NOX4 Regulates ROS Levels Under Normoxic and Hypoxic Conditions, Triggers Proliferation, and Inhibits Apoptosis in Pulmonary Artery Adventitial Fibroblasts

Abstract

The NADPH oxidases are involved in vascular remodeling processes and oxygen sensing. Hypoxia-induced pulmonary arterial remodeling results in thickening of the vessel wall and reduction of the area of vessel lumen, leading to pulmonary hypertension and cor pulmonale. The proliferation of pulmonary artery adventitial fibroblasts (PAFB) is critically involved in this process. In this study, we analyzed the role of the non-phagocytic NADPH oxidase subunits NOX1 and NOX4 in PAFB. NOX4 was predominantly expressed in comparison to NOX1 at mRNA levels. Under hypoxic conditions, NOX4 was significantly upregulated at mRNA and protein levels. Silencing of NOX4 by siRNA caused reduction of ROS levels under both normoxic and hypoxic (24 h) conditions and suppressed the significant hypoxic-induced ROS increase. PAFB proliferation was significantly decreased in cells transfected with NOX4 siRNA, whereas apoptosis was enhanced. Also, the expression of NOX4 was studied in PAFB isolated from the lungs of patients with idiopathic pulmonary arterial hypertension (IPAH). Interestingly, a significant increase of NOX4 mRNA expression was observed under hypoxic conditions in PAFB from the lungs with IPAH compared to healthy donors. In conclusion, NOX4 maintains ROS levels under normoxic and hypoxic conditions and enhances proliferation and inhibits apoptosis of PAFB.

Reactive Oxygen Species in Inflammation and Tissue Injury

“…NOX4 is the predominant homolog expressed in human lung microvascular and human pulmonary arterial endothelial cells compared with NOX1, NOX2, NOX3, or NOX5 (338). Inflammatory stimuli LPS, TNF a, hyperoxia, TGF-b, and hypoxia were demonstrated to enhance ROS generation via NOX4 (29,191,242,295,335,338). The expression of intercellular adhesion molecule-1 (ICAM-1), IL-8, and MCP-1 in endothelial cells in response to LPS was demonstrated to be dependent on NOX4 activation (335).…”

Reactive oxygen species and vascular biology: implications in human hypertension

“…99 Nox2 is the major source of ROS in endothelial cells under basal conditions and in pathological conditions Nox1 and Nox4 may be upregulated. 100 Nox2, Nox4 and Nox5 seem to localize primarily in the perinuclear area associated with membranes on the endoplasmic reticulum and nucleus although Nox2 is also found in the plasma membrane within cholesterol-enriched domains, which may serve as signaling platforms for ROS generation in vascular disease. 101 Vascular smooth muscle cells possess Nox2 (in human resistance arteries) and Nox4, which are major sources of ROS.…”

NOX Enzymes and Pulmonary Disease

“…A role for NOX2 in hypoxiainduced endothelial dysfunction involving intrapulmonary arteries has been demonstrated (33). In pulmonary artery adventitial fibroblasts, hypoxia significantly upregulates NOX4 expression at the mRNA and protein levels, whereas silencing of NOX4 by siRNA reduces ROS levels and decreases cellular proliferation (65). Hypoxia-dependent development of PAH in mice has been linked to increased NOX4 expression in pulmonary artery smooth muscle cells (SMCs) (79), suggesting a key role for NOX4 in the vascular remodeling associated with hypoxia-induced PAH.…”