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2 Department of Cell and Developmental Biology and Department of Pharmacology, The University of North Carolina, Chapel Hill, NC 27599
Address correspondence to Cam Patterson, 5109 Neuroscience Research Bldg., CB#7126, The Carolina Cardiovascular Biology Center, The University of North Carolina, Chapel Hill, NC 27599-7126. Tel.: (919) 843-6477. Fax: (919) 843-4585. E-mail: cpatters{at}med.unc.edu
Abstract
A vital step in growth factordriven angiogenesis is the coordinated engagement of endothelial integrins with the extracellular matrix. The molecular mechanisms that partner growth factors and integrins are being elucidated, revealing an intricate interaction of surface receptors and their signaling pathways.
Key Words: angiogenesis; growth factors; growth factor receptors; integrins; signal transduction pathways
Blood vessel formation is a dynamic process that involves interactions between soluble mediators, adhesive substrates, and endothelial cell surface receptors. Endothelial cell activation is a necessary first step in angiogenesis, which triggers the recruitment of smooth muscle cells and pericytes to newly formed vessels. Two growth factor families activate this initiating pathway in angiogenesis, the vascular endothelial growth factors (VEGFs)* and fibroblast growth factors (FGFs) (for review see Cross and Claesson-Welsh, 2001). VEGF-A, a factor that was initially identified based on its ability to increase vascular permeability and endothelial cell proliferation, is required for angiogenesis during development and is a necessary stimulus for hypoxia-induced angiogenesis. Four alternatively spliced isoforms of VEGF-A exist that bind two receptor tyrosine kinases, VEGF receptor (VEGFR)-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), on the surface of endothelial cells. The FGF family is even more fecund, consisting of at least 20 members that act on four separate receptors. Binding of VEGFs and FGFs to their respective receptors triggers receptor tyrosine phosphorylation followed by recruitment of intracellular adaptor proteins and activation of signaling molecules (Fig. 1). Through alterations in lipid metabolism, intracellular calcium levels, and protein kinase and phosphatase activities, growth factors elicit the pleiotrophic events necessary for new vessels to sprout from preexisting ones.
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Distinct angiogenic pathways as defined by specific growth factorintegrin pairs
The discovery by Cheresh and coworkers that antagonists specific for Vß3 or
Vß5 selectively block angiogenesis induced by bFGF and VEGF, respectively, provided some of the first support for a role of
V integrins in angiogenesis and led them to postulate the existence of two separate angiogenic pathways (Friedlander et al., 1995). They observed that antibody antagonists of
Vß3 abolished basic FGF (bFGF)- and tumor necrosis factor
stimulated angiogenesis but only partially affected the response to VEGF, whereas antagonists of
Vß5 inhibited VEGF-, TGF
-, and phorbol esterinduced angiogenesis. Cheresh and coworkers further distinguished the pathways based on pharmacologic susceptibility by demonstrating that inhibitors of PKC (Friedlander et al., 1995) and the tyrosine kinase Src (Eliceiri et al., 1999) block VEGF- but not bFGF-induced angiogenesis. This elegant but simple initial model of growth factor
V integrin coupling in angiogenesis has now evolved into a complicated picture of intricate interactions between growth factor receptors and integrins.
How do growth factors influence V integrinmediated function?
Although they share few structural similarities and recognize widely different ligands, growth factor receptors and integrins elicit overlapping and, in some cases, additive intracellular effects (Fig. 1). Synergy between integrins and growth factors may occur in signaling complexes that cluster along the cell surface (Plopper et al., 1995). Substantial evidence points to both a physical and functional association between integrins and VEGFR-2 that may be regulated by VEGF. VEGF-induced tyrosine phosphorylated of VEGFR-2 and cell proliferation is augmented in endothelial cells adherent to the Vß3 ligand vitronectin (Soldi et al., 1999). After VEGF stimulation, tyrosine-phosphorylated VEGFR-2 coimmunoprecipitates with
Vß3 but not with integrin ß1 or ß5. Moreover, function-blocking antibodies to
V and ß3 inhibit VEGF-stimulated phosphorylation of VEGFR-2 and activation of the regulatory subunit of phosphatidylinositol (PI) 3-kinase (Soldi et al., 1999). In CHO cells, VEGFR-2 immunoprecipitates with
Vß3 but not with integrin ß5 (Borges et al., 2000) apparently through interactions involving the extracellular domain of integrin ß3. Deletion or alteration of the ß3 cytoplasmic domain does not affect the association (Borges et al., 2000), suggesting that the interaction does not require focal adhesion formation.
