PLZF promotes compensatory lung growth by increasing HPMEC proliferation and angiogenesis

Abstract

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.

Fig 1. PLZF is significantly downregulated after PNX.

A: Heatmap of significantly different genes. B: Volcano map of significantly different genes. C: The expression level of PLZF in lung tissue was determined by RT‒qPCR. D: Western blotting was used to assess the expression level of PLZF in lung tissue. The data are expressed as the mean ± SD (n = 5). ***P < 0.001 vs. control.

Fig 2. PLZF overexpression promotes HPMECs proliferation and angiogenesis.

A: Transfection efficiency of PcDNA3.1-PLZF in HPMECs was assessed by Western blot; B: CCK-8 was used to assess the proliferation ability of HPMECs following overexpression of PLZF; C: EdU staining was used to assess the proliferation activity of HPMECs and quantification of the percentage of EdU-positive cells, scale bars: 100 px; D: Tube formation experiment for the vascularization ability of HPMECs, scale bars: 100 μm; E: Flow cytometry was used to assess apoptosis of HPMECs; F: Western blot was used to assess the level of related proteins in HPMECs. The data are expressed as the mean ± SD (n = 3). **P < 0.01, ***P < 0.001 vs. Control.

Fig 3. PLZF promotes HPMECs proliferation and angiogenesis by inhibiting Keap1.

A, B: The Cistrome DB database predicted PLZF binding sites and binding sequences in Keap1 promoter; C: ChIP was used to detect the binding of PLZF to the promoter region of Keap1 gene; D: Coimmunoprecipitation was used to assess the interaction between PLZF and Keap1; E: RT‒qPCR was used to assess the expression level of Keap1 in HPMECs; F: Western blotting was used to assess the expression level of Keap1 in HPMECs; G: The transfection efficiency of PcDNA3.1‒Keap1 was assessed by Western blot; H: The proliferation ability of HPMECs was assessed by CCK-8; I: EdU staining was used to assess the proliferation activity of HPMECs and quantify the percentage of EdU-positive cells, scale bars: 100 px; J: A tube formation assay was used to assess the angiogenic ability of HPMECs, scale bars: 100 μm; K: The apoptosis of HPMECs was assessed by flow cytometry; L: Expression levels of the angiogenesis markers VEGF, bFGF, CD31, and CD34 were determined by Western blot. The data are expressed as the mean ± SD (n = 3). ^**P < 0.01, ^***P < 0.001 vs. Control; ^#P < 0.05, ^###P < 0.001 vs. PcDNA3.1-PLZF.

Fig 4. PLZF promotes angiogenesis without affecting HPMECs proliferation by activating the HIF-1

α/VEGF signaling pathway. A: Western blotting was used to assess the expression levels of VEGF, HIF-1α and VEGFR-2 in HPMECs; B: RT‒qPCR was used to assess the expression level of HIF-1α; C: The expression level of HIF-1α was assessed by Western blotting; D: The cells were treated with the proteasome inhibitor MG132, and the expression of HIF-1α was detected by Western Blot; E: CCK-8 was used to assess the proliferation ability of HPMECs; F: HPMECs proliferation was assessed by EdU staining and the percentage of EdU-positive cells was quantified, scale bars: 100 px; G: A tube formation assay was used to assess the angiogenic ability of HPMECs, scale bars: 100 μm; H: HPMECs apoptosis was assessed by flow cytometry; I: Western blotting was used to assess the expression levels of the angiogenesis markers VEGF, bFGF, CD31, and CD34; J: Co-immunoprecipitation was used to detect the interaction between Keap1 and HIF-1α. The data are expressed as the mean ± SD (n = 3). ^** P < 0.01, ^*** P < 0.001 vs. Control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. PcDNA3.1-PLZF.

Fig 5. PLZF promotes HPMECs proliferation and angiogenesis via activation of the Nrf2 signaling pathway-mediated HIF-1

α/VEGF. A: Western blotting was used to assess the expression levels of Nrf2, HO-1 and NQO1 in HPMECs; B: The expression level of Nrf2 was assessed via RT‒qPCR; C: Western blotting was used to assess the expression level of Nrf2; D: CCK‒8 was used to assess the proliferation ability of HPMECs; E: EdU staining was used to assess the proliferation activity of HPMECs and quantify the percentage of EdU-positive cells, scale bars: 100 px; F: A tube formation assay was used to assess the angiogenic ability of HPMECs, scale bars: 100 μm; G: HPMECs apoptosis was assessed by flow cytometry; H: Western blotting was used to assess the expression levels of Keap1, HIF-1α, HO-1, NQO1, and VEGF in HPMECs. The data are expressed as the mean ± SD (n = 3). ^** P < 0.01, ^*** P < 0.001 vs. Control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. PcDNA3.1-PLZF.

Fig 6. PLZF overexpression promotes CLG after PNX in mice.

A: Representative image of the remaining lung; B: Ratio of lung weight to body weight; C: Immunofluorescence was used to assess the proliferation of pulmonary endothelial cells, scale bars: 100 μm; D: Western blot analysis of the levels of PLZF, Keap1, Nrf2, HIF-1α and VEGF in lung tissue. The data are expressed as the mean ± SD (n = 5). ** P < 0.01, *** P < 0.01 vs. PNX + PcDNA3.1 vector.