Raffaella Soldi

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.

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.

Actions of molecules which regulate hemopoiesis on endothelial cells: memoirs of common ancestors?

The proliferation and differentiation of hematopoietic stem cells (hematopoiesis) takes place in close contact with stromal cells and matrix in bone marrow. Hematopoiesis requires cytokines, collectively termed colony stimulating factors (CSFs), which act on progenitor cell populations and induce their commitment to a specific lineage. For instance, leukemia, inhibitor factor and stem cell factor act on pluripotent cells and immature progenitors, granulocyte-macrophage colony stimulating factor (GM-CSF) acts at early stages of the development of myelomonocytic lineage, whereas granulocyte-colony stimulating factor (G-CSF) and macrophage-colony stimulating factor (M-CSF) act on more mature cells of the same lineage and are only required later during the differentiation of this cell lineage. A second important element for the hematopoietic process is the presence of extracellular matrix proteins, which bind CSFs and correctly present the molecules to specific receptors present on the surface of the progenitor cells. Finally, stromal cells (i.e. fibroblasts, endothelial cells and adipocytes) which support the growth of hematopoietic stem cells in vitro, are crucial for the production of CSFs and protein matrix and regulate the passage of mature cells from bone marrow to bloodstream. Idiopathic myelofibrosis is an example of the relevance of microenvironment in hematopoiesis. This disease is characterized by fibroblast and basement membrane accumulation, appearance of myofibroblasts and modification of the capillary network and provokes a bone marrow aplasia. In this article we review recent studies on the role of hemopoietic cytokines on stromal cells, in particular on endothelial cells, and propose a double role for CSFs in hematopoiesis: to induce the commitment of progenitor cells and to maintain the behavior of bone marrow endothelial cells.