Direct Evidence for Endothelial Vascular Endothelial Growth Factor Receptor-1 Function in Nitric Oxide–Mediated Angiogenesis
http://www.100md.com
Shakil Ahmad, Peter W. Hewett, Ping Wang
参见附件。
the Department of Reproductive and Vascular Biology (S.A., P.W.H., B.A.-A., M.C., T.F., A.A.), Institute for Biomedical Research, The Medical School, University of Birmingham, UK
Department of Biochemistry and Molecular Biology (P.W., L.W., D.M.), Mayo Clinic Foundation, Rochester, Minn
Department for Molecular Biomedical Research (J.J.H.), Flanders Interuniversity Institute of Biotechnology, Ghent University, Belgium
Max Delbrueck Center for Molecular Medicine (F.l.N.), Berlin, Germany.
Abstract
Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase C1. Consistent with these findings, the VEGFR-1–specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2–activated phospholipase C1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.
Key Words: angiogenesis nitric oxide eNOS VEGF VEGF receptors
Introduction
Vascular endothelial growth factor (VEGF) is essential for vascular development, angiogenesis, and revascularization after endothelial injury caused by angioplasty or stent placement.1 Mice lacking VEGF receptor-2 (VEGFR-2) die in utero at embryonic day 8.5 (E8.5) because of a defect in vasculogenesis resulting from a failure of the migration and expansion of hematopoietic/endothelial progenitors,2–4 whereas VEGFR-1–null mice die between E8.5 to E9.0 because of excess formation of endothelial cells that abnormally coalesce into disorganized tubules.5 Deletion of the tyrosine kinase domain of VEGFR-1 does not impair angiogenesis, suggesting that VEGFR-1 acts as a decoy to regulate the bioavailability of VEGF for VEGFR-2 activation during development.6,7 However, there is now increasing evidence for the involvement of VEGFR-1 in adult angiogenesis,8–10 which was proposed to be largely associated with the presence of VEGFR-1 in inflammatory cells,8 or attributable to the amplification of VEGFR-2 signaling.11 Nevertheless, direct evidence for endothelial VEGFR-1 signaling is lacking, and the current concept persists that VEGFR-2 is the critical receptor for angiogenesis.
Vasodilatation is an initiating event in sprouting angiogenesis, and nitric oxide (NO) has been identified as a key mediator in this process.12 Independently, our laboratory and that of Sessa were the first to show that VEGF stimulates the release of NO from human umbilical vein endothelial cells (HUVECs).13,14 Several reports indicate that VEGFR-2 activation results in the upregulation and phosphorylation of endothelial NO synthase (eNOS)15,16 and increased production of cyclic GMP.17,18 Activation of VEGFR-1 also induces NO release and promotes reorganization of endothelial cells into capillary-like tube networks.10 VEGF receptor activation leads to phosphorylation of specific tyrosine residues, and, via different adaptor molecules, signal transduction cascades are initiated. An array of phosphorylation sites in VEGFR-1 and VEGFR-2 have been identified, and some have been shown to associate with specific signal transduction proteins.19 Indeed, mice carrying a phenylalanine substitution of VEGFR-2 Y1175 die in utero.20 Previous studies showed that these signaling events involve the activation of phospholipase C (PLC), protein kinase C (PKC), the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt).19 Despite these studies, it is not known whether VEGFR-1 or VEGFR-2 use a common or alternative signaling pathways in eNOS activation and angiogenesis, and the specific tyrosine residues of the VEGF receptors involved in these processes have yet to be identified.
Using a chimeric receptor system in primary endothelial cells, we demonstrate that the independent activation of either VEGFR-2 or VEGFR-1 stimulates capillary-like tube network formation on growth factor–reduced Matrigel in an NO-dependent manner. A major advantage of this chimeric approach is that it negates the possibility of chimeric receptors dimerizing with the endogenous VEGF receptors in endothelial cells, leading to a mixed-phenotype response. Here we provide direct evidence for a new paradigm of VEGFR-1 and VEGFR-2 signaling in the initiation of NO-driven in vitro angiogenesis and identify Tyr794 of VEGFR-1 as a key residue in this process.
Materials and Methods
Materials
Recombinant VEGF and epidermal growth factor (EGF) were obtained from PeproTech-EC (London, UK). Mouse anti-eNOS monoclonal antibody was obtained from BD Transduction Laboratories (Lexington, KY), and rabbit polyclonal antibodies against phospho-eNOS at serine-1177 (p-eNOSSer1177) and eNOS were purchased from Cell Signaling (Beverly, Mass). Wortmannin, LY294002, U73122, and nitro-L-arginine (L-NNA) were purchased from Calbiochem (Nottingham, UK). All other reagents were obtained from Sigma (Dorset, UK).
Cell Culture
Primary HUVECs were isolated and cultured as described previously.10 Cells were used at passage 3 or 4 for experiments and serum starved in endothelial cell serum-free medium (Gibco-BRL, UK) supplemented with 0.2% BSA for 24 hours before stimulation.
Transduction of Chimeric Receptors in HUVECs
The chimeric VEGF receptors used in this study comprise the intracellular and transmembrane domains of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) fused to the extracellular domain of the human EGF receptor. As EGF does not bind to VEGF receptors, it will not activate the endogenous VEGF receptors. The chimeric receptors EGLT and EGDR and their tyrosine-to-phenylalanine and deletion mutants (EGLT-Y794F, EGLT-793S, EGDR-Y951F, EGDR-Y1054F, and EGDR-Y1059F) were generated and cloned into the pMMP retroviral vector, and retrovirus-containing cell supernatant was harvested and used immediately to infect HUVECs.21 Following 16 hours of incubation, the medium was replaced with fresh growth medium and the HUVECs used 48 hours after infection. The expression of chimeric receptors in HUVECs following retroviral transduction was demonstrated by immunoprecipitation and Western blotting (see Figure 1A).
