Ninjurin2 Promotes Pulmonary Endothelial Barrier Dysfunction Through VEGF/VEGFR2 Signaling Pathway

Previous studies have established an association between Ninjurin2 and diseases related to vascular endothelial dysfunction, such as ischemic stroke and coronary heart disease. Given that impaired pulmonary endothelial barrier function is a critical feature of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), we hypothesized that Ninjurin2 may also participate in the regulation of lung vascular permeability. Therefore, this study investigates the role of Ninjurin2 in regulating endothelial cell function and dysfunction, aiming to elucidate the underlying mechanisms in ALI and ARDS. This study utilized both animal models and in vitro experiments to investigate the role of Ninjurin2 in regulating endothelial cell function and dysfunction. Endothelial-specific Ninjurin2 overexpressing (NINJ2-TG^EC) mice were generated and compared with control mice (NINJ2^flox/flox). To assess vascular permeability across various organs, we employed Evans Blue dye extravasation assays. At the cellular level, fluorescein isothiocyanate-dextran (FITC-dextran) flux assays were utilized to evaluate endothelial barrier integrity. Additionally, co-immunoprecipitation (Co-IP) and Western blotting were performed to investigate the interaction between Ninjurin2 and VEGFR2 and to analyze the activation of the VEGF/VEGFR2 signaling pathway. NINJ2-TG^EC mice developed severe lung injury with significantly increased vascular permeability in multiple organs. Overexpression of Ninjurin2 inhibited VE-cadherin and ZO-1 expression, leading to disruption of endothelial adherens and tight junctions and increased endothelial permeability in vivo and in vitro. Ninjurin2 bound directly to VEGFR2 and activated VEGFR2/AKT signaling and enhanced VEGF165-induced barrier dysfunction. Pharmacological inhibition or genetic silencing of VEGFR2 reversed Ninjurin2-mediated vascular leakage and pulmonary pathology. Furthermore, Ninjurin2 overexpression induced ROS accumulation, AKT/NF-κB/ICAM-1 activation, and monocyte-endothelial adhesion. These data establish that Ninjurin2 compromises endothelial barrier integrity and promotes vascular inflammation through a VEGFR2-dependent mechanism. Ninjurin2 exacerbates vascular endothelial hyperpermeability, particularly in the lungs, by interacting with VEGFR2 and activating the VEGF/VEGFR2 signaling pathway. The results suggest that Ninjurin2 could be a useful therapeutic target for ALI and ARDS.