SnRNA-seq reveals cellular heterogeneity and proliferation mechanisms in limb venous malformations.

backgroundVenous malformations (VMs) are congenital vascular anomalies characterized by abnormal vascular proliferation, with limb VMs often leading to functional impairment and physical discomfort. However, the cellular heterogeneity and underlying molecular mechanisms driving pathological proliferation in limb VMs remain incompletely elucidated.

methodsIn this study, we collected 10 tissue samples including specimens from 5 limb VM patients and 5 normal control tissues and performed single-nucleus RNA sequencing (snRNA-seq) to comprehensively map the cellular landscape of VMs. We first identified distinct cell subpopulations covering vascular endothelial cells, vascular smooth muscle cells, and immune cells, and compared gene expression levels between VM and normal tissues. Afterward, we conducted weighted gene co-expression network analysis (hdWGCNA) and constructed the protein-protein interaction (PPI) network to screen critical proliferation-related genes in VMs. We further analyzed the signaling pathways associated with these candidate genes and carried out subsequent functional validation experiments to explore the biological role of core gene TEK in proliferative vascular endothelial cells (PVECs). Besides, we also analyzed the characteristic pathways of the PVEC subpopulation to clarify its proliferation-related molecular features.

resultsWe found obvious expression differences of genes in various cell subpopulations between VM and normal tissues. Three genes, namely tyrosine protein kinase receptor (TEK), Fms-like tyrosine kinase 1 (FLT1), and EGF-like domain multiple 7 (EGFL7), were identified as key proliferation-related genes with significant upregulation in VM lesions, and these three genes were closely associated with the activation of PI3K/AKT/mTOR, IL6/JAK/STAT3, and TNF-α/NF-κB pathways. Functional experimental results showed that TEK knockdown could significantly inhibit proliferative vascular endothelial cell (PVEC) proliferation and promote cell apoptosis, and reverse the abnormal activation of inflammatory pathways. As a vital pathogenic cell subpopulation, PVECs facilitated abnormal vascular proliferation through activating pathways including the G2/M checkpoint and E2F targets.

conclusionsCollectively, our study systematically elucidated the cellular heterogeneity framework and proliferation mechanisms of limb VMs, identifying TEK, FLT1, and EGFL7 as key regulators of pathological proliferation. These findings provide new insights into the pathogenesis of VMs and lay a foundation for developing precise therapeutic strategies targeting proliferation-related pathways.