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Biomedical subjects

Federico Bussolino

Publications and source records attributed to Federico Bussolino.

At least 19 recordsLinked to original sources
PubMed2006

Comparative genome analysis of the neurexin gene family in Danio rerio: insights into their functions and evolution.

Neurexins constitute a family of proteins originally identified as synaptic transmembrane receptors for a spider venom toxin. In mammals, the 3 known Neurexin genes present 2 alternative promoters that drive the synthesis of a long (alpha) and a short (beta) form and contain different sites of alternative splicing (AS) that can give rise to thousands of different transcripts. To date, very little is known about the significance of this variability, except for the modulation of binding to some of the Neurexin ligands. Although orthologs of Neurexins have been isolated in invertebrates, these genes have been studied mostly in mammals. With the aim of investigating their functions in lower vertebrates, we chose Danio rerio as a model because of its increasing importance in comparative biology. We have isolated 6 zebrafish homologous genes, which are highly conserved at the structural level and display a similar regulation of AS, despite about 450 Myr separating the human and zebrafish species. Our data indicate a strong selective pressure at the exonic level and on the intronic borders, in particular on the regulative intronic sequences that flank the exons subject to AS. Such a selective pressure could help conserve the regulation and consequently the function of these genes along the vertebrates evolutive tree. AS analysis during development shows that all genes are expressed and finely regulated since the earliest stages of development, but mark an increase after the 24-h stage that corresponds to the beginning of synaptogenesis. Moreover, we found that specific isoforms of a zebrafish Neurexin gene (nrxn1a) are expressed in the adult testis and in the earliest stages of development, before the beginning of zygotic transcription, indicating a potential delivery of paternal RNA to the embryo. Our analysis suggests the existence of possible new functions for Neurexins, serving as the basis for novel approaches to the functional studies of this complex neuronal protein family and more in general to the understanding of the AS mechanism in low vertebrates.

Alternative Splicing↗
PubMed2006

Embryonic cleavage modeling as a computational approach to sphere packing problem.

Embryonic cleavage depends on the distribution in the cytosol of molecular signals that influence mitotic spindle positioning. By implementing the New Kind of Science (NKS) theory in which a simple rule can evolve with a complex overall behavior, here we propose a new theoretical approach that is capable of modeling the mechanisms of early embryonic cleavage dynamics in the ascidian Styela Partita. The model shows that the same spindle pole orientation rule governs the first five embryonic cleavages, which progressively allow the transition from one to thirty-two daughter cells. Finally, we present evidence of a strong similarity between Kepler's Sphere Packing Problem and embryonic cleavage, which thus represents a prominent example of natural computing.

Algorithms↗
PubMed2006

Loss of inhibitory semaphorin 3A (SEMA3A) autocrine loops in bone marrow endothelial cells of patients with multiple myeloma.

Vascular endothelial growth factor165 (VEGF165) and semaphorin3A (SEMA3A) elicit pro- and antiangiogenic signals respectively in endothelial cells (ECs) by binding to their receptors VEGFR-2, neuropilin-1 (NRP1), and plexin-A1. Here we show that the VEGF165-driven angiogenic potential of multiple myeloma (MM) ECs is significantly higher than that of monoclonal gammopathy of undetermined significance (MGUS) ECs (MGECs) and human umbilical vein (HUV) ECs. This is probably due to a constitutive imbalance of endogenous VEGF165/SEMA3A ratio, which leans on VEGF165 in MMECs but on SEMA3A in MGECs and HUVECs. Exogenous VEGF165 induces SEMA3A expression in MGECs and HUVECs, but not in MMECs. Moreover, by counteracting VEGF165 activity as efficiently as an anti-VEGFR-2 antibody, exogenous SEMA3A restrains the over-angiogenic potential of MMECs. Our data indicate that loss of endothelial SEMA3A in favor of VEGF165 could be responsible for the angiogenic switch from MGUS to MM.

Bone Marrow Cells↗
PubMed2005

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 SHP2(C459S) 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 SHP2(C459S) 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.

Collagen Type I↗
PubMed2005

Integrins and angiogenesis: a sticky business.

