ROS-induced endothelial dysfunction in pulmonary arterial hypertension

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PROJECT SUMMARY The objectives of this K08 proposal are two-fold: 1) foster the development of essential scientific skills that will allow the candidate to become an independent physician-scientist focused on the role of ROS and intracellular calcium in abnormal endothelial function and 2) investigate mechanisms underlying abnormal endothelial migration/proliferation in pulmonary arterial hypertension (PAH). Through laboratory experience, coursework and peer review, Dr. Suresh and his mentor at Johns Hopkins University, Dr. Larissa Shimoda, have designed a specific training plan that will provide Dr. Suresh with the research skills needed to pursue independent investigation of endothelial function in lung diseases including PAH. PAH is a lethal disease characterized by abnormal migration and proliferation of endothelial cells (ECs) in the distal blood vessels of the lung. There are currently no therapies that target the underlying endothelial dysfunction in PAH. Reactive oxygen species (ROS) and intracellular calcium (Ca2+) are important mediators of migration and proliferation in ECs and both are known to be elevated in PAH, but the mechanisms that link ROS and Ca2+ influx to the transformation of normal ECs to the abnormal phenotype seen in PAH is unknown. Our prior published work and preliminary data in ECs isolated from humans with PAH (hPAH-ECs) and rats undergoing Sugen/Hypoxia (SuHx), an experimental form of PAH (rPAH-ECs), suggest that in ECs: 1) elevations in ROS increase [Ca2+]i by activating the calcium channel TRPV4; 2) regulation of TRPV4 phosphorylation by the Src kinase Fyn tethered to the cell membrane by its anchor, CD36, is critical for activation of TRPV4; 3) baseline ROS levels, cytosolic Ca2+, migration and proliferation are elevated in rPAH- and hPAH-ECs, and attenuated by quenching of ROS or inhibition of TRPV4; 4) rPAH-ECs exhibit evidence of mitochondrial dysfunction that may represent the source of ROS elevations in PAH; and 5) loss of CD36 or Fyn attenuates development of PAH in a murine SuHx model. Thus, we hypothesize that phosphorylation of TRPV4 by CD36-tethered Fyn is required for activation of this channel by elevated cytosolic ROS that occur due to mitochondrial dysfunction, promoting EC migration and proliferation. Using rat and human PAH-ECs (and normoxic controls) we propose the following aims: 1) Determine whether CD36 and Fyn are required for increased basal Ca2+ levels, migration and proliferation in PAH-EC in vitro; 2) Evaluate whether quenching mitochondrial ROS restores normal EC function in PAH-ECs; and 3) identify the contribution of the CD36/Fyn/TRPV4 pathway of ROS-induced Ca2+ influx towards development/progression of PAH in vivo. Methods for studying these aims include fluorescent live cell imaging of intracellular Ca2+ and ROS, genetic knockdown techniques, in vitro migration and proliferation assays, and an in vivo model of PAH with physiologic and histologic measurements. Completing these aims will provide a rigorous training program for Dr. Suresh and uncover mechanisms of endothelial dysfunction in PAH that could be translated into future therapies for this and other vascular diseases.