Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: Human VV phenotypes. (A) VVs (arrowheads) were visualized in controls and in patients carrying mutations in the indicated genes, without (left) and with (right) power Doppler imaging of blood flow during a calf squeeze. An asterisk (*) marks stagnant/reversed flow in a VV sinus. Blood flow is from left to right. Bars, 1 mm. (B and C) Number (B) and length (C) of VV identified in patients carrying mutations in FOXC2 ( n = 8 patients), GJC2 ( n = 3), GJA1 ( n = 1), and healthy controls ( n = 10). See Tables S1, S2, and S3. ***, P < 0.0005 (ANOVA with Bonferroni correction). Error bars represent means ± SEM. (D) Immunolocalization (arrowheads) of FOXC2 and CX43 in adult human VV leaflets. Bar, 20 µm. Mut, mutation.

Article Snippet: Antibodies were raised in rabbits to Cx43 (3512; Cell Signaling Technology), sheep to FOXC2 (AF5044; R&D Systems), Foxc2 (AF6989; R&D Systems), and goat to Integrinα9 (BAF3827; R&D Systems).

Techniques: Imaging, Mutagenesis

Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: Mean number of valves in controls and patients

Article Snippet: Antibodies were raised in rabbits to Cx43 (3512; Cell Signaling Technology), sheep to FOXC2 (AF5044; R&D Systems), Foxc2 (AF6989; R&D Systems), and goat to Integrinα9 (BAF3827; R&D Systems).

Techniques: Control

Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: Mean VV leaflet length in controls and patients

Article Snippet: Antibodies were raised in rabbits to Cx43 (3512; Cell Signaling Technology), sheep to FOXC2 (AF5044; R&D Systems), Foxc2 (AF6989; R&D Systems), and goat to Integrinα9 (BAF3827; R&D Systems).

Techniques: Control

Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: VV development in the proximal FV. (A and B) Micrographs before (A) and after (B) dissection to perform whole-mount imaging of the proximal FV valve (boxed region). IVC, inferior vena cava; FA, femoral artery. Bar, 200 µm. (C) Localization of Prox1 (red), Foxc2 (green), and PECAM1 (blue) at the indicated time points. An asterisk (*) marks single commissure at stage 3, and two commissures at stage 4. Arrowheads, reoriented and elongated VFCs. FE, free edge. Bars, 20 µm. n ≥ 6 VV/stage. (D) Corresponding schematics of stages in VV development: Prox1 hi free-edge cells (red), leaflets (green), and leaflet attachment to the vein wall (blue). Arrows, blood flow direction. (E) A peri-VV reduction (between dotted lines) in SMA-expressing cells (red). Bar, 20 µm. n > 6. (F) Quantification of elongated Prox1 hi valve cells on the anterior and posterior vein wall at P0. n = 9 (paired t test). (G) Arrowheads, anterior wall VFCs. Bar, 20 µm. Blood flow left to right in C–G.

Article Snippet: Antibodies were raised in rabbits to Cx43 (3512; Cell Signaling Technology), sheep to FOXC2 (AF5044; R&D Systems), Foxc2 (AF6989; R&D Systems), and goat to Integrinα9 (BAF3827; R&D Systems).

Techniques: Dissection, Imaging, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: Regulation of initial VV organization by Foxc2 and Nfatc1. (A) Colocalization of Foxc2 (red) and Nfatc1 (green) in VFCs at P0. Foxc2 and Nfatc1 localization are also shown separately. n = 4. Bar, 20 µm. (B–E) The proportion of VVs identified at stage 0 (white) and stage 1 (gray) at P0 after deletion of Foxc2 (B) or Ppp3r1 (CnB1; C) from E15, or treatment with cyclosporin from E17 (D), or with combined Foxc2 deletion and cyclosporin treatment (E). The number of VVs analyzed for each condition is indicated above each bar. ***, P < 0.0005, χ 2 . (E) Localization of Prox1 (red) is shown at P0 after combined Foxc2 deletion and cyclosporin treatment. Bar, 20 µm.

Article Snippet: Antibodies were raised in rabbits to Cx43 (3512; Cell Signaling Technology), sheep to FOXC2 (AF5044; R&D Systems), Foxc2 (AF6989; R&D Systems), and goat to Integrinα9 (BAF3827; R&D Systems).

