Novel FOXF1-Stabilizing Compound TanFe Stimulates Lung Angiogenesis in Alveolar Capillary Dysplasia

Abstract

Rationale: Alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) is linked to heterozygous mutations in the FOXF1 (Forkhead Box F1) gene, a key transcriptional regulator of pulmonary vascular development. There are no effective treatments for ACDMPV other than lung transplant, and new pharmacological agents activating FOXF1 signaling are urgently needed. Objectives: Identify-small molecule compounds that stimulate FOXF1 signaling. Methods: We used mass spectrometry, immunoprecipitation, and the in vitro ubiquitination assay to identify TanFe (transcellular activator of nuclear FOXF1 expression), a small-molecule compound from the nitrile group, which stabilizes the FOXF1 protein in the cell. The efficacy of TanFe was tested in mouse models of ACDMPV and acute lung injury and in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV. Measurements and Main Results: We identified HECTD1 as an E3 ubiquitin ligase involved in ubiquitination and degradation of the FOXF1 protein. The TanFe compound disrupted FOXF1-HECTD1 protein-protein interactions and decreased ubiquitination of the FOXF1 protein in pulmonary endothelial cells in vitro. TanFe increased protein concentrations of FOXF1 and its target genes Flk1, Flt1, and Cdh5 in LPS-injured mouse lungs, decreasing endothelial permeability and inhibiting lung inflammation. Treatment of pregnant mice with TanFe increased FOXF1 protein concentrations in lungs of Foxf1^+/- embryos, stimulated neonatal lung angiogenesis, and completely prevented the mortality of Foxf1^+/- mice after birth. TanFe increased angiogenesis in human vascular organoids derived from induced pluripotent stem cells of a patient with ACDMPV with FOXF1 deletion. Conclusions: TanFe is a novel activator of FOXF1, providing a new therapeutic candidate for treatment of ACDMPV and other neonatal pulmonary vascular diseases.

Keywords: FOXF1; alveolar capillary dysplasia; neonatal pulmonary disease; pulmonary angiogenesis; pulmonary endothelium.

Figure 1.

The TanFe (transcellular activator of nuclear FOXF1 expression) small-molecule compound increases FOXF1 (Forkhead Box F1) protein amounts in vitro. (A) Chemical structure of the TanFe compound. (B) Western blots show amounts of endogenous FOXF1 protein and other transcription factors in TanFe-treated fetal lung endothelial Mouse Fetal Lung Mesenchyme-91 U (MFLM-91U) cells. Cells were treated with either vehicle alone (control) or 20 μM of TanFe and harvested 24 hours after the treatment. Protein samples were pooled from five independent cell cultures (left panels). Three independent Western blots were quantified and normalized to β-Actin (right graph). (C) TanFe increases nuclear FOXF1 fluorescence. MFLM-91 U endothelial cells were fixed and stained for FOXF1 (green). DAPI was used to visualize cell nuclei (blue). Inserts show high-magnification images of FOXF1-stained cells. (D and E) Western blots show the dose response and the time course of TanFe treatment in MFLM-91 U cells (n = 3 independent experiments). Half maximal effective concentration (EC₅₀) for TanFe is 3.6 μM (dotted lines). (F) Luciferase (Luc) assay shows increased FOXF1 transcriptional activity in TanFe-treated MFLM-91 U cells. Cells were transfected with either FOXF1-specific LUC reporter plasmid (FOXF1-LUC) or Empty-LUC plasmid (control) and treated with TanFe for 24 hours (n = 5 for each group). (G) TanFe treatment does not change Foxf1 mRNA. Quantitative RT-PCR was used to measure Foxf1 mRNA in MFLM-91 U cells after treatment with different concentrations of TanFe (n = 3). Expression levels were normalized to β-Actin (Actb mRNA). *P < 0.05 and ****P < 0.0001. FACT 140 = facilitates chromatin transcription complex subunit SPT16 -140 kDa; NF-κB = nuclear factor-κB; n.s. = not significant.

Figure 2.

