Soledad Negrotto

Regulation of neutrophil extracellular trap formation by anti-inflammatory drugs

The formation of neutrophil extracellular traps (NETs) is a newly described phenomenon that increases the bacteria-killing ability and the inflammatory response of neutrophils. Because NET generation occurs in an inflammatory microenvironment, we examined its regulation by anti-inflammatory drugs. Treatment of neutrophils with dexamethasone had no effect, but acetylsalicylic acid (ASA) treatment prevented NET formation. NETosis was also abrogated by the presence of BAY 11-7082 [(E)-3-[4-methylphenylsulfonyl]-2-propenenitrile] and Ro 106-9920 [6-(phenylsulfinyl)tetrazolo[1,5-b]pyridazine], two structurally unrelated nuclear factor-κB (NF-κB) inhibitors. The decrease in NET formation mediated by ASA, BAY-11-7082, and Ro 106-9920 was correlated with a significant reduction in the phosphorylation of NF-κB p65 subunit, indicating that the activation of this transcription factor is a relevant signaling pathway involved in the generation of DNA traps. The inhibitory effect of these drugs was also observed when NET generation was induced under acidic or hyperthermic conditions, two stress signals of the inflammatory microenvironment. In a mouse peritonitis model, while pretreatment of animals with ASA or BAY 11-7082 resulted in a marked suppression of NET formation along with increased bacteremia, dexamethasone had no effect. Our results show that NETs have an important role in the local control of infection and that ASA and NF-κB blockade could be useful therapies to avoid undesired effect of persistent neutrophil activation.

Acidosis downregulates platelet haemostatic functions and promotes neutrophil proinflammatory responses mediated by platelets

Acidosis is one of the hallmarks of tissue injury such as trauma, infection, inflammation, and tumour growth. Although platelets participate in the pathophysiology of all these processes, the impact of acidosis on platelet biology has not been studied outside of the quality control of laboratory aggregation assays or platelet transfusion optimization. Herein, we evaluate the effect of physiologically relevant changes in extracellular acidosis on the biological function of platelets, placing particular emphasis on haemostatic and secretory functions. Platelet haemostatic responses such as adhesion, spreading, activation of αIIbβ3 integrin, ATP release, aggregation, thromboxane B2 generation, clot retraction and procoagulant activity including phosphatidilserine exposure and microparticle formation, showed a statistically significant inhibition of thrombin-induced changes at pH of 7.0 and 6.5 compared to the physiological pH (7.4). The release of alpha granule content was differentially regulated by acidosis. At low pH, thrombin or collagen-induced secretion of vascular endothelial growth factor and endostatin were dramatically reduced. The release of von Willebrand factor and stromal derived factor-1α followed a similar, albeit less dramatic pattern. In contrast, the induction of CD40L was not changed by low pH, and P-selectin exposure was significantly increased. While the generation of mixed platelet-leukocyte aggregates and the increased chemotaxis of neutrophils mediated by platelets were further augmented under acidic conditions in a P-selectin dependent manner, the increased neutrophil survival was independent of P-selectin expression. In conclusion, our results indicate that extracellular acidosis downregulates most of the haemostatic platelet functions, and promotes those involved in amplifying the neutrophil-mediated inflammatory response.

Regulation of platelet responses triggered by Toll-like receptor 2 and 4 ligands is another non-genomic role of nuclear factor-kappaB.

