Sine oculis homeobox 1 drives endothelial dysfunction in preclinical pulmonary arterial hypertension

Endothelial dysfunction plays a critical role in the initiation and progression of vascular remodeling and pulmonary arterial hypertension (PAH). Sine oculis homeobox 1 (<i>SIX1</i>) is a developmentally restricted transcription factor, and its expression ceases upon the completion of embryonic development. Deletion of <i>Six1</i> impairs the differentiation of pulmonary vascular endothelial cells, resulting in pulmonary vascular defects and postnatal death. In this study, we found that SIX1 was increased in the endothelia of pulmonary arteries from patients with PAH and from patients with idiopathic pulmonary fibrosis-associated pulmonary hypertension (PH). Silencing <i>SIX1</i> using siRNA inhibited hypoxia-induced endothelial dysfunction in vitro, including proliferation, endothelial-to-mesenchymal transition, and endothelin-1 release. Endothelial-specific <i>Six1</i> knockout improved pulmonary hemodynamics, endothelial dysfunction, pulmonary artery remodeling, and right ventricular function in SU5416/hypoxia (SuHx)-induced PH mice. Moreover, endothelial-specific <i>Six1</i> overexpression mediated by adeno-associated virus induced a spontaneous PH in wild-type C57BL/6 mice. Structure-based virtual screening and surface plasmon resonance analysis demonstrated that zafirlukast was an inhibitor of SIX1 transcriptional activity. Zafirlukast suppressed hypoxia-induced endothelial dysfunction in vitro and reversed SuHx-induced PH in Sprague-Dawley rats. Mechanistically, multiomics profiling of hypoxia-induced endothelial dysfunction in vitro revealed that microtubule-associated serine/threonine kinase family member 4 (<i>MAST4</i>) was a target of SIX1. MAST4 phosphorylated the Thr^185/202 sites of mitogen-activated protein kinases 1 and 3 (MAPK1/3), resulting in endothelial dysfunction. Overall, we found that SIX1 was a driver for endothelial dysfunction and PH through regulating <i>MAST4</i> transcription and subsequently MAPK1/3 activation. Targeting SIX1 may be a promising strategy for PAH treatment and drug development.