TanFe (transcellular activator of nuclear FOXF1 expression) increases FOXF1 (Forkhead Box F1) protein amounts in vivo. (A) Schematic shows treatment of C57Bl/6 mice with LPS and TanFe. LPS was administered intratracheally on Day 0. TanFe or vehicle was injected intraperitoneally on Days 0, 1, and 2 (5 mg/kg body weight). (B) TanFe increases FOXF1 protein amounts in LPS-injured mouse lungs. Total lung protein was prepared 6 days after LPS injury and analyzed by Western blot for FOXF1, FLK1, FLT1, CDH5 (VE cadherin), and β-Actin. Samples were obtained from individual mice (n = 3 mice per group). (C and D) Immunostaining for endomucin shows that TanFe increases the capillary density in alveolar regions of LPS-treated mice. Mice were harvested on Day 6 after LPS injury and lung paraffin sections were stained for endomucin (green). Slides were counterstained with DAPI (blue). Ten random slides were used to quantitate the staining (n = 3 mice per group). Magnification is ×400. (E) Endothelial permeability assay shows that TanFe protects endothelial barrier function after LPS lung injury. Evans blue dye was injected intraperitoneally, and lung tissue was collected 4 hours after injection (n = 5 mice in each group). Before the lung harvest, vasculature was perfused with saline to remove intravascular Evans blue dye. (F–H) TanFe inhibits accumulation of inflammatory cells in BAL fluid (BALF) of LPS-injured mice. BALF was collected on Day 3 after LPS lung injury and analyzed for total number of cells and for neutrophil counts (n = 5 mice in each group). (I) Hematoxylin and eosin staining shows that TanFe decreases lung inflammation in the alveolar region of LPS-injured mice. Mice were harvested on Day 6 after LPS injury (n = 3 mice per group). TanFe or vehicle was given on Days 0, 1, and 2. Magnification is ×400. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s. = not significant.

Figure 3.

TanFe (transcellular activator of nuclear FOXF1 expression) disrupts FOXF1 (Forkhead Box F1)–HECTD1 protein–protein interactions. (A) Immunoprecipitation (IP) shows that HECTD1 interacts with endogenous FOXF1 protein. IP was performed with cell lysates from Mouse Fetal Lung Mesenchyme-91 U (MFLM-91U) cells using the HECTD1 antibody or IgG control. Protein samples were pooled from five independent cell cultures. (B) IP shows that FOXF1 interacts with HECTD1 protein. IP was performed with cell lysates from MFLM-91 U cells stably expressing His-Flag–tagged mouse FOXF1. Cells were treated with 20 μM of TanFe or vehicle (Dimethyl sulfoxide) for 24 hours. FOXF1-interacting proteins were purified using anti-Flag M2 agarose beads and Talon metal affinity resin and were analyzed by Western blot. (C) The in vitro ubiquitination assay shows that HECTD1 ubiquitinates the FOXF1 protein. HA-HECTD1 or HA-HECTD1 C2579G mutants were expressed in Human Embryonic Kidney (HEK) 293T cells, purified using HA magnetic beads, and eluted using HA peptide. The ubiquitination assay was performed in the presence of FLAG-FOXF1 immobilized magnetic beads and the purified HA-HECTD1 proteins. Binding of biotinylated Ub to FLAG-FOXF1 protein was detected by Western blot using Horseradish peroxidase (HRP) streptavidin. (D) Western blot shows that increasing TanFe concentrations interfere with binding of purified HECTD1 and FOXF1 proteins. Endogenous HECTD1 was immunoprecipitated from MFLM-91 U cells and incubated with the purified FLAG-FOXF1 protein immobilized on agarose beads. Protein samples were pooled from three cell cultures. Ub = ubiquitin; UBE3A = Ubiquitin protein ligase E3A.

Figure 4.

TanFe (transcellular activator of nuclear FOXF1 expression) inhibits FOXF1 (Forkhead Box F1) ubiquitination. (A) TanFe decreases ubiquitination of the FOXF1 protein. IP was performed in protein extracts from Mouse Fetal Lung Mesenchyme-91 U (MFLM-91U) cells stably expressing His-Flag–tagged FOXF1 (HF-FOXF1) using the FLAG antibody followed by Western blot with ubiquitin antibodies. TanFe treatment (20 μM) was performed for 24 hours. Equal protein loading is shown by Western blot for FOXF1 (n = 3 pooled cell cultures). (B) Chromatin fractionation shows that TanFe increases FOXF1 amounts in the chromatin fraction. TanFe does not change HECTD1 amounts in cell fractions. Chromatin fractionation was performed in MFLM-91 U cells treated with 20 μM of TanFe for 24 hours. Cell lysates were pooled from three independent cell cultures. (C) Western blot shows that inhibition of HECTD1 by siRNA (siHECTD1) increases FOXF1 protein amounts in MFLM-91 U cells. siCtrl is scrambled siRNA (n = 3 pooled cell cultures). (D) Overexpression of HECTD1 decreases FOXF1 expression. Cells were transfected with the HA-HECTD1 expression vector, and the Western blot was performed 48 hours after transfection (n = 3 pooled cell cultures). Chrom = chromatin; Ctrl = control; Cyto-Nucleo = cytoplasmic and nuclear extract; IB = Immunoblot; IP = immunoprecipitation.

Figure 5.

