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A sequence of events in vascular and stromal cells maintained in a highly coordinated manner regulates angiogenesis and tissue remodeling. These processes are mediated by the ability of cells to respond to environmental cues and activate surface integrins. Physiological and pathological processes in vascular biology are dependent on the specificity of important signaling mechanisms that are activated through the association between growth factors, their receptors, integrins, and their specific extracellular matrix ligands. A large body of evidence from in vitro and in vivo models demonstrates the importance of coordination of signals from the extracellular environment that activates specific tyrosine kinase receptors and integrins in order to regulate angiogenic processes in vivo. In addition to complex formation between growth factor receptors and integrins, growth factors and cytokines also directly interact with integrins, depending upon their concentration levels in the environment, and differentially regulate integrin-related processes. Recent studies from a number of laboratories including ours have provided important novel insights into the involvement of many signaling events that improve our existing knowledge on the cross-talk between growth factor receptors and integrins in the regulation of angiogenesis. In this review, our focus will be on updating the recent developments in the field of integrin-growth factor receptor associations and their implications in the vascular processes.
Angiogenesis, the process of formation of new blood vessels from the pre-existing vasculature , is necessary for physiological processes, including embryogenesis, wound healing, and the normal response to hypoxic conditions and additionally underlies a number of pathologies, such as tumor development, rheumatoid arthritis, diabetic microvascular disease, macular degeneration, ischemia, and inflammation . Development and remodeling of the vascular system and associated tissues require complex interactions of signals and physical forces orchestrating the activities of endothelial cells, pericytes, fibroblasts, and smooth muscle cells . Endothelium, the vasculature’s inner lining, is responsible for the initiation of angiogenesis, and crucial for this process is the ability of endothelium to attach to extracellular matrix (ECM) proteins and migrate to form endothelial tubes . Formation of the initial endothelial tubes is followed by recruitment of stromal and inflammatory cells ultimately resulting in a network of matured blood vessels . These series of events are regulated by a number of growth factors, cytokines, and ECM proteins and their receptors . Initial endothelial tube formation requires the coordinated integration of mitogenic and migratory signals elicited by pro-angiogenic growth factors such as vascular endothelial growth factor (VEGF) . Induction of endothelial cell migration by VEGF also depends on interactions between endothelium and various ECM proteins mediated by integrins . Major integrins on endothelial cells include αvβ3, αvβ5, α5β1, and α6β4, which serve as receptors for vitronectin, fibronectin, and laminin, respectively . Among these, integrin αvβ3 has been shown to be particularly important for the angiogenic stimulation by VEGF and other growth factors [8–10].
In the last decade, an increasing body of evidences demonstrate that integrins are not mere ECM receptors, but major regulators of the biological activity of several signaling systems within the cell. A number of laboratories, including ours, have demonstrated that the specificity of molecular signaling in the endothelium during development and during pathological processes is determined by a synergism between growth factor receptors and cell adhesion molecules [11–16]. Integrins and growth factor receptors are known to independently regulate angiogenesis [17, 18]. However, the combined effects of growth factors and ECM proteins via their respective receptors appear to be essential for endothelial activation and neovascularization [19, 20]). For the better understanding of the molecular mechanisms underlying physiological and pathological angiogenesis, analysis of cross-talk between growth factor receptors and integrins with emphasis on the signaling cascades elicited by this interaction would be essential. Therefore, in this review, we will focus on the recent developments in our understanding of the interactions between growth factor receptors and integrins and the functional consequences of their synergism.
