DNA methylation plays a fundamental role in regulating transcription during development and differentiation. However, its functional role in the regulation of endothelial cell (EC) transcription during state transition, meaning the switch from an angiogenic to a quiescent cell state, has not been systematically studied. Here, we report the longitudinal changes of the DNA methylome over the lifetime of the murine pulmonary vasculature. We identified prominent alterations in hyper- and hypomethylation during the transition from angiogenic to quiescent ECs. Once a quiescent state was established, DNA methylation marks remained stable throughout EC aging. These longitudinal differentially methylated regions correlated with endothelial gene expression and highlighted the recruitment of de novo DNA methyltransferase 3a (DNMT3A), evidenced by its motif enrichment at transcriptional start sites of genes with methylation-dependent expression patterns. Loss-of-function studies in mice revealed that the absence of DNMT3A-dependent DNA methylation led to the loss of active enhancers, resulting in mild transcriptional changes, likely due to loss of active enhancer integrity. These results underline the importance of DNA methylation as a key epigenetic mechanism of EC function during state transition. Furthermore, we show that DNMT3A-dependent DNA methylation appears to be involved in establishing the histone landscape required for accurate transcriptome regulation.
© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.

The inflammatory response after myocardial infarction (MI) is a precisely regulated process that greatly affects subsequent wound healing and remodeling. However, understanding about the process is still limited. Macrophages are critically involved in inflammation resolution after MI. Krüppel-like factor 9 (Klf9) is a C2H2 zinc finger-containing transcription factor that has been implicated in glucocorticoid regulation of macrophages. However, the contribution of Klf9 to macrophage phenotype and function in the context of MI remains unclear. Our study revealed that KLF9 deficiency resulted in higher mortality and cardiac rupture rate, as well as a considerable exacerbation in cardiac function. Single-cell RNA sequencing and flow cytometry analyses revealed that, compared with WT mice, Klf9-/- mice displayed excessive neutrophil infiltration, insufficient macrophage infiltration, and a reduced proportion of monocyte-derived CD206+ macrophages after MI. Moreover, the expression of IFN-γ/STAT1 pathway genes in Klf9-/- cardiac macrophages was dysregulated, characterized by insufficient expression at 1 day post-MI and excessive expression at day 3 post-MI. Mechanistically, Klf9 directly binds to the promoters of Stat1 gene, regulating its transcription. Overall, these findings indicate that Klf9 beneficially influences wound healing after MI by modulating macrophage recruitment and differentiation by regulating the IFN-γ/STAT1 signaling pathway.

Endothelial dysfunction and senescence are pivotal in pulmonary diseases, including chronic obstructive pulmonary disease (COPD). Protein arginine methyltransferase 1 (PRMT1) is the major enzyme responsible for asymmetric arginine dimethylation and plays a role in diverse biological processes, including cardiovascular function. Yet, its role in endothelial cells (ECs) remains poorly understood. Here, the role of PRMT1 is investigated in ECs, particularly in the context of COPD pathogenesis. Endothelial-specific PRMT1 knockout mice exhibit pulmonary hemorrhage, inflammation, barrier disruption, and apoptosis, accompanied by hyperactivation of nuclear factor kappa B (NF-κB). Bulk RNA sequencing of whole lungs and single-cell RNA sequencing of pulmonary ECs reveal that endothelial PRMT1 ablation results in a major alteration in inflammation-related gene expression. In a COPD model, PRMT1 deficiency aggravates the COPD phenotypes, including enlarged alveolar spaces, increased cell death, and senescence. PRMT1 inhibition in ECs exacerbates tumor necrosis factor alpha-triggered EC senescence and dysfunction attributable to NF-κB hyperactivation. PRMT1 as a critical regulator of pulmonary EC function, preventing NF-κB-driven endothelial dysfunction and senescence is highlighted here.
© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.

Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) remains a major challenge in the treatment of lung cancer. Cancer associated fibroblasts (CAFs) play a key role in promoting resistance to anti-cancer therapies. This study identified a subpopulation of CAFs characterized by the overexpression of collagen triple helix repeat-containing 1 (CTHRC1) through single-cell RNA sequencing of lung cancer patients undergoing EGFR-TKI treatment. These CTHRC1+ CAFs were enriched in drug-resistant tumors. Mechanistically, CTHRC1+ CAFs enhance the glycolytic activity of cancer cells by activating the TGF-β/Smad3 signaling pathway. Excess lactate produced in the process of glycolysis further upregulates CTHRC1 expression in CAFs through histone lactylation, creating a positive feedback loop that sustains EGFR-TKI resistance. The study also demonstrated that Gambogenic Acid, a natural compound, can disrupt this feedback loop, thereby improving the efficacy of EGFR-TKI therapy. Additionally, the presence of CTHRC1+ CAFs in tumor tissues could serve as a biomarker for predicting the response to EGFR-TKI therapy and patient prognosis. Overall, this study highlights the significant role of CAFs in EGFR-TKI resistance and suggests that targeting CTHRC1+ CAFs could be a promising strategy to overcome drug resistance in lung cancer.
© 2025. The Author(s).

Abstract Lipedema is a hereditary disorder characterized by excessive accumulation of subcutaneous adipose tissue in the limbs. The genetic causes and mechanisms underlying abnormal adipocyte expansion in lipedema, however, remain unknown. Here, we identify compound heterozygous mutations in the PROC gene in three lipedema patients from two unrelated consanguineous families. In vitro studies demonstrate the wild-type Protein C (PC), encoded by PROC, plays an inhibitory role in adipogenesis; conversely, the identified PC mutants, p.R271Q and p.R272H, fail to inhibit this process. In mice, the receptor of PC (PROCR) marks adipocyte progenitors, and conditional deletion of PROCR in these cells leads to an increased number of newborn adipocytes within white adipose tissue (WAT). Transcriptomic analysis alongside chemical blockage tests identifies HIF-1α as a primary downstream transcription factor mediating PC–PROCR signaling in adipogenesis. Furthermore, adipose biopsy samples from the patients’ thighs exhibit hyperplastic expansion of adipocytes, while single-nucleus RNA sequencing confirms increased adipogenic capacity and down-regulated HIF-1α activity in affected subjects. These findings establish PROC as the first causal gene for human lipedema and unveil a previously unexpected role of the PC–PROCR axis in orchestrating adipogenesis.