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. 2003 Jan;23(1):14-25.
doi: 10.1128/MCB.23.1.14-25.2003.

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis

Zhao-Jun Liu  1 , Takashi Shirakawa, Yan Li, Akinobu Soma, Masahiro Oka, G Paolo Dotto, Ronald M Fairman, Omaida C Velazquez, Meenhard Herlyn
Affiliations

Regulation of Notch1 and Dll4 by vascular endothelial growth factor in arterial endothelial cells: implications for modulating arteriogenesis and angiogenesis

Zhao-Jun Liu et al. Mol Cell Biol. 2003 Jan.

Abstract

Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.

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Figures

FIG. 1.
FIG. 1.
Effect of angiogenic factors on induction of Notch and Delta genes. Total RNA was extracted from various endothelial cells transduced with recombinant adenoviruses and subjected to reverse transcription-PCR. The specific PCR bands were separated in 2% agarose gels and stained with ethidium bromide. β-Actin mRNA was amplified as a control. Results are from a representative experiment of three performed.
FIG. 2.
FIG. 2.
Induction of Notch1/Dll4 by recombinant human VEGF165 in HIAECs. (a) Dose effect of VEGF on Notch1/Dll4 induction. Cells were harvested 24 h after stimulation with recombinant human VEGF165 at the indicated doses, and total RNA was extracted and subjected to reverse transcription-PCR analyses. Intensity of Notch1/Dll4 bands obtained from cells stimulated with 100 ng of recombinant human VEGF165 per ml was normalized as 100, and amounts of Notch1/Dll4 mRNA are given relative to this value. Data are means ± standard deviations of three independent experiments. (b) Specific effect of VEGF on Notch1/Dll4 induction. Cells were harvested 24 h after stimulation with recombinant human VEGF165, bFGF, and acidic FGF at the indicated doses, and total RNA was extracted and subjected to reverse transcription-PCR analyses. Results are from a representative experiment of two performed. (c) Kinetics of Notch1/Dll4 induction by recombinant human VEGF165. Subconfluent HIAECs were stimulated with 100 ng of recombinant human VEGF165 per ml, harvested at the indicated times, and analyzed for Notch1/Dll4 expression by reverse transcription-PCR. Results are from a representative experiment of two performed. (d) Kinetics of Notch1/Dll4 induction by recombinant human VEGF165. The same samples obtained as in panel c were analyzed for Notch1/Dll4 expression by Northern blotting. 28s rRNA was stained with methylene blue. Results are from a representative experiment of two performed. (e) Specificities of VEGF induction of Notch1/Dll4. Cells were transduced with VEGF-Trap/Ad5 and Fc/Ad5 for 48 h and stimulated with 100 ng of recombinant human VEGF165 per ml for 24 h. RNA was isolated and subjected to reverse transcription-PCR. β-Actin mRNA was amplified as a control. Results are from a representative experiment of three performed.
FIG. 3.
FIG. 3.
Requirement for VEGFR1/R2 in Notch1/Dll4 expression. (a) Expression of VEGFR1, VEGFR2, but not VEGFR3 mRNA in HIAECs. RNA from HIAECs and 293 cells was subjected to Northern blot analyses. Membranes were hybridized with 32P-labeled VEGFR1, -R2, and -R3 probes at an activity of 106 cpm/ml. Data for R1 was obtained by PhosphoImager scanning, while those for R2 and R3 were obtained by exposure to Kodak film for 4 days. (b) Both VEGFR1 and -R2 are involved in VEGF-induced Notch1/Dll4 expression. HIAECs were transduced with the different recombinant adenoviruses indicated and harvested at 48 h, and RNA was subjected to reverse transcription-PCR. Results are from a representative experiment of three performed.
FIG. 4.
FIG. 4.
Phosphatidylinositol 3-kinase is involved in VEGF-induced Notch1/Dll4 expression. (a) Effect of VEGF and PD98059 on MAPK activation. HIAECs were left untreated or treated with PD98059 for 5 min before addition of recombinant human VEGF165. Cells were lysed 15 min later and analyzed by Western blotting. Specific bands were immunoblotted with anti-phospho-MAPK antibody, stripped, and reblotted with anti-MAPK antibody. (b) Effect of VEGF and exogenous phosphatidylinositol 3-kinase mutants on phosphatidylinositol 3-kinase activation. HIAECs were transduced with adenoviruses for 48 h and then either stimulated with recombinant human VEGF165 for 15 min or left unstimulated. The phosphatidylinositol 3-kinase assay was performed as described in the text. The PIP3 product, separated by thin-layer chromatography, and