Human lymphoblastoid cells produce extracellular matrix-degrading enzymes and induce endothelial cell proliferation, migration, morphogenesis, and angiogenesis

Human lymphoproliferative diseases can be hypothesized to invade locally and to metastatize via mechanisms similar to those developed by a variety of solid tumors, i.e., the secretion of extracellular matrix-degrading enzymes and stimulation of angiogenesis. To assess this hypothesis, Namalwa, Raji, and Daudi cell lines (Burkitt’s lymphoma), LIK and SB cell lines (B-cell lymphoblastic leukemia), CEM and Jurkat cell lines (T-cell lymphoblastic leukemia), and U266 cell line (multiple myeloma) were evaluated for their capacity to produce matrix metalloproteinase-2 and -9, and urokinase-type plasminogen activator. These cell lines were also assessed for their ability: (1) to produce the angiogenic basic fibroblast growth factor and vascular endothelial growth factor; (2) to induce an angiogenic phenotype in cultured endothelial cells, represented by cell proliferation, chemotaxis, and morphogensis; (3) to stimulate angiogenesis in different in vivo experimental models. All cell lines expressed the mRNA for one or both metalloproteinases. Namalwa, Raji, LIK, SB, and U266 cells secreted the active form of both metalloproteinases, while Daudi, CEM, and Jurkat cells produced metalloproteinase-2 but not -9. In contrast, urokinase-type plasminogen activator was secreted only by SB cells. While Raji, LIK, SB, CEM, and Jurkat cells secreted both basic fibroblast growth factor and vascular endothelial growth factor, Daudi and U266 cells produced only the former, and Namalwa cells only the latter. Accordingly, the conditioned medium of all cell lines stimulated cell proliferation and/or chemotaxis in cultured endothelial cells, with the exception of that of Namalwa cells which was ineffective. The conditioned medium of CEM and Jurkat cells induced morphogenesis in cultured endothelial cells grown on a reconstituted basement membrane (Matrigel). Lastly, Namalwa, Raji, LIK, SB, U266, CEM, and Jurkat cells induced angiogenesis and mononuclear cell recruitment in the murine Matrigel sponge model and in a chick embryo chorioallantoic membrane assay. The extent of angiogenesis in both models was strictly correlated with the density of the mononuclear cell infiltrate. The results indicate that human lymphoproliferative disease cells possess both local and remote invasive ability via the secretion of matrix-degrading enzymes and the induction of angiogenesis which is fostered by host inflammatory cells and by an intervening ensemble of angiogenic factors.     

Neutropenia induced by platelet-activating factor (PAF-acether) released from neutrophils: The inhibitory effect of prostacyclin (PGI2)

Soluble and phagocytic stimuli released PAF-acether from PMN leucocytes, as determined by chromatography and bioassay by platelet aggregation. The same material caused aggregation of human and rabbit PMN leucocytesin vitro which was inhibited by ETYA and PGI₂. PGI₂ also inhibited PAF-acether release by PMN leucocytes and,in vivo, PGI₂ abolished not only PAF-acether-induced, but also immune complex or C5a-induced thrombocytopenia and neutropenia in rabbits. These data suggest that PAF-acether may be involved in activation of both platelets and PMN leucocytesin vivo.     

Role of TGFβ1 and WNT6 in FGF2 and BMP4-driven endothelial differentiation of murine embryonic stem cells

Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

     

In vivo effect of human granulocyte-macrophage colony-stimulating factor on megakaryocytopoiesis.

The effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on megakaryocytopoiesis and platelet production was investigated in patients with normal hematopoiesis. Three findings indicated that GM-CSF plays a role in megakaryocytopoiesis. During treatment with GM-CSF (recombinant mammalian, glycosylated; Sandoz/Schering-Plough, 5.5 micrograms protein/kg/d, subcutaneously for 3 days) the percentage of megakaryocyte progenitors (megakaryocyte colony forming unit [CFU-Mk]) in S phase (evaluated by the suicide technique with high 3H-Tdr doses) increased from 31% +/- 16% to 88% +/- 11%; and the maturation profile of megakaryocytes was modified, with a relative increase in more immature stage I-III forms. Moreover, by autoradiography (after incubation of marrow cells with 125I-labeled GM-CSF) specific GM-CSF receptors were detectable on megakaryocytes. Nevertheless, the proliferative stimulus induced on the progenitors was not accompanied by enhanced platelet production (by contrast with the marked granulomonocytosis). It may be suggested that other cytokines are involved in the regulation of the intermediate and terminal stages of megakaryocytopoiesis in vivo and that their intervention is an essential prerequisite to turn the GM-CSF-induced proliferative stimulus into enhanced platelet production.     

