Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity - PubMed (original) (raw)

Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity

Yasuhiro Funahashi et al. Vasc Cell. 2011.

Abstract

Background: In the vasculature, Notch signaling functions as a downstream effecter of Vascular Endothelial Growth Factor (VEGF) signaling. VEGF regulates sprouting angiogenesis in part by inducing and activating matrix metalloproteases (MMPs). This study sought to determine if VEGF regulation of MMPs was mediated via Notch signaling and to determine how Notch regulation of MMPs influenced endothelial cell morphogenesis.

Methods and results: We assessed the relationship between VEGF and Notch signaling in cultured human umbilical vein endothelial cells. Overexpression of VEGF-induced Notch4 and the Notch ligand, Dll4, activated Notch signaling, and altered endothelial cell morphology in a fashion similar to that induced by Notch activation. Expression of a secreted Notch antagonist (Notch1 decoy) suppressed VEGF-mediated activation of endothelial Notch signaling and endothelial morphogenesis. We demonstrate that Notch mediates VEGF-induced matrix metalloprotease activity via induction of MMP9 and MT1-MMP expression and activation of MMP2. Introduction of a MMP inhibitor blocked Notch-mediated endothelial morphogenesis. In mice, analysis of VEGF-induced dermal angiogenesis demonstrated that the Notch1 decoy reduced perivascular MMP9 expression.

Conclusions: Taken together, our data demonstrate that Notch signaling can act downstream of VEGF signaling to regulate endothelial cell morphogenesis via induction and activation of specific MMPs. In a murine model of VEGF-induced dermal angiogenesis, Notch inhibition led to reduced MMP9 expression.

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Figures

Figure 1

Figure 1

VEGF activated Notch signaling in HUVEC. A) HUVEC were transduced with increasing amounts of Ad-VEGF as indicated, and Notch/CSL reporter activity determined. B) The VEGFR inhibitor (SU5416) perturbed Notch signal activation downstream of VEGF. HUVEC were transduced with Ad-VEGF at 40 MOI and increasing amounts of SU5416 added to HUVEC prior to determining CSL reporter activity. C) Notch1 decoy blocked VEGF-induced Notch signal activation. HUVEC were transduced with Ad-VEGF at 20 MOI and increasing amounts of Ad-Notch1 (N1) decoy as indicated. Ad-LacZ was added to transductions to normalize the MOI between conditions. Notch/CSL reporter luciferase data are represented as fold induction relative to Ad-LacZ controls. Experiments were performed in triplicate, and data of a representative experiment presented.

Figure 2

Figure 2

VEGF-dependent HUVEC morphogenic changes are mediated by Notch. A) HUVEC were transduced with Ad-LacZ or Ad-VEGF, with or without Ad-Notch1 (N1) decoy at 40 MOI. Ad-LacZ was used to normalize MOI to 80. VEGF induced HUVEC morphogenesis (lower left panel) that was blocked by N1 decoy (lower right panel). B) Quantification of Ad-VEGF-induced morphogenesis, measured as numbers of HUVEC with cellular extensions. HUVEC were transduced with increasing amounts of Ad-VEGF as indicated and Ad-LacZ was used to normalize MOI to 40. C) N1 decoy blocked VEGF-induced HUVEC morphological changes without affecting total cell number. HUVEC were transduced with Ad-VEGF at 40 MOI and increasing amounts of Ad-N1 decoy as indicated. Ad-LacZ was used to normalize the MOI to 100. Experiments were performed in triplicate, and data of a representative experiment presented.

Figure 3

Figure 3

Notch mediates VEGF-induced fibrinolysis and gelatinase activity. A) N1 decoy blocked VEGF-induced fibrinolytic activity. HUVEC were transduced with Ad-VEGF, Ad-N1decoy or both at 40 MOI and Ad-LacZ was used as a control and to normalize the MOI to 80. Transductants were seeded on fibrin gels and fibrin degradation determined after 5 days. B) N1decoy blocked VEGF-induced MMP2 activation. HUVEC were transduced with Ad-VEGF at 20 MOI and increasing amounts of Ad-N1decoy as indicated. Ad-LacZ was used as a control and to normalize the MOI to 80. Four days post-infection, supernatants were collected and subjected to gelatinase zymography. C) N1 decoy blocked VEGF-induced expression and activation of MMP9. HUVEC were transduced with Ad-VEGF at 40 MOI, with or without Ad-N1decoy at 40 MOI and Ad-LacZ was used as a control and to normalize the MOI to 80. Supernatants from transduced HUVEC were collected at 2, 4, 6, and 8 days post-infection and subjected to gelatinase zymography. D) Cross-section of skin from DAS assay stained for PECAM (red) and MMP9 (green). White boxes highlight the large vessels within the subcutaneous adipose depot. Yellow boxes highlight the capillaries within the dermal smooth muscle cell layer. Representative photographs are shown.

