doi: 10.1161/CIRCRESAHA.107.160937
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© 2007 American Heart Association, Inc.
Editorials |
Substrate Matters
Reciprocally Stimulatory Integrin and VEGF Signaling in Endothelial Cells
From Inserm U644, Institute for Biomedical Research, Rouen University Medical School, Rouen, France.
Correspondence to Dr Ebba Brakenhielm, Inserm U644, UFR de Médicine-Pharmacie, 22 Boulevard Gambetta, 76 183 Rouen, France. E-mail Ebba.Brakenhielm{at}univ-rouen.fr
See related article, pages 570–580
Key Words: angiogenesis • extracellular matrix • signaling pathways • src kinase • VEGF receptor
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Introduction |
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 Top Introduction Common Intracellular Signaling...ECM Influences on Growth...References |
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The integrin family of transmembrane receptors is composed of heterodimers of different
and ß subunits that direct cellular adhesion to extracellular matrix (ECM) components such as collagens, laminins, fibronectin, and vitronectin. Certain members of the integrin family, such as 2ß1, 5ß1, vß3, and vß5 integrins, are specifically upregulated in endothelial cells during angiogenesis, eg, during vascular remodeling and growth associated with inflammation, wound healing, ischemic injury, or tumor growth.1 Functional inhibition of these integrins, via blocking antibodies or cyclic peptide antagonists, has been found to reduce angiogenic responses to growth factors, ischemia, and tumors.2 Thus, in addition to their involvement in steady-state maintenance of blood vessel integrity, integrins seem to play an essential role in the carry-out of angiogenic programs in activated endothelial cells. The integrin signaling is bidirectional and encompasses both outside-in signals, eg, activation of second messenger pathways in response to changes in ECM composition, and inside-out signals, eg, changes in cellular adhesion and motility in response to growth factor stimulation.3 In addition, there is a degree of interdependence between the cellular circuits processing ECM-derived signals and soluble growth factor–derived signals. For instance, growth factor–induced responses are modulated by cellular adhesion, and integrin-mediated outside-in signals, dependent on the specific type of integrin molecule(s) engaged by the ECM, seem to directly influence growth factor receptor signaling pathways.4,5
The reciprocal communication between integrins and growth factor receptors most likely acts to integrate and coordinate cellular activity in response to different types of stimuli. However, the molecular mechanisms involved and the direct functional consequences of the cross-talks between integrins and growth factor receptors have only recently begun to unravel. Interestingly, several growth factor receptors, including IGFR, EGFR, PDGFR-ß, and VEGFR-2, have been found to immunoprecipitate with activated integrin molecules, in particular with ß1 or ß3 integrins.4 For example, the association of PDGFR-ß or VEGFR-2 with vß3 integrin has been shown to depend mainly on the extracellular domain of the ß3 chain.6 Although most studies have indicated that ligand-induced receptor activation is a necessary prerequisite for the association of the growth factor receptors with integrins such as vß3,7,8 other reports have revealed that, under conditions of elevated expression levels of both components, the interaction between growth factor receptors and integrins may occur even in the absence of soluble growth factor.6,9 For instance, ligand-independent PDGFR-ß phosphorylation was observed in fibroblasts attached to ß1 integrin substrates,10 suggesting that ECM-induced integrin-mediated activation of growth factor receptors may bypass the requirement of growth factors for inducing a proliferative or migratory cellular response.
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Common Intracellular Signaling Pathways |
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TopIntroductionCommon Intracellular Signaling...ECM Influences on Growth...References |
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Because the colocalization of growth factor receptors and integrins often occurs in focal contacts where the integrins cluster on binding to their ECM ligands, molecular exchanges between their respective signaling pathways may be facilitated. Additionally, and as a further indication of the potentially coordinated mechanisms regulating cellular activation, many of the signaling pathways induced by growth factor receptors are also engaged after ECM-induced activation of integrins. For instance, several kinases such as FAK, c-Src, Jnk, PKC, and MAP kinases are stimulated by
vß3 integrin clustering.11 Similarly, on binding of VEGF-A to its main endothelial signaling receptor VEGFR-2, the receptor autophosphorylates, leading to the activation of multiple signaling cascades via the recruitment of various initiator components, eg, adaptor molecules such as Grb2 and the Src family of tyrosine kinases (c-Src, Fyn, Yes); Ras and Rho GTPases; as well as tyrosine phosphatases (SHP-1, SHP-2).12,13 Further downstream effector molecules include fokal adhesion kinase (FAK), PI3K, Jnk, and MAP kinases, which contribute to the launching of cellular programs regulating cell morphology and chemotaxis as well as increased survival or proliferation, all concordant with an angiogenic cellular phenotype.
