ROC‐325 Attenuates Hypoxia‐Induced Pulmonary Hypertension Through Dual Inhibition of Autophagy and Hypoxia‐Inducible Factor 2α Signaling

The activation of autophagy in endothelial cells is a key mechanism underlying vascular remodeling, leading to the development and progression of pulmonary hypertension (PH). In our previous studies, we developed ROC‐325, a novel lysosomal autophagy inhibitor, and demonstrated its potent therapeutic effects in severe pulmonary arterial hypertension (PAH). However, its role in chronic hypoxia‐induced PH (HPH) remains unknown. Here, we observed increased LC3B‐II/I and decreased p62/SQSTM1 in lung tissues of hypoxic mice, indicative of autophagy activation. ROC‐325 treatment suppressed this activation by inhibiting LC3B‐II accumulation and restoring p62 levels. ROC‐325 significantly reduced right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), and pulmonary artery medial thickening and muscularization, indicating a significantly protective effect against HPH. Endothelial hypoxia‐inducible factor 2α (HIF‐2α) upregulation, resulting from prolyl hydroxylase domain protein 2 (PHD2) deficiency, was implicated in autophagy activation. Endothelial conditional PHD2 knockout mice, a spontaneous PH model, exhibited elevated LC3B‐II/I and HIF‐2α levels, both of which were reduced by ROC‐325, leading to significant PH attenuation. In vitro, ROC‐325 inhibited the proliferation of pulmonary microvascular endothelial cells derived from PHD2‐deficient mice and enhanced HIF‐2α degradation in human pulmonary artery endothelial cells. These findings, in summary, identify ROC‐325 as a promising therapeutic agent for hypoxia‐induced PH by modulating both autophagy and endothelial HIF‐2α signaling.