Research ArticleVASCULAR BIOLOGY

FOXF1 maintains endothelial barrier function and prevents edema after lung injury

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Science Signaling  19 Apr 2016:
Vol. 9, Issue 424, pp. ra40
DOI: 10.1126/scisignal.aad1899
  • Fig. 1 Deletion of Foxf1 from endothelial cells causes respiratory insufficiency and lethality in adult mice.

    (A) H&E staining of lung paraffin sections from Tam-treated endFoxf1−/− mice shows lung congestion, infiltration of inflammatory cells from blood vessels (V) into the lung tissue, occlusion of pulmonary bronchioles (Br), and pulmonary hemorrhage (shown with arrowheads). Tam was given 15 days before the lung harvest. Trichrome staining shows fibrosis in Tam-treated endFoxf1−/− lungs. Scale bars, 50 μm. Images are representative of five mice per genotype. (B) Kaplan-Meier survival analysis shows uniform mortality in Tam-treated endFoxf1−/− mice (n = 20 mice per genotype). (C) Tam-treated endFoxf1−/− lungs were visibly edematous (left panel) (images are representative of five mice per genotype) and had a higher lung wet/dry weight ratio (right panel) (n = 5 mice per genotype). (D) Protein concentration in BALF was increased in Tam-treated endFoxf1−/− mice (n = 5 mice per genotype). (E) Tam-treated endFoxf1−/− mice exhibited decreased airway compliance, but airway resistance and respiratory elasticity were increased (n = 6 mice per genotype). *P < 0.05; **P < 0.01.

  • Fig. 2 Deletion of Foxf1 increases lung inflammation.

    (A) H&E staining shows accumulation of inflammatory cells in endFoxf1−/− lungs at different time points after Tam administration (n = 5 mice per genotype and time point). (B and C) Immunostaining with Mac-3 antibody shows increased numbers of macrophages after deletion of Foxf1. Mac-3–positive cells were counted in 10 random microscope fields of Tam-treated endFoxf1−/− and Foxf1fl/fl lungs (n = 5 mice per genotype). (D) Diff-Quik staining shows increased numbers of macrophages in BALF of Tam-treated endFoxf1−/− mice (n = 5 mice per genotype). Scale bar, 20 μm. *P < 0.05; **P < 0.01.

  • Fig. 3 Foxf1 deletion from endothelial cells decreases S1pr1, Clnd5, and Cdh5 mRNAs.

    (A) Differentially expressed genes in Tam-treated endFoxf1−/− lungs were identified using RNA-seq analysis. Heat map analysis shows the two-dimensional clustering of mRNAs that were significantly altered in Tam-treated endFoxf1−/− lungs compared to Tam-treated Foxf1fl/fl controls. Samples from three mice per genotype were combined together before the RNA-seq analysis. (B) Biological processes influenced by endothelial Foxf1 deletion were identified using ToppGene Suite (http://toppgene.cchmc.org). The statistical significance of each biological process was presented using negative log2 transformation of P value. (C) mRNA changes and P values in Tam-treated endFoxf1−/− lungs are shown in the table. (D) RNA-seq data were confirmed by qRT-PCR of total lung RNA (n = 5 mice per genotype). mRNA abundance was normalized to β-actin mRNA. (E and F) FACS was used to isolate endothelial cells (CD45CD31+eGFP+) from endFoxf1−/− lungs. Endothelial cells from Tam-treated Pdgfb-iCreER/Foxf1+/+ mice were used as a control. qRT-PCR shows reduced Foxf1 and S1pr1 mRNAs in FACS-sorted endothelial cells from Tam-treated endFoxf1−/− lungs (n = 3 mice per genotype). *P < 0.05; ***P < 0.001. SSC-A, side-scatter area; FSC-A, forward-scatter area.

  • Fig. 4 Knockdown of FOXF1 increases endothelial permeability.

