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Identification of Flk-1 target genes in vasculogenesis: Pim-1 is required for endothelial and mural cell differentiation in vitro

Alessio Zippo, Alessandra De Robertis, Monia Bardelli, Federico Galvagni and Salvatore Oliviero

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    ES cells differentiate into mature ECs and SMCs by VEGF-A treatment. Indirect immunofluorescence analysis by double staining of endothelial and mural precursors. (A-C) At 5.5 days of ES cell differentiation, ECs stained with PECAM1 (green) and α-SMA-positive cells (red) form a network on different focal planes. Nuclei were stained with DAPI as indicated. Scale bar, 40 μm. (D-E) From day 5.5, EC precursors stained with PECAM1 (green) form primitive capillary tubes, which at 8.5 days are surrounded by SMCs stained with α-SMA (red). Scale bar, 16 μm. (F-G) At 8.5 days of differentiation, ECs are positive for VE-cadherin, which is a late marker of differentiation, and are able to uptake the acetylated low-density lipoprotein (Ac-LDL) particles. Green fluorescent Ac-LDL particles were added directly to the medium of differentiated ECs and analyzed at the microscope. Scale bar, 200 μm. (H) Differentiated ECs at 8.5 days plated on matrigel form capillary-like structure in vitro. Differentiated ECs from VEGF-A-induced EBs were disaggregated and plated on Matrigel. (I) SMCs showed fully differentiated α-SMA fibers. The field was at the periphery of the EBs to observe SMCs. Scale bar, 24 μm.

  • Figure 2.

    Flk-1 mutant ES cells do not differentiate into ECs and SMCs in chemically defined medium. Immunostaining of Flk-1 knock-out (Flk-1-/-), heterozygous (Flk-1+/-), and wild type (Flk-1+/+) cells at 5.5 days of differentiation with VEGF-A as indicated. (A-C) DAPI staining of cell nuclei. (D-F) PECAM1 staining of EC precursors. (G-I) α-SMA staining for mural precursors. Scale bar, 800 μm. (L) Quantitative RT-PCR of PECAM1 and α-SMA mRNAs and at 8.5 days of differentiation with VEGF-A in serum-free medium of Flk-1 wild-type, heterozygous, and knock-out cells as indicated. (M) Quantitative RT-PCR of PECAM1 and α-SMA mRNAs at 8.5 days of differentiation in FCS of Flk-1 wild-type, heterozygous, and knock-out cells as indicated. The data shown in panels L-M are the results of 3 independent experiments. Error bars indicate standard deviation.

  • Figure 3.

    Genes expressed in Flk-1-positive endothelial precursors are transiently up-regulated during angiogenesis in vivo. Time-course analysis of transcripts in hormonally induced ovaries. Vertical bars indicate the fold induction of each transcript as indicated from RNA collected from untreated (1) or hormonally induced ovaries during the follicular phase (FP) at 5 hours (2), 20 hours (3), and 44 hours (4) after pregnant mare's serum gonadotropin (PMS-G) stimulation and luteic phase (LP) at 8 hours (5), 16 hours (6), and 32 hours (7) after human chorionic gonadotropin (hCG) stimulation. Super ovulation in mice was induced as previously described,21 and the induction was quantified by real time RT-PCR and normalized on GAPDH expression. The data shown are the results of 3 independent experiments.

  • Figure 4.

    Genes expressed in Flk-1-positive endothelial precursors are expressed in endothelial cells of the ovary during angiogenesis. Immunofluorescence analysis on frozen ovary sections at 8 hours of the LP. Endothelial-positive cells were identified by staining with PECAM1 (green) antibody, and specific colocalization was valued by double staining with specific antibodies (red) as indicated. Colocalization analysis was performed with a confocal microscope. CL indicates corpus luteum. Scale bar, 40 μm.

  • Figure 5.

    Pim-1 is transiently expressed in ECs of the ovary during angiogenesis. Immunofluorescence analysis on frozen ovary sections at ovaries during the follicular phase (FP) and luteic phase (LP) at the time indicated. Endothelial-positive cells were identified by staining with PECAM1 (green) antibody, and specific colocalization was valued by double staining with Pim-1 antibodies (red) as indicated. Colocalization analysis was performed with a confocal microscope. Scale bar, 40 μm.

  • Figure 6.

    Genes expressed in Flk-1-positive endothelial precursors are induced by VEGF-A in HUVECs. Time-course analysis of the human orthologues of the mouse genes as in Figure 3, as indicated. Vertical bars for each transcript, as indicated, measure the fold induction at the time indicated. Induction analysis was performed with RT-PCR and normalized on GAPDH expression. The data shown are the results of 3 independent experiments.

