Human Aquaporin 1 (AQP1) mutants confer apoptosis resistance in pulmonary arterial smooth muscle cells (PASMCs)

RATIONALE: Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling, with PASMC hyperplasia and formation of vaso-occlusive lesions. PASMCs from patients with PAH are resistant to apoptosis, a feature which may propagate disease as abnormal PASMCs evade death. Whole genome sequencing has identified over-representation of 13 AQP1 variants in patients with PAH who are negative for previously reported disease associated genes, suggesting these are disease driving mutations. Furthermore, patients with PAH-associated AQP1 mutations appear to develop early and severe disease. Our lab has established that overexpression of wild type AQP1 (WTAQP1) promotes PASMC apoptosis resistance, likely via a direct interaction with the pro-apoptotic enzyme caspase-3 (casp3). However, the role of human AQP1 mutants in PASMC apoptosis resistance has yet to be explored. In this study we hypothesized that human AQP1 mutants confer apoptosis resistance to at least the degree of, if not more than, WTAQP1. METHODS: Adenoviral constructs were designed containing the two most common human AQP1 mutants: Arg126Cys (AQP1H1) and Arg195Trp (AQP1H2). These mutants were overexpressed in control PASMCs isolated from the distal pulmonary arterioles of rats, and cell lysates immunoblotted for expression. As these mutations are located within the membrane spanning portion of the protein potentially involved in AQP1 canonical water channel transport, water permeability was tested. PASMCs plated on glass coverslips were infected with enhanced green fluorescent protein (eGFP, control), WTAQP1, AQP1H1, or AQP1H2. Calcein was added (5 µM, 30 m at 37°C), then coverslips placed in a closed chamber and perfused with isotonic, hypotonic, and hypertonic fluids in rapid succession. Change in calcein fluorescence was fit with a single exponential to calculate the time constant (), reflecting the rate of water transport. Next, PASMCs infected with eGFP (control), WTAQP1, AQP1H1, or AQP1H2 were treated with PBS or pro-apoptotic hydrogen peroxide (H 2 O 2 , 500 mM, 24 h). First, caspase-3 (casp3) activity was measured in total cell lysates using a luminescent caspase-3/7 activity assay (Caspase-Glo® 3/7) and second, morphological assessment of apoptosis was measured via visualization of nuclear condensation and fragmentation of cells with Hoechst 33342 stain. RESULTS: PASMCs infected with human AQP1 mutants demonstrated increased water permeability to a similar extent as WTAQP1 when compared to eGFP infected cells. Following H 2 O 2 treatment, casp3 activity was lower in PASMCs overexpressing WTAQP1 compared to eGFP, and even lower still with overexpression of human AQP1 mutants. Moreover, following stimulation of apoptosis, overexpression of human AQP1 mutants rendered PASMCs resistant to apoptosis to a similar extent as those infected with WTAQP1, with a trend towards enhanced apoptosis resistance. CONCLUSIONS: Our data suggest that the two most common AQP1 human mutations do not alter the canonical water transport function of the protein, but rather, may blunt casp3 activation and confer apoptosis resistance in PASMCs. Future studies characterizing the mechanism by which human AQP1 mutants alter casp3 activation and apoptosis are essential to understanding the pathobiology of disease and development of novel targeted therapeutics focused on restoring PASMC apoptosis susceptibility. NIH R01s HL159906 and HL126514, NIH T32 HL007534, NIH F32 HL165766, Bauernschmidt Award. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.