Extracellular histones reduce survival and angiogenic responses of late outgrowth progenitor and mature endothelial cells.

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.

Stimulation of PAR-1 or PAR-4 promotes similar pattern of VEGF and endostatin release and pro-angiogenic responses mediated by human platelets.

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.

Expression and functionality of Toll-like receptor 3 in the megakaryocytic lineage.

BACKGROUND: In addition to their key role in hemostasis, platelets and megakaryocytes regulate immune and inflammatory responses, in part through their expression of Toll-like receptors (TLRs). Among the TLRs, TLR3 recognizes dsRNA associated with viral infection. Thrombocytopenia is a frequent complication of viral infection. However, the expression and functionality of TLR3 in megakaryocytes and platelets is not yet well understood. OBJECTIVE: To study the expression and functionality of TLR3 in the megakaryocytic lineage. METHODS AND RESULTS: RT-PCR, flow cytometric and immunofluorescence assays showed that TLR3 is expressed in CD34(+) cells, megakaryocytes, and platelets. Immunoblotting assays showed that stimulation of megakaryocytes with two synthetic agonists of TLR3, Poly(I:C) and Poly(A:U), activated the nuclear factor-κB (NF-κB), phosphoinositide 3-kinase (PI3K)/Akt, extracellular signal-related kinase (ERK)1/2 and p38 pathways. TLR3-megakaryocyte activation resulted in reduced platelet production in vitro and interferon-ß release through the PI3K-Akt and NF-κB signaling pathways. TLR3 ligands potentiated the aggregation mediated by classic platelet agonists. This effect was also observed for ATP release, but not for P-selectin or CD40L membrane exposure, indicating that TLR3 activation was not involved in α-granule release. In addition, TLR3 agonists induced activation of the NF-κB, PI3K-Akt and ERK1/2 pathways in platelets. Reductions in platelet production and platelet fibrinogen binding mediated by Poly(I:C) or Poly(A:U) were prevented by the presence of an inhibitor of the TLR3-dsRNA complex. CONCLUSIONS: Our findings indicate that functional TLR3 is expressed in CD34(+) cells, megakaryocytes, and platelets, and suggest a potential role for this receptor in the megakaryopoiesis/thrombopoiesis alterations that occur in viral infections.

Platelets interact with Coxsackieviruses B and have a critical role in the pathogenesis of virus-induced myocarditis.

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.

Functional responses and molecular mechanisms involved in histone-mediated platelet activation.

Histones are highly alkaline proteins found in cell nuclei and they can be released by either dying or inflammatory cells. The recent observations that histones are major components of neutrophil extracellular traps and promote platelet aggregation and platelet-dependent thrombin generation have shown that these proteins are potent prothrombotic molecules. Because the mechanism(s) of platelet activation by histones are not completely understood, we explored the ability of individual recombinant human histones H1, H2A, H2B, H3 and H4 to induce platelet activation as well as the possible molecular mechanisms involved. All histones were substrates for platelet adhesion and spreading and triggered fibrinogen binding, aggregation, von Willebrand factor release, P-selectin and phosphatidylserine (PS) exposure and the formation of platelet-leukocyte aggregates; however, H4 was the most potent. Histone-mediated fibrinogen binding, P-selectin and PS exposure and the formation of mixed aggregates were potentiated by thrombin. Histones induced the activation of ERK, Akt, p38 and NFκB. Accordingly, histone-induced platelet activation was significantly impaired by pretreatment of platelets with inhibitors of ERK (U 0126), PI3K/Akt (Ly 294002), p38 (SB 203580) and NFκB (BAY 11-7082 and Ro 106-9920). Preincubation of platelets with either aspirin or dexamethasone markedly decreased fibrinogen binding and the adhesion mediated by histones without affecting P-selectin exposure. Functional platelet responses induced by H3 and H4, but not H1, H2A and H2B, were partially mediated through interaction with Toll-like receptors -2 and -4. Our data identify histones as important triggers of haemostatic and proinflammatory platelet responses, and only haemostatic responses are partially inhibited by anti-inflammatory drugs.

Platelet-mediated angiogenesis is independent of VEGF and fully inhibited by aspirin.

