Fig 1: Ligand-specific and redundant functions of Angpt4 and ANGPT4.(A) Angiopoietin-induced TIE2 translocation and activation in cell–cell junctions. TIE2-WT HUVECs were left non-stimulated (control) or stimulated with recombinant angiopoietins as indicated for 1 hr, fixed, and stained for total TIE2 (green), phosphorylated TIE2 (pTIE2, red) and nuclei (DAPI, blue). White cropping indicates examples of ROIs (sites where cell contacts occurred based on microscopic examination) for TIE2 and pTIE2 intensity measurement from randomly selected cells. (B) Quantification of TIE2 activation (pTIE2/total TIE2) in cell–cell junctions (n = 2 to 7 stimulations, total 330 to 915 cell junctions/stimulation were measured). (C) Sparse TIE2-GFP HUVECs were stimulated with angiopoietins overnight, fixed and imaged. In ANGPT1-stimulated cells, majority of TIE2-GFP is ECM bound (arrows). ANGPT2 promoted TIE2 translocation to the retracting cell edges (arrows) and less in ECM (dotted line indicates leading edge of the cell). Angpt4 and ANGPT4 did not induce long-term effect on TIE2 clustering and translocation into ECM. (D) Angiopoietin binding to acellularized ECM fraction from cultured HUVECs (n = 4 run in triplicate). (E) TIE2-WT HUVECs spreading on fibronectin, ANGPT1, ANGPT2, Angpt4 or ANGPT4. Cells were let to spread on coated coverslips for 1 hr and stained for actin. Quantification of image data is shown in (F). Cell area was normalized to fibronectin (FN) (n = 3 to 9 experiments, 333 to 1778 cells per coating were analyzed). (G) Angiopoietin-TIE2 binding affinity measured by ELISA. ANGPT1 binding to TIE2 (250 ng/ml) was set to one at ANGPT1 concentration 500 ng/ml (n = 4 experiments). (H) Angiopoietin binding to TIE2 in surface plasmon resonance assay. Human purified TIE2-Fc was immobilized on CM5 chip, and angiopoietins were injected onto the TIE2-Fc surface at 100 nM concentration. Mean ±SD, ANOVA followed by the Bonferroni post hoc test. ***p<0.001, **p<0.01, *p<0.05 vs. control; †††p<0.001, ††p<0.01 vs. ANGPT1; ###p<0.001, ##p<0.01, #p<0.05 vs. ANGPT2.
Fig 2: mRNA expression of angiopoietins and Tie2 in retinal vein development.mRNA expression was detected in whole mount retinas from WT mice by in situ hybridization. (A) At P3, astrocytes expressing Angpt4 precede the front of the developing vasculature (red blood cells indicated by arrows). (B) At P12, Angpt4 expression is notable around the developing vein in the peripheral retina (V, asterisks) and colocalizes with the astrocyte marker GFAP (white overlay in insert). (C) Angpt1 expression is not detected at P12 by whole mount in situ hybridization in the peripheral retina. (D) Angpt2 is expressed in retinal neurons (arrowhead) in the intermediate retinal plexus. (E) Expression of Tie2 is detected in the endothelium of arteries (A), veins and capillaries at P12. OR, ora serrata.
Fig 3: Tie2 phosphorylation is reduced under oxidative conditions. (A) HUVECs were starved for 6–8 h and treated with angiopoietin-1 (Ang1; 250 ng/mL) and/or H2O2 (1 mM) for 30 min, then stained with actin (red) and VE-cadherin. White asterisks indicate the empty space between endothelial cells. Scale bar, 20 μm. (B) Serum-starved HUVEC monolayer on the upper chamber of the transwell was treated with Ang1 (250 ng/mL) and/or H2O2 (1 mM) along with FITC-dextran for 30 min. (C) PBS in the lower chamber of the transwell, containing FITC-dextran that leaked from the upper chamber, was collected, and the fluorescence measured. Error bars indicate the mean ± SEM (n = 4 independent experiments). ∗p < 0.05, ∗∗∗p < 0.001 (One-way ANOVA and Bonferroni's comparison test). (D) HUVECs were starved and treated with Ang1 (250 ng/mL) and/or H2O2 (1 mM), then stained with anti-Tie2 (green), anti-phosphorylated Tie2 (pTie2; red), and Hoechst. Scale bar, 20 μm. (E) Tie2 was harvested via immunoprecipitation using an anti-Tie2 extracellular domain (ECD) antibody, and then blotted with anti-pTie2 (upper) and anti-Tie2 intracellular domain (ICD) antibodies. (F) Representative images of skin sections stained with anti-pTie2 (red), anti-CD31 (green), and Hoechst. Scale bar, 100 μm. (G) Boxed regions in (F) were digitally magnified, and images of Tie2 and CD31 staining, as well as merged images, presented. Scale bar, 5 μm. (H) Fluorescence intensities of pTie2 within CD31+ regions were measured and normalized against those of CD31. Dots represent CD31+ regions (n = 18) pooled from stained sections of 5 different mice per condition. Error bars indicate the mean ± SEM. ∗∗∗p < 0.001 (unpaired t-test).
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