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Biomedical subjects

F Bussolino

Publications and source records attributed to F Bussolino.

At least 19 recordsLinked to original sources
PubMed2003

Percolation, morphogenesis, and burgers dynamics in blood vessels formation.

Experiments of in vitro formation of blood vessels show that cells randomly spread on a gel matrix autonomously organize to form a connected vascular network. We propose a simple model which reproduces many features of the biological system. We show that both the model and the real system exhibit a fractal behavior at small scales, due to the process of migration and dynamical aggregation, followed at large scale by a random percolation behavior due to the coalescence of aggregates. The results are in good agreement with the analysis performed on the experimental data.

Computer Simulation↗
PubMed2002

Recombinant AAV vector encoding human VEGF165 enhances wound healing.

Delivery of therapeutic genes represents an appealing possibility to accelerate healing of wounds that are otherwise difficult to treat, such as those in patients with metabolic disorders or infections. Experimental evidence indicates that in such conditions potentiation of neo-angiogenesis at the wound site might represent an important therapeutic target. Here we explore the efficacy of gene therapy of wound healing with an adeno-associated virus (AAV) vector expressing the 165 amino acid isoform of vascular endothelial growth factor-A (VEGF-A). By gene marker studies, we found that AAV vectors are highly efficient for gene transfer to the rat skin, displaying an exquisite tropism for the panniculus carnosus. Gene expression from these vectors is sustained and persistent over time. Delivery of VEGF165 to full thickness excisional wounds in rats resulted in remarkable induction of new vessel formation, with consequent reduction of the healing time. Histological examination of treated wounds revealed accelerated remodeling of epidermis and dermis, with formation of a thick granular layer, containing numerous newly formed capillaries, as well as vessels of larger size. These data underline the importance of neo-angiogenesis in the healing process and indicate that VEGF gene transfer might represent a novel approach to treat wound healing disorders.

Animals↗
PubMed2001

Cytoadherence of Plasmodium falciparum-infected erythrocytes is mediated by a redox-dependent conformational fraction of CD36.

The adherence of Plasmodium falciparum-infected RBC (IRBC) to postcapillary venular endothelium is an important determinant of the pathogenesis of severe malaria complications. Cytoadherence of IRBC to endothelial cells involves specific receptor/ligand interactions. The glycoprotein CD36 expressed on endothelial cells is the major receptor involved in this interaction. Treatment of CD36-expressing cells with reducing agents, such as DTT and N-acetylcysteine, was followed by CD36 conformational change monitorable by the appearance of the Mo91 mAb epitope. Only a fraction of the surface expressed CD36 molecules became Mo91 positive, suggesting the presence of two subpopulations of molecules with different sensitivities to reduction. The Mo91 epitope has been localized on a peptide (residues 260-279) of the C-terminal, cysteine-rich region of CD36. Treatment with reducing agents inhibited the CD36-dependent cytoadherence of IRBC to CD36-expressing cells and dissolved pre-existent CD36-mediated IRBC/CD36-expressing cell aggregates. CD36 reduction did not impair the functionality of CD36, since the reactivity of other anti-CD36 mAbs as well as the binding of oxidized low density lipoprotein, a CD36 ligand, were maintained. The modifications induced by reduction were reversible. After 14 h CD36 was reoxidized, the cells did not express the Mo91 epitope, and cytoadherence to IRBC was restored. The results indicate that IRBCs bind only to a redox-modulated fraction of CD36 molecules expressed on the cell surface. The present data indicate the therapeutic potential of reducing agents, such as the nontoxic drug N-acetylcysteine, to prevent or treat malaria complications due to IRBC cytoadhesion.

Acetylcysteine↗
PubMed2001

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-gamma 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-gamma 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.

Angiogenesis Inhibitors↗
PubMed2001

HIV-1 Tat protein stimulates in vivo vascular permeability and lymphomononuclear cell recruitment.

