Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis

Caitriona O'Leary, Mary K McGahon, Sadaf Ashraf, Jennifer McNaughten, Thomas Friedel, Patrizia Cincolà, Peter Barabas, Jose A Fernandez, Alan W Stitt, J Graham McGeown, Tim M Curtis

Research output: Contribution to journalArticle

1 Citation (Scopus)
144 Downloads (Pure)

Abstract

Purpose: We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis.

Methods: Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca2+ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording.

Results: TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model.

Conclusions: TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina.

Original languageEnglish
Pages (from-to)3297-3309
JournalInvestigative ophthalmology & visual science
Volume60
Issue number10
DOIs
Publication statusPublished - 01 Aug 2019

Fingerprint

Endothelial Cells
Ligation
Retina
Retinal Neovascularization
Staining and Labeling
Oxygen
Retinal Vessels
Vascular Endothelial Growth Factor A
Cell Movement
Cytoplasm
Western Blotting
Cell Proliferation
Cell Membrane
Polymerase Chain Reaction
Messenger RNA
In Vitro Techniques
Proteins

Cite this

O'Leary, Caitriona ; McGahon, Mary K ; Ashraf, Sadaf ; McNaughten, Jennifer ; Friedel, Thomas ; Cincolà, Patrizia ; Barabas, Peter ; Fernandez, Jose A ; Stitt, Alan W ; McGeown, J Graham ; Curtis, Tim M. / Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis. In: Investigative ophthalmology & visual science. 2019 ; Vol. 60, No. 10. pp. 3297-3309.
@article{4181f1fe619c434584c5e09748cd9ab6,
title = "Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis",
abstract = "Purpose: We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis.Methods: Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca2+ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording.Results: TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model.Conclusions: TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina.",
author = "Caitriona O'Leary and McGahon, {Mary K} and Sadaf Ashraf and Jennifer McNaughten and Thomas Friedel and Patrizia Cincol{\`a} and Peter Barabas and Fernandez, {Jose A} and Stitt, {Alan W} and McGeown, {J Graham} and Curtis, {Tim M}",
year = "2019",
month = "8",
day = "1",
doi = "10.1167/iovs.18-26344",
language = "English",
volume = "60",
pages = "3297--3309",
journal = "Investigative Opthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "10",

}

Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis. / O'Leary, Caitriona; McGahon, Mary K; Ashraf, Sadaf; McNaughten, Jennifer; Friedel, Thomas; Cincolà, Patrizia; Barabas, Peter; Fernandez, Jose A; Stitt, Alan W; McGeown, J Graham; Curtis, Tim M.

In: Investigative ophthalmology & visual science, Vol. 60, No. 10, 01.08.2019, p. 3297-3309.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Involvement of TRPV1 and TRPV4 Channels in Retinal Angiogenesis

AU - O'Leary, Caitriona

AU - McGahon, Mary K

AU - Ashraf, Sadaf

AU - McNaughten, Jennifer

AU - Friedel, Thomas

AU - Cincolà, Patrizia

AU - Barabas, Peter

AU - Fernandez, Jose A

AU - Stitt, Alan W

AU - McGeown, J Graham

AU - Curtis, Tim M

PY - 2019/8/1

Y1 - 2019/8/1

N2 - Purpose: We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis.Methods: Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca2+ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording.Results: TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model.Conclusions: TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina.

AB - Purpose: We investigate the contribution of TRPV1 and TRPV4 channels to retinal angiogenesis.Methods: Primary retinal microvascular endothelial cells (RMECs) were used for RT-PCR, Western blotting, immunolabeling, Ca2+ signaling, and whole-cell patch-clamp studies while localization of TRPV1 also was assessed in retinal endothelial cells using whole mount preparations. The effects of pharmacologic blockers of TRPV1 and TRPV4 on retinal angiogenic activity was evaluated in vitro using sprout formation, cell migration, proliferation, and tubulogenesis assays, and in vivo using the mouse model of oxygen-induced retinopathy (OIR). Heteromultimerization of TRPV1 and TRPV4 channels in RMECs was assessed using proximity ligation assays (PLA) and electrophysiologic recording.Results: TRPV1 mRNA and protein expression were identified in RMECs. TRPV1 labelling was found to be mainly localized to the cytoplasm with some areas of staining colocalizing with the plasma membrane. Staining patterns for TRPV1 were broadly similar in endothelial cells of intact vessels within retinal flat mounts. Functional expression of TRPV1 and TRPV4 in RMECs was confirmed by patch-clamp recording. Pharmacologic inhibition of TRPV1 or TRPV4 channels suppressed in vitro retinal angiogenesis through a mechanism involving the modulation of tubulogenesis. Blockade of these channels had no effect on VEGF-stimulated angiogenesis or Ca2+ signals in vitro. PLA and patch-clamp studies revealed that TRPV1 and TRPV4 form functional heteromeric channel complexes in RMECs. Inhibition of either channel reduced retinal neovascularization and promoted physiologic revascularization of the ischemic retina in the OIR mouse model.Conclusions: TRPV1 and TRPV4 channels represent promising targets for therapeutic intervention in vasoproliferative diseases of the retina.

U2 - 10.1167/iovs.18-26344

DO - 10.1167/iovs.18-26344

M3 - Article

C2 - 31369032

VL - 60

SP - 3297

EP - 3309

JO - Investigative Opthalmology and Visual Science

JF - Investigative Opthalmology and Visual Science

SN - 0146-0404

IS - 10

ER -