Abstract Rationale Acute respiratory distress syndrome (ARDS) and ventilator induced lung injury (VILI) share key pathological features, including pulmonary microvascular endothelial dysfunction and excessive inflammation. N6 methyladenosine (m6A) RNA methylation has emerged as a critical regulator of cellular stress responses during ARDS/VILI, however, its functional role and regulatory mechanisms in lung endothelium remain undefined. Methods Single-cell RNA sequencing (scRNA-seq) was performed on lungs from LPS challenged mice (2 mg/kg, intratracheal). In vitro studies utilized endothelial stimulation with LPS or 18% cyclic stretch, endothelial barrier measurement (ECIS), m6A dot blot, Western blot, nuclear cytoplasmic fractionation, and mass spectrometry for modification site identification. Functional validation included the METTL3 inhibitor STM2457 and endothelium specific METTL3 knockout mice (Mettl3fl/fl × Tie2-Cre). Lung injury was assessed by BAL cell count, protein, cytokine levels, and histological scoring. VILI was modeled by high tidal volume ventilation (40 mL/kg, 4 h). Results scRNA-seq revealed that Mettl3 is selectively upregulated in pulmonary endothelial cells following LPS challenge. At the cellular level, LPS enhanced METTL3 expression, promoted its nuclear translocation, increased global m6A methylation, and disrupted endothelial barrier integrity. Both METTL3 siRNA and STM2457 suppressed LPS or stretch induced m6A elevation and preserved barrier function. In vivo, STM2457 significantly attenuated VILI induced increases in BAL protein, cell counts, and inflammatory cytokines. Mass spectrometry identified phosphorylation at S43 of METTL3, a potential ERK target, suggesting that mechanical stress stabilizes METTL3 through the ERK-METTL3 signaling axis. Endothelial specific METTL3 deletion markedly reduced LPS induced lung inflammation, histological injury, and cytokine production. Conclusion Endothelial METTL3 acts as a central integrator of inflammatory and mechanical signals, amplifying m6A-dependent proinflammatory responses that drive endothelial dysfunction in ARDS/VILI. Mechanical stress induced phosphorylation of METTL3 at S43 enhances its stability and nuclear localization, facilitating inflammation amplification. Both genetic deletion and pharmacological inhibition of METTL3 mitigate lung injury, identifying METTL3 as a promising upstream therapeutic target for ARDS/VILI. This abstract is funded by: NIH
Dong et al. (Fri,) studied this question.