  • Figure 7.

    Pim-1 is required for vasculogenesis and angiogenesis in vitro. (A) Western blot analysis of Pim-1 silencing obtained by infecting ES cells with a lentivirus vector expressing Pim-1 siRNA. (B) FACS analysis of differentiated ES cells at 5.5 and 8.5 days using specific antibodies for each marker as indicated. Green lines indicate the positive cell population, and numbers above each curve indicate the percentage of positive cells compared with red lines, which represent negative controls. ES cells were infected with a lentivirus vector generating, under the control of the U6 promoter, siRNA for renilla luciferase gene (control) or the murine Pim-1. (C) HUVECs were infected with the lentiviral vector expressing siRNA to silence the renilla luciferase (control) or human PIM-1 under the control of the U6 polymerase promoter. Pim-1 expression in HUVECs was valued by Western blot at 2 hours after VEGF-A induction. (D) PIM-1 silencing affects both proliferation and migration of HUVECs in vitro. Vertical bars represent the fold induction as indicated. Proliferation and migration tests were performed as described.24 The data are the results of 3 independent experiments. (E) Pim-1 silencing inhibited the angiogenesis assay in vitro. A representative experiment is shown. The assay was performed by plating HUVECs either infected with the mock vector or infected with the siRNA lentiviral vector on Matrigel, and HUVECs were induced with VEGF-A as described.24 (F) PIM-1 silencing inhibited sprouting of ECs. A representative experiment is shown. The sprouting assay was performed as described.35 Original magnification × 40.

Tables

  • Table 1.

    Comparison of the expression levels of transcripts differentially expressed in Flk1+/+ versus Flk1−/− ES cells at 5.5 days of differentiation in vitro by macroarray analysis and real-time quantitative RT-PCR

    Category/geneFold increase by macroarrayFold increase by RT-PCR
    Nuclear factor
    Cbp 4.3 ± 0.6 7.6 ± 0.5
    Hec 6.4 ± 1.2 3.4 ± 0.2
    Nm23M1 3.1 ± 0.8 3.8 ± 0.7
    Nm23M2 3.9 ± 0.6 2.1 ± 0.3
    Pim-1 2.9 ± 0.9 3.2 ± 0.2
    pp32 3.7 ± 0.8 3.2 ± 0.5
    Set 5.2 ± 0.8 2.4 ± 0.2
    Slug 3.4 ± 0.3 5.9 ± 0.5
    Sox18 5.7 ± 0.2 4.4 ± 0.7
    Growth factor
    Sfrp1 (FrzA) 4.9 ± 0.3 11.3 ± 1.2
    Receptors
    Edg5 4.6 ± 0.3 3.1 ± 0.5
    EphB3 (Sek4) 3.5 ± 0.2 2.2 ± 0.3
    Signal transduction
    Ship-1 9.9 ± 0.8 7.0 ± 0.4
    Btk 3.0 ± 0.3 3.2 ± 0.2
    Unknown
    Mesdc2 > 20 13.6 ± 3.1
    • Data are presented as mean ± SEM. The fold change detected by quantitative RT-PCR represents the average between the results of 3 independent experiments.

  • Table 2.

    Expression levels in ES cells infected with Pim-1 RNAi versus Flk1+/+ ES cells at 5.5 and 8.5 days of differentiation in vitro

    Fold increase in Pim-1 RNAi
    GeneDay 5.5Day 8.5
    Flk-1 −2.7 ± 0.4 −14.2 ± 21
    PECAM1 −2.8 ± 0.3 −4.5 ± 0.5
    VE-cadherin −8.8 ± 1.2 −42 ± 6.3
    Tie-2 −2.9 ± 0.3 −35.0 ± 4.4
    Flt4 −5.9 ± 1.0 −1.4 ± 0.1
    VEGF-A −12.3 ± 1.1 −1.3 ± 0.1
    VEGF-D −1.6 ± 0.2 −2.5 ± 0.4
    α-SMA −4.6 ± 0.6 −2.5 ± 0.3
    SM22α −4.5 ± 0.5 −2.6 ± 0.4
    Calponin-h1 −7.3 ± 0.9 −1.7 ± 0.2
    SM-MHC −3.7 ± 0.3 +1.7 ± 0.1
    Brachyury −37.3 ± 4.9 +1.0 ± 0.1
    α 1-fetoprotein −1.1 ± 0.1 +143 ± 18
    Nestin +1.4 ± 0.2 −3.5 ± 0.5
    Myogenin +81.2 ± 11 +129.0 ± 15
    • Data are presented as mean ± SEM. The fold change detected by quantitative RT-PCR represents the average among the results of 4 independent measurements.