Adenovirus Infection of HUVECs
Recombinant adenovirus encoding dominant-negative Akt (Akt-dn) and PTEN (Phosphatase and TENsin homolog deleted on chromosome 10) under the control of the CMV early promoter and an empty control virus were propagated in HEK293 cells, purified on CsCl gradients, titered, and stored at –80°C in 4% sucrose buffer. HUVECs were plated on 6-well dishes and infected with adenovirus in low-serum growth medium at a multiplicity of infection (moi) of 100 overnight.
Western Blotting
Cells lysates were immunoblotted as described previously.21 Membranes were probed with an anti-eNOS monoclonal antibody (1:1000) or rabbit polyclonal antibodies against phospho-eNOS-Ser1177 (1:1000) at 4°C overnight. Proteins were visualized using the ECL detection kit (Amersham-Pharmacia, UK).
NO Release
Total NO in conditioned media was assayed as nitrite, the stable breakdown product of NO, using a Sievers NO chemiluminescence analyzer (Analytix, Sunderland, UK) as described previously.10
Tube Formation Assay
The formation of capillary-like structures was examined on growth factor–reduced Matrigel in 24-well plates as described previously.10 Tube formation was quantified by measuring the total tube length in five random x200 power fields per well using a Nikon phase-contrast inverted microscope with Image ProPlus image analysis software (Media Cybernetics, Silver Spring, Md). Mean total tube length was calculated from 3 independent experiments performed in duplicate.
Statistics
All data are expressed as the mean (±SEM). Statistical comparisons were performed using 1-way ANOVA followed by the Student–Newman–Keuls test as appropriate. Statistical significance was set at a value of P<0.05.
Results
Tyrosine 794 of VEGFR-1 Is Essential for Activation of eNOS
The stimulation of HUVECs with VEGF induced a rapid transient increase in phosphorylation of eNOS at Ser1177, which reached a maximum at 2 minutes (Figure 1B). HUVECs expressing EGLT (HUVECEGLT) or EGDR (HUVECEGDR) produced similar patterns of eNOS phosphorylation in response to EGF, indicating that both VEGFR-1 and VEGFR-2 receptors are capable of activating eNOS. No response was observed in control HUVECs expressing LacZ or uninfected HUVECs following EGF stimulation (data not shown). Stimulation of HUVECEGLT or HUVECEGDR cells with EGF produced a similar time-dependent increase in NO release compared with VEGF-induced release in wild-type HUVECs (Figure 1C). These results were confirmed using the VEGF receptor–specific ligands placenta growth factor-1 (PlGF-1) and VEGF-E (Figure IIB in the online data supplement, available at http://circres.ahajournals.org).
To identify the tyrosine residues of VEGFR-1 involved in the activation of eNOS, we used VEGFR-1 mutant (EGLT-Y794F), where Tyr794 was replaced with phenylalanine, and a truncated mutant (EGLT-793S), where a stop codon was introduced at amino acid 794. EGF had little effect on eNOS phosphorylation (Figure 1D) or NO production (Figure 1E) in HUVECEGLT-Y795F or HUVECEGLT-793S, indicating that Tyr794 of VEGFR-1 is essential for VEGF-mediated eNOS activation and NO release.
In the VEGFR-2 mutants, HUVECEGDR-Y951F showed decreased phosphorylation of eNOS at Ser1177 (Figure 1F) and a greater than 50% reduction in NO release at 10 and 30 minutes of stimulation with EGF (Figure 1G). Mutations in the VEGFR-2 catalytic domain at Tyr1054 decreased NO release without apparent reduction in eNOS phosphorylation at Ser1177, whereas loss of Tyr1059 inhibited both NO release and eNOS phosphorylation in HUVECEGDR-Y1059F (Figure 1F and 1G).
Tyrosine 794 of VEGFR-1 Is Essential for In Vitro Angiogenesis
To investigate the relative activity of VEGFR-1 and VEGFR-2 on endothelial cell capillary-like tube formation, HUVECs were plated onto growth factor-reduced Matrigel and mean total tube length was quantified. EGF treatment of either HUVECEGLT or HUVECEGDR led to an increase in the formation of tubular networks (Figure 2A and 2B). To determine whether NO is an essential mediator of both VEGFR-1– and VEGFR-2–induced angiogenesis, HUVECEGLT or HUVECEGDR were preincubated with the NOS inhibitor L-NNA before stimulation with EGF. The addition of the 500 μmol/L L-NNA completely abolished EGF-stimulated tube formation in both HUVECEGLT and HUVECEGDR (Figure 2A and 2B).
To demonstrate that VEGFR-1 Tyr794 is also required for capillary tube formation, HUVECEGLT-Y794F or HUVECEGLT-793S were stimulated with EGF for 24 hours and the formation of tubular networks was assessed. Neither HUVECEGLT-Y794F nor HUVECEGLT-793S responded to EGF stimulation, indicating that Tyr794 of VEGFR-1 is essential for in vitro angiogenesis (Figure 2C; see also supplemental Figure IA). In addition, when NO donor (glyco-S-nitroso-N-penicillamine [glyco-SNAP], 10 μmol/L) or a membrane-permeable stable cGMP analog (8-bromo-cGMP, 100 μmol/L) was added to HUVECEGLT-Y794F or HUVECEGLT-793S, they formed capillary-like tubes (Figure 2D; supplemental Figure IB).
Interestingly, HUVECEGDR-Y951F and HUVECEGDR-Y1054F induced the formation of tubular networks on stimulation with EGF despite the fact that these mutations resulted in greater than 50% reduction in NO release (Figure 2E; supplemental Figure IC). This suggests that there may be a critical threshold level of NO, below which tube formation fails to occur. As expected, HUVECEGDR-Y1059F, which did not induce NO release, failed to undergo angiogenesis (Figure 2E). HUVECEGDR-Y1059F formed capillary networks when exposed to the NO donor glyco-SNAP or 8-bromo-cGMP (Figure 2F; supplemental Figure ID).
