Abstract
Background:
Venous malformations (VMs) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of-function somatic mutations in TIE2 (endothelial tyrosine kinase receptor) have been identified as the leading driver of VM pathogenesis. The aim of this study was to determine whether the aberrant venous lumen expansion is caused by recruitment of wild-type (WT) endothelial cells (EC) to the lesion or by TIE2-mutant EC clonal expansion.
Methods:
To investigate the contribution of TIE2-mutant EC and WT EC to the aberrant venous lumen expansion, we used a xenograft murine model of VM generated with a combination of TIE2-mutant EC and WT EC. To perform longitudinal studies, we used a 3-dimensional fibrin gel lumen formation assay and a migration assay, both using WT EC in competition or confrontation with TIE2-mutant EC. To investigate the mechanisms implicated in VM lumen expansion, we used RNA-sequencing and short-hairpin RNA silencing in the TIE2-mutant EC.
Results:
We demonstrate here that in the VM xenograft model, the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, and WT EC were rarely found. Functionally, the TIE2-mutant EC exerted a competitive advantage over WT EC by inhibiting WT EC sprouting. In line with these findings, TIE2-mutant EC promoted repulsion of WT EC. Short-hairpin RNA-mediated silencing of Sema (Semaphorin) 3A or Sema3F in TIE2-mutant EC rescued this chemorepellent phenotype and restored the ability of WT EC to migrate, sprout, and form lumens. Furthermore, knockdown of Sema3A or Sema3F in TIE2-mutant EC normalized the blood vessel size in vivo.
Conclusions:
Our results demonstrate that WT EC are not recruited to the aberrant veins, suggesting that VM pathogenesis is fueled by clonal expansion of TIE2-mutant EC. Mechanistically, we show that Sema3A and Sema3F are overexpressed in TIE2-mutant EC and play a crucial role in the pathological vascular lumen expansion in VM.