Loss of endothelial ALK1 signaling induces the emergence of a KIT+ angiogenic endothelial cluster driving brain arteriovenous malformations II

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), 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 perineural (PNVP) and intraneural (INVP) vascular plexuses, which differ in vessel composition and flow dynamics, at P8. Single-cell RNA sequencing (scRNAseq) was performed to characterize EC heterogeneity and identify transcriptomic changes in both vascular plexus of mutant mice. Results Loss of endothelial ALK1 signaling triggered bAVM formation predominantly in 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.