Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR

Renata Colavitti, Giovanni Pani, Barbara Bedogni, Rosanna Anzevino, Silvia Borrello, Johannes Waltenberger, Tommaso Galeotti

Research output: Contribution to journalArticle

297 Citations (Scopus)

Abstract

Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitricoxide synthase inhibitors diphenyleneiodonium and NGmethyl-L-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-γ-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.

Original languageEnglish (US)
Pages (from-to)3101-3108
Number of pages8
JournalJournal of Biological Chemistry
Volume277
Issue number5
DOIs
StatePublished - Feb 1 2002
Externally publishedYes

Fingerprint

Vascular Endothelial Growth Factor Receptor-2
Reactive Oxygen Species
Vascular Endothelial Growth Factor A
Phosphatidylinositol 3-Kinase
Phosphorylation
Endothelial cells
Oxidants
Endothelial Cells
Antioxidants
Chemical activation
Lipoxygenases
Masoprocol
Scavenger Receptors
Lipoxygenase Inhibitors
Vascular Endothelial Growth Factor Receptor
Monomeric GTP-Binding Proteins
Peroxides
Receptor Protein-Tyrosine Kinases
Type C Phospholipases
Glutathione Transferase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR. / Colavitti, Renata; Pani, Giovanni; Bedogni, Barbara; Anzevino, Rosanna; Borrello, Silvia; Waltenberger, Johannes; Galeotti, Tommaso.

In: Journal of Biological Chemistry, Vol. 277, No. 5, 01.02.2002, p. 3101-3108.

Research output: Contribution to journalArticle

Colavitti, Renata ; Pani, Giovanni ; Bedogni, Barbara ; Anzevino, Rosanna ; Borrello, Silvia ; Waltenberger, Johannes ; Galeotti, Tommaso. / Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR. In: Journal of Biological Chemistry. 2002 ; Vol. 277, No. 5. pp. 3101-3108.
@article{d5571f6c6cbc4921a5769abd31890a25,
title = "Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR",
abstract = "Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitricoxide synthase inhibitors diphenyleneiodonium and NGmethyl-L-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-γ-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.",
author = "Renata Colavitti and Giovanni Pani and Barbara Bedogni and Rosanna Anzevino and Silvia Borrello and Johannes Waltenberger and Tommaso Galeotti",
year = "2002",
month = "2",
day = "1",
doi = "10.1074/jbc.M107711200",
language = "English (US)",
volume = "277",
pages = "3101--3108",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "5",

}

TY - JOUR

T1 - Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR

AU - Colavitti, Renata

AU - Pani, Giovanni

AU - Bedogni, Barbara

AU - Anzevino, Rosanna

AU - Borrello, Silvia

AU - Waltenberger, Johannes

AU - Galeotti, Tommaso

PY - 2002/2/1

Y1 - 2002/2/1

N2 - Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitricoxide synthase inhibitors diphenyleneiodonium and NGmethyl-L-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-γ-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.

AB - Recent evidence shows the involvement of reactive oxygen species (ROS) in the mitogenic cascade initiated by the tyrosine kinase receptors of several growth factor peptides. We have asked whether also the vascular endothelial growth factor (VEGF) utilizes ROS as messenger intermediates downstream of the VEGF receptor-2 (VEGFR-2)/KDR receptor given that the proliferation of endothelial cells during neoangiogenesis is physiologically regulated by oxygen and likely by its derivative species. In porcine aortic endothelial cells stably expressing human KDR, receptor activation by VEGF is followed by a rapid increase in the intracellular generation of hydrogen peroxide as revealed by the peroxide-sensitive probe dichlorofluorescein diacetate. Genetic and pharmacological studies suggest that such oxidant burst requires as upstream events the activation of phosphatidylinositol 3-kinase and the small GTPase Rac-1 and is likely initiated by lipoxygenases. Interestingly, ROS generation in response to VEGF is not blocked but rather potentiated by endothelial nitricoxide synthase inhibitors diphenyleneiodonium and NGmethyl-L-arginine, ruling out the possibility of nitric oxide being the oxidant species here detected in VEGF-stimulated cells. Inhibition of KDR-dependent generation of ROS attenuates early signaling events including receptor autophosphorylation and binding to a phospholipase C-γ-glutathione S-transferase fusion protein. Moreover, catalase, the lipoxygenase inhibitor nordihydroguaiaretic acid, the synthetic ROS scavenger EUK-134, and phosphatidylinositol 3-kinase inhibitor wortmannin all reduce ERK phosphorylation in response to VEGF, and antioxidants prevent VEGF-dependent mitogenesis. Finally, cell culture and stimulation in a nearly anoxic environment mimic the effect of ROS scavenger on receptor and ERK phosphorylation, reinforcing the idea that ROS are necessary components of the mitogenic signaling cascade initiated by KDR. These data identify ROS as a new class of intracellular angiogenic mediators and may represent a potential premise for new antioxidant-based antiangiogenic therapies.

UR - http://www.scopus.com/inward/record.url?scp=0036479214&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036479214&partnerID=8YFLogxK

U2 - 10.1074/jbc.M107711200

DO - 10.1074/jbc.M107711200

M3 - Article

VL - 277

SP - 3101

EP - 3108

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 5

ER -