title: Microvascular retinal endothelial and pericyte cell apoptosis in vitro: role of hedgehog and Notch signaling.
abstract: OBJECTIVE
  Aberrant retinal blood flow is a hallmark of retinopathies and may be a causative factor in their pathophysiology. In this study, the effects of pulsatile flow on hedgehog and Notch control of retinal endothelial cell and pericyte apoptosis were examined.
  
  
  METHODS
  The levels of hedgehog and Notch signaling components in bovine retinal endothelial cells (BRECs) and pericytes (BRPs) were examined in vitro under static conditions and after exposure to pulsatile flow, with a perfused transcapillary co-culture system. Notch and hedgehog signaling was examined by immunocytochemistry, immunoblot, and real-time PCR.
  
  
  RESULTS
  Notch and hedgehog proteins were present in BRECs and BRPs in vitro and in human retinal vasculature in vivo. Inhibition of hedgehog with cyclopamine and Notch with DAPT decreased hedgehog target gene levels and Notch intracellular receptor expression, respectively, concomitant with an increase in BREC and BRP apoptosis. Sonic hedgehog (Shh) mediated upregulation of Notch1 receptor levels was attenuated after cyclopamine treatment in both cell types. Exposure of co-cultured BRECs and BRPs to pulsatile flow increased apoptosis in the BRPs while concurrently decreasing apoptosis in the BRECs. These changes were concomitant with increased expression of Notch and hedgehog signaling components in the BRECs and reduced expression in the BRPs. The flow-induced decrease in apoptosis in the BRECs was associated with increased Notch receptor expression and was reversed after inhibition of hedgehog signaling with cyclopamine and inhibition of Notch signaling after ectopic expression of the CBF-1/RBP-Jκ-binding protein, RPMS-1.
  
  
  CONCLUSIONS
  Pulsatile flow promotes BREC survival and enhances BRP apoptosis through modulation of Notch and hedgehog pathways. These interactions have important implications for the pathogenesis of retinopathies.
authors.affiliation: Vascular Health Research Centre, Faculty of Science and Health, Dublin City University, Dublin, Ireland.
authors.forename: Tony E
authors.initials: TE
authors.lastname: Walshe
authors.forename: Paul
authors.initials: P
authors.lastname: Connell
authors.forename: Lorna
authors.initials: L
authors.lastname: Cryan
authors.forename: Gail
authors.initials: G
authors.lastname: Ferguson
authors.forename: Tom
authors.initials: T
authors.lastname: Gardiner
authors.forename: David
authors.initials: D
authors.lastname: Morrow
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Redmond
authors.forename: Colm
authors.initials: C
authors.lastname: O'Brien
authors.forename: Paul A
authors.initials: PA
authors.lastname: Cahill
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D042783:Endothelial Cells / Q000166:cytology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D053823:Hedgehog Proteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D020286:Pericytes / Q000166:cytology* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D051880:Receptors, Notch / Q000096:biosynthesis / Q000235:genetics*; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D053823:Hedgehog Proteins; D051880:Receptors, Notch; D004247:DNA