HIV-1 Tat protein released by infected cells is a chemotactic molecule for leukocytes and induces a proinflammatory program in endothelial cells (EC) by activating vascular endothelial growth factor (VEGF) receptors expressed on both cell types. Its potential role in causing vascular permeability and leukocyte recruitment was studied in vivo following its s.c. injection in mice. Tat caused a dose-dependent early (15 min) and late (6 h) wave of permeability that were inhibited by a neutralizing Ab anti-VEGF receptor type 2. Tissue infiltration of lymphomononuclear cells, mainly monocytes (76%), was evident at 6 h and persisted up to 24 h. WEB2170, a platelet activating factor (PAF) receptor antagonist, reduced the early leakage by 70-80%, but only slightly inhibited the late wave and cell recruitment. In vitro, Tat induced a dose-dependent flux of albumin through the EC monolayer that was inhibited by Ab anti-vascular VEGF receptor type 2 and WEB2170, and PAF synthesis in EC that was blocked by the Ab anti-VEGF receptor type 2. Lastly, an anti-monocyte chemotactic peptide-1 (MCP-1) Ab significantly reduced the lymphomononuclear infiltration elicited by Tat. In vitro, Tat induced a dose-dependent production of MCP-1 by EC after a 24-h stimulation. These results highlighted the role of PAF and MCP-1 as secondary mediators in the onset of lymphomononuclear cell recruitment in tissues triggered by Tat.

Animals↗
PubMed2001

Tat-induced platelet-activating factor synthesis contributes to the angiogenic effect of HIV-1 Tat.

This study shows that human umbilical cord vein-derived endothelial cells (HUVEC) stimulated with HIV-1 Tat synthesized platelet-activating factor (PAF), a phospholipid mediator of inflammation that possesses angiogenic properties. The synthesis of PAF by HUVEC stimulated with Tat was dose and time dependent. Moreover, in vitro experiments were performed to evaluate whether migration of HUVEC induced by Tat was dependent on the synthesis of PAF. It was found that the cell motility induced by Tat was inhibited by WEB 2170, a specific PAF receptor antagonist. In vivo, the neoangiogenesis induced by Tat was also inhibited by WEB 2170 in a murine model, in which matrigel subcutaneously injected was used as substratum for angiogenesis. These results suggest that the synthesis of PAF by endothelial cells mediates, at least in part, the angiogenic activity of Tat by promoting the endothelial cell migration.

Angiogenesis Inducing Agents↗
PubMed2001

Involvement of a serine protease, but not of neutrophil elastase, in tumor necrosis factor-induced lethal hepatitis and induction of platelet-activating factor.

BACKGROUND/AIMS: Tumor necrosis factor (TNF) plays an essential role in several types of acute and chronic hepatitis. Our aims in the present study were to elucidate the mechanism by which TNF leads to acute lethal hepatitis, thereby focusing on the role of serine proteases and platelet-activating factor (PAF). METHODS: All experiments were performed in a model of acute hepatitis, induced by TNF in combination with D-(+)-galactosamine (GalN). Neutrophil elastase (NE)-deficient mice, generated by gene targeting were used in the studies. RESULTS: We found that a serine protease plays an essential mediating role in the in vivo TNF effect as alpha1-antitrypsin (alpha1-AT), soybean trypsin inhibitor (STI) and turkey trypsin inhibitor (TTI), confer complete protection. alpha1-AT and TTI, but not STI, reduce PAF blood levels, induced by TNF/GalN, which is compatible with an elastase-like serine protease involvement in PAF synthesis. In our search for relevant serine proteases we believed that NE was an excellent candidate protease. However, we found that TNF/GalN-induced lethality is not attenuated in mice deficient in NE. CONCLUSIONS: The data suggest that TNF-induced lethal hepatitis is accompanied by increases in circulating PAF and plasma clotting time, and mediated by a serine protease, but not by NE.

Acute Disease↗
PubMed2001

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 Kaposi's 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.

