doi: 10.1161/01.RES.0000245430.24075.a4
© 2006 American Heart Association, Inc.
Editorials |
Vascular Endothelial Growth Factor Signaling to Endothelial Nitric Oxide Synthase
More than a FLeeTing Moment
From the Yale School of Medicine (W.C.S., M.I.L.), Vascular Biology & Transplantation Program, Boyer Center for Molecular Medicine, New Haven, Conn.
Correspondence to William C. Sessa, Yale School of Medicine, Vascular Biology & Transplantation Program, Boyer Center for Molecular Medicine, 295 Congress Ave, New Haven, CT 06536. E-mail william.sessa{at}yale.edu
See related article, pages 715–722
Key Words: VEGF • receptors • NO • chimeric • angiogenesis
There is substantial evidence supporting the idea that the process of angiogenesis requires the synthesis of endothelium-derived nitric oxide (NO). NO, characterized as a major endothelial-derived relaxing factor, exerts paracrine and autocrine roles in maintaining cardiovascular homeostasis, vascular tone, and microvascular permeability. During the early steps of angiogenesis in which new blood vessels sprout from existing vascular beds, there is a persistence of vasodilation and increase in vascular permeability, suggesting that these hemodynamic changes in the existing vasculature are indispensable during an angiogenic process. A number of angiogenic factors can triggers the release of NO, synthesized by the endothelial isoform of NO synthase (eNOS). One such factor is the vascular endothelial growth factor (VEGF), which as its name implies, is a critical mitogenic, chemoattractant, and survival factor for endothelial cells1 in addition to being characterized as a potent vascular permeability factor.2 Early studies have demonstrated that VEGF can readily stimulate NO production in cultured cells and isolated blood vessels and that NO is essential in mediating the VEGF-induced endothelial cell proliferation and organization into tubes in 3D cultures.3,4 Subsequent in vivo studies have also demonstrated that eNOS knockout mice had significantly attenuated VEGF and ischemia induced angiogenesis and vascular permeability.5,6 These studies place eNOS derived NO as a key mediator of VEGF induced angiogenesis in postnatal mice.
Since the discovery of VEGF, or VEGF-A, and subsequent members of the VEGF family (VEGF-B, -C, -D, -E, and placenta growth factor, PlGF), intense research has been performed to elucidate their modes of action. These growth factor ligands bind to 3 receptor tyrosine kinases (RTKs), namely VEGF receptor-1, -2, and -3 as well as to coreceptors such as heparan sulfate proteoglycans or neuropilins. Like many other RTKs, these receptors are able to form homodimers or heterodimers depending on the binding properties of the ligand. On dimerization of the receptors, activation of the tyrosine kinase follows leading to autophosphorylation of the receptor, thus creating docking sites for subsequent binding and activation of downstream signaling molecules.