Interactions between cells and ECM proteins are crucial for virtually all tissue responses, including cell movement, proliferation, and matrix remodeling . Integrins represent that most important family of ECM receptors  and are found in all multicellular organisms ranging from sponges to mammals . The biological significance of the range of ECM-integrin specificities during cell adhesion and migration remains an important issue. Integrins are present on the cell surface as heterodimers, each composed of non-homologous trans-membrane α and β subunits, both of which are involved in the recognition of ligands and regulate bi-directional signaling . ‘Outside–in signaling’ provides the cells cues from the extracellular environment, while ‘inside–out signaling’ results in changes in the conformation of integrins, thus promoting modifications in their functional activity . Vascular cells, which include endothelial cells, pericytes, fibroblasts, and smooth muscle cells, express numerous integrins including α1β1, α2β1, α4β1, α5β1, αvβ1, αvβ3, αvβ5, α6β1, and α6β4  and, therefore, are able to interact with a wide range of ligands. Thus, integrins have been widely implicated in the regulation of angiogenesis .
Changes in ECM environment result in the induction of multiple cellular signaling pathways in vascular cells via integrins . These include activation of Ras, PI3 Kinase-Akt signaling, MAP kinase, Src and Rac, Rho, cdc42 GTPases [26–29]. Furthermore, recognition of ECM by some integrins such as αvβ3 results in the phosphorylation of integrin cytoplasmic tyrosine residues, which, in turn, promotes recruitment of intracellular adaptor proteins [15, 30].
In many instances, activation of the intracellular signaling cascade triggered by growth factors is substantially amplified if endothelial cells are attached to ECM . This clearly indicates that integrin- and growth factor-mediated cellular responses synergize and function to coordinate the biochemical responses of multiple cell types.
A role of growth factors and their tyrosine kinase receptors in integrin-dependent processes such as ECM recognition, migration, matrix assembly, tissue remodeling, and angiogenesis is gradually emerging. Optimal cell stimulation with epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin like growth factor-1 (IGF-1), or VEGF [15, 32–34], is greatly influenced by integrin-mediated cell adhesion to the respective ECM protein. In vascular smooth muscle cells, EGF or IGF-1 responses depend on integrin αvβ3 [35–37], whereas functions of EGF-stimulated kidney epithelial cells depends on β1 integrins . The physiological importance of IGF-1/αvβ3 association is further supported by the fact that treatment with αvβ3 antagonists reduce IGF-1 signaling and development of atherosclerotic lesions [38, 39]. Detailed information on interaction between growth factor receptors and integrins is provided in Table 1.
The capacity of growth factor stimulation to synergize with the ECM ligand associated signals may be due to the co-clustering of these receptors on the cell surface. Co-immunoprecipitation has been an important approach to identify physical interactions between growth factor receptors and integrins in vascular cells. Integrin αvβ3 has been reported to interact with the PDGF receptor (PDGFR) and VEGFR-2 [13, 15, 40, 41]; as well as IRS-1, a cyto-plasmic signal transduction mediator of insulin and IGF receptors . Additionally, integrins α6β4 and α6β1 interact with ErB-2 receptor in human breast carcinoma epithelial cells upon EGF stimulation [43, 44]. An integrin-activating anti-α6 antibody promotes association between α6β4 and ErB-2, which, in turn, might be responsible for enhanced cell proliferation and invasion as a result of stimulation by the antibody . The most recent data also indicate that integrin α6β4 induces ErB-3 expression in breast carcinoma cells .
The mitogen VEGF regulates key steps in the initiation of angiogenesis such as endothelial cell activation, vascular permeability, trans-migration of inflammatory cells, endothelial migration, and proliferation . VEGFs are a family of secreted polypeptides which, similar to PDGF, has a highly conserved cysteine-knot structure that is responsible for receptor binding. VEGF-A, the founder member of the family, is generally described as VEGF or vascular permeability factor (VPF). Endothelial responses by VEGF are exerted through binding to two homologous membrane tyrosine kinase receptors present on the surface of endothelial cells: VEGFR-1, encoded by the gene Flt1, and VEGFR-2, encoded by Flk1/KDR . On endothelial cells, neuropilin is another receptor for VEGF that increases the affinity of VEGFR-2 for VEGF . Gene knockout of VEGFR-1 and VEGFR-2 in mice results in lethality [49, 50] indicating that these receptors are essential for the angiogenesis process during embryogenesis. A recent study also shows that partial knockdown of VEGFR-1 regulates excessive angiogenesis in response to VEGFR-2 activation .