VEGFR-1–Mediated eNOS Activation Is Dependent on PI3K Activity
To investigate the role of PI3K signaling in eNOS activation via VEGFR-1 or VEGFR-2, both pharmacological inhibitors and the dominant-negative p85 subunit (p85-dn) of PI3K were used. HUVECEGLT and HUVECEGDR were pretreated with 100 nmol/L Wortmannin or 15 μmol/L LY294002 for 10 minutes and stimulated with EGF for 2 minutes to determine the level of eNOS phosphorylation. Both Wortmannin and LY294002 strongly inhibited EGF-induced eNOS phosphorylation in HUVECEGLT but not in HUVECEGDR (Figure 3A). To confirm conclusively that PI3K is essential for VEGFR-1–mediated eNOS phosphorylation, p85-dn was coexpressed with EGLT and EGDR and the cells stimulated with EGF. No increase in eNOS phosphorylation was detected in the p85-dn–expressing HUVECEGLT, confirming that eNOS activation is via PI3K (Figure 3B). In contrast, the expression of p85-dn in HUVECEGDR did not inhibit EGF-induced eNOS phosphorylation (Figure 3B). Consistent with the observed lack of eNOS phosphorylation in LY294002-treated HUVECEGLT, inhibition of PI3K significantly decreased the release of NO in HUVECEGLT but had no effect on HUVECEGDR (Figure 3C), demonstrating the requirement for PI3K in VEGFR-1–mediated eNOS activation. These findings were confirmed using HUVECEGLT and HUVECEGDR infected with adenoviruses expressing wild-type PTEN (PTEN-wt) or empty control virus. EGF-stimulated NO release from HUVECEGLT, but not HUVECEGDR, was blocked by the overexpression of PTEN-wt (Figure 3D).
VEGFR-2 Activates eNOS via PLC1
The activation of VEGFR-2, but not VEGFR-1, with receptor selective VEGF ligands increased the phosphorylation PLC1 (supplemental Figure IIA). As VEGFR-2 activity was independent of PI3K, HUVECEGLT or HUVECEGDR were preincubated with the PLC inhibitor U73122 before stimulation with EGF. PLC inhibition prevented EGF-induced eNOS phosphorylation in HUVECEGDR but had no effect on eNOS activation in HUVECEGLT (Figure 4A). In addition, pretreatment with U73122 significantly attenuated EGF-mediated release of NO in HUVECEGDR (Figure 4B). However, U73122 had no effect on EGF-induced NO release in HUVECEGLT, demonstrating that VEGFR-2–mediated eNOS activation is regulated by PLC (Figure 4B). These findings were confirmed by RNA interference-mediated knockdown of PLC1, which inhibited the release of NO stimulated via VEGFR-2 but not by VEGFR-1 (supplemental Figure IIB).
Akt Activation Is Essential for VEGF-Mediated NO Release and Angiogenesis
As the phosphorylation of eNOS at Ser1177 is associated with Akt activation,22 we investigated whether Akt is a common mediator of both VEGFR-1– and VEGFR-2–driven eNOS activation and tube formation. Both the VEGFR-1– and VEGFR-2–selective ligands PlGF-1 and VEGF-E induced a rapid increase in Akt phosphorylation at Ser473, consistent with its activation (see supplemental Figure IIIA). To examine the requirement of Akt for VEGFR-1– or VEGR-2–mediated eNOS activity and tube formation, HUVECEGLT or HUVECEGDR were infected with adenoviruses expressing either Akt-dn or constitutively active myristylated Akt (myr-Akt) or empty vector control at an moi of 100 for 18 hours before stimulation with EGF. The overexpression of Akt-dn blocked NO release from EGF-stimulated HUVECEGLT and HUVECEGDR to control levels (Figure 5A) and also inhibited in vitro angiogenesis (Figure 5B; supplemental Figure IIIC). In contrast myr-Akt strongly promoted NO release and tube formation, independent of EGF stimulation and chimeric receptor stimulation (data not shown). These findings were confirmed using a pharmacological Akt inhibitor (Akt-i) (supplemental Figure IIIB). Collectively, these results demonstrate that both eNOS and Akt are required for VEGFR-1– and VEGFR-2–mediated angiogenesis.
Discussion
In this study, we show conclusively that VEGFR-1 is a signaling receptor for eNOS activation and in vitro angiogenesis. We demonstrate, to our knowledge, for the first time that chimeric VEGF receptor mutants lacking the ability to produce NO fail to induce capillary network formation, providing direct evidence for NO as a second messenger for VEGF-driven angiogenesis. Furthermore, both the activation of eNOS and endothelial tube network formation are dependent on Tyr794 of VEGFR-1 and mediated via the PI3K pathway, whereas VEGFR-2 uses PLC to stimulate these activities and Tyr951 is partially required. However, VEGF receptor pathways converge on Akt, which appears to be the common mediator of VEGF-stimulated eNOS activation and in vitro angiogenesis (Figure 6). This is consistent with a recent report showing that the loss of Akt1, but not Akt2, markedly reduces VEGF-stimulated NO accumulation in murine lung endothelial cells.23
Relatively little is known about the signaling proteins that associate with VEGFR-1. Earlier studies reported that VEGFR-1 could activate PLC in NIH3T3 fibroblasts24,25 and monocytes.26 One study reported PLC activation,27 although another failed to detect this in porcine aortic endothelial cells overexpressing VEGFR-1.28 Here we demonstrate not only the importance of Tyr794 of VEGFR-1 for NO-mediated angiogenesis but also show that this process is mediated in a PI3K-dependent and PLC-independent manner. In a yeast 2-hybrid system, Tyr794 and Tyr1169 interact with PLC N-SH2 binding domains.29,30 Ito and colleagues identified 2 major (Tyr1213, Tyr1242) and minor (Tyr1327, Tyr1333) VEGFR-1 phosphorylation sites, and, of these, Tyr1213 and 1333 were found to associate with of PLC, Grb2, Shp2, Crk, and Nck in vitro and increase the phosphorylation of PLC, Shp2, and Crk in porcine aortic endothelial cells.27,31 Although no association of p85 with VEGFR-1 was detected, the juxtamembrane region of VEGFR-1 containing Tyr794 and Tyr815 was not examined in these studies.27,31 VEGF failed to induce the activation of PI3K in porcine aortic endothelial cells overexpressing VEGFR-1.28,31 In contrast to these studies, p85 SH2 domain was shown to bind Tyr1213 of VEGFR-1 in vitro,29 which is reported to be a key site activated by VEGF.11 Our study demonstrates the role of PI3K in VEGFR-1–mediated eNOS activation as transduction of dominant-negative p85 subunit of PI3K into EGLT-expressing endothelial cells inhibits eNOS phosphorylation. Indeed, using chimeric receptor approaches, we showed that VEGFR-1 activates PI3K to negatively regulate VEGFR-2–induced proliferation.32,33 This activation of PI3K is dependent on VEGF Tyr794,21 which is also responsible for NO-mediated angiogenesis in vitro. One explanation for the discrepancy in VEGFR-1 signaling detected in fibroblasts, monocytes, and transformed porcine aortic endothelial cells compared with the primary endothelial cell cultures used in this study is that the former may lack the appropriate repertoire of adaptor proteins necessary to recapitulate the exact signal transduction events.25
