Interaction between integrin αvβ3 and extracellular matrix is crucial for endothelial cells sprouting from capillaries and for angiogenesis. Furthermore, integrin‐mediated outside‐in signals co‐operate with growth factor receptors to promote cell proliferation and motility. To determine a potential regulation of angiogenic inducer receptors by the integrin system, we investigated the interaction between αvβ3 integrin and tyrosine kinase vascular endothelial growth factor receptor‐2 (VEGFR‐2) in human endothelial cells. We report that tyrosine‐phosphorylated VEGFR‐2 co‐immunoprecipitated with β3 integrin subunit, but not with β1 or β5, from cells stimulated with VEGF‐A165. VEGFR‐2 phosphorylation and mitogenicity induced by VEGF‐A165 were enhanced in cells plated on the αvβ3 ligand, vitronectin, compared with cells plated on the α5β1 ligand, fibronectin or the α2β1 ligand, collagen. BV4 anti‐β3 integrin mAb, which does not interfere with endothelial cell adhesion to vitronectin, reduced (i) the tyrosine phosphorylation of VEGFR‐2; (ii) the activation of downstream transductor phosphoinositide 3‐OH kinase; and (iii) biological effects triggered by VEGF‐A165. These results indicate a new role for αvβ3 integrin in the activation of an in vitro angiogenic program in endothelial cells. Besides being the most important survival system for nascent vessels by regulating cell adhesion to matrix, αvβ3 integrin participates in the full activation of VEGFR‐2 triggered by VEGF‐A, which is an important angiogenic inducer in tumors, inflammation and tissue regeneration.

The bone morphogenic protein antagonist gremlin is expressed during embryonic development and under different pathologic conditions, including cancer. Gremlin is a proangiogenic protein belonging to the cystine-knot superfamily that includes transforming growth factor-β proteins and the angiogenic vascular endothelial growth factors (VEGFs). Here, we demonstrate that gremlin binds VEGF receptor-2 (VEGFR2), the main transducer of VEGF-mediated angiogenic signals, in a bone morphogenic protein–independent manner. Similar to VEGF-A, gremlin activates VEGFR2 in endothelial cells, leading to VEGFR2-dependent angiogenic responses in vitro and in vivo. Gremlin thus represents a novel proangiogenic VEGFR2 agonist distinct from the VEGF family ligands with implications in vascular development, angiogenesis-dependent diseases, and tumor neovascularization.

The chromosomal high mobility group box-1 (HMGB1) protein acts as a proinflammatory cytokine when released in the extracellular environment by necrotic and inflammatory cells. In the present study, we show that HMGB1 exerts proangiogenic effects by inducing MAPK ERK_1/2 activation, cell proliferation, and chemotaxis in endothelial cells of different origin. Accordingly, HMGB1 stimulates membrane ruffling and repair of a mechanically wounded endothelial cell monolayer and causes endothelial cell sprouting in a three-dimensional fibrin gel. In keeping with its in vitro properties, HMGB1 stimulates neovascularization when applied in vivo on the top of the chicken embryo chorioallantoic membrane whose blood vessels express the HMGB1 receptor for advanced glycation end products (RAGE). Accordingly, RAGE blockade by neutralizing Abs inhibits HMGB1-induced neovascularization in vivo and endothelial cell proliferation and membrane ruffling in vitro. Taken together, the data identify HMGB1/RAGE interaction as a potent proangiogenic stimulus.

In vivo IL-12-dependent tumor inhibition rests on the ability of IL-12 to activate a CD8-mediated cytotoxicity, inhibit angiogenesis, and cause vascular injury. Although in vivo studies have shown that such inhibition stems from complex interactions of immune cells and the production of IFN-γ and other downstream angiostatic chemokines, the mechanisms involved are still poorly defined. Here we show that IL-12 activates an anti-angiogenic program in Con A-activated mouse spleen cells (activated spc) or human PBMC (activated PBMC). The soluble factors they release in its presence arrest the cycle of endothelial cells (EC), inhibit in vitro angiogenesis, negatively modulate the production of matrix metalloproteinase-9, and the ability of EC to adhere to vitronectin and up-regulate ICAM-1 and VCAM-1 expression. These effects do not require direct cell-cell contact, yet result from continuous interaction between activated lymphoid cells and EC. We used neutralizing Abs to show that the IFN-inducible protein-10 and monokine-induced by IFN-γ chemokines are pivotal in inducing these effects. Experiments with nu/nu mice, nonobese diabetic-SCID mice, or activated spc enriched in specific cell subpopulations demonstrated that CD4⁺, CD8⁺, and NK cells are all needed to mediate the full anti-angiogenetic effect of IL-12.