Tiny dancers : the integrin–growth factor nexus in angiogenic signalin
http://www.100md.com
《细胞学杂志》
1 Department of Medicine (Cardiology) and The Carolina Cardiovascular Biology Center, The University of North Carolina, Chapel Hill, NC 27599
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@med.unc.edu
Abstract
A vital step in growth factor–driven 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.
Figure 1. Cross-talk between growth factors and integrins in endothelial cells. VEGF binding to VEGFR-2 results in receptor dimerization and phosphorylation of specific tyrosine residues within the intracellular domain of the receptor. PLC, Sck, and VRAP (not depicted) all interact directly with VEGFR-2. The mechanism of activation of FAK, Src, MAPK, PI 3-kinase, and AKT by VEGFR-2 is less clearly understood. Recent work indicates that VEGF-mediated Src activation promotes FAK association with V?5. The extracellular domain of ?3 directs association of V?3 with VEGFR-2. Engagement of either VEGFR-2 or V?3 enhances the function of the reciprocal receptor. Binding of ECM proteins to V?3 triggers phosphorylation of tyrosine residues located in the intracellular domain of the ?3 chain and induces receptor clustering. Signaling molecules activated by ligation and/or clustering of V?3 include FAK, Src, MAPK, PI 3-kinase, and Rho family members. Recent evidence suggests a role for an unidentified arachidonic acid (AA) metabolite in V?3 activation of Rac. Phosphorylation of intracellular tyrosine residues of VEGFR-2 occurs in response to V?3 ligation. The p53/bax pathway linked to apoptosis is suppressed by V?3 engagement. The proapoptotic mediator caspase 8 may be activated by unligated V?3-dependent membrane recruitment. Additionally, ligation of either V?3 or V?5 may influence the function of the reciprocal receptor.
Endothelial cell migration, proliferation, and the formation of new lumen during angiogenesis require coordinated interactions with the extracellular matrix (ECM). Growth factors regulate these interactions, in part, by stimulating the secretion of enzymes that degrade and alter ECM. Endothelial cell adhesion molecules, such as the integrins, are also required to coordinate interactions with the ECM. Integrins are heterodimeric cell surface adhesion receptors that mediate cell–cell and cell–ECM interactions and control cell migration, differentiation, proliferation, and survival by generating intracellular signals similar to those evoked by growth factors (for review see Aplin et al., 1998). Two lines of evidence suggest that endothelial V integrins in particular play a key role in regulating angiogenesis. First, exogenously administered antibody and small molecule inhibitors of V integrins decrease or prevent angiogenesis of tumors, retinal vessels, skin, and arthritic joints in a wide variety of animal and experimental models. Second, V integrins appear to serve as cellular receptors for several endogenous pro- and antiangiogenic proteins. In this mini-review, we examine recent insights derived from studies of the integrin–growth factor receptor nexus that help to understand how convergent and divergent signaling pathways mediate angiogenic events. We also consider studies which indicate that angiogenic integrins serve as a crucial switch to regulate endothelial cell survival and destruction.
Distinct angiogenic pathways as defined by specific growth factor–integrin pairs
The discovery by Cheresh and coworkers that antagonists specific for V?3 or V?5 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 V?3 abolished basic FGF (bFGF)- and tumor necrosis factor –stimulated angiogenesis but only partially affected the response to VEGF, whereas antagonists of V?5 inhibited VEGF-, TGF-, and phorbol ester–induced 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 integrin–mediated 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 V?3 ligand vitronectin (Soldi et al., 1999). After VEGF stimulation, tyrosine-phosphorylated VEGFR-2 coimmunoprecipitates with V?3 but not with integrin ?1 or ?5. Moreover, function-blocking antibodies to V and ?3 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 V?3 but not with integrin ?5 (Borges et al., 2000) apparently through interactions involving the extracellular domain of integrin ?3. Deletion or alteration of the ?3 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 V?3 (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 TGF? (Enenstein et al., 1992; Sepp et al., 1994). The effects of individual growth factors are integrin specific, since TGF? heightens expression of the more abundant ?1 integrins, whereas bFGF's effects are restricted to V?3. VEGF binding to VEGFR-2 activates multiple integrins, including V?3, V?5, 5?1, and 2?1, 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 V?3 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 V?3 and V?5, 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 V?3 but not V?5 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 V?3-dependent endothelial migration and angiogenesis. Dormond et al. (2001) demonstrated recently that inhibition of cyclooxygenase-2 prevents V?3-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 V?3-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 V?3, 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 V?3 in the control of cell survival and proliferation. Administration of V?3 antibody antagonists results in apoptosis of angiogenic but not quiescent vascular cells (Brooks et al., 1994b). Immobilization of endothelial cells on plates coated with V?3 antibodies suppresses p53 and the bax cell death pathway, and inhibition of V?3- but not V?5- or ?1-mediated cell adhesion activates p53 (Str?mblad 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 (Str?mblad et al., 2002). NF-B also plays an important role in V?3-mediated endothelial cell survival after