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ECM Influences on Growth Factor–Induced Cellular Responses |
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TopIntroductionCommon Intracellular Signaling...ECM Influences on Growth...References |
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Among the integrins,
vß3 integrins seem to occupy a particular function in angiogenesis. Whereas in vitro experiments have shown that vß3 integrins are upregulated by VEGF-A in microvascular endothelial cells,14 in vivo studies have frequently revealed elevated levels of vß3 integrins at sites of active angiogenesis.15 In addition, blocking antibodies or small molecule inhibitors (RGD cyclic peptide) against vß3 display potent antiangiogenic effects.15 Indeed, a neutralizing antibody against vß3, Vitaxin, has been tested in clinical trials against metastatic melanoma or prostate cancer based on its antiangiogenic properties.16
Interestingly, the binding of vß3 integrin to the ECM has been shown to potentiate the proliferative and migratory cellular response to various growth factors such as insulin, EGF, PDGFs, and VEGF-A.7,8,17–19 For instance, cellular attachment to vitronectin or fibrinogen, both specific ligands for vß3 integrins, increases basal HUVEC proliferation by 2.5-fold and VEGF-A–stimulated proliferation by 4.7-fold, as compared with cells attached to BSA.8 At the same time, other types of ECM, such as the 5ß1 ligand fibronectin and the 2ß1 ligand collagen-1, were significantly less effective in stimulating VEGF-induced endothelial responses in these cells. Further support for the specific regulatory function of the VEGFR-2/vß3 integrin complex is given by the demonstration that blocking ß3 integrin antibodies, but not anti-ß1 antibodies, inhibit VEGF-A–induced VEGFR-2 phosphorylation, and leads to drastically reduced cellular migration and proliferation.8 Thus, vß3 integrins seem to play a special role in coordinating the activity of several growth factor receptor systems with links to angiogenesis.
In this issue of Circulation Research, Mahabeleshwar et al20 expand on the specific features of the interactions occurring between integrins and the VEGFR-2 growth factor receptor in primary human endothelial cells (HUVECs). Similar to previous studies,8 they show that cellular adhesion to the vß3 integrin ligand vitronectin, rather than to the 2ß1 ligand collagen or the 6ß1/6ß4 ligand laminin, dramatically enhances VEGF-A–induced VEGFR-2 phosphorylation, and leads to a complex formation between VEGFR-2 and vß3 integrin molecules. Further, this ECM-derived potentiation of VEGF signaling is significantly reduced by blocking antibodies against either v or ß3 integrin chains, indicating the specific involvement of vß3 integrins. Conversely, Mahabeleshwar et al20 also demonstrate that VEGF-A stimulation increases vitronectin-induced ß3 integrin phosphorylation. Importantly, they reveal an essential role of c-Src in mediating both growth factor–induced as well as ECM-stimulated phosphorylation of the ß3 integrin. Further, c-Src is found to associate with both VEGFR-2 and ß3 integrin, and c-Src–mediated ß3 phosphorylation is shown to be required for the formation of the VEGFR-2/vß3 integrin complex.
On a molecular level, the authors convincingly demonstrate direct phosphorylation by recombinant purified c-Src of the tyrosine residues Y747 and Y759 in the ß3 integrin cytoplasmic tail.20 Furthermore, they show that these same residues are targeted during VEGF-A–induced ß3 integrin phosphorylation. Although the potential role of FAK, previously shown to form a dual kinase signaling complex with c-Src,21 was not investigated in this current study, previous work has suggested that VEGF-A–induced c-Src phosphorylation specifically mediates the association between FAK and vß5 rather than with vß3 integrin.22 Of note, the presence of both c-Src and ß5, but not ß3 integrin, was found to be critical for the VEGF-A–induced vascular permeability effects in mice.22,23
On a functional level, the studies performed by Mahabeleshwar et al20 reveal that inactivation of c-Src is associated with significantly decreased endothelial adhesion to vitronectin, but not to collagen or laminin, indicative of the importance of c-Src in vß3-dependent cellular adhesion. Moreover, the c-Src–mediated structural modification of ß3 results in activation of vß3 integrins, leading to augmented soluble ligand (fibrinogen) binding capacity, as well as to enhanced chemotaxis and precapillary tube formation of endothelial cells in response to VEGF-A stimulation. Additionally, cellular adhesion to vitronectin, leading to vß3 integrin activation, was found to be associated with increased basal as well as VEGF-A–stimulated endothelial proliferation as compared with cells grown on collagen or laminin. This observation could be partially explained by a synergistic stimulation of the Akt pathway occurring in cells attached to vitronectin and stimulated by VEGF-A.20 However, whether c-Src is equally essential for VEGF-A–induced integrin-mediated endothelial responses in vivo remains to be further investigated.