Endothelium, Vascular↗
PubMed2001

Dynamic modules and heterogeneity of function: a lesson from tyrosine kinase receptors in endothelial cells.

An important unresolved issue related to tyrosine kinase receptor signaling pathways is the lack of specificity of the molecular effectors involved. The specificity of the biological responses that are nevertheless elicited may be explained by differences in activation thresholds, as well as by temporal (transient versus sustained) and topographical aspects of receptor activation. On the basis of recent lessons from endothelial cells, we argue that an additional strategy can be adopted to generate specificity, i.e. tyrosine kinase receptors may form distinct signaling modules with other transmembrane proteins, such as adhesive receptors, to elicit different biological programs in stimulated cells.

Animals↗
PubMed2001

Expression of angiopoietin-1 in human glioblastomas regulates tumor-induced angiogenesis: in vivo and in vitro studies.

To define a role for the angiopoietin/Tie2 system in astrocytoma angiogenesis, we examined the expression of angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) in these tumors by immunohistochemistry and in situ hybridization. Furthermore, we studied in vitro the effects elicited by glioblastoma cell-secreted Ang1 or by recombinant Ang1 on functions of endothelial cells (ECs). Our observations of astrocytomas show that a stage-specific induction of angiopoietins occurs and is correlated with angiogenic phases of different intensity. Ang1 expression was found in a few astrocytes scattered in the tumor at all stages of astrocytoma progression. In blood vessels, Ang1 mRNA increased progressively in high-grade glioblastomas, in which the number of vessels was higher than in low-grade tumors. Ang2 was detected in tumor cells and in ECs in high-grade astrocytomas, whereas its expression was negligible in low-grade tumors. Coculture of glioblastoma cell lines producing Ang1 with endothelium demonstrated a key role of this ligand in the control of EC network organization. We found that recombinant Ang1 in vitro induces EC spreading and reorganization of the cell monolayer into cordlike structures. These results suggest that Ang1 directly acts on ECs by modulating cell-cell and cell-matrix associations and promoting the differentiation phase of angiogenesis.

Angiogenesis Inducing Agents↗
PubMed2000

Human immunodeficiency virus transactivator protein (Tat) stimulates chemotaxis, calcium mobilization, and activation of human polymorphonuclear leukocytes: implications for Tat-mediated pathogenesis.

The extracellular activities of the human immunodeficiency virus (HIV) transactivator protein (Tat) include induction of angiogenesis and stimulation of monocyte migration. Here it is shown that polymorphonuclear leukocytes (PMNL), mostly neutrophils, rapidly invade in response to Tat in vivo and initiate the formation of new vessels. In vitro, Tat was chemotactic for PMNL and induced calcium (Ca(2+)) mobilization. Tat proteins with inactivating substitutions in the arginine-glycine-aspartic acid or basic domain were still active in inducing PMNL migration, whereas Tat peptides mapped the migration and Ca(2+) mobilization activity to a cysteine-rich core domain, previously described as a Tat "chemokine-like" region (peptide CysL(24-51)). Tat and the CysL(24-51) peptide also induced PMNL superoxide production and the release of the angiogenic factors interleukin-8 and vascular endothelial growth factor from PMNL. CysL(24-51) did not induce endothelial cell migration but was angiogenic in vivo. These data indicate that the Tat activity on PMNL is mediated by its chemokine-like region and that PMNL recruitment by Tat is linked to angiogenesis.

Amino Acid Substitution↗
PubMed2000

Cu(II) and Zn(II) complexes with hyaluronic acid and its sulphated derivative. Effect on the motility of vascular endothelial cells.

