<i>Ex vivo</i> lung perfusion enhances donor lung preservation in mice <i>via</i> Hippo signaling activation

Ex vivo lung perfusion (EVLP) is a promising technique that allows organ preservation and repair, while the molecular mechanisms remain unknown. This study aimed to establish a translational murine EVLP model and to unveil the molecular mechanisms responsible for EVLP beneficial effects. We developed a murine EVLP system with four experimental groups: (<i>1</i>) without ischemia or EVLP (control), (<i>2</i>) 45 min EVLP followed by 135 min cold ischemia (EVLP-CI), (<i>3</i>) 135 min cold ischemia followed by 45 min EVLP (CI-EVLP), and (<i>4</i>) 180 min cold ischemia (CI). Following 3-hour preservation, changes in lung weight (Δweight) and lung vascular filtration coefficient (<i>K</i>_f) were measured. Complementary <i>in vitro</i> studies utilized human pulmonary microvascular endothelial cells under simulated perfusion conditions. Compared to CI group, both EVLP intervention groups exhibited superior preservation outcomes, with an attenuated Δweight and <i>K</i>_f, and histological and microscopic evidence of lung damage. Proteomic profiling on mouse lungs revealed that EVLP regulated the Hippo signaling in response to CI. Pharmacological inhibition (TDI-011536 or Lats-IN-1) or genetic deletion of Yap1 or Lats1 specifically in endothelial cells (<i>Yap1</i>^EN-KO or <i>Lats1</i>^EN-KO) abrogated EVLP-mediated endothelial barrier protection. EVLP efficacy in lung preservation was enhanced by Yap1 phosphorylation activation using AICAR or metformin. <i>In vitro</i> perfusion models recapitulated these findings, where barrier function was disrupted with Yap1 phosphorylation inhibitor, with a decreased cytoplasmic localization of Yap1. Our findings establish the functional murine EVLP model and first demonstrate that mechanical perfusion preserves donor lung viability through Hippo signaling-mediated endothelial barrier stabilization.