Gosemann Jan-H, Friedmacher Florian, Hunziker Manuela, Alvarez Luis, Corcionivoschi Nicolae, Puri Prem
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
Pediatr Surg Int. 2013 Jan;29(1):3-8. doi: 10.1007/s00383-012-3209-0.
Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). NADPH oxidases (Nox) are the main source of superoxide production in vasculature. Nox4 is highly expressed in the smooth muscle and endothelial cells of the vascular wall and increased activity has been reported in the pulmonary vasculature of both experimental and human pulmonary hypertension. Peroxisome proliferator-activated receptor (PPARγ) is a key regulator of Nox4 expression. Targeted depletion of PPARγ results in pulmonary hypertension phenotype whereas activation of PPARγ attenuates pulmonary hypertension and reduces Nox4 production. The nitrofen-induced CDH model is an established model to study the pathogenesis of pulmonary hypertension in CDH. It has been previously reported that PPARγ-signaling is disrupted during late gestation and H(2)O(2) production is increased in nitrofen-induced CDH. We designed this study to investigate the hypothesis that Nox4 expression and activation is increased and vascular PPARγ is decreased in nitrofen-induced CDH.
Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into control and CDH. RT-PCR, western blotting and confocal-immunofluorescence-double-staining were performed to determine pulmonary expression levels of PPARγ, Nox4 and Nox4-activation (p22(phox)).
There was a marked increase in medial and adventitial thickness in pulmonary arteries of all sizes in CDH compared to controls. Pulmonary Nox4 levels were significantly increased whereas PPARγ levels were decreased in nitrofen-induced CDH compared to controls. Western blotting revealed increased pulmonary protein expression of the Nox4-activating subunit p22(phox) and decreased protein expression of PPARγ in CDH compared to controls. Confocal-microscopy confirmed markedly increased pulmonary expression of the Nox4 activating subunit p22(phox) accompanied by decreased perivascular PPARγ expression in lungs of nitrofen-exposed fetuses compared to controls.
To our knowledge, the present study is the first to report increased Nox4 production in the pulmonary vasculature of nitrofen-induced CDH. Down-regulation of the PPARγ-signaling pathway may lead to increased superoxide production, resulting in pulmonary vascular dysfunction and contributing to pulmonary hypertension in the nitrofen-induced CDH model. PPARγ-activation inhibiting Nox4 production may therefore represent a potential therapeutic approach for the treatment of pulmonary hypertension in CDH.
持续性肺动脉高压仍然是先天性膈疝（CDH）患者死亡和发病的主要原因。NADPH氧化酶（Nox）是脉管系统中超氧化物产生的主要来源。Nox4在血管壁的平滑肌和内皮细胞中高表达，并且在实验性和人类肺动脉高压的肺血管系统中均有报道其活性增加。过氧化物酶体增殖物激活受体（PPARγ）是Nox4表达的关键调节因子。靶向敲除PPARγ会导致肺动脉高压表型，而激活PPARγ可减轻肺动脉高压并减少Nox4的产生。硝基芬诱导的CDH模型是研究CDH中肺动脉高压发病机制的成熟模型。此前已有报道称，在妊娠晚期PPARγ信号被破坏，并且在硝基芬诱导的CDH中过氧化氢（H₂O₂）的产生增加。我们设计本研究以探究以下假设：在硝基芬诱导的CDH中，Nox4的表达和激活增加，而血管PPARγ减少。
在妊娠第9天（D9）给怀孕大鼠注射硝基芬或赋形剂。在D21处死胎儿，并分为对照组和CDH组。进行逆转录聚合酶链反应（RT-PCR）、蛋白质印迹法和共聚焦免疫荧光双重染色，以确定肺组织中PPARγ、Nox4和Nox4激活（p22phox）的表达水平。
与对照组相比，CDH组所有大小肺动脉的中膜和外膜厚度均显著增加。与对照组相比，硝基芬诱导的CDH组肺组织中Nox4水平显著升高，而PPARγ水平降低。蛋白质印迹法显示，与对照组相比，CDH组肺组织中Nox4激活亚基p22phox的蛋白表达增加，而PPARγ的蛋白表达减少。共聚焦显微镜检查证实，与对照组相比，暴露于硝基芬的胎儿肺组织中Nox4激活亚基p22phox的肺组织表达显著增加，同时血管周围PPARγ表达减少。
据我们所知，本研究首次报道了硝基芬诱导的CDH肺血管系统中Nox4产生增加。PPARγ信号通路的下调可能导致超氧化物产生增加，从而导致肺血管功能障碍，并促成硝基芬诱导的CDH模型中的肺动脉高压。因此，激活PPARγ抑制Nox4产生可能是治疗CDH中肺动脉高压的一种潜在治疗方法。
