Vascular cells express numerous integrins that are able to interact with a wide range of ligands.
ECM ligand associated signals synergize with growth factor stimulation thanks to the co-clustering of their receptors on the cell surface.
Direct interactions between growth factor receptors and integrins.
Proof of ανβ3/VEGFR-2 interaction
ανβ3 is expressed at low levels on quiescent endothelial cells in vivo, but is significantly elevated during angiogenesis, thus suggesting a role in angiogenic process.
To examine the role of cell adhesion on VEGFR-2 activation, the phosphorylation of the receptor was evaluated in adherent or suspended endothelial cells treated with VEGF-A. Cells were lysed and immunoprecipitated with anti-VEGFR-2 Ab, then immunoprecipitate was analyzed by SDS–PAGE followed by immunoblotting with anti-phosphotyrosine mAb. The results show that the phosphorylation of VEGFR-2 is enhanced in adherent cells. In suspended cells, the effect of VEGF-A165 is markedly lower.
Subsequent experiments have been performed to test whether a specific matrix protein has a role in VEGFR-2 activation. Endothelial cells stimulated with VEGF-A165 were plated on vitronectin, fibronectin or collagen I, which are the ligands for αvβ3, α5β1 and α2β1, respectively. VEGFR-2 is more phosphorylated in VEGF-A165-stimulated endothelial cells plated on vitronectin, than in cells plated on fibronectin or collagen, suggesting a specific involvement of avb3 integrin in the activation of VEGFR-2.
Next it was evaluated whether mAbs raised against integrin subunits β3 and αv could interfere with the VEGFR-2 phosphorylation stimulated by VEGF-A165. Confluent and quiescent endothelial cells were preincubated with mAbs against αv, β3 and β1 integrin subunits, washed and then stimulated with VEGF-A165. Cell lysates were immunoprecipitated by anti-VEGFR-2 Ab, and the proteins separated by SDS–PAGE were probed with anti-phosphotyrosine mAb. Anti-αv and anti-β3 mAbs inhibited the VEGFR-2 phosphorylation, but anti-β1 mAb was ineffective. Thus, these experiments indicate that αvβ3 integrin, but not b1, is crucial in mediating tyrosine phosphorylation of VEGFR-2.
Soldi R., The Embo Journal, 1999.
The interaction between ανβ3 and VEGFR-2 was evaluated in CHO cells transfected with integrin subunits and VEGFR-2. The β3 integrin was precipitated with antibodies directed against its cytoplasmic tail, and the immunoprecipitates were probed for the presence of the RTKs by immunoblotting. VEGFR-2 could only be detected in anti- β3 precipitates from cells that had been transfected with the αν and β3 subunits, thus indicating that ανβ3 and VEGFR-2 coimmunoprecipitates.
Borges E., The Journal Of Biological Chemistry, 2000.
Mouse models
Pharmacological model
Blockade of αvβ3 integrin with monoclonal antibodies or low-molecular-weight antagonists inhibits blood vessel formation in a variety of in vivo models, including tumor angiogenesis and neovascularization during oxygen-induced retinopathy. Taken together, these inhibition data suggest critical roles for αvβ3 in angiogenesis, and highlight its importance as potential target in anti-angiogenic therapy.
Knock-out model
In contrast with these inhibitor studies, mice lacking αv or β3 integrins exhibit extensive developmental angiogenesis and enhanced tumor growth. β3-null mice show enhanced VEGF-induced angiogenic responses, due to the elevated VEGFR-2 levels observed in β3-deficient endothelial cells.
Reynolds L.E., Nature Medicine, 2002
Knock-in model
To elucidate the role of β3 integrin and its complexes, mice that express a mutant form of β3 integrin, but not the WT form, were characterized. In the mutant, the two tyrosine residues known to be involved in integrin signaling, Tyr747 and Tyr759, were substituted to phenylalanines. The mutant is unable to undergo phosphorylation of cytoplasmic domain, resulting in deficient integrin signaling. In these mice, named DiYF, the mutant β3 integrin is physically present on all cell types, which should prevent any developmental compensatory changes. Frequently, the loss of a particular protein results in increased expression of other members of the same family, revealing the functional redundancy.
Lack of β3integrin phosphorylation resulted in impaired angiogenic response. As a result of reduced vascularization, tumor growth was significantly inhibited. At the same time, DiYF mutation did not affect embryogenesis, organogenesis, and overall vascular development, indicating that integrin phosphorylation is crucial for pathological but not normal vascularization.
Mahabeleshwar G. H., The Journal Experimental Medicine, 2006.
VEGFR-2/ανβ3 complex formation
During angiogenesis, endothelial cells sprouting out of existing blood vessels adhere to a provisional ECM rich in vitronectin via integrin ανβ3. Upon VEGF-A stimulation, VEGFR-2 recruits and activates c-src, which phosphorylates the cytosolic tail of β3 integrin. This post-translational modification promotes the formation of the VEGFR-2/ ανβ3 complex, that increases the integrin affinity for ECM and the VEGFR-2 responsiveness to VEGF.
Serini G., Cardiovascular Research, 2008.
Payaningal R, Angiogenesis, 2009.
In ECs from DiYF mice, VEGF-induced functional responses (cell adhesion, spreading, migration, and capillary tube formation) were defective compared with WT. Lack of β3integrin lead to disruption of the VEGFR-2/ανβ3 complex and lack of VEGFR-2 phosphorylation in response to VEGF. Furthermore, VEGF induced integrin activation (inside-out signaling) was suppressed.
Since VEGF (via VEGFR-2 as its major functional receptor on ECs) induces phosphorylation of ανβ3 and phosphorylation of ανβ3, in turn, is required for complete and sustained phosphorylation of VEGFR-2, it can be concluded that these two receptors are able to cross activate each other in ECs, therefore forming a functional partnership that is essential for successful angiogenesis. ανβ3 and its inside-out and outside-in signaling is essential for pathological angiogenesis and undoubtedly represents a promising target for pharmaceutical approaches. However, when developing new inhibitors for angiogenesis, one should take into consideration the complexity of ανβ3 regulation, its interactions with other rec