Intrinsic tyrosine kinase activity in VEGFRs is triggered by ligand-induced homodimerization and oligomerization. Although reports suggest the formation of heterodimers between VEGFR-1 and VEGFR-2 [6, 52], its role in endothelial cells is still ambiguous. Although VEGFR-1 knockout in mice results in embryonic lethality , knockouts expressing VEGFR-1 lacking its kinase domain possesses normal vasculature . In contrast, strong phosphorylation of tyrosine residues in VEGFR-2 has been observed upon homodimerization . Residues Y1175 and Y1214 have been described as major autophosphorylation sites on VEGFR-2 . Other residues such as Y951 and Y996 in the kinase insert domain and Y1054 and Y1059 in the kinase catalytic domain  are putative phosphorylation sites that may be important for VEGFR-2’s interaction with other molecules or receptors.
Recent studies demonstrate that the role of VEGFR-1 in postnatal angiogenesis is more complicated than initially recognized. Treatment with placental growth factor (PlGF), a VEGFR-1 specific ligand, promotes angiogenesis in vitro and in vivo . Over-expression of PlGF also results in enhanced angiogenesis in tumor and skin . It has been reported that PlGF stimulates oligomerization of VEGFR-1 and VEGFR-2, leading to trans-activation of VEGFR-2 and promotion of angiogenesis . Meantime, over-expression of PlGF in many cancer cell lines inhibited angiogenesis and tumor growth via inhibition of homodimerization of VEGFR-2 . A recent study also shows that partial knockdown of VEGFR-1 regulates excessive angiogenesis in response to VEGFR-2 activation .
During initiation of angiogenesis, endothelial cells sprouting out of existing blood vessels adhere to a provisional ECM rich in vitronectin and fibrinogen via integrin αvβ3 [15, 59]. Stimulation of endothelial cells with VEGF results in the formation of a complex between VEGFR-2 and integrin αvβ3 . No similar effect was observed for collagen receptor α2β1 and laminin receptors α6β1 and α6β4 [19, 20]. Moreover, collagen, a ligand for α2β1 and α1β1 integrins, exerted an inhibitory effect on VEGFR-2 . Endothelial cell adhesion to collagen reduced VEGF-induced VEGFR-2 phosphorylation by recruiting tyrosine phosphatase SHP2 to the phosphorylated tyrosine 1117 residue of VEGFR-2. One of the key functional consequences of an interaction between αvβ3 and VEGFR-2 is augmentation of integrin αvβ3 activity or its activation.
An anti-β3 monoclonal antibody BV4, which inhibits adhesion to vitronectin (prototypic ligand for αvβ3) decreased phosphorylation of VEGFR-2 suggesting an important function of αvβ3 in the activation of the angiogenic program in endothelial cells . In general, the blockade of αvβ3 results in diminished autophosphorylation of VEGFR-2 while integrin activation with LIBS-like activating antibodies or engagement by natural ligand promotes VEGFR-2 phosphorylation . Our recent study showed that VEGFR-2 forms a complex with β3 integrin subunit, but not with β1 or β5, when stimulated with VEGF [13, 14] (Fig. 1). This complex can be observed in vivo as well as in cell culture . Stimulation of endothelial cells by VEGF in the presence of ECM proteins induces phosphorylation of the β3 cytoplasmic tyrosine residues, which, in turn, is required for association between VEGFR-2 and β3 . Mutations of both tyrosines within αvβ3 cytoplasmic domain to phenylalanine abolish the complex formation between αvβ3 and VEGFR-2 resulting in diminished VEGFR-2 activation by VEGF . Other studies demonstrate that the extracellular but not the cytoplasmic domain of VEGFR-2 is crucial for integrin-VEGFR-2 cross signaling . Although the structural details of the complex between αvβ3 and VEGFR-2 remain unclear, its formation might be dependent on both the extracellular and cytoplasmic domains of the β3 subunit [14, 15, 34].