In this study, we demonstrate that VEGFR-2–mediated NO release is dependent on PLC activity as U73122 blocked EGF-induced NO release in HUVECEGDR but not in HUVECEGLT. In addition, we showed that Tyr771 of PLC1 is phosphorylated by the VEGFR-2–specific ligand VEGF-E (and not VEGFR-1), leading to NO release that is inhibited by PLC1 knockdown (supplemental Figure II). The phosphorylation of PLC was reported to be associated with the activation of VEGFR-2 expressed in NIH3T3 fibroblasts,25 and VEGF increases phosphorylation of PLC1 Tyr771 and eNOS Ser1177 in HUVECs.16 Several autophosphorylation sites have been identified in the cytoplasmic domain of VEGFR-2,34,35 and the binding of PLC Src homology (SH2) domains is associated with various tyrosine residues on this receptor.29 Tyr951 is important for VEGFR-2–mediated RhoA activation and the migration but not proliferation of endothelial cells.36 However, its role in NO release and angiogenesis had not been investigated. Here we show that a loss of this residue results in a greater than 50% reduction in NO release. Tyr951, which lies in the kinase insert domain, is a docking site for PLC37 and the VEGF receptor–associated phosphatase/T-cell–specific adapter molecule.38 Recently, the phosphorylation of Tyr951 was shown regulate F-actin filament reorganization and cell migration via VEGF receptor–associated phosphatase/T-cell–specific adapter molecule.39 Interestingly, Tyr951 is phosphorylated in a subpopulation of endothelial cells predominantly in vessels lacking pericytes, suggesting that it may be associated with sites of active angiogenesis.39 As the loss of Tyr951 resulted in a partial reduction of NO release, it is likely that other tyrosine residues in VEGFR-2 are responsible for the generation of NO. VEGFR-2 Tyr1175, which also binds and activates PLC to promote endothelial cell migration,35 is likely to be involved. Indeed, mice carrying a phenylalanine substitution of this residue die in utero because of a failure of endothelial cell proliferation and differentiation.20 Tyr1054 and Tyr1059 lie within the catalytic domain of VEGFR-2 and are important for maximal receptor activation, as their combined mutation leads to a 90% reduction in receptor phosphorylation.34 As expected, these residues are essential for full receptor activity and loss of Tyr1059 appeared to be the most critical in VEGFR-2–driven NO-mediated in vitro angiogenesis.
During murine yolk sac vasculogenesis, endoderm-derived NO is important for the formation of the primary capillary plexus,40 and eNOS has been linked to vasculogenesis in the adult. Endothelial NOS–null mice exhibit impaired mobilization of bone marrow progenitor cells in ischemia, which cannot be restored by bone marrow transplantation.41 VEGF promotes the survival of endothelial progenitor cells and hematopoietic stem cells via VEGFR-2 in the bone marrow42,43 and exogenous VEGF or PlGF markedly potentiate megakaryocyte maturation, suggesting a role for VEGFR-1 in these processes.44 VEGFR-1 is involved in the mobilization of bone marrow–derived circulating progenitors during neoangiogenesis45,46 and the homing of bone marrow–derived hematopoietic progenitor cells to tumor-specific premetastatic sites.47 Our identification of VEGFR-1 Tyr794 as key residue for the activation of eNOS and neovascularization suggests that it may also hold true for VEGFR-1–mediated signaling in mobilization of bone marrow progenitor cells. Collectively, these observations, together with our findings, indicate that blockade of VEGFR-2 alone might be insufficient to achieve maximal therapeutic benefit in angiogenesis-driven pathologies.
Acknowledgments
The pSUPER-PLC1 plasmid was a gift from Prof Yun Soo Bae (Center for Cell Signaling Research, Ewha Womans University, Seoul, Korea). The adenoviruses expressing Akt-dn was kindly provided by Prof Kenneth Walsh (Tufts University School of Medicine, Boston, Mass) and PTEN by Dr Christopher D. Kontos (Duke University Medical Center, Durham, NC).
Sources of Funding
This work was supported by grants from the British Heart Foundation and Medical Research Council. S.A., P.W.H., and A.A. belong to the European Vascular Genomics Network, a Network of Excellence supported by the European Community Sixth Framework Programme for Research Priority 1 "Life Sciences, Genomics and Biotechnology for Health" (contract LSHM-CT-2003-503254). The NIH and the American Cancer Society supported P.W. and D.M.
Disclosures
None.
Footnotes
These authors contributed equally to this work and should be considered first authors.
Both authors contributed equally to this work.
Original received December 9, 2005; resubmission received June 5, 2006; revised resubmission received August 15, 2006; accepted August 23, 2006.
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the Department of Reproductive and Vascular Biology (S.A., P.W.H., B.A.-A., M.C., T.F., A.A.), Institute for Biomedical Research, The Medical School, University of Birmingham, UK
Department of Biochemistry and Molecular Biology (P.W., L.W., D.M.), Mayo Clinic Foundation, Rochester, Minn
Department for Molecular Biomedical Research (J.J.H.), Flanders Interuniversity Institute of Biotechnology, Ghent University, Belgium
Max Delbrueck Center for Molecular Medicine (F.l.N.), Berlin, Germany.
