ROBO4 triggers pulmonary arterial hypertension through promoting the progressive reduction of pulmonary micro-vessels and the remodeling of distal pulmonary arterioles

Background Pulmonary arterial hypertension (PH) is characterized by increased pulmonary vascular resistance and elevated pulmonary arterial pressure, ultimately leading to heart failure. Pulmonary vascular remodeling is the primary pathological basis of PH, which is closely associated with the remodeling of distal pulmonary arterioles and the progressive reduction of pulmonary micro-vessels. ROBO4, a member of the Roundabout (ROBO) family, is mainly involved in embryonic vascular growth and pathological angiogenesis. Recently, it has been found to be up-regulated in pulmonary vascular endothelial cells based on single-cell sequencing data from patients with PH. Purpose Given that ROBO4 regulates angiogenesis and its potential high expression in the pulmonary vascular endothelium related to PH, we explored whether ROBO4 might play a crucial role in regulating pulmonary vascular remodeling and its underlying mechanism. Results The expression levels of ROBO4 in pulmonary microvascular endothelial cells (PMVECs) and remodeling distal pulmonary arterial endothelial cells (PAECs) from PH patients were higher than those in samples from healthy volunteers. Overexpression of ROBO4 in the pulmonary endothelium of mice induced pulmonary vascular remodeling and PH. Endothelial knockout of ROBO4 alleviated Su/hypo-induced PH in mice. ROBO4 blocks the proliferation, migration, and tubulogenesis of PMVECs as well as the pseudopodia formation of endothelial organoids by directly binding to the Fyn protein and blocking the VEGFR2/p38/CDC42 signaling pathway. Furthermore, ROBO4 also promotes the apoptosis of endothelial cells by directly binding to the Fyn protein and inhibiting its phosphorylation, which in turn inhibits the phosphorylation of NOX4 and induces the release of ROS. Methods Lung samples from PH patients and Su/hypo-induced PH mice were used to analyze the expression of ROBO4. Tie2-Cre-AAV9-Robo4 mice with endothelial expression of ROBO4 were used to investigate the role of ROBO4 overexpression in PH. Cdh5-P2A-CreERT2/Robo4flox/flox mice with endothelial knockout of ROBO4 were used to examine the role of ROBO4 in vivo in PH. Echocardiography, right heart intubation, HE/IHC/EVG/IF staining were used to examine the hemodynamic and histomorphological indices. 3D endothelial organoid models, PMVECs, and PAECs were used to analyze the role of ROBO4 in angiogenesis and apoptosis in vitro. Molecular docking, SPR, Co-IP, Western blot, qRT-PCR, and other methods were used to verify the underlying mechanism. Conclusions These results suggest that ROBO4 is a key inducer in triggering the remodeling of distal pulmonary arterioles and the progressive reduction of pulmonary micro-vessels in PH by mediating VEGFR2/p38/CDC42 and NOX4-associated ROS release.