TIE2 Interacts with Major Vault Protein and Modulates Angiogenic Function in Human Retinal Microvascular Endothelial...

Human retinal microvascular endothelial cell (HRMEC) dysfunction contributes to retinal vascular diseases. This study aimed to elucidate the molecular role of TIE2 in HRMEC angiogenesis and identify its intracellular regulatory mechanisms.

TIE2 knockdown HRMECs were established using lentiviral shRNA. Proliferation was assessed by CCK-8 assay, angiogenesis by Matrigel tube formation, apoptosis by flow cytometry, and transcriptome by RNA sequencing. TIE2-major vault protein (MVP) interaction was validated by co-immunoprecipitation and immunofluorescence. PI3K/AKT pathway involvement was examined using a specific inhibitor.

TIE2 knockdown significantly suppressed HRMEC proliferation (A450 nm; 24h: 0.97 ± 0.04, 0.98 ± 0.03, 0.86 ± 0.03, p > 0.05; 48h: 1.87 ± 0.16, 1.86 ± 0.04, 1.57 ± 0.02, p < 0.0001; 72h: 2.14 ± 0.05, 2.15 ± 0.04, 1.90 ± 0.04, p < 0.001) and tube formation (total number of tubes: 10.00 ± 1.00, 9.67 ± 0.58, 6.33 ± 0.58, p < 0.01) while promoting apoptosis (%: 3.55 ± 0.16, 3.96 ± 0.37, 7.25 ± 1.13, p < 0.01). Transcriptomic analysis revealed extensive gene expression changes associated with TIE2 knockdown, with enrichment in pathways related to ion channel activity, neuroactive ligand-receptor interaction, and calcium signaling. Co-immunoprecipitation identified MVP as a TIE2-interacting protein, while immunofluorescence provided supportive evidence for a partially overlapping distribution of detectable TIE2 and MVP signals in control HRMECs. Functionally, MVP knockdown attenuated TIE2 agonist-induced angiogenesis (total number of tubes: 22.67 ± 1.16 vs. 18.33 ± 0.58, p < 0.0001), whereas MVP overexpression partially rescued angiogenic defects caused by TIE2 knockdown (9.00 ± 1.00 vs. 20.33 ± 2.08, p < 0.0001). These effects were dependent on activation of the PI3K/AKT signaling pathway (total number of tubes: 28.33 ± 0.58 vs. 23.33 ± 1.53, p < 0.0001).

TIE2 is an important regulator of HRMEC angiogenic function. Our findings suggest that TIE2 interacts with MVP and modulates angiogenic responses, at least in part through PI3K/AKT signaling. These results provide mechanistic insight into retinal endothelial biology and identify the TIE2-MVP axis as a potential therapeutic target in retinal vascular diseases.