Consensus clustering methodology to improve molecular stratification of non-small cell lung cancer.

Recent advances in machine learning research, combined with the reduced sequencing costs enabled by modern next-generation sequencing, paved the way to the implementation of precision medicine through routine multi-omics molecular profiling of tumours. Thus, there is an emerging need of reliable models exploiting such data to retrieve clinically useful information. Here, we introduce an original consensus clustering approach, overcoming the intrinsic instability of common clustering methods based on molecular data. This approach is applied to the case of non-small cell lung cancer (NSCLC), integrating data of an ongoing clinical study (PROMOLE) with those made available by The Cancer Genome Atlas, to define a molecular-based stratification of the patients beyond, but still preserving, histological subtyping. The resulting subgroups are biologically characterized by well-defined mutational and gene-expression profiles and are significantly related to disease-free survival (DFS). Interestingly, it was observed that (1) cluster B, characterized by a short DFS, is enriched in KEAP1 and SKP2 mutations, that makes it an ideal candidate for further studies with inhibitors, and (2) over- and under-representation of inflammation and immune systems pathways in squamous-cell carcinomas subgroups could be potentially exploited to stratify patients treated with immunotherapy.

Gelatin-based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering.

Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF-controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4-D6P2T) by assessing VEGFR-2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF-releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin-based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.

Case report: the importance of oral manifestations in diagnosing iron deficiency in childhood.

AIM: The aim of this article is to report a case of iron deficiency diagnosed in a child after routine oral examination. CASE REPORT: A 5-year-old male child of African descent was brought to the paediatric dental clinic of a public university in Rio de Janeiro, Brazil. His mother's main complaint was her child's decayed teeth and sensitivity in the tongue every time he ate spicy or hot food. Anamnesis revealed chronic respiratory problems due to allergy, two previous episodes of anaemia and hospitalization about 15 months before the dental visit because of severe primary herpetic gingivostomatitis. Soft tissue examination revealed his tongue had various patches of atrophic mucosa characterizing absence of papillae in these areas. The child's dietary assessment indicated that he never ate meat or vegetables. Haematological investigation showed that the child probably had an iron deficiency, although the full blood count was not totally compatible with anaemia. A rapid initial recovery was quite noticeable after the beginning of oral therapy with ferrous sulphate, as remission of tongue sensitivity as well as papillae neoformation were observed.

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.

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.

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.

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.

Tumor necrosis factor-alpha regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-alpha does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-alpha up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-alpha increased the cellular amounts of VEGFR-2 protein and tripled the high affinity 125I-VEGF-A165 capacity without affecting the Kd of ligand-receptor interaction. As a consequence, TNF-alpha enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-alpha is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-alpha-mediated pathological settings.

Tat-human immunodeficiency virus-1 induces human monocyte chemotaxis by activation of vascular endothelial growth factor receptor-1.

Human immunodeficiency virus-1 (HIV-1) Tat protein can be released by infected cells and activates mesenchymal cells. Among these, monocytes respond to Tat by migrating into tissues and releasing inflammatory mediators. In the present study, we have examined the molecular mechanism of monocyte activation by Tat, showing that this viral protein signals inside the cells through the tyrosine kinase receptor for vascular endothelial growth factor encoded by fms-like tyrosine kinase gene (VEGFR-1/Flt-1). Subnanomolar concentrations of Tat induced monocyte chemotaxis, which was inhibited by cell preincubation with vascular-endothelial growth factor-A (VEGF-A). This desensitisation was specific for VEGF-A, because it not was observed with FMLP. In addition, the soluble form of VEGFR-1 specifically inhibited polarization and migration induced by Tat and VEGF-A, thus confirming the common use of this receptor. Binding studies performed at equilibrium by using radiolabeled Tat showed that monocytes expressed a unique class of binding site, with a kd of approximately 0.2 nmol/L. The binding of radiolabeled Tat to monocyte surface and the cross-linking to a protein of 150 kD was inhibited specifically by an excess of cold Tat or VEGF-A. Western blot analysis with an antibody anti-VEGFR-1/Flt-1 performed on monocyte phosphoproteins immunoprecipitated by an monoclonal antibody anti-phosphotyrosine showed that Tat induced a rapid phosphorylation in tyrosine residue of the 150-kD VEGFR-1/Flt-1. Taken together, these results suggest that biologic activities of HIV-1 Tat in human monocytes may, at least in part, be elicited by activation of VEGFR-1/Flt-1.

Activation of JAK2 in human vascular endothelial cells by granulocyte-macrophage colony-stimulating factor.

