Rational Design and Therapeutic Potential of a Novel Nox1 Inhibitor for the Treatment of Pulmonary Hypertension: In Vitro and In Vivo Effects of Nox1 Inhibition

摘要

NADPH oxidases (Noxes) represent a family of enzymes who produce reactive oxygen species. Excessive Nox activity is associated with multiple pathological conditions, including hypertension. Despite Nox1’s association with morbidity, there is a paucity of specific Nox1 inhibitors. The overarching hypothesis of this project was that Nox1 promotes endothelial phenotypes contributing to pulmonary hypertension and associated cardiac dysfunction. Pharmacological Nox1 inhibition testing this hypothesis was performed via designing the first specific peptidic Nox1 inhibitor (NoxA1ds). Our results show that Nox1 is key to endothelial O2•- and VEGF-stimulated migration and that Nox1 contributes to left ventricle cardiac dysfunction.udFunctional Nox1 is activated in part by association of Nox1 with one of its essential cytosolic subunits NOXA1. NoxA1ds was designed to mimic a putative activation domain in NOXA1 with a single F199A amino acid mutation. NoxA1ds specifically inhibited Nox1 but not Nox2, Nox4, Nox5 in reconstituted cell-free systems. Mechanistically, we found that NoxA1ds binds to Nox1 and disrupts Nox1:NOXA1 association and thus enzyme assembly.udTo identify the relative roles of Nox1 and Nox2 in human pulmonary artery endothelial cell (HPAEC) physiology, the relative specificity of Nox2ds for Nox2 vs Nox1 required validation. In part, this thesis established Nox2ds as specific for Nox2 over canonical, hybrid, or inducible Nox1 and Nox4. NoxA1ds and Nox2ds were then used to establish that Nox1, but not Nox2, is responsible for hypoxia-induced O2•- in HPAEC and VEGF-stimulated HPAEC migration. Additional data revealed that VEGF stimulates Nox1:NOXA1 association and identified fibroblasts as a source for hypoxic VEGF production.udThe role of Nox1 in HPAEC O2- and migration suggested that Nox1 may contribute to of the development of pulmonary arterial hypertension. Treatment of pulmonary hypertensive rats with aerosolized NoxA1ds improved left ventricular dilation but displayed minimal benefit in the right ventricle, indicating Nox1 may play a predominant role in the systemic vs pulmonary vasculature.udMajor contributions of this study include the design and characterization a novel Nox1 inhibitor (NoxA1ds), the identification of pulmonary endothelial phenotypes mediated by Nox1 rather than Nox2, and that the contribution of Nox1 to left ventricular dilation in the context of severe PAH.