Groenman Freek A, Rutter Martin, Wang Jinxia, Caniggia Isabella, Tibboel Dick, Post Martin
CIHR Group in Lung Development, Hospital for Sick Children Research Institute, Department of Pediatrics and Institute of Medical Sciences, University of Toronto, Toronto, Ontario, Canada.
Am J Physiol Lung Cell Mol Physiol. 2007 Sep;293(3):L557-67. doi: 10.1152/ajplung.00486.2006. Epub 2007 Jun 1.
Low oxygen stimulates pulmonary vascular development and airway branching and involves hypoxia-inducible factor (HIF). HIF is stable and initiates expression of angiogenic factors under hypoxia, whereas normoxia triggers hydroxylation of the HIF-1alpha subunit by prolyl hydroxylases (PHDs) and subsequent degradation. Herein, we investigated whether chemical stabilization of HIF-1alpha under normoxic (20% O(2)) conditions would stimulate vascular growth and branching morphogenesis in early lung explants. Tie2-LacZ (endothelial LacZ marker) mice were used for visualization of the vasculature. Embryonic day 11.5 (E11.5) lung buds were dissected and cultured in 20% O(2) in the absence or presence of cobalt chloride (CoCl(2), a hypoxia mimetic), dimethyloxalylglycine (DMOG; a nonspecific inhibitor of PHDs), or desferrioxamine (DFO; an iron chelator). Vascularization was assessed by X-gal staining, and terminal buds were counted. The fine vascular network surrounding the developing lung buds seen in control explants disappeared in CoCl(2)- and DFO-treated explants. Also, epithelial branching was reduced in the explants treated with CoCl(2) and DFO. In contrast, DMOG inhibited branching but stimulated vascularization. Both DFO and DMOG increased nuclear HIF-1alpha protein levels, whereas CoCl(2) had no effect. Since HIF-1alpha induces VEGF expression, the effect of SU-5416, a potent VEGF receptor (VEGFR) blocker, on early lung development was also investigated. Inhibition of VEGFR2 signaling in explants maintained under hypoxic (2% O(2)) conditions completely abolished vascularization and slightly decreased epithelial branching. Taken together, the data suggest that DMOG stabilization of HIF-1alpha during early development leads to a hypervascular lung and that airway branching proceeds without the vasculature, albeit at a slower rate.
低氧刺激肺血管发育和气道分支，涉及缺氧诱导因子（HIF）。HIF在缺氧条件下稳定并启动血管生成因子的表达，而常氧则触发脯氨酰羟化酶（PHD）对HIF-1α亚基的羟基化及随后的降解。在此，我们研究了在常氧（20% O₂）条件下HIF-1α的化学稳定是否会刺激早期肺外植体中的血管生长和分支形态发生。使用Tie2-LacZ（内皮LacZ标记）小鼠来可视化脉管系统。解剖胚胎第11.5天（E11.5）的肺芽，并在20% O₂中培养，分别添加或不添加氯化钴（CoCl₂，一种缺氧模拟物）、二甲基乙二酰甘氨酸（DMOG；PHD的非特异性抑制剂）或去铁胺（DFO；一种铁螯合剂）。通过X-gal染色评估血管形成，并计数终末芽。在对照外植体中可见的围绕发育中肺芽的精细血管网络在CoCl₂和DFO处理的外植体中消失。此外，用CoCl₂和DFO处理的外植体中上皮分支减少。相比之下，DMOG抑制分支但刺激血管形成。DFO和DMOG均增加核HIF-蛋白水平，而CoCl₂无此作用。由于HIF-1α诱导VEGF表达，还研究了强效VEGF受体（VEGFR）阻滞剂SU-5416对早期肺发育的影响。在低氧（2% O₂）条件下维持的外植体中抑制VEGFR2信号完全消除了血管形成，并轻微降低了上皮分支。综上所述，数据表明在早期发育过程中DMOG使HIF-1α稳定导致肺血管增生，并且气道分支在没有脉管系统的情况下仍能进行，尽管速率较慢。