With the aim of improving the compatibility of biomaterials to be used for the construction of cardiovascular prosthesis, we have designed bioactive macromolecules resulting from chemical modifications of hyaluronic acid (Hyal). The stability constants of Cu(II) and Zn(II) complexes with the sulphated derivative of hyaluronic acid (HyalS3.5) were evaluated. Two different complexes have been found for each metal ion, CuL, Cu(OH)2L and ZnL, Zn(OH)2L (L means the disaccharide unit of the ligands) in aqueous solution at 37 degrees C. The dihydroxo Cu(II) complex was present in high percentage at pH=7.4. On the contrary, the Zn(II) ion was present with a relatively low percentage of both complexes. The ability to stimulate endothelial cell adhesion and migration was evaluated for Hyal, HyalS3.5 and their complexes with Cu(II) and Zn(II) ions. The results revealed that Hyal and [Cu(OH)2HyalS3.5](4.5)- induced cell adhesion, while [ZnHyalS3.5](2.5)- and [Zn(OH)2HyalS3.5](4.5)- inhibited the process. The chemotactic activity of increasing concentrations of the above complexes was also evaluated, demonstrating that [Cu(OH)2HyalS3.5](4.5)- complex at 1 microM concentration was the most active in inducing cell migration. These results have been also strengthened by analysing adherent cell migration in agarose. In conclusion, sulphated hyaluronic acid coordinated to Cu(II) seems to be a promising matrix molecule for the construction of cardiovascular prosthesis.

Animals↗
PubMed2000

Src-mediated activation of alpha-diacylglycerol kinase is required for hepatocyte growth factor-induced cell motility.

Diacylglycerol kinases are involved in cell signaling, either as regulators of diacylglycerol levels or as intracellular signal-generating enzymes. However, neither their role in signal transduction nor their biochemical regulation has been elucidated. Hepatocyte growth factor (HGF), upon binding to its tyrosine kinase receptor, activates multiple signaling pathways stimulating cell motility, scattering, proliferation and branching morphogenesis. Herein we demonstrate that: (i) the enzymatic activity of alpha-diacylglycerol kinase (alphaDgk) is stimulated by HGF in epithelial, endothelial and alphaDgk-transfected COS cells; (ii) cellular expression of an alphaDgk kinase-defective mutant inhibits activation of endogenous alphaDgk acting as dominant negative; (iii) specific inhibition of alphaDgk prevents HGF-induced cell movement of endothelial cells; (iv) HGF induces the association of alphaDgk in a complex with Src, whose tyrosine kinase activity is required for alphaDgk activation by HGF; (v) Src wild type stimulates alphaDgk activity in vitro; and (vi) alphaDgk can be tyrosine phosphorylated in intact cells.

Animals↗
PubMed2000

Human endothelial cells expressing polyoma middle T induce tumors.

The middle T oncogene of murine polyomavirus (PymT) rapidly transforms and immortalizes murine embryonic endothelial cells (EC), leading to the formation of vascular tumors in newborn mice, by recruitment of host, non-transformed EC. These tumors are reminiscent of human vascular tumors like cavernous hemangioma, Kaposi's sarcoma or those characterizing Kasabach-Merrit syndrome. Here we investigate the in vitro and in vivo behavior of human primary umbilical cord vein EC expressing PymT. While PymT has been unable to transform human fibroblasts in earlier experiments or controls done here, mT expressing EC (PymT-EC) derived by infection with pLX-PymT retrovirus induce hemangiomas in nu/nu mice. These tumors contain not only human cells but also recruited mouse EC as shown by the presence of human and murine CD31 positive EC. In vitro analysis shows that PymT-EC retain endothelial specific markers like CD31, Von Willebrand factor, and VE-cadherin, and reach the confluence without signs of overgrowth. They are also responsive to vascular endothelial growth factor-A. However, their proliferation rate is increased. The balance between urokinase-type plasminogen activator and plasminogen activator inhibitor-1 is modified; RNA and catalytic activity for the former are elevated while PAI-1 RNA is reduced. In contrast with murine model, where the PymT EC cells become immortal, the effects induced by PymT in human EC are transient. After 12-15 passages, human PymT EC stop proliferating, assume a senescent phenotype, and lose the ability to induce hemangiomas. At the same time both the amount of middle T protein and the level of activation of pp60c-src lower.

