Abstract 4359059: Hemolysis-Triggered CLIC1/RALY Axis Promotes Endothelial-to-Smooth Muscle Cell Crosstalk in Pulmonary Hypertension

Abstract

Background: Pulmonary hypertension (PH) is a progressive and life-threatening vascular disorder characterized by elevated pulmonary arterial pressure, and right ventricular (RV) hypertrophy. Our previous study identified hemolysis as a key contributor to PH. Free heme released during hemolysis induces oxidative stress, inflammation and disrupts endothelial cell (EC) barrier integrity, contributing to vascular dysfunction. Chloride intracellular channel-1 (CLIC1), a redox-sensitive ion channel, is known to regulate critical cellular processes and has recently been implicated in pulmonary vascular remodeling. Hypothesis: We hypothesize that in PH, hemolysis-derived free heme activates CLIC1 in pulmonary endothelial cells (ECs), which is then transferred to smooth muscle cells (SMCs) and promotes SMC proliferation. Methods: We utilized two in vivo hemolytic PH models: Sugen5416/hypoxia (Su/Hx) rats and G6PD knockdown (G6PD-KD) mice. In vitro assays were performed in human and murine lung ECs exposed to free heme. CLIC1 expression, oxidative stress, mitochondrial function, and signaling targets were analyzed. Recombinant CLIC1 and immunoprecipitation (IP)-mass spectrometry were used to investigate downstream interactions. Results: G6PD-KD mice developed hemolysis-associated PH, evidenced by elevated RV systolic pressure (RVSP), extensive pulmonary vascular remodeling, and increased EC-specific CLIC1 expression. Similar CLIC1 upregulation was observed in Su/Hx rats and lung tissues from IPAH patients. In vitro, heme stimulation enhanced CLIC1 expression, increased ROS, and upregulated the transcription factor EGR1 in ECs. As a result, CLIC1 was released from ECs and trafficked to SMCs, promoting SMC proliferation; this was further validated by rCLIC1 treatment. Proteomics following CLIC1-IP identified the heterogeneous nuclear ribonucleoprotein RALY as a CLIC1-interacting partner. In SMCs, the CLIC1/RALY axis triggered Akt signaling (p<0.001), promoting a phenotypic shift toward proliferation. Pharmacological inhibition of CLIC1 with IAA-94 significantly reduced RVSP (p<0.001), RV hypertrophy, and vascular remodeling and restored mitochondrial function and ATP production in G6PD-KD ECs. Conclusion: These findings uncover a novel CLIC1/RALY signaling axis mediates EC/SMC communication and promotes pulmonary vascular remodeling in PH. Targeting CLIC1 with IAA-94 effectively ameliorates disease features, positioning CLIC1 as a potential therapeutic target for PH.