the origin are indicated. (c) Effect of specific kinase inhibitors on VEGF-induced Notch1/Dll4 expression. HIAECs were treated for 5 min with various inhibitors before addition of recombinant human VEGF165. Cells were harvested 24 h later, and extracted RNA was subjected to reverse transcription-PCR. Results are from a representative experiment of three performed. (d) Enforced expression of DN-Δp85 in HIAECs. Cells transduced with adenoviruses as indicated for 48 h or left untreated were lysed, and whole-cell lysates were subjected to Western blot (IB) analysis. The endogenous wild-type p85 (Wp85) and exogenous mutant of p85 (Δp85) are indicated. (e) Effect of phosphatidylinositol 3-kinase mutants on VEGF-induced Notch1/Dll4 expression. HIAECs transduced with adenoviruses for 48 h were either stimulated with recombinant human VEGF165 or left untreated. Cells were harvested at 24 h, and extracted total RNA was subjected to reverse transcription-PCR. Results are from a representative experiment of three performed.
FIG. 5.
FIG. 5.
Involvement of Akt in phosphatidylinositol 3-kinase-mediated signaling. (a) Activation of endogenous Akt by activated phosphatidylinositol 3-kinase. Cell lysates used in Fig. 4d, which contained equal amounts of proteins, were subjected to Western blot analysis with anti-phospho-Akt antibody. (b) Enforced expression of Myr-Akt in HIAECs. Cells transduced with Myr-Akt/Ad5 for 48 h or left untreated were harvested, lysed, and analyzed by Western blotting. Membranes were first immunoblotted with anti-Akt antibody to demonstrate overexpression of Myr-Akt and then reblotted with anti-phospho-Akt antibody to detect Akt phosphorylation. (c) Effect of Myr-Akt on VEGF-induced Notch1/Dll4 expression. HIAECs transduced with Myr-Akt/Ad5 for 48 h were either stimulated with recombinant human VEGF165 or left untreated. The experiment was performed as described for Fig. 4e. Results are from a representative experiment of three performed.
FIG. 6.
FIG. 6.
Effects of NICD and HES1 on cell proliferation and survival. (a) [3H]thymidine uptake by HIAECs. Cells were transduced with NICD/Ad5, HES1/Ad5, or LacZ/Ad5. The assay was performed as described in the text. Results are means ± standard deviations of three independent experiments. *, P < 0.05, Student's t test. (b) Prolongation of cell survival by NICD and HES1. Adenovirus-transduced cells were cultured in serum-free medium, and live cells were counted. Results are means ± standard deviations of three independent experiments. Inset: Western blot assay of NICD and HES1 (exogenous HES1 in HES/HIAECs and induced endogenous HES1 in NICD/HIAECs) expression in HIAECs. β-Actin was used as a control for equal loading of proteins.
FIG. 7.
FIG. 7.
Enhancement of network and cord formation in HIAECs by NICD and HES-1. (a) Stabilization of HIAEC network formation on Matrigel. Transduced HIAECs were seeded on Matrigel and photographed in representative areas at the indicated times. Experiments were repeated three times. Magnification, ×10. (b) Enhancement of HIAEC network and cord formation in an in vitro three-dimensional model. Fluorescent staining of endothelial cells in whole-mount collagen gels is shown for VEGF/FF plus LacZ/HIAECs (panel 1), VEGF/FF plus NICD/HIAECs (panel 2), VEGF/FF plus HES/HIAECs (panel 3), LacZ/FF plus LacZ/HIAECs (panel 4), LacZ/FF plus NICD/HIAECs (panel 5), and LacZ/FF plus HES/HIAECs (panel 6). Magnification, ×10. The scale bar is shown in panel 4.
FIG. 8.
FIG. 8.
Suppressive effect of RBP-Jκ (R218H) on VEGF-driven network and cord formation in the three-dimensional model. (a) Exogenous expression of RBP-Jκ (R218H) in HIAECs. Equal numbers (106) of cells transfected with either pEFBOS-RBP-Jκ (R218H)-Myc-tag or pEFBOS for 48 h were harvested, lysed, and analyzed by blotting (IB) with anti-Myc tag antibody. (b) Inhibition of HIAEC network and cord formation in an in vitro three-dimensional model. Fluorescent staining of endothelial cells in whole-mount collagen gels was done with anti-vWF. Magnification, ×10. (c) Quantitative representation of network and cord formation in an in vitro three-dimensional model. The numbers of networks and cords were counted in at least three low-power fields (LPF) per sample. Magnification, ×10. Experiments were performed in quadruplicate, and results are means ± standard deviations of three independent experiments. *, P < 0.05, Student's t test.
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