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Adhesion events in angiogenesis Brian P Eliceiri and David A Cheresh Recent work from several laboratories indicates that the coordination of endothelial cell adhesion events with growth factor receptor inputs regulates endothelial cell responses during angiogenesis. Analyses of the signaling pathways downstream of integrins, cadherins and growth-factor receptors are providing an insight into the molecular basis of known anti-angiogenic strategies, as well as into the design of novel therapies. Addresses The Scripps Research Institute, IMM-24, 10550 North Torrey Pines Road, La Jolla, California 92037, USA Correspondence: David A Cheresh; e-mail: [email protected]

to blood vessel formation during development (vasculogenesis) and neovascularization in tumor/growth factor models (angiogenesis), specific integrins may regulate distinct endothelial responses. The evidence for a general role for integrin-mediated endothelial cell adhesion during angiogenesis comes from recent findings suggesting that the anti-angiogenic effects of Endostatin and other ECM fragments (Table 1) appear to involve specific integrin interaction(s). In combination, these studies suggest that an understanding of the molecular interactions between endothelial cells and the ECM will be important in the design of anti-angiogenic strategies with therapeutic applications in humans.

Current Opinion in Cell Biology 2001, 13:563–568 0955-0674/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations bFGF basic fibroblast growth factor ECM extracellular matrix EGF epidermal growth factor FAK focal adhesion kinase PDGF platelet-derived growth factor PKC protein kinase C VEGF vascular endothelial growth factor

Introduction Angiogenesis depends on endothelial cell interactions with the extracellular matrix. The coordination of integrins and growth factor inputs provides specificity during neovascularization associated with development and pathological processes. Evidence from various experimental systems demonstrates the physiological importance of the coordination of signals from growth factors and the extracellular matrix (ECM) to support cell proliferation and migration. Several examples of cross-talk between these two important classes of receptors indicate that integrin ligation is required for growth-factor-induced biological processes. Integrins can directly associate with growth factor receptors, thereby regulating the capacity of integrin–growth-factor-receptor complexes to propagate downstream signaling. In addition to cell–ECM interactions, regulation of cell–cell adhesion by VE (vascular endothelial)-cadherins is critical during angiogenesis. For example, VE-cadherins mediate endothelial barrier function, angiogenesis and can also support cross-talk with VEGF (vascular endothelial growth factor) receptors [1,2]. As the role of VE-cadherins in angiogenesis has been recently reviewed [3–5], our review will focus on the recent progress in the study of integrins and growth-factor receptors during endothelial cell signaling leading to adhesion-dependent migration, survival and angiogenesis. The anti-angiogenic effect of αv integrin antagonists indicates a central role for integrins and cell adhesion during angiogenesis. Although other integrins clearly contribute

Integrins and the extracellular matrix Cell adhesion to the extracellular matrix is mediated by integrins, a family of heterodimeric transmembrane proteins comprising at least 16 α and 8 β subunits in mammals [6]. Different combinations of single α and β subunits dimerize to form approximately 24 different receptors with distinct and often overlapping specificity for ECM proteins. The biological significance of the range of ECM–integrin specificities during cell adhesion is not known. Although integrins support specific cell–ECM interactions for endothelial cell adhesion and migration, the identification of the underlying mechanisms by which specific subsets of integrins mediate development, wound healing and angiogenesis remains a challenge [6–9]. Integrins are widely recognized as important molecules for the transduction of positional cues from the ECM to the intracellular signaling machinery. For example, integrin ligation induces a wide range of intracellular signaling events, including the activation of Ras, MAP kinase, focal adhesion kinase (FAK), Src, Rac/Rho/cdc42 GTPases, PKC and PI3K (phosphatidylinositol 3-kinase) [7,10–12]. In addition, integrin ligation increases intracellular pH and calcium levels, inositol lipid synthesis, cyclin synthesis and the expression of immediate early genes [13] and promotes cell survival [14–17]. Interestingly, many of the signaling pathways and effectors activated by integrin ligation are also activated following growth-factor stimulation. This suggests that integrin and growth-factor-mediated cellular responses may synergize and coordinate biochemical responses. The physiological importance of integrins during angiogenesis has been most extensively studied in the case of the αv integrins. Antagonists of integrins αvβ3 and αvβ5 block growth-factor- and tumor-induced angiogenesis in multiple animal models [15,18]. Furthermore, recent data from clinical trials suggest that antagonists of αvβ3 and/or αvβ5 may have a clinical benefit in humans with solid tumors [19•]. Direct genetic approaches using knockout mouse models indicates that targeted deletion of the αv gene is lethal,

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Cell-to-cell contact and extracellular matrix

Table 1

Table 2

Integrin-mediated endothelial/ECM interactions as targets for endogenous anti-angiogenesis inhibitors.

