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1.
J Thromb Haemost ; 14(2): 397-410, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26663311

ABSTRACT

UNLABELLED: ESSENTIALS: Extracellular histones are highly augmented in sites of neovessel formation, such as regeneration tissues. We studied histone effect on survival and angiogenic activity of mature and progenitor endothelial cells. Extracellular histones trigger apoptosis and pyroptosis and reduce angiogenesis in vivo and in vitro. Histone blockade can be useful as a therapeutic strategy to improve angiogenesis and tissue regeneration. BACKGROUND: Extracellular histones are highly augmented in sites of neovessel formation, like regeneration tissues. Their cytotoxic effect has been studied in endothelial cells, although the mechanism involved and their action on endothelial colony-forming cells (ECFCs) remain unknown. OBJECTIVE: To study the effect of histones on ECFC survival and angiogenic functions and compare it with mature endothelial cells. METHODS AND RESULTS: Nuclear morphology analysis showed that each human recombinant histone triggered both apoptotic-like and necrotic-like cell deaths in both mature and progenitor endothelial cells. While H1 and H2A exerted a weak toxicity, H2B, H3 and H4 were the most powerful. The percentage of apoptosis correlated with the percentage of ECFCs exhibiting caspase-3 activation and was zeroed by the pan-caspase inhibitor Z-VAD-FMK. Necrotic-like cell death was also suppressed by this compound and the caspase-1 inhibitor Ac-YVAD-CMK, indicating that histones triggered ECFC pyroptosis. All histones, at non-cytotoxic concentrations, reduced migration and H2B, H3 and H4 induced cell cycle arrest and impaired tubulogenesis via p38 activation. Neutrophil-derived histones exerted similar effects. In vivo blood vessel formation in the quail chorioallantoic membrane was also reduced by H2B, H3 and H4. Their cytotoxic and antiangiogenic effects were suppressed by unfractioned and low-molecular-weight heparins and the combination of TLR2 and TLR4 blocking antibodies. CONCLUSIONS: Histones trigger both apoptosis and pyroptosis of ECFCs and inhibit their angiogenic functions. Their cytotoxic and antiangiogenic effects are similar in mature endothelial cells and disappear after heparin addition or TLR2/TLR4 blockade, suggesting both as therapeutic strategies to improve tissue regeneration.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Endothelial Progenitor Cells/drug effects , Histones/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Neovascularization, Physiologic/drug effects , Animals , Apoptosis/drug effects , Caspase 1/metabolism , Cell Cycle Checkpoints/drug effects , Cell Line , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Endothelial Progenitor Cells/metabolism , Endothelial Progenitor Cells/pathology , Human Umbilical Vein Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/pathology , Humans , Pyroptosis/drug effects , Quail , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases/metabolism
2.
Platelets ; 26(8): 799-804, 2015.
Article in English | MEDLINE | ID: mdl-26082997

ABSTRACT

BACKGROUND: Platelets mediate angiogenesis through the secretion of several factors, including the pro-angiogenic vascular endothelial growth factor (VEGF) and the anti-angiogenic endostatin. Although previous findings indicated that these molecules are packed into different alpha-granules and selectively released by specific stimulation of protease-activated receptor (PAR)-1 or PAR-4, recent evidences are against this hypothesis. OBJECTIVES: To elucidate the controversies about the VEGF and endostatin release and the overall angiogenic effect of PARs-stimulated platelets. METHODS: VEGF and endostatin were quantified by enzyme linked-immunosorbent assay (ELISA). Endothelial proliferation (pNPP assay), wound healing (scratch assay) and tubule formation (matrigel) of human microvascular endothelial cells (HMEC-1) and endothelial progenitor cells (EPC) were determined using supernatants from PAR-1- or PAR-4-stimulated platelets. RESULTS: Activation of washed platelets (WPs) by PAR-1- or PAR-4-activating peptide (AP) promoted the VEGF and endostatin secretion in a concentration-dependent manner, being PAR-1-AP more potent than PAR-4-AP. The release of both molecules was abrogated by pre-incubation of platelets with PAR antagonists. Activation of platelet-rich plasma (PRP) with either PAR-1-AP or PAR-4-AP induced a significant VEGF secretion. Quantification of platelet-endostatin secretion was not possible in PRP due to the high levels of plasmatic endostatin vs. platelet content. Releasates from PAR-1- or PAR-4-activated WPs promoted similar pattern of angiogenic responses of HMEC-1 or EPC. Moreover, proliferation of HMEC-1 mediated by PAR-stimulated PRP releasates was delayed and significantly lower compared with that induced by PAR-stimulated WPs. CONCLUSIONS: Our results are in contrast with the previously described differential release of VEGF and endostatin induced by the selective PAR-1 or PAR-4 stimulation, and support the notion that while circulating endostatin accounts for the maintenance of a systemic anti-angiogenic state, locally, the release of platelet alpha-granule content promotes angiogenesis.