Besides the regulation of hematopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF)induces the expression of a functional program in endothelial cells (ECs) related to angiogenesis and to their survival in the bone marrow microenvironment. ECs express specific GM-CSF high-affinity binding sites, which mediate the proliferative and migratory response. We now report that ECs express the alpha and beta subunits of GM-CSF receptor (GM-CSFR), and that GM-CSF is able to activate the Janus kinase (JAK)2, a member of the cytosolic tyrosine kinase family, which is known to mediate signals of several non-tyrosine kinase receptors. JAK2 tyrosine phosphorylation, as well as activation of its catalytic activity, is induced by subnanomolar concentrations of GM-CSF and occurs within 3 minutes of stimulation and persists at least for 10 minutes. The effect is specific as inferred by the lack of effect of heat-inactivated GM-CSF or neutralized by specific antibodies and by the finding that interleukin-5, which utilizes a specific alpha chain and the same beta chain of GM-CSFR, does not phosphorylate JAK2. Furthermore, we show that the amount of JAK2 physically associated with GM-CSFR beta chain is increased after GM-CSF stimulation and that GM-CSF triggers both beta chain and JAK2 tyrosine phosphorylation. Taken together, these results suggest that biologic activities of GM-CSF in vascular endothelium may, in part, be elicited by GM-CSFR-mediated JAK2 activation.

The angiogenesis induced by HIV-1 tat protein is mediated by the Flk-1/KDR receptor on vascular endothelial cells.

The HIV-1 Tat protein transactivates HIV, viral and some host cell genes. Tat can be released by infected cells and acts extracellularly in the microenvironment, regulating functions of immunocompetent and mesenchymal cells. One of the most striking effects of Tat is the induction of a functional program in vascular cells related to angiogenesis and inflammation (migration, proliferation and expression of plasminogen activator inhibitor-1 and E selectin). Tat induces growth of Kaposi's sarcoma (KS) spindle cells and is angiogenic in vivo and in transgenic mice10-12. We previously reported that Tat is a direct angiogenic factor and noted the Tat arginine- and lysine-rich sequence is similar to that of other potent angiogenic growth factors, such as vascular endothelial growth factor-A (VEGF-A). It is possible that Tat mimics one of these factors by interacting with its growth factor tyrosine kinase receptor. Here we demonstrate that Tat specifically binds and activates the Flk-1/kinase insert domain receptor (Flk-1/KDR), a VEGF-A tyrosine kinase receptor (for review see ref. 13), and that Tat-induced angiogenesis is blocked by agents blocking the Flk-1/KDR receptor. Endothelial cell stimulation by Tat occurs in the absence of activation of FLT-1, another VEGF-A tyrosine kinase receptor.

IL-6 is an in vitro and in vivo autocrine growth factor for middle T antigen-transformed endothelial cells.

Murine endothelial cells immortalized with the middle-size Ag of polyomavirus (PmT) cause vascular tumors in syngenic mice by recruitment of host normal endothelial cells. This pathogenic process is similar to that occurring in Kaposi's sarcoma, in which the core of the lesion is constituted by "spindle cells," which recruit normal vascular mesenchymal cells. In murine endothelial cells, PmT induces modification of the expression of genes, including that of IL-6. Since IL-6 is a pleiotrophic cytokine that also regulates endothelial cell functions related to angiogenesis, we studied the relevance of IL-6 in the tumorigenicity of PmT-endothelial cells. In vitro studies demonstrated that the spontaneous PmT-endothelial cell proliferation rate was slow during the first 6 days of culture and then increased rapidly and paralleled the IL-6 release. The addition of recombinant IL-6 during the first days of culture induced a marked proliferation in a dose-dependent manner. PmT-endothelial cells expressed on their surface a high-affinity binding site for IL-6 constituted by both IL-6Ralpha and gp130 transmembrane receptors. The growth-promoting effect of exogenous IL-6 or that released by PmT-endothelial cells was abrogated by mAbs anti-IL-6Ralpha, whereas a mAb recognizing the endothelial cell CD31 molecule was inactive. 15A7 mAb anti-murine IL-6Ralpha was also active in vivo, reducing the number of metastases forming after transplantation of PmT-endothelial cells in DBA/2 mice. 15A7 mAb also increased the survival of mice bearing vascular tumors. We conclude that IL-6 is involved in the progression of vascular tumors induced by PmT, and that the blockage of IL-6-mediated intercellular circuits could be useful in the management of human vascular tumors, including Kaposi's sarcoma.

Platelet-activating factor (PAF) induces the early tyrosine phosphorylation of focal adhesion kinase (p125FAK) in human endothelial cells.