Evidence for crosstalk between growth factor receptors and integrins. Direct growth factor receptor associations are listed.

Angiogenesis inhibitor

Growth factor receptor

Integrin

References

Endostatin (fragment of type XVII collagen)

αv,α5

[52•]

Tumstatin (NC1 domain of type IV collagen α3 chain)

αvβ3

[26,28•,56]

αvβ3,α3β1

[35,57,58]

Angiostatin (fragment of plasminogen)

αvβ3

*

PEX (noncatalytic fragment of MMP2)

αvβ3

[66–68]

Thrombospondin

*T Tarui, L Miles, Y Takada, Third International Symposium on AntiAngiogenic Agents, Irving, Texas, January 2001.

resulting in extensive blood vessel hemorrhage in a subset of organs. In contrast, mice deficient in integrin subunits β3 or β5 develop apparently normal blood vessels [20,21], although adult β5- but not β3-deficient mice appear to have VEGF-mediated vascular permeability defects (BP Eliceri, D Sheppard, DA Cheresh, unpublished data). These knockout mouse studies provide an interesting context in which to further examine, using inhibitor strategies, the role of αv integrins in development compared with the role of αv integrins in pathological processes in adult mice. Data from several laboratories indicate that the administration of antagonists of receptors for the ECM (i.e. αv integrins), as well as administration of endogenous components of the ECM, that is Endostatin [22], Angiostatin [23], thrombospondin [24], canstatin [25,26•], Arresten [27] and tumstatin [26•,28•], can suppress angiogenesis in vivo. Although the molecular basis for these findings remains poorly understood, we will focus on recent advances in the understanding of how specific integrins can coordinate with growth factor receptors in cultured endothelial cells and in vivo.

Integrin and growth factor receptor cross-talk in cultured cells Although integrins are responsible for mediating cell adhesion to the ECM, a role for growth factors in adhesion is gradually emerging. Growth-factor-induced cell proliferation, adhesion and migration in cultured cell models often require specific integrins. For example, optimal cell stimulation with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin or VEGF [29,30,31•] depends on integrin-mediated cell adhesion to the appropriate ECM (reviewed in [6,9]). Furthermore, integrin αvβ3 co-immunoprecipitates with VEGFR-2 [31•,32], as well as with the PDGF receptor (PDGFR) [31•]. Recently, VEGF has been shown to promote the adhesion and migration of cultured endothelial cells via integrins αvβ3, αvβ5 and β1 [33•]. Interestingly, basic fibroblast growth factor (bFGF),

Integrin

References

PDGFR

αvβ3

[29,31•]

VEGFR-2

αvβ3

[31•]

IR

αvβ3

[29]

ErB-2

α6β4

[65]

but not insulin-like growth factor (IGF) or PDGF, enhances endothelial cell adhesion and migration in vitro [33•]. Integrin αvβ3 can also couple with thrombospondin by a direct interaction with integrin-associated protein (IAP or CD47) to mediate enhanced cell spreading on vitronectin [34]. Other integrins, including α3β1, can also interact with thrombospondin [35]. Taken together, these findings suggest that although a wide variety of cell types may depend on integrin–growth-factor-receptor cross-talk for integrinmediated cell adhesion and migration, discrete growth factor receptors may be required to provide cell-type-specific biological responses.