Techniques:

Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: Maturation of leaflets and commissures. (A) Immunolocalization at P6 of Nfatc1 (green) and Prox1 (red) in VV of WT mice administered control solvent or cyclosporin ( n = 5 versus 8 VVs, P < 0.05, χ 2 ). (B) Immunolocalization of Foxc2 (green) and Prox1 (red) in VVs of mice of the indicated genotypes. n ≥ 6 per condition. (C and D) Quantification of leaflet length and stage of VV development in mice of the indicated genotypes. Error bars represent means ± SEM. t test for leaflet length. (C, D, G, H, and I) Colors represent developmental stage (see key). The number of VVs analyzed for each condition is given above each bar. *, P < 0.05; **, P < 0.005 (χ 2 for developmental stage). (E) Immunolocalization of Cx37 (arrowheads) and Cx43 in VV leaflets at P6. n ≥ 4. (F) Localization of Prox1, Foxc2, and SMA at P6 in Gja4 −/− vein. n ≥ 6 (G and H) Stages of VV development reached in mice of the indicated genotypes. (I) Localization of Prox1, GFP, and SMA in Gjc2 GFP/GFP KO reporter vein at P2 ( n = 4), and stages of VV development reached in mice of the indicated genotypes. (J) Localization of Cx47-expressing VV cells (arrowheads) of Gjc2 GFP/+ reporter mice at P6. n = 4. Multichannel images are reproduced in supplementary data. Bars, 20 µm. (A–J) Blue stain is PECAM1.

Article Snippet: Antibodies were raised in rabbits to Cx43 (3512; Cell Signaling Technology), sheep to FOXC2 (AF5044; R&D Systems), Foxc2 (AF6989; R&D Systems), and goat to Integrinα9 (BAF3827; R&D Systems).

Techniques: Control, Solvent, Expressing, Staining

Journal: The Journal of Experimental Medicine

Article Title: Human venous valve disease caused by mutations in FOXC2 and GJC2

doi: 10.1084/jem.20160875

Figure Lengend Snippet: Role of blood flow in valve growth and the regulation of VV maintenance. (A and B) Representative images at P0 and P6 after single FV ligation (arrowheads in A) or double ligation and FV division (arrowheads in B) at P0, and quantification of the proportion of valves reaching each developmental stage at P6 in VVs of operated and unoperated limbs. The number of VVs analyzed is given above bars. An asterisk (*) indicates collateral vessel. Bars, 500 µm. **, P < 0.005, χ 2 . Dotted boxes X and Y indicate regions examined by immunofluorescence at P6. In X, arrowhead indicates suture, and the arrow shows the remodeling FV. In Y, the asterisk (*) indicates large collaterals, and the arrow shows the proximal FV. Bars, 20 µm in micrographs. (C) Localization of Foxc2 (green) and quantification of VV area in VV exposed to reduced flow ( n = 8) and controls ( n = 12). ***, P < 0.0005, t test. Bar, 20 µm. (D and E) Analysis of VV length (Hematoxylin-eosin) at 2 wk (D) and 24 wk (E) after Foxc2 deletion. P = NS (ANOVA). The number of VVs analyzed is given below each bar.

Article Snippet: Antibodies were raised in rabbits to Cx43 (3512; Cell Signaling Technology), sheep to FOXC2 (AF5044; R&D Systems), Foxc2 (AF6989; R&D Systems), and goat to Integrinα9 (BAF3827; R&D Systems).

Techniques: Ligation, Immunofluorescence

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation regulates FOXC2 function in vivo. (A) Endothelial cell-specific gain-of-function models for the analysis of FOXC2 phosphorylation. (B) Both models express comparable levels of the transgene, as evidenced by RT-PCR analysis of the indicated mRNAs from E15.5 lungs. Transgene expression was initiated at E13.5. (C) Macroscopic appearances of FOXC2ecGOF, pmFOXC2ecGOF, and control E15.5 embryos. (D and E) FOXC2 overexpression does not affect capillary sprouting. (F) Overexpression of FOXC2 but not pmFOXC2 promotes vascular remodeling in maturing capillaries. Note the increased capillary branching and density in FOXC2ecGOF embryos. Whole-mount staining of E15.5 head skin for pan-endothelial marker CD31 (green) and the transgene (red). The transgene expression was detected using anti-Myc antibody. Scale bars: 100 μm (D), 38 μm (E), 35 μm (F). (G) Quantification of vascular branching, density, and sprouting at the vascular front in the control, pmFOXC2ecGOF, and FOXC2ecGOF embryos. n = 3 per genotype. *, P < 0.05. n.s., nonsignificant.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Staining, Marker

