Ismail Saleh, Sturrock Anne, Wu Ping, Cahill Barbara, Norman Kimberly, Huecksteadt Thomas, Sanders Karl, Kennedy Thomas, Hoidal John
Division of Respiratory, Critical Care, and Occupational Pulmonary Medicine, University of Utah Health Sciences Centerand VA Medical Center, Salt Lake City, Utah 84132, USA.
Am J Physiol Lung Cell Mol Physiol. 2009 Mar;296(3):L489-99. doi: 10.1152/ajplung.90488.2008. Epub 2008 Nov 26.
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.
持续性缺氧可导致肺动脉高压，这可能与肺动脉的显著重塑有关，包括平滑肌细胞增殖和肥大。我们之前证明，NADPH氧化酶同系物NOX4通过转化生长因子-β1（TGF-β1）介导人肺动脉平滑肌细胞（HPASMC）增殖。我们现在发现，缺氧可增加体外HPASMC增殖，同时伴随着活性氧生成增加和NOX4基因表达增加，且抗氧化剂、黄素酶抑制剂二苯基碘鎓（DPI）和NOX4基因沉默可抑制这种增殖。当将缺氧HPASMC的培养基添加到在常氧条件下生长的HPASMC中时，也可观察到HPASMC增殖和NOX4表达，提示存在自分泌刺激。缺氧HPASMC培养基中的TGF-β1和胰岛素样生长因子结合蛋白-3（IGFBP-3）均增加，且缺氧HPASMC中IGFBP-3基因表达增加。用抗TGF-β1抗体处理可减弱NOX4和IGFBP-3基因表达、IGFBP-3蛋白在培养基中的积累以及增殖。用小干扰RNA（siRNA）抑制IGFBP-3表达可降低NOX4基因表达和缺氧增殖。相反，NOX4沉默不会降低缺氧IGFBP-3基因表达或分泌蛋白。Smad抑制无效，但磷脂酰肌醇3-激酶（PI3K）信号通路抑制剂LY-294002可抑制NOX4和IGFBP-3基因表达、IGFBP-3分泌以及缺氧导致的细胞增殖。缺氧HPASMC的免疫印迹显示，TGF-β1介导的丝氨酸/苏氨酸激酶（Akt）磷酸化增加，这与缺氧诱导PI3K/Akt信号通路激活以促进增殖一致。我们得出结论，缺氧的HPASMC产生TGF-β1，其以自分泌方式通过PI3K/Akt诱导IGFBP-3。IGFBP-3增加NOX4基因表达，导致HPASMC增殖。这些观察结果加深了我们对缺氧性肺血管重塑的理解。