TanFe (transcellular activator of nuclear FOXF1 expression) increases lung angiogenesis and prevents mortality in Foxf1^+/− (Forkhead Box F1) mice. (A) Schematic shows a breeding strategy and TanFe treatments of pregnant mice on Embryonic Days E11.5, E13.5, and E15.5 (10 mg/kg body weight). (B) TanFe increases FOXF1 protein amounts in lungs of Foxf1^+/− embryos. Immunoblots show the concentrations of FOXF1, FLK1, PECAM-1, FAAP100, and β-Actin in lung extracts from E18.5 embryos treated with either TanFe or vehicle. Protein lysates from three WT lungs were pooled together before the analysis. Foxf1^+/− lungs (Het) were analyzed individually (left panels). Quantification of two independent Western blots shows that TanFe does not change FAAP100 protein concentrations but increases FOXF1, FLK1, and PECAM1 in Foxf1^+/− embryos collected at E18.5 to P1 (n = 3–6 embryos in each group) (right graphs). (C) TanFe increases capillary density in Foxf1^+/− lungs. Images show endomucin staining (green) of E18.5 lungs after TanFe treatment. DAPI (blue) was used to stain cell nuclei. Magnification is ×400. Quantification of endomucin staining was performed using ImageJ software in 10 random images from three mouse lungs in each group. (D) TanFe increases survival of Foxf1^+/− mice after birth. Mice were treated with TanFe (10 mg/kg body weight) or vehicle on Embryonic Days E11.5, E13.5, and E15.5. Survival rates of vehicle-treated Foxf1^+/− mice (n = 175) and TanFe-treated Foxf1^+/− mice (n = 49) were determined at Postnatal Day 30 (P30). (E) TanFe improves alveolarization in Foxf1^+/− mice after birth. Hematoxylin and eosin staining shows that TanFe decreases alveolar simplification in Foxf1^+/− mice at P35 (n = 7–13 in each group). Magnification is ×100. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s. = not significant; WT = wild-type.

Figure 6.

TanFe (transcellular activator of nuclear FOXF1 expression) increases angiogenesis in FOXF1 (Forkhead Box F1)-deficient endothelial cells in vitro. (A) Western blot shows that TanFe treatment increases FOXF1 protein concentrations but does not affect Vinculin in FOXF1-deficient endothelial Mouse Fetal Lung Mesenchyme-91 U (MFLM-91U) cells. Inhibition of FOXF1 was achieved by transient transfection with FOXF1-specific siRNA (siFOXF1). siRNA against a nontargeted RNA sequence was used as a control (siCtrl). (B) Quantification of FOXF1 protein concentrations was performed using densitometric analysis of Western blot images and normalized to Vinculin (n = 3). (C–E) In vitro angiogenesis assay shows that TanFe increases the formation of endothelial sprouts in endothelial Human umbilical vein endothelial cells (HUVECs) infected with shFOXF1 lentivirus. Lentiviral particles with shRNA against a nontargeted RNA sequence were used as a control (shCtrl). Scale bars, 100 μm. The complexity of the vascular network in Matrigel was quantitated by measurements of sprout length and counts of the sprout junctions (n = 4 for each group). *P < 0.05. n.s. = not significant.

Figure 7.

TanFe (transcellular activator of nuclear FOXF1 expression) increases angiogenesis in human vascular organoids (VOs) derived from a patient with ACDMPV. (A) Schematic diagram shows the differentiation protocol used to generate VOs from human induced pluripotent stem cells (hiPSCs). Brightfield images show morphological changes undergone by hiPSCs during VO differentiation. Scale bars for Days D0–D7, 500 μm. Scale bar for D15, 250 μm. (B) Images show immunostaining of whole-mount D15 VOs for CD31 (gray, endothelial cells) and PDGFRb (red, mural cells/pericytes). Nuclei were stained with DAPI. Scale bar, 200 μm. (C) TanFe treatment increases the percentages of CD31⁺ and PDGFRb⁺ cells in vascular organoids at D15 (n = 3 in each group). (D) Time course gene expression profile of FOXF1 during D3–D15 VO differentiation from human iPSCs. (E) Images show immunostaining of D15 VO sections for CD31 (gray) and FOXF1 (red). Scale bar, 200 μm. (F) High-magnification images of CD31-stained sections show lumen-like structures in D15 VOs (arrowheads). Scale bar, 50 μm. (G) Quantification of the endothelial density within VOs (n = 3). (H) Quantification of the number of lumen-like structures within VOs (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. ACDMPV = alveolar capillary dysplasia with misalignment of pulmonary veins; BMP4 = bone morphogenetic protein 4; EB = embryoid body; FBS = fetal bovine serum; FGF2 = fibroblast growth factor 2; UD = undifferentiated human iPSCs; VEGF = vascular endothelial growth factor; YHWZ = tyrosine 3/tryptophan 5 -monooxygenase activation protein.