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

Persistent hypoxia can cause pulmonary arterial hypertension (PAH) 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 homologue NOX4 mediates human pulmonary artery smooth muscle cell (HPASMC) proliferation by transforming growth factorβ1 (TGF-β1). We now show that hypoxia increases HPASMC proliferation in vitro, accompanied by increased ROS generation and NOX4 gene expression, and 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 is added to HPASMC grown in normoxic conditions, suggesting autocrine stimulation. TGF-β1 and insulinlike 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-β1 antibody attenuates NOX4 and IGFBP-3 gene expression, accumulation of IGFBP-3 protein in media, and proliferation. Inhibition of IGFBP-3 expression with 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 LY294002 does inhibit NOX4 and IGFBP-3 gene expression, IGFBP3 secretion and cellular proliferation resulting from hypoxia. Immunoblots from hypoxic HPASMC reveal increased TGF-β1-mediated phosphorylation of the serine/threonine kinase (Akt), consistent with hypoxia-induced activation of PI3-K/Akt signaling pathways to promote proliferation. We conclude that hypoxic HPASMC produce TGF-β1 that acts in an autocrine fashion to induce IGFBP3. IGFBP-3 through PI3-K/Akt increases NOX4 gene expression, resulting in HPASMC proliferation. These observations add to our understanding hypoxic pulmonary vascular remodeling. IGFBP-3 and NOX4 Mediate Hypoxic HPASMC Proliferation 3