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 qPCR 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.

Early‐onset hypertension associated with extensive cutaneous capillary malformations harboring postzygotic variants in GNAQ and GNA11

“…Recently, molecular investigations have found that human endothelial cells of CMs harboring the GNAQ p.R183Q variant lead to constitutive activation of phospholipase C (PLC), promoting the expression of proangiogenic downstream targets of the nuclear factor kappa B (NF‐κB) signaling pathway, most importantly angiopoietin‐2 (ANGPT2) 8 . ANGPT2 was shown to be a critical driver of vessel enlargement and distortion that may impact blood flow and permeability in blood vessels comprised of cells expressing GNAQ p.R183Q.…”

“…Postzygotic variations in these genes have been implicated in dysregulated endothelial cell signaling. 7,8 We present a series of five patients with cutaneous CMs, postzygotic GNAQ/11 variants, and early-onset hypertension. We review possible mechanisms to explain the association between the development of CMs and early-onset hypertension.…”

“…The genes GNAQ and GNA11 encode for highly conserved members of the G‐protein subunit alpha‐q family, Gq and G11, respectively. Postzygotic variations in these genes have been implicated in dysregulated endothelial cell signaling 7,8 …”

GNAQ mutations drive port wine birthmark-associated Sturge-Weber syndrome: A review of pathobiology, therapies, and current models

“…Additional in vivo and in vitro complex studies like those performed by Huang et al are needed to advance our understanding of PWB. The authors in this study identified Angpt2 as a downstream factor increased by endothelial GNAQ p.R183Q signaling that caused vessel dilation; this feature was reversed through GNAQ inhibition using YM-254890 or shRNA knockdown of Angpt2 (Huang et al, 2022). Thorough studies that identify direct controllers of PWB vessel dysfunction are rare.…”

Pathogenesis of Port-Wine Stains: Directions for Future Therapies

“…One such study revealed that GNAQ (R183Q) mutations modestly increased the activity of ERK. To further elucidate the underlying mechanism of GNAQ (R183Q) mutations, experiments on these types of mutations were performed in vitro and in vivo [ 52 ]. Endothelial colony-forming cell lines were transfected with lentivirus expressing p.R183Q GNAQ (EC-R183Q).…”