A novel mechanism regulating apoptosis in pulmonary arterial smooth muscle cells

About This Grant

PROJECT SUMMARY The objectives of this K08 proposal are twofold: 1) development of a specific skillset that will facilitate transition to an independent clinician scientist focused on mechanisms driving pulmonary vascular remodeling in PAH and 2) investigate a novel mechanism of apoptosis resistance in pulmonary arterial smooth muscle cells (PASMCs). Incorporating hands on laboratory experience, enrollment in didactic course work, and activities to enhance career development, Dr. Niedermeyer and her mentors, Dr. Larissa Shimoda and Dr. Mahendra Damarla, have designed a 5-year training plan providing the research and career skills needed to transition to an independent investigator. Pulmonary arterial hypertension (PAH) remains a disease of high morbidity and mortality with a 3-yr survival of only 55% in high-risk patients despite available therapies, and there are no current therapies which target the underlying pathobiology driving pulmonary vascular remodeling. Extensive work exploring underlying mechanisms driving apoptosis resistance in other diseases has shed light on the cell membrane protein aquaporin 1 (AQP1). Our lab was the first to show AQP1 protein is present in PASMCs28 and significantly upregulated in PASMCs isolated from the Sugen-Hypoxia (SuHx) PH model. Furthermore, we found SuHx PASMCs are resistant to apoptosis and suppressing AQP1 restores apoptosis susceptibility. The exact role of AQP1 in the development of apoptosis resistance remains unclear, but I have substantial evidence to indicate it may be via a direct interaction with the pro-apoptotic enzyme caspase-3 (casp3). Using proximity-based biotinylation assays, I established a novel interaction between AQP1 and caspase-3. Based on preliminary data, I hypothesize that in PASMCs with increased and/or mutant AQP1, binding of pro-casp3 to AQP1 reduces the amount of activatable casp3, conferring apoptosis resistance and driving vascular remodeling in PH. To test this hypothesis, I propose three specific aims: 1) determine the structural and functional components of casp3 that regulate binding to AQP1, 2) investigate features of AQP1 that influence casp3 binding and activation, and 3) determine whether modulating AQP1 abundance and/or casp3 binding alters vascular remodeling and PH. I will utilize a combination of techniques including unique protein constructs in complementary binding assays, measurements of casp3 activity in total cell lysates and subcellular compartments paired with advanced live cell imaging, and a novel transgenic murine model which allows for conditional knockout of AQP1 in PASMCs. Completing these aims will provide a rigorous training program for Dr. Niedermeyer and uncover mechanisms of PASMC apoptosis resistance that could provide potential therapeutic targets.