Figure 4

Figure 4

Notch induced MMP9 and MT1-MMP expression downstream of VEGF. A) Transduction of an activated form of Notch1 (N1IC) induced MMP9 transcripts in HUVEC. RT-PCR analysis of MMP2 and MMP9 compared to control GAPDH. B) Transduction of N1IC induced MT1-MMP. RT-PCR analysis of MT1-MMP and MT2-MMP (left panels) compared to control GAPDH (see A). Western analysis of MT1-MMP (right panel). C) N1decoy blocked VEGF-mediated induction of MMP9 and MT1-MMP determined by RT-PCR; GAPDH serves as a control. HUVEC were transduced with Ad-VEGF at 40 MOI, with or without Ad-N1decoy at 40 MOI and Ad-LacZ was used as a control and to normalize the MOI to 80. For RT-PCR experiments, the number of PCR cycles is indicated to the right of the figures. Experiments were performed in triplicate, and data of a representative experiment presented.

Figure 5

Figure 5

Notch-induced HUVEC morphogenesis was mediated via its induction of MMPs. A) Notch-induced HUVEC morphogenesis on collagen was blocked by addition of an MMP inhibitor. HUVEC were transduced with either Ad-LacZ or Ad-N1IC at 40 MOI and seeded on type 1 collagen gels in the absence or presence of 50 μM GM6001. Images are representative of 5 days post-infection. B) Quantification of Ad-N1IC-induced morphogenesis on collagen, measured as numbers of HUVEC with cellular extensions. C) Notch-induced HUVEC morphogenesis on fibrin was blocked by addition of an MMP inhibitor, but not a serine-protease inhibitor. HUVEC were transduced with either Ad-LacZ or Ad-N1IC at 40 MOI and seeded on fibrin gels in the absence or presence of the matrix metalloprotease inhibitor (50 μM GM6001), or the serine-protease inhibitor, (10 mM eACE). Images are representative of 5 days post-infection. D) Quantification of Ad-N1IC-induced morphogenesis on fibrin. Experiments were performed in triplicate, and data of a representative experiment presented. NT (No Treatment).

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References

    1. Wagner S, Fueller T, Hummel V, Rieckmann P, Tonn JC. Influence of VEGF-R2 inhibition on MMP secretion and motility of microvascular human cerebral endothelial cells (HCEC) J Neurooncol. 2003;62:221–231. doi: 10.1023/A:1023335732264. - DOI - PubMed
    1. Dias S, Hattori K, Zhu Z, Heissig B, Choy M, Lane W, Wu Y, Chadburn A, Hyjek E, Gill M. et al.Autocrine stimulation of VEGFR-2 activates human leukemic cell growth and migration. J Clin Invest. 2000;106:511–521. doi: 10.1172/JCI8978. - DOI - PMC - PubMed
    1. Wang H, Olszewski B, Rosebury W, Wang D, Robertson A, Keiser JA. Impaired angiogenesis in SHR is associated with decreased KDR and MT1-MMP expression. Biochem Biophys Res Commun. 2004;315:363–368. doi: 10.1016/j.bbrc.2004.01.059. - DOI - PubMed
    1. Deryugina EI, Quigley JP. Pleiotropic roles of matrix metalloproteases in tumor angiogenesis: contrasting, overlapping and compensatory functions. Biochim Biophys Acta. 2010;1803:103–120. doi: 10.1016/j.bbamcr.2009.09.017. - DOI - PMC - PubMed
    1. Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteases: regulators of the tumor microenvironment. Cell. 2010;141:52–67. doi: 10.1016/j.cell.2010.03.015. - DOI - PMC - PubMed

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