Once engaged by multivalent or immobilized ligands such as fibrin or fibrinogen, integrin αvβ3 is able to form clusters on the cell surface, an ability important for recruitment of additional components of this multireceptor complex that include VEGFR-2 and PI3K [61, 62]. Illustration of the impact of αvβ3 integrin clustering on this complex was demonstrated using anti-β3 antibody, which interferes with αvβ3 clustering but not with cell adhesion to ECM . In addition to perturbing the formation of the complex, this antibody markedly inhibited VEGFR-2-mediated phosphorylation, PI3 kinase activation, focal adhesion dynamics, and migration of endothelial cells in response to VEGF. And vice versa, the clustering of integrins might promote certain cellular responses . The complex between VEGFR-2 and αvβ3 has also been implicated in mediating the pro-angiogenic effects of Factor XIII (FXIII) of the coagulation cascade . This activated transglutaminase crosslinks integrin β3 to VEG-FR-2, thus enhancing their interaction and resulting in complex formation . Interestingly, VEGFR-2 is not the only VEGF receptor known to interact with integrins. Stimulation of VEGFR-3 by VEGF-C or VEGF-D results in selective association between VEGFR-3 and integrin α5β1 in lymphatic endothelial cells and this interaction appears to be involved in the regulation of lymphangiogenesis [66, 67].
Research during the last decade has resulted in the development and characterization of a number of knockout mouse models related to cardiovascular biology. This has provided important insights into the importance of integrins and growth factor receptors, associating to transduce downstream signals to regulate vascular development in a number of angiogenesis dependent processes. For example, in mice the lack of fibronectin or integrin α5β1 results in embryonic lethality, indicating the importance of this integrin and its ligand in embryonic vasculogenesis . This was further supported by studies involving gene ablation of integrin α5 in mice, where endothelial structure formation is impaired in embryoid bodies [68, 69]. In combination, these studies indicate the importance of fibronectin receptor in embryonic angiogenesis. In contrast, although integrin αv gene knockout in mice is lethal , ablation of integrins β5 and/or β3 results in normal development of embryo and normal postnatal angiogenesis . Both integrins β5 and/or β3 null mice have extensive tumor induced angiogenesis in vivo . Since much of the other vasculature development appears to be normal, it clearly indicates that a compensatory mechanism does exist in these knockout mice. Endothelial cells in integrins β5 and/ or β3 mouse vasculature express higher amounts of VEG-FR-2, thus demonstrating a possible compensatory mechanism. In addition, this effect also indicates a close association between VEGFR-2 and integrin αvβ3 in endothelial cell activation and angiogenesis. A true function of integrin αvβ3 has been revealed by a knockin mouse model (DiYF mice) with two integrin β3 cytoplasmic tyrosine residues mutated to phenylalanine (see below) . In this case, pathological angiogenesis induced by tumor implantation is significantly reduced in DiYF mice compared to WT. Besides this, study also reveals that mutations in two integrin β3 cytoplasmic tyrosine residues block the formation of complex between VEGFR-2 and integrin upon stimulation with VEGF. Another study reports that although mice lacking integrin β5 develop normal vasculature, these mice have specific VEGF-induced vascular permeability defects . Collectively, studies in these transgenic mice clearly demonstrate the importance of association between VEGFR-2 and integrin αvβ3 or integrin αvβ5 in VEGF induced angiogenic responses.
