UNLABELLED

Capillary malformations (CM) are slow-flow vascular abnormalities present at birth and predominantly manifest as cutaneous lesions. In the rare neurocutaneous disorder known as Sturge Weber Syndrome (SWS), individuals exhibit CM not only on the skin but also within the leptomeninges of the brain and the choroid of the eye. >90% of CM are caused by a somatic R183Q mutation in the gene encoding Gαq - a heterotrimeric G-protein subunit. The somatic mutation is notably enriched in endothelial cells (ECs) isolated from CM-affected regions. Here we show blood vessels in cutaneous and leptomeningeal SWS lesions exhibit extravascular fibrin indicating a compromised endothelial barrier. Longitudinal MRI of the brain in one SWS patient further suggests vascular permeability. To explore this pathological phenotype, we employed the trans-endothelial electrical resistance (TEER) assay to measure permeability of the EC-EC barrier . Human EC CRISPR edited to create a R183Q allele (EC-R183Q) exhibited a reduced barrier compared to mock edited EC (EC-WT). We sought to identify signaling molecules needed for EC barrier formation. Knockdown of angiopoietin-2 (ANGPT2), known to be significantly increased in EC-R183Q and in CM, partially yet significantly restored the barrier, while an anti-ANGPT2 function blocking antibody did not. We next tested the MEK1,2 inhibitor (Trametinib) because MAPK signaling is increased by mutation. MEK1,2 inhibitors partially restored the EC barrier, implicating involvement of MAPK/ERK signaling. The combination of ANGPT2 knockdown and Trametinib significantly restored the EC barrier to near EC-WT levels. The additive impacts of ANGPT knockdown and MEK1,2 inhibition indicate the two operate in separate pathways. In summary, we discovered that p.R183Q ECs exhibit compromised endothelial barrier formation, reflecting the compromised EC barrier in CM lesions, and that ANGPT2 knockdown combined with Trametinib effectively restores the EC-EC barrier.

NOVELTY AND SIGNIFICANCE

The mutant Gαq-R183Q in endothelial cells activates phospholipase β3, contributing to increased angiopoietin-2, a pro-angiogenic, proinflammatory molecule that contributes to vascular permeability.Endothelial Gαq-R183Q is sufficient to drive formation of enlarged blood vessels akin to what is observed in CM. ANGPT2 shRNA knockdown prevented the enlarged vessel phenotype in a xenograft model.An EC-specific GNAQ p.R183Q mouse model showed permeability in brain vessels, detected by perfusion of Evans Blue dye, indicating reduced vascular integrity. Reduced vascular integrity in CM is confirmed by Martius Scarlet Blue staining and longitudinal MRI imaging of SWS brain. p.R183Q EC form a weaker endothelial barrier compared to control ECs. The weakened endothelial barrier in the mutant ECscan be rescued by Gαq inhibitor, YM254890, confirming the compromised barrier is a consequence of the mutant Gαq. Titration experiments modeling the mosaic nature of the p.R183Q in CMshow that 5- 10% p.R183Q EC in the monolayer is sufficient to reduce endothelial barrier formation. Knockdown of ANGPT2 or MEK1,2 inhibition partially restored the endothelial barrier in p.R183Q EC. Combining knockdown of ANGPT2 and addition of a MEK inhibitor, Trametinib, restored the endothelial barrier to near what is seen in wild type ECs. Sturge Weber Syndrome (SWS) is a neurocutaneous disorder that involves atypical blood vessel overgrowth in the skin, brain and eye. It is associated with facial CM (aka port wine birthmark), leptomeningeal CM in the brain visible with MRI, and glaucoma. Theneurological sequalae involve seizures, cerebral atrophies and calcification, and intellectual disorders. Currently there are no molecularly targeted therapies for non-syndromic CM or SWS. Our study shows the involvement of MAPK pathway and the proinflammatory molecule ANGPT2 in endothelial permeability and suggests a path to target p.R183Q driven CM.