BACKGROUND AND PURPOSE: Platelets are major players in every step of vessel development through the local delivery of angiogenesis-modulating factors, including the pro-angiogenic protein VEGF and the anti-angiogenic endostatin. Although thrombin is a potent agonist and is highly elevated in angiogenesis-related diseases, studies regarding its action on the release of platelet angiogenic factors are scarce and controversial. Herein, we have investigated the role of thrombin not only in VEGF and endostatin release but also in net platelet angiogenic activity. EXPERIMENTAL APPROACH: Human platelets were stimulated with thrombin in the presence of the various inhibitors of the signalling pathways involved in platelet activation. Supernatants/releasates were used to determine the levels of angiogenic molecules and to induce angiogenic responses. KEY RESULTS: We found that thrombin induced the secretion of both VEGF and endostatin; however, the overall effect of the releasates was pro-angiogenic as they promoted tubule-like formation and increased the proliferation of endothelial cells. Both responses were only slightly suppressed in the presence of a VEGF receptor-neutralizing antibody. Pharmacological studies revealed that while inhibitors of PKC, p38, ERK1/2, Src kinases or PI3K/Akt exerted only partial inhibitory effects, aspirin fully blocked the pro-angiogenic activity of the releasate. CONCLUSIONS AND IMPLICATIONS: In contrast to current belief, platelet pro-angiogenic responses are independent of VEGF and appear to be the result of the combined action of several molecules. Moreover, our data reinforce the notion that aspirin is a good candidate for a therapeutic agent to treat angiogenesis-related diseases.

Esophagojejunal reconstruction after total gastrectomy for gastric cancer using a transorally inserted anvil delivery system.

INTRODUCTION: Total gastrectomy (TG) is commonly performed for the treatment of patients with gastric cancer. However, reconstruction of the esophagojejunal (EJ) anastomosis can be technically demanding, with reported anastomotic leak rates in the Western world still approaching 10-15%. We report our experience using the transoral anvil delivery system (OrVil™) for creation of the EJ anastomosis after TG. METHODS: From 2007 to 2011, 48 consecutive patients with gastric cancer underwent open (n=31) or laparoscopic (n=17) TG. EJ reconstruction was performed with the transoral anvil deliver system (OrVil™) in an end-to-side fashion. Demographic, clinic, and perioperative data were obtained from a prospectively maintained database. RESULTS: Of the 48 patients, 83% were male. Median age at resection was 64 years. Median body mass index was 27.1 kg/m2. Seventy-nine percent (n=38) of patients had at least one comorbidity. Fifteen patients (31%) had at least one perioperative complication. There was one perioperative death (2%) following a duodenal stump leak. There were four EJ leaks (8%) and two EJ stenoses (independent of leak; 4%). There was one EJ leak (6%) and one EJ stenosis (6%) following a case that was first attempted laparoscopically. There were no deaths as a consequence of an EJ leak. CONCLUSIONS: The use of the transoral anvil delivery system during EJ reconstruction is a safe and effective option for reconstruction after open or laparoscopic TG with acceptable mortality and morbidity. The anastomotic leak rate appears to be comparable to that of other techniques.

Expression and functionality of type I interferon receptor in the megakaryocytic lineage.

BACKGROUND: Type I interferons (IFN-I) negatively regulate megakaryo/thrombopoiesis. However, expression of the IFN-I receptor (IFNAR) in the megakaryocytic lineage is poorly characterized. OBJECTIVES: To study the expression and functionality of IFNAR in the megakaryocytic lineage. METHODS AND RESULTS: Although IFNAR mRNA was found in every cell type studied, its protein expression showed differences between them. According to flow cytometry and immunofluorescence, IFNAR1 was observed in Meg-01, Dami, CD34+ cells and megakaryocytes, but not in proplatelets or platelets. Immunoblotting assays showed that IFNAR1 and IFNAR2 were highly expressed in all cell types, except in platelets where it was barely detectable. Regarding IFNAR1, 130- and 90-kDa bands were detected in Meg-01 and Dami, whereas 130- and 60-kDa bands were found in CD34+ cells and megakaryocytes. Activation of megakaryocytic IFNAR by IFN-ß induced pSTAT1/2 and upregulated the antiviral genes IRF7 and MXA. The latter response was completely suppressed by IFNAR blockade. In contrast, the low levels of IFNAR in platelets were not functional as pSTAT1/2, aggregation and P-selectin expression were not induced by IFN-I. In addition, megakaryocytes increased IFN-I transcript levels and produced IFN-ß upon stimulation with PolyI:C, a synthetic dsRNA that mimics viral infection. CONCLUSIONS: Early progenitors and mature megakaryocytes, but not platelets, express functional IFNAR and synthetize/release IFN-ß, revealing not only that megakaryo/thrombopoiesis regulation by IFN-I is associated with a specific interaction with its receptor, but also that megakaryocytes may play a role in the antiviral defense by being both IFN producers and responders.

