NOX4 mediates hypoxia-induced proliferation of human pulmonary artery smooth muscle cells: the role of autocrine production of transforming growth factor-β1 and insulin-like growth factor binding protein-3

Abstract

Persistent hypoxia can cause pulmonary arterial hypertension that may be associated with significant remodeling of the pulmonary arteries, including smooth muscle cell proliferation and hypertrophy. We previously demonstrated that the NADPH oxidase homolog NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factor-beta1 (TGF-beta1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased reactive oxygen species generation and NOX4 gene expression, and is inhibited by antioxidants, the flavoenzyme inhibitor diphenyleneiodonium (DPI), and NOX4 gene silencing. HPASMC proliferation and NOX4 expression are also observed when media from hypoxic HPASMC are added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-beta1 and insulin-like growth factor binding protein-3 (IGFBP-3) are both increased in the media of hypoxic HPASMC, and increased IGFBP-3 gene expression is noted in hypoxic HPASMC. Treatment with anti-TGF-beta1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with small interfering RNA (siRNA) decreases NOX4 gene expression and hypoxic proliferation. Conversely, NOX4 silencing does not decrease hypoxic IGFBP-3 gene expression or secreted protein. Smad inhibition does not but the phosphatidylinositol 3-kinase (PI3K) signaling pathway inhibitor LY-294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP-3 secretion, and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-beta1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-beta1 that acts in an autocrine fashion to induce IGFBP-3 through PI3K/Akt. IGFBP-3 increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling.

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, Vascular Noxs, and Hypertension: Focus on Translational and Clinical Research

“…Nox4 appears to generate H 2 O 2 , although the primary product is probably O 2 -, which is rapidly dismutated to H 2 O 2 (202,232). Nox4 contributes to basal ROS production through its constitutive activity and to increased ROS generation when induced by Ang II, glucose, tumor necrosis factor a, and growth factors (90,127). Recent studies have identified a 28-kDa Splice Variant of Nox4 located in the nucleus of vascular cells, which may be important in pathophysiologic effects through modulation of nuclear signaling and DNA damage (9).…”

Activation of peroxisome proliferator-activated receptor γ ameliorates monocrotaline-induced pulmonary arterial hypertension in rats

“…Several signaling pathways are associated with the growth and apoptosis of PASMCs during PAH (12,13). For example, stimulation of human PASMCs with platelet-derived growth factor induced PI3K-dependent activation of Akt, p70 S6 kinase and ribosomal protein S6, suggesting that PI3K signaling is necessary to mediate human PASMCs proliferation and the activation of PI3K may play an important role in vascular remodeling (14).…”