Notch modulates VEGF action in endothelial cells by inducing Matrix Metalloprotease activity
© Funahashi et al; licensee BioMed Central Ltd. 2011
Received: 29 December 2010
Accepted: 18 January 2011
Published: 18 January 2011
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.
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.
Angiogenesis, the formation of new blood vessels from existing vasculature, is a multi-step process that plays a central role in embryogenesis and pathological phenomena. Vascular Endothelial Growth Factor (VEGF) is a key regulator of angiogenesis and is important for the degradation of extracellular matrix (ECM), as well as the subsequent proliferation, migration, and survival of endothelial cells. ECM components, including fibrins, collagens, and laminins, form a lamina around existing vasculature that must be degraded in order to form new vessels.
VEGF signaling via VEGFR-2 induces the expression of endothelial cell-derived matrix metalloproteases (MMPs), including MMP2 , MMP9 , and MT1-MMP , which degrade the matrix to allow for endothelial sprouting. MMPs are thus essential for angiogenesis, and their loss from either endothelium or inflammatory cells has been associated with severe angiogenic defects. At the same time, increased MMP activity has significant vascular consequences. MMPs are antagonized by Tissue Inhibitors of Matrix Metalloproteases (TIMPs), and a pathological increase in endothelial MMP over TIMP activity has been proposed to contribute to vessel wall thickening, abdominal aortic aneurysm formation, varicose veins, hypertension and preeclampsia [4–6]. MMPs are not, however, strictly proangiogenic; other more recently elucidated functions of this protein family include the mediation of vascular regression, as well as the generation of ECM fragments with antiangiogenic properties.
MMP2 and MMP9 belong to the class of gelatinases with collagen and fibrin as substrates and are expressed as inactive pro-proteins. MMP2 and MMP9 are processed to activate their metalloprotease activity. MT1-MMP is a member of the membrane associated or membrane-type MMPs [7–9]. In the vasculature, MT-MMPs are thought to be involved in the localized pericellular release of bioactive growth factors such as bFGF, VEGF, and TGF-beta, processing of pro-MMP2 into an activated MMP2, and degradation of ECM components including collagens and fibrins [10, 11]. MT1-MMP has been shown to be the most potent fibrinolytic MMP and has a critical role in the creation of channels in the ECM, into which endothelial cells migrate during sprouting angiogenesis [12, 13]. MT1-MMP function is also required for proper lumenization of the new vessel .
Notch signaling is an evolutionarily conserved pathway that regulates cell fate decisions. Notch proteins, Notch 1 through 4, act as receptors and their ligands Jagged (Jagged-1, 2) and Delta-like (Dll-1, 3, 4), are all transmembrane proteins . Upon ligand binding, the cytoplasmic domain of Notch is released by proteolytic cleavage via presenilin/γ-secretase , translocates to the nucleus, and interacts with the transcriptional repressor CSL (CBF1/Su(H)/Lag2), converting it to a transcriptional activator . An essential role for Notch signaling in arterial differentiation and vascular remodeling has been demonstrated by genetic studies of mice with targeted mutations in either Notch (Notch1 -/- , and Notch1 -/- ;Notch4 -/- ), or Notch ligands (Jagged1 -/- and Dll4 -/+ or Dll4 -/- ), reviewed in Shawber et al. . In addition, targeted activation of Notch4 specifically in endothelial cells disrupted vascular remodeling, resulting in embryonic lethality . These studies demonstrate that proper levels of Notch signaling are essential for patterning of the vasculature during a period of embryonic development that is known to be critically dependent on VEGF [14, 18].
Signaling via both VEGF and Notch is indispensable in vascular development, and it has become evident that these two pathways are interconnected. Heterozygous deficiency of Dll4 results in embryonic lethality with profound vascular defects, including defective arterial branching from the aorta and arterial regression, processes that also depend on VEGF signaling [19–21] suggesting Dll4 and VEGF work in concert. In cultured human arterial endothelial cells, VEGF, but not bFGF, induced expression of Notch1 and Dll4 . Furthermore, expression of Dll4 reduced VEGF/VEGFR-2 signaling, likely via downregulation of VEGFR-2 expression in cultured endothelial cells . A role for Notch signaling in tumor angiogenesis was originally hypothesized from the observation that VEGF induced Dll4 in the angiogenic endothelium of tumor xenografts  and blocking Dll4 functions resulted in dysregulated non-productive angiogenesis [24, 25].
In this study, we demonstrate that Notch mediates VEGF-induced MMP activity in endothelial cells. A Notch antagonist, called Notch1 decoy, blocked VEGF activation of Notch/CSL signaling, VEGF-induced HUVEC morphogenesis on both collagen and fibrin gels, and VEGF-induced fibrinolysis. Notch signaling upregulated the expression of MMP9 and MT1-MMP, and activated MMP2 and MMP9 in endothelial cells. Accordingly, we found that the Notch1 decoy-mediated suppression of HUVEC morphogenesis occurred via inhibition of MMP activity. Finally, Notch1 decoy suppressed endothelial MMP9 expression in an in vivo neovascularization model in mice. These data demonstrate that Notch directly regulates the endothelial cell response to VEGF via induction of MMPs.
