RATIONALE: Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, with PASMC hyperplasia and formation of vaso-occlusive lesions. New data show PASMCs isolated from robust animal models of PH are resistant to apoptosis, a feature which may propagate disease as abnormal PASMCs evade death. Apoptosis is a highly regulated pathway resulting in caspase-3 (casp3) activation and translocation to the nucleus, where cleavage of proteins results in cell death. In several cancers the membrane protein aquaporin 1 (AQP1) plays a role in apoptosis resistance, although the mechanism remains unknown. We showed AQP1 is expressed in PASMCs and increased in rat models of PH. Therefore, we hypothesized increased AQP1 contributes to apoptosis resistance in PASMCs. METHODS: The Sugen-hypoxia (SuHx) rat model of PH was used. Rats were injected with vascular endothelial growth factor receptor type 2 inhibitor Sugen-5416 and placed in 10% O2 for 3 wk, then returned to 21% O2 for 2 wk. Control animals received vehicle injection and were kept in 21% O2. Distal PASMCs were isolated via enzymatic digestion. AQP1 was depleted in SuHx PASMCs with small interfering RNA (siAQP1), with non-targeting siRNA as control. AQP1 was overexpressed in control PASMCs with adenoviral constructs containing wild type AQP1 (AdAQP1) or green fluorescent protein (control). Cells were treated with PBS or pro-apoptotic hydrogen peroxide (500 μM; 24 h). Apoptosis was measured by Hoechst staining. Caspase activity was measured via luminescent caspase-3/7 activity assay (Caspase-Glo® 3/7) in whole cell lysates and nuclear fractions. RESULTS: Depleting AQP1 via siAQP1 restored apoptosis susceptibility in SuHx PASMCs, and overexpressing AQP1 via AdAQP1 reduced susceptibility to apoptosis in control PASMCs. At baseline, there was no difference in casp3 activity between SuHx and control total cell lysates. However, casp3 activity was lower in SuHx nuclear fractions. Under stimulated conditions, SuHx PASMCs exhibited significantly less total and nuclear casp3 activity, with nuclear activity similar to levels observed in unstimulated controls. To explore an underlying mechanism for these findings, in silico analysis of AQP1 was performed revealing 3 potential casp3 cleavage sites, suggesting the possibility these proteins could interact. To investigate a potential protein-protein interaction, proximity-based biotinylation assays were performed in PASMCs using AdAQP1 (control) or AQP1 fused to biotin ligase. Cells were incubated with cell-permeable biotin (50 μM), lysed, and biotinylated proteins precipitated and immunoblotted for casp3 and other cytosolic proteins not expected to interact with AQP1. To confirm bidirectionality, similar biotinylation assays were performed using adenoviral constructs with wild-type (control) or biotin ligase-fused casp3 and immunoblotting for AQP1. Results showed a specific AQP1/casp3 interaction in PASMCs. Furthermore, the predominant form of casp3 interacting with AQP1 appears to be inactive (37kD). CONCLUSION: We speculate that increased AQP1 in SuHx PASMCs interacts with casp3 preventing its activation and/or nuclear translocation, conferring apoptosis resistance. Future studies further characterizing the interaction between AQP1 and casp3 could provide the basis for novel therapeutic targets focused on restoring PASMC apoptosis susceptibility. F32 HL165766-01, T32 HL007534, Bauernschmidt Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.