Growth factors modify the signals necessary for angiogenesis by altering the levels of integrins and their affinity for ligands. The normally low endothelial expression of Vß3 (Brooks et al., 1994a) can be upregulated by bFGF (Enenstein et al., 1992; Brooks et al., 1994a; Sepp et al., 1994) and VEGF (Senger et al., 1996) but not by TGFß (Enenstein et al., 1992; Sepp et al., 1994). The effects of individual growth factors are integrin specific, since TGFß heightens expression of the more abundant ß1 integrins, whereas bFGF's effects are restricted to
Vß3. VEGF binding to VEGFR-2 activates multiple integrins, including
Vß3,
Vß5,
5ß1, and
2ß1, to enhance cell adhesion and migration (Byzova et al., 2000). Particular tumors with high adhesive properties display autocrine/paracrine integrin activation by VEGF (Byzova et al., 2000). Finally, by activating the small GTP-binding protein Rac, bFGF enhances Rac-dependent recruitment of activated
Vß3 to lamellipodia where the receptor directs cell migration (Kiosses et al., 2001).
What specific signaling pathways are coupled to V integrins?
Integrins play complex roles in controlling cell migration, growth, differentiation, and apoptosis. Because of the redundancy in the matrix proteins that are recognized by Vß3 and
Vß5, delineating their contribution to endothelial cell biology has relied in large part on the use of inhibitors or matrix molecules that appear to specifically target one or the other integrin. For example, Del1, an ECM protein and potent angiogenic factor whose expression is restricted to endothelial cells (Hidai et al., 1998), binds
Vß3 but not
Vß5 and triggers focal adhesion formation and phosphorylation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) (extracellular-regulated kinase), and Shc (Penta et al., 1999). Arachidonic acid metabolism may also be critical to
Vß3-dependent endothelial migration and angiogenesis. Dormond et al. (2001) demonstrated recently that inhibition of cyclooxygenase-2 prevents
Vß3-mediated endothelial cell spreading, migration, and activation of Cdc 42 and Rac. These effects are overcome by prostaglandins, phorbol esters, and constitutively active Cdc42 or Rac, suggesting that an unidentified arachidonic acid metabolite may play a critical role in
Vß3-mediated activation of Rac. In an angiogenesis model, constitutively active Rac restored bFGF-induced angiogenesis in the presence of cyclooxygenase-2 inhibition. This exciting link between
Vß3, arachidonic acid metabolism, and angiogenesis provides a novel mechanistic explanation for the observation that nonsteroid antiinflammatory agents protect against cancer development and progression.
Several observations implicate Vß3 in the control of cell survival and proliferation. Administration of
Vß3 antibody antagonists results in apoptosis of angiogenic but not quiescent vascular cells (Brooks et al., 1994b). Immobilization of endothelial cells on plates coated with
Vß3 antibodies suppresses p53 and the bax cell death pathway, and inhibition of
Vß3- but not
Vß5- or ß1-mediated cell adhesion activates p53 (Strömblad et al., 1996). Furthermore,
V antagonists appear to require the presence of p53 to inhibit retinal neovascularization, in that p53-deficient mice are protected from their effects (Strömblad et al., 2002). NF-
B also plays an important role in
Vß3-mediated endothelial cell survival after serum deprivation (Scatena et al., 1998). Moreover,
Vß3 antagonists block sustained endothelial MAPK activity in bFGF-treated chick chorioallantoic membranes. (Eliceiri et al., 1998). A recent article demonstrated that endothelial
Vß3 elicits an "integrin-mediated death" pathway in cells grown in an environment devoid of
Vß3 ligands (Stupack et al., 2001). Unligated
Vß3 appears to initiate apoptosis by recruiting and activating caspase-8, an effect that is mimicked by the proximal regions of the cytoplasmic domains of both ß3 and ß1 but not ß5. Limited calpain-dependent cleavage of the cytoplasmic domain of ß3 has been observed early in the course of suspension-induced apoptosis in endothelial cells (Meredith et al., 1998). Whether calpain cleavage of ß3 disrupts prosurvival signals generated by
Vß3 and/or facilitates the recruitment of caspase 8 in nonadherent endothelial cells remains to be determined.
Several endogenous angiogenesis inhibitors may exert their antiproliferative effects, in part, via Vß3 (see Table I for a more complete list). Endothelial cell attachment to immobilized endostatin (a 20-kd collagen COOH terminus cleavage product) is mediated by
Vß3,
5ß1, and
Vß5 (Rehn et al., 2001); adhesion to immobilized tumstatin (NC1 domain of the
3 chain of type IV collagen) is inhibited by antibodies to
Vß3, ß1, and
6 (Maeshima et al., 2000). Both soluble endostatin and tumstatin inhibit endothelial cell proliferation, but endostatin elicits minimal apoptosis (25% cells), whereas soluble tumstatin and tumstatin peptide derivatives induce apoptosis at levels comparable to tumor necrosis factor
(Maeshima et al., 2001). Tumstatin also prevents
Vß3-dependent activation of FAK, PI 3-kinase, protein kinase B (Akt), and cap-dependent protein synthesis in endothelial cells (Maeshima et al., 2002). The separate effects of endostatin and tumstatin on endothelial cell function may be mediated by distinct conformations assumed by
Vß3 upon binding these two ligands or may be the result of additional signals generated by integrins other than
Vß3 (e.g.,
Vß5 in the case of endostatin). The interaction of fibroblasts with CYR61, an angiogenic matrix molecule that binds both
Vß5 and
Vß3, demonstrates that
V integrins are able to mediate discrete functions upon binding the same ligand: CYR61 engagement of
Vß5 promotes fibroblast migration, but engagement of
Vß3 is required to enhance bFGF-induced proliferation (Grzeszkiewicz et al., 2001).
