Identification of a Pathological CD133+ Endothelial Cells in Venous Malformations

Abstract

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.