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

BackgroundHereditary 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. MethodsWe 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. ResultsLoss 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. ConclusionOur 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. Clinical PerspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIUsing endothelial-specific Alk1 knockout mouse models and single-cell transcriptomics, we identified a novel angiogenic endothelial cell population as a key driver of brain AVM formation. C_LIO_LIThis angiogenic EC cluster shares molecular features with human bAVM cells, including high expression of KIT, which we identified as a new direct transcriptional target of BMP9-ALK1 signaling. C_LIO_LIPharmacological inhibition of KIT using small molecules or blocking antibodies effectively prevents AVM formation in vivo, establishing KIT as a promising therapeutic target. C_LI What are the clinical implications?O_LIOur findings support the therapeutic potential of targeting KIT with FDA-approved drugs such as Imatinib to treat HHT2-associated brain AVMs, offering a non-invasive alternative to surgical intervention. C_LIO_LICharacterizing AVM-specific endothelial subtypes may enable the development of targeted and personalized therapies, improving patient outcomes and minimizing treatment-associated risks in HHT. C_LI