Abstract
Vascular endothelial growth factor-A (VEGF) is critical for angiogenesis but fails to induce neovascularization in ischemic tissue lesions in mice lacking endothelial nitric oxide synthase (eNOS). VEGF receptor-2 (VEGFR-2) is critical for angiogenesis, although little is known about the precise role of endothelial VEGFR-1 and its downstream effectors in this process. Here we have used a chimeric receptor approach in which the extracellular domain of the epidermal growth factor receptor was substituted for that of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) and transduced into primary cultures of human umbilical vein endothelial cells (HUVECs) using a retroviral system. Activation of HUVECs expressing EGLT or EGDR induced rapid phosphorylation of eNOS at Ser1177, release of NO, and formation of capillary networks, similar to VEGF. Activation of eNOS by VEGFR-1 was dependent on Tyr794 and was mediated via phosphatidylinositol 3-kinase, whereas VEGFR-2 Tyr951 was involved in eNOS activation via phospholipase C1. Consistent with these findings, the VEGFR-1–specific ligand placenta growth factor-1 activated phosphatidylinositol 3-kinase and VEGF-E, which is selective for VEGFR-2–activated phospholipase C1. Both VEGFR-1 and VEGFR-2 signal pathways converged on Akt, as dominant-negative Akt inhibited the NO release and in vitro tube formation induced following activation of EGLT and EGDR. The identification Tyr794 of VEGFR-1 as a key residue in this process provides direct evidence of endothelial VEGFR-1 in NO-driven in vitro angiogenesis. These studies provide new sites of modulation in VEGF-mediated vascular morphogenesis and highlight new therapeutic targets for management of vascular diseases.
Key Words: angiogenesis nitric oxide eNOS VEGF VEGF receptors
Introduction
Vascular endothelial growth factor (VEGF) is essential for vascular development, angiogenesis, and revascularization after endothelial injury caused by angioplasty or stent placement.1 Mice lacking VEGF receptor-2 (VEGFR-2) die in utero at embryonic day 8.5 (E8.5) because of a defect in vasculogenesis resulting from a failure of the migration and expansion of hematopoietic/endothelial progenitors,2–4 whereas VEGFR-1–null mice die between E8.5 to E9.0 because of excess formation of endothelial cells that abnormally coalesce into disorganized tubules.5 Deletion of the tyrosine kinase domain of VEGFR-1 does not impair angiogenesis, suggesting that VEGFR-1 acts as a decoy to regulate the bioavailability of VEGF for VEGFR-2 activation during development.6,7 However, there is now increasing evidence for the involvement of VEGFR-1 in adult angiogenesis,8–10 which was proposed to be largely associated with the presence of VEGFR-1 in inflammatory cells,8 or attributable to the amplification of VEGFR-2 signaling.11 Nevertheless, direct evidence for endothelial VEGFR-1 signaling is lacking, and the current concept persists that VEGFR-2 is the critical receptor for angiogenesis.
Vasodilatation is an initiating event in sprouting angiogenesis, and nitric oxide (NO) has been identified as a key mediator in this process.12 Independently, our laboratory and that of Sessa were the first to show that VEGF stimulates the release of NO from human umbilical vein endothelial cells (HUVECs).13,14 Several reports indicate that VEGFR-2 activation results in the upregulation and phosphorylation of endothelial NO synthase (eNOS)15,16 and increased production of cyclic GMP.17,18 Activation of VEGFR-1 also induces NO release and promotes reorganization of endothelial cells into capillary-like tube networks.10 VEGF receptor activation leads to phosphorylation of specific tyrosine residues, and, via different adaptor molecules, signal transduction cascades are initiated. An array of phosphorylation sites in VEGFR-1 and VEGFR-2 have been identified, and some have been shown to associate with specific signal transduction proteins.19 Indeed, mice carrying a phenylalanine substitution of VEGFR-2 Y1175 die in utero.20 Previous studies showed that these signaling events involve the activation of phospholipase C (PLC), protein kinase C (PKC), the mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt).19 Despite these studies, it is not known whether VEGFR-1 or VEGFR-2 use a common or alternative signaling pathways in eNOS activation and angiogenesis, and the specific tyrosine residues of the VEGF receptors involved in these processes have yet to be identified.
Using a chimeric receptor system in primary endothelial cells, we demonstrate that the independent activation of either VEGFR-2 or VEGFR-1 stimulates capillary-like tube network formation on growth factor–reduced Matrigel in an NO-dependent manner. A major advantage of this chimeric approach is that it negates the possibility of chimeric receptors dimerizing with the endogenous VEGF receptors in endothelial cells, leading to a mixed-phenotype response. Here we provide direct evidence for a new paradigm of VEGFR-1 and VEGFR-2 signaling in the initiation of NO-driven in vitro angiogenesis and identify Tyr794 of VEGFR-1 as a key residue in this process.
Materials and Methods
Materials
Recombinant VEGF and epidermal growth factor (EGF) were obtained from PeproTech-EC (London, UK). Mouse anti-eNOS monoclonal antibody was obtained from BD Transduction Laboratories (Lexington, KY), and rabbit polyclonal antibodies against phospho-eNOS at serine-1177 (p-eNOSSer1177) and eNOS were purchased from Cell Signaling (Beverly, Mass). Wortmannin, LY294002, U73122, and nitro-L-arginine (L-NNA) were purchased from Calbiochem (Nottingham, UK). All other reagents were obtained from Sigma (Dorset, UK).
Cell Culture
Primary HUVECs were isolated and cultured as described previously.10 Cells were used at passage 3 or 4 for experiments and serum starved in endothelial cell serum-free medium (Gibco-BRL, UK) supplemented with 0.2% BSA for 24 hours before stimulation.
Transduction of Chimeric Receptors in HUVECs
The chimeric VEGF receptors used in this study comprise the intracellular and transmembrane domains of VEGFR-1 (EGLT) or VEGFR-2 (EGDR) fused to the extracellular domain of the human EGF receptor. As EGF does not bind to VEGF receptors, it will not activate the endogenous VEGF receptors. The chimeric receptors EGLT and EGDR and their tyrosine-to-phenylalanine and deletion mutants (EGLT-Y794F, EGLT-793S, EGDR-Y951F, EGDR-Y1054F, and EGDR-Y1059F) were generated and cloned into the pMMP retroviral vector, and retrovirus-containing cell supernatant was harvested and used immediately to infect HUVECs.21 Following 16 hours of incubation, the medium was replaced with fresh growth medium and the HUVECs used 48 hours after infection. The expression of chimeric receptors in HUVECs following retroviral transduction was demonstrated by immunoprecipitation and Western blotting (see Figure 1A).