Platelet-activating factor (PAF) is a potent activator of angiogenesis and controls the motility and the shape of vascular endothelium. The mechanism(s) whereby PAF exerts its action are in part known. Here we report that the biological active (R)PAF enantiomer administrated to cultured endothelial cells induces the early phosphorylation in tyrosine residues of focal adhesion kinase (p125FAX) and paxillin, two molecules involved in the early signaling and cytoskeleton assembly in cells that undergo integrin-mediated adhesion or are challenged by neuropeptides or lysophosphatidic acid. The phenomenon is rapidly turned on, lasts for a few minutes and is adhesion-independent indicating that the chain of events induced by (R)PAF, including p125FAK activation, precedes adhesion. The inhibitory effect of WEB2086, a PAF receptor antagonist, and the lack of activity exerted by the (S)PAF enantiomer, indicate that (R)PAF-mediated p125FAK activation, is PAF receptor-dependent. Calphostin C, an inhibitor of protein kinase C blocks the effect of (R)PAF on p125FAK phosphorylation suggesting that protein kinase C activation is up-stream the activation of this tyrosine kinase. When endothelial cells are exposed to a substratum that allows adhesion and spreading. (R)PAF-stimulated cells, change their adhesive phenotype and start migrating. Inhibitors of tyrosine kinases, like 3-(1,4,-dihydroxytetralyl) methylen-2-oxindole and herbimycin A, reduce the cells migration, the transendothelial flux of albumin and the enhancement of p125FAK activity induced by (R)PAF. The observation that increased tyrosine phosphorylation of p125FAK and its ensuring association with focal adhesion occurs rapidly upon (R)PAF challenge indicates that this signaling molecule has a primary and independent role also in the signaling cascade initiated by (R)PAF.

Platelet activating factor produced in vitro by Kaposi's sarcoma cells induces and sustains in vivo angiogenesis.

Imbalance in the network of soluble mediators may play a pivotal role in the pathogenesis of Kaposi's sarcoma (KS). In this study, we demonstrated that KS cells grown in vitro produced and in part released platelet activating factor (PAF), a powerful lipid mediator of inflammation and cell-to-cell communication. IL-1, TNF, and thrombin enhanced the synthesis of PAF. PAF receptor mRNA and specific, high affinity binding site for PAF were present in KS cells. Nanomolar concentration of PAF stimulated the chemotaxis and chemokinesis of KS cells, endothelial cells, and vascular smooth muscle cells. The migration response to PAF was inhibited by WEB 2170, a hetrazepinoic PAF receptor antagonist. Because neoangiogenesis is essential for the growth and progression of KS and since PAF can activate vascular endothelial cells, we examined the potential role of PAF as an instrumental mediator of angiogenesis associated with KS. Conditioned medium (CM) from KS cells (KS-CM) or KS cells themselves induced angiogenesis and macrophage recruitment in a murine model in which Matrigel was injected subcutaneously. These effects were inhibited by treating mice with WEB 2170. Synthetic PAF or natural PAF extracted from plasma of patients with classical KS also induced angiogenesis, which in turn was inhibited by WEB 2170. The action of PAF was amplified by expression of other angiogenic factors and chemokines: these included basic and acidic fibroblast growth factor, placental growth factor, vascular endothelial growth factor and its specific receptor flk-1, hepatocyte growth factor, KC, and macrophage inflammatory protein-2. Treatment with WEB 2170 abolished the expression of the transcripts of these molecules within Matrigel containing KS-CM. These results indicate that PAF may cooperate with other angiogenic molecules and chemokines in inducing vascular development in KS.

Platelet activation and inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07.

The surface glycoprotein CD36 (GPIV) is known to mediate the adhesion of Plasmodium falciparum malaria-infected red blood cells and to be a receptor for extracellular matrix proteins such as collagen and thrombospondin. The murine monoclonal IgM antibody NL07, which is specific for CD36, has now been shown to also be a potent inhibitor of the adhesion of P falciparum malaria-infected red blood cells to C32 melanoma cells. Treatment of platelets with NL07 monoclonal antibody resulted in rapid degranulation, release of ATP and serotonin, increase in [Ca2+]i, and tyrosine phosphorylation of a substrate protein of 130 kD. In about one-half of the experiments, activation with NL07 resulted in the formation of small aggregates of 10 to 30 platelets, whereas in the other half of the experiments, large aggregates were seen similar to those induced by adenosine diphosphate (ADP) and these large aggregates could be converted to the small aggregates by ATP alpha S or by AP-2 or other antibodies against GPIIb and/or IIIa. Microaggregates of 2 to 5 platelets were seen with Glanzmann's platelets that constitutively lack GPIIb/IIIa. Aggregate formation was not seen with heat-treated serum, in the presence of anti C1q antibodies, or when using C5-, C8-, or C9-deficient human sera. Although activation of platelets with purified complement components results in a slow morphologic change without aggregation, involvement of CD36 results in rapid complement-mediated activation leading to formation of small aggregates that is largely independent of GPIIb/IIIa and that, under certain circumstances, proceeds to the formation of large ADP-dependent aggregates.