Nitric oxide modulates the angiogenic phenotype of middle-T transformed endothelial cells.

The role of nitric oxide (NO) in the induction of angiogenesis was evaluated in a murine heart endothelioma cell line (H.end.FB) carrying the mT oncogene. Two clonal derivatives of H.end.FB, H80 and H73, exhibiting different NO synthase (NOS) activities were selected and used in the study. The relationship among NOS activity and tumor cell behaviour (growth, and angiogenic capacity) and the molecular control of gene expression were investigated. H.end.FB and H80 on one side and H73 on the other side exhibited the highest and lowest NOS activity, respectively. Cell growth was inversely correlated to the amount of NO produced by the cell lines. Conversely, in the avascular rabbit cornea assay, H.end.FB and H80 cells were strongly angiogenic, while H73 were poorly angiogenic, indicating that the ability of the cells to induce neovascularization was associated with the extent of NO produced. Consistently, systemic administration to rabbits of the NOS inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the angiogenicity of H.end.FB cells. RT-PCR evidenced that H.end.FB expressed mRNA for TGF-beta1 and all VEGF isoforms, VEGF165 being predominantly expressed. NOS inhibition reduced the basal expression of VEGF isoforms, while it markedly potentiated TGF-beta1 expression. These results indicate that the endogenous production of NO in tumor cells can serve as an autocrine/paracrine signalling mechanism of progression, by controlling angiogenic factor/modulator expressio

Heparan Sulfate Proteoglycans Mediate the Angiogenic Activity of the Vascular Endothelial Growth Factor Receptor-2 Agonist Gremlin.

OBJECTIVE: Heparan sulfate proteoglycans (HSPGs) modulate the interaction of proangiogenic heparin-binding vascular endothelial growth factors (VEGFs) with signaling VEGF receptor-2 (VEGFR2) and neuropilin coreceptors in endothelial cells (ECs). The bone morphogenic protein antagonist gremlin is a proangiogenic ligand of VEGFR2, distinct from canonical VEGFs. Here we investigated the role of HSPGs in VEGFR2 interaction, signaling, and proangiogenic capacity of gremlin in ECs. METHODS AND RESULTS: Surface plasmon resonance demonstrated that gremlin binds heparin and heparan sulfate, but not other glycosaminoglycans, via N-, 2-O, and 6-O-sulfated groups of the polysaccharide. Accordingly, gremlin binds HSPGs of the EC surface and extracellular matrix. Gremlin/HSPG interaction is prevented by free heparin and heparan sulfate digestion or undersulfation following EC treatment with heparinase II or sodium chlorate. However, at variance with canonical heparin-binding VEGFs, gremlin does not interact with neuropilin-1 coreceptor. On the other hand, HSPGs mediate VEGFR2 engagement and autophosphorylation, extracellular signaling-regulated kinase(1/2) and p38 mitogen-activated protein kinase activation, and consequent proangiogenic responses of ECs to gremlin. On this basis, we evaluated the gremlin-antagonist activity of a panel of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. The results demonstrate that the highly N,O-sulfated derivative K5-N,OS(H) binds gremlin with high potency, thus inhibiting VEGFR2 interaction and angiogenic activity in vitro and in vivo. CONCLUSIONS: HSPGs act as functional gremlin coreceptors in ECs, affecting its productive interaction with VEGFR2 and angiogenic activity. This has allowed the identification of the biotechnological K5-N,OS(H) as a novel angiostatic gremlin antagonist

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

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

Nanoliter contact angle probes tumor angiogenic ligand-receptor protein interactions

