MFGE8 regulates the EndoMT of HLMECs through the BMP signaling pathway and fibrosis in acute lung injury

Figures

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  Fig. 1. LPS treatment of HLMECs induces EndoMT. (A) HLMECs were induced with 20 µg/ml LPS for 96 h, and subsequently the morphology of cells was observed under a microscope. Magnification, × 100; scale bar, 50 μm. (B) HLMECs were induced with 20 µg/ml LPS for 48 h, and a Transwell migration assay was used to examine cell invasion. Magnification, × 100; scale bar, 50 μm. (C) HLMECs were induced with 20 µg/ml LPS for 24 h, and a wound-healing assay was used to assess cell migration. Magnification, × 100; scale bar, 50 μm. (D) HLMECs were induced with 20 µg/ml LPS for 96 h, and fluorescence microscopy was used to visualize CD31 and α-SMA expression. Magnification, × 200; scale bar, 20 μm. (E) HLMECs were induced with 20 µg/ml LPS for 96 h, and the protein expression of CD31 and α-SMA was examined using western blotting. β-Actin was used as the loading control. (F) HLMECs were induced with 20 µg/ml LPS for 96 h, and the mRNA expression levels of the CD31 and α-SMA genes were examined. Data are presented as the mean ± SD of three repeats. *P < 0.05 vs. control. HLMEC, Human lung microvascular endothelial cell; EndoMT, endothelial-mesenchymal transition

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  Fig. 2. Analysis of the knockdown efficiency of MFGE8. (A) Western blotting was used to examine the expression of MFGE8 in LPS-induced HLMECs. β-Actin was used as the loading control. (B) mRNA expression levels of MFGE8 in LPS-induced HLMECs. (C and D) For knockdown three specific siRNAs and a non-targeting siRNA control were transfected into HLMECs to knock down MFGE8 expression, and the efficiency of knockdown was assessed using western blotting (C) and RT-qPCR (D). Data are presented as the mean ± SD of three repeats. *P < 0.05 vs. control. HLMEC, Human lung microvascular endothelial cell; MFGE8, Milk Fat Globule Epidermal Growth Factor 8; siRNA, small interfering

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  Fig. 3. Knockdown of MFGE8 exacerbated LPS-induced EndoMT in HLMECs. (A) Microscopy was used to observe the morphology of HLMECs. Magnification, x100; scale bar, 50 μm. (B) A Transwell migration assay was used to examine cell invasion. Magnification, × 100; scale bar, 50 μm. (C) A wound-healing assay was used to assess cell migration. Magnification, x100; scale bar, 50 μm. (D) Fluorescence microscopy was used to examine the protein expression of CD31 and α-SMA. Magnification, × 200; scale bar, 20 μm. (E) Protein and (F) mRNA expression levels of CD31 and α-SMA. Data are presented as the mean ± SD of three repeats. *P < 0.05. siRNA, small interfering RNA; HLMEC, Human lung microvascular endothelial cell; MFGE8, Milk Fat Globule Epidermal Growth Factor 8; LPS, lipopolysaccharide

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  Fig. 4. rhMFGE8 attenuated LPS-induced EndoMT in HLMECs. (A) Microscopy was used to observe the morphology of HLMECs. Magnification, x100; scale bar, 50 μm. (B) Transwell migration assays were used to examine cell invasion. Magnification, × 100; scale bar, 50 μm. (C) A wound-healing assay was used to assess cell migration. Magnification, x100; scale bar, 50 μm. (D) Fluorescence microscopy was used to analyze the protein expression of CD31 and α-SMA. Magnification, x200; scale bar, 20 μm. (E) Protein and (F) mRNA expression levels of CD31 and α-SMA. Data are presented as the mean ± SD of three repeats. *P < 0.05. siRNA, small interfering RNA; HLMEC, Human lung microvascular endothelial cell; MFGE8, Milk Fat Globule Epidermal Growth Factor 8; LPS, lipopolysaccharide; rh, recombinant human; EndoMT, endothelial-mesenchymal transition

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  Fig. 5. LPS activated the BMP/Smad1/5/Smad1/4 signaling pathway. (A and C) Protein and mRNA expression levels of BMP, Smad1, Smad5, Smad4, Snail, and pSmad1/5. (B) The BMP inhibitor LDN-212,854 was used to confirm the relationship between LPS and the BMP signaling pathway. Western blotting was used to detect the protein expression levels of Smad1, Smad5, and Smad4. Data are presented as the mean ± SD of three repeats. *P < 0.05. HLMEC, Human lung microvascular endothelial cell; MFGE8, Milk Fat Globule Epidermal Growth Factor 8; LPS, lipopolysaccharide; rh, recombinant human; siRNA, small interfering RNA

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  Fig. 6. rhMFGE8 attenuated ECM deposition and exerted a protective role in the early stages of EndoMT. A total of 45 mice were divided into three groups: Control group, LPS group, and LPS + rhMFGE8 group. Mice were sacrificed after 7, 14, or 28 days. (A) Hematoxylin and eosin staining was used to observe the LPS-induced changes in cell morphology and the alveolar septum in the lung. Magnification, x100; scale bar, 100 μm. (B) Masson staining was used to examine ECM deposition in the lung tissues. Magnification, × 100; scale bar, 100 μm. (C) Fluorescence microscopy was used to analyze the protein expression levels of CD31 and α-SMA in lung tissue. CD31 is shown by red fluorescence, and α-SMA is shown by green fluorescence. Magnification, × 100; scale bar, 20 μm). (D-F) Western blotting was used to examine the protein expression levels of CD31 and α-SMA after 7,14, and 28 days. Data are presented as the mean ± SD of five repeats. (G-I) Collagen were measured by Sircol assay.*P < 0.05. HLMEC, Human lung microvascular endothelial cell; MFGE8, Milk Fat Globule Epidermal Growth Factor 8; LPS, lipopolysaccharide; rh, recombinant human; siRNA, small interfering RNA; ECM, extracellular matrix;…