Ex vivo Lung Perfusion Enhances Donor Lung Preservation in Mice via Hippo Signaling Activation

Abstract 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: (1) without ischemia or EVLP (control), (2) 45 min EVLP followed by 135 min cold ischemia (EVLP-CI), (3) 135 min cold ischemia followed by 45 min EVLP (CI-EVLP), and (4) 180 min cold ischemia (CI). Following 3-hour preservation, changes in lung weight (Δweight) and lung vascular filtration coefficient (Kf) were measured. Complementary in vitro 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 Kf, 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 (Yap1EN-KO or Lats1EN-KO) abrogated EVLP-mediated endothelial barrier protection. EVLP efficacy in lung preservation was enhanced by Yap1 phosphorylation activation using AICAR or metformin. In vitro 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.