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Analysis of FOXC2 phosphorylation. (A) Endogenous and recombinant human FOXC2 are similarly phosphorylated in primary LECs and immortalized cell lines. Cell lysates were treated (+) or not treated (−) with lambda protein phosphatase (λ-PPase) and analyzed by Western blotting with anti-FOXC2 or anti-Myc antibodies. (B) Schematic representation of FOXC2 phosphorylation sites. FHD, forkhead domain; TA, transactivation domains (5, 34); PD, phosphorylated domain. Phosphorylation sites identified by LC-MS/MS are shaded in red; phosphorylation sites identified by mutagenesis are shaded in yellow. Peptides detected by MS in tryptic and Glu-C digests are underlined in cyan and green, respectively. Amino acid numbering is the same as in the endogenous protein (NP_005242). (C) Substitution of eight phosphorylation sites in Myc-FOXC2 with alanine abolishes the phosphorylation-dependent electrophoretic mobility shift. Lysates of cells transfected with a plasmid expressing the phosphorylation-deficient mutant Myc-pmFOXC2 were treated (+) or not treated (−) with λ-PPase and analyzed by Western blotting with anti-Myc antibody. (D) FOXC2 phosphorylation-deficient mutant (pm) has increased electrophoretic mobility compared to the wild-type (wt) protein. Shown is Western blot analysis of lysates from HepG2 cells transduced with adenoviruses expressing Myc-FOXC2 or Myc-pmFOXC2.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Recombinant, Western Blot, Liquid Chromatography with Mass Spectroscopy, Mutagenesis, Electrophoretic Mobility Shift Assay, Transfection, Plasmid Preparation, Expressing, Transduction

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: FOXC2 interacts with peptidyl-prolyl cis/trans isomerase PIN1, alpha isoform of the regulatory subunit B of the protein phosphatase PP2A (PPP2R2A), and ERK1/2 protein kinases. (A) Coimmunoprecipitation assays with anti-Myc antibody demonstrate the association of Myc-FOXC2 with endogenous PIN1 in HeLa cells transduced with recombinant Ad-Myc-FOXC2. Shown is Western blot (WB) of anti-Myc immunoprecipitates consecutively probed with anti-Myc and anti-PIN1 antibodies. Control immunoprecipitation was performed from extracts of HeLa cells transduced with recombinant adenovirus expressing bacterial β-galactosidase (Ad-LacZ). (B) Myc-FOXC2 binds to endogenous PPP2R2A and ERK1/2 in HepG2 cells transduced with recombinant Ad-Myc-FOXC2. Coimmunoprecipitation assays were performed and analyzed as in A, except that anti-PPP2R2A and anti-total ERK1/2 antibodies were used for immunoblotting. (C) Immunocomplex kinase assays demonstrate that Myc-FOXC2 is phosphorylated in vitro by the coprecipitating endogenous ERK1/2 kinases. Shown is Western blot (WB) of anti-Myc immunoprecipitates incubated in the presence of [γ-32P]ATP and phosphorimage of the corresponding membrane. The blot was consecutively probed with anti-Myc, anti-total ERK1/2 and anti-active ERK1/2 (p-ERK1/2) antibodies. The identity of ERK1/2 was confirmed by immunocomplex kinase assays with anti-Myc antibody using lysates of HepG2 cells stimulated with PMA in the presence or absence of 10 mM U0126, a selective inhibitor of upstream MEK. (D) Inhibition of ERK1/2 does not modify the electrophoretic mobility of endogenous FOXC2 in LECs. Shown is a Western blot of total LEC lysates consecutively probed with anti-FOXC2 and anti-active ERK1/2 antibodies. (E) The electrophoretic mobility of endogenous FOXC2 changes after release from serum starvation-induced cell cycle arrest in LECs, suggesting CDK involvement in FOXC2 phosphorylation.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Transduction, Recombinant, Western Blot, Immunoprecipitation, Expressing, In Vitro, Incubation, Inhibition

