Abstract 096: Sequential Activation of Nox1 and Gremlin1 Leads to Endothelial Proliferation in Human Pulmonary Arterial Hypertension

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Pulmonary arterial hypertension (PAH) is a rapidly degenerating and devastating disease of increased pulmonary vessel resistance leading to eventual right heart failure. Until now, palliative modalities have targeted the reduction of vascular tone with little success. Recent studies have delved into the mechanisms regulating increased pulmonary vascular resistance: aberrant vascular remodeling and occlusion. However, little is known of the molecular mechanisms responsible for endothelial proliferation, a root cause of PAH-associated vascular remodeling. We provide the first evidence to our knowledge of an upregulation of NADPH oxidase 1 (Nox1) at the transcript and protein (2.1±0.62 -fold, P < 0.05) level in resistance vessels from PAH vs. non-PAH subjects. This coincided with an increase in bone morphogenetic protein (BMP) antagonist Gremlin1 protein expression (2.3±0.47 [[Unable to Display Character: –]]fold vs. non-PAH, P < 0.05) and reactive oxygen species (ROS) production (iodonium-inhibitable hydrogen peroxide production: 0.69±0.06 vs. 0.43±0.032 nmol/min/mg protein for PAH vs. non-PAH, respectively, P < 0.05). In vitro studies in human pulmonary artery endothelial cells (HPAEC) demonstrate that hypoxia (24 hr, 1 % O 2 ) drives Nox1 subunit expression (Nox1 protein: 1.4±0.075-fold vs. normoxia, P < 0.05), assembly and oxidase activity (superoxide production, nmol/min/mg protein: 14.0±1.9 vs. 6.00±0.94 for normoxia, P < 0.01) leading to elevation in sonic hedgehog (SHH; 1.5±0.011 fold, P < 0.05) and Gremlin1 (1.90±0.32-fold, P < 0.01) expression. Nox1 gene-silencing in hypoxia-exposed HPAEC abrogated this cascade. Moreover, hypoxia-induced endothelial cell proliferation (1.18±0.038-fold vs. normoxia, P < 0.05) was attenuated with loss of either Nox1 or Gremlin1. Finally, incubation of normoxic HPAEC with conditioned media from hypoxia-exposed HPAEC resulted in increased proliferation, which was abrogated by Nox1 suppression of donor cells. Together these data support a Nox1-Gremlin1 signaling axis in pulmonary vascular endothelium that is likely to contribute to pathophysiological endothelial proliferation and the progression of pulmonary hypertension. The findings also support targeting of Nox1 as a viable therapeutic option to combat PAH.