Single-cell sequencing reveal transcriptional cell lineage heterogeneity in extracranial arteriovenous malformation

Extracranial arteriovenous malformations (eAVMs) are rare congenital vascular anomalies consisting of abnormal artery-vein bypass with no intervening capillary network and can lead to disability and death. In this study, an integration of two cutting-edge technologies, single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST), was applied to uncover the underlying pathophysiology of eAVMs. Patient-matched eAVM tissue samples and normal tissue control data were analyzed. We identified different cell states of endothelial cells (ECs), perivascular cells and immune cells in eAVMs and characterized their spatial distributions with multiple immunofluorescence staining (mIF), immunostaining and spatial transcriptomics. Dysregulated cell-to-cell interactions among ECs, perivascular cells and immune cells that are associated with disease progression were cataloged. By performing scRNA-seq on 117,360 individual cells, we identified 14 transcriptomically defined cell lineages from eAVMs. We uncovered the presence of MAFB+ nidus ECs. Moreover, mesenchymal activation was observed in ECs, characterized by high expression of POSTN or FN1 and the upregulation of the NOTCH and TGF-β pathways. mIF and ST verified the location of MAFB+ nidus ECs and mesenchymal activation of eAVM ECs. We identified transcriptional variation within perivascular cells and the presence of smooth muscle–like pericytes in eAVMs. Ligand‒receptor-based cell‒cell interaction analysis indicated that Nd ECs might specifically communicate with perivascular cells through the MDK, VEGF, SEMA3 and ANGPT pathways, and this cellular crosstalk was supported by colocalization analysis of spatial transcriptomics. In addition, we found that hypoxia plays an important role in the development of eAVMs, and hyperbaric oxygen therapy (HBOT) was used to treat one large pelvic eAVM. Thus, correcting the hypoxic microenvironment could be a promising therapeutic strategy.