From an evolutionary point of view, the development of a cardiovascular system allowed vertebrates to nourish the several organs that compose their wider multicellular organism and to survive. Acquisition of new genes encoding for extracellular matrix (ECM) proteins and their cognate integrin receptors as well as secreted pro- and anti-angiogenic factors proved to be essential for the development of vascular networks in the vertebrate embryo. Postnatal tissue neo-vascularization plays a key role during wound healing and pathological angiogenesis as well. There is now clear evidence that building blood vessels in the embryo and in the adult organism relies upon different endothelial integrins and ECM ligands. A successful vascular development depends on fibronectin and its major receptor alpha5beta1 integrin, but not on alphavbeta3, alphavbeta5, and alpha6beta4 integrins that are instead central regulators of postnatal tumor angiogenesis. Here, endothelial alphavbeta3 elicits anti- or pro-angiogenic signals depending respectively on whether it is occupied by a soluble (e.g. type IV collagen derived tumstatin) or an insoluble (vitronectin) ECM ligand. The laminin-5 receptor alpha6beta4 integrin, expressed only by endothelial cells of mature blood vessels, controls the invasive phase of tumor angiogenesis in the adult organism. Finally, regulation of vascular morphogenesis relies upon the fine modulation of integrin activation by chemoattractant and chemorepulsive cues, such as angiogenic growth factors and semaphorins.

Animals↗
PubMed2005

Diffusion-limited phase separation in eukaryotic chemotaxis.

The ability of cells to sense spatial gradients of chemoattractant factors governs the development of complex eukaryotic organisms. Cells exposed to shallow chemoattractant gradients respond with strong accumulation of the enzyme phosphatidylinositol 3-kinase (PI3K) and its D3-phosphoinositide product (PIP(3)) on the plasma membrane side exposed to the highest chemoattractant concentration, whereas PIP(3)-degrading enzyme PTEN and its product PIP(2) localize in a complementary pattern. Such an early symmetry-breaking event is a mandatory step for directed cell movement elicited by chemoattractants, but its physical origin is still mysterious. Here, we propose that directional sensing is the consequence of a phase-ordering process mediated by phosphoinositide diffusion and driven by the distribution of chemotactic signal. By studying a realistic reaction-diffusion lattice model that describes PI3K and PTEN enzymatic activity, recruitment to the plasma membrane, and diffusion of their phosphoinositide products, we show that the effective enzyme-enzyme interaction induced by catalysis and diffusion introduces an instability of the system toward phase separation for realistic values of physical parameters. In this framework, large reversible amplification of shallow chemotactic gradients, selective localization of chemical factors, macroscopic response timescales, and spontaneous polarization arise naturally. The model is robust with respect to order-of-magnitude variations of the parameters.

Chemotaxis↗
PubMed2005

Gorham-Stout syndrome: a monocyte-mediated cytokine propelled disease.

UNLABELLED: We studied the biological features and the immunophenotype of a cell culture established from the lesion of soft tissues of a woman affected by Gorham-Stout syndrome. We found that these cells belonged to a monocytic lineage with some characteristics of immature osteoclasts and were able to release large amounts of osteoclastogenic and angiogenic molecules that may contribute to disease progression. INTRODUCTION: Gorham-Stout syndrome is a rare disease characterized by osteolysis and proliferation of vascular or lymphatic vessels, with a severe outcome. Its etiology and the identification of the cell types involved are completely unknown. MATERIALS AND METHODS: A cell culture from a lesion of soft tissues was established, and its behavior in vitro and in immunodeficient mice was studied. We analyzed (1) the cell phenotype by flow cytometry; (2) the adhesive and migratory properties on different substrates; (3) the ability to differentiate into mature osteoclasts; (4) the production of osteclastogenic and angiogenic molecules; (5) the in vivo angiogenic activity of the cells subcutaneously implanted in mouse in a Matrigel plug; and (6) the ability to recapitulate the disease when transplanted in nude mice. RESULTS AND CONCLUSIONS: The established culture consisted of a morphologically homogeneous cell population belonging to a monocytic lineage having some features of an osteoclast-like cell type. Cells had an invasive phenotype, were angiogenic, and produced osteoclastogenic (IL-6, TGF-beta1, IL-1beta) and angiogenic (vascular endothelial growth factor-A [VEGF-A], CXCL-8) molecules when challenged with inflammatory cytokines. Immunodeficient mice injected with these cells did not show any bone lesions or vascular alteration, but had high amounts of circulating human IL-6 and VEGF-A. Cells isolated from a cutaneous lymphangiomatosis did not show any of these findings. These data suggest that cells of monocyte-macrophage lineage play an essential role in the pathogenesis of Gorham-Stout disease, whose progression is propelled by cytokine circuits that accelerate angiogenesis and osteoclastogenesis.