Adenovirus Infection of HUVECs
Recombinant adenovirus encoding dominant-negative Akt (Akt-dn) and PTEN (Phosphatase and TENsin homolog deleted on chromosome 10) under the control of the CMV early promoter and an empty control virus were propagated in HEK293 cells, purified on CsCl gradients, titered, and stored at –80°C in 4% sucrose buffer. HUVECs were plated on 6-well dishes and infected with adenovirus in low-serum growth medium at a multiplicity of infection (moi) of 100 overnight.
Western Blotting
Cells lysates were immunoblotted as described previously.21 Membranes were probed with an anti-eNOS monoclonal antibody (1:1000) or rabbit polyclonal antibodies against phospho-eNOS-Ser1177 (1:1000) at 4°C overnight. Proteins were visualized using the ECL detection kit (Amersham-Pharmacia, UK).
NO Release
Total NO in conditioned media was assayed as nitrite, the stable breakdown product of NO, using a Sievers NO chemiluminescence analyzer (Analytix, Sunderland, UK) as described previously.10
Tube Formation Assay
The formation of capillary-like structures was examined on growth factor–reduced Matrigel in 24-well plates as described previously.10 Tube formation was quantified by measuring the total tube length in five random x200 power fields per well using a Nikon phase-contrast inverted microscope with Image ProPlus image analysis software (Media Cybernetics, Silver Spring, Md). Mean total tube length was calculated from 3 independent experiments performed in duplicate.
Statistics
All data are expressed as the mean (±SEM). Statistical comparisons were performed using 1-way ANOVA followed by the Student–Newman–Keuls test as appropriate. Statistical significance was set at a value of P<0.05.
Results
Tyrosine 794 of VEGFR-1 Is Essential for Activation of eNOS
The stimulation of HUVECs with VEGF induced a rapid transient increase in phosphorylation of eNOS at Ser1177, which reached a maximum at 2 minutes (Figure 1B). HUVECs expressing EGLT (HUVECEGLT) or EGDR (HUVECEGDR) produced similar patterns of eNOS phosphorylation in response to EGF, indicating that both VEGFR-1 and VEGFR-2 receptors are capable of activating eNOS. No response was observed in control HUVECs expressing LacZ or uninfected HUVECs following EGF stimulation (data not shown). Stimulation of HUVECEGLT or HUVECEGDR cells with EGF produced a similar time-dependent increase in NO release compared with VEGF-induced release in wild-type HUVECs (Figure 1C). These results were confirmed using the VEGF receptor–specific ligands placenta growth factor-1 (PlGF-1) and VEGF-E (Figure IIB in the online data supplement, available at http://circres.ahajournals.org).
To identify the tyrosine residues of VEGFR-1 involved in the activation of eNOS, we used VEGFR-1 mutant (EGLT-Y794F), where Tyr794 was replaced with phenylalanine, and a truncated mutant (EGLT-793S), where a stop codon was introduced at amino acid 794. EGF had little effect on eNOS phosphorylation (Figure 1D) or NO production (Figure 1E) in HUVECEGLT-Y795F or HUVECEGLT-793S, indicating that Tyr794 of VEGFR-1 is essential for VEGF-mediated eNOS activation and NO release.
In the VEGFR-2 mutants, HUVECEGDR-Y951F showed decreased phosphorylation of eNOS at Ser1177 (Figure 1F) and a greater than 50% reduction in NO release at 10 and 30 minutes of stimulation with EGF (Figure 1G). Mutations in the VEGFR-2 catalytic domain at Tyr1054 decreased NO release without apparent reduction in eNOS phosphorylation at Ser1177, whereas loss of Tyr1059 inhibited both NO release and eNOS phosphorylation in HUVECEGDR-Y1059F (Figure 1F and 1G).
Tyrosine 794 of VEGFR-1 Is Essential for In Vitro Angiogenesis
To investigate the relative activity of VEGFR-1 and VEGFR-2 on endothelial cell capillary-like tube formation, HUVECs were plated onto growth factor-reduced Matrigel and mean total tube length was quantified. EGF treatment of either HUVECEGLT or HUVECEGDR led to an increase in the formation of tubular networks (Figure 2A and 2B). To determine whether NO is an essential mediator of both VEGFR-1– and VEGFR-2–induced angiogenesis, HUVECEGLT or HUVECEGDR were preincubated with the NOS inhibitor L-NNA before stimulation with EGF. The addition of the 500 μmol/L L-NNA completely abolished EGF-stimulated tube formation in both HUVECEGLT and HUVECEGDR (Figure 2A and 2B).
To demonstrate that VEGFR-1 Tyr794 is also required for capillary tube formation, HUVECEGLT-Y794F or HUVECEGLT-793S were stimulated with EGF for 24 hours and the formation of tubular networks was assessed. Neither HUVECEGLT-Y794F nor HUVECEGLT-793S responded to EGF stimulation, indicating that Tyr794 of VEGFR-1 is essential for in vitro angiogenesis (Figure 2C; see also supplemental Figure IA). In addition, when NO donor (glyco-S-nitroso-N-penicillamine [glyco-SNAP], 10 μmol/L) or a membrane-permeable stable cGMP analog (8-bromo-cGMP, 100 μmol/L) was added to HUVECEGLT-Y794F or HUVECEGLT-793S, they formed capillary-like tubes (Figure 2D; supplemental Figure IB).
Interestingly, HUVECEGDR-Y951F and HUVECEGDR-Y1054F induced the formation of tubular networks on stimulation with EGF despite the fact that these mutations resulted in greater than 50% reduction in NO release (Figure 2E; supplemental Figure IC). This suggests that there may be a critical threshold level of NO, below which tube formation fails to occur. As expected, HUVECEGDR-Y1059F, which did not induce NO release, failed to undergo angiogenesis (Figure 2E). HUVECEGDR-Y1059F formed capillary networks when exposed to the NO donor glyco-SNAP or 8-bromo-cGMP (Figure 2F; supplemental Figure ID).