Two angiogenic pathways are characterized by distinct αv integrins in vivo Angiogenic growth factors such as bFGF and VEGF induce angiogenesis through somewhat distinct signaling cascades [36]. bFGF- and VEGF-induced angiogenesis are each inhibited by antagonists of the distinct yet functionally related αv integrins, αvβ3 and αvβ5, respectively [36]. In vivo studies, using both the rabbit corneal eye pocket and the chick chorioallantoic membrane angiogenesis assay, reveal that an anti-αvβ3 monoclonal antibody blocks bFGF-induced angiogenesis, whereas an anti-αvβ5 antibody blocks VEGFinduced angiogenesis [36]. Furthermore, inhibition of Src kinase [37] or protein kinase C (PKC) [36] disrupts VEGFinduced angiogenesis specifically but does not affect bFGF-induced angiogenesis. Although anti-αvβ5 antagonists do not affect bFGF-induced neovascularization, anti-αvβ3 antagonists can inhibit up to 50% of VEGF-induced angiogenesis [36]. This observation is consistent with findings that VEGF can promote αvβ3- and αvβ5-mediated endothelial cell adhesion and migration in vitro ([38]; BP Eliceri, DA Cheresh, unpublished data). Although both VEGFR-2 and PDGFR associate with αvβ3 in cultured cells in vitro [31], VEGF, but not PDGF, promotes αvb3-dependent endothelial cell migration in vitro [33]. Integrins containing β1 subunits are implicated in growthfactor-induced angiogenesis. For example, antagonists of α1β1 or α2β1 block VEGF-induced angiogenesis [39], whereas αvβ3-mediated endothelial cell migration and angiogenesis depend on the ligation state of α5β1 to fibronectin [40•]. Mice lacking fibronectin or α5β1 die early in development, indicating an important role for

Adhesion events in angiogenesis Eliceiri and Cheresh

α5β1 during development [41,42]. Antagonists of αvβ3 or α5β1 block bFGF- but not VEGF-induced angiogenesis, suggesting that α5β1 and αvβ3 may regulate a similar pathway of angiogenesis [40]. Several reports provide additional evidence that ligation of integrins α5β1 and αvβ3 during cell adhesion are important mechanisms of integrin cross-talk [43–46].

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Table 3 Evidence for crosstalk between growth factor receptors and integrins. Integrin-mediated growth factor responses are listed. Integrin

Response

References

PDGF

αvβ3

Proliferation, migration

[29]

bFGF

αvβ3,α5β1

Angiogenesis, migration

[36,40•]

The findings discussed in this review suggest that the ECM provides instructional cues to cells within tissues undergoing remodeling and development, as well as providing physical support and barrier functions. The study of these processes during angiogenesis is important for understanding the coordination of signaling between integrins and growth factor receptors. For example, several recent studies suggest that homodimers and heterodimers of individual VEGFRs, VEGFR-1 and VEGFR-2, can propagate distinct signals in cultured endothelial cells [47–49]. Although the cytoplasmic domain of VEGFR-1 is dispensable for mouse development [50], VEGFR-1–VEGFR-2 heterodimers, as well as VEGFR-2 homodimers, are important for endothelial cell migration [48,49]. Furthermore, specific juxtamembrane domains within VEGFR-1 suppress VEGF-induced PI3K activation and cell migration [51•]. Future studies may reveal a regulation of downstream signaling in the vascular endothelium by αv integrins, coordinating with specific combinations of VEGFR heterodimers and/or homodimers in vivo. They may also lead to better understanding of how endogenous constituents of the ECM regulate angiogenesis. For example, the capacity of endogenous angiogenesis inhibitors to interact with α5 or αv integrins presents an intriguing challenge in the understanding of how neovascularization may be regulated in vivo.

VEGF

αvβ5

Angiogenesis

[36]

VEGF

αvβ5,αvβ3,β1

Adhesion, migration

[33•]

Endostatin and tumstatin interact with αv integrins in cultured endothelial cells

Clinical relevance of cell–ECM interactions and angiogenesis

Endostatin, a 20 kDa carboxy-terminal cleavage product of collagen XVII, was originally described by O’Reilly and colleagues [22] as a potent angiogenesis inhibitor in vivo. Recent findings by Vuori and co-workers [52••] suggest that αv as well as α5 integrins are important targets for Endostatin function in endothelial cells. Recombinant Endostatin interacts with integrins α5 and αv on the endothelial cell surface, mediating cell survival and migration. Interestingly, Endostatin can block bFGF- but not VEGF-induced angiogenesis [52••,53,54], although Endostatin apparently disrupts VEGF responses in other models [55]. Although the molecular interactions between Endostatin and integrins remain to be characterized, these findings are consistent with a role for integrins α5β1 [40•] and αvβ3 [36] in angiogenesis.

