Drape, E.; Carrier, L.; Cagnone, G.; Drissi Touzani Walali, H.; Bizou, M.; Van Vliet, P. P.; Andelfinger, G.; Larrivee, B.; Dubrac, A.

Background: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is a genetic disorder caused by mutations in the ALK1 (ACVRL1) gene, encoding a receptor for Bone Morphogenetic Proteins 9 and 10 (BMP9/BMP10). HHT2 patients frequently develop brain arteriovenous malformations (bAVMs), which are abnormal connections between arteries and veins. Currently, surgical resection is the only treatment, associated with significant risks and complications. Despite evidence suggesting endothelial cell (EC) heterogeneity in bAVMs, it remains poorly characterized, limiting our ability to identify new therapeutic avenues. Methods: We employed endothelial cell-specific and inducible Alk1 knockout mice (Alk1iECKO) with tamoxifen-induced deletion at postnatal day 6 (P6). We separately analyzed the P8 perineural (PNVP) and intraneural (INVP) vascular plexuses, which differ in vessel composition and flow dynamics. Single-cell RNA sequencing (scRNAseq) was performed to characterize EC heterogeneity and identify transcriptomic changes in both vascular plexuses of mutant versus wild type mice. Results: Loss of endothelial ALK1 signaling triggered bAVM formation predominantly in the PNVP vascular network. scRNAseq revealed that Alk1 deletion promoted brain capillaries differentiation into angiogenic-1 ECs, whereas it drove PNVP venules EC proliferation and the emergence of the unique angiogenic-2 cluster. The latter shares transcriptomic features with human AVM ECs, including angiogenic tip cell markers and a strong glycolytic signature. Among its defining markers, Kit emerged as a direct downstream target of BMP9-ALK1 signaling. Pharmacological KIT inhibition using Masitinib, Imatinib, or KIT-blocking antibodies prevented bAVM formation in Alk1iECKO mice. Conclusion: Our study uncovers a previously unrecognized EC population, the angiogenic-2 cluster, as a key contributor to bAVM development. We identify Kit as a central regulator of this cluster, establishing it as a promising therapeutic target for preventing bAVMs in HHT2.