Subject(s)
Blood Platelets/metabolism , Endostatins/metabolism , Receptor, PAR-1/agonists , Receptors, Thrombin/agonists , Vascular Endothelial Growth Factor A/metabolism , Blood Platelets/drug effects , Endothelial Cells/metabolism , Humans , Neovascularization, Physiologic , Oligopeptides/pharmacology , Platelet Activation/drug effects
3.
J Thromb Haemost ; 13(2): 271-82, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25393316

ABSTRACT

BACKGROUND: To further understand the role of platelets in the pathogenesis of viral infections we explored platelet interaction with Coxsackieviruses B (CVB) 1 and 3. CVB is a group of viruses that cause the majority of human enterovirus-related viral myocarditis; their receptor (CAR) is expressed on the platelet surface and there is a well-characterized CVB3-induced myocarditis murine model. METHODS: Human platelets were infected with CVB1 and 3 and viruses were detected in pellets and in supernatants. C57BL/6J mice with or without platelet depletion were inoculated with CVB3 and peripheral blood and heart samples collected at different times post-infection. RESULTS: CVB1 and 3 RNA and a capsid protein were detected in infected platelets. Despite the fact that titration assays in Vero cells showed increasing infectivity titers over time, supernatants and pellets from infected platelets showed similar levels, suggesting that platelets were not susceptible to a replicative infectivity cycle. CVB binding was CAR-independent and resulted in P-selectin and phosphatidylserine (PS) exposure. CVB3-infected mice showed a rapid thrombocytopenia that correlated with an increase in platelet PS exposure and platelet-leukocyte aggregates without modification of platelet P-selectin expression or von Willebrand factor levels. Mortality, viremia, heart viral titers and myocarditis were significantly higher in platelet-depleted than normal animals. Type I IFN levels were not changed but IgG levels were lower in infected and platelet-depleted mice. CONCLUSIONS: Our data reveal that platelets play a critical role in host survival and immune response against CVB3 infection.


Subject(s)
Blood Platelets/virology , Coxsackievirus Infections/blood , Coxsackievirus Infections/virology , Enterovirus B, Human/pathogenicity , Myocarditis/blood , Myocarditis/virology , Animals , Blood Platelets/immunology , Blood Platelets/metabolism , Capsid Proteins/blood , Capsid Proteins/genetics , Chlorocebus aethiops , Coxsackievirus Infections/immunology , Disease Models, Animal , Enterovirus B, Human/genetics , Enterovirus B, Human/immunology , Enterovirus B, Human/metabolism , Female , Host-Pathogen Interactions , Humans , Immunoglobulin G/blood , Male , Mice, Inbred C57BL , Myocarditis/immunology , P-Selectin/blood , Phosphatidylserines/blood , RNA, Viral/blood , Thrombocytopenia/blood , Thrombocytopenia/virology , Time Factors , Vero Cells , Virus Replication
4.
Cell Death Dis ; 5: e1507, 2014 Nov 06.
Article in English | MEDLINE | ID: mdl-25375372

ABSTRACT

Solid tumors are infiltrated by immune cells where macrophages and senescent T cells are highly represented. Within the tumor microenvironment, a cross-talk between the infiltrating cells may occur conditioning the characteristic of the in situ immune response. Our previous work showed that tumors induce senescence of T cells, which are powerful suppressors of lympho-proliferation. In this study, we report that Tumor-Induced Senescent (TIS)-T cells may also modulate monocyte activation. To gain insight into this interaction, CD4+ or CD8+TIS-T or control-T cells were co-incubated with autologous monocytes under inflammatory conditions. After co-culture with CD4+ or CD8+TIS-T cells, CD14+ monocytes/macrophages (Mo/Ma) exhibit a higher expression of CD16+ cells and a reduced expression of CD206. These Mo/Ma produce nitric oxide and reactive oxygen species; however, TIS-T cells do not modify phagocyte capacity of Mo/Ma. TIS-T modulated-Mo/Ma show a higher production of pro-inflammatory cytokines (TNF, IL-1ß and IL-6) and angiogenic factors (MMP-9, VEGF-A and IL-8) and a lower IL-10 and IP-10 secretion than monocytes co-cultured with controls. The mediator(s) present in the supernatant of TIS-T cell/monocyte-macrophage co-cultures promote(s) tubulogenesis and tumor-cell survival. Monocyte-modulation induced by TIS-T cells requires cell-to-cell contact. Although CD4+ shows different behavior from CD8+TIS-T cells, blocking mAbs against T-cell immunoglobulin and mucin protein 3 and CD40 ligand reduce pro-inflammatory cytokines and angiogenic factors production, indicating that these molecules are involved in monocyte/macrophage modulation by TIS-T cells. Our results revealed a novel role for TIS-T cells in human monocyte/macrophage modulation, which may have deleterious consequences for tumor progression. This modulation should be considered to best tailor the immunotherapy against cancer.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , CD40 Ligand/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cytokines/biosynthesis , Membrane Proteins/metabolism , Monocytes/metabolism , CD4-Positive T-Lymphocytes/cytology , CD40 Ligand/genetics , CD8-Positive T-Lymphocytes/cytology , Cell Communication , Cell Survival , Cellular Senescence , Coculture Techniques , Cytokines/metabolism , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Gene Expression Regulation , HeLa Cells , Hepatitis A Virus Cellular Receptor 2 , Humans , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Mannose Receptor , Mannose-Binding Lectins/genetics , Mannose-Binding Lectins/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Membrane Proteins/genetics , Monocytes/cytology , Nitric Oxide/metabolism , Primary Cell Culture , Reactive Oxygen Species/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, IgG/genetics , Receptors, IgG/metabolism , Signal Transduction , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
5.
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