1 Department of Pediatrics, Stanford University School of Medicine, Stanford, California; and.
2 Department of Pediatric and Adolescent Medicine, Center of Molecular Medicine Cologne, University Hospital of Cologne, Cologne, Germany.
Am J Respir Cell Mol Biol. 2018 Nov;59(5):623-634. doi: 10.1165/rcmb.2017-0332OC.
Mechanical ventilation with O-rich gas (MV-O) inhibits alveologenesis and lung growth. We previously showed that MV-O increased elastase activity and apoptosis in lungs of newborn mice, whereas elastase inhibition by elafin suppressed apoptosis and enabled lung growth. Pilot studies suggested that MV-O reduces lung expression of prosurvival factors phosphorylated epidermal growth factor receptor (pEGFR) and Krüppel-like factor 4 (Klf4). Here, we sought to determine whether apoptosis and lung growth arrest evoked by MV-O reflect disrupted pEGFR-Klf4 signaling, which elafin treatment preserves, and to assess potential biomarkers of bronchopulmonary dysplasia (BPD). Five-day-old mice underwent MV with air or 40% O for 8-24 hours with or without elafin treatment. Unventilated pups served as controls. Immunoblots were used to assess lung pEGFR and Klf4 proteins. Cultured MLE-12 cells were exposed to AG1478 (EGFR inhibitor), Klf4 siRNA, or vehicle to assess effects on proliferation, apoptosis, and EGFR regulation of Klf4. Plasma elastase and elafin levels were measured in extremely premature infants. In newborn mice, MV with air or 40% O inhibited EGFR phosphorylation and suppressed Klf4 protein content in lungs (vs. unventilated controls), yielding increased apoptosis. Elafin treatment inhibited elastase, preserved lung pEGFR and Klf4, and attenuated the apoptosis observed in lungs of vehicle-treated mice. In MLE-12 studies, pharmacological inhibition of EGFR and siRNA suppression of Klf4 increased apoptosis and reduced proliferation, and EGFR inhibition decreased Klf4. Plasma elastase levels were more than twofold higher, without a compensating increase of plasma elafin, in infants with BPD, compared to infants without BPD. These findings indicate that pEGFR-Klf4 is a novel prosurvival signaling pathway in lung epithelium that MV disrupts. Elafin preserves pEGFR-Klf4 signaling and inhibits apoptosis, thereby enabling lung growth during MV. Together, our animal and human data raise the question: would elastase inhibition prevent BPD in high-risk infants exposed to MV-O?
富氧机械通气（MV-O）抑制肺泡发生和肺生长。我们之前的研究表明，MV-O 增加了新生小鼠肺部的弹性蛋白酶活性和细胞凋亡，而弹性蛋白酶抑制剂 Elafin 抑制了细胞凋亡并促进了肺生长。初步研究表明，MV-O 降低了肺组织中促生存因子磷酸化表皮生长因子受体（pEGFR）和 Krüppel 样因子 4（Klf4）的表达。在此，我们试图确定 MV-O 引起的细胞凋亡和肺生长停滞是否反映了被 Elafin 治疗保留的 pEGFR-Klf4 信号通路的中断，并评估支气管肺发育不良（BPD）的潜在生物标志物。5 日龄的小鼠接受空气或 40%氧气的 MV 通气 8-24 小时，同时给予或不给予 Elafin 治疗。未通气的幼仔作为对照。免疫印迹用于评估肺 pEGFR 和 Klf4 蛋白。将 MLE-12 细胞暴露于 AG1478（EGFR 抑制剂）、Klf4 siRNA 或载体中，以评估对增殖、凋亡和 EGFR 对 Klf4 的调节的影响。测量极低出生体重儿的血浆弹性蛋白酶和 Elafin 水平。在新生小鼠中，空气或 40%氧气的 MV 抑制了 EGFR 的磷酸化，并抑制了肺中的 Klf4 蛋白含量（与未通气的对照组相比），导致细胞凋亡增加。Elafin 治疗抑制弹性蛋白酶，保留肺 pEGFR 和 Klf4，并减轻了载体处理的小鼠肺中观察到的细胞凋亡。在 MLE-12 研究中，EGFR 的药理学抑制和 Klf4 的 siRNA 抑制增加了细胞凋亡并降低了增殖，并且 EGFR 抑制降低了 Klf4。与没有 BPD 的婴儿相比，患有 BPD 的婴儿的血浆弹性蛋白酶水平高出两倍多，而血浆 Elafin 没有相应增加。这些发现表明，pEGFR-Klf4 是肺上皮中的一种新的促生存信号通路，MV 会破坏该信号通路。Elafin 保留了 pEGFR-Klf4 信号通路并抑制了细胞凋亡，从而在 MV 期间促进了肺生长。我们的动物和人类数据共同提出了一个问题：在接受 MV-O 的高危婴儿中，弹性蛋白酶抑制是否可以预防 BPD？
