Microvascular retinal endothelial and pericyte cell apoptosis in vitro: role of hedgehog and Notch signaling

Tony E Walshe, Paul Connell, Lorna Cryan, Gail Ferguson, Tom Gardiner, David Morrow, Eileen M Redmond, Colm O'Brien, Paul A Cahill

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

PURPOSE: 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.

Original languageEnglish
Pages (from-to)4472-83
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume52
Issue number7
DOIs
Publication statusPublished - 23 Jun 2011

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Pericytes
Endothelial Cells
Apoptosis
Pulsatile Flow
Notch Receptors
Notch1 Receptor
Hedgehog Proteins
In Vitro Techniques
Coculture Techniques
Real-Time Polymerase Chain Reaction
Cell Survival
Carrier Proteins
Up-Regulation
Immunohistochemistry

Keywords

  • Animals
  • Apoptosis/physiology
  • Blotting, Western
  • Cattle
  • Cells, Cultured
  • DNA/genetics
  • Endothelial Cells/cytology
  • Endothelium, Vascular/cytology
  • Hedgehog Proteins/biosynthesis
  • Humans
  • Immunohistochemistry
  • Pericytes/cytology
  • Polymerase Chain Reaction
  • Receptors, Notch/biosynthesis
  • Retina/cytology
  • Signal Transduction

Cite this

Walshe, Tony E ; Connell, Paul ; Cryan, Lorna ; Ferguson, Gail ; Gardiner, Tom ; Morrow, David ; Redmond, Eileen M ; O'Brien, Colm ; Cahill, Paul A. / Microvascular retinal endothelial and pericyte cell apoptosis in vitro : role of hedgehog and Notch signaling. In: Investigative Ophthalmology and Visual Science. 2011 ; Vol. 52, No. 7. pp. 4472-83.
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abstract = "PURPOSE: 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.",
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author = "Walshe, {Tony E} and Paul Connell and Lorna Cryan and Gail Ferguson and Tom Gardiner and David Morrow and Redmond, {Eileen M} and Colm O'Brien and Cahill, {Paul A}",
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Walshe, TE, Connell, P, Cryan, L, Ferguson, G, Gardiner, T, Morrow, D, Redmond, EM, O'Brien, C & Cahill, PA 2011, 'Microvascular retinal endothelial and pericyte cell apoptosis in vitro: role of hedgehog and Notch signaling', Investigative Ophthalmology and Visual Science, vol. 52, no. 7, pp. 4472-83. https://doi.org/10.1167/iovs.10-7061

Microvascular retinal endothelial and pericyte cell apoptosis in vitro : role of hedgehog and Notch signaling. / Walshe, Tony E; Connell, Paul; Cryan, Lorna; Ferguson, Gail; Gardiner, Tom; Morrow, David; Redmond, Eileen M; O'Brien, Colm; Cahill, Paul A.

In: Investigative Ophthalmology and Visual Science, Vol. 52, No. 7, 23.06.2011, p. 4472-83.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Microvascular retinal endothelial and pericyte cell apoptosis in vitro

T2 - role of hedgehog and Notch signaling

AU - Walshe, Tony E

AU - Connell, Paul

AU - Cryan, Lorna

AU - Ferguson, Gail

AU - Gardiner, Tom

AU - Morrow, David

AU - Redmond, Eileen M

AU - O'Brien, Colm

AU - Cahill, Paul A

PY - 2011/6/23

Y1 - 2011/6/23

N2 - PURPOSE: 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.

AB - PURPOSE: 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.

KW - Animals

KW - Apoptosis/physiology

KW - Blotting, Western

KW - Cattle

KW - Cells, Cultured

KW - DNA/genetics

KW - Endothelial Cells/cytology

KW - Endothelium, Vascular/cytology

KW - Hedgehog Proteins/biosynthesis

KW - Humans

KW - Immunohistochemistry

KW - Pericytes/cytology

KW - Polymerase Chain Reaction

KW - Receptors, Notch/biosynthesis

KW - Retina/cytology

KW - Signal Transduction

U2 - 10.1167/iovs.10-7061

DO - 10.1167/iovs.10-7061

M3 - Article

C2 - 21498615

VL - 52

SP - 4472

EP - 4483

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 7

ER -