Animals↗
PubMed2005

Stable interaction between alpha5beta1 integrin and Tie2 tyrosine kinase receptor regulates endothelial cell response to Ang-1.

During angiogenic remodeling, Ang-1, the ligand of Tie2 tyrosine kinase, is involved in vessel sprouting and stabilization through unclear effects on nascent capillaries and mural cells. In our study, we hypothesized that the Ang-1/Tie2 system could cross-talk with integrins, and be influenced by the dynamic interactions between extracellular matrix and endothelial cells (ECs). Here, we show that alpha5beta1 specifically sensitizes and modulates Tie2 receptor activation and signaling, allowing EC survival at low concentrations of Ang-1 and inducing persistent EC motility. Tie2 and alpha5beta1 interact constitutively; alpha5beta1 binding to fibronectin increases this association, whereas Ang-1 stimulation recruits p85 and FAK to this complex. Furthermore, we demonstrate that Ang-1 is able to mediate selectively alpha5beta1 outside-in FAK phosphorylation. Thus, Ang-1 triggers signaling pathways through Tie2 and alpha5beta1 receptors that could cross-talk when Tie2/alpha5beta1 interaction occurs in ECs plated on fibronectin. By using blocking antibodies, we consistently found that alpha5beta1, but not alphavbeta3 activation, is essential to Ang-1-dependent angiogenesis in vivo.

Angiopoietin-1↗
PubMed2005

Direct recruitment of CRK and GRB2 to VEGFR-3 induces proliferation, migration, and survival of endothelial cells through the activation of ERK, AKT, and JNK pathways.

Vascular endothelial growth factor receptor-3 (VEGFR-3) plays a key role for the remodeling of the primary capillary plexus in the embryo and contributes to angiogenesis and lymphangiogenesis in the adult. However, VEGFR-3 signal transduction pathways remain to be elucidated. Here we investigated VEGFR-3 signaling in primary human umbilical vein endothelial cells (HUVECs) by the systematic mutation of the tyrosine residues potentially involved in VEGFR-3 signaling and identified the tyrosines critical for its function. Y1068 was shown to be essential for the kinase activity of the receptor. Y1063 signals the receptor-mediated survival by recruiting CRKI/II to the activated receptor, inducing a signaling cascade that, via mitogen-activated protein kinase kinase-4 (MKK4), activates c-Jun N-terminal kinase-1/2 (JNK1/2). Inhibition of JNK1/2 function either by specific peptide inhibitor JNKI1 or by RNA interference (RNAi) demonstrated that activation of JNK1/2 is required for a VEGFR-3-dependent prosurvival signaling. Y1230/Y1231 contributes, together with Y1337, to proliferation, migration, and survival of endothelial cells. Phospho-Y1230/Y1231 directly recruits growth factor receptor-bonus protein (GRB2) to the receptor, inducing the activation of both AKT and extracellular signal-related kinase 1/2 (ERK1/2) signaling. Finally, we observed that Y1063 and Y1230/Y1231 signaling converge to induce c-JUN expression, and RNAi experiments demonstrated that c-JUN is required for growth factor-induced prosurvival signaling in primary endothelial cells.

Cell Movement↗
PubMed2005

Identification of CD36 molecular features required for its in vitro angiostatic activity.

Thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, acts directly on endothelial cells (EC) via CD36 to inhibit their migration and morphogenesis induced by basic fibroblast growth factor. Here we show that CD36 triggered by TSP-1 inhibits in vitro angiogenesis stimulated by vascular endothelial growth factor-A (VEGF-A). To demonstrate that the TSP-1 inhibitory signal was mediated by CD36, we transduced CD36 in CD36-deficient endothelial cells. Both TSP-1 and the agonist anti-CD36 mAb SMO, which mimics TSP-1 activity, reduced the VEGF-A165-induced migration and sprouting of CD36-ECs. To address the mechanisms by which CD36 may exert its angiostatic function, we investigated the functional components of the C-terminal cytoplasmic tail by site-directed mutagenesis. Our results indicate that C464, R467, and K469 of CD36 are required for the inhibitory activity of TSP-1. In contrast, point mutation of C466 did not alter TSP-1 ability to inhibit EC migration and sprouting. Moreover, we show that activation of CD36 by TSP-1 down-modulates the VEGF receptor-2 (VEGFR-2) and p38 mitogen-associated protein kinase phosphorylation induced by VEGF-A165, and this effect was specifically abolished by point mutation at C464. These results identify specific amino acids of the C-terminal cytoplasmic tail of CD36 crucial for the in vitro angiostatic activity of TSP-1 and extend our knowledge of regulation of VEGFR-2-mediated biological activities on ECs.