VEGFR-1–Mediated eNOS Activation Is Dependent on PI3K Activity
To investigate the role of PI3K signaling in eNOS activation via VEGFR-1 or VEGFR-2, both pharmacological inhibitors and the dominant-negative p85 subunit (p85-dn) of PI3K were used. HUVECEGLT and HUVECEGDR were pretreated with 100 nmol/L Wortmannin or 15 μmol/L LY294002 for 10 minutes and stimulated with EGF for 2 minutes to determine the level of eNOS phosphorylation. Both Wortmannin and LY294002 strongly inhibited EGF-induced eNOS phosphorylation in HUVECEGLT but not in HUVECEGDR (Figure 3A). To confirm conclusively that PI3K is essential for VEGFR-1–mediated eNOS phosphorylation, p85-dn was coexpressed with EGLT and EGDR and the cells stimulated with EGF. No increase in eNOS phosphorylation was detected in the p85-dn–expressing HUVECEGLT, confirming that eNOS activation is via PI3K (Figure 3B). In contrast, the expression of p85-dn in HUVECEGDR did not inhibit EGF-induced eNOS phosphorylation (Figure 3B). Consistent with the observed lack of eNOS phosphorylation in LY294002-treated HUVECEGLT, inhibition of PI3K significantly decreased the release of NO in HUVECEGLT but had no effect on HUVECEGDR (Figure 3C), demonstrating the requirement for PI3K in VEGFR-1–mediated eNOS activation. These findings were confirmed using HUVECEGLT and HUVECEGDR infected with adenoviruses expressing wild-type PTEN (PTEN-wt) or empty control virus. EGF-stimulated NO release from HUVECEGLT, but not HUVECEGDR, was blocked by the overexpression of PTEN-wt (Figure 3D).
VEGFR-2 Activates eNOS via PLC1
The activation of VEGFR-2, but not VEGFR-1, with receptor selective VEGF ligands increased the phosphorylation PLC1 (supplemental Figure IIA). As VEGFR-2 activity was independent of PI3K, HUVECEGLT or HUVECEGDR were preincubated with the PLC inhibitor U73122 before stimulation with EGF. PLC inhibition prevented EGF-induced eNOS phosphorylation in HUVECEGDR but had no effect on eNOS activation in HUVECEGLT (Figure 4A). In addition, pretreatment with U73122 significantly attenuated EGF-mediated release of NO in HUVECEGDR (Figure 4B). However, U73122 had no effect on EGF-induced NO release in HUVECEGLT, demonstrating that VEGFR-2–mediated eNOS activation is regulated by PLC (Figure 4B). These findings were confirmed by RNA interference-mediated knockdown of PLC1, which inhibited the release of NO stimulated via VEGFR-2 but not by VEGFR-1 (supplemental Figure IIB).
Akt Activation Is Essential for VEGF-Mediated NO Release and Angiogenesis
As the phosphorylation of eNOS at Ser1177 is associated with Akt activation,22 we investigated whether Akt is a common mediator of both VEGFR-1– and VEGFR-2–driven eNOS activation and tube formation. Both the VEGFR-1– and VEGFR-2–selective ligands PlGF-1 and VEGF-E induced a rapid increase in Akt phosphorylation at Ser473, consistent with its activation (see supplemental Figure IIIA). To examine the requirement of Akt for VEGFR-1– or VEGR-2–mediated eNOS activity and tube formation, HUVECEGLT or HUVECEGDR were infected with adenoviruses expressing either Akt-dn or constitutively active myristylated Akt (myr-Akt) or empty vector control at an moi of 100 for 18 hours before stimulation with EGF. The overexpression of Akt-dn blocked NO release from EGF-stimulated HUVECEGLT and HUVECEGDR to control levels (Figure 5A) and also inhibited in vitro angiogenesis (Figure 5B; supplemental Figure IIIC). In contrast myr-Akt strongly promoted NO release and tube formation, independent of EGF stimulation and chimeric receptor stimulation (data not shown). These findings were confirmed using a pharmacological Akt inhibitor (Akt-i) (supplemental Figure IIIB). Collectively, these results demonstrate that both eNOS and Akt are required for VEGFR-1– and VEGFR-2–mediated angiogenesis.
