Canonical and noncanonical signaling. GTP-bound Gαq can exert canonical signaling through phospholipase C-β (PLCβ) (71). Phosphorylation of PLCβ catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 can then activate its receptor on the endoplasmic reticulum, stimulating the release of calcium into the cytoplasm. The concomitant production of DAG with the release of calcium leads to the activation of PKC. PKC can in turn activate RAS, a GTPase that can promote recruitment of RAF kinase on the membrane. Activated RAF acts on MEK, which phosphorylates the kinases ERK1 and -2 (Figure 2). Additional MEK effectors are c-Jun, JNK, and p38 kinase (72).

Mutant GNAQ p.R183Q signaling in ECs. Schematic of the molecular pathways involved in CM. The GNAQ activating mutation p.R183Q promotes disassembly of the heterotrimeric complex subunit αq from the β and γ subunits, impairing the hydrolysis of GTP to GDP. The Gαq bound to GTP promotes phospholipase C-β3 (PLCβ3) signaling, which catalyzes the conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol trisphosphate (IP3) and diacylglycerol (DAG). DAG leads to the activation of protein kinase C (PKC), which can in turn induce RAF (rapidly accelerated fibrosarcoma) kinase activation and MEK, which phosphorylates ERK1 and -2, promoting translocation into the nucleus and gene transcription. PKC can also induce NF-κB translocation into the nucleus, inducing ANGPT2 expression to promote proangiogenic signaling. The IP3 metabolite can activate its receptor, IP3R, on the endoplasmic reticulum, stimulating the release of calcium into the cytoplasm.

In ECs expressing GNAQ p.R183Q, PLCβ3 is constitutively activated by phosphorylation at Ser537 (60). Although increased MEK and ERK1/2 phosphorylation was documented in SWS patient tissue biopsies (73), the activation of the MAPK/ERK pathway in cultured ECs with the p.R183Q mutation is mild compared with ECs expressing GNAQ p.Q209L (19, 36, 54). Furthermore, the GNAQ p.R183Q substitution does not activate p38 or JNK in the same way that p.Q209L does (19, 54). The moderate activation of ERK, and differential effect on p38 and JNK pathways in ECs during fetal development, may explain the CM phenotype, as opposed to a vascular tumor.

Parallel to the canonical PLCβ-mediated signaling axis, Gαq has been shown to control noncanonical signaling through members of the Trio family of guanine nucleotide exchange factors for Rho, such as Trio, Kalirin, p63RhoGEF (74–76), and G protein receptor kinase 2 (GRK2), which bind to activated Gαq with high affinity (77, 78). In melanocytes expressing GNAQ p.Q209L, the activation of Trio leads to the phosphorylation of FAK, promoting the aberrant activation of YAP and PI3K pathways to drive tumor growth (79–83). To date, the activation of the noncanonical pathways, including YAP and PI3K, has not been established in ECs with hyperactive mutant GNAQ.

Calcium signaling. The role of mutant Gαq protein family members in calcium signaling is highlighted by the presence of inherited germline GNA11 mutations in patients with hypoparathyroidism and hypocalciuric hypercalcemia. Different types of amino acid substitution in GNA11 have been shown to contribute to the altered sensitivity of cells to extracellular calcium levels (84). In particular, loss-of-function GNA11 mutations lead to elevated serum calcium concentrations, while gain-of-function mutations result in the opposite calcium signaling phenotype, which consists of low levels of calcium in the serum. Mice with biallelic germline deletion of Gna11 and parathyroid-specific loss of Gnaq alleles also developed hypercalcemia, strongly implicating GNAQ/11 signaling in extracellular calcium homeostasis in both humans and mice (85). Molecular analysis of the mutation GNA11 p.R60L, which was identified in a patient with hypoparathyroidism, revealed it is less activating than GNA11 p.Q209L (86). This supports the hypothesis that GNA11 p.R60L leads to a less severe phenotype that is compatible with life when expressed in the germline compared with substitutions at p.Q209 and most likely at p.R183.

It is important to note that approximately 50% of patients with hypocalcemia type 1 are affected by seizures, which is a phenotypic manifestation of SWS (84). In most of these patients, seizures were associated with ectopic and basal ganglia calcifications. Furthermore, serum hypocalcemia has been associated with seizures, as low calcium concentrations in the cerebrospinal fluid can lead to increased excitability in the central nervous system (87).

Brain cortical calcifications have been reported in the majority of patients with SWS, as well as in other vascular anomalies such as cerebral cavernous malformation (CCM) and in genetic disorders affecting the RAS/MAPK pathway such as neurofibromatosis (88, 89).

In past years, conflicting reports made it unclear whether the calcifications in SWS patients are localized in the walls of the angiomatous vessels or are free parenchymal deposits within the superficial or deep cortex (90, 91). A recent study by Knöpfel and colleagues revealed that SWS patients have an abnormal calcium metabolic profile resulting in hypocalcemia (92), which correlated with neurological symptoms. Histopathological analysis of tissue biopsies determined that the calcium deposits were mostly in the vessel wall of cortical capillaries and small venules.

Furthermore, Zecchin and colleagues determined that expression of the GNAQ or GNA11 p.R183Q mutation in human ECs increased the GPCR-induced intracellular calcium signaling, which is potentiated by the calcium release–activated calcium (CRAC) channels (93). Treatment with a short interfering RNA (siRNA) targeting the mutant allele or with a CRAC channel inhibitor promoted rescue of the increased calcium signaling.