Amino Acid Sequence↗
PubMed2005

Involvement of chemokine receptor 4/stromal cell-derived factor 1 system during osteosarcoma tumor progression.

Despite intensive chemotherapy and surgery treatment, lung and bone metastasis develop in about 30% of patients with osteosarcoma. Mechanisms for this preferential metastatic behavior are largely unknown. We investigated the role of the chemokine receptor 4 (CXCR4)/stromal cell-derived factor 1 (SDF-1) system to drive the homing of osteosarcoma cells. We analyzed the expression of the CXCR4 and SDF-1 proteins on several osteosarcoma cell lines and the effects of SDF-1 on migration, adhesion, and proliferation of these cancer cells. In vitro assays showed that the migration of osteosarcoma cells expressing CXCR4 receptor follows an SDF-1 gradient and that their adhesion to endothelial and bone marrow stromal cells is promoted by SDF-1 treatment. Moreover, the production of matrix metalloproteinase-9 is increased after SDF-1 exposure. We finally proved in a mouse model our hypothesis of the CXCR4/SDF-1 axis involvement in the metastatic process of osteosarcoma cells. Development of lung metastasis after injection of osteosarcoma cells was prevented by the administration of a CXCR4 inhibitor, the T134 peptide. These data show a possible explanation for the preferential osteosarcoma metastatic development into the lung, where SDF-1 concentration is high, and suggest that molecular strategies aimed at inhibiting the CXCR4/SDF-1 pathway, such as small-molecule inhibitors or anti-CXCR4 antibodies, might prevent the dissemination of osteosarcoma cells.

Animals↗
PubMed2005

Sema4D induces angiogenesis through Met recruitment by Plexin B1.

Semaphorins, a large family of membrane-bound and secreted proteins, signal through their transmembrane receptors, the plexins. Semaphorins and plexins share structural homologies with scatter factor receptors, a family of tyrosine kinase receptors for which Met is the prototype. Semaphorins have been studied primarily in the developing nervous system, where they act as repelling cues in axon guidance. However, they are widely expressed in several tissues, and their role in epithelial morphogenesis has been recently established. Not much is known about their role in angiogenesis, a key step during embryonic development and adulthood. Here we demonstrate that a semaphorin, Sema4D, is angiogenic in vitro and in vivo and that this effect is mediated by its high-affinity receptor, Plexin B1. Moreover, we prove that biologic effects elicited by Plexin B1 require coupling and activation of the Met tyrosine kinase. In sum, we identify a proangiogenic semaphorin and provide insight about the signaling machinery exploited by Plexin B1 to control angiogenesis.

Animals↗
PubMed2004

Cell surface-associated Tat modulates HIV-1 infection and spreading through a specific interaction with gp120 viral envelope protein.

Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transactivator of viral gene expression, has the unusual property of being released by infected cells. Recent studies suggest that extracellular Tat is partially sequestered by heparan sulfate proteoglycans. As a consequence, Tat is concentrated on the cell surface and protected from proteolytic degradation, thus remaining in a biologically active form. We show that Tat binds the surfaces of both HIV-1-infected and surrounding uninfected cells. We provide evidence for a specific interaction between Tat and the HIV-1 glycoprotein 120 (gp120) envelope protein, which enhances virus attachment and entry into cells. We map the interacting sites of both Tat and gp120 and show that synthetic peptides mimicking the gp120 site inhibit HIV-1 infection. Our data demonstrate that membrane-associated Tat is a novel modulator of virus entry and suggest that the Tat-gp120 interaction represents a critical step in HIV-1 spreading during the course of infection.

Gene Products, tat↗
PubMed2004

Inhibition of vascular endothelial growth factor receptor 2-mediated endothelial cell activation by Axl tyrosine kinase receptor.

