Synergistic inhibition of platelet function by NADPH oxidase inhibitors and clopidogrel

Previous studies have shown platelets play an important role in prothrombotic complications due to several factors such as hyperglycemia, oxidative stress, and hypercholesterolemia, which affected platelets reactivity. Platelets activation involves ADP stimulation via P2Y12 receptor, whereas reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox) act as the second messenger, which involved in platelets activation and may contribute to thrombus formation. The aim of the present study was to investigate the influence of Nox on the purinergic receptor (P2Y12 receptor) in activation of human platelets function stimulated by platelets agonist. This research explored the effects of Nox inhibitors and clopidogrel either alone or in combination, on various agonist-stimulated human platelets, including platelets aggregation and adhesion measured by modified LTA, expression of platelets activation markers, and calcium mobilization using flow cytometry, and ROS formation, NADPH oxidase activity, as well as cAMP levels by chemiluminescence assay. Taken together, findings from these experiments suggest that the combination of clopidogrel and Nox inhibitors synergistically reduced platelets aggregation, platelets adhesion, and expression of platelets activation marker during late activation, ROS formation, NADPH oxidase activity, calcium mobilization and increased cAMP levels in vitro. This combination showed that P2Y12receptor reactivity was influenced by the activation of NADPH oxidase. Thus, these data demonstrated a potential combination therapy to reduce the risk of thrombosis formation.

The TGF-β /NADPH Oxidases Axis in the Regulation of Liver Cell Biology in Health and Disease

The Transforming Growth Factor-beta (TGF-β) pathway plays essential roles in liver development and homeostasis and become a relevant factor involved in different liver pathologies, particularly fibrosis and cancer. The family of NADPH oxidases (NOXs) has emerged in recent years as targets of the TGF-β pathway mediating many of its effects on hepatocytes, stellate cells and macrophages. This review focuses on how the axis TGF-β/NOXs may regulate the biology of different liver cells and how this influences physiological situations, such as liver regeneration, and pathological circumstances, such as liver fibrosis and cancer. Finally, we discuss whether NOX inhibitors may be considered as potential therapeutic tools in liver diseases.

Mitochondrial dynamics and reactive oxygen species initiate thrombopoiesis from mature megakaryocytes

Blood platelets are essential for controlling hemostasis. They are released by megakaryocytes (MKs) located in the bone marrow, upon extension of cytoplasmic protrusions into the lumen of bone marrow sinusoids. Their number increases in postpulmonary capillaries, suggesting a role for oxygen gradient in thrombopoiesis (ie, platelet biogenesis). In this study, we show that initiation of thrombopoiesis from human mature MKs was enhanced under hyperoxia or during pro-oxidant treatments, whereas antioxidants dampened it. Quenching mitochondrial reactive oxygen species (mtROS) with MitoTEMPO decreased thrombopoiesis, whereas genetically enhancing mtROS by deacetylation-null sirtuin-3 expression increased it. Blocking cytosolic ROS production by NOX inhibitors had no impact. Classification according to the cell roundness index delineated 3 stages of thrombopoiesis in mature MKs. Early-stage round MKs exhibited the highest index, which correlated with low mtROS levels, a mitochondrial tubular network, and the mitochondrial recruitment of the fission activator Drp1. Intermediate MKs at the onset of thrombopoiesis showed high mtROS levels and small, well-delineated mitochondria. Terminal MKs showed the lowest roundness index and long proplatelet extensions. Inhibiting Drp1-dependent mitochondrial fission of mature MKs by Mdivi-1 favored a tubular mitochondrial network and lowered both mtROS levels and intermediate MKs proportion, whereas enhancing Drp1 activity genetically had opposite effects. Reciprocally, quenching mtROS limited mitochondrial fission in round MKs. These data demonstrate a functional coupling between ROS and mitochondrial fission in MKs, which is crucial for the onset of thrombopoiesis. They provide new molecular cues that control initiation of platelet biogenesis and may help elucidate some unexplained thrombocytopenia.

5-Hydroxytryptamine Modulates Maturation and Mitochondria Function of Human Oligodendrocyte Progenitor M03-13 Cells

Inside the adult CNS, oligodendrocyte progenitor cells (OPCS) are able to proliferate, migrate and differentiate into mature oligodendrocytes (OLs) which are responsible for the production of myelin sheet and energy supply for neurons. Moreover, in demyelinating diseases, OPCs are recruited to the lesion areas where they undergo differentiation and myelin synthesis. Serotonin (5-hydroxytryptamine, 5-HT) is involved in OLs’ development and myelination, but so far the molecular mechanisms involved or the effects of 5-HT on mitochondria function have not yet been well documented. Our data show that 5-HT inhibits migration and proliferation committing cells toward differentiation in an immortalized human oligodendrocyte precursor cell line, M03-13. Migration blockage is mediated by reactive oxygen species (ROS) generation since antioxidants, such as Vit C and Cu-Zn superoxide dismutase, prevent the inhibitory effects of 5-HT on cell migration. 5-HT inhibits OPC migration and proliferation and increases OL phenotypic markers myelin basic protein (MBP) and Olig-2 via protein kinase C (PKC) activation since the inhibitor of PKC, bis-indolyl-maleimide (BIM), counteracts 5-HT effects. NOX inhibitors as well, reverse the effects of 5-HT, indicating that 5-HT influences the maturation process of OPCs by NOX-dependent ROS production. Finally, 5-HT increases mitochondria function and antioxidant activity. The identification of the molecular mechanisms underlying the effects of 5-HT on maturation and energy metabolism of OPCs could pave the way for the development of new treatments for autoimmune demyelinating diseases such as Multiple Sclerosis where oligodendrocytes are the primary target of immune attack.

