Mechanistic study of ANXA3-mediated endoplasmic reticulum stress promoting M1 macrophage polarization in pulmonary arterial hypertension based on bioinformatics and nine machine learning algorithms.

BACKGROUND

Endoplasmic reticulum stress (ERS) plays a critical role in inflammation-related disorders, but its immunoregulatory mechanisms in pulmonary arterial hypertension (PAH) remain unclear. This study aimed to identify key ERS-associated genes and clarify their roles in immune modulation during PAH progression.

METHODS

Transcriptomic data from three Gene Expression Omnibus (GEO) datasets comprising peripheral blood mononuclear cells (PBMCs) of PAH patients (n = 100) and healthy controls (n = 61) were integrated to screen differentially expressed ERS-related genes. Nine machine learning algorithms were applied, and four candidate genes were identified and validated by quantitative real-time PCR (qRT-PCR) in PBMCs from 20 idiopathic PAH (IPAH) patients and in two PAH rat models: monocrotaline (MCT)-induced and SU5416/hypoxia (SuHx)-induced. Immune infiltration analysis, consensus clustering, single-cell RNA sequencing (scRNA-seq), and immunofluorescence (IF) were used to determine immune-specific localization. Functional experiments were then performed to investigate the role of the key gene in ERS regulation, immune responses, macrophage function, and macrophage-pulmonary artery smooth muscle cell (PASMC) interactions.

RESULTS

Fifty differentially expressed ERS-related genes were identified. Least absolute shrinkage and selection operator (LASSO) regression achieved the best diagnostic performance and identified four key candidates: BCL2 Like 1 (BCL2L1), Tumor Necrosis Factor Receptor Superfamily Member 17 (TNFRSF17), DnaJ Heat Shock Protein Family Member B1 (DNAJB1), and Annexin A3 (ANXA3). qRT-PCR confirmed altered expression of these genes in PBMCs and lung tissues from both PAH models. ANXA3 was specifically overexpressed in PAH-associated macrophages and co-localized with Cluster of Differentiation 68-positive (CD68+) macrophages in SuHx rat lungs. Functionally, ANXA3 silencing improved right ventricular function and reduced pulmonary vascular remodeling in vivo, while in vitro knockdown suppressed lipopolysaccharide (LPS)-induced M1 polarization, decreased secretion of proinflammatory cytokines including interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-18 (IL-18), and inhibited ERS markers such as glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), and activating transcription factor 4 (ATF4). These inhibitory effects were reversed by the ERS agonist thapsigargin (TG) but enhanced by the ERS inhibitor 4-phenylbutyric acid (4-PBA). In addition, ANXA3 knockdown alleviated macrophage-induced PASMC proliferation and migration triggered by inflammatory stimuli.

CONCLUSION

This study demonstrates that ANXA3 regulates ERS to drive M1 macrophage polarization and inflammation, which subsequently promotes PASMC function and promotes PAH progression. ANXA3 may serve as a novel immunoinflammatory target and potential therapeutic candidate.