Abstract

Pulmonary arterial hypertension (PAH) is a devastating disease in which reactive oxygen species (ROS) play an important role. The disease is characterized by lung arterial cell proliferation (inward remodeling) leading to right ventricle (RV) failure. We postulated that NADPH oxidase 1 (NOX1)-derived ROS mediate endothelial cell (EC) proliferation and hemodynamic changes in PAH. Indeed, we found increased NOX1 (1.6±0.10-fold; n=7-9, p<0.01) in human pulmonary artery ECs (HPAECs) exposed to 24 hr hypoxia (Hx) vs. normoxia (Nx) (1% v. 21% O 2 , in vitro PAH phenotype). To test an in vivo NOX1 role, C57BL/6J (WT), mice were implanted with pumps delivering a NOX1-selective peptidic inhibitor (NoxA1ds) or scrambled control (Scr) and exposed to Nx (21% O 2 ) or Hx (10% O 2 ) for 3 wks plus vehicle or VEGF receptor antagonist Sugen 5416. Sugen/Hx (Su/Hx) increased RV max pressure (RVMP) 2-fold ± 0.20-fold (n=7-9, p<0.001) along with elevated mean pulmonary artery pressure (mPAP, 2-fold 0.25-fold, n=7-9, p<0.001), pulmonary vascular resistance (PVR, 2-fold±0.2-fold, n=7-9, p<0.001), and right ventricular max dP/dt (RVM dP/dt, 2-fold±0.2-fold, n=7-9, p<0.001) vs. Nx. NoxA1ds infusion significantly attenuated these rises. In parallel, RNA-Seq of HPAECs treated with NOX1 siRNA and exposed to Nx or Hx revealed the protein kinase R-like ER kinase (PERK) branch of the unfolded protein response (UPR) as NOX1-mediated. UPR promotes tumor growth in cancer due to positive effects on cell survival and proliferation. Indeed, phospho-PERK expression measured via WB increased by 1.5-fold ± 0.3-fold (n=9, p<0.01) in HPAECs exposed to Hx, which was completely reversed by siNOX1. Further, downstream members of the PERK-UPR pathway followed similar trends including phospho-eukaryotic translation initiation factor 2A (eIF2α, 3-fold ± 0.6-fold, n=9, p<0.05), activating transcription factor 4 (ATF4, 2.1-fold ± 0.4-fold, n=9, p<0.01), and protein disulfide-isomerase (PDI, 2.3-fold ± 0.65-fold, n=9, p<0.01). We corroborated these findings in cross-sections of Su/Hx mouse lungs and in autopsied iPAH patient lungs. The findings are consistent with NOX1 mediating activation of the UPR through PERK, which, in turn, induces EC hyperproliferation in PAH.