    (A) Pulmonary vascular permeability was determined by Evans blue dye extravasation. The amounts of Evans blue dye in BALF of endFoxf1−/− mice were unchanged at day 3 but increased at days 6 and 15 after Tam administration (n = 5 mice per genotype and time point). (B) Foxf1, Cdh5, and S1pr1 mRNAs were decreased in FOXF1-depleted HUVECs as shown by qRT-PCR. Bcl2 and Bax mRNAs were unchanged. β-Actin mRNA was used for normalization (n = 3 independent cultures). (C) Colocalization studies show efficient depletion of FOXF1 (red) from the nuclei of HUVECs [stained with endothelial cell–specific marker Erg (green)] transduced with a lentivirus containing FOXF1-specific shRNA (Lenti-shFoxf1). Images are representative of three independent experiments. Scale bars, 20 μm. (D) Immunofluorescence staining of VE-cadherin (green) and F-actin (red) shows loss of VE-cadherin in the adherens junctions of FOXF1-depleted HUVECs (arrowheads). Images are representative of three independent experiments. Scale bars, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole. (E) FOXF1 depletion by Lenti-shFoxf1 increased transendothelial albumin leak (representative of three independent experiments). (F) The number of apoptotic cells was not changed after depletion of FOXF1. Forty-eight hours after lentiviral transduction, HUVECs were stained with annexin V–APC and 7AAD. Percentages of necrotic (7AAD+annexin V) and apoptotic cells (annexin V+) were determined by flow cytometry (representative of three independent experiments). (G) FOXF1 depletion in HUVECs did not influence cell survival and metabolic activity as demonstrated by the WST-1 assay (representative of three independent experiments). *P < 0.05; ***P < 0.001.

  • Fig. 5 Deletion of Foxf1 does not induce apoptosis of endothelial cells in the lung.

    (A and B) Flow cytometry was used to identify apoptotic cells in Tam-treated Foxf1fl/fl and endFoxf1−/− lungs at day 6 after Tam treatment. CD31+ endothelial cells were costained with annexin V and 7AAD. Percentages of live (annexin V/7AAD), early apoptotic (annexin V+/7AAD), late apoptotic (annexin V+/7AAD+), and necrotic (dead) cells (annexin V/7AAD+) are shown on scatter plots (A) and histograms (B). Three mice were used in each group. (C) Immunostaining for cleaved caspase-3 shows the presence of apoptotic cells in endFoxf1−/− lungs at day 15 after Tam treatment. Apoptosis was not detected in endothelial cells (arrowheads) (n = 3 mice per genotype). Scale bars, 50 μm.

  • Fig. 6 FOXF1 directly binds to and induces transcriptional activity of the S1pr1 and Cdh5 promoters.

    (A) Schematic drawing of the mouse S1pr1 promoter region with potential FOXF1 DNA binding sites (white boxes). FOXF1 protein binds to the −5141/−4815-bp and −3880/−3876-bp S1pr1 promoter regions, the former of which has three potential FOXF1 binding sites. FOXF1 binding to both S1pr1 promoter regions was decreased in Tam-treated endFoxf1−/− lungs. FOXF1 binding to genomic DNA was normalized to immunoglobulin G (IgG) control antibodies. FOXF1 did not bind to the −2772/−2759-bp S1pr1 promoter region or HoxA11 genomic DNA that lacked potential FOXF1 binding motifs (negative controls) (n = 3 mice per genotype). (B) Schematic drawing of the pGL2-mS1PR1pr-Luc construct containing the S1pr1 promoter region. The CMV-FOXF1 plasmid increases luciferase (Luc) activity of this reporter construct in MFLM-91U cells. CMV-empty plasmid was used as a negative control. Data are representative of three independent experiments. (C and D) Schematic drawings, ChIP, and Luc assay of the mouse VE-cadherin (Cdh5) promoter. FOXF1 binds to and activates the Cdh5 promoter through the −788/−775-bp promoter region (representative of three independent experiments). (E) Schematic drawing of the mouse Cldn5 promoter region. Locations of potential FOXF1 DNA binding sites are indicated (white boxes). FOXF1 does not bind Cldn5 promoter DNA as shown by ChIP (representative of three independent experiments). **P < 0.01.

  • Fig. 7 Pharmacological treatment with S1P protects heterozygous endFoxf1+/− mice from BHT-mediated lung injury.