The Src family of kinases is a group of non-receptor tyrosine kinases that are activated both by activation of growth factor receptors and integrins through ‘outside–in signaling’ . Major members of this family include cSrc, Fyn, Lyn, and Yes . Many previous reports have suggested the Src family of kinases as potential regulators of integrin-growth factor receptor association in vascular cells . Src has been reported to interact with EGFR to regulate cell proliferation , and in association with PDGFR, it induces integrin dependent cell adhesion and migration . A functional overlap between the members in the same cell types is evident from specific knockout studies in mice. Although cSrc null mice develop normal blood vessels, they exhibit osteoporosis and impaired vascular permeability in response to VEGF [78, 79]. Interestingly, mice deficient in Yes also exhibit defects in VEGF induced vascular permeability , suggesting non-redundant functions of these two Src kinases in the regulation of vascular permeability. In contrast, Fyn-deficient mice do not exhibit a defect in vascular leakage induced by VEGF . Mice lacking Src kinases cSrc, Yes, and Fyn (SYF) develop blood-filled islands in the embryo leading to lethality . These reports demonstrate that, if not all, many of the functional requirements of the Src family of tyrosine kinases are compensated for in the absence of one. However, together, they are absolutely necessary for the normal development of embryonic and post-natal vasculature.
A recent study in our lab has demonstrated the non-redundant function of cSrc in vascular cells in the regulation of integrin αvβ3 and VEGFR-2 complex formation via inside–out signaling . Phosphorylation by VEGF stimulation of both Y747 and Y759 in the integrin β3 cytoplasmic domain was observed to be enhanced when endothelial cells are plated on vitronectin compared to other ECM proteins. Antibodies that specifically block the function of αv or β3, but not β1 or β5, inhibited VEGF stimulated tyrosine phosphorylation of both integrin β3 and VEGFR-2. Immunoprecipitation analysis revealed that stimulation with VEGF results in complex formation between VEGFR-2 and integrin β3 and not with β1 or β5 . Treatment with Src inhibitors completely blocked VEGF mediated complex formation between VEGFR-2 and integrin β3 (Fig. 1). At basal activity, only Yes is associated with integrin β3 and treatment with VEGF had no effect on modulating its interaction with integrin β3. In contrast, cSrc interaction with integrin β3 was enhanced upon VEGF stimulation. Fyn did not interact with integrin β3 in either of the conditions. These data suggested the non-overlapping role of cSrc in the regulation of VEGFR-2 and integrin β3 association.
Further analysis revealed the importance of Src kinases in the phosphorylation of integrin β3 cytoplasmic tyrosine residues . Phosphorylation of integrin β3 was significantly reduced in SYF null mice. While treatment with Src inhibitor or expression with dominant negative cSrc inhibited phosphorylation of integrin β3 cytoplasmic tyrosine residues, expression with the constitutively active form of cSrc resulted in enhanced phosphorylation of both Y747 and Y759. Most importantly, in vitro phosphorylation assay demonstrated that recombinant cSrc phosphorylates integrin β3 at the cytoplasmic tyrosine residues. Specific inactivation of cSrc results in inhibition of complex formation between VEGFR-2 and integrin β3 resulting in impaired binding to the specific αvβ3 ligand vitronectin, but not to other integrin ligands like collagen and laminin. These facts point out the direct and specific involvement of c-Src in the regulation of αvβ3 interaction with ECM. These induced structural changes caused by β3 phosphorylation promoted signaling that resulted in pre-capillary tube formation and enhanced chemotaxis upon VEGF stimulation.
Studies demonstrating the importance of cSrc in regulating the association between VEGFR-2 and integrin β3 indicated the importance of integrin β3 tyrosine residues in their complex formation. This report was further strengthened by the data derived from knockin mice, where the two tyrosine residues Y747 and Y759 are mutated to phenylalanine . In the DiYF (two Y to F mutations in the integrin β3 cytoplasmic domain) knockin mouse model, phosphorylation of VEGFR-2 in response to VEGF is impaired. DiYF mutations in integrin β3 cytoplasmic domain also resulted in the blockade of complex formation between integrin β3 and VEGFR-2.