Taken together, these results emphasize importance of ECM in influencing, via integrin outside-in signals, growth factor–induced signaling pathways of crucial importance to angiogenesis, such as endothelial migration, tube formation, and proliferation. Vice versa, the study by Mahabeleshwar et al20 reveals the direct effects of activated growth factor receptors on integrin functional status that is closely linked to cell motility. Indeed, the relationship between VEGFR-2 and ß3 integrin in endothelial cells seems to be reciprocally stimulatory. The molecular interactions, mainly regulated by c-Src, largely influences the endothelial responsiveness to VEGF-A as well as to integrin ligation of the ECM, and may thus be of crucial importance for the regulation of angiogenesis and of relevance for strategies aiming at therapeutic stimulation of neovascularization in ischemic organs. An indication of other potential ways that vß3 integrins may influence growth factor–mediated signals is the observation that integrins are necessary for the second wave of MAP kinase activation after soluble growth factor (FGF-2) stimulation.24 Additionally, vß3 integrins may regulate the cell cycle by suppressing gene expression of molecules such as p53 and p21WAF/CIP1, and by increasing Bax:Bcl-2 ratios, resulting in cellular protection against apoptosis.25 In support, activated vß3 integrins constitute an important survival signal for nascent blood vessels, and conversely vß3 integrin inhibitors have been found to induce apoptosis in endothelial cells.2
Some key questions still remain with regard to the potential differences between the role of vß3 integrins in regulation of physiological versus pathological angiogenesis, particularly as data generated from different transgenic mouse models have lead to some discordant findings. Whereas it was found that the majority of v integrin knock-out mice die before birth because of vascular defects,26 the ß3 knock-out mice appear grossly normal, with the exception of a recently published phenotype of reduced coronary capillary vessel maturity in adult male mice,27 suggestive of a specific role for vß3 integrins in postnatal cardiac remodeling. In addition, these mice display an increased pathological angiogenic phenotype,28 with enhanced sensitivity to VEGF-A–stimulated angiogenesis as well as rapid tumor growth. This shows that, although vß3 integrin antagonists have clearly antiangiogenic effects,15 the ß3 integrin is not necessary for physiological or pathological angiogenesis. One possible explanation for this unexpected finding is that VEGFR-2 levels may be elevated in blood vessels by way of overcompensation in the absence of ß3 integrin expression.29 Similarly, whereas VEGF-A induces vß3 integrin expression in vitro in microvascular endothelial cells,13 the activation of vß3 integrin has been found to reduce VEGFR-2 expression levels in endothelial cells.28,29 This feed-back loop may constitute a protection against unrestrained angiogenic stimulation, ensuring that the blood vessel response remains finely tuned to the specific requirements of the tissue.
In contrast, a more recently generated mouse knock-in model, expressing a ß3 negative mutant that cannot be phosphorylated, displayed as expected an impaired pathological angiogenic phenotype with features of reduced VEGF-A sensitivity.30 Conversely, mice overexpressing constitutively active ß3 integrin were also reported to exhibit impaired pathological angiogenic responses characterized by reduced tumor vessel density with more immature and small vessels, leading to reduced tumor growth.29 In the wake of these different studies, the vß3 integrin have been proposed by some to play an antiangiogenic role,1,31 rather than a priming or facilitating function for angiogenic growth factor–mediated signaling. Thus, the study by Mahabeleshwar et al20 adds valuable further mechanistic insight to the issue and ensures that the debate regarding the role of integrins in angiogenesis will go on more informed.
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Acknowledgments |
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Sources of Funding
E.B. acknowledges financial support from the Wenner-Gren Foundations, Sweden, and from the Inserm organisation, France.
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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References |
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Related Article:
Circ. Res. 2007 101: 570-580.
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