    (A) Deletion of one Foxf1 allele in adult heterozygous endFoxf1+/− mice did not influence lung structure as shown by H&E staining (left panels). Increased inflammation was found in Tam-treated endFoxf1+/− lungs after BHT administration (right panels). Images are representative of five mice per genotype. (B) Kaplan-Meier survival analysis shows that administration of S1P partially rescues mortality in BHT-treated endFoxf1+/− mice (n = 15 mice per genotype and treatment). (C) qRT-PCR shows that BHT injury decreased Foxf1 and S1pr1 mRNAs in Tam-treated endFoxf1+/− lungs compared to Tam-treated Foxf1+/fl controls. β-Actin mRNA was used for normalization (n = 5 mice per genotype and time point). (D and E) S1P decreases macrophage inflammation in BHT-treated endFoxf1+/− mice as shown by Mac-3 staining. Mac-3–positive cells were counted in 10 random microscope fields (n = 5 mice per genotype). (F) Increased vascular permeability in endFoxf1+/− mice after BHT-induced lung injury. Administration of S1P decreased vascular permeability in BHT-treated endFoxf1+/− mice. The amounts of Evans blue dye were measured in BALF (n = 5 mice per genotype and treatment). (G) FOXF1 is decreased in human patients with ALI. FOXF1 staining was performed using lung paraffin sections from patients with severe bacterial pneumonia (n = 7). Normal human lungs (patients without lung injury) were used as controls (n = 6). FOXF1 abundance is decreased in pulmonary endothelial cells from patients with ALI (arrowheads in inserts). Arrows show FOXF1-positive cells in alveolar region. Scale bars, 50 μm. Ar, artery. *P < 0.05.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/424/ra40/DC1

    Fig. S1. Breeding strategy, Tam administration protocol, and analysis of FOXF1 in pulmonary macrophages.

    Fig. S2. Deletion of Foxf1 from endothelial cells does not influence the histological structure of the liver, heart, and kidney.

    Fig. S3. FOXF1 is reduced in pulmonary endothelial cells of Tam-treated endFoxf1−/− mice.

    Fig. S4. Tam treatment reduces the number of FOXF1-positive endothelial cells in endFoxf1−/− lungs.

    Fig. S5. Cdh5, Flk1, and Pecam-1 mRNAs are present in FACS-sorted endothelial cells.

    Fig. S6. Transplantation of bone marrow does not affect the lung phenotype of endFoxf1−/− mice.

    Fig. S7. Deletion of Foxf1 from endothelial cells reduces the abundance of VE-cadherin and S1PR1 in lung tissue.

    Fig. S8. Experimental protocol for the administration of S1P to BHT-treated heterozygous endFoxf1+/− mice.

    Fig. S9. S1P does not prevent mortality in Tam-treated homozygous endFoxf1−/− mice.

    Fig. S10. CYM5442 decreases lung inflammation in LPS-treated endFoxf1+/− mice.

    Table S1. RNA-seq analysis of FOXF1-deficient lungs.

    Table S2. TaqMan primers for qPCR.

    Table S3. Primers used for the ChIP and dual-luciferase assays.

  • Supplementary Materials for:

    FOXF1 maintains endothelial barrier function and prevents edema after lung injury

    Yuqi Cai, Craig Bolte, Tien Le, Chinmayee Goda, Yan Xu, Tanya V. Kalin,* Vladimir V. Kalinichenko*

    *Corresponding author. E-mail: vladimir.kalinichenko{at}cchmc.org (V.V.K.); tatiana.kalin{at}cchmc.org (T.V.K.)

    This PDF file includes:

    • Fig. S1. Breeding strategy, Tam administration protocol, and analysis of FOXF1 in pulmonary macrophages.
    • Fig. S2. Deletion of Foxf1 from endothelial cells does not influence the histological structure of the liver, heart, and kidney.
    • Fig. S3. FOXF1 is reduced in pulmonary endothelial cells of Tam-treated endFoxf1−/− mice.
    • Fig. S4. Tam treatment reduces the number of FOXF1-positive endothelial cells in endFoxf1−/− lungs.
    • Fig. S5. Cdh5, Flk1, and Pecam-1 mRNAs are present in FACS-sorted endothelial cells.
    • Fig. S6. Transplantation of bone marrow does not affect the lung phenotype of endFoxf1−/− mice.
    • Fig. S7. Deletion of Foxf1 from endothelial cells reduces the abundance of VE-cadherin and S1PR1 in lung tissue.
    • Fig. S8. Experimental protocol for the administration of S1P to BHT-treated heterozygous endFoxf1+/− mice.
    • Fig. S9. S1P does not prevent mortality in Tam-treated homozygous endFoxf1−/− mice.
    • Fig. S10. CYM5442 decreases lung inflammation in LPS-treated endFoxf1+/− mice.
    • Table S1. RNA-seq analysis of FOXF1-deficient lungs.
    • Table S2. TaqMan primers for qPCR.
    • Table S3. Primers used for the ChIP and dual-luciferase assays.

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    Citation: Y. Cai, C. Bolte, T. Le, C. Goda, Y. Xu, T. V. Kalin, V. V. Kalinichenko, FOXF1 maintains endothelial barrier function and prevents edema after lung injury. Sci. Signal. 9, ra40 (2016).

    © 2016 American Association for the Advancement of Science

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