Reduced interaction between VEGFR-2 and integrin β3, impaired VEGF signaling and reduced VEGFR-2 phosphorylation in DiYF endothelial cells resulted in their impaired adhesion and migration on vitronectin and entactin, two major αvβ3 ligands in the tissues and basement membrane, respectively . In vivo, tumor angiogenesis was impaired in DiYF mice resulting in reduced tumor growth . Overall, these studies in DiYF knockin mice reveal the importance of the two tyrosine residues in integrin β3 in the formation of complex with VEGFR-2, which then results in conformational activation via inside–out signaling, which enhances its affinity for the ECM substrate, vitronectin.
Interaction between VEGFR-2 and αvβ3 integrin regulates VEGF signaling and interactions of endothelial cells with vitronectin, fibrin, and entactin/nidogen [14, 84]. However, pro-angiogenic activities are not limited to VEGF and these selected components of matrix. Originally, it had been shown that neutralizing antibody against αvβ3 integrin diminished bFGF stimulated vascular survival and endothelial cell migration, suggesting the cooperation between FGF-RTK and integrin αvβ3 in angiogenesis . Also, similar to VEGF, stimulation of endothelial cells with bFGF might also promote activation of αvβ3 . However, the molecular mechanisms underlying the cooperation between these receptors are not yet elucidated, with the exception of a study showing the importance of endothelial cell engagement by fibrinogen during complex formation of FGF-RTK and αvβ3 upon bFGF stimulation . In addition, FGF receptor-3 has also been implicated to interact with various types of integrins .
Besides VEGFR and FGF-R, a third major receptor tyrosine kinase that has been implicated in the regulation of endothelial function and angiogenesis is Tie-2 receptor, which binds to its ligands Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2) . Both angiopoietins interact with the same receptor, Tie-2, and reciprocally regulate vascular stability . While Ang-1 is essential for the maturation of blood vessels and reduction in vascular permeability, thereby stabilizing the newly formed blood vessels , Ang-2, competes with Ang-1 for the Tie-2 receptor and enhances vascular permeability and destabilization of blood vessels , which is a prerequisite for VEGF mediated vascular permeability and extravasation .
A recent report suggests that in order for Ang-1 to regulate vascular stabilization, its receptor Tie-2 has to be associated with integrin α5β1  (Fig. 2). Engagement of integrin α5β1 by fibronectin increases integrin interaction with Tie-2 in a time and concentration dependent manner. This results in Tie-2 phosphorylation in response to threshold levels of Ang-1. While in complex with integrin α5β1, Tie-2 activity is sustained for longer than an hour compared to transient effects upon Ang-1 stimulation in the absence of fibronectin. Thus, similar to αvβ3 and VEGFR-2, there is a functional alliance between integrin α5β1 and Tie-2 receptor (Fig. 2). In endothelial cells attached to fibronectin, Ang-1 stimulates recruitment of p85 to Tie-2  and FAK to the cytoplasmic tails of integrin α5β1  within focal adhesions. This complex formation between Tie-2 and integrin α5β1 appears to involve the activation of PI-3 kinase signaling downstream of FAK activation [88, 94]. In addition to its interaction with integrin α5β1, FAK is also reported to regulate vascular permeability in response to VEGF activation involving integrin αvβ5 . Moreover, another report shows that Ang-1 can directly bind to integrin α5β1 even in the absence of Tie-2 receptor . Overall, these studies show that Tie-2 and integrin α5β1 form complexes in order to regulate vascular stabilization.
