Raw data 5.

Angiogenic signaling pathway activation has been shown to accelerate compensatory lung growth (CLG) after unilateral pneumonectomy (PNX). Therefore, studying specific genes regulating angiogenic signaling pathways is a novel strategy to promote CLG. EdU, flow cytometry and tube formation experiments were performed to test the metabolism of human pulmonary microvascular endothelial cells (HPMECs). Western blotting was used to analyze the levels of promyelocytic leukemia zinc finger protein (PLZF), kelch-like ECH-associated protein 1 (Keap1), hypoxia-inducible factor-1α (HIF-1α), hemeoxygenase-1 (HO-1), quinone oxidoreductase (NQO1), nuclear factor E2-related factor 2 (Nrf2) and other proteins. The proliferation of pulmonary endothelial cells was assessed by Ki67 double staining. A unilateral PNX mouse model was constructed, and changes in lung volume and weight were assessed. Our bioinformatics results suggested that PLZF showed a clear downward trend after unilateral PNX. PLZF overexpression significantly promoted HPMECs proliferation and angiogenesis and inhibited their apoptosis. Further studies revealed that both Keap1 overexpression and Nrf2 silencing altered the effects of PLZF overexpression on HPMECs and inhibited their apoptosis. Notably, HIF-1α silencing reversed the effect of PLZF overexpression on HPMECs angiogenesis but not on proliferation or apoptosis. Knockdown of Nrf2 not only affected HPMECs proliferation and apoptosis but also affected angiogenesis. An in vivo study confirmed that PLZF overexpression promoted an increase in residual lung volume and lung weight in mice after unilateral PNX and significantly promoted the proliferation of lung endothelial cells. In conclusion, our study revealed that PLZF promotes HPMECs proliferation and angiogenesis and accelerates CLG by inhibiting Keap1 activation of the Nrf2 and HIF-1α/VEGF signaling pathways.

血管生成信号通路激活已被证实可加速单侧肺切除术（unilateral pneumonectomy, PNX）后代偿性肺生长（compensatory lung growth, CLG）。因此，研究调控血管生成信号通路的特定基因，是促进代偿性肺生长的全新策略。本研究采用EdU、流式细胞术及管形成实验，检测人肺微血管内皮细胞（human pulmonary microvascular endothelial cells, HPMECs）的代谢状态；通过蛋白质印迹法（Western blotting）分析早幼粒细胞白血病锌指蛋白（promyelocytic leukemia zinc finger protein, PLZF）、Kelch样ECH相关蛋白1（kelch-like ECH-associated protein 1, Keap1）、缺氧诱导因子-1α（hypoxia-inducible factor-1α, HIF-1α）、血红素氧合酶-1（hemeoxygenase-1, HO-1）、醌氧化还原酶（quinone oxidoreductase, NQO1）及核因子E2相关因子2（nuclear factor E2-related factor 2, Nrf2）等蛋白的表达水平。采用Ki67双染色法评估肺内皮细胞的增殖情况。构建单侧肺切除术小鼠模型，检测残肺体积与重量的变化。生物信息学分析结果显示，单侧肺切除术后PLZF的表达水平呈现显著下降趋势。PLZF过表达可显著促进人肺微血管内皮细胞的增殖与血管生成，并抑制其凋亡。进一步研究表明，Keap1过表达与Nrf2沉默均可逆转PLZF过表达对人肺微血管内皮细胞的作用，并抑制细胞凋亡。值得注意的是，HIF-1α沉默可逆转PLZF过表达对人肺微血管内皮细胞血管生成的影响，但对其增殖与凋亡无显著作用。敲低Nrf2不仅可影响人肺微血管内皮细胞的增殖与凋亡，还可影响其血管生成过程。体内实验证实，PLZF过表达可促进单侧肺切除术后小鼠残肺体积与重量的增加，并显著促进肺内皮细胞的增殖。综上，本研究揭示PLZF可通过抑制Keap1介导的Nrf2及HIF-1α/VEGF信号通路激活，促进人肺微血管内皮细胞增殖与血管生成，进而加速代偿性肺生长。