Angiogenesis mediates critical aspects of several disease states including cancer, rheumatoid arthritis, psoriasis, diabetic retinopathy, age-related macular degeneration, atherosclerosis and restenosis. Extensive preclinical data indicate that endogenous constituents of the ECM as well as integrin antagonists suppress angiogenesis during tumor growth in vivo. Organic small-molecule antagonists specific for αv integrins, as well as a humanized monoclonal antibody to integrin αvβ3 (Vitaxin), block angiogenesis and tumor growth in multiple orthotopic and heterotopic animal tumor models [18,61–63]. While the results of clinical trials with anti-αvβ3/αvβ5 small-molecule antagonists are being examined in late stage cancer patients, the first results of human cancer trials with Vitaxin have been described [19•]. In this Phase I trial, 8 of the 14 patients showed disease stabilization and/or some objective tumor reduction, with no evidence of toxicity in many of these patients. Phase II clinical trials are ongoing to determine the efficacy of Vitaxin during long-term treatment. The development of orally bioavailable integrin-antagonists, along with other angiogenesis inhibitors, may become clinically relevant therapies. While

Recent findings from two laboratories demonstrate that another important endogenous angiogenesis inhibitor, Tumstatin (the NC1 domain of the α3 chain of type IV collagen), can interact with αv integrins in vitro [56] and suppress angiogenesis in vivo [26•,28•]. In addition to Endostatin and

Growth factor

VEGF

α2β1,α1β1,αvβ3 Angiogenesis, migration

[38,39]

Tumstatin, thrombospondin is another angiogenesis inhibitor [57,58] that can interact with integrins. Therefore, several endogenous angiogenesis inhibitors appear to mediate their anti-angiogenic effects by direct or indirect αv integrin interactions. Although additional novel endogenous angiogenesis inhibitors have been described within the past two years, including Arresten (NC1 domain of α1 chain of type IV collagen) [27] and Canstatin (fragment of α2 chain of type IV collagen) [25,26•], their capacity to associate with specific integrins remains unknown. Although another angiogenesis inhibitor, Angiostatin (a plasminogen fragment), appears to function through a different mechanism(s) [59,60], recent unpublished observations indicate that Angiostatin can also interact with integrin αvβ3 (T Tarui, L Miles, Y Takada, abstract Third International Symposium on Anti-Angiogenic Agents, Irving, Texas, 19-20 January, 2001). Therefore, it appears likely that many anti-angiogenesis inhibitors, including antagonists of matrix metalloproteases, may block angiogenesis by interfering with integrin-mediated endothelial cell adhesion and migration.

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a review of the range of anti-angiogenic strategies is beyond the scope of this review (see [64]), the initial findings with Vitaxin are an important example of the clinical value of disrupting pathways of endothelial cell adhesion and survival during angiogenesis.

Conclusions Recent evidence from several laboratories suggests that the coordination of inputs from growth factors and the ECM regulate key aspects of angiogenesis. Further study of the basic cell biological mechanisms underlying mechanisms of integrin-mediated cell adhesion and survival during angiogenesis will continue to provide insight into how to target tumorassociated vasculature during angiogenesis to block tumor growth and metastasis and to prevent other diseases. The elucidation of the molecular basis of angiogenesis remains a challenge because of the complex interactions between the ECM and cells and between cells that must be temporally and spatially coordinated. For example, examination of the signaling events transduced by cell adhesion molecules to endothelial cells may reveal mechanisms in which cells can process growth factor stimuli to impact changes in intracellular phosphorylation cascades, gene expression levels and ECM-associated enzymatic activities. The coordinated response of these inputs may direct the processes of cell migration, proliferation and differentiation in vivo.

Acknowledgements We thank our colleagues for their discussion and input on this review. BPE was supported by a Scientist Development Grant from the American Heart Association and DAC by grants CA50286, CA45726 and CA78045 from the National Institutes of Health.

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