Reagents and Expression Vectors (adenovirus/retrovirus)
GM6001 was used at 50 μM (Elastin Products Company). eACA was used at 10 mM (Sigma). SU5416 is from Eisai Co. LTD. Notch1 decoy (N1decoy) encodes the extracellular domain of rat Notch1 (bp 241-4229, accession# X57405) fused in frame to human IgG Fc, as described . The constitutively active Notch1 adenovirus (N1IC) encodes the cytoplasmic domain of human Notch1 as described . LacZ, human VEGF165, and Notch1 decoy cDNAs were engineered into pAdlox, recombinant adenoviruses generated and stocks produced as described .
Cells and Adenoviral Infections
HUVEC were isolated from human umbilical vein as described  and grown using EGM-2 Bullet kit (complete medium, LONZA). Porcine type I collagen is from Nitta Gelatin (Osaka, Japan). Fibrin gels were made by combining 2 mg/ml fibrinogen (Sigma) and 0.0625 U/ml thrombin (Sigma) at 4°C, followed by a one-hour incubation at 37°C. Adenovirus infections were as described  at indicated dosage (expressed as MOI - multiplicity of infection).
HUVEC were cultured on type I collagen-coated plates for 5 days in complete media, then starved in serum free media (SFM, Invitrogen) for 48 hours and cell lysates collected in TENT buffer (50 mM Tris pH8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100). Westerns were conducted using an anti-MT1-MMP antibody (Ab-1, EMD Biosciences).
HUVEC were seeded on type I collagen gels 2 days after infection and cultured post-confluence for 5 days before isolation of total RNA. Total RNA was isolated with RNeasy mini kit (Qiagen) and First-strand cDNA was synthesized using SuperScript II™ (Invitrogen). PCR primers were designed to recognize transcripts for GAPDH, Jagged1, Dll4, Notch1, Notch4, MMP2, MMP9, MT1-MMP and MT2-MMP. PCR used Platinum Taq DNA polymerase (Invitrogen) and reactions removed at noted cycle number and product analyzed as described . The following oligos were used for RT-PCR:
Luciferase Reporter Assay
HUVEC (3.0×105) were infected with adenovirus encoding human VEGF165(Ad-VEGF) and two days later transfected with Effectene (Qiagen) using 475 ng CSL luciferase reporter (pGA981-6)  25 ng Renilla luciferase pRL-SV40 (Promega, Madison, WI) for normalizing transfection efficiency. The following day Luciferase and Renilla activity was determined with Dual-Luciferase® Reporter Assay System (Promega) and Berthold dual-injection luminometer. Notch1 decoy effects on VEGF-activity were evaluated with reporter assays, as above, after co-transduction of HUVEC with adenovirus encoding Notch1 decoy (Ad-N1ECDFc) and Ad-VEGF. All assays were performed in triplicate.
HUVEC Morphogenesis Assay
HUVEC morphogenesis was assessed via microscopy and scored as cells with single or multiple processes after 5 days of culturing on porcine type I collagen gels, as described . Adenovirus infections were performed 2 days prior to seeding on collagen gels. GM6001 was added to medium 1 hr after HUVEC seeding. Cell number was measured using the Cell Counting Kit-8 (Dojindo Molecular Technologies).
HUVEC were seeded as a monolayer on bovine fibrin gels in complete medium in 24 well plate. After 5 days, wells were incubated with 330 ng/ml Thiazolyl Blue Tetrazolium Bromide (4) (Sigma) for 2 hours. Reagent was removed and wells documented by digital photography. Experiments were performed in triplicate.
Gelatin Zymography was performed on HUVEC conditioned media as described. HUVEC were infected with Ad-VEGF at a MOI of 40 or co-infected with Ad-VEGF and Ad-N1ECDFc each at a MOI of 40. Adenovirus encoding LacZ (Ad-LacZ) was used to normalize infections to 100 MOI. Adenovirus-transduced-HUVEC were cultured on collagen gels, conditioned medium collected at indicated days for MMP9 and day 4 for MMP2.
Dorsal Air Sac (DAS) Assay
The DAS assay was performed as described . KP1/VEGF121 cells were transduced with Ad-GFP or Ad-Notch1 decoy at 60 MOI and packed into Millipore chambers that were transplanted into a DAS of C57BL/6 mice. Mice were sacrificed four days after implantation, and dorsal skin removed and embedded in OCT. Each group consisted of 3-5 mice, and experiments were done in duplicate. Dermal cross-sections were immunostained as described with antibodies against PECAM (Pharminigen) and MMP9 (ABcam) and visualized and imaged by fluorescent microscopy at 20× magnification.
