Identification of pathological CD133+ endothelial cells in venous malformations

Introduction Venous malformations (VMs) are congenital malformations of the venous system. Histologically, they are composed of dilated vascular channels. Prior studies have demonstrated that CD31 + endothelial cells (ECs) in VMs have pathogenic variants. Recent studies by our group found that the EC progenitor marker, CD133+, was expressed on VM endothelium in patient tissues. We hypothesized that a CD133+ VM endothelial cells contributes to VM pathobiology. Methods VM cells were isolated from resected venous malformation tissues or fluid using CD133 as a marker. Isolated VM populations were characterized by quantative RT-PCR, fluorescence-activated cell sorting (FACS) and immunofluorescence staining (IF) for the expression of progenitor and mature EC genes/proteins. Cells underwent whole exome sequencing (WES) to probe for genetic variants. AKT and ERK activation status was assessed by Western blot and IF, and cell proliferation determined. Isolated CD133+ cells were xenografted in mice and their ability to recapitulate VM phenotype was assessed by histological analysis, IF and colormetric staining. Results CD133+ cells isolated from VMs expressed progenitor and mature EC genes and proteins, and we termed them CD133+ VM endothelial cells (CD133+ VMECs). WES revealed CD133+ VMECs had pathogenic variants and variants of uncertain significance in genes reported in VMs, PIK3CA and TEK . CD133+ VMECs had increase proliferation and a subset had increase nuclear phospho-AKT. When implanted into a xenograft model, CD133+ VMECs with PIK3CA and TEK variants recapitulated clinical VM phenotypes. Conclusion We have identified a novel cell type in VMs, CD133+ VMECs that express EC progenitor proteins, demonstrating incomplete or misdirected differentiation down the EC lineage and are capable of recapitulate the phenotype in a mouse model.