Liquid-liquid phase separation of EphB4 drives pulmonary hypertension via YAP activation

Vascular extracellular matrix stiffening is an early and pervasive driver of pulmonary hypertension (PH), though its mechanistic links to pathological cellular responses remain unclear. This study identifies Eph receptor B4 (EphB4), a receptor tyrosine kinase, as a key mediator of stiffness-induced responses in pulmonary artery smooth muscle cells (PASMCs). EphB4 was significantly upregulated in PH, and its smooth muscle-specific deficiency alleviated PH in rats. Matrix stiffening promoted conformational extension of the EphB4 C-terminal intrinsically disordered region (IDR), inducing its liquid-liquid phase separation (LLPS) into biomolecular condensates. These condensates sequestered Yes-associated protein (YAP)-regulating proteins, including ANXA2 and YWHA, disrupting YAP cytoplasmic retention and promoting its nuclear translocation to drive PASMC proliferation. Targeting the IDR of EphB4 with a retro-reversed peptide inhibited LLPS. Finally, delivering this peptide via VAPG-modified nanoparticles effectively attenuated PH progression. Our work establishes a novel EphB4-ANXA2/YWHA-YAP axis and highlights EphB4 phase separation as a promising therapeutic target for PH.