serum deprivation (Scatena et al., 1998). Moreover, V?3 antagonists block sustained endothelial MAPK activity in bFGF-treated chick chorioallantoic membranes. (Eliceiri et al., 1998). A recent article demonstrated that endothelial V?3 elicits an "integrin-mediated death" pathway in cells grown in an environment devoid of V?3 ligands (Stupack et al., 2001). Unligated V?3 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 ?3 and ?1 but not ?5. Limited calpain-dependent cleavage of the cytoplasmic domain of ?3 has been observed early in the course of suspension-induced apoptosis in endothelial cells (Meredith et al., 1998). Whether calpain cleavage of ?3 disrupts prosurvival signals generated by V?3 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 V?3 (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 V?3, 5?1, and V?5 (Rehn et al., 2001); adhesion to immobilized tumstatin (NC1 domain of the 3 chain of type IV collagen) is inhibited by antibodies to V?3, ?1, and 6 (Maeshima et al., 2000). Both soluble endostatin and tumstatin inhibit endothelial cell proliferation, but endostatin elicits minimal apoptosis (2–5% cells), whereas soluble tumstatin and tumstatin peptide derivatives induce apoptosis at levels comparable to tumor necrosis factor– (Maeshima et al., 2001). Tumstatin also prevents V?3-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 V?3 upon binding these two ligands or may be the result of additional signals generated by integrins other than V?3 (e.g., V?5 in the case of endostatin). The interaction of fibroblasts with CYR61, an angiogenic matrix molecule that binds both V?5 and V?3, demonstrates that V integrins are able to mediate discrete functions upon binding the same ligand: CYR61 engagement of V?5 promotes fibroblast migration, but engagement of V?3 is required to enhance bFGF-induced proliferation (Grzeszkiewicz et al., 2001).
Table I. Endogenous angiogenic proteins/proteolytic fragments that may exert their effects, at least in part, via integrins
Although V?3 appears to play a key role in regulating endothelial cell survival, proliferation, and apoptosis, V?5 has been linked to Src-dependent pathways stimulated by VEGF. In stroke models where VEGF contributes to cerebral vascular permeability, brain edema, and injury (van Bruggen et al., 1999), Src-deficient mice and wild-type mice treated with Src inhibitors display reduced vascular permeability and smaller infarct volumes (Paul et al., 2001). In a recent article, Eliceiri et al. (2002) demonstrated that mice deficient in integrin ?5 are similarly protected from the effects of VEGF. More importantly, they elucidate the first mechanistic link between VEGF-stimulated Src activity and V?5 by demonstrating that VEGF promotes Src-dependent association of FAK with V?5 in endothelial cells. VEGF stimulation dramatically increases the coimmunoprecipitation of FAK with V?5, an event that involves Src-mediated phosphorylation of FAK at tyrosine residue 861. The effects are selective in that VEGF does not alter the constitutive association of V?3 and FAK, and bFGF does not promote V?5–FAK complex formation. These findings provide a novel mechanism for VEGF regulation of V?5 signaling, which places Src upstream rather than downstream of FAK activation and integrin association.
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 V?3 function. Antagonists of integrin 5?1, which block growth factor- and tumor-induced angiogenesis, inhibit V?3-promoted human umbilical vein endothelial cell migration and focal contact formation via a protein kinase A–dependent pathway (Kim et al., 2000). Interestingly, in the same cells V?3 antagonists have been shown to block 5?1-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, ?3, or ?5 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 ?3 (Hodivala-Dilke et al., 1999) or ?5 (Huang et al., 2000) undergo normal angiogenesis, and the pattern of retinal neovascularization in ?3-null mice is indistinguishable from that in wild-type mice. In a follow-up to the original characterization of the ?3-/- mice, Hodivala-Dilke's group reported that tumors grown in ?3-/- mice or in mice with a combined deficiency of ?3 and ?5 are larger in size and display enhanced angiogenesis (Reynolds et al., 2002). They observed an augmented angiogenic response to VEGF in ?3-/- endothelial cells that corresponded to increased levels of VEGFR-2, suggesting that upregulation of VEGF signaling may enhance tumor angiogenesis in ?3-deficient mice. Both Eliceiri's latest work and recent studies in the ?3-/- mice establish that a closer examination of the ?5-/- and ?3-/- 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 V?3 are linked to proliferative and/or apoptotic pathways, whereas Eliceiri et al. (2002) convincingly tie V?5 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 V?3 and/or V?5 inhibitors with the apparent normal developmental angiogenesis and enhanced tumor angiogenesis in the ?3- and combined ?3/?5-deficient mice. Does compensatory upregulation of VEGFR-2 account for normal developmental angiogenesis in the ?3-/- mice? In the absence of V?3, does VEGF enhance the affinity of alternate compensatory integrins? Or, do V?3 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 V?3 antagonists by upregulating receptor levels? One hypothesis that reconciles the antiangiogenic effects of V inhibitors with enhanced tumor angiogenesis in mice lacking V?3 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 V?3 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, V?3. In this scenario, the lack of basal V?3-mediated endothelial inhibition in ?3-/- mice could result in enhanced proliferation in response to VEGF or other factors. Treatment with V antagonists may maintain the initial V?3-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 integrin–growth factor nexus have not been fully revealed.
