Stefania Mitola

Role of alphavbeta3 integrin in the activation of vascular endothelial growth factor receptor-2.

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.

Cutting Edge: Extracellular High Mobility Group Box-1 Protein Is a Proangiogenic Cytokine

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 ERK1/2 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.

IL-12 Inhibition of Endothelial Cell Functions and Angiogenesis Depends on Lymphocyte-Endothelial Cell Cross-Talk

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.

Gremlin is a novel agonist of the major proangiogenic receptor VEGFR2.

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.

Identification of Specific Molecular Structures of Human Immunodeficiency Virus Type 1 Tat Relevant for Its Biological Effects on Vascular Endothelial Cells

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Tat transactivates viral genes and is released by infected cells, acting as a soluble mediator. In endothelial cells (EC), it activates a proangiogenic program by activating vascular endothelial growth factor receptor type 2 (VEGFR-2) and integrins. A structure-activity relationship study was performed by functional analysis of Tat substitution and deletion variants to define the Tat determinants necessary for EC activation. Variants were made (i) in the basic and (ii) in the cysteine-rich domains and (iii) in the C-terminal region containing the RGD sequence required for integrin recognition. Our results led to the following conclusions. (i) Besides a high-affinity binding site corresponding to VEGFR-2, EC express low-affinity binding sites. (ii) The basic and the cysteine-rich variants bind only to the low-affinity binding sites and do not promote tyrosine phosphorylation of VEGFR-2. Furthermore, they have a reduced ability to activate EC in vitro, and they lack angiogenic activity. (iii) Mutants with mutations in the C-terminal region are partially defective for in vitro biological activities and in vivo angiogenesis, but they activate VEGFR-2 as Tat wild type. In conclusion, regions encoded by the first exon of tat are necessary and sufficient for activation of VEGFR-2. However, the C-terminal region, most probably through RGD-mediated integrin engagement, is indispensable for full activation of an in vitro and in vivo angiogenic program.

Type I Collagen Limits VEGFR-2 Signaling by a SHP2 Protein-Tyrosine Phosphatase–Dependent Mechanism 1

During angiogenesis, a combined action between newly secreted extracellular matrix proteins and the repertoire of integrins expressed by endothelial cells contributes in the regulation of their biological functions. Extracellular matrix-engaged integrins influence tyrosine kinase receptors, thus promoting a regulatory cross-talk between adhesive and soluble stimuli. For instance, vitronectin has been reported to positively regulate VEGFR-2. Here, we show that collagen I downregulates VEGF-A–mediated VEGFR-2 activation. This activity requires the tyrosine phosphatase SHP2, which is recruited to the activated VEGFR-2 when cells are plated on collagen I, but not on vitronectin. Constitutive expression of SHP2C459S mutant inhibits the negative role of collagen I on VEGFR-2 phosphorylation. VEGFR-2 undergoes internalisation, which is associated with dynamin II phosphorylation. Expression of SHP2C459S impairs receptor internalisation suggesting that SHP2-dependent dephosphorylation regulates this process. These findings demonstrate that collagen I in provisional extracellular matrix surrounding nascent capillaries triggers a signaling pathway that negatively regulates angiogenesis.

Activation of diacylglycerol kinase alpha is required for VEGF-induced angiogenic signaling in vitro.

Vascular endothelial growth factor-A (VEGF-A) promotes angiogenesis by stimulating migration, proliferation and organization of endothelium, through the activation of signaling pathways involving Src tyrosine kinase. As we had previously shown that Src-mediated activation of diacylglycerol kinase-α (Dgk-α) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-α is involved in the transduction of angiogenic signaling. In PAE-KDR cells, an endothelial-derived cell line expressing VEGFR-2, VEGF-A165, stimulates the enzymatic activity of Dgk-α: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-α forms a complex. Conversely in HUVEC, VEGF-A165-induced activation of Dgk is only partially sensitive to R59949, suggesting that also other isoforms may be activated, albeit still dependent on Src tyrosine kinase. Specific inhibition of Dgk-α, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-α dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-α by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-α in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-α generates a signal essential for both proliferative and migratory response to VEGF-A165, suggesting that it may constitute a novel pharmacological target for angiogenesis control.