Much attention has focused on the signaling events elicited by VEGF-A (herein after referred to as VEGF), which can bind to the receptors, VEGFR-1 (also called Flt-1) and VEGFR-2 (also called Flk-1 in mouse or KDR in human). Gene ablation of either of these 2 receptors revealed important and distinct functional differences during vascular development: whereas the Flk-1 null mice die between embryonic days 8.5 to 9.5 as a result of decreased vasculogenesis because of early defect in hematopoietic and endothelial cells in blood islands,7 the Flt-1 null mice die between embryonic days 8.5 to 9, attributable to an obstruction of vessels by an overgrowth of endothelial cells.8 These studies suggest that whereas Flk-1 functions as a positive regulator for endothelial cell proliferation and differentiation, Flt-1 functions as the negative regulator. Interestingly, a subsequent study examining mice which were lacking the Flt-1 tyrosine-kinase domain revealed normal vascular development, which raised an interesting hypothesis that Flt-1 functions to sequester VEGF from Flk-1 during embryogenesis.9 This is supported by the evidence that Flt-1 binds to VEGF with much higher affinity than Flk-1 although exhibiting 
10-fold less kinase activity, thus suggesting that Flt-1 may serve to "trap" VEGF, limiting Flk-1 signaling. Despite the fact that the tyrosine kinase domain of Flt-1 is dispensable during embryonic vasculogenesis/angiogenesis, other cells from these mice are impaired and experiments using chimeras of Flt-1 and the juxtamembrane region of Flk-1 (and vice versa) have revealed an uncoupling of signaling pathways between migratory and proliferative responses to the same VEGF stimulus.10
Because of the complexity of VEGF signaling, it remains difficult to delineate the signaling mechanism that lies downstream of either Flt-1 or Flk-1 as both receptors can signal through common ligands. Furthermore, there is increasing evidence that suggest these 2 receptors may interact and possibly regulate each other (reviewed by Matsumoto and Claesson-Welsh11). Many studies have used overexpression of either receptor in porcine aortic endothelial cells (PAECs), which do not normally express these receptors12; antibodies directed against either receptor to abolish individual downstream signaling pathways13; or overexpression of a chimeric receptor fusing the intracellular and juxtamembrane domains of Flk-1 or Flt-1 to different extracellular domain such as colony-stimulating factor-1 receptor,14 or epidermal growth factor (EGF) receptor (EGFR).15 The latter system allows independent activation of either receptor (EGFR–Flt-1, called EGLT, or EGFR–Flk-1, called EGDR) using EGF as an agonist to stimulate the kinase activity of Flt or Flk without activating the endogenous VEGF receptors. This may be advantageous in bypassing issues in which endogenous receptors can heterodimerize and complicate the signaling pathways downstream from each receptor.
In this present study in Circulation Research, Ahmed et al described that the activation of eNOS via VEGF signaling can occur via both Flt-1 and Flk-1. They demonstrated that eNOS phosphorylation on Ser1177 via a phosphatidylinositol 3'-kinase (PI3K) mechanism can occur through EGF activation of the chimeric EGLT receptor and this promotes in vitro angiogenesis. Several key tyrosine residues have been identified on both Flt-1 and Flk-1 whereby on receptor dimerization, autophosphorylation of these residues can lead to binding and activation of downstream signaling molecules. Of particular interest, the tyrosine residues at 794 and 1169 have been identified as major binding sites for phospholipase C gamma (PLC
) whereby the amino acids spanning these residues contain consensus sequence for the binding of the N-terminal Src homology 2 (SH2) domain in PLC.16,17 However, very little is known about the downstream signaling events from these residues as well as other tyrosine residues identified to date.
Ahmed et al demonstrated that the activation of eNOS via EGLT is dependent on the phosphorylation on the Tyr794 residue on Flt-1, as the truncated mutant, EGLT-793S, abolished the EGF stimulated eNOS phosphorylation on Ser1177. Furthermore, introduction of a nonphosphorylatable mutant Tyr794Phe into HUVECs significantly diminished NO release on EGF stimulation.15 An earlier report published by the same group demonstrated that VEGF mediated in vitro endothelial cell tube formation was dependent on Flt-1 signaling and that this was NO dependent as exogenous NO donor partially reversed the decrease in tube formation when an antibody against Flt-1 was added to neutralize the effect of VEGF.13 Using the same EGLT and EGDR chimeric receptor system, it was previously demonstrated that Tyr794 was critical in the inhibitory actions by which Flt-1 can exert on Flk-1 in stimulating endothelial cell proliferation and that this was because of the passive trapping of ligand by Flt-1.18 This inhibitory action of Flt-1 was also dependent on the p85 regulatory subunit of PI3K.18 Interestingly, the negative effect of Flt-1 on Flk-1 does not alter endothelial cell migration, suggesting the existence of a divergent regulatory mechanism by these receptors on different endothelial functions.18 These studies contrast a previous report in which the juxtamembrane domain (in which Tyr794 is located) of Flt-1 was swapped with that from Flk-1 and this domain negatively regulated Flk-1 downstream signaling events, such as p85 phosphorylation and endothelial cell migration, but not phosphorylation of mitogen activated protein kinase (MAPK).19 Despite the importance of the Tyr794 residue in mediating several endothelial functions, it remains to be elucidated whether or not the negative regulation of Flt-1 on Flk-1 also applies to the subsequent activation of eNOS.
