Fig 2: The central mechanism is that upregulated PEAR1 promotes AARS1‐mediated HIF‐1α lactylation at K172, enhances HIF‐1α interaction with importin α and nuclear translocation, and subsequently drives glycolysis and lactate production. The accumulated lactate further promotes H3K18 lactylation at the Pear1 promoter, thereby enhancing Pear1 transcription and establishing a positive feedback loop.

Fig 3: Glycolysis‐Derived Lactate Promotes PEAR1 Expression through H3K18la during S‐ALI. (A) WB analysis of Pan‐lysine lactylation (Kla) protein levels in lung tissues from SHAM and CLP groups (n = 5 per group). (B) Representative IF staining images and relative intensity quantification of Pan‐Kla (green) in lung tissues from SHAM and CLP groups (n = 5 per group). Vascular endothelium was labeled with CD31 (red), and nuclei were stained with DAPI (blue). Scale bar, 20 µm. (C) Proteomic lactylation analysis revealed significantly altered histone lactylation sites in lung tissue from SHAM and CLP groups (n = 3 per group). (D) WB analysis of H3K18la protein levels in lung tissues from SHAM and CLP groups (n = 5 per group). (E) Representative IF staining images and relative intensity quantification of H3K18la (green) in lung tissues from SHAM and CLP groups (n = 5 per group). Vascular endothelium was labeled with CD31 (red), and nuclei were stained with DAPI (blue). Scale bar, 20 µm. (F) WB analysis of H3K18la protein levels in MPMVECs with or without LPS stimulation (10 µg/mL for 12 h) (n = 5 per group). (G) Representative IF staining images and relative intensity quantification of H3K18la (green) in MPMVECs with or without LPS stimulation (10 µg/mL for 12 h) (n = 5 per group). Nuclei were stained with DAPI (blue). Scale bar, 20 µm. (H) Heat maps of the genome occupancy of H3K18la and H3K18ac ± 3 kb flanking transcription start sites in MPMVECs with or without LPS stimulation (10 µg/mL for 12 h) from CUT&Tag analysis (n = 3 per group). (I) IGV tracks for Pear1 from CUT&Tag analysis in MPMVECs. (J) ChIP‐qPCR assays of H3K18la and H3K18ac occupancy rates in the promoter region of Pear1 in MPMVECs with or without LPS stimulation (10 µg/mL for 12 h) (n = 5 per group). (K) MPMVECs were pre‐treated with sodium oxamate (OXA) (20 mM for 3 h), lactate (8 mM for 7 h) or PBS, followed stimulated by LPS (10 µg/mL for 12 h). H3K18la and PEAR1 protein levels were detected by WB analysis (n = 5 per group). All data were represented as the means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant.

Fig 4: PEAR1 Regulates Glycolysis in PMVECs in a Model of S‐ALI. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes (DEGs) from RNA‐seq of MPMVECs transfected with si‐NC or si‐Pear1 and subsequent stimulation with LPS (10 µg/mL, 12 h) (n = 3 per group). (B, C) Heatmaps of DEGs in the glycolysis pathway and pyruvate metabolism from MPMVECs transfected with si‐NC or si‐Pear1 and subsequent stimulation with LPS (n = 3 per group). (D) WB analysis of key glycolytic enzymes (HK2, PFKP, PKM2, and LDHA) in MPMVECs transfected with si‐NC or si‐Pear1 and subsequent stimulation with or without LPS (n = 5 per group). (E) RT‐qPCR analysis of key glycolytic enzyme mRNA levels in MPMVECs in different groups (n = 5 per group). (F) Representative IF staining images and relative intensity quantification of LDHA (red), PKM2 (green), PFKP (red) and HK2 (green) in MPMVECs in different groups. Nuclei were stained with DAPI (blue) (n = 5 per group). Scale bar, 20 µm. (G) Extracellular acidification rate (ECAR) was measured in MPMVECs in absence of exogenous glucose, after sequential addition of 10 mM glucose followed by oligomycin (1 µM), and 2‐DG (50 mM). And basal glycolysis, glycolytic capacity, and glycolytic reserve were presented as bar graphs in different groups (n = 3 per group). (H) Oxygen consumption rate (OCR) was measured in MPMVECs during the sequential injection of oligomycin (1.5 µM), FCCP (1.5 µM), and a mixture of rotenone and antimycin A (0.5 µM). And basal respiration, ATP‐linked respiration, and Maximal respiration were presented as bar graphs in different groups (n = 3 per group). All data were represented as the means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant.

