The Relationship Between NOX4 And ENOS Expression In Pulmonary Vasculature In Mouse Model Of Hypoxia Pulmonary Hypertension

Background: Chronic hypoxia induces constriction of pulmonary vasculature causing irreversible changes of vascular remodeling, characterized by medial and adventitial thickening of the muscular and elastic vessels and muscularization of previously nonmuscularized more distal small vessels. This is the basis of pulmonary arterial hypertension (PAH).Nicotinamide adenine dinucleotide phosphate oxidase(NADPH oxidase, NOX) is one of the major sources of reactive oxygen species (ROS) in vasculature. The important role of NADPH oxidase in nonphagocytic was emphasized in recent years. Six homologs of the cytochrome subunit gp91phox of the phagocyte NADPH oxidase were found: NOX1, NOX3, NOX4, NOX5, DUOX1, and DUOX2. Together with the phagocyte NADPH oxidase itself (NOX2/gp91phox), the homologs are now referred to as the NOX family of NADPH oxidases. These enzymes share the capacity to transport electrons across the plasma membrane and to generate superoxide and other downstream ROS. The physiological functions of NOX family enzymes include host defense, post-translational processing of proteins, cellular signaling, regulation of gene expression, cell differentiation, hyperplasia, apoptosis, in particular hypertrophy, remodeling, and angiogenesis in the vascular system. Mittal and colleagues first reported all family members of NOX expressed in pulmonary vasculature. NOX4 appeared to be the most prominent NOX expressed in pulmonary arteries. They suggested that NOX4 play an important role in the chronic hypoxia responses of pulmonary vasculature and the development of PAH.Nitric oxide (NO) is a highly reactive gas that produces a variety of physiological and pathophysiological effects, including inhibition of vascular smooth muscle proliferation and vascular remodeling. It plays an important role in the development of chronic hypoxia induced PAH. Endothelium-derived NO,produced by endothelial nitric oxide synthase (eNOS) acts on soluble guanylyl cyclase in the vascular smooth muscle to stimulate guanosine 3',5'-cyclic monophosphate (cGMP) formation, which leads to vasodilation. Under chronic hypoxia condition, endothelium-derived NO act as antiproliferation in vascular smooth muscle.To date, only limited studies have been reported about the expression and regulation of NOX4 in the pathogenesis of chronic hypoxia induced PAH. Those studies about the expression of eNOS in lung vascular smooth muscle were inconsistent. There was no report about the relationship between the expression change of NOX4 and eNOS in mouse lung vascular following chronic hypoxia. In this study we investigated the effect of chronic hypoxia on the expression of eNOS and NOX4 in mouse lung by real time PCR and immunohistochemisty. We also studied the express difference between WT (+/+) and eNOS(-/- )mouse. The relationship between NOX4 and eNOS expressions were also analyzed.Methods: WT(+/+)C57BL/6J male mice were exposed to either normoxia or hypoxia (10±0.5% 02) for 2, 4, 6 days or 2 weeks. Following the intraperitoneal injection with sodium pentobarbital lung, lung tissues were harvested. Left lung was fixed with 10% neutral formalin and processed for paraffin-embedding. The immunohistochemisty assay was used for evaluating SM a-actin or eNOS expressions. The right lung lobe was flash-frozen in liquid nitrogen and stored at -70℃until assay. The real time PCR with primers of eNOS or NOX4 were performed. Eight WT(+/+)C57BL/6J or eNOS (-/-) mouse male mice in normoxia condition were also injected intraperitoneally with sodium pentobarbital lung and lung tissues were harvested for real time PCR to detect NOX4 mRNA expressions.Result: WT(+/+) mice exposed to hypoxia exhibited significant vascular remodeling characterized by medial and adventitial thickening of the muscular and elastic vessels and muscularization of previously nonmuscularized more distal small vessels. The expressions of mRNA of eNOS or NOX4 were significantly increased in the lungs in chronically hypoxic rats compared with normoxia controls. The immunohistochemical staining using an isoform-specific eNOS antibody demonstrated an significant increase in eNOS expression in the lung vascular endothelium. In normoxia controls,NOX4 mRNA expression in eNOS (-/-) lung tissue was significantly increased compared with WT(+/+) mice.Conclusions:1 In mouse model, chronic hypoxia induces significant pulmonary vascular remodeling. The expression of eNOS and NOX4 mRNA in lung vascular was significantly increased. The eNOS protein expressions in the lung vascular endothelial cell were also increased.2 A positive correlation between NOX4 and eNOS mRNA expression in pulmonary vasculature could be found in mouse model of hypoxia pulmonary hypertension. Knockout eNOS gene leads to an increased NOX4 expression in mouse lung tissue.