Our findings reveal FLRT3, a critical regulator of endothelial barrier function during IRI through the RND3-RhoA pathway, is a potential therapeutic target for pulmonary IRI.

We established both a mouse lung I/R model and a hypoxia/reoxygenation (H/R) cell culture model using human pulmonary microvascular endothelial cells (HPMECs). The effects of FLRT3 manipulation were assessed through lentiviral-mediated overexpression and knockdown approaches. Lung injury was evaluated by histological analysis, immunohistochemistry, and lung injury scoring. Endothelial barrier function was assessed using transmission electron microscopy, Evans blue extravasation, and endothelial permeability assays.

Pulmonary ischemia/reperfusion injury (IRI) causes endothelial barrier dysfunction and increased vascular permeability. Fibronectin leucine-rich transmembrane protein-3 (FLRT3) is known to regulate endothelial cell function, but its role in pulmonary IRI remains unexplored.

FLRT3 expression was predominantly localized in pulmonary endothelial cells and was downregulated following I/R injury. Lentiviral vectors overexpressing FLRT3 (LV-FLRT3, 1 × 109 TU/ml) via tail vein injection before I/R surgery. FLRT3 overexpression effectively protected against lung injury by maintaining vascular integrity and reducing edema formation in I/R-challenged mice. In H/R-treated HPMECs, we identified that FLRT3 protein underwent autophagic-lysosomal degradation. Mechanistically, FLRT3 preserved endothelial barrier function through interaction with Rho family GTPase 3 (RND3), which prevented RhoA pathway-mediated cytoskeletal disruption. FLRT3 overexpression in HPMECs promoted cell migration, maintained cytoskeletal structure, and reduced endothelial hyperpermeability under H/R conditions. Importantly, RND3 knockdown in vivo significantly attenuated FLRT3's protective effects against I/R injury, as evidenced by increased lung injury scores, vascular permeability, and RhoA pathway activation. Conclusions: Our findings reveal FLRT3, a critical regulator of endothelial barrier function during IRI through the RND3-RhoA pathway, is a potential therapeutic target for pulmonary IRI.