Ahmed et al report regulation of eNOS activation by via Tyr951 phosphorylation on Flk-1. Flk-1 was long been thought to be the major mediator of physiological and pathological effects of VEGF (reviewed by Cross et al20). Several tyrosine residues on Flk-1 have been mapped and examined in the context of signal transduction. In particular, Tyr1054 and Tyr1059 have been found to be required for maximal activation of the Flk-1 tyrosine kinase.21 Furthermore, Tyr951 has been shown to be important in VEGF mediated migration but not proliferation.22 Indeed, the phosphorylation of Tyr951 can also mediate the binding of the VEGF receptor associated protein (VRAP), which can serve as an adaptor protein for the binding of PLC and PI3K,23 or mediate the direct binding to the N-terminal or C-terminal SH2 domain in PLC.24 Thus, it is interesting that Ahmed et al observed that this residue is also important in the activation of eNOS through a PLC dependent pathway. PLC activation downstream of Flk-1 has been thought to be mainly attributable to Tyr1175 autophosphorylation,25 leading to subsequent activation of MAP kinase, resulting in increased endothelial cell proliferation.26 Moreover, mice which express the nonphosphorylatable phenylalanine mutant of this residue (Tyr1173Phe in mice) die in utero at embryonic day 8.5 to 9.5, similar to Flk-1 null mice, suggesting that phosphorylation of Tyr1175 and subsequent signaling downstream from this residue are critical during vascular development.27 This residue has also been thought to be essential for the activation of PI3K and protein kinase B (Akt) activities supported by the evidence that Tyr1175Phe mutant had decreased association of the Flk-1 receptor to the p85 regulatory subunit of PI3K.28 Thus, Tyr1175 is also likely to be involved in Flk-1 activation of eNOS through a PI3K/Akt pathway as, Ahmed et al demonstrated, and both receptors converge on the activation of Akt leading to eNOS phosphorylation. Introduction of a dominant negative form of Akt completely abolished EGF induced NO release and in vitro tube formation in HUVEC expressing either EGLT or EGDR.
As a final thought, it remains enigmatic what the relative contributions of Flt-1 versus Flk-1 signaling are to VEGF biology, as VEGF binds with very high affinity to Flt-1 (Kd 1 to 20 pM) compared with that of Flk-1 (Kd 50 to 770 pM); yet, Flt-1 receptor phosphorylation in response to VEGF is quite low, and can only be readily observed in overexpression models. Although studies in which the VEGF receptors or chimeric receptors are overexpressed can offer insights into dissecting the signaling mechanisms through individual receptors without confounding factors, it remains difficult to validate how these models mimic the physiological situation. For instance, it has been estimated that the expression of Flt-1 is 3,000 copies per cell whereas Flk-1 is expressed at 40,000 copies per cell. The overexpression of these chimeric receptors may not resemble the natural frequency or distribution of endogenous Flt-1 versus Flk-1. Despite this caveat, the study by Ahmed et al showing that eNOS activation by VEGF can occur through Flt-1 adds another level of complexity to the VEGF receptor signaling story. Precisely how these 2 receptors respond to the same ligand to trigger different signaling events, including cross talk or feedback and eventually converge on the activation of eNOS through Akt still needs to be thoroughly investigated.
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Sources of Funding
W.C.S. is supported by grants from the National Institute of Health (R01 HL64793, RO1 HL61371, R01 HL57665, PO1 HL70295, and contract No. N01-HV-28186 [NHLBI-Yale Proteomics Contract]).
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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