Abstract:

Objective To investigate the effects of TEK receptor tyrosine kinase (Tie2) L914F mutation on the biological behavior of vascular endothelial cells and the changes of related signaling pathways in patients with venous malformations. Methods Gene sequencing was used to detect Tie2-L914F mutations in venous malformations. HE staining and immunohistochemical staining were used to detect the expression of platelet derived growth factor subunit B gene (PDGFB) and α-smooth muscle actin (α-SMA) in venous malformations caused by the mutation. Lentivirus overexpressing Tie2-wild type (Tie2-WT), Tie2-L914F and Tie2-GFP (green fluorescent protein, GFP) infected human umbilical vein endothelial cells (HUVECs). Real-time fluorescence quantitative PCR was used to detect the expression level of Tie2 in endothelial cells expressing exogenous Tie2 and Flag-tagged protein Tie2 protein was detected by western blotting to verify transfection efficiency. The proliferation, apoptosis, migration and tube-forming ability of the cells were determined by CCK-8, flow cytometry, Transwell migration assay and Matrigel matrix gel tube-forming assay. Western blotting was used to detect the expression levels of Protein Kinase B (PKB/AKT), FOXO1 and their phosphorylation, and the expression level of PDGFB was detected by ELISA. Results The number of patients with venous malformations with L914F mutations was about 33.3%. HE and immunohistochemical staining showed that the expressions of PDGFB and α-SMA were significantly down-regulated in venous malformation tissues with Tie2-L914F mutation, and were positively correlated with the decrease in cell coverage of the tube wall. Compared with Tie2-WT endothelial cells, the apoptosis number of Tie2-L914F endothelial cells was significantly reduced, while the proliferation and migration ability was significantly increased, and the tube-forming ability was significantly decreased. Western blotting and ELISA showed that the phosphorylation levels of AKT and FOXO1 downstream of Tie2 signaling pathway in endothelial cells expressing Tie2-L914F were significantly increased, and the expression level of PDGFB was significantly decreased. Conclusion In venous malformations, Tie2-L914F mutation may downregulate the expression of PDGFB through AKT signaling pathway, which affects the biological behavior of vascular endothelial cells.

Key words: venous malformation, TEK receptor tyrosine kinase (Tie2), AKT pathway, Tie2-L914F mutations