Abstract Rationale Acute respiratory distress syndrome (ARDS) remains a major cause of ICU mortality, often aggravated by ventilator-induced lung injury (VILI). Sphingosine-1-phosphate receptor 3 (S1PR3) is upregulated by mechanical stress and contributes to endothelial barrier disruption in ARDS/VILI. However, the post-transcriptional mechanisms regulating S1PR3 remain poorly understood. This study investigates the role of m6A RNA methylation, mediated by METTL3, in controlling S1PR3 expression and its impact on endothelial mitochondrial homeostasis during ARDS/VILI. Methods Human pulmonary artery endothelial cells (HPAECs) were transfected with adenoviral S1PR3. Mitochondrial structure (TOMM20 immunofluorescence), reactive oxygen species (MitoSOX), membrane potential (TMRM), and cellular respiration (Seahorse XF) were evaluated. RhoA activation was measured and inhibited using Fasudil. S1PR3 mRNA levels, stability, and m6A methylation were assessed by RT-PCR and MeRIP-seq. METTL3 was inhibited pharmacologically (STM-2457) or genetically via endothelial-specific Mettl3 knockout mice. For in vivo studies, endothelial S1PR3 was deleted using CRISPR/Cas9 plasmid delivery (JetPEI), followed by high tidal volume ventilation (30 mL/kg, 4 h) to induce VILI. Lung injury was evaluated by bronchoalveolar lavage (BAL) cell counts, protein leakage, cytokine levels, and histopathology. Results S1PR3 overexpression in endothelial cells caused mitochondrial fragmentation, increased ROS generation, decreased membrane potential, and reduced oxidative phosphorylation efficiency. These effects were accompanied by enhanced RhoA activation, which was reversed by Fasudil treatment. LPS exposure increased S1PR3 mRNA stability and m6A methylation. Single-cell RNA sequencing of LPS-challenged mouse lungs revealed co-upregulation of Mettl3 and S1PR3 in endothelium. METTL3 inhibition or knockout suppressed S1PR3 expression and protected against LPS-induced lung injury, evidenced by reduced BAL cellularity, protein levels, cytokines (IL-6, TNF-α), and histological injury. Similarly, endothelial S1PR3 knockout markedly attenuated VILI-associated lung inflammation. Conclusions This study identifies a novel METTL3-m6A-S1PR3 axis driving mitochondrial dysfunction and endothelial injury in VILI. METTL3-mediated m6A methylation stabilizes S1PR3 mRNA, promoting RhoA/ROCK-dependent mitochondrial fission and bioenergetic impairment. Targeting either METTL3 or S1PR3 offers a promising therapeutic strategy for mitigating VILI and ARDS. This abstract is funded by: National Institutes of Health (NIH), United States
Liang et al. (Fri,) studied this question.
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