References
Aplin, A.E., A. Howe, S.K. Alahari, and R.L. Juliano. 1998. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol. Rev. 50:197–263.
Bader, B.L., H. Rayburn, D. Crowley, and R.O. Hynes. 1998. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all v integrins. Cell. 95:507–519.
Borges, E., Y. Jan, and E. Ruoslahti. 2000. Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the ?3 integrin through its extracellular domain. J. Biol. Chem. 275:39867–39873.
Bornstein, P. 2001. Thrombospondins as matricellular modulators of cell function. J. Clin. Invest. 107:929–934.
Brooks, P.C., R.A. Clark, and D.A. Cheresh. 1994a. Requirement of vascular integrin v?3 for angiogenesis. Science. 264:569–571.
Brooks, P.C., A.M. Montgomery, M. Rosenfeld, R.A. Reisfeld, T. Hu, G. Klier, and D.A. Cheresh. 1994b. Integrin V?3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell. 79:1157–1164.
Byzova, T.V., C.K. Goldman, N. Pampori, N. Thomas, A. Bett, S.J. Shattil, and E.F. Plow. 2000. A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol. Cell. 6:851–860.
Camenisch G., M.T. Pisabarro, D. Sherman, J. Kowalski, M. Nagel, P. Hass, A. Gurney, S.C. Bodary X.H. Liang, K.R. Clark, et al. 2002. ANGPTL3 stimulates endothelial cell adhesion and migration via integrin V?3 and induces blood vessel formation in vivo. J. Biol. Chem. 277:17281–17290.
Carlson, T.R., Y. Feng, P.C. Maisonpierre, M. Mrksich, and A.O. Morla. 2001. Direct cell adhesion to the angiopoietins mediated by integrins. J. Biol. Chem. 276:26516–26525.
Cross, M.J., and L. Claesson-Welsh. 2001. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol. Sci. 22:201–207.
Dormond, O., A. Foletti, C. Paroz, and C. Ruegg. 2001. NSAIDs inhibit V?3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis. Nat. Med. 7:1041–1047.
Eliceiri, B.P., R. Klemke, S. Str?mblad, and D.A. Cheresh. 1998. Integrin V?3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis. J. Cell Biol. 140:1255–1263.
Eliceiri, B.P., R. Paul, R.L. Schwartzberg, J.D. Hood, J. Leng, and D.A. Cheresh. 1999. Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. Mol. Cell. 4:915–924.
Eliceiri, B.P., X.S. Puente, J.D. Hood, D.G. Stupack, D.D. Schlaepfer, X.Z. Huang, D. Sheppard, and D.A. Cheresh. 2002. Src-mediated coupling of Fak to integrin v?5 in VEGF signaling. J. Cell Biol. 157:149–160.
Enenstein J., N.S. Waleh, and R.H. KrAm. 1992. Basic FGF and TGF-? differentially modulate integrin expression of human microvascular endothelial cells. Exp. Cell Res. 203:499–503.
Friedlander M., P.C. Brooks, R.W. Shaffer, C.M. Kincaid, J.A. Varner, and D.A. Cheresh. 1995. Definition of two angiogenic pathways by distinct v integrins. Science. 1995. 270:1500–1502.
Grzeszkiewicz, T.M., D.J. Kirschling, N. Chen, and L.F. Lau. 2001. CYR61 stimulates human skin fibroblast migration through integrin v?5 and enhances mitogenesis through integrin v?3, independent of its carboxyl-terminal domain. J. Biol. Chem. 276:21943–21950.
Hidai, C., T. Zupancic, K. Penta, A. Mikhail, M. Kawana, E.E. Quertermous, Y. Aoka, M. Fukagawa, Y. Matsui, D. Platika, et al. 1998. Cloning and characterization of developmental endothelial locus-1: an embryonic endothelial cell protein that binds the v?3 integrin receptor. Genes Dev. 12:21–33.
Hodivala-Dilke, K.M., X. McHugh, D.A. Tsakiris, H. Rayburn, D. Crowley, M. Ullman-Cullere, F.P. Ross, B.S. Coller, S. Teitelbaum, and R.O. Hynes. 1999. ?3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J. Clin. Invest. 103:229–238.
Huang, X., M. Griffiths, J. Wu, R.V. Farese, and D. Sheppard. 2000. Normal development, wound healing, and adenovirus susceptibility in ?5-deficient mice. Mol. Cell. Biol. 20:755–759.
Kim, S., M. Harris, and J.A. Varner. 2000. Regulation of integrin V?3-mediated endothelial cell migration and angiogenesis by integrin 5?1 and protein kinase A. J. Biol. Chem. 275:33920–33928.
Kiosses, W.B., S.J. Shattil, N. Pampori, and M.A. Schwartz. 2001. Rac recruits high-affinity integrin v?3 to lamellipodia in endothelial cell migration. Nat. Cell Biol. 3:316–320.