In addition to the major tyrosine kinase receptors discussed above, angiogenesis is also under the tight control of many pleiotropic molecules such as hepatocyte growth factor (HGF), which interacts with and activates Met, a receptor tyrosine kinase . A number of in vitro and in vivo models demonstrate that HGF activates Met on the endothelial cell surface and promotes angiogenesis . Importantly, HGF regulates matrix recognition by endothelial integrins . HGF, largely synthesized and secreted by platelets, forms hetero-complexes with vitronectin and fibronectin, two important ligands for integrins αvβ3 and α5β1 . These hetero-complexes, VN/HGF and FN/HGF, trigger association between Met and integrins αvβ3 and α5β1, respectively  (Fig. 3). As a result, autophosphorylation of Met in the presence of ECM is enhanced compared to that in the absence of VN or FN. Moreover, another report demonstrates that complex formation between Met and integrin α6β4, a laminin receptor , is necessary for the regulation of tumor angiogenesis , thus demonstrating the importance of Met/integrin α6β4 association in pathological angiogenesis.
Semaphorins (Sema) are a family of secreted and membrane bound cytokines that signal through four classes of plexins (type A–D), a family of membrane receptors characterized by the presence in their cytosolic tail, two domains with homology to the R-Ras GTPase activating proteins (GAPs), separated by a linker region that can bind other small GTPases, such as Rnd-1 and Rac1 [19, 20]. Sema of secreted class 3 group also employ Neurophilin (Nrp-1 or -2) as co-receptors in association with type A or type D plexins [19, 20]. Nrp-1 was initially shown to act as a VEGFR-2 co-receptor with implications in cardiovascular development [48, 102–104]. Observations in vitro and in vivo that endothelial cells exhibit autocrine loops of several Sema 3 other than Sema 3A , suggest that multiple Sema 3 could cooperate to regulate angiogenesis. In endothelial cells, plexinD1 and to a lesser extend plexinA2, are the most abundant plexins, which possesses significantly higher affinities for Sema3A and Sema3C .
At the level of endothelial cells, both Sema3A and Sema3F have shown to inhibit adhesion and migration via inhibition of integrins . This was later confirmed in other cell types as well . In contrast, Sema3C, which possesses an Arg-Gly-Asp (RGD) motif, has been reported to serve as a ligand for integrins and promote endothelial cell adhesion and migration . Endothelial cell adhesion and migration is also regulated by Sema4D via PlexinB1 stimulation and suppression of R-Ras activity, which in turn inhibits integrin β1 activation . Sema4D thus promotes association between PlexinB1 and Met tyrosine kinase receptor and prevents interaction between integrin β1 and Met . Collectively, these reports suggest that semaphorins, via interaction with multiple growth factors, might inhibit growth factor stimulated activation of integrins and cross-talk.
The complex ECM, which has a very high affinity for many soluble growth factors, has been demonstrated to act as sequestering machinery for growth factors such as TGFβ . Recent reports demonstrate that many pro- and anti-angiogenic molecules may be entrapped in the ECM and be directly recognized by integrins. Integrin α9β1 has been shown to recognize immobilized VEGF-A165 and VEGF-A121 [113, 114]. In response to these immobilized growth factors, VEGF, VEGFR-2, and integrin α9β1 form a complex and transduce signals in an adaptive manner . The same integrin had later been shown to interact with VEGF-C, VEGF-D, and HGF, and the lack of these interactions might explain the abnormal lymphatic development in integrin α9 knockout mice . Additionally, integrins α3β1 and αvβ3 have been reported to interact with VEGF-A165 and VEGF-A189, but not with VEGF-A121 . A recent report indicates that bFGF might also directly bind to integrin αvβ3 and regulate bFGF-induced endothelial activation and angiogenesis involving ERK and Akt activation . Detailed information on interaction between angiogenic modulators and integrins is provided in Table 2.
