Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells

Michael H. Davies, David O. Zamora, Justine R. Smith, Michael (Mike) Powers

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Purpose: EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. Methods: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 μL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). Results: Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2 ± VEGF compared to IgG control ± VEGF (P <0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. Conclusions: These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.

Original languageEnglish (US)
Pages (from-to)382-386
Number of pages5
JournalMicrovascular Research
Volume77
Issue number3
DOIs
StatePublished - May 2009

Fingerprint

Ephrin-B2
Retinal Neovascularization
Endothelial cells
Caspase 3
Endothelial Cells
Apoptosis
In Situ Nick-End Labeling
Pathologic Neovascularization
Vascular Endothelial Growth Factor A
EphB4 Receptor
Immunoglobulin G
Cells
Ephrins
Oxygen
Blood Vessels
von Willebrand Factor
Immunohistochemistry
Modulation
Ligands
Inbred C57BL Mouse

Keywords

  • Angiogenesis
  • Apoptosis
  • EphB4
  • EphrinB2
  • Neovascularization
  • Retinopathy of prematurity

ASJC Scopus subject areas

  • Biochemistry
  • Cardiology and Cardiovascular Medicine
  • Cell Biology

Cite this

Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells. / Davies, Michael H.; Zamora, David O.; Smith, Justine R.; Powers, Michael (Mike).

In: Microvascular Research, Vol. 77, No. 3, 05.2009, p. 382-386.

Research output: Contribution to journalArticle

Davies, Michael H. ; Zamora, David O. ; Smith, Justine R. ; Powers, Michael (Mike). / Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells. In: Microvascular Research. 2009 ; Vol. 77, No. 3. pp. 382-386.
@article{08725c6c0eb5459a8576c4351d4b10cd,
title = "Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells",
abstract = "Purpose: EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. Methods: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75{\%} oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 μL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). Results: Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2 ± VEGF compared to IgG control ± VEGF (P <0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. Conclusions: These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.",
keywords = "Angiogenesis, Apoptosis, EphB4, EphrinB2, Neovascularization, Retinopathy of prematurity",
author = "Davies, {Michael H.} and Zamora, {David O.} and Smith, {Justine R.} and Powers, {Michael (Mike)}",
year = "2009",
month = "5",
doi = "10.1016/j.mvr.2009.01.013",
language = "English (US)",
volume = "77",
pages = "382--386",
journal = "Microvascular Research",
issn = "0026-2862",
publisher = "Academic Press Inc.",
number = "3",

}

TY - JOUR

T1 - Soluble ephrin-B2 mediates apoptosis in retinal neovascularization and in endothelial cells

AU - Davies, Michael H.

AU - Zamora, David O.

AU - Smith, Justine R.

AU - Powers, Michael (Mike)

PY - 2009/5

Y1 - 2009/5

N2 - Purpose: EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. Methods: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 μL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). Results: Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2 ± VEGF compared to IgG control ± VEGF (P <0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. Conclusions: These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.

AB - Purpose: EphB4 receptors and their ephrinB2 ligands are essential for vascular development, but also play a role in pathological neovascularization (NV). We previously reported that soluble (s) forms of EphB4 and ephrinB2 significantly reduced retinal NV in a model of oxygen-induced retinopathy. This study investigates if these molecules suppress retinal NV by stimulation of endothelial cell (EC) apoptosis. Methods: C57BL/6 mice at postnatal day 7 (P7) were exposed to 75% oxygen for 5 days (P12) and allowed to recover in room air to induce retinal NV. One eye was injected intravitreally with 150 ng in 1.5 μL of sEphB4 or sEphrinB2 on P12 and P14, while contralateral eyes were injected with IgG antibody as control. Eyes were enucleated for histological analysis. At P16 TUNEL analysis and caspase-3 immunohistochemistry was performed on retinal sections to compare the apoptotic response between sEphB4 or sEphrinB2 injected eyes and controls. In vitro studies were performed with human retinal microvascular EC (HREC). Results: Quantification of TUNEL positive vascular cells, located in areas of retinal NV, revealed approximately 2.5-fold increase in apoptosis in sEphrinB2 injected eyes compared to control eyes. Immunohistochemistry studies revealed co-localization of both TUNEL positive cells and caspase-3 positive cells with the endothelial marker, von Willebrand factor. Cultured HREC demonstrated significantly higher caspase-3 activity after a 3 h stimulation with sEphrinB2 ± VEGF compared to IgG control ± VEGF (P <0.005). sEphB4 stimulation had no significant effect on caspase-3 activity in HREC cultures. Conclusions: These data suggest that modulation of the endogenous ephrin signaling mechanism by sEphrinB2 may induce suppression of retinal NV via induction of apoptosis. Results of the in vitro studies suggest that sEphrinB2 may directly induce apoptosis of EC during pathological neovascularization.

KW - Angiogenesis

KW - Apoptosis

KW - EphB4

KW - EphrinB2

KW - Neovascularization

KW - Retinopathy of prematurity

UR - http://www.scopus.com/inward/record.url?scp=64049096339&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=64049096339&partnerID=8YFLogxK

U2 - 10.1016/j.mvr.2009.01.013

DO - 10.1016/j.mvr.2009.01.013

M3 - Article

C2 - 19232363

AN - SCOPUS:64049096339

VL - 77

SP - 382

EP - 386

JO - Microvascular Research

JF - Microvascular Research

SN - 0026-2862

IS - 3

ER -