Human Immunodeficiency Virus Type 1 Tat Regulates Endothelial Cell Actin Cytoskeletal Dynamics through PAK1 Activation and Oxidant Production

ABSTRACT Human immunodeficiency virus type 1 Tat exerts prominent angiogenic effects which may lead to a variety of vasculopathic conditions in AIDS patients. Because endothelial cells undergo prominent cytoskeletal rearrangement during angiogenesis, we investigated the specific effects of Tat on the endothelial cell actin cytoskeleton. Glutathione S-transferase (GST)-Tat, at a level of 200 ng/ml (equivalent to 52 ng of Tat/ml), caused stress fiber disassembly, peripheral retraction, and ruffle formation in human umbilical vein endothelial cells (HUVEC) and human lung microvascular endothelial cells. At 600 ng of GST-Tat/ml (157 ng of Tat/ml), actin structures were lost, and severe cytoskeletal collapse occurred. In contrast, GST-Tat harboring mutations within either the cysteine-rich or basic domains exerted minimal effects on the endothelial cytoskeleton. HUVEC expressing a DsRed-Tat fusion protein displayed similar actin rearrangements, followed by actin collapse, whereas neighboring nontransfected cells retained normal actin structures. Because active mutants of p21-activated kinase 1 (PAK1) induce identical changes in actin dynamics, we hypothesized that Tat exerts its cytoskeletal effects through PAK1. GST-Tat activated PAK1 within 5 min, and adenovirus delivery of a kinase-dead PAK1 [PAK1(K298A)] completely prevented cytoskeletal collapse induced by GST-Tat or DsRed-Tat and also blocked downstream activation of c-Jun N-terminal kinase. Further, GST-Tat increased phosphorylation of the NADPH oxidase subunit p47phox and caused its rapid redistribution to membrane ruffles. PAK1(K298A) blocked p47phox phosphorylation, and interference with NADPH oxidase function through superoxide scavenging or through expression of a transdominant inhibitor, p67(V204A), prevented GST-Tat-induced alterations in the actin cytoskeleton. We conclude that Tat induces actin cytoskeletal rearrangements through PAK1 and downstream activation of the endothelial NADPH oxidase.

Interactions between endothelial cells and HIV-1

Endothelial cells (EC) participate in inflammatory and immune reactions by producing and responding to soluble mediators. Human immunodeficiency virus (HIV)-1 profoundly alters the features of EC. In some anatomical districts, they are infected by the virus and may represent a relevant reservoir. During lymphomononuclear cell diapedesis, EC activate virus replication in crossing cells. Direct or indirect damage of EC is particularly relevant in central nervous system, where blood-brain barrier perturbation is pivotal in neuronal degeneration. The observed alterations of EC adhesive properties contribute in altered leukocyte traffic from blood to lymphoid organs and tissues and play a role in the onset of immune surveillance alteration. These alterations of EC functions are relevant for the general vasculopathy, which marks the acquired immunodeficiency syndrome, and in particular are instrumental in the pathogenesis of Kaposis sarcoma. Here we discuss the biological and molecular activation of EC in HIV-1 infection that represents the basis to understand the pathogenesis of HIV-1 associated vascular diseases.

IL-12 Regulates an Endothelial Cell-Lymphocyte Network: Effect on Metalloproteinase-9 Production

IL-12 is key cytokine in innate immunity and participates in tumor rejection by stimulating an IFN-γ-mediated response characterized by CD8+ mediated-cytotoxicity, inhibition of angiogenesis, and vascular injury. We previously demonstrated that activated lymphocytes stimulated with IL-12 induced an angiostatic program in cocultured vascular endothelial cells. In this study, we have extended this observation showing that a reciprocal modulation of cellular responses occurs. Actually, the presence of endothelial cells enhanced the inhibitory effect of IL-12 on metalloproteinase-9 expression in activated PBMC as well as their ability to transmigrate across an extracellular matrix. IL-12 triggered intracellular signaling, as indicated by STAT-1 activation, appeared to mainly operative in activated CD4 + cells challenged with IL-12, but it was also initiated in CD8+ lymphocytes in the presence of endothelial cells. On the other hand, stimulated PBMC reduced the expression and the activity of metalloproteinase-9, up-regulated that of tissue inhibitor metalloproteinase-1, and stimulated the STAT-1 pathway in cocultured endothelial cells. We used neutralizing Abs to show that the IFN-inducible protein 10 (CXCL10) and monokine-induced by IFN-γ (CXCL9) chemokines produced by both PBMC and endothelial cells are pivotal in inducing these effects. Altogether these results suggest the existence of an IL-12-regulated circuit between endothelium and lymphocytes resulting in a shift of proteolytic homeostasis at site of tissue injury.