A104-12 Decoding the Earliest Features of Ventilator-Induced Alveolar Epithelial Injury in the Ex Vivo Perfused Human Lung

Abstract Rationale High positive pressure is a major risk factor for ARDS in ventilated patients, yet little is known about the mechanisms of VILI. One obstacle to progress is that biopsies are not performed, so patient lung tissue is not available for study. A second obstacle is that samples like blood and bronchoalveolar lavage (BAL) fluid from one patient with ARDS must be compared to a different patient, making it difficult to disentangle whether molecular differences are from injury- or patient-specific variables. To overcome these obstacles, we are employing ex vivo lung perfusion (EVLP) of paired human lungs from the same donor and applying higher- versus lower-pressure ventilation as the sole variable. Methods We performed EVLP of unclaimed donor human lungs using our well-established model (Maishan et al., JCI Insight, 2025). Both lungs from each donor received identical perfusion and ventilation parameters over 2 hours (including PEEP of 5 cmH2O), except that the PIP was set to 5 cmH2O (lower-pressure ventilation, LPV) for one lung and PIP set to 15 cmH2O (higher-pressure ventilation, HPV) for the other lung. These studies were done with the paired lungs from six different donors. We used % weight gain during perfusion as a measure of edema, BAL total protein concentration as an index of barrier permeability, and RAGE levels in the BAL and in the circulating perfusate as a measure of epithelial barrier injury. Fixed tissue was immunostained for RAGE (AT1 cells), CLAUDIN-4 (epithelial junctions), and KERATIN-8 (AT2 to AT1 differentiation intermediates). Results HPV induced lung injury with significantly greater weight gain (Figure 1A), BAL total protein concentration (Figure 1B), and RAGE in the BAL (Figure 1C) and circulating perfusate (Figure 1D) compared to the donor-matched LPV lung. Immunostaining analysis revealed substantial loss of AT1 cells (Figure 1E, F), significantly reduced CLAUDIN-4 expression (Figure 1E-G), and significantly increased KERATIN-8 expression (Figure 1H-J) in alveoli of injured HPV lung tissue compared to the donor-matched LPV lung. Conclusions HPV in the human lung caused rapid alveolar epithelial barrier disruption and edema formation, along with AT1 cell death, RAGE accumulation in the air spaces and circulation, and compromised CLAUDIN-4 epithelial cell junctions. Despite accumulation of KERATIN-8 positive alveolar epithelial cells after HPV, regeneration of AT1 cells by AT2 cells was limited. These results emphasize that VILI can develop at an early timepoint in human lungs. This abstract is funded by: NIH 1R01HL170601-01A1