SIX1 mediates pulmonary arterial hypertension endothelial dysfunction through the IL-6/STAT3 axis

Pulmonary arterial hypertension (PAH) is a life-threatening and cardiovascular disease characterized by elevated pulmonary artery pressure leading to right ventricular hypertrophy. Its features include endothelial dysfunction and vascular remodeling. Inflammation is a key factor in PAH progression. This study focuses on the role and mechanism of sine oculis homeobox 1 (SIX1) in PAH through transcriptomics analysis, as well as validation in vivo and in vitro models, aiming to identify novel therapeutic strategies.

To analyze the transcriptomic data from mouse lung tissues (control vs. PAH), we employed an integrated strategy that combined Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment with weighted gene co-expression network analysis (WGCNA). This approach identified SIX1 as a key factor in PAH. We predicted potential binding sites between SIX1 and STAT3 using the Joint Academic Site for Protein And Regulatory (JASPAR) database. Through an integrated approach leveraging both in vivo and in vitro PAH models, we uncovered a pivotal role for SIX1 in disease pathogenesis, including hemodynamics, histopathology, protein expression (Western blot) and gene expression [reverse transcription quantitative polymerase chain reaction (RT-qPCR)], immunofluorescence (IF), and cellular phenotypes (knockdown, viability, migration, proliferation, apoptosis).

Through linear regression analysis in the transcriptome sequencing data, we found that SIX1 expression positively correlated with clinical and pathological indices of PAH. Subsequently, KEGG pathway analysis revealed that STAT3 was enriched in multiple pathways. Using JASPAR, multiple binding sites between SIX1 and STAT3 were predicted. Consistent with our transcriptomic results, SIX1 was significantly induced in vivo, with corresponding increases observed not only in messenger RNA (mRNA) but also in protein, and IF staining confirmed its predominant localization in endothelial cells. In vitro, the inflammatory cytokine interleukin-6 (IL-6), a key driver of PAH, significantly induced SIX1 expression and activated STAT3 in human umbilical vein endothelial cells (HUVECs). Notably, SIX1 knockdown effectively reversed the IL-6-driven proliferation and migration of endothelial cells. This phenotypic rescue was accompanied by a significant inhibition of STAT3 phosphorylation and a disruption of SIX1-STAT3 protein colocalization.

We conclude SIX1 aggravates endothelial dysfunction via the IL-6/STAT3 axis, thereby identifying SIX1 as a therapeutic target for PAH.