Vasoactive factors, Nox isoforms and redox biology in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterised by elevated pulmonary arterial pressures and obstructive lesions in the distal vasculature. As a result, the right ventricle is placed under excessive strain resulting in adaptive hypertrophy, progressing to maladaptive hypertrophy and failure. Women develop PAH more frequently than men. It is postulated that 17β-estradiol (E2) plays a role in disease pathogenesis and/or the E2 metabolic axis may be dysregulated in PAH. Growing evidence also implicates a role for ROS and oxidative stress in PAH, yet mechanisms linking these systems are elusive. We hypothesised that either E2 or the E2 metabolite, 16α-hydroxyestrone (16αOHE1), stimulates Nox-induced ROS generation and proliferative responses in human pulmonary artery smooth muscle cells (hPASMC) and that, in PAH, aberrant growth signaling promotes vascular remodeling. The pathophysiological significance of E2-Nox-dependent processes was studied in female Nox1-/- and Nox4-/- mice exposed to chronic hypoxia. HPASMCs from female non-PAH individuals (control hPASMC) and female PAH patients (PAH-hPASMC) were exposed to E2 and 16αOHE1 in the presence/absence of inhibitors of Nox1, Nox2 and Nox4, cytochrome P450 1B1 (CYP1B1) and estrogen receptors (ER), ERα, ERβ and G-protein coupled estrogen receptor (GPER). E2, through ERβ, increased Nox1 and Nox4-derived O2- and redox-sensitive growth in control hPASMCs. 16αOHE1, through ERα activation, stimulated O2- production in control hPASMCs and PAH-hPASMCs. E2- -stimulated O2- production was inhibited by CYP1B1 blockade. Basal expression of Nox1 and Nox4 was potentiated in PAH-hPASMCs. In control hPASMCs, 16αOHE1 increased p47phox and poldip2 and Nox1 expression. In PAH-hPASMCs, 16αOHE1 decreased nuclear factor erythroid-2-related factor-2 (Nrf-2) activity and expression of Nrf-2-regulated antioxidant genes in PAH-hPASMCs. Female Nox1-/-, but not Nox4-/- mice were protected against chronic hypoxia-induced pulmonary hypertension and vascular remodeling. Expression of CYP1B1 was increased in pulmonary arteries of wild-type and Nox4-/- mice exposed to hypoxia, yet this induction in CYP1B1 expression was absent in those arteries from hypoxic Nox1-/- mice. Findings detailed in Chapter 3 show that in PAH-hPASMCs, 16αOHE1 stimulates redox-sensitive cell growth through both Nox1 and Nox4. In vivo studies exhibited protection against pulmonary hypertension specifically in Nox1-/- mice. This study provides new insights through Nox1/ROS and Nrf-2 whereby 16αOHE1 influences hPASMC function, which when upregulated may contribute to vascular injury in PAH