Ribosomal protein L36-mediated selective loading of microRNA-4432 into extracellular vesicles contributes to perivascular cell dysfunction in venous malformations

BACKGROUND: Venous malformations (VMs), predominantly arising from activating mutations of TIE2 in endothelial cells (ECs), are characterized by dilated and tortuous vessels with a paucity of perivascular cells (PCs). The mechanisms of interaction between mutant ECs and PCs remain largely elusive. OBJECTIVES: To investigate the characteristics of extracellular vesicles (EVs) from VM ECs, especially the microRNAs (miRNAs) carried and their roles in crosstalk between ECs and PCs in VM pathogenesis. METHODS: miRNA profiles of human umbilical vein endothelial cells overexpressing TIE2L914F (L914F cells) and TIE2WT [wildtype (WT) cells], along with their EVs, were analysed by RNA sequencing. In vitro studies using umbilical cord stem cells (UCSCs) were done to carry out functional assays of VM EVs and their enriched miRNA-4432 (miR-4432). miRNA pulldown and RNA interference techniques were used to identify the sorting regulator of miR-4432 into VM EVs. RESULTS: RNA secretion was upregulated in L914F EVs vs. WT EVs. miRNA sequencing revealed a distinct profile of L914F EVs vs. L914F cells, WT cells and WT EVs, identifying miR-4432 as being preferentially encapsulated in EVs from L914F cells. Functional assays demonstrated that VM EVs and EV-carried miR-4432 inhibited the differentiation, adhesion and proliferation of UCSCs. Furthermore, ribosomal protein L36 (RPL36) was identified as an RNA binding protein and sorting regulator of miR-4432 during the EV secretion process in L914F cells. CONCLUSIONS: This study, for the first time, identified an interaction between VM ECs and PCs via EVs, and offers valuable data on the miRNA profiles of VM ECs and normal ECs, along with their EVs. Our findings suggest that the RPL36-mediated selective loading of miR-4432 into EVs may contribute to the aberrant PC coverage in VMs, providing novel insights into VM pathogenesis and potential treatment strategies.