C26-05 Distinct Mechanisms of Hemolysis-induced Signaling in Proximal and Distal Pulmonary Artery Endothelial Cells

Abstract Rationale Pulmonary hypertension (PH) is a progressive vascular disorder characterized by elevated pulmonary vascular remodeling. Our previous work identified intravascular hemolysis as a major contributor to vascular remodeling, where free heme induces oxidative stress, inflammation, and endothelial barrier disruption. Chloride intracellular channel 1 (CLIC1), a redox-sensitive ion channel, regulates proliferation and differentiation and has been implicated in pulmonary vascular remodeling. While hemolysis-mediated CLIC1 activation has been described in proximal pulmonary artery endothelial cells (PAECs), its effects on distal capillary endothelial cells, particularly aerocytes, the specialized gas-exchange ECs, remain unknown. Hypothesis: We hypothesize that hemolysis-derived free heme differentially activates CLIC1 signaling in proximal and distal pulmonary endothelial populations. In PAECs, CLIC1 is released and transferred to smooth muscle cells (SMCs), promoting proliferation via RALY-Akt signaling. In contrast, in capillary aerocytes, heme-induced CLIC1 activation drives a phenotypic transition from a specialized gas-exchange to a mesenchymal-like state, contributing to microvascular remodeling. Methods Two in vivo models of PH were utilized: Sugen5416/hypoxia (Su/Hx) rats and glucose-6-phosphate dehydrogenase knockdown (G6PD-KD) mice. In vitro assays were performed in human and murine pulmonary ECs and in isolated aerocytes (CD31+/Ednrb+) treated with free heme (50 µM, 6h). CLIC1 expressions, oxidative stress, EGR1 activation, and downstream signaling were analyzed using hemodynamic studies, single-cell RNA seq, immunoblotting, and proteomics. Results G6PD-KD mice and Su/Hx rats displayed elevated RV systolic pressure (RVSP), severe vascular remodeling, and increased EC-specific CLIC1 expression. In PAECs, heme-induced CLIC1 release promoted SMC proliferation through RALY-Akt signaling (p 0.001). Proteomics confirmed RALY as a CLIC1-binding partner. Pharmacological inhibition of CLIC1 with IAA-94 markedly reduced RVSP (p 0.001), attenuated remodeling, and improved mitochondrial function. Single-cell transcriptomics from the lungs of PH patients revealed a marked reduction in the aerocyte population. In Su/Hx lungs and isolated aerocytes, heme exposure upregulated CLIC1 and RALY while downregulating the gas-exchange enzyme carbonic anhydrase 4 (CA4) and increasing mesenchymal markers α-SMA and SM22, consistent with a CLIC1-dependent phenotypic shift. These findings suggest that hemolysis-induced CLIC1 activation contributes to capillary rarefaction in PH lungs. Conclusion Hemolysis activates CLIC1-dependent pathways in proximal and distal pulmonary endothelium. While in PAECs, CLIC1 mediates EC-SMC crosstalk via RALY-Akt signaling, in aerocytes, it drives a mesenchymal-like reprogramming and loss of gas-exchange function. Targeting CLIC1 may therefore protect both conduit and capillary vascular compartments, offering a unified therapeutic approach to PH. This abstract is funded by: NIH