P03.27 Role of NOX2 for hypoxia-induced chemoresistance in acute myeloid leukemia

BackgroundRelapse of acute myeloid leukemia (AML) may arise from residual chemoresistant leukemic cells. A hypoxic tumor microenvironment, such as the bone marrow, is known to enhance chemoresistance in various forms of cancer, including AML. Hypoxia inducible factor 1 alpha (HIF-1α) is an important mediator of cellular adaptation to hypoxia. HIF-1α is a constitutively expressed transcription factor that is rapidly degraded under normoxic conditions after hydroxylation by oxygen sensors, such as the HIF prolyl hydroxylases (PHDs). However, under hypoxic conditions the oxygen sensors lose the ability to induce the degradation of HIF-1α resulting in its stabilization and translocation to the nucleolus where it induces the transcription of genes associated with glucose metabolism, angiogenesis, and cell survival. This may result in proliferation of malignant cells, impaired tumor cell differentiation and chemoresistance. Reactive oxygen species (ROS) have been shown to inhibit PHDs and may thereby stabilize HIF-1α, and may thus contribute to chemoresistance. AML cells may generate ROS via the myeloid NADPH oxidase NOX2. We therefore hypothesized that NOX inhibitors would decrease chemoresistance in a hypoxic environment.Materials and MethodsThe wild type (WT) AML cell line PLB-985 and its NOX2 knocked out (KO) counterpart were cultured for five days in hypoxia (1% oxygen) or normoxia (21% oxygen) in the presence or absence of the NOX inhibitors histamine dihydrochloride (HDC), diphenyleneiodonium (DPI) and GSK2795039. Thereafter cells were exposed to the chemotherapeutic agent daunorubicin for 48 hours (in hypoxia or normoxia) and cell death was determined using the XTT assay. Stabilization of HIF-1α was measured either by western blot or flow cytometry. Differentiation of cells was quantified by measuring the expression of CD14 and CD11b by flow cytometry.ResultsHypoxia reduced the sensitivity of WT PLB-985 cells to daunorubicin induced cell death (P < 0.05, n=4) whereas NOX2 KO cells were equally sensitive to daunorubicin in hypoxia and normoxia (P > 0.5, n=4). Furthermore, NOX2 KO AML cells displayed increased sensitivity to daunorubicin induced killing compared with PLB WT cells in a hypoxic environment (P < 0.05, n=4). Preliminary results show that pharmacological NOX inhibition using DPI enhanced the sensitivity of WT AML cells to daunorubicin induced killing. These results suggests that functional NOX2 contributes to chemoresistance in a hypoxic environment. As expected, hypoxia stabilized the expression of HIF-1α in AML cells. Preliminary results suggest that HIF-1α expression was reduced in the presence of NOX inhibitors.ConclusionsGenetic deletion or pharmacological inhibition of NOX2 sensitized AML cells to daunorubicin induced killing in hypoxic environments. NOX2 may thus be a target for overcoming chemoresistance in AML cells in the hypoxic bone marrow environment.SupportThis work was supported by Assar Gabrielsson’s Foundation( FB19-64) and other grants used by the research group.Disclosure InformationS. Paul: None. H. Grauers Wiktorin: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Patent. R. Kiffin: None. K. Hellstrand: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Patent. A. Martner: E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Patent.

VAS2870 and VAS3947 attenuate platelet activation and thrombus formation via a NOX-independent pathway downstream of PKC

NADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by treatment with ML171, GSK2795039, and GKT136901/GKT137831 did not affect thrombin and U46619-induced platelet aggregation. These findings suggest that VAS compounds may inhibit platelet aggregation via a NOX-independent manner. Thus, we aimed to investigate the detailed antiplatelet mechanisms of VAS compounds. The data revealed that VAS compounds blocked various agonist-induced platelet aggregation, possibly via blocking PKC downstream signaling, including IKKβ and p38 MAPK, eventually reducing platelet granule release, calcium mobilization, and GPIIbIIIa activation. In addition, VAS compounds inhibited mouse platelet aggregation-induced by collagen and thrombin. The in vivo study also showed that VAS compounds delayed thrombus formation without affecting normal hemostasis. This study is the first to demonstrate that, in addition to inhibiting NOX activity, VAS compounds reduced platelet activation and thrombus formation through a NOX-independent pathway downstream of PKC. These findings also indicate that VAS compounds may be safe and potentially therapeutic agents for treating patients with cardiovascular diseases.

VAS2870 Inhibits Histamine-Induced Calcium Signaling and vWF Secretion in Human Umbilical Vein Endothelial Cells

In this study, we investigated the effects of NAD(P)H oxidase (NOX) inhibitor VAS2870 (3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine) on the histamine-induced elevation of free cytoplasmic calcium concentration ([Ca2+]i) and the secretion of von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) and on relaxation of rat aorta in response to histamine. At 10 μM concentration, VAS2870 suppressed the [Ca2+]i rise induced by histamine. Inhibition was not competitive, with IC50 3.64 and 3.22 μM at 1 and 100 μM concentrations of histamine, respectively. There was no inhibition of [Ca2+]i elevation by VAS2870 in HUVECs in response to the agonist of type 1 protease-activated receptor SFLLRN. VAS2870 attenuated histamine-induced secretion of vWF and did not inhibit basal secretion. VAS2870 did not change the degree of histamine-induced relaxation of rat aortic rings constricted by norepinephrine. We suggest that NOX inhibitors might be used as a tool for preventing thrombosis induced by histamine release from mast cells without affecting vasorelaxation.