Impaired reannealing of the endothelial barrier after an increase in lung vascular permeability induced by PAR-1 activation in FoxM1 CKO mice. FoxM1 CKO mice or WT littermates were treated with either saline or a PAR-1 agonist peptide (i.v., 5 mg/kg BW). Lungs were isolated at the indicated times and perfused for K_f,c measurements. K_f,c was also measured at baseline (time 0). Data are expressed as mean ± SD (error bars; n = 3). *, P < 0.05 versus WT/agonist. Agonist, PAR-1 agonist peptide. Three independent experiments were performed with 20 WT and 21 FoxM1 CKO mice at the age of 3.5–4 mo old. FoxM1 CKO lungs exhibited persistent microvessel leakage after PAR-1 activation.

Requirement of FoxM1 for reforming a restrictive barrier in ECs after PAR-1 activation. (A) TER assay of endothelial AJ integrity in response to thrombin challenge (PAR-1 activation). HMVEC-L transfected with either human FoxM1 siRNA (siRNA) or scRNA, or mock-transfected (CTL), were plated to confluency on electrodes. AT 65 h after transfection, TER of each monolayer at baseline was recorded, and it was then monitored for 3 h after thrombin challenge (4 U/ml). TER values of each monolayer were normalized to their values at basal levels. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus either CTL or scRNA. FoxM1 deficiency induced a defective recovery of the endothelial barrier function after thrombin challenge. (B) Western blot analysis demonstrating siRNA-mediated knockdown of FoxM1 in HMVEC-L. At 65 h after transfection, HMVEC-L were lysed, and 15 µg of each lysate per lane was loaded for detection of FoxM1 protein level with anti-FoxM1 antibody. The same membrane was blotted with anti-actin antibody for loading control. The experiment was performed three times with similar results.

Confocal microscopy of endothelial junctions after PAR-1 activation. (A) Representative micrographs of anti–VE-cadherin staining. After transfection with either human FoxM1 siRNA (siRNA) or scRNA, HMVEC-L were plated in 24-well plates with a glass coverslip in each chamber at 100% confluency. At 65 h after transfection, the cells were treated with 4 U/ml thrombin in triplicate, and fixed with 4% paraformaldehyde at the indicated times. The fixed cells were immunostained with anti–VE-cadherin antibody (green) to visualize junctions, and nuclei were counterstained with DAPI (blue). FoxM1-deficient HMVEC-L (siRNA-transfected) exhibited defective reannealing of AJs at 2 and 4 h after thrombin challenge, in contrast to scRNA-transfected controls (scRNA). Arrows indicate failure of reannealing of AJs. The experiment was performed three times with similar data. Bar, 50 µm. (B) Quantification of intercellular AJ gap area. The area of intercellular AJ gaps was quantified using MetaMorph 7.1.0 by manually outlining cells and selecting for gaps. Values are expressed as the percentage of total surface area. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA at 120 and 240 min after thrombin challenge.

Decreased expression of β-catenin in FoxM1-deficient ECs. (A) Quantitative RT-PCR analysis of expression of components of endothelial AJs. At 65 h after transfection of either FoxM1 siRNA or scRNA, confluent HMVEC-L were lysed for RNA isolation. mRNA levels of the indicated genes were quantified by quantitative RT-PCR analysis. Data are expressed as mean ± SD (error bars; n = 3). *, P < 0.05 versus scRNA. The experiment was performed three times with similar results. (B and C) Western blot analysis demonstrating decreased protein levels of β-catenin in FoxM1-deficient HMVEC-L. At 65 h after transfection with either human FoxM1 siRNA (siRNA) or scRNA, confluent HMVEC-L were lysed for Western blot analysis of each protein. Anti-actin was used as a loading control (B). The experiment was performed three times with similar results. β-catenin expression was quantified by densitometry analysis (C). Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA. (D and E) Subcellular localization of endothelial AJ proteins at baseline and after thrombin challenge. At 65 h after transfection, confluent HMVEC-L monolayers were collected for cell fractionation. Each membrane (M) or cytosolic (C) fraction (7.5 µg per lane) was loaded for detection of each protein (D). The experiment was performed three times with similar results. β-catenin localization in the membrane fraction versus cytosolic fraction was quantified by densitometry (E). Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.01 versus membrane expression at time 0 (basal); #, P < 0.05 versus cytosolic expression at time 0; ƒ, P < 0.05 versus scRNA-membrane expression at time 0.

Rescue of defective reannealing of endothelial AJ barrier of FoxM1-deficient ECs by restoration of β-catenin protein. (A and B) Decreased β-catenin expression is responsible for impaired reannealing of the endothelial AJ barrier in FoxM1-deficient HMVEC-L. HMVEC-L were transfected with human FoxM1 scRNA, siRNA, and siRNA plus plasmid DNA expressing human β-catenin (siRNA⁺β-cat). At 65 h after transfection, the monolayers were challenged with 4 U/ml thrombin, and TER was recorded for 3 h. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA. (C and D) β-catenin expression rescued defective reannealing of the endothelial AJ barrier of FoxM1-deficient HMVEC-L in a dose-dependent manner. HMVEC-L were transfected with either FoxM1 scRNA, siRNA, or siRNA plus plasmid DNA expressing human β-catenin at the indicated amounts. At 65 h after transfection, monolayers were challenged with 4 U/ml thrombin, and TER was recorded for 3 h. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA. (E) Western blots demonstrating increased β-catenin expression in FoxM1-deficient HMVEC-L by plasmid DNA transfection. The experiment was performed three times with similar results.

