Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels

Lan Huang; Colette Bichsel; Alexis L. Norris; Jeremy Thorpe; Jonathan Pevsner; Sanda Alexandrescu; Anna Pinto; David Zurakowski; Robin J. Kleiman; Mustafa Sahin; Arin K. Greene; Joyce Bischoff

doi:10.1161/ATVBAHA.121.316651

Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels

Lan Huang, Colette Bichsel, Alexis L. Norris, Jeremy Thorpe, Jonathan Pevsner, Sanda Alexandrescu, Anna Pinto, David Zurakowski, Robin J. Kleiman, Mustafa Sahin, Arin K. Greene, Joyce Bischoff

Kennedy Krieger Institute

Research output: Contribution to journal › Article › peer-review

Abstract

OBJECTIVE: Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. APPROACH AND RESULTS: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. CONCLUSIONS: Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.

Original language	English (US)
Pages (from-to)	E27-E43
Journal	Arteriosclerosis, thrombosis, and vascular biology
Volume	42
Issue number	1
DOIs	https://doi.org/10.1161/ATVBAHA.121.316651
State	Published - Jan 1 2022

Keywords

Angiopoietin-2
Endothelial cells
GTP-binding protein alpha subunits
Port wine stain
Sturge-Weber syndrome

ASJC Scopus subject areas

Cardiology and Cardiovascular Medicine

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10.1161/ATVBAHA.121.316651

Cite this

Huang, L., Bichsel, C., Norris, A. L., Thorpe, J., Pevsner, J., Alexandrescu, S., Pinto, A., Zurakowski, D., Kleiman, R. J., Sahin, M., Greene, A. K., & Bischoff, J. (2022). Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels. Arteriosclerosis, thrombosis, and vascular biology, 42(1), E27-E43. https://doi.org/10.1161/ATVBAHA.121.316651

Huang, L, Bichsel, C, Norris, AL, Thorpe, J, Pevsner, J, Alexandrescu, S, Pinto, A, Zurakowski, D, Kleiman, RJ, Sahin, M, Greene, AK & Bischoff, J 2022, 'Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels', Arteriosclerosis, thrombosis, and vascular biology, vol. 42, no. 1, pp. E27-E43. https://doi.org/10.1161/ATVBAHA.121.316651

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title = "Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels",

abstract = "OBJECTIVE: Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. APPROACH AND RESULTS: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. CONCLUSIONS: Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.",

keywords = "Angiopoietin-2, Endothelial cells, GTP-binding protein alpha subunits, Port wine stain, Sturge-Weber syndrome",

author = "Lan Huang and Colette Bichsel and Norris, {Alexis L.} and Jeremy Thorpe and Jonathan Pevsner and Sanda Alexandrescu and Anna Pinto and David Zurakowski and Kleiman, {Robin J.} and Mustafa Sahin and Greene, {Arin K.} and Joyce Bischoff",

note = "Publisher Copyright: {\textcopyright} 2021 American Heart Association, Inc.",

year = "2022",

month = jan,

day = "1",

doi = "10.1161/ATVBAHA.121.316651",

language = "English (US)",

volume = "42",

pages = "E27--E43",

journal = "Arteriosclerosis, thrombosis, and vascular biology",

issn = "1079-5642",

publisher = "Lippincott Williams and Wilkins",

number = "1",

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TY - JOUR

T1 - Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels

AU - Huang, Lan

AU - Bichsel, Colette

AU - Norris, Alexis L.

AU - Thorpe, Jeremy

AU - Pevsner, Jonathan

AU - Alexandrescu, Sanda

AU - Pinto, Anna

AU - Zurakowski, David

AU - Kleiman, Robin J.

AU - Sahin, Mustafa

AU - Greene, Arin K.

AU - Bischoff, Joyce

PY - 2022/1/1

Y1 - 2022/1/1

N2 - OBJECTIVE: Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. APPROACH AND RESULTS: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. CONCLUSIONS: Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.

AB - OBJECTIVE: Capillary malformation (CM) occurs sporadically and is associated with Sturge-Weber syndrome. The somatic mosaic mutation in GNAQ (c.548G>A, p.R183Q) is enriched in endothelial cells (ECs) in skin CM and Sturge-Weber syndrome brain CM. Our goal was to investigate how the mutant Gαq (G-protein αq subunit) alters EC signaling and disrupts capillary morphogenesis. APPROACH AND RESULTS: We used lentiviral constructs to express p.R183Q or wild-type GNAQ in normal human endothelial colony forming cells (EC-R183Q and EC-WT, respectively). EC-R183Q constitutively activated PLC (phospholipase C) β3, a downstream effector of Gαq. Activated PLCβ3 was also detected in human CM tissue sections. Bulk RNA sequencing analyses of mutant versus wild-type EC indicated constitutive activation of PKC (protein kinase C), NF-κB (nuclear factor kappa B) and calcineurin signaling in EC-R183Q. Increased expression of downstream targets in these pathways, ANGPT2 (angiopoietin-2) and DSCR (Down syndrome critical region protein) 1.4 were confirmed by quantitative PCR and immunostaining of human CM tissue sections. The Gαq inhibitor YM-254890 as well as siRNA targeted to PLCβ3 reduced mRNA expression levels of these targets in EC-R183Q while the pan-PKC inhibitor AEB071 reduced ANGPT2 but not DSCR1.4. EC-R183Q formed enlarged blood vessels in mice, reminiscent of those found in human CM. shRNA knockdown of ANGPT2 in EC-R183Q normalized the enlarged vessels to sizes comparable those formed by EC-WT. CONCLUSIONS: Gαq-R183Q, when expressed in ECs, establishes constitutively active PLCβ3 signaling that leads to increased ANGPT2 and a proangiogenic, proinflammatory phenotype. EC-R183Q are sufficient to form enlarged CM-like vessels in mice, and suppression of ANGPT2 prevents the enlargement. Our study provides the first evidence that endothelial Gαq-R183Q is causative for CM and identifies ANGPT2 as a contributor to CM vascular phenotype.

KW - Angiopoietin-2

KW - Endothelial cells

KW - GTP-binding protein alpha subunits

KW - Port wine stain

KW - Sturge-Weber syndrome

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JO - Arteriosclerosis, thrombosis, and vascular biology

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ER -

Endothelial GNAQ p.R183Q Increases ANGPT2 (Angiopoietin-2) and Drives Formation of Enlarged Blood Vessels

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