Hyperthermia inhibits platelet hemostatic functions and selectively regulates the release of alpha-granule proteins.

BACKGROUND: Hyperthermia is one of the main disturbances of homeostasis occurring during sepsis or hypermetabolic states such as cancer. Platelets are important mediators of the inflammation that accompanies these processes, but very little is known about the changes in platelet function that occur at different temperatures. OBJECTIVES: To explore the effect of higher temperatures on platelet physiology. METHODS: Platelet responses including adhesion, spreading (fluorescence microscopy), α(IIb)ß(3) activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A(2) generation, alpha-granule protein secretion (ELISA) and protein phosphorylation from different signaling pathways (immunoblotting) were studied. RESULTS: Preincubation of platelets at temperatures higher than 37 °C (38.5-42 °C) inhibited thrombin-induced hemostasis, including platelet adhesion, aggregation, ATP release and thromboxane A(2) generation. The expression of P-selectin and CD63, as well as vascular endothelial growth factor (VEGF) release, was completely inhibited by hyperthermia, whereas von Willebrand factor (VWF) and endostatin levels remained substantially increased at high temperatures. This suggested that release of proteins from platelet granules is modulated not only by classical platelet agonists but also by microenvironmental factors. The observed gradation of response involved not only antiangiogenesis regulators, but also other cargo proteins. Some signaling pathways were more stable than others. While ERK1/2 and AKT phosphorylation were resistant to changes in temperature, Src, Syk, p38 phosphorylation and IkappaB degradation were decreased in a temperature-dependent fashion. CONCLUSIONS: Higher temperatures, such as those observed with fever or tissue invasion, inhibit the hemostatic functions of platelets and selectively regulate the release of alpha-granule proteins.

The glycan-binding protein galectin-1 controls survival of epithelial cells along the crypt-villus axis of small intestine.

Intestinal epithelial cells serve as mechanical barriers and active components of the mucosal immune system. These cells migrate from the crypt to the tip of the villus, where different stimuli can differentially affect their survival. Here we investigated, using in vitro and in vivo strategies, the role of galectin-1 (Gal-1), an evolutionarily conserved glycan-binding protein, in modulating the survival of human and mouse enterocytes. Both Gal-1 and its specific glyco-receptors were broadly expressed in small bowel enterocytes. Exogenous Gal-1 reduced the viability of enterocytes through apoptotic mechanisms involving activation of both caspase and mitochondrial pathways. Consistent with these findings, apoptotic cells were mainly detected at the tip of the villi, following administration of Gal-1. Moreover, Gal-1-deficient (Lgals1(-/-)) mice showed longer villi compared with their wild-type counterparts in vivo. In an experimental model of starvation, fasted wild-type mice displayed reduced villi and lower intestinal weight compared with Lgals1(-/-) mutant mice, an effect reflected by changes in the frequency of enterocyte apoptosis. Of note, human small bowel enterocytes were also prone to this pro-apoptotic effect. Thus, Gal-1 is broadly expressed in mucosal tissue and influences the viability of human and mouse enterocytes, an effect which might influence the migration of these cells from the crypt, the integrity of the villus and the epithelial barrier function.

NF-kappaB inhibitors impair platelet activation responses.

BACKGROUND: Although platelets are anucleated cells, they express several transcription factors that exert non-genomic functions, including the positive and negative regulation of platelet activation. NF-kappaB is a major transcriptional regulator of genes involved in survival, proliferation and inflammation. OBJECTIVE: Because platelets play a critical role not only in hemostasis, but also in inflammation and tumor progression, we evaluated the role of NF-kappaB in platelet physiology. RESULTS: Immunofluorescence, Western blotting and ELISA studies revealed that platelets express IkappaBalpha and NF-kappaB, and that stimulation with thrombin triggers IkappaBalpha phosphorylation and degradation and the binding of platelet NF-kappaB p65 subunit to synthetic oligonucleotides containing the consensus sequence for NF-kappaB. Two specific unrelated inhibitors of NF-kappaB activation, BAY 11-7082 and Ro 106-9920, reduced PAC-1 and fibrinogen binding to integrin alpha(IIb)beta3 and restricted platelet spreading on immobilized fibrinogen. Both inhibitors impaired aggregation mediated by ADP, epinephrine, collagen or thrombin, but not arachidonic acid. ATP release, TXB2 formation, P-selectin expression, ERK phosphorylation and cPLA2 activity stimulated by thrombin were reduced in BAY 11-7082- or Ro 106-9920-treated platelets. Although bleeding time was not affected, ADP-induced platelet aggregation was impaired in mice treated with BAY 11-7082. CONCLUSIONS: Our results suggest that NF-kappaB may be a novel mediator of platelet responses. The blockade of platelet function by NF-kappaB inhibitors might be relevant in those clinical situations where these drugs are being considered for anti-tumor and/or anti-inflammatory therapy.

