Pulmonary Hypertension Group, Center for Perinatal Research, Research Institute at Nationwide Children's Hospital, and Department of Pediatrics, Ohio State University, Columbus, Ohio, USA.
Am J Physiol Lung Cell Mol Physiol. 2010 Apr;298(4):L600-6. doi: 10.1152/ajplung.00122.2009. Epub 2010 Feb 5.
We hypothesized that hypoxia would activate epidermal growth factor receptor (EGFR) tyrosine kinase, leading to increased arginase expression and resulting in proliferation of human pulmonary microvascular endothelial cell (hPMVEC). To test this hypothesis, hPMVEC were incubated in normoxia (20% O(2), 5% CO(2)) or hypoxia (1% O(2), 5% CO(2)). Immunoblotting for EGFR and proliferating cell nuclear antigen was done, and protein levels of both total EGFR and proliferating cell nuclear antigen were greater in hypoxic hPMVEC than in normoxic hPMVEC. Furthermore, hypoxic hPMVEC had greater levels of EGFR activity than did normoxic hPMVEC. Hypoxic hPMVEC had a twofold greater level of proliferation compared with normoxic controls, and this increase in proliferation was prevented by the addition of AG-1478 (a pharmacological inhibitor of EGFR). Immunoblotting for arginase I and arginase II demonstrated a threefold induction in arginase II protein levels in hypoxia, with little change in arginase I protein levels. The hypoxic induction of arginase II protein was prevented by treatment with AG-1478. Proliferation assays were performed in the presence of arginase inhibitors, and hypoxia-induced proliferation was also prevented by arginase inhibition. Finally, treatment with an EGFR small interfering RNA prevented hypoxia-induced proliferation and urea production. These findings demonstrate that hypoxia activates EGFR tyrosine kinase, leading to arginase expression and thereby promoting proliferation in hPMVEC.
我们假设缺氧会激活表皮生长因子受体（EGFR）酪氨酸激酶，导致精氨酸酶表达增加，从而促进人肺微血管内皮细胞（hPMVEC）的增殖。为了验证这一假设，将 hPMVEC 在常氧（20% O(2)，5% CO(2))或低氧（1% O(2)，5% CO(2))下孵育。进行 EGFR 和增殖细胞核抗原的免疫印迹，发现低氧 hPMVEC 中的总 EGFR 和增殖细胞核抗原的蛋白水平均高于常氧 hPMVEC。此外，低氧 hPMVEC 的 EGFR 活性水平高于常氧 hPMVEC。低氧 hPMVEC 的增殖水平比常氧对照组高两倍，而这种增殖的增加可以通过添加 AG-1478（EGFR 的药理学抑制剂）来预防。免疫印迹分析表明，低氧条件下精氨酸酶 II 的蛋白水平增加了三倍，而精氨酸酶 I 的蛋白水平变化不大。用 AG-1478 处理可预防低氧诱导的精氨酸酶 II 蛋白表达。在存在精氨酸酶抑制剂的情况下进行增殖测定，并且低氧诱导的增殖也可以通过精氨酸酶抑制来预防。最后，用 EGFR 小干扰 RNA 处理可预防低氧诱导的增殖和尿素生成。这些发现表明，缺氧激活 EGFR 酪氨酸激酶，导致精氨酸酶表达，从而促进 hPMVEC 的增殖。
