Metabolic regulation of Ca2+ entry and endothelial-mesenchymal transition in pulmonary arterial hypertension

PROJECT SUMMARY Pulmonary arterial hypertension (PAH) is a lethal disease characterized by abnormal proliferation of microvascular endothelial cells (MVECs) in the distal blood vessels of the lung. There are currently no therapies that target the underlying endothelial dysfunction in PAH. Mitochondrial dysfunction, increased intracellular calcium ([Ca2+]i) and endothelial-mesenchymal transition (EndMT) are important pathogenic abnormalities observed in MVECs isolated from patients with PAH, but the mechanisms that link these cellular abnormalities is unknown. In MVECs isolated from rats undergoing Sugen/Hypoxia (SuHx), an experimental form of PAH (SuHx-MVECs), our prior work and current preliminary data suggest a) mitochondrial dysfunction recapitulating those seen in human PAH ECs, b) increased mitochondrial reactive oxygen species (mtROS) and β-hydroxybutyrate (BOHB), c) increased activation of the transient receptor potential vanilloid-4 (TRPV4) channel and increased intracellular calcium ([Ca2+]i), d) EndMT and e) increased proliferation. Further, we also observe specific shifts in metabolism (increased use of anaerobic respiration as well as an increase in fatty acid oxidation), suggesting a metabolic basis for mitochondrial dysfunction in SuHx- MVECs. Our preliminary data now suggest specific roles for two metabolic byproducts of increased fatty acid oxidation – BOHB and mtROS - in sensitizing and activating TRPV4, respectively. Thus, we hypothesize that, in PAH, a shift in MVEC mitochondrial metabolism from glycolysis to fatty acid oxidation promotes mtROS and BOHB generation, leading to TRPV4 activation and consequently, Ca2+-dependent activation of EndMT and proliferation. Using MVECs isolated from rats, mice and humans with and without PAH, as well as in vivo experiments utilizing various novel transgenic rats and mice, we propose the three independent aims centered around the following questions: 1) How does increased fatty acid oxidation promote TRPV4 activation in MVECs; 2) Is TRPV4 activation necessary and sufficient to induce EndMT; and 3) What is the impact of TRPV4 loss or BOHB supplementation on EndMT development in vivo. To accomplish these aims, we plan on utilizing a variety of methods ranging from fluorescent live-cell imaging of MVECs isolated from WT and transgenic mice and rats, in vitro studies in human MVECs from PAH patients (and controls), and in vivo lineage-tracing studies to examine EndMT development in rodent models of PAH. Completion of these aims will provide novel insight into the interplay between fatty acid metabolism, Ca2+ homeostasis and EndMT in normal MVECs and role of the metabolic dysfunction and increased [Ca2+]I and EndMT in PAH.