NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes postischaemic revascularisation

Karla M O'Neill, David C Campbell, Kevin S Edgar, Eleanor K Gill, Arya Moez, Kiran J McLoughlin, Christina L O'Neill, Margaret Dellett, Ciarán J Hargey, Rawan A Abudalo, Michael O'Hare, Philip Doyle, Tinrui Toh, Joshua Khoo, June Wong, Cian M McCrudden, Marco Meloni, Coy Brunssen, Henning Morawietz, Mervin C YoderDenise M McDonald, Chris J Watson, Alan W Stitt, Andriana Margariti, Reinhold J Medina, David J Grieve

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Abstract

AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy, and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function.

METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4 which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs upregulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair.

CONCLUSIONS: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective postischaemic revascularisation.

Original languageEnglish
JournalCardiovascular Research
Early online date01 Apr 2019
DOIs
Publication statusEarly online date - 01 Apr 2019
Externally publishedYes

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Fetal Blood
Reactive Oxygen Species
Blood Vessels
Acetates
NADPH Oxidase
Angiogenesis Inducing Agents
Proteome
Hindlimb
Lectins
Superoxides
Small Interfering RNA
Superoxide Dismutase
Cell Movement
Blood Cells
Flow Cytometry
Lasers
Plasmids
Homeostasis
Fluorescence

Bibliographical note

© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

Cite this

O'Neill, Karla M ; Campbell, David C ; Edgar, Kevin S ; Gill, Eleanor K ; Moez, Arya ; McLoughlin, Kiran J ; O'Neill, Christina L ; Dellett, Margaret ; Hargey, Ciarán J ; Abudalo, Rawan A ; O'Hare, Michael ; Doyle, Philip ; Toh, Tinrui ; Khoo, Joshua ; Wong, June ; McCrudden, Cian M ; Meloni, Marco ; Brunssen, Coy ; Morawietz, Henning ; Yoder, Mervin C ; McDonald, Denise M ; Watson, Chris J ; Stitt, Alan W ; Margariti, Andriana ; Medina, Reinhold J ; Grieve, David J. / NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes postischaemic revascularisation. In: Cardiovascular Research. 2019.
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abstract = "AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy, and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function.METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4 which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs upregulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair.CONCLUSIONS: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective postischaemic revascularisation.",
author = "O'Neill, {Karla M} and Campbell, {David C} and Edgar, {Kevin S} and Gill, {Eleanor K} and Arya Moez and McLoughlin, {Kiran J} and O'Neill, {Christina L} and Margaret Dellett and Hargey, {Ciar{\'a}n J} and Abudalo, {Rawan A} and Michael O'Hare and Philip Doyle and Tinrui Toh and Joshua Khoo and June Wong and McCrudden, {Cian M} and Marco Meloni and Coy Brunssen and Henning Morawietz and Yoder, {Mervin C} and McDonald, {Denise M} and Watson, {Chris J} and Stitt, {Alan W} and Andriana Margariti and Medina, {Reinhold J} and Grieve, {David J}",
note = "{\circledC} The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.",
year = "2019",
month = "4",
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doi = "10.1093/cvr/cvz090",
language = "English",
journal = "Cardiovascular Research",
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O'Neill, KM, Campbell, DC, Edgar, KS, Gill, EK, Moez, A, McLoughlin, KJ, O'Neill, CL, Dellett, M, Hargey, CJ, Abudalo, RA, O'Hare, M, Doyle, P, Toh, T, Khoo, J, Wong, J, McCrudden, CM, Meloni, M, Brunssen, C, Morawietz, H, Yoder, MC, McDonald, DM, Watson, CJ, Stitt, AW, Margariti, A, Medina, RJ & Grieve, DJ 2019, 'NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes postischaemic revascularisation', Cardiovascular Research. https://doi.org/10.1093/cvr/cvz090

NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes postischaemic revascularisation. / O'Neill, Karla M; Campbell, David C; Edgar, Kevin S; Gill, Eleanor K; Moez, Arya; McLoughlin, Kiran J; O'Neill, Christina L; Dellett, Margaret; Hargey, Ciarán J; Abudalo, Rawan A; O'Hare, Michael; Doyle, Philip; Toh, Tinrui; Khoo, Joshua; Wong, June; McCrudden, Cian M; Meloni, Marco; Brunssen, Coy; Morawietz, Henning; Yoder, Mervin C; McDonald, Denise M; Watson, Chris J; Stitt, Alan W; Margariti, Andriana; Medina, Reinhold J; Grieve, David J.

In: Cardiovascular Research, 01.04.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes postischaemic revascularisation

AU - O'Neill, Karla M

AU - Campbell, David C

AU - Edgar, Kevin S

AU - Gill, Eleanor K

AU - Moez, Arya

AU - McLoughlin, Kiran J

AU - O'Neill, Christina L

AU - Dellett, Margaret

AU - Hargey, Ciarán J

AU - Abudalo, Rawan A

AU - O'Hare, Michael

AU - Doyle, Philip

AU - Toh, Tinrui

AU - Khoo, Joshua

AU - Wong, June

AU - McCrudden, Cian M

AU - Meloni, Marco

AU - Brunssen, Coy

AU - Morawietz, Henning

AU - Yoder, Mervin C

AU - McDonald, Denise M

AU - Watson, Chris J

AU - Stitt, Alan W

AU - Margariti, Andriana

AU - Medina, Reinhold J

AU - Grieve, David J

N1 - © The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.

PY - 2019/4/1

Y1 - 2019/4/1

N2 - AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy, and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function.METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4 which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs upregulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair.CONCLUSIONS: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective postischaemic revascularisation.

AB - AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy, and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function.METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4 which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs upregulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair.CONCLUSIONS: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective postischaemic revascularisation.

U2 - 10.1093/cvr/cvz090

DO - 10.1093/cvr/cvz090

M3 - Article

C2 - 30937452

JO - Cardiovascular Research

JF - Cardiovascular Research

SN - 0008-6363

ER -