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Clearly, V integrin-dependent events do not occur in isolation, and other endothelial integrins may influence angiogenic events. Additionally, integrin cross-talk, the phenomena in which ligation of one integrin influences the behavior of a second integrin on the same cell, may regulate endothelial
Vß3 function. Antagonists of integrin
5ß1, which block growth factor- and tumor-induced angiogenesis, inhibit
Vß3-promoted human umbilical vein endothelial cell migration and focal contact formation via a protein kinase Adependent pathway (Kim et al., 2000). Interestingly, in the same cells
Vß3 antagonists have been shown to block
5ß1-mediated migration (Simon et al., 1997), suggesting that endothelial integrin cross-talk may be bidirectional.
What has target gene deletion in mice revealed about the role of V integrins in angiogenesis?
Studies in mice with targeted gene deletions of either V, ß3, or ß5 were initially less informative than anticipated with regard to the role of these integrins in angiogenesis. Embryos deficient in
V develop normally until E9.5 and have unimpaired vasculogenesis and angiogenesis in many organs. Approximately 80% of the embryos die, apparently as the result of placental defects. The mice that survive suffer lethal intracranial and intestinal hemorrhage (Bader et al., 1998). Mice lacking either integrin ß3 (Hodivala-Dilke et al., 1999) or ß5 (Huang et al., 2000) undergo normal angiogenesis, and the pattern of retinal neovascularization in ß3-null mice is indistinguishable from that in wild-type mice. In a follow-up to the original characterization of the ß3-/- mice, Hodivala-Dilke's group reported that tumors grown in ß3-/- mice or in mice with a combined deficiency of ß3 and ß5 are larger in size and display enhanced angiogenesis (Reynolds et al., 2002). They observed an augmented angiogenic response to VEGF in ß3-/- endothelial cells that corresponded to increased levels of VEGFR-2, suggesting that upregulation of VEGF signaling may enhance tumor angiogenesis in ß3-deficient mice. Both Eliceiri's latest work and recent studies in the ß3-/- mice establish that a closer examination of the ß5-/- and ß3-/- mice is warranted and may reveal fascinating information about the relationship between integrins and growth factors.
Bringing together V integrins, growth factors, and angiogenesis
A wealth of data indicates that growth factor receptors and V integrins interact physically and functionally to generate the signals necessary for angiogenesis (Fig. 1). Many of the responses modulated by
Vß3 are linked to proliferative and/or apoptotic pathways, whereas Eliceiri et al. (2002) convincingly tie
Vß5 with pathways involving FAK and Src. However, several questions remain. The chief question is how to resolve the discrepancies in the antiangiogenic effects of antibody and small molecule
Vß3 and/or
Vß5 inhibitors with the apparent normal developmental angiogenesis and enhanced tumor angiogenesis in the ß3- and combined ß3/ß5-deficient mice. Does compensatory upregulation of VEGFR-2 account for normal developmental angiogenesis in the ß3-/- mice? In the absence of
Vß3, does VEGF enhance the affinity of alternate compensatory integrins? Or, do
Vß3 antagonists mediate their effects through distinct signaling mechanisms such as by recruitment of caspase 8 with subsequent activation of apoptotic pathways or by transdominant integrin inhibition? Do growth factors paradoxically induce endothelial cell susceptibility to
Vß3 antagonists by upregulating receptor levels? One hypothesis that reconciles the antiangiogenic effects of
V inhibitors with enhanced tumor angiogenesis in mice lacking
Vß3 is the receptor can either promote or inhibit endothelial cell survival/proliferation depending on the presence of external stimuli and the composition of the ECM. Thus, under certain conditions
Vß3 may assume a conformation that generates signals to maintain endothelial cells in a quiescent state. Tumor-induced alterations in growth factors and matrix may shift the conformation of, and signaling pathways generated by,
Vß3. In this scenario, the lack of basal
Vß3-mediated endothelial inhibition in ß3-/- mice could result in enhanced proliferation in response to VEGF or other factors. Treatment with
V antagonists may maintain the initial
Vß3-mediated inhibitory signals and/or may trigger signals for apoptosis or transdominant inhibition of other integrins. Although investigations in this field have made rapid progress, the complexities of the integringrowth factor nexus have not been fully revealed.
Footnotes
* Abbreviations used in this paper: bFGF, basic FGF; ECM, extracellular matrix; FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; PI, phosphatidylinositol; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
Acknowledgments
Due to space limitations, we were unable to cite all of the pertinent references. We apologize to those whose work was not included.
This work was supported by a Junior Faculty Award from the American Society of Hematology to S.S. Smyth, and by the National Institutes of Health grant HL067935 to S.S. Smyth and grants HL03658, HL61656, AG10514, GM61728, and HL65619 to C. Patterson.
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