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation regulates FOXC2-mediated transcription in primary LECs. (A) Immunofluorescent staining for Myc (green), lymphatic marker PROX1 (red), and DNA (blue) of LECs transduced with adenoviruses expressing wild-type Myc-FOXC2, phosphorylation-deficient mutant Myc-pmFOXC2, or control bacterial β-galactosidase (LacZ). Note that wild-type and mutant FOXC2 have similar expression levels and subcellular localization. Bars, 20 μm. (B) Phosphorylation regulates FOXC2 transcriptional activity. Gene expression profiling was performed on the adenovirus-transduced LECs shown in panel A. Genes whose expression changed >2-fold in response to the loss of FOXC2 phosphorylation (FDR < 0.01) are shown in orange (upregulated) and purple (downregulated) in the Volcano plot of significance against the fold change in gene expression. Vertical dotted lines mark the 2-fold change limits. (C) RT-PCR validation of the gene expression profiling results. Genes upregulated or downregulated >2-fold in response to the loss of FOXC2 phosphorylation are shown in orange and purple, respectively; genes affected <2-fold are shown in gray. No change in FOXC2 expression reflects equally efficient cell transduction with Ad-Myc-FOXC2 and Ad-Myc-pmFOXC2. The data are presented as log2-transformed fold change in gene expression normalized to a housekeeping gene (GAPDH). Horizontal dotted lines mark the 2-fold change limits. Shown are the means and standard deviations of triplicate determinations in a single experiment representative of two independent experiments. (D) Heat map representation of the differences in gene expression in response to the loss of FOXC2 phosphorylation. The left heat map shows expression levels of 57 of 59 genes downregulated >2-fold (FDR < 0.01) in Ad-Myc-pmFOXC2-transduced LECs compared to Ad-Myc-FOXC2-transduced LECs. The right heat map shows expression levels of 57 out of 88 genes upregulated >2-fold (FDR < 0.01) in the same cells. Three biological replicates are shown for each condition. The color key at the lower left corresponds to the mean-centered, arctan-transformed log2-fold change in gene expression falling within the range from −π/2 to π/2. Blue denotes genes with relative decreased expression; red denotes genes with relative increased expression.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Staining, Marker, Transduction, Expressing, Mutagenesis, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation differentially regulates FOXC2 binding to genomic target sites in the context of native chromatin but not in vitro. We used genome-wide ChIP-chip to compare the binding of adenovirus-expressed wild-type Myc-FOXC2 and the phosphorylation-deficient mutant Myc-pmFOXC2 to physiological binding sites occupied by endogenous FOXC2 in primary LECs. Endogenous FOXC2 enrichment profiles are shown at the top of each panel. Purple peaks indicate FOXC2-enriched regions; their relative occupancies by Myc-FOXC2 and Myc-pmFOXC2 are shown in callout boxes in green and orange, respectively. Vertical axes represent MAT score. Binding sites are numbered as in Norrmén et al. (8); genomic coordinates refer to the hg18 human genome assembly. An unbound control region is shown in the lower right panel. The ChIP-chip results were validated by ChIP-qPCR with primers flanking ∼100-bp sequences within the FOXC2-enriched regions. The results are presented as the fold enrichment over the unbound control region. Green and orange bars correspond to wild-type Myc-FOXC2 and Myc-pmFOXC2, respectively. Shown are the means and standard deviations of triplicate determinations. An EMSA was used to compare the in vitro binding of adenovirus-expressed wild-type Myc-FOXC2, Myc-pmFOXC2, and deletion mutant Myc-FOXC2 D219-366 to naked dsDNA from the ChIP-enriched regions or the unbound control region. Binding specificity was controlled with adenovirus-expressed bacterial β-galactosidase (LacZ) and anti-Myc antibody. Asterisks indicate the positions of the antibody-supershifted complexes.

Article Snippet: The immunoprecipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and either immunoblotted with sheep anti-human FOXC2 antibody (R&D Systems) or subjected to autoradiography.

Techniques: Binding Assay, In Vitro, Genome Wide, ChIP-chip, Mutagenesis