Hyperthermia inhibits angiogenesis by a plasminogen activator inhibitor 1-dependent mechanism

Hyperthermia (HT) associated with radiotherapy or chemotherapy is a promising method for cancer treatment, although the molecular mechanisms of this process are not well understood. HT exhibits various antitumor effects, including damage of tumor vasculature. Here, we investigate the effect of HT on in vitro and in vivo angiogenesis. We show that heat treatment of endothelial cells (ECs) affect their differentiation into capillary-like structures in two models of in vitro angiogenesis. Furthermore, the formation of new vessels promoted by angiogenic inducers in the chick embryo chorioallantoic membrane assay is impaired after heat treatment. These effects cannot be explained by direct cytotoxicity but are dependent on modulation of angiogenesis-involved genes. Gene expression profile of ECs subjected to heat shock demonstrates that plasminogen activator inhibitor 1 (PAI-1), a protein involved in the control of extracellular matrix degradation, is specifically up-regulated. The use of anti-PAI-1-neutralizing antibodies reverts the effect of HT on the in vitro EC morphogenesis and in vivo vessel formation. Moreover, microvessel outgrowth from PAI-1(-/-) aortic rings was not affected by HT compared with aortic rings from PAI-1(+/+) mice. Heat treatment of murine mammary adenocarcinomas results in inhibition of tumor growth, associated with a reduction of microvessel number and an increase of PAI-1 expression. These results indicate that heat-mediated PAI-1 induction is an important pathway by which HT exerts its antitumor activity and may represent a rationale for a combined cancer therapy based on HT associated with antiangiogenic molecules.     

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.     

Incidence of food anaphylaxis in Piemonte region (Italy): data from registry of Center for Severe Allergic Reactions

There are wide differences in estimated incidence and prevalence of anaphylaxis because of the absence, until recently, of a universal consensus on the definition of anaphylaxis and the different source of collected data. We aimed to estimate the incidence of food anaphylaxis based on the database of Piemonte Region (Italy) Reference Center for Severe Allergic Reactions. All cases of severe food allergic reactions reported in 2010 were studied. Clinical data associated to the reports were evaluated according to National Institute of Allergy and Infectious Disease and Food Allergy and Anaphylaxis Network diagnostic criteria of anaphylaxis. 75 % of the 778 cases were classified as food anaphylaxis (incidence of 13/100,000 person-years, ranging from 9.9 in adults to 29/100,000 person-years in children). Nuts were the most frequent foods causing anaphylaxis. Milk and eggs were responsible for anaphylaxis more often in children, while peach, vegetables and crustaceans were in adults. Cardiovascular symptoms were more frequent in adults. Gastrointestinal involvement was more frequent in children. A high prevalence of respiratory allergic comorbidities was observed. Food is an important cause of anaphylaxis, particularly in subjects with respiratory allergic comorbidities. Children and adults differ in triggers and clinical presentation of anaphylaxis.     

Regulation of nitric oxide synthesis in uraemia

Nitric oxide (NO) is a cell-to-cell mediator involved in theregulation of vascular tone and in the mechanisms of host defence.Since uraemic syndrome is characterized by abnormalities inblood pressure and flow and by impairment of white cell function,we studied the regulation of nitric oxide synthase (NOS) activityby uraemic plasma. We used three different cellular types havingdifferent levels of NOS activity: tEnd. 1 murine endothelialcell line transformed by mT oncogene of polyomavirus had a highNOS activityand expressed endothelial-NOS (eNOS) and inducible-NOS(iNOS) isoforms; human endothelial cells from cord umbilicalvein (HUVEC) had low enzymatic activity and expressed only eNOS;finally, J774 murine macrophage line was characterised by iNOSinduced after treatment with cytokines. We demonstrated thatmost (79%) of end-stage uraemic plasma studied inhibited NOSactivity in tEnd.1 and in cytokine induced -J774, whereas theywere ineffective on HUVEC. Twenty percent of plasma samples(14 of 67) activated NOS activity in tEnd.1 and in J774 cells,but not in HUVEC, suggesting the presence of molecule(s) whichinfluence iNOS. The effect of plasma was not dependent on thetype of haemodialysis treatment. A great number of plasmas frompatients with moderate renal failure also inhibited NOS activityin tEnd.1, suggesting that the accumulation of molecules affectingNOS was caused by the renal failure rather than the haemodialytictreatment. However, the haemodialysis modified the effect of plasmas onNOS activity. Plasma taken after haemodialysis session showeda reduced inhibitory activity in tEnd.1 and in some cases itenhanced NOS activity. Simultaneously, molecules reducing NOSactivity accumulated in the ultrafiltrate. The plasma concentrationof N^G-N^G dimethyl-L-arginine (asymmetrical dimethylarginine,ADMA), an inhibitor of NOS, increased in end-stage uraemic patientsand was reduced by haemodialysis. However, the concentrationsreached in uraemic plasmas were lower than the ADMA ICM₅₀ ontEnd.1 NOS, indicating that this compound contributes with othermolecules to the inhibitory effect of uraemic plasma. Haemodialysisreduced also the enhanced effect exerted by some plasmas onNOS in J774. Therefore, the effect of endstage uraemic plasmaon NOS activity derive from the balance between inhibitors andactivators.