Adult↗
PubMed2000

Aberrantly glycosylated IgA molecules downregulate the synthesis and secretion of vascular endothelial growth factor in human mesangial cells.

To gain insight into the glomerular capillary repair mechanisms in immunoglobulin A (IgA) nephropathy, we focused on vascular endothelial growth factor (VEGF-A) and nitric oxide (NO). Because abnormal glycosylation of serum IgA has been shown in IgA nephropathy, we examined whether VEGF-A and NO production by mesangial cells (MCs) could be modulated by aberrantly glycosylated (desialylated or degalactosylated) IgA. VEGF-A and NO synthase (NOS) gene expression were examined by reverse-transcriptase polymerase chain reaction (RT-PCR) or Northern blot analysis, and VEGF-A peptide, by capture enzyme-linked immunosorbent assay and NOS activity as production of tritium ([(3)H]) citrulline from [(3)H] arginine. Semiquantitative densitometric analysis of RT-PCR experiments showed a significant downregulation of VEGF-A messenger RNA (mRNA) in MCs incubated with aberrantly glycosylated IgA. This resulted in decreased release of VEGF-A in culture medium (P: < 0. 01). NOS activity and inducible NOS (iNOS) mRNA were enhanced by aberrantly glycosylated IgA (both P: < 0.01). No modulation of constitutive NOS mRNA was found. The depression of the VEGF-A production induced by aberrantly glycosylated IgA was mediated by NO because it was completely reversed by the NOS inhibitor, N:omega-nitro-L-arginine methyl ester. The NO donor, sodium nitroprusside, induced a bimodal modulation of VEGF; although low concentrations (0.0001 nmol/L) increased VEGF-A synthesis, greater concentrations (1,000 nmol/L) depressed it. In conclusion, we report negative control of VEGF-A synthesis in MCs by aberrantly glycosylated IgA, mediated by enhanced iNOS activity. We speculate that both increased iNOS activity and depressed VEGF-A synthesis might have a role in impairing vascular repair and favor sclerosis in IgA nephropathy.

Down-Regulation↗
PubMed2000

Identification of specific molecular structures of human immunodeficiency virus type 1 Tat relevant for its biological effects on vascular endothelial cells.

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.

Base Sequence↗
PubMed1999

Vascular endothelial growth factor-C stimulates the migration and proliferation of Kaposi's sarcoma cells.

Recent evidence suggesting vascular endothelial growth factor-C (VEGF-C), which is a regulator of lymphatic and vascular endothelial development, raised the question whether this molecule could be involved in Kaposi's sarcoma (KS), a strongly angiogenic and inflammatory tumor often associated with infection by human immunodeficiency virus-1. This disease is characterized by the presence of a core constituted of three main populations of "spindle" cells, having the features of lymphatic/vascular endothelial cells, macrophagic/dendritic cells, and of a mixed macrophage-endothelial phenotype. In this study we evaluated the biological response of KS cells to VEGF-C, using an immortal cell line derived from a KS lesion (KS IMM), which retains most features of the parental tumor and can induce KS-like sarcomas when injected subcutaneously in nude mice. We show that VEGFR-3, the specific receptor for VEGF-C, is expressed by KS IMM cells grown in vitro and in vivo. In vitro, VEGF-C induces the tyrosine phosphorylation of VEGFR-2, a receptor also for VEGF-A, as well as that of VEGFR-3. The activation of these two receptors in KS IMM cells is followed by a dose-responsive mitogenic and motogenic response. The stimulation of KS IMM cells with a mutant VEGF-C unable to bind and activate VEFGR-2 resulted in no proliferative response and in a weak motogenic stimulation, suggesting that VEGFR-2 is essential in transducing a proliferative signal and cooperates with VEGFR-3 in inducing cell migration. Our data add new insights on the pathogenesis of KS, suggesting that the involvement of endothelial growth factors may not only determine KS-associated angiogenesis, but also play a critical role in controlling KS cell growth and/or migration and invasion.

Animals↗