GAS6, the product of a growth arrest specific (GAS) gene, is the ligand of the tyrosine kinase receptor Axl. GAS6 and Axl are both expressed in endothelial cells, where they are involved in many processes such as leukocyte transmigration through capillaries and neointima formation in injured vessels. Here, we show that Axl stimulation by GAS6 results in inhibition of the ligand-dependent activation of vascular endothelial growth factor (VEGF) receptor 2 and the consequent activation of an angiogenic program in vascular endothelial cells. GAS6 inhibits chemotaxis of endothelial cells stimulated by VEGF-A isoforms, but not that triggered by fibroblast growth factor-2 or hepatocyte growth factor. Furthermore, it inhibits endothelial cell morphogenesis on Matrigel and VEGF-A-dependent vascularization of chick chorion allantoid membrane. GAS6 activates the tyrosine phosphatase SHP-2 (SH2 domain-containing tyrosine phosphatase 2), which is instrumental in the negative feedback exerted by Axl on VEGF-A activities. A dominant-negative SHP-2 mutant, in which Cys 459 is substituted by Ser, reverted the effect of GAS6 on stimulation of VEGF receptor 2 and endothelial chemotaxis triggered by VEGF-A. These studies provide the first demonstration of a cross talk between Axl and VEGF receptor 2 and add new information on the regulation of VEGF-A activities during tissue vascularization.

Animals↗
PubMed2004

Vasculogenic potential of long term repopulating cord blood progenitors.

In the adult, involvement of bone marrow-derived circulating endothelial progenitor cells (EPCs) in tissue revascularization (vasculogenesis) and the cooperation of hematopoietic cell subsets in supporting this process have been described in different experimental animal models. However, the effective contribution of such cells in restoring organ vascularization in a clinical setting needs to be clarified. In this study, a mouse transplantation model was engrafted by human cord blood hematopoietic stem and progenitor cells to follow the behavior of donor-derived endothelial and hematopoietic cells in the presence of a localized source of an angiogenic inducer. Human endothelial markers (CD31+/CD45-, VE-cadherin+) were always detectable in the bone marrow of transplanted mice, while they were only randomly detectable in peripheral neovascularization sites. To investigate the ability of human transplanted hematopoietic stem cells to support new vessel formation in response to altered homeostatic conditions, chimeric mice were further treated by systemic injection of human mononuclear cells (MNCs). Our data indicate that MNC administration in transplanted mice enhances vasculogenesis in the newly formed vessels. Taken together these results suggest that human-derived EPCs, long-term engrafting a xenotransplantation model, have hematopoietic and endothelial developmental potential, which can be modulated by altering the physiological conditions of host microenvironment.

Animals↗
PubMed2004

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-alpha (Dgk-alpha) is required for hepatocytes growth factor-stimulated cell migration, we asked whether Dgk-alpha 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-alpha: activation is inhibited by R59949, an isoform-specific Dgk inhibitor, and is dependent on Src tyrosine kinase, with which Dgk-alpha 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-alpha, obtained in both cells by R59949 and in PAE-KDR by expression of Dgk-alpha dominant-negative mutant, impairs VEGF-A165-dependent chemotaxis, proliferation and in vitro angiogenesis. In addition, in HUVEC, specific downregulation of Dgk-alpha by siRNA impairs in vitro angiogenesis on matrigel, further suggesting the requirement for Dgk-alpha in angiogenic signaling in HUVEC. Thus, we propose that activation of Dgk-alpha 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.

Animals↗
PubMed2004

Aminopeptidase A is a functional target in angiogenic blood vessels.

We show that a membrane-associated protease, aminopeptidase A (APA), is upregulated and enzymatically active in blood vessels of human tumors. To gain mechanistic insight, we evaluated angiogenesis in APA null mice. We found that, although these mice develop normally, they fail to mount the expected angiogenic response to hypoxia or growth factors. We then isolated peptide inhibitors of APA from a peptide library and show that they specifically bind to and inhibit APA, suppress migration and proliferation of endothelial cells, inhibit angiogenesis, and home to tumor blood vessels. Finally, we successfully treated tumor-bearing mice with APA binding peptides or anti-APA blocking monoclonal antibodies. These data show that APA is a regulator of blood vessel formation, and can serve as a functional vascular target.

Amino Acid Motifs↗