Decreased membrane expression of β-catenin in confluent FoxM1-deficient ECs. (A) Representative micrographs of immunofluorescent staining with anti–β-catenin antibody. At 65 h after transfection with either human FoxM1 scRNA, siRNA, or siRNA plus plasmid DNA expressing human β-catenin (siRNA⁺β-catenin, 1.5 µg/10⁶ cells), monolayers were fixed and immunostained with anti–β-catenin antibody to visualize β-catenin (red). Nuclei were counterstained with DAPI (blue). The experiment was performed three times with similar results. Bar, 20 µm. (B) Quantification of fluorescent intensity demonstrating reduced membrane expression of β-catenin in FoxM1-deficient HMVEC-L, which was restored by exogenous β-catenin expression. Accumulation of β-catenin at the membrane was quantified using 12-bit depth confocal z-series images. Maximum pixel values from different z sections were projected to a single plane using MetaMorph 7.1.0. Integrated fluorescence intensity of threshold projected images was calculated for β-catenin. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA. **, P < 0.01 versus siRNA. (C) Western blot analysis demonstrating decreased β-catenin protein levels in the membrane fraction in confluent FoxM1-deficient HMVEC-L. At 65 h after transfection, confluent HMVEC-L monolayers were collected for cell fractionation. Each membrane fraction or cytosolic fraction (7.5 µg per lane) was loaded for detection of β-catenin with anti–β-catenin antibody. The experiment was performed three times with similar results.

Knockdown of β-catenin mimics the defective reannealing phenotype of endothelial junctions seen in FoxM1-deficient monolayers after PAR-1 activation. (A and B) siRNA dose response of knockdown of β-catenin. At 48 h after transfection with β-catenin scRNA, or siRNA at indicated doses, the confluent HMVEC-L were lysed for Western blot analysis of β-catenin protein levels. The same membrane was blotted with anti-actin for loading control (A). The experiment was performed three times with similar results. Densitometry was used to quantify the protein levels of β-catenin under each condition (B). Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA. 1.5 µmol/L of β-catenin siRNA induces ∼50% knockdown of β-catenin protein level. (C) TER assay demonstrating that partial knockdown of β-catenin results in impaired recovery of endothelial AJ function after thrombin challenge. HMVEC-L transfected with either human β-catenin siRNA (siRNA, 1.5 µmol/L) or scRNA were plated on electrodes at confluency. At 48 h after transfection, TER of each monolayer at baseline was recorded and monitored for 6 h after the thrombin challenge (4 U/ml). The TER value of each monolayer was normalized to its value at baseline. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus scRNA.

FoxM1 regulation of β-catenin transcription. (A) Schematic drawing of the 6-kb promoter region of the human ctnnb1 gene. FoxM1 binding sites are indicated (open box); the first exon is also show (shaded box). The three luciferase reporter constructs with various deletions of FoxM1 binding sites are also shown. (B) ChIP assay demonstrating that FoxM1 directly binds to two promoter regions of the ctnnb1 gene. Cross-linked chromatin from either mock-transfected or FoxM1 siRNA- or scRNA- transfected HMVEC-L was immunoprecipitated with either anti-FoxM1 antibody or IgG control. After immunoprecipitation, genomic DNA was analyzed for the amount of ctnnb1 promoter DNA using quantitative real-time PCR with primers specific for each region. FoxM1 binding to genomic DNA was normalized to IgG control. Data are shown as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus either mock or scRNA. (C) Luciferase reporter assay demonstrating that FoxM1 induced the transcriptional activity of the human ctnnb1 gene. After transfection with either luciferase reporter constructs under the control of a promoter of the human ctnnb1 gene with indicated deletions (a, b, and c) or empty vector (vector), HMVEC-L were plated at subconfluent (40%) or confluent conditions. At 40 h after transfection, the cells were collected for analysis of luciferase activity. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.05 versus either construct a or b at a confluent condition; #, P < 0.01 confluent versus subconfluent conditions; **, P < 0.005 versus either construct a or b at a subconfluent condition. (D) Western blot analysis demonstrating markedly decreased FoxM1 expression in confluent HMVEC-L. Cell lysates of HMVEC-L at 100% confluency and 50–70% confluency were used for Western blotting of FoxM1 expression. The experiment was performed three times with similar results.

Normalization of the endothelial AJ barrier by restoration of β-catenin expression after increased vascular permeability in FoxM1 CKO lungs. (A) Quantitative RT-PCR analysis of gene expression. After 2 d in culture, microvascular ECs isolated from WT or FoxM1 CKO mouse lungs were lysed for total RNA isolation. Non-EC cultures (fibroblasts) were also lysed for total RNA isolation. mRNA levels of the indicated genes were quantified by quantitative RT-PCR. Data are expressed as mean ± SD (error bars; n = 3 independent experiments). *, P < 0.01 versus WT-EC; #, P < 0.05 versus WT-EC. (B) Western blotting demonstrating decreased β-catenin expression in FoxM1 CKO lungs compared with WT lungs and restoration of β-catenin expression by liposome-mediated transduction of plasmid DNA expressing β-catenin. The experiment was performed three times with similar results. CKO, FoxM1 CKO. (C) Normalization of vascular junctional repair by restoration of β-catenin expression in FoxM1 CKO lungs. Plasmid DNA expressing β-catenin or empty vector were transduced in FoxM1 CKO lungs through i.v. injection of the liposome–DNA complex. WT mice transduced with empty vector DNA were used as controls. At 40 h after transduction, mice were challenged with a PAR-1 agonist peptide (i.v., 5 mg/kg BW). Lungs were isolated at 2 h after challenge and perfused for K_f,c measurement. Data are expressed as mean ± SD (error bars; n = 3–4 animals/group from three independent experiments). *, P < 0.05 versus either WT + vector, or CKO + β-catenin.