Histones Induce the Procoagulant Phenotype of Endothelial Cells through Tissue Factor Up-Regulation and Thrombomodulin Down-Regulation

<div><p>The high circulating levels of histones found in various thrombotic diseases may compromise the anticoagulant barrier of endothelial cells. We determined how histones affect endothelial procoagulant tissue factor (TF) and anticoagulant thrombomodulin (TM). Surface antigens, soluble forms, and mRNA levels of TF and TM were measured by flow cytometry, ELISA, and real-time RT-PCR, respectively. TF and TM activity were measured using procoagulant activity, thrombin generation, or chromogenic assays. Involvement of the toll-like receptor (TLR) was assessed using the neutralizing antibodies. Histones dose-dependently induced surface antigens, activity and mRNA levels of endothelial TF. Histone-treated endothelial cells significantly shortened the lag time and enhanced the endogenous thrombin potential of normal plasma, which was normalized by a TF neutralizing antibody. Histones induced phosphatidylserine and protein-disulfide isomerase expression in endothelial cells. Histones also reduced the surface antigen, activity, and mRNA levels of endothelial TM. Polysialic acid and heparin reversed the histone-induced TF up-regulation and TM down-regulation. Activated protein C did not affect the TF up-regulation, but interrupted TM down-regulation. TLR2, and TLR4 inhibitors partially blocked the TF up-regulation. Histones induced the endothelial procoagulant phenotype through TF up-regulation and TM down-regulation. The effects of histones were partly mediated by TLR2, TLR4. Strategies to inhibit the harmful effects of histones in endothelial cells may be required in order to prevent a thrombotic environment.</p></div

Histones induced TF expression in endothelial cells.

<p>(A) After EA.hy926 cells were stimulated with various levels of calf thymus histones for 4 h, the surface expression of TF antigens was determined by flow cytometry. A summary of the TF surface expression stimulated by calf thymus histones is shown in the right panel. (B) The procoagulant activity of TF was measured in EA.hy926 cells incubated with various levels of calf thymus histones for 4 h. (C) The expression of TF mRNA was quantified using real-time RT-PCR in EA.hy926 cells incubated with various levels of calf thymus histones for 3 h. (D) EA.hy926 cells were either stimulated with or without 50 μg/mL calf thymus histones for 4 h, and incubated with PBS (control), mouse isotype IgG (Iso-IgG, 30 μg/mL), or inhibitory TF antibody (anti-TF, 30 μg/mL) for 10 minutes. The ETP and lag time were analyzed using a thrombin generation assay. (E) EA.hy926 cells were stimulated with individual human recombinant histone (20 μg/mL H1, H2A/H2B, H3.3, H4) for 4 h, and the expression of surface TF antigens was determined by flow cytometry. All data are presented as mean ± SEM. The data were combined data from 3 or more different experiments. * <i>P<</i>0.05 vs. control (calf thymus histones not treated).</p

Neutralization of the histone effect by PSA, heparin, and APC.

<p>Calf thymus histones were pretreated with or without 62.5 μM PSA, 100 IU/mL heparin, and 100 nM APC for 15 min. The pretreated calf thymus histones (50 μg/mL) were added to the EA.hy926 cells for 4 h. The surface expression levels of TF (A) and TM (B) were measured by flow cytometry. All data are presented as mean ± SEM. The data were combined data from 3 or more different experiments. * <i>P<</i>0.05 vs. control (calf thymus histones not treated), ^† <i>P<</i>0.05 vs. histone-treated.</p

Histones increased PDI expression in endothelial cells.

<p>(A) EA.hy926 cells were stimulated with various levels of calf thymus histones for 4 h, and the levels of surface PDI expression were determined by flow cytometry. (B) EA.hy926 cells were pre-incubated with PBS (control) or inhibitors, such as anti-PDI antibody (RL90, 10 μg/mL), glutathione (7.5 mM), and quercetin (200 μM) for 1 h. The cells were then stimulated with or without 50 μg/mL calf thymus histones for 4 h. The ETP and lag time were analyzed using a thrombin generation assay. All data are presented as mean ± SEM. The data were combined data from 3 or more different experiments. * <i>P<</i>0.05 vs. control (calf thymus histones not treated), ^† <i>P<</i>0.05 vs. histone-treated cells.</p

Inhibition of TLR in the histone-induced procoagulant phenotype.

