GATA6 expression in endothelial cells. A, quantitative real-time PCR analysis of GATA6 mRNA in HUVECs, HUAECs, and HCMECs is shown. The results are representative of three independent experiments (each with n = 3 per cell type). B, a representative Western blot for GATA6 from HUVEC, HUAEC, and HCMEC total cell lysates is shown. Actin was used as a loading control. C, in situ hybridization of mouse heart and kidney sections stained for GATA6 mRNA (blue) is shown. Specific expression of GATA6 in endothelial cells is indicated by arrows. GATA6 expression was also detected in the adjacent myocardium and in smooth muscle cells. Staining for Semaphorin 7A was used as a negative control. Samples were visualized using an Axiovert 200M microscope (Carl Zeiss) with a 40×/0.75 lens (Carl Zeiss).

GATA6 activation in endothelial cells. A, immunofluorescence staining for GATA6 (red) in HUVECs that were unstimulated or stimulated for 4 h either with growth factor mixture or hypoxia. Samples were visualized using an Axiovert 200M microscope (Carl Zeiss) with a 40×/0.75 lens (Carl Zeiss). The results are representative of three independent experiments. B, shown is a ChIP assay in unstimulated or growth factor-stimulated HUVECs. The immunoprecipitation (IP) was performed with GATA6 antibodies or a nonspecific IgG; recovered DNA as well as total input DNA (Input) were analyzed by PCR using primers that bind in the 5′ promoter region of the PECAM1, EDN1, or NOS3 genes, respectively. The results are representative of three independent experiments. C, shown is a schematic illustration of the GATA-luciferase (Ad.GATA-Luc) construct that was adenovirally delivered for reporter assays to measure total GATA-dependent transcriptional activity. D, shown is real-time PCR analysis of GATA6 mRNA in HUVECs transfected with a control siRNA or siRNA against GATA6. Expression in cells transfected with a control siRNA was used as reference and set to 1. The results are representative of three independent experiments (with n = 2–3 per condition in each). E, representative Western blot analysis of GATA2, -3, and -6 protein abundance in HUVECs transfected with a control siRNA or siRNA against GATA6 is shown. Actin was used as a loading control. F, luciferase activity in HUVECs that were infected with Ad.GATA-Luc and transfected either with a control (Co) siRNA or a siRNA against GATA6 (G6) and that either remained unstimulated (Unstim) or stimulated with growth factor mixture (GF) or hypoxia. Results from a representative experiment are shown (n = 3 per condition). The experiment was repeated twice with similar results. *, p < 0.01 versus siRNA control, unstimulated; #, p < 0.01 versus siRNA control with growth factor stimulation; §, p < 0.05 versus siRNA control with hypoxia stimulation.

GATA6 is essential for endothelial cell function. A, endothelial cell proliferation (assessed by BrdU incorporation with a spectrophotometric absorbance assay) in HUVECs or HCMECs transfected with a control siRNA or siRNA against GATA6 is shown. B, shown is quantification and representative images (C) of vessel-like structure formation on Matrigel in HUVECs and HCMECs when treated with a control siRNA or siRNA against GATA6. HUVECs were visualized using an Axiovert 200M microscope (Carl Zeiss) with a 5×/0.12 lens (Carl Zeiss). D, shown is a migration assay (assessed the amount of cells that successfully migrated along a growth factor gradient) with HUVECs or HCMECs transfected with a control siRNA or siRNA against GATA6. E, shown is a Western blot for GATA6 and actin (loading control) in HUVECs in which GATA6 protein in GATA6 siRNA-treated cells was restored using adenoviral GATA6 (Ad.GATA6) expression. The β-galactosidase-expressing adenovirus (Ad.βgal) was used as a control. F, endothelial function was assessed by proliferation, vessel-like structure formation, and migration assays in cells that were treated like described under E. OD, optical density; HPF, high power field. For A, B, D, and F, data from a representative experiment are shown (n = 3–5 per condition). The experiment was repeated twice with similar results. *, p < 0.05 versus siRNA Control; #, p < 0.05 versus siRNA-GATA6 + Ad.GATA6.

GATA6 overexpression enhances endothelial cell function. A, shown is a representative Western blot analysis of GATA6 expression in HUVECs infected with an adenovirus expressing GATA6 (Ad.GATA6) or β-galactosidase (Ad.βgal). Actin was used as a loading control. B, immunofluorescence staining for GATA6 (red) in HUVECs infected with an adenovirus expressing GATA6 (Ad.GATA6) or β-galactosidase (Ad.βgal) is shown. Samples were visualized using an Axiovert 200M microscope (Carl Zeiss) with a 40×/0.75 lens (Carl Zeiss). C and D, quantification of vessel-like structure formation (C) and migration (D) in HUVECs and HCMECs 48 h after infection with Ad.βgal or Ad.GATA6 is shown. HPF, high power field. Data from a representative experiment are shown (n = 3 per condition). The experiment was repeated twice with similar results. *, p < 0.05 versus Ad.βgal.