Maeshima, Y., P.C. Colorado, and R. Kalluri. 2000. Two RGD-independent V?3 integrin binding sites on tumstatin regulate distinct anti-tumor properties. J. Biol. Chem. 275:23745–23750.
Maeshima Y., U.L. Yerramalla, M. Dhanabal, K.A. Holthaus, S. Barbashov, S. Kharbanda, C. Reimer, M. Manfredi, W.M. Dickerson, and R. Kalluri. 2001. Extracellular matrix-derived peptide binds to v?3 integrin and inhibits angiogenesis. J. Biol. Chem. 276:31959–31968.
Maeshima, Y., A. Sudhakar, J.C. Lively, K. Ueki, S. Kharbanda, C.R. Kahn, N. Sonenberg, R.O. Hynes, and R. Kalluri. 2002. Tumstatin, an endothelial cell-specific inhibitor of protein synthesis. Science. 295:140–143.
Meredith, J., Z. Mu, T. Saido, and X. Du. 1998. Cleavage of the cytoplasmic domain of the integrin ?3 subunit during endothelial cell apoptosis. J. Biol. Chem. 273:19525–19531.
Paul, R., Z.G. Zhang, B.P. Eliceiri, Q. Jiang, A.D. Boccia, R.L. Zhang, M. Chopp, and D.A. Cheresh. 2001. Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke. Nat. Med. 7:222–227.
Penta K., J.A. Varner, L. Liaw, C. Hidai, R. Schatzman, and T. Quertermous 1999. Del1 induces integrin signaling and angiogenesis by ligation of v?5. J. Biol. Chem. 274:11101–11109.
Plopper, G.E., H.P. McNamee, L.E. Dike, K. Bojanowski, and D.E. Ingber. 1995. Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex. Mol. Biol. Cell. 6:1349–1365.
Rehn, M., T. Veikkola, E. Kukk-Valdre, H. Nakamura, M. Ilmonen, C. Lombardo, T. Pihlajaniemi, K. Alitalo, and K. Vuori. 2001. Interaction of endostatin with integrins implicated in angiogenesis. Proc. Natl. Acad. Sci. USA. 98:1024–1029.
Reynolds, L.E., L. Wyder, J.C. Lively, D. Taverna, S.D. Robinson, X. Huang, D. Sheppard, R.O. Hynes, and K.M. Hodivala-Dilke. 2002. Enhanced pathological angiogenesis in mice lacking ?3 integrin or ?3 and ?5 integrins. Nat. Med. 8:27–34.
Scatena, M., M. Almeida, M.L. Chaisson, N. Fausto, R.F. Nicosia, and C.M. Giachelli. 1998. NF-kappaB mediates v?3 integrin-induced endothelial cell survival. J. Cell Biol. 141:1083–1093.
Sepp N.T., L.J. Li, K.H. Lee, E.J. Brown, S.W. Caughman, T.J. Lawley, and R.A. Swerlick. 1994. Basic fibroblast growth factor increases expression of the V?3 integrin complex on human microvascular endothelial cells. J. Invest. Dermatol. 103:295–299.
Senger, D.R., S.R. Ledbetter, K.P. Claffey, A. Papadopoulos-Sergiou, C.A. Peruzzi, and M. Detmar. 1996. Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the v?3 integrin, osteopontin, and thrombin. Am. J. Pathol. 149:293–305.
Simon K.O., E.M. Nutt, D.G. Abraham, G.A. Rodan, and L.T. Duong. 1997. The V?3 integrin regulates 5?1-mediated cell migration toward fibronectin. J. Biol. Chem. 272:29380–29389
Soldi R., S. Mitola, M. Strasly, P. Defilippi, G. Tarone, and F. Bussolino. 1999. Role of v?3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J. 18:882–892.
Str?mblad S., J.C. Becker, M. Yebra, P.C. Brooks, and D.A. Cheresh. 1996. Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin V?3 during angiogenesis. J. Clin. Invest. 98:426–433.
Str?mblad, S., A. Fotedar, H. Brickner, C. Theesfeld, E. Aguilar de Diaz, M. Friedlander, and D.A. Cheresh. 2002. Loss of p53 compensates for V-integrin function in retinal neovascularization. J. Biol. Chem. 277:13371–13374.
Stupack, D.G., X.S. Puente, S. Boutsaboualoy, C.M. Storgard, and D.A. Cheresh. 2001. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J. Cell Biol. 155:459–470.
Tarui, T., L.A. Miles, and Y. Takada. 2001. Specific interaction of angiostatin with integrin V?3 in endothelial cells. J. Biol. Chem. 276:39562–39568.
van Bruggen, N., H. Thibodeaux, J.T. Palmer, W.P. Lee, L. Fu, B. Cairns, D. Tumas, R. Gerlai, S.P. Williams, M. van Lookeren Campagne, et al. 1999. VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain. J. Clin. Invest. 104:1613–1620.(Susan S. Smyth1 and Cam Patterson1,2)
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@med.unc.edu
Abstract
A vital step in growth factor–driven 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.