VEGF-induced HUVEC morphogenesis is mediated by Notch
Notch mediates VEGF-induced fibrinolysis
Notch regulates VEGF-induced matrix metalloprotease activity
The gelatinases MMP2 and MMP9 function to degrade both fibrin and collagen matrices produced by endothelial cells . VEGF signaling promotes the secretion and activation of MMPs, such as MMP2 and MMP9 . As Notch signaling mediated VEGF-induced HUVEC morphogenesis, we determined if the induction of MMP2 and MMP9 activity by VEGF was mediated by Notch. HUVEC were infected with Ad-VEGF and increasing amounts of Ad-Notch1 decoy, and conditioned media was used in zymographic analysis. After four days, the level of MMP2 pro-form was unchanged, while VEGF converted MMP2 to an active form (Figure 3B). VEGF-induced MMP9 activity 4 days post-transduction, and this activity increased over the following four days (Figure 3C). The VEGF-mediated MMP activation we observed was consistent with previous reports . To examine the role of Notch in the VEGF induced MMP activities, we assessed MMPs after expression of the Notch1 decoy. Co-expression of VEGF with Notch1 decoy inhibited the activation of MMP2 and blocked the transcriptional induction and biochemical activation of MMP9 by VEGF (Figure 3B, C). Thus, VEGF activated MMP2 and MMP9 gelatinase activity by a Notch-dependent mechanism.
To determine if Notch influenced MMP9 expression during VEGF-induced angiogenesis, we evaluated the expression of MMP9 in vivo using a dorsal air sac angiogenesis (DAS) model . In the DAS model, a chamber containing VEGF-expressing KP1 cells is implanted in the air sac under the dorsal skin. VEGF is released from the chamber and induces neo-angiogenesis in the overlying skin. Using this model, we have previously demonstrated that Notch1 decoy perturbed VEGF-driven angiogenesis and suppressed endothelial VEGFR-1 expression . To evaluate MMP9 expression in this model, cross-sections of skin from DAS assays using either control VEGF/Fc- or VEGF/Notch1 decoy-expressing KP1 cells were co-immunostained with antibodies against MMP9 and the endothelial cell marker PECAM. In the control Fc-expressing tissues, MMP9 expression was observed around the vessels within both the smooth muscle cell layer and subcutaneous fat depot (Figure 3D). In the Notch1-decoy DAS tissues, there was a reduction in PECAM staining indicating reduced vasculature, as we previously reported . We also observed a loss of endothelial-associated MMP9 expression in the Notch1 decoy-expressing DAS tissues relative to Fc control tissue. Thus, we demonstrate that blocking Notch signaling downstream of VEGF suppressed endothelial MMP9 expression in vivo.
Notch signaling induces MMP9 and MT1-MMP expression in HUVEC
Next, we asked whether Notch activity is required for the VEGF-induction of MMP9 and MT1-MMP. Ectopic expression of VEGF increased both MMP9 and MT1-MMP transcripts (Figure 4C). Co-infections of Ad-Notch1 decoy with Ad-VEGF suppressed the expression of both MMP9 and MT1-MMP transcripts. Taken together, these data demonstrate that Notch1 activation increased MMP2 and MMP9 activity. Thus, Notch functions downstream of VEGF to regulate MMPs via distinct mechanisms; Notch upregulated MMP9 activity at the transcriptional level, while it regulated MMP2 activity at the cell surface via the induction of MT1-MMP.
Notch-induced HUVEC morphogenesis is mediated by MMPs
In this study, we describe a new means by which Notch functions downstream of VEGF to regulate the angiogenic process. We identified MMPs as novel targets of Notch signaling in endothelial cells. Our data suggest that VEGF activates Notch signaling by upregulating the expression of Dll4 and Notch4, consistent with other reports . Notch1 decoy blocked VEGF-activation of Notch/CSL signaling, VEGF-induced HUVEC morphogenesis on collagen and fibrin gels, VEGF-dependent fibrinolysis and VEGF-induced dermal angiogenesis in vivo. Notch signaling, in turn, mediated VEGF-induced matrix metalloprotease activity by upregulating the expression of MMP9 and MT1-MMP, likely leading to activation of MMP9 and MMP2. These MMPs function as key regulators of angiogenesis in both physiological and pathological settings . During angiogenesis, MMP activity functions to promote ECM degradation, lumen formation, the activation of membrane receptors, and release of ECM/membrane-associated pro-angiogenic growth factors, such as VEGF, bFGF and TGF-β . MMPs also have anti-angiogenic functions, as they are capable of generating matrix protein fragments that suppress angiogenesis . Taken altogether, we propose that Notch signaling functions downstream of VEGF to affect angiogenesis in part by inducing endothelial cell localized matrix metalloprotease activity.