Discussion
In this study, we show conclusively that VEGFR-1 is a signaling receptor for eNOS activation and in vitro angiogenesis. We demonstrate, to our knowledge, for the first time that chimeric VEGF receptor mutants lacking the ability to produce NO fail to induce capillary network formation, providing direct evidence for NO as a second messenger for VEGF-driven angiogenesis. Furthermore, both the activation of eNOS and endothelial tube network formation are dependent on Tyr794 of VEGFR-1 and mediated via the PI3K pathway, whereas VEGFR-2 uses PLC to stimulate these activities and Tyr951 is partially required. However, VEGF receptor pathways converge on Akt, which appears to be the common mediator of VEGF-stimulated eNOS activation and in vitro angiogenesis (Figure 6). This is consistent with a recent report showing that the loss of Akt1, but not Akt2, markedly reduces VEGF-stimulated NO accumulation in murine lung endothelial cells.23
Relatively little is known about the signaling proteins that associate with VEGFR-1. Earlier studies reported that VEGFR-1 could activate PLC in NIH3T3 fibroblasts24,25 and monocytes.26 One study reported PLC activation,27 although another failed to detect this in porcine aortic endothelial cells overexpressing VEGFR-1.28 Here we demonstrate not only the importance of Tyr794 of VEGFR-1 for NO-mediated angiogenesis but also show that this process is mediated in a PI3K-dependent and PLC-independent manner. In a yeast 2-hybrid system, Tyr794 and Tyr1169 interact with PLC N-SH2 binding domains.29,30 Ito and colleagues identified 2 major (Tyr1213, Tyr1242) and minor (Tyr1327, Tyr1333) VEGFR-1 phosphorylation sites, and, of these, Tyr1213 and 1333 were found to associate with of PLC, Grb2, Shp2, Crk, and Nck in vitro and increase the phosphorylation of PLC, Shp2, and Crk in porcine aortic endothelial cells.27,31 Although no association of p85 with VEGFR-1 was detected, the juxtamembrane region of VEGFR-1 containing Tyr794 and Tyr815 was not examined in these studies.27,31 VEGF failed to induce the activation of PI3K in porcine aortic endothelial cells overexpressing VEGFR-1.28,31 In contrast to these studies, p85 SH2 domain was shown to bind Tyr1213 of VEGFR-1 in vitro,29 which is reported to be a key site activated by VEGF.11 Our study demonstrates the role of PI3K in VEGFR-1–mediated eNOS activation as transduction of dominant-negative p85 subunit of PI3K into EGLT-expressing endothelial cells inhibits eNOS phosphorylation. Indeed, using chimeric receptor approaches, we showed that VEGFR-1 activates PI3K to negatively regulate VEGFR-2–induced proliferation.32,33 This activation of PI3K is dependent on VEGF Tyr794,21 which is also responsible for NO-mediated angiogenesis in vitro. One explanation for the discrepancy in VEGFR-1 signaling detected in fibroblasts, monocytes, and transformed porcine aortic endothelial cells compared with the primary endothelial cell cultures used in this study is that the former may lack the appropriate repertoire of adaptor proteins necessary to recapitulate the exact signal transduction events.25
In this study, we demonstrate that VEGFR-2–mediated NO release is dependent on PLC activity as U73122 blocked EGF-induced NO release in HUVECEGDR but not in HUVECEGLT. In addition, we showed that Tyr771 of PLC1 is phosphorylated by the VEGFR-2–specific ligand VEGF-E (and not VEGFR-1), leading to NO release that is inhibited by PLC1 knockdown (supplemental Figure II). The phosphorylation of PLC was reported to be associated with the activation of VEGFR-2 expressed in NIH3T3 fibroblasts,25 and VEGF increases phosphorylation of PLC1 Tyr771 and eNOS Ser1177 in HUVECs.16 Several autophosphorylation sites have been identified in the cytoplasmic domain of VEGFR-2,34,35 and the binding of PLC Src homology (SH2) domains is associated with various tyrosine residues on this receptor.29 Tyr951 is important for VEGFR-2–mediated RhoA activation and the migration but not proliferation of endothelial cells.36 However, its role in NO release and angiogenesis had not been investigated. Here we show that a loss of this residue results in a greater than 50% reduction in NO release. Tyr951, which lies in the kinase insert domain, is a docking site for PLC37 and the VEGF receptor–associated phosphatase/T-cell–specific adapter molecule.38 Recently, the phosphorylation of Tyr951 was shown regulate F-actin filament reorganization and cell migration via VEGF receptor–associated phosphatase/T-cell–specific adapter molecule.39 Interestingly, Tyr951 is phosphorylated in a subpopulation of endothelial cells predominantly in vessels lacking pericytes, suggesting that it may be associated with sites of active angiogenesis.39 As the loss of Tyr951 resulted in a partial reduction of NO release, it is likely that other tyrosine residues in VEGFR-2 are responsible for the generation of NO. VEGFR-2 Tyr1175, which also binds and activates PLC to promote endothelial cell migration,35 is likely to be involved. Indeed, mice carrying a phenylalanine substitution of this residue die in utero because of a failure of endothelial cell proliferation and differentiation.20 Tyr1054 and Tyr1059 lie within the catalytic domain of VEGFR-2 and are important for maximal receptor activation, as their combined mutation leads to a 90% reduction in receptor phosphorylation.34 As expected, these residues are essential for full receptor activity and loss of Tyr1059 appeared to be the most critical in VEGFR-2–driven NO-mediated in vitro angiogenesis.
During murine yolk sac vasculogenesis, endoderm-derived NO is important for the formation of the primary capillary plexus,40 and eNOS has been linked to vasculogenesis in the adult. Endothelial NOS–null mice exhibit impaired mobilization of bone marrow progenitor cells in ischemia, which cannot be restored by bone marrow transplantation.41 VEGF promotes the survival of endothelial progenitor cells and hematopoietic stem cells via VEGFR-2 in the bone marrow42,43 and exogenous VEGF or PlGF markedly potentiate megakaryocyte maturation, suggesting a role for VEGFR-1 in these processes.44 VEGFR-1 is involved in the mobilization of bone marrow–derived circulating progenitors during neoangiogenesis45,46 and the homing of bone marrow–derived hematopoietic progenitor cells to tumor-specific premetastatic sites.47 Our identification of VEGFR-1 Tyr794 as key residue for the activation of eNOS and neovascularization suggests that it may also hold true for VEGFR-1–mediated signaling in mobilization of bone marrow progenitor cells. Collectively, these observations, together with our findings, indicate that blockade of VEGFR-2 alone might be insufficient to achieve maximal therapeutic benefit in angiogenesis-driven pathologies.
Acknowledgments
The pSUPER-PLC1 plasmid was a gift from Prof Yun Soo Bae (Center for Cell Signaling Research, Ewha Womans University, Seoul, Korea). The adenoviruses expressing Akt-dn was kindly provided by Prof Kenneth Walsh (Tufts University School of Medicine, Boston, Mass) and PTEN by Dr Christopher D. Kontos (Duke University Medical Center, Durham, NC).
Sources of Funding
This work was supported by grants from the British Heart Foundation and Medical Research Council. S.A., P.W.H., and A.A. belong to the European Vascular Genomics Network, a Network of Excellence supported by the European Community Sixth Framework Programme for Research Priority 1 "Life Sciences, Genomics and Biotechnology for Health" (contract LSHM-CT-2003-503254). The NIH and the American Cancer Society supported P.W. and D.M.
Disclosures
None.
Footnotes
These authors contributed equally to this work and should be considered first authors.
Both authors contributed equally to this work.
Original received December 9, 2005; resubmission received June 5, 2006; revised resubmission received August 15, 2006; accepted August 23, 2006.
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