Figure 1. Cross-talk between growth factors and integrins in endothelial cells. VEGF binding to VEGFR-2 results in receptor dimerization and phosphorylation of specific tyrosine residues within the intracellular domain of the receptor. PLC, Sck, and VRAP (not depicted) all interact directly with VEGFR-2. The mechanism of activation of FAK, Src, MAPK, PI 3-kinase, and AKT by VEGFR-2 is less clearly understood. Recent work indicates that VEGF-mediated Src activation promotes FAK association with V?5. The extracellular domain of ?3 directs association of V?3 with VEGFR-2. Engagement of either VEGFR-2 or V?3 enhances the function of the reciprocal receptor. Binding of ECM proteins to V?3 triggers phosphorylation of tyrosine residues located in the intracellular domain of the ?3 chain and induces receptor clustering. Signaling molecules activated by ligation and/or clustering of V?3 include FAK, Src, MAPK, PI 3-kinase, and Rho family members. Recent evidence suggests a role for an unidentified arachidonic acid (AA) metabolite in V?3 activation of Rac. Phosphorylation of intracellular tyrosine residues of VEGFR-2 occurs in response to V?3 ligation. The p53/bax pathway linked to apoptosis is suppressed by V?3 engagement. The proapoptotic mediator caspase 8 may be activated by unligated V?3-dependent membrane recruitment. Additionally, ligation of either V?3 or V?5 may influence the function of the reciprocal receptor.
Endothelial cell migration, proliferation, and the formation of new lumen during angiogenesis require coordinated interactions with the extracellular matrix (ECM). Growth factors regulate these interactions, in part, by stimulating the secretion of enzymes that degrade and alter ECM. Endothelial cell adhesion molecules, such as the integrins, are also required to coordinate interactions with the ECM. Integrins are heterodimeric cell surface adhesion receptors that mediate cell–cell and cell–ECM interactions and control cell migration, differentiation, proliferation, and survival by generating intracellular signals similar to those evoked by growth factors (for review see Aplin et al., 1998). Two lines of evidence suggest that endothelial V integrins in particular play a key role in regulating angiogenesis. First, exogenously administered antibody and small molecule inhibitors of V integrins decrease or prevent angiogenesis of tumors, retinal vessels, skin, and arthritic joints in a wide variety of animal and experimental models. Second, V integrins appear to serve as cellular receptors for several endogenous pro- and antiangiogenic proteins. In this mini-review, we examine recent insights derived from studies of the integrin–growth factor receptor nexus that help to understand how convergent and divergent signaling pathways mediate angiogenic events. We also consider studies which indicate that angiogenic integrins serve as a crucial switch to regulate endothelial cell survival and destruction.
Distinct angiogenic pathways as defined by specific growth factor–integrin pairs
The discovery by Cheresh and coworkers that antagonists specific for V?3 or V?5 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 V?3 abolished basic FGF (bFGF)- and tumor necrosis factor –stimulated angiogenesis but only partially affected the response to VEGF, whereas antagonists of V?5 inhibited VEGF-, TGF-, and phorbol ester–induced 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 integrin–mediated 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 V?3 ligand vitronectin (Soldi et al., 1999). After VEGF stimulation, tyrosine-phosphorylated VEGFR-2 coimmunoprecipitates with V?3 but not with integrin ?1 or ?5. Moreover, function-blocking antibodies to V and ?3 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 V?3 but not with integrin ?5 (Borges et al., 2000) apparently through interactions involving the extracellular domain of integrin ?3. Deletion or alteration of the ?3 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 V?3 (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 TGF? (Enenstein et al., 1992; Sepp et al., 1994). The effects of individual growth factors are integrin specific, since TGF? heightens expression of the more abundant ?1 integrins, whereas bFGF's effects are restricted to V?3. VEGF binding to VEGFR-2 activates multiple integrins, including V?3, V?5, 5?1, and 2?1, 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 V?3 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 V?3 and V?5, 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 V?3 but not V?5 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 V?3-dependent endothelial migration and angiogenesis. Dormond et al. (2001) demonstrated recently that inhibition of cyclooxygenase-2 prevents V?3-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 V?3-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 V?3, 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 V?3 in the control of cell survival and proliferation. Administration of V?3 antibody antagonists results in apoptosis of angiogenic but not quiescent vascular cells (Brooks et al., 1994b). Immobilization of endothelial cells on plates coated with V?3 antibodies suppresses p53 and the bax cell death pathway, and inhibition of V?3- but not V?5- or ?1-mediated cell adhesion activates p53 (Str?mblad 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 (Str?mblad et al., 2002). NF-B also plays an important role in V?3-mediated endothelial cell survival after serum deprivation (Scatena et al., 1998). Moreover, V?3 antagonists block sustained endothelial MAPK activity in bFGF-treated chick chorioallantoic membranes. (Eliceiri et al., 1998). A recent article demonstrated that endothelial V?3 elicits an "integrin-mediated death" pathway in cells grown in an environment devoid of V?3 ligands (Stupack et al., 2001). Unligated V?3 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 ?3 and ?1 but not ?5. Limited calpain-dependent cleavage of the cytoplasmic domain of ?3 has been observed early in the course of suspension-induced apoptosis in endothelial cells (Meredith et al., 1998). Whether calpain cleavage of ?3 disrupts prosurvival signals generated by V?3 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 V?3 (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 V?3, 5?1, and V?5 (Rehn et al., 2001); adhesion to immobilized tumstatin (NC1 domain of the 3 chain of type IV collagen) is inhibited by antibodies to V?3, ?1, and 6 (Maeshima et al., 2000). Both soluble endostatin and tumstatin inhibit endothelial cell proliferation, but endostatin elicits minimal apoptosis (2–5% cells), whereas soluble tumstatin and tumstatin peptide derivatives induce apoptosis at levels comparable to tumor necrosis factor– (Maeshima et al., 2001). Tumstatin also prevents V?3-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 V?3 upon binding these two ligands or may be the result of additional signals generated by integrins other than V?3 (e.g., V?5 in the case of endostatin). The interaction of fibroblasts with CYR61, an angiogenic matrix molecule that binds both V?5 and V?3, demonstrates that V integrins are able to mediate discrete functions upon binding the same ligand: CYR61 engagement of V?5 promotes fibroblast migration, but engagement of V?3 is required to enhance bFGF-induced proliferation (Grzeszkiewicz et al., 2001).
