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 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

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

Histological studies of OA knee joints induced by surgical induction techniques.

<p>(A-C) Macroscopic images of hematoxylin and eosin staining of the rat animal model. (D-F) Macroscopic images of safranin O staining of the rat animal model. (G, H) Macroscopic images of hematoxylin and eosin staining of the rabbit animal model. (I, J) Macroscopic images of alcian blue staining of the rabbit animal model. (Scale bars: 200 μm).</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

Overview of purine metabolism by HGPRT.

The purine metabolic system represents a pathway by which hypoxanthine and guanine can be salvaged by HGPRT. HGPRT serves to catalyze the salvage synthesis of IMP and GMP from the purine bases hypoxanthine and guanine. HGPRT also prevents the accumulation of substrates that are converted to uric acid.</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

Effects of <i>S</i>. <i>thermophilus</i> IDCC 2201 on gut microbiota in SD rats with PO-induced hyperuricemia.

Distribution of gut microbiota at the (A) phylum and (B) order levels. (C) Taxonomic levels from the phylum to the genus (LDA score > 3.5, p S. thermophilus IDCC 2201 (1×108 CFU/ day); G6: L. plantarum CR1+S. thermophilus IDCC 2201 (1×108 CFU/ day); and G9: Allopurinol (50 mg/kg rat/day). A significant difference from G1 is denoted as * p † p †† p # p ## p < 0.01.</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

OA induction procedures for anterior cruciate ligament transection (ACLT) in the animal models.

<p>(A-D) The ACLT images for the rat animal model. (A) A vertical midline incision was made in the skin, followed by the elimination of the fat pad, and then, the anterior cruciate ligament (ACL) was exposed in the knee joint. (B) The ACL was dissected with a surgical scissor. (C) The medial meniscus was completely removed, and the medial collateral ligament (MCL) was transected too. (D) The image shows the extracted medial meniscus from the knee joint of rats. (E-H) The ACLT images for the rabbit animal model. (E) After a vertical midline incision, the patella was pushed laterally to expose the ACL. (F) The exposed ACL was cut in the rabbit model. (G) The medial meniscus was removed, and the MCL was transected without injury to the cartilage. (H) The image shows the completely extracted medial meniscus from the rabbit knee joint.</p