Schrenk Sandra, Sherpa Chhiring, Bischoff Lindsay J, Cai Yuqi, Boscolo Elisa
Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
bioRxiv. 2025 Jul 24:2025.07.18.665640. doi: 10.1101/2025.07.18.665640.
Venous malformation (VM) are developmental defects of the vasculature characterized by tremendously enlarged and dysfunctional veins. Gain-of-function somatic mutations in the endothelial-specific tyrosine kinase receptor TIE2 have been identified as 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 endothelial cells (EC) to the lesion or by TIE2-mutant EC clonal expansion.
To investigate the contribution of TIE2-mutant EC and wild-type EC to the aberrant venous lumen expansion, we used a xenograft murine model of VM generated with a combination of TIE2-mutant EC and wild-type EC. To perform longitudinal studies, we employed a three-dimensional (3D) fibrin gel lumen formation assay and a migration assay, both using wild-type EC in competition or confrontation with TIE2-mutant EC. To investigate the mechanisms implicated in VM lumen expansion we used RNA-sequencing and short interference (sh)RNA in the TIE2-mutant EC.
We demonstrate here that in the VM xenograft model, the aberrant blood vessels were lined almost exclusively by TIE2-mutant EC, and wild-type EC were rarely found. Functionally, the TIE2-mutant EC exerted a competitive advantage over wild-type EC by inhibiting wild-type EC sprouting. In line with these findings, TIE2-mutant EC promoted repulsion of wild-type EC. ShRNA-mediated silencing of Sema3A or Sema3F in TIE2-mutant EC rescued this chemorepellent phenotype and restored the ability of wild-type EC to migrate, sprout and form lumens. Furthermore, knock-down of Sema3A or 3F in TIE2-mutant EC normalized the blood vessel size in vivo.
Our results demonstrate that wild-type EC are not recruited to the aberrant veins suggesting VM pathogenesis is fueled by clonal expansion of TIE2-mutant EC. Mechanistically, we show that Sema3A and 3F are overexpressed in TIE2-mutant EC and play a crucial role in the pathological vascular lumen expansion in VM.
静脉畸形（VM）是血管的发育缺陷，其特征是静脉极度扩张且功能失调。内皮特异性酪氨酸激酶受体TIE2的功能获得性体细胞突变已被确定为VM发病机制的主要驱动因素。本研究的目的是确定异常静脉腔扩张是由野生型内皮细胞（EC）募集到病变部位引起的，还是由TIE2突变型EC的克隆性扩张引起的。
为了研究TIE2突变型EC和野生型EC对异常静脉腔扩张的作用，我们使用了由TIE2突变型EC和野生型EC组合构建的VM异种移植小鼠模型。为了进行纵向研究，我们采用了三维（3D）纤维蛋白凝胶管腔形成试验和迁移试验，两者均使用野生型EC与TIE2突变型EC竞争或对抗。为了研究与VM管腔扩张相关的机制，我们在TIE2突变型EC中使用了RNA测序和短发夹（sh）RNA。
我们在此证明，在VM异种移植模型中，异常血管几乎完全由TIE2突变型EC构成，很少发现野生型EC。在功能上，TIE2突变型EC通过抑制野生型EC发芽而对野生型EC具有竞争优势。与这些发现一致，TIE2突变型EC促进了对野生型EC的排斥。在TIE2突变型EC中，shRNA介导的Sema3A或Sema3F沉默挽救了这种化学排斥表型，并恢复了野生型EC迁移、发芽和形成管腔的能力。此外，在TIE2突变型EC中敲低Sema3A或3F可使体内血管大小正常化。
我们的结果表明，野生型EC不会募集到异常静脉，这表明VM发病机制是由TIE2突变型EC的克隆性扩张推动的。从机制上讲，我们表明Sema3A和3F在TIE2突变型EC中过表达，并在VM病理性血管腔扩张中起关键作用。