GATA6 is necessary to maintain viability in endothelial cells. A, quantification of viable (surviving) HUVECs 48 h after treatment with a control siRNA or siRNA against GATA6 is shown. The cells were kept in a growth factor mixture-containing medium. Data from a representative experiment are shown (n = 2–3 per condition). The experiment was repeated twice with similar results. B, flow cytometric analysis of HUVECs treated as described under A and stained with annexin-V and 7AAD is shown. The numbers in the lower right quadrant of the plots indicate the number of annexin-V-positive cells (early apoptotic cells), whereas in the upper right quadrant the number of cells staining positive for both annexin-V and 7AAD (late apoptotic/necrotic cells) is indicated. Data from a representative experiment are shown; the experiment was repeated twice with similar results. C, shown is quantification of viable (surviving) HUVECs 17 h after growth factor withdrawal was started and when treated with a control siRNA or siRNA against GATA6. Data from a representative experiment are shown (n = 3 per condition). The experiment was repeated twice with similar results. *, p < 0.01 versus siRNA control. D, flow cytometric analysis of HUVECs stained with annexin-V and 7AAD 17 h after growth factor withdrawal was started is shown. Data from a representative experiment are shown; the experiment was repeated twice with similar results. HPF, high power field.

GATA6 suppresses TGFβ in endothelial cells. A, RNA isolated from HUVECs treated with a control (Co) siRNA (columns 1 and 2) or siRNA against GATA6 (G6, columns 3 and 4) was subjected to gene expression array analysis. The heat map plots represent the expression level of genes: green, down-regulation; red, up-regulation relative to the control (black). B, quantitative real-time PCR analysis of the indicated genes in HUVECs that were regulated as indicated in the cells treated with GATA6 siRNA versus control siRNA. The expression level in cells transfected with a control siRNA was used as a reference and set to 1. The -fold increase in the mRNA level is indicated. Data from at least two independent experiments (each with n = 2–3 per condition) are shown. *, p < 0.01 versus sicontrol; #, p < 0.05 versus sicontrol. C, GATA6 directly regulates the Tgfb1 promoter. Relative luciferase activity from rat heart endothelial (RHE-A) cells transfected with a Tgfb1 promoter luciferase construct and co-transfected with a GATA6-expressing or a control plasmid is shown. As the Tgfb1 promoter construct, either the −1079 bp (TGFβ1–1079-Luc) or the −406 bp (TGFβ1–406-Luc) construct was used. A schematic representation of the Tgfb1 promoter constructs is shown above the bar graph. Data from a representative experiment are shown (n = 3); the experiment was repeated twice with similar results. *, p < 0.01 versus TGFβ1–1079-Luc.

GATA6 facilitates endothelial cell function by suppressing ALK5/SMAD2 signaling. A, shown are proliferation and vessel structure formation assays when HUVECs transfected with control (Co) siRNA or GATA6 (G6) siRNA were additionally treated with a TGFβ neutralizing antibody (AB) or control IgG. Data from a representative experiment are shown (n = 3 per condition). The experiment was repeated twice with similar results. *, p < 0.05 versus other groups. B, shown is a representative Western blot analysis of phospho (P) or total SMAD2 and SMAD1/5 proteins from HUVECs transfected with a control siRNA or GATA6 siRNA and additionally treated with a specific ALK5 inhibitor (SB-431542, 10 μm) as indicated. Each condition is represented in duplicate. Actin was used as a loading control. C, shown is a representative Western blot analysis of phospho (P) or total SMAD2 and SMAD1/5 proteins from HUVECs treated with recombinant TGFβ1 protein for 90 min as indicated. Each condition is represented in duplicate. Actin was used as a loading control. D, proliferation, vessel-like structure formation, cell migration, and cell death assays when HUVECs transfected with a control siRNA or GATA6 siRNA were additionally treated with the ALK5 inhibitor (SB-431542, 10 μm) as indicated. Data from a representative experiment are shown (n = 3 per condition). The experiment was repeated twice with similar results. *, p < 0.05 versus other groups; #, p < 0.05 versus siRNA control (Co). E, shown is a schematic representation of the functional role of GATA6 in endothelial cells. Left side, GATA6 suppresses TGFβ1/β2 expression and thereby keeps ALK5-SMAD2 activation at a low level. ALK1-SMAD1/5 signaling was previously shown to enhance angiogenic function and survival in endothelial cells (28, 38). Right side, down-regulation of GATA6 results in increased production of TGFβ, enhanced activation of the ALK5-SMAD2 cascade, and inhibition of endothelial function and survival. Activation of ALK1-SMAD1/5 is not influenced by GATA6.