Integrins are also known to partially mediate pro-angiogenic effects of Ang-1 and Ang-2 via direct interactions . The region of Ang-1 and -2 that binds to integrins is located within the fibrinogen-like domain (QHREDGS), which resembles the integrin recognition motifs KRLDGS or REDV of fibrinogen and fibronectin, respectively [93, 118–120]. Both endothelial cells and fibroblasts adhere to Ang-1 and Ang-2. However, only Ang-2 is able to promote cell spreading and cytoskeletal remodeling . Ang-1 has been reported to serve as an important ligand for integrin α5β1 in the absence of Tie-2, a well characterized receptor for both Ang-1 and -2 . These observations combined with the cooperation between Tie-2 and integrin α5β1 discussed above indicate that Ang-1 has the potential to trigger both inside–out and outside–in signals. In many cell types lacking Tie-2, Ang-1 and -2 interact directly with integrins. In cardiomyocytes integrins α2β1, α5β1, α6β1, α6β4, αvβ1, and αvβ3 have been shown to bind to Ang-1, resulting in Akt activation and cell survival . Glioma cells have also been reported to attach to Ang-2 using many of these receptors, with the exception for αvβ1 . Breast cancer epithelial cells bind to Ang-2 and promote migration involving integrin α5β1 . Altogether, these reports indicate that direct interaction between integrins and Ang-1 and -2 modulate the process of angiogenesis.
Proteolytic degradation of ECM results in the release of a number of anti-angiogenic molecules that have the ability to bind and inactivate endothelial integrins, thus resulting in inhibition of endothelial tube formation and angiogenesis . While ECM ligands interact with and activate integrins on the endothelial cell surface, thus protecting the cells from anoikis , integrins engaged by the ECM fragments after proteolytic degradation induce apoptosis and inhibit proliferation and migration of endothelial cells . One such molecule is endostatin, which is a C-terminal non-collagenous domain of type XVIII collagen that exerts inhibitory effects on integrin α5β1 through an Argrich peptide at its N-terminus . Proteolytic degradation of non-collagenous domain of the α-chain of type IV collagen results in three different anti-angiogenic molecules namely tumstatin, arresten, and canstatin . Among these, tumstatin binds to integrin αvβ3 and promotes apoptosis, as a result of inhibition of FAK, Akt, and mTOR signaling [127, 128]. Arresten, generated from the non-collagenous domain of the α1 chain of type IV collagen, competes with collagen IV binding to integrin α1β1 thus inhibiting endothelial cell interaction with collagen and vitronectin [21, 129]. Canstatin, derived from the α2 chain of type IV collagen, binds to integrin αvβ3 and αvβ5 resulting in endothelial cell activation of caspases and promotion of endothelial cell apoptosis . Altogether, these reports indicate that ECM can promote and inhibit endothelial integrin activity, which is dependent on its intact or fragmented nature.
Angiostatin is another known anti-angiogenic molecule that is proteolytically derived internal fragment of serine protease plasminogen. This contains various members of the five plasminogen “kringle” domains, depending on the sites of proteolysis. Different forms of angiostatin exhibit different activities. A number of groups have sought to identify the native cell surface binding site(s) for angiostatin, resulting in at least five different binding sites proposed for angiostatin on the surface of endothelial cells . Angiostatins have been reported to bind to integrin αvβ3 thereby inhibiting angiogenesis .
Anti-angiogenic therapy is emerging as an effective tool for the treatment of many different types of cancers. Modulation of angiogenesis can also be useful for other angiogenesis-dependent anomalies such as ischemia, myocardial infarction, and post-surgery wound complications as well as physiological processes such as embryogenesis. These associations that regulate the assembly and remodeling of ECM by the vascular cells are also necessary for the integrity and remodeling of injured tissue. Many of the current day methods for targeting angiogenesis are via inhibitors and monoclonal antibodies against integrins, growth factors and growth factor receptors. Many of these therapeutic approaches have suffered major setbacks, mainly due to the uncertainties in the complex signaling networks these receptors regulate in vascular cells and the injured tissue. A complete analysis of the signaling pathways regulating the complex association between multiple growth factors, their receptors and integrins will be extremely useful in order to improve the existing methods of targeting angiogenesis involving these receptors. Hence, further validation of existing hypotheses in the mechanistic aspects of integrin-growth factor receptor association by the next generation of researchers is essential.