INTRODUCTION Platelets express Toll-like receptors (TLRs) that recognise molecular components of pathogens and, in nucleated cells, elicit immune responses through nuclear factor-kappaB (NF-κB) activation. We have shown that NF-κB mediates platelet activation in response to classical agonists, suggesting that this transcription factor exerts non-genomic functions in platelets. The aim of this study was to determine whether NF-κB activation is a downstream signal involved in TLR2 and 4-mediated platelet responses. MATERIAL AND METHODS Aggregation and ATP release were measured with a Lumi-aggregometer. Fibrinogen binding, P-selectin and CD40 ligand (CD40L) levels and platelet-neutrophil aggregates were measured by cytometry. I kappa B alpha (IκBα) degradation and p65 phosphorylation were determined by Western blot and von Willebrand factor (vWF) by ELISA. RESULTS Platelet stimulation with Pam3CSK4 or LPS resulted in IκBα degradation and p65 phosphorylation. These responses were suppressed by TLR2 and 4 blocking and synergised by thrombin. Aggregation, fibrinogen binding and ATP and vWF release were triggered by Pam3CSK4. LPS did not induce platelet responses per se, except for vWF release, but it did potentiate thrombin-induced aggregation, fibrinogen binding and ATP secretion. Pam3CSK4, but not LPS, induced P-selectin and CD40L expression and mixed aggregate formation. All of these responses, except for CD40L expression, were inhibited in platelets treated with the NF-κB inhibitors BAY 11-7082 or Ro 106-9920. CONCLUSION TLR2 and 4 agonists trigger platelet activation responses through NF-κB. These data show another non-genomic function of NF-κB in platelets and highlight this molecule as a potential target to prevent platelet activation in inflammatory or infectious diseases.

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

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.

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

Summary.  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), αIIbβ3 activation (flow cytometry), aggregation (turbidimetry), ATP release (luminescence), thromboxane A2 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 A2 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.

Binding of galectin-1 to αIIbβ3 integrin triggers “outside-in” signals, stimulates platelet activation, and controls primary hemostasis

Understanding noncanonical mechanisms of platelet activation represents an important challenge for the identification of novel therapeutic targets in bleeding disorders, thrombosis, and cancer. We previously reported that galectin‐1 (Gal‐1), a β‐galactoside‐binding protein, triggers platelet activation in vitro. Here we investigated the molecular mechanisms underlying this function and the physiological relevance of endogenous Gal‐1 in hemostasis. Mass spectrometry analysis, as well as studies using blocking antibodies against the anti‐αIIb subunit of αIIbβ3 integrin or platelets from patients with Glanzmanns thrombasthenia syndrome (αIIbβ3 deficiency), identified this integrin as a functional Gal‐1 receptor in platelets. Binding of Gal‐1 to platelets triggered the phosphorylation of β3‐integrin, Syk, MAPKs, PI3K, PLCγ2, thromboxane (TXA2) release, and Ca2+ mobilization. Not only soluble but also immobilized Gal‐1 promoted platelet activation. Gal‐1‐deficient (Lgals1–/–) mice showed increased bleeding time (P< 0.0002, knockout vs. wild type), which was not associated with an abnormal platelet count. Lgals1–/– platelets exhibited normal aggregation to PAR4, ADP, arachidonic acid, or collagen but abnormal ATP release at low collagen concentrations. Impaired spreading on fibrinogen and clot retraction with normal levels of αIIbβ3 was also observed in Lgals1–/– platelets, indicating a failure in the “outside‐in” signaling through this integrin. This study identifies a noncanonical mechanism, based on galectin‐integrin interactions, for regulating platelet activation.—Romaniuk, M. A., Croci, D. O., Lapponi, M. J., Tribulatti, M. V., Negrotto, S., Poirier, F., Campetella, O., Rabinovich, G. A., Schattner, M. Binding of galectin‐1 to αIIbβ3 integrin triggers “outside‐in” signals, stimulates platelet activation, and controls primary hemostasis. FASEB J. 26, 2788–2798 (2012). www.fasebj.org

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

Summary.  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‐β 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‐β 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‐β, 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.

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

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.

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

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.

The low viability of human CD34+ cells under acidic conditions is improved by exposure to thrombopoietin, stem cell factor, interleukin-3, or increased cyclic adenosine monophosphate levels

BACKGROUND: Transplanted hematopoietic progenitor cells (CD34+) have shown great promise in regenerative medicine. However, the therapeutic potential of transplanted cells is limited by their poor viability. It is well known that the microenvironment in which progenitors reside substantially affects their behavior. Because extracellular acidosis is a common feature of injured tissues or the tumor microenvironment and is a critical regulator of cell survival and activation, we evaluated the impact of acidosis on CD34+ cell biology.