title: Long-term treatment with TGFbeta1 impairs mechanotransduction in bovine aortic endothelial cells.
abstract: OBJECTIVE
  Vascular endothelial cells play a role in the physiological response to mechanical stress. Transforming growth factor beta1 (TGFbeta1) induces morphological changes in endothelial cells, and this may alter their mechanosensitive responses. The aim of this study was to examine the effects of TGFbeta1 on hypotonic stress (HTS)-induced responses in bovine aortic endothelial cells (BAECs).
  
  
  METHODS
  Cultured BAECs were treated with 3 ng ml(-1) TGFbeta1 for 24 h (24h-TGFbeta1) or 7 days (7d-TGFbeta1). Cytosolic actin fibres were stained with rhodamine-phalloidin. Intracellular Ca2+ concentration was measured using fura2. Tyrosine phosphorylation and RhoA expression were assessed by Western blotting. Expression of RhoA mRNA was assessed by real-time PCR.
  
  
  RESULTS
  BAECs developed pseudopod-like processes within 24 h and showed a fibroblast-like appearance after 7 days. HTS induced Ca2+ transients via endogenous ATP release in both control and 24h-TGFbeta1 BAECs but not in 7d-TGFbeta1 BAECs. We have previously shown that HTS-induced ATP release is mediated by sequential activation of RhoA and tyrosine kinases. The basal amount of membrane-bound RhoA was significantly lower in 7d-TGFbeta1 than in 24h-TGFbeta1 or control BAECs. HTS increased the membrane-bound RhoA to the same fractional level in 24h-TGFbeta1 and control BAECs, but its net maximal amount was significantly lower in 7d-TGFbeta1. HTS-induced downstream signals of RhoA activation, i.e. the tyrosine phosphorylation of FAK and paxillin, were markedly suppressed in 7d-TGFbeta1 BAECs.
  
  
  CONCLUSIONS
  These results indicate that long-term treatment with TGFbeta1 does not impair mechanoreception in BAECs but impairs mechanotransduction by affecting RhoA membrane translocation.
authors.affiliation: Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Watanabe
authors.forename: M
authors.initials: M
authors.lastname: Oike
authors.forename: Y
authors.initials: Y
authors.lastname: Ohta
authors.forename: Y
authors.initials: Y
authors.lastname: Ito
mesh_terms: D000199:Actins / Q000502:physiology; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000502:physiology; D000818:Animals; D001011:Aorta / Q000166:cytology; D001013:Aorta, Thoracic / Q000166:cytology / Q000187:drug effects; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D020013:Calcium Signaling / Q000187:drug effects; D002417:Cattle; D003600:Cytosol / Q000187:drug effects / Q000648:ultrastructure; D042783:Endothelial Cells / Q000187:drug effects* / Q000648:ultrastructure; D005456:Fluorescent Dyes; D016257:Fura-2; D007038:Hypotonic Solutions; D008465:Mechanoreceptors / Q000187:drug effects*; D010766:Phosphorylation; D010812:Physical Stimulation; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D007038:Hypotonic Solutions; D012333:RNA, Messenger; D053773:Transforming Growth Factor beta1; D014443:Tyrosine; D000255:Adenosine Triphosphate; D020742:rhoA GTP-Binding Protein; D002118:Calcium; D016257:Fura-2