Table I. Endogenous angiogenic proteins/proteolytic fragments that may exert their effects, at least in part, via integrins
Although V?3 appears to play a key role in regulating endothelial cell survival, proliferation, and apoptosis, V?5 has been linked to Src-dependent pathways stimulated by VEGF. In stroke models where VEGF contributes to cerebral vascular permeability, brain edema, and injury (van Bruggen et al., 1999), Src-deficient mice and wild-type mice treated with Src inhibitors display reduced vascular permeability and smaller infarct volumes (Paul et al., 2001). In a recent article, Eliceiri et al. (2002) demonstrated that mice deficient in integrin ?5 are similarly protected from the effects of VEGF. More importantly, they elucidate the first mechanistic link between VEGF-stimulated Src activity and V?5 by demonstrating that VEGF promotes Src-dependent association of FAK with V?5 in endothelial cells. VEGF stimulation dramatically increases the coimmunoprecipitation of FAK with V?5, an event that involves Src-mediated phosphorylation of FAK at tyrosine residue 861. The effects are selective in that VEGF does not alter the constitutive association of V?3 and FAK, and bFGF does not promote V?5–FAK complex formation. These findings provide a novel mechanism for VEGF regulation of V?5 signaling, which places Src upstream rather than downstream of FAK activation and integrin association.
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 V?3 function. Antagonists of integrin 5?1, which block growth factor- and tumor-induced angiogenesis, inhibit V?3-promoted human umbilical vein endothelial cell migration and focal contact formation via a protein kinase A–dependent pathway (Kim et al., 2000). Interestingly, in the same cells V?3 antagonists have been shown to block 5?1-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, ?3, or ?5 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 ?3 (Hodivala-Dilke et al., 1999) or ?5 (Huang et al., 2000) undergo normal angiogenesis, and the pattern of retinal neovascularization in ?3-null mice is indistinguishable from that in wild-type mice. In a follow-up to the original characterization of the ?3-/- mice, Hodivala-Dilke's group reported that tumors grown in ?3-/- mice or in mice with a combined deficiency of ?3 and ?5 are larger in size and display enhanced angiogenesis (Reynolds et al., 2002). They observed an augmented angiogenic response to VEGF in ?3-/- endothelial cells that corresponded to increased levels of VEGFR-2, suggesting that upregulation of VEGF signaling may enhance tumor angiogenesis in ?3-deficient mice. Both Eliceiri's latest work and recent studies in the ?3-/- mice establish that a closer examination of the ?5-/- and ?3-/- 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 V?3 are linked to proliferative and/or apoptotic pathways, whereas Eliceiri et al. (2002) convincingly tie V?5 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 V?3 and/or V?5 inhibitors with the apparent normal developmental angiogenesis and enhanced tumor angiogenesis in the ?3- and combined ?3/?5-deficient mice. Does compensatory upregulation of VEGFR-2 account for normal developmental angiogenesis in the ?3-/- mice? In the absence of V?3, does VEGF enhance the affinity of alternate compensatory integrins? Or, do V?3 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 V?3 antagonists by upregulating receptor levels? One hypothesis that reconciles the antiangiogenic effects of V inhibitors with enhanced tumor angiogenesis in mice lacking V?3 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 V?3 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, V?3. In this scenario, the lack of basal V?3-mediated endothelial inhibition in ?3-/- mice could result in enhanced proliferation in response to VEGF or other factors. Treatment with V antagonists may maintain the initial V?3-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 integrin–growth factor nexus have not been fully revealed.
References
Aplin, A.E., A. Howe, S.K. Alahari, and R.L. Juliano. 1998. Signal transduction and signal modulation by cell adhesion receptors: the role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol. Rev. 50:197–263.