Pericellular matrix metalloproteases regulate the local availability of bioactive angiogenic factors, such as VEGF and bFGF. It has been proposed that matrix metalloproteases function to increase VEGF activity to promote pathological angiogenesis during tumorigenesis . Consistent with this hypothesis, deletion of MMP9 in a mouse model of pancreatic β-cell carcinoma, Rip1Tag2, suppressed tumor progression . In these mice, the level of VEGF was unaltered, but mobilization of VEGF from the extracellular matrix was inhibited. Thus, endothelial upregulation of MMP9 by Notch may lead to the localized release and activation of matrix-bound growth factors.
Besides its role in processing and activating MMP2, MT1-MMP also functions to degrade the surrounding fibrin matrix during physiological and pathological angiogenesis. The breakdown of the local fibrin matrix is essential in the initiation and propagation of angiogenic responses, and generates a provisional matrix that can sustain the formation of nascent vessels during wound healing, inflammation and tumor growth. MT1-MMP localizes at the leading edge of migrating endothelial cells and is thought to aid in the degradation of the extracellular matrix to facilitate endothelial cell invasion . Although MT1-MMP may be the most potent fibrinolytic MMP, the gelatinases MMP2 and MMP9 also recognize fibrin as a substrate. More recently, Stratman et al demonstrated that the formation of vascular guidance tunnels within collagen gels occurred via a MT1-MMP-dependent proteolytic process and that these tunnels become conduits for EC motility in sprouting angiogenesis  Thus, the induction of MT1-MMP by Notch may lead to the activation of MMP2 that together with MT1-MMP and MMP9 promote extracellular matrix degradation and endothelial cell morphogenesis.
MT1-MMP activity has also been shown to be critical for endothelial migration during sprouting angiogenesis [42, 49]; however Notch signal activation is a potent inhibitor of endothelial cell migration [50, 51], which suggests that Notch signaling has both anti- and pro-angiogenic functions. Thus, Notch may have an early pro-angiogenic function in the induction and activation MMPs to promote the degradation of the pericellular matrix, but it subsequently needs to be turned off to allow for endothelial cell migration and tube formation to progress. MT1-MMP activity has also been shown to be critical for endothelial lumen formation . Taking this finding into consideration, Notch may be turned off in tip cells and induces MMPs in stalk cells, which may be important for remodeling the matrix during the process of lumen formation. The latter model fits well with our current understanding of Notch function in the restriction of endothelial sprouting by blocking tip cell differentiation .
The molecular mechanism by which Notch regulates MMP9 and MT1-MMP expression in HUVEC is unclear. In lung metastases of Rip1Tag2 mice, MMP9 expression, within the endothelium, was regulated by VEGFR-1 . Since Notch induces VEGFR-1 expression in endothelial cells [43, 54], VEGFR-1 signaling may mediate the induction of MMP9 by Notch. However, Notch1 has also been shown to regulate the expression and activation of MMP9 through NF-κB in pancreatic cancer cells , suggesting that the regulation of MMP9 expression and activity in endothelial cells by Notch may be mediated by NF-κB signaling. Finally, the activation of MMP9 at the cell surface is dependent on the uPA/plasmin pathway. In human prostatic cancer cells, knock-down of Notch1 resulted in a downregulation of MMP9 and uPA and its receptor, uPAR . From our data it is unclear whether Notch regulates MMP9 solely at the level of transcription, and the possibility remains that it may regulate the uPA/plasmin pathway to activate MMP9 at the cell surface. This is a subject of future investigation.
We propose that Notch activates MMP2 via induction of MT1-MMP, which is present in its active form at the cell surface. In the tumor microenvironment, an imbalance may arise from an increase in MMP expression/activity and a decrease in the expression of the MT-MMP inhibitors, TIMP-2 or TIMP-3 [4, 5]. This imbalance would result in a pro-angiogenic state. A similar imbalance in MMP/TIMP activity ratio has also been implicated in aortic aneurysm, varicose veins, hypertension and preeclampsia . As inhibition of Notch signaling with Notch1 decoy blocked the induction of MMP9 and MT1-MMP transcripts in HUVEC, blocking Notch signaling in pathological settings may perturb angiogenesis and may prove therapeutically useful in the treatment of vascular disorders.
List of Abbreviations
vascular endothelial growth factor
vascular endothelial cell growth factor receptor
human umbilical vein endothelial cell
membrane type matrix metalloprotease
multiplicity of infection
tissue inhibitor of metalloproteases
Supported by NIH 5RO1HL062454 (JK), NIH 5R01CA136673 (JK, CJS.) and from the Department of Defense W81XWH-07-1-0362 (J.K.). We thank Valeriya Borisenko for technical assistance.
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