Combined, these findings suggest that the progressive mineral deposition in the microvasculature contributes to the abnormal cerebral perfusion and neurological symptoms of SWS.

Angiogenesis pathway signaling. G-protein noncanonical functions include RTK signaling. VEGF-A signaling is a well-known regulator of EC mitogenic responses and promotes vascular migration and permeability by binding to its receptor KDR (kinase insert domain receptor). KDR was shown to be overexpressed in CM vessels, suggesting increased pathway activation that could contribute to the proangiogenic phenotype (94). Gαq/11 proteins can play a role in VEGF-induced EC migration by promoting KDR-mediated RhoA and Rac1 activation. Upon VEGF stimulation, KDR can form a rapid but transient complex with Gaq/11 (95). Next, upon Gαq/11 activation, the release of free Gβγ subunits can phosphorylate PLC and subsequently PKC, which in turn, leads to RhoA activation (96). Interaction between Gαq/11 proteins and KDR has also been shown to promote EC proliferation by mediating concomitant MAPK activation and intracellular calcium mobilization. While KDR activation by VEGF-A can promote Gαq/11 signaling, the stimulation of the bradykinin B2 receptor, a GPCR coupled with Gαq/11, induces tyrosine phosphorylation of KDR, and can promote increased endothelial nitric oxide (NO) synthase activity, which is a known regulator of vasodilation (97). This body of literature documents the important role of Gαq signaling during vascular development and supports the notion that excessive or reduced Gαq signaling leads to vascular abnormalities.

Angiogenesis and vascular permeability are also mediated by angiopoietin-2 (ANGPT2) (98, 99). ANGPT2 is expressed at low levels in quiescent ECs and is increased during angiogenesis and in response to inflammatory mediators, which leads to permeable and destabilized blood vessels. Although the precise molecular regulation of ANGPT2 by Gαq/11 is not known, several studies showed the overexpression of ANGPT2 in ECs expressing hyperactive mutant GNAQ (28, 60, 65). Knockdown of ANGPT2 in GNAQ-mutant (R183Q) ECs reduced blood vessel diameter in a xenograft model of CM, indicating an important role for ANGPT2 in the dilated vascular phenotype. Importantly, ANGPT2 has also been implicated in several other vascular anomalies such as arteriovenous malformations (AVMs) (100), CCM (101), and kaposiform lymphangiomatosis (KLA) (102, 103). Combined, these studies establish ANGPT2 as a promising potential common target for these vascular diseases.

ANGPT2 is also overexpressed during inflammation (104). Inflammatory markers such as NF-κB, IL-1β, and E-selectin were reported to be increased in ECs expressing GNAQ p.R183Q or p.Q209L (28, 60, 65). This suggests that EC-autonomous inflammatory processes are a common feature of ECs expressing hyperactive GNAQ and may in turn regulate ANGPT2 expression. Although different GNAQ mutations such as p.R183Q and p.Q209L are associated with different classes of vascular anomalies (as CM and vascular tumors, respectively), recent studies highlighted that the transcriptional consequences of these mutations in ECs are similar and include upregulation of pathways such as MAPK, angiogenesis, inflammation, and upregulation of ANGPT2 (28, 60, 65). This would suggest that the differences between hyperactive GNAQ mutation types may affect the level of activation/expression rather than the specific downstream targets (28).

Finally, while the precise mediators of NF-κB activation in the context of CM are not known, one interesting possibility is that it can be activated by shear stress upon Gαq activation by mechanosensing GPCRs (105).

Mechanosensing. ECs can sense hemodynamic forces in a process termed mechanosensing, which is indispensable for vascular function. Disruptions in hemodynamics or in EC mechanosensing could significantly contribute to blood vessel dilation and/or CM lesion formation. Gαq/11-coupled GPCRs (GqPCRs) can participate in the mediation of mechanochemical signaling by (a) direct activation of PI3K/AKT, which can promote vasodilation via NO synthesis; (b) via PIP2, DAG, and IP3 metabolites that can modulate ion channels; and (c) by inducing membrane hyperpolarization.

Shear stress can activate bradykinin 2 (B2) receptors (106), leading to an increase in intracellular calcium levels that promote increased NO production or membrane hyperpolarization (107, 108). G protein–coupled receptor 68 (GPR68) and histamine H1 receptor (H1R) can instead mediate flow-induced vasodilation by enhancing NO production (109, 110).

GqPCR-initiated changes in intracellular metabolite levels can signal via EC-specific ion channels such as TRPV4, TREK-1, and Kir2.1 (111). Endothelial PIEZO1 is a mechanosensory ion channel activated by laminar flow. PIEZO1 can influence GqPCR activity and subsequent NO production by the release of ATP (105). Recent studies have implicated PIEZO1 in endothelial calcium signaling in the brain microvasculature (112). Therefore, dysregulation of PIEZO1 may be involved in CM vessel blood flow abnormalities and in vascular calcifications identified in SWS patients.

Studies on the effects of the GNAQ mutations in EC mechanosensors and mechanotransductors could reveal important associations and dysfunctions. Devising in vitro microfluidic systems with the use of GNAQ p.Q183R mutant ECs subjected to different types of flow dynamics and shear stress could advance our understanding of the regulation of the mechanosensing machinery in CM.