Bader, B.L., H. Rayburn, D. Crowley, and R.O. Hynes. 1998. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all v integrins. Cell. 95:507–519.
Borges, E., Y. Jan, and E. Ruoslahti. 2000. Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the ?3 integrin through its extracellular domain. J. Biol. Chem. 275:39867–39873.
Bornstein, P. 2001. Thrombospondins as matricellular modulators of cell function. J. Clin. Invest. 107:929–934.
Brooks, P.C., R.A. Clark, and D.A. Cheresh. 1994a. Requirement of vascular integrin v?3 for angiogenesis. Science. 264:569–571.
Brooks, P.C., A.M. Montgomery, M. Rosenfeld, R.A. Reisfeld, T. Hu, G. Klier, and D.A. Cheresh. 1994b. Integrin V?3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell. 79:1157–1164.
Byzova, T.V., C.K. Goldman, N. Pampori, N. Thomas, A. Bett, S.J. Shattil, and E.F. Plow. 2000. A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol. Cell. 6:851–860.
Camenisch G., M.T. Pisabarro, D. Sherman, J. Kowalski, M. Nagel, P. Hass, A. Gurney, S.C. Bodary X.H. Liang, K.R. Clark, et al. 2002. ANGPTL3 stimulates endothelial cell adhesion and migration via integrin V?3 and induces blood vessel formation in vivo. J. Biol. Chem. 277:17281–17290.
Carlson, T.R., Y. Feng, P.C. Maisonpierre, M. Mrksich, and A.O. Morla. 2001. Direct cell adhesion to the angiopoietins mediated by integrins. J. Biol. Chem. 276:26516–26525.
Cross, M.J., and L. Claesson-Welsh. 2001. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition. Trends Pharmacol. Sci. 22:201–207.
Dormond, O., A. Foletti, C. Paroz, and C. Ruegg. 2001. NSAIDs inhibit V?3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis. Nat. Med. 7:1041–1047.
Eliceiri, B.P., R. Klemke, S. Str?mblad, and D.A. Cheresh. 1998. Integrin V?3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis. J. Cell Biol. 140:1255–1263.
Eliceiri, B.P., R. Paul, R.L. Schwartzberg, J.D. Hood, J. Leng, and D.A. Cheresh. 1999. Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability. Mol. Cell. 4:915–924.
Eliceiri, B.P., X.S. Puente, J.D. Hood, D.G. Stupack, D.D. Schlaepfer, X.Z. Huang, D. Sheppard, and D.A. Cheresh. 2002. Src-mediated coupling of Fak to integrin v?5 in VEGF signaling. J. Cell Biol. 157:149–160.
Enenstein J., N.S. Waleh, and R.H. KrAm. 1992. Basic FGF and TGF-? differentially modulate integrin expression of human microvascular endothelial cells. Exp. Cell Res. 203:499–503.
Friedlander M., P.C. Brooks, R.W. Shaffer, C.M. Kincaid, J.A. Varner, and D.A. Cheresh. 1995. Definition of two angiogenic pathways by distinct v integrins. Science. 1995. 270:1500–1502.
Grzeszkiewicz, T.M., D.J. Kirschling, N. Chen, and L.F. Lau. 2001. CYR61 stimulates human skin fibroblast migration through integrin v?5 and enhances mitogenesis through integrin v?3, independent of its carboxyl-terminal domain. J. Biol. Chem. 276:21943–21950.
Hidai, C., T. Zupancic, K. Penta, A. Mikhail, M. Kawana, E.E. Quertermous, Y. Aoka, M. Fukagawa, Y. Matsui, D. Platika, et al. 1998. Cloning and characterization of developmental endothelial locus-1: an embryonic endothelial cell protein that binds the v?3 integrin receptor. Genes Dev. 12:21–33.
Hodivala-Dilke, K.M., X. McHugh, D.A. Tsakiris, H. Rayburn, D. Crowley, M. Ullman-Cullere, F.P. Ross, B.S. Coller, S. Teitelbaum, and R.O. Hynes. 1999. ?3-integrin-deficient mice are a model for Glanzmann thrombasthenia showing placental defects and reduced survival. J. Clin. Invest. 103:229–238.
Huang, X., M. Griffiths, J. Wu, R.V. Farese, and D. Sheppard. 2000. Normal development, wound healing, and adenovirus susceptibility in ?5-deficient mice. Mol. Cell. Biol. 20:755–759.
Kim, S., M. Harris, and J.A. Varner. 2000. Regulation of integrin V?3-mediated endothelial cell migration and angiogenesis by integrin 5?1 and protein kinase A. J. Biol. Chem. 275:33920–33928.
Kiosses, W.B., S.J. Shattil, N. Pampori, and M.A. Schwartz. 2001. Rac recruits high-affinity integrin v?3 to lamellipodia in endothelial cell migration. Nat. Cell Biol. 3:316–320.
Maeshima, Y., P.C. Colorado, and R. Kalluri. 2000. Two RGD-independent V?3 integrin binding sites on tumstatin regulate distinct anti-tumor properties. J. Biol. Chem. 275:23745–23750.
