:Protein Kinase C-delta / Q000378:metabolism; D051789:Proto-Oncogene Protein c-fli-1 / Q000235:genetics* / Q000378:metabolism; D016315:Proto-Oncogene Proteins c-abl / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D017466:Receptors, Endothelin / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012595:Scleroderma, Systemic / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D014652:Vascular Diseases
chemical_list: D000199:Actins; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; C497426:Fli1 protein, mouse; D051789:Proto-Oncogene Protein c-fli-1; D012333:RNA, Messenger; D017466:Receptors, Endothelin; D013449:Sulfonamides; C544013:alpha-smooth muscle actin, mouse; D016315:Proto-Oncogene Proteins c-abl; D051745:Protein Kinase C-delta; D000077300:Bosentan