Any molecular recognition reaction supported by a solid-phase drives a specific change of the solid-solution interfacial tension. Sessile Contact Angle (CA) experiments can be readily used to track this thermodynamic parameter, prompting this well-known technique to be reinvented as an alternative, easy-access and label-free way to probe and study molecular recognition events. Here we deploy this technique, renamed for this application CONAMORE (CONtact Angle MOlecular REcognition), to study the interaction of the tumor-derived pro-angiogenic vascular endothelial growth factor-A (VEGF-A) with the extracellular domain of its receptor VEGFR2. We show that CONAMORE recognizes the high affinity binding of VEGF-A at nanomolar concentrations to surface-immobilized VEGFR2 regardless of the presence of a ten folds excess of a non specific interacting protein, and that it further proofs its specificity and reliability on competitive binding experiments involving neutralizing anti-VEGF-A antibodies. Finally, CONAMORE shows the outstanding capability to detect the specific interaction between VEGFR2 and low molecular weight ligands, such as Cyclo-VEGI, a VEGFR2 antagonist cyclo-peptide, that weights about 2 kDa

Integrins: A flexible platform for endothelial vascular tyrosine kinase receptors.

Compared to lower metazoans, vertebrates built up an exclusively new set of adhesion-related genes involved in the tissue development and in their functions. They include a large variety of extracellular matrix proteins and their heterodimeric integrin adhesive receptors. Integrins control the adhesive state of the cell through complex molecular mechanisms. Outside-in signalling informs the cell about the extracellular matrix environment, while Inside-out signalling results in changes in integrin functional activity. In the last 10 years it has well established a reciprocal integration of signals originating from integrins and receptors for soluble growth factors. This review summarizes the current understanding of this connection in vascular endothelial cells and highlights how integrins regulate a genetic program triggered by angiogenic inducers during embryo development and in adult life

Acetate intolerance is mediated by enhanced synthesis of nitric oxide by endothelial cells.

The clinical picture of acetate intolerance strictly mimics the nitric oxide (NO) effect, including smooth muscle relaxation and extreme vasodilation. Because acetate induces production of cAMP, which is a powerful stimulus of NO synthase (NOS), we evaluated the effect of different dialysate solutions with and without acetate on NOS activity in endothelial cells (EC). NOS activity of EC, evaluated as H3-citrulline produced from H3-arginine, was modulated by the dialysate composition (e.g., 38 mmol/L acetate produced an increase of 3.2 +/- 0.39-fold compared with basal values (P < 0.0005), and the small amount of acetate (4 mmol/L) in 35 mmol/L bicarbonate solution increased the NOS activity by 2 +/- 0.49-fold (P < 0.05). Conversely, the acetate-free solution produced no effect on NOS activity. The mRNA encoding for inducible NOS was highly expressed in EC incubated with acetate buffer and also with acetate in bicarbonate dialysis buffer. The EC proliferative index was depressed by acetate (P < 0.0005), and tumor necrosis factor synthesis was increased (P < 0.0005) compared with acetate-free buffer. This study suggests that dialytic "acetate intolerance" can be induced by the activation, through cAMP and tumor necrosis factor release, of NOS. The small amount of acetate in bicarbonate dialysate, although capable of inducing in vitro NOS activation, is likely to be rapidly metabolized, whereas the large amounts of this anion in acetate fluids overwhelm metabolism by the liver. Acetate-free dialysate is the only solution that provides an acceptable level of biocompatibility both in vivo and in vitro

An analysis on decentralized adaptive MAC protocols for Cognitive Radio networks

The scarcity of bandwidth in the radio spectrum has become more vital since the demand for more and more wireless applications has increased. Most of the spectrum bands have been allocated although many studies have shown that these bands are significantly underutilized most of the time. The problem of unavailability of spectrum and inefficiency in its utilization has been smartly addressed by the Cognitive Radio (CR) Technology which is an opportunistic network that senses the environment, observes the network changes, and then using knowledge gained from the prior interaction with the network, makes intelligent decisions by dynamically adapting their transmission characteristics. In this paper some of the decentralized adaptive MAC protocols for CR networks have been critically analyzed and a novel adaptive MAC protocol for CR networks, DNG-MAC which is decentralized and non-global in nature, has been proposed. The results show the DNG-MAC out performs other CR MAC protocols in terms of time and energy efficiency

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.

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