Abstract Rationale Excessive endothelial cell (EC) death and inflammation are the defining hallmarks of lethal respiratory diseases, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS). Therapies that can promote survival of EC during injury, vital for restoring lung vascular integrity, remain elusive. Epigenetic mechanisms, particularly DNA methylation, play a crucial role in regulating cellular responses to inflammation and infection. Here, we tested the hypothesis that pathogens alter the epigenome of the EC to switch from survival to an apoptotic fate during lung injury. Methods We employed an endotoxemia model of lung injury using lipopolysaccharide (LPS) (10 mg/kg i.p) to profile the DNA methylation landscape of EC. We used Reduced Representation Bisulfite Sequencing (RRBS) to profile genome-wide DNA methylation patterns and dynamics at single-base resolution during both injury and resolution phases. DNA methylation was quantified using super-resolution imaging of nuclear architecture at the single-cell level, S-adenosylmethionine (SAM) generation, an ELISA-based colorimetric assay, and methylated DNA immunoprecipitation (MeDIP) to quantify 5-methylcytosine (5mC). EC apoptosis and proliferation were analyzed by immunostaining and flow cytometry. The causal role of DNMT3a in regulating the endothelial epigenome during injury was assessed by tracing the lineage of EC using inducible tdTomatocdh5-creERT2 mice, along with EC-specific DNMT3a-knockout (td-EC-DNMT3a−/−) mice. Results Endotoxemia induced heterogeneous differentially methylated regions (DMRs) patterns during injury. We identified 1,101 hypomethylated and 2,664 hypermethylated genes during the injury phase, suggesting a potential connection between aberrant DNA methylation and lung injury. LPS also enhanced SAM production in EC in a time-dependent manner. Loss of DNMT3a in EC markedly reduced global and site-specific methylation levels. Moreover, EC-specific loss of DNMT3a in mice or pharmacological inhibition of DNMT3a markedly reduced edema formation, secretion of pro-inflammatory cytokines, and EC death. Conclusion These findings identify DNMT3a-dependent DNA methylation as a central regulatory mechanism governing EC apoptosis and inflammatory injury, thereby highlighting DNMT3a as a potential therapeutic target for promoting vascular repair and restoration of lung-fluid homeostasis in ALI. This abstract is funded by: American Lung Association
Akhter et al. (Fri,) studied this question.