Fig 5: PEAR1 Increases HIF‐1α Binding to Importin α via K172 Lactylation to Promote HIF‐1α Nuclear Localization. (A) WB analysis of HIF‐1α, c‐Myc, p‐STAT3, and p‐NF‐κB p65 in MPMVECs transfected with si‐NC or si‐Pear1 and subsequent stimulation with or without LPS (n = 5 per group). (B) RT‐qPCR analysis of Hif1a, Myc, Stat3, and Rela mRNA levels in MPMVECs in different groups (n = 5 per group). (C) Representative IF staining images of HIF‐1α (green) in MPMVECs in different groups. And nuclei were stained with DAPI (blue). Scale bar: 20 µm (main) and 4 µm (inset). (D) WB analysis of cytoplasmic and nuclear HIF‐1α expression in MPMVECs in different groups (n = 5 per group). (E) Co‐immunoprecipitation (Co‐IP) was performed to examine the interaction between HIF‐1α and importin α in MPMVECs transfected with si‐NC or si‐Pear1 and subsequent stimulation with LPS (10 µg/mL for 12 h). (F) Co‐IP was performed to examine the interaction between HIF‐1α and importin α in LPS‐stimulated MPMVECs with or without overexpressing Pear1 (19‐754 aa). (G) Co‐IP was performed to examine the interaction between HIF‐1α and importin α in LPS‐stimulated MPMVECs with or without overexpressing Pear1 (776‐1034 aa). (H) Co‐IP was performed to examine lactylation of HIF‐1α in MPMVECs after treatment with or without LPS (10 µg/mL for 12 h). (I) Co‐IP was performed to examine lactylation of HIF‐1α in MPMVECs transfected with si‐NC or si‐Pear1 and subsequent stimulation with or without LPS (10 µg/mL for 12 h). (J) Illustration of possible lactylation sites of HIF‐1α in the MPMVECs analyzed via IP‐LC‐MS/MS. Three possible lactylation sites of HIF‐1α observed are shown. (K, L) MPMVECs were transfected with His‐Hif1a‐WT or single‐site mutant (K11R, K12R, and K172R) (K) multi‐site mutant plasmids (K11R/K12R, K11R/K172R, K12R/K172R, and K11R/K12R/K172R) (L). Immunoprecipitation of exogenously expressed HIF‐1α was performed using an anti‐His antibody, and lactylation levels were assessed with an anti‐Pan‐Kla antibody. (M) Co‐IP was performed to examine the interaction between HIF‐1α and importin α after overexpressed His‐Hif1a‐WT or His‐Hif1a‐K172R in LPS‐stimulated MPMVECs with or without Pear1 silencing. (N) Interaction between HIF‐1α and importin α detected by PLA experiments in LPS‐stimulated MPMVECs in different groups. Red fluorescent puncta indicate PLA signals, representing interaction or close proximity between HIF‐1α and importin α. Nuclei were stained with DAPI (blue). Scale bar, 20 µm. (O) WB analysis of cytoplasmic and nuclear HIF‐1α expression in LPS‐stimulated MPMVECs in different groups. (P) WB analysis of HK2, PFKP, PKM2, and LDHA in LPS‐stimulated MPMVECs in different groups (n = 5 per group). All data were represented as the means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns, not significant.