Role of NADPH oxidases and KDR channels in the pathophysiology of hypoxia induced pulmonary hypertension

摘要

The pulmonary vasculature has the unique ability to undergo vasoconstriction in response to acute hypoxia, a physiological mechanism known as hypoxic pulmonary vasoconstriction (HPV). Sustained or chronic hypoxia, however, leads to proliferation of vascular smooth muscle cells of pulmonary arterioles, which causes a permanent increase in pulmonary vascular resistance, and may lead to right heart dysfunction. The underlying mechanisms of vascular proliferation under chronic hypoxia have not been fully defined. The NADPH oxidases are one family of recently discovered molecules which generate reactive oxygen species (ROS) and have been suggested to be important for cellular signaling under physiological conditions. However, NADPH oxidase generated oxidative stress can also lead to inflammation, vascular smooth muscle cell proliferation and endothelial damage under pathological conditions. Many homologs of NADPH oxidases exist, the classical homolog is gp91phox or NOX2, and the recently discovered homologs include NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2. Superoxide production by classical gp91phox is induced by assembly of the cytosolic subunits such as p40phox, p47phox and p67phox with membrane-bound gp91phox complex. A previous report from our laboratory has shown that the knockout mice of p47phox subunit exhibit reduced acute HPV as compared to wild type mice suggesting an essential role of NADPH oxidases in regulation of vascular tone in acute hypoxia. Against this background, the current thesis aimed to elucidate the role of NADPH oxidases in vascular remodeling in chronic hypoxia, and its possible downstream mediators. Screening of NADPH oxidase expression revealed that all subunits were expressed in the lung homogenate and that NOX4 was prominently up-regulated under chronic hypoxia. The NOX4 mRNA was also up-regulated in the microdissected vessels of mice exposed up to three weeks of chronic hypoxia. In addition, a functional interference with NOX4 using NOX4 siRNA resulted in reduced ROS production and reduced proliferation of pulmonary arterial smooth muscle cells (PASMC) revealing an important contribution of NOX4 in PASMC proliferation and particularly in hypoxia induced pulmonary hypertension. Intriguingly, a similar reflection was found in lungs of patients with idiopathic pulmonary hypertension that underwent lung transplantation. Further experiments demonstrated that NOX4 inhibited voltage-gated delayed rectifier K+ channels (KDR) under hypoxia. Pharmacological inhibition with apocynin and genetic ablation with NOX4siRNA resulted in increased KDR current under hypoxia. In addition, the current study demonstrated that NOX4 is essential for ET-1 mediated calcium influx in PASMC as NOX4 knockdown using NOX4 siRNA abolished the ET-1 mediated calcium influx under chronic hypoxia. Thus, the NOX4-ROS-KDR-[Ca2+] pathway may contribute to the development of pulmonary hypertension.