Nitric oxide: news from stem cells to platelets.

Nitric oxide (NO) is a diffusible, short-lived, diatomic free radical ubiquitously produced by mammalian cells. The generation of NO from L-arginine is enzymatically regulated by three different isoforms of NO synthases. The NO signaling pathway involves mainly the activation of soluble guanylyl cyclase to produce cyclic GMP (cGMP) as a second messenger and downstream mediator. In addition, the free radical activity of NO can cause cellular damage through a phenomenon known as nitrosative stress. NO is a pleiotropic biomodulator in several systems, including the cardiovascular, nervous and immune systems. In the hematopoietic system, NO is thought to be an autocrine or paracrine messenger but also an intracellular effector molecule. Megakaryopoiesis and subsequent thrombopoiesis occur through complex biologic steps that involve hematopoietic stem cell commitment to megakaryocytic lineage, megakaryocyte maturation and finally, platelet release. Here, we summarize the current knowledge regarding the role of exogenous and endogenous NO in hematopoietic stem cell biology, megakaryocyte development and platelet biogenesis as well as relevance of platelet-derived NO generation on platelet function. Dysregulation of NO synthesis has been observed in several diseases, and the evaluation of a series of pharmacological agents with the ability to modulate the NO/cGMP pathway in platelets will also be discussed.

Oncocalyxone A inhibits human platelet aggregation by increasing cGMP and by binding to GP Ibalpha glycoprotein.

BACKGROUND AND PURPOSE: Oncocalyxone A (OncoA) has a concentration-dependent anti-platelet activity. The present study aimed to further understand the mechanisms related to this effect. EXPERIMENTAL APPROACH: Human platelet aggregation was measured by means of a turbidimetric method. OncoA (32-256 microM) was tested against several platelet-aggregating agents, such as adenosine diphosphate (ADP), collagen, arachidonic acid (AA), ristocetin and thrombin. KEY RESULTS: OncoA completely inhibited platelet aggregation with a calculated mean inhibitory concentration (IC50-microM) of 122 for ADP, 161 for collagen, 159 for AA, 169 for ristocetin and 85 for thrombin. The anti-aggregatory activity of OncoA was not inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). OncoA, at a concentration that caused no significant anti-aggregatory activity, potentiated sodium nitroprusside (SNP) anti-aggregatory activity (18.8+/-2.9%-SNP vs 85.0+/-8.2%-SNP+OncoA). The levels of nitric oxide (NO) or cAMP were not altered by OncoA while cGMP levels were increased more than 10-fold by OncoA in resting or ADP-activated platelets. Flow cytometry revealed that OncoA does not interact with receptors for fibrinogen, collagen or P-selectin. Nevertheless, OncoA decreased the binding of antibodies to GP Ibalpha, a glycoprotein that is related both to von Willebrand factor and to thrombin-induced platelet aggregation. CONCLUSION AND IMPLICATIONS: OncoA showed anti-aggregatory activity in platelets that was associated with increased cGMP levels, not dependent on NO and with blocking GP Ibalpha glycoprotein. This new mechanism has the prospect of leading to new anti-thrombotic drugs.

Expression and characterization of HlyX hemolysin from Leptospira interrogans serovar Copenhageni Potentiation of hemolytic activity by LipL32

The HlyX, a putative hemolysin identified from the Leptospira genomes, was cloned, expressed in Escherichia coli, purified, and its hemolytic activity was confirmed. Mouse polyclonal antiserum against the recombinant HlyX recognized HlyX-related antigens in a panel of Leptospira species extracts and it was also able to abolish the hemolytic activity of HlyX. A mixture of HlyX and LipL32, a known hemolysin from Leptospira, induced hemolysis in a synergistic way that was fully inhibited by antiserum against either protein. Moreover, sera from patients with leptospirosis also recognized the recombinant HlyX, showing that it is presented to the host immune system during Leptospira infection.