Maeshima Y., U.L. Yerramalla, M. Dhanabal, K.A. Holthaus, S. Barbashov, S. Kharbanda, C. Reimer, M. Manfredi, W.M. Dickerson, and R. Kalluri. 2001. Extracellular matrix-derived peptide binds to v?3 integrin and inhibits angiogenesis. J. Biol. Chem. 276:31959–31968.
Maeshima, Y., A. Sudhakar, J.C. Lively, K. Ueki, S. Kharbanda, C.R. Kahn, N. Sonenberg, R.O. Hynes, and R. Kalluri. 2002. Tumstatin, an endothelial cell-specific inhibitor of protein synthesis. Science. 295:140–143.
Meredith, J., Z. Mu, T. Saido, and X. Du. 1998. Cleavage of the cytoplasmic domain of the integrin ?3 subunit during endothelial cell apoptosis. J. Biol. Chem. 273:19525–19531.
Paul, R., Z.G. Zhang, B.P. Eliceiri, Q. Jiang, A.D. Boccia, R.L. Zhang, M. Chopp, and D.A. Cheresh. 2001. Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke. Nat. Med. 7:222–227.
Penta K., J.A. Varner, L. Liaw, C. Hidai, R. Schatzman, and T. Quertermous 1999. Del1 induces integrin signaling and angiogenesis by ligation of v?5. J. Biol. Chem. 274:11101–11109.
Plopper, G.E., H.P. McNamee, L.E. Dike, K. Bojanowski, and D.E. Ingber. 1995. Convergence of integrin and growth factor receptor signaling pathways within the focal adhesion complex. Mol. Biol. Cell. 6:1349–1365.
Rehn, M., T. Veikkola, E. Kukk-Valdre, H. Nakamura, M. Ilmonen, C. Lombardo, T. Pihlajaniemi, K. Alitalo, and K. Vuori. 2001. Interaction of endostatin with integrins implicated in angiogenesis. Proc. Natl. Acad. Sci. USA. 98:1024–1029.
Reynolds, L.E., L. Wyder, J.C. Lively, D. Taverna, S.D. Robinson, X. Huang, D. Sheppard, R.O. Hynes, and K.M. Hodivala-Dilke. 2002. Enhanced pathological angiogenesis in mice lacking ?3 integrin or ?3 and ?5 integrins. Nat. Med. 8:27–34.
Scatena, M., M. Almeida, M.L. Chaisson, N. Fausto, R.F. Nicosia, and C.M. Giachelli. 1998. NF-kappaB mediates v?3 integrin-induced endothelial cell survival. J. Cell Biol. 141:1083–1093.
Sepp N.T., L.J. Li, K.H. Lee, E.J. Brown, S.W. Caughman, T.J. Lawley, and R.A. Swerlick. 1994. Basic fibroblast growth factor increases expression of the V?3 integrin complex on human microvascular endothelial cells. J. Invest. Dermatol. 103:295–299.
Senger, D.R., S.R. Ledbetter, K.P. Claffey, A. Papadopoulos-Sergiou, C.A. Peruzzi, and M. Detmar. 1996. Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the v?3 integrin, osteopontin, and thrombin. Am. J. Pathol. 149:293–305.
Simon K.O., E.M. Nutt, D.G. Abraham, G.A. Rodan, and L.T. Duong. 1997. The V?3 integrin regulates 5?1-mediated cell migration toward fibronectin. J. Biol. Chem. 272:29380–29389
Soldi R., S. Mitola, M. Strasly, P. Defilippi, G. Tarone, and F. Bussolino. 1999. Role of v?3 integrin in the activation of vascular endothelial growth factor receptor-2. EMBO J. 18:882–892.
Str?mblad S., J.C. Becker, M. Yebra, P.C. Brooks, and D.A. Cheresh. 1996. Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin V?3 during angiogenesis. J. Clin. Invest. 98:426–433.
Str?mblad, S., A. Fotedar, H. Brickner, C. Theesfeld, E. Aguilar de Diaz, M. Friedlander, and D.A. Cheresh. 2002. Loss of p53 compensates for V-integrin function in retinal neovascularization. J. Biol. Chem. 277:13371–13374.
Stupack, D.G., X.S. Puente, S. Boutsaboualoy, C.M. Storgard, and D.A. Cheresh. 2001. Apoptosis of adherent cells by recruitment of caspase-8 to unligated integrins. J. Cell Biol. 155:459–470.
Tarui, T., L.A. Miles, and Y. Takada. 2001. Specific interaction of angiostatin with integrin V?3 in endothelial cells. J. Biol. Chem. 276:39562–39568.
van Bruggen, N., H. Thibodeaux, J.T. Palmer, W.P. Lee, L. Fu, B. Cairns, D. Tumas, R. Gerlai, S.P. Williams, M. van Lookeren Campagne, et al. 1999. VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain. J. Clin. Invest. 104:1613–1620.(Susan S. Smyth1 and Cam Patterson1,2)