<p>EA.hy926 cells were pre-incubated with mouse isotype IgG (Iso-IgG, 50 μg/mL), mouse anti-human TLR2 (aTLR2, 50 μg/mL), or anti-human TLR4 (aTLR4, 50 μg/mL) for 30 min, and then stimulated with or without 50 μg/mL calf thymus histones for 4 h. The surface expression levels of TF and TM were determined by flow cytometry. All data are presented as mean ± SEM. The data were combined data from 3 or more different experiments. * <i>P<</i>0.05 vs. control (calf thymus histones not treated), ^† <i>P<</i>0.05 vs. histone-treated, ^‡ <i>P<</i>0.1 vs. histone-treated.</p

Histones induced phosphatidylserine exposure in endothelial cells.

<p>(A) EA.hy926 cells were stimulated with various levels of calf thymus histones for 4 h, and the surface phosphatidylserine level was determined by flow cytometer. (B) EA.hy926 cells were stimulated with or without 50 μg/mL calf thymus histones for 4 h, and incubated with PBS (vehicle) or 10 μg/mL annexin V for 15 min. The ETP and lag time were analyzed using a thrombin generation assay. All data are presented as mean ± SEM. The data were combined data from 3 or more different experiments. * <i>P<</i>0.05 vs. control (calf thymus histones not treated).</p

Extracellular Histone Released from Leukemic Cells Increases Their Adhesion to Endothelium and Protects them from Spontaneous and Chemotherapy-Induced Leukemic Cell Death

<div><p>Introduction</p><p>When leukocytes are stimulated by reactive oxygen species (ROS), they release nuclear contents into the extracellular milieu, called by extracellular traps (ET). The nuclear contents are mainly composed of the histone–DNA complex and neutrophil elastase. This study investigated whether leukemic cells could release ET and the released histone could induce endothelial activation, eventually resulting in leukemic progression.</p><p>Methods</p><p>The circulating ET were measured in 80 patients with hematologic diseases and 40 healthy controls. ET formation and ROS levels were investigated during leukemic cell proliferation <i>in vitro</i>. Histone-induced endothelial adhesion molecules expression and cell survival were measured by flow cytometry.</p><p>Results</p><p>Acute leukemia patients had high levels of ET, which correlated with peripheral blast count. Leukemic cells produced high ROS levels and released extracellular histone, which was significantly blocked by antioxidants. Histone significantly induced 3 endothelial adhesion molecules expression, and promoted leukemic cell adhesion to endothelial cells, which was inhibited by histone inhibitors (heparin, polysialic acid, and activated protein C), neutralizing antibodies against these adhesion molecules, and a Toll like receptor(TLR)9 antagonist. When leukemic cells were co-cultured with endothelial cells, adherent leukemic cells showed better survival than the non-adherent ones, demonstrating that histone-treated endothelial cells protected leukemic cells from both spontaneous and chemotherapy-induced death.</p><p>Conclusion</p><p>Our data demonstrate for the first time that extracellular histone can be released from leukemic cells through a ROS-dependent mechanism. The released histone promotes leukemic cell adhesion by inducting the surface expression of endothelial adhesion molecules and eventually protects leukemic cells from cell death.</p></div

The baseline characteristics and laboratory results of the study populations.

<p>The baseline characteristics and laboratory results of the study populations.</p

Multiple regression analyses for determination of contributing factors to histone-DNA complex, cell free dsDNA and neutrophil elastase levels in patients

<p>Multiple regression analyses for determination of contributing factors to histone-DNA complex, cell free dsDNA and neutrophil elastase levels in patients</p

Circulating histone levels are increased in patients with acute leukemia and correlate with peripheral blast counts.

<p>(A) Circulating levels of the histone–DNA complex in normal controls (n = 40) and in patients with acute leukemia (n = 21), myeloproliferative neoplasms (MPN) with neutrophilia (n = 13), MPN without neutrophilia (n = 32), and aplastic anemia (n = 14). (B, C, D) Correlation of peripheral blast count with circulating levels of histone–DNA complex, cell-free double-stranded DNA (dsDNA), and neutrophil elastase in acute leukemia patients.</p