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Background Patients undergoing deep vein thrombosis (VT) have over 30% recurrence, directly increasing their risk of post-thrombotic syndrome. Current murine models of inferior vena cava (IVC) VT model host one thrombosis event. Objective We aimed to develop a murine model to study IVC recurrent VT in mice. Materials and Methods An initial VT was induced using the electrolytic IVC model (EIM) with constant blood flow. This approach takes advantage of the restored vein lumen 21 days after a single VT event in the EIM demonstrated by ultrasound. We then induced a second VT 21 days later, using either EIM or an IVC ligation model for comparison. The control groups were a sham surgery and, 21 days later, either EIM or IVC ligation. IVC wall and thrombus were harvested 2 days after the second insult and analysed for IVC and thrombus size, gene expression of fibrotic markers, histology for collagen and Western blot for citrullinated histone 3 (Cit-H3) and fibrin. Results Ultrasound confirmed the first VT and its progressive resolution with an anatomical channel allowing room for the second thrombus by day 21. As compared with a primary VT, recurrent VT has heavier walls with significant up-regulation of transforming growth factor-β (TGF-β), elastin, interleukin (IL)-6, matrix metallopeptidase 9 (MMP9), MMP2 and a thrombus with high citrullinated histone-3 and fibrin content. Conclusion Experimental recurrent thrombi are structurally and compositionally different from the primary VT, with a greater pro-fibrotic remodelling vein wall profile. This work provides a VT recurrence IVC model that will help to improve the current understanding of the biological mechanisms and directed treatment of recurrent VT.

Objective: Antiphospholipid syndrome (APS) is a leading acquired cause of thrombotic events. While antiphospholipid antibodies have been shown to promote thrombosis in mice, the role of neutrophils has not been explicitly studied. Here, we characterized neutrophils in the context of a new model of antiphospholipid antibody-mediated venous thrombosis. Methods: Mice were administered IgG fractions prepared from patients with APS. At the same time, flow through the inferior vena cava was reduced by a standard stenosis. Resulting thrombi were characterized for size and neutrophil content. Circulating factors and the vessel wall were also assessed. Results: As measured by both thrombus weight and thrombosis frequency, mice treated with IgG from patients with APS demonstrated exaggerated thrombosis as compared with controls. APS thrombi were enriched for citrullinated histone H3 (a marker of neutrophil extracellular traps/NETs). APS mice also demonstrated elevated levels of circulating cell-free DNA and human IgG bound to the neutrophil surface. In contrast, circulating neutrophil numbers and markers of vessel wall activation were not appreciably different between APS and control mice. Regarding therapy, treatment with either deoxyribonuclease (which dissolves NETs) or a neutrophil-depleting antibody reduced thrombosis in APS mice to the level seen in controls. Conclusion: These data support a mechanism whereby circulating neutrophils are primed by antiphospholipid antibodies to accelerate thrombosis. This line of investigation suggests new, immunomodulatory approaches for the treatment of APS. This article is protected by copyright. All rights reserved.

SummaryVenous thromboembolism is a major cause of death during and immediately post-sepsis. Venous thrombosis (VT) is mediated by cell adhesion molecules and leukocytes, including neutrophil extracellular traps (NETs). Sepsis, or experimentally, endotoxaemia, shares similar characteristics and is modulated via toll like receptor 4 (TLR4). This study was undertaken to determine if endotoxaemia potentiates early stasis thrombogenesis, and secondarily to determine the role of VT TLR4, ICAM-1 and neutrophils (PMNs). Wild-type (WT), ICAM-1-/- and TLR4-/- mice underwent treatment with saline or LPS (10 mg/kg i.p.) alone, or followed by inferior vena cava (IVC) ligation to generate stasis VT. In vivo microscopy of leukocyte trafficking was performed in non-thrombosed mice, and tissue and plasma were harvested during early VT formation. Pre-thrombosis, circulating ICAM-1 was elevated and increased leukocyte adhesion and rolling occurred on the IVC of LPS-treated mice. Post-thrombosis, endotoxaemic mice formed larger, platelet-poor thrombi. Endotoxaemic TLR4-/- mice did not have an augmented thrombotic response and exhibited significantly decreased circulating ICAM-1 compared to endotoxaemic WT controls. Endotoxaemic ICAM-1-/- mice had significantly smaller thrombi compared to controls. Hypothesising that PMNs localised to the inflamed endothelium were promoting thrombosis, PMN depletion using anti-Ly6G antibody was performed. Paradoxically, VT formed without PMNs was amplified, potentially related to endotoxaemia induced elevation of PAI-1 and circulating FXIII, and decreased uPA. Endotoxaemia enhanced early VT occurs in a TLR-4 and ICAM-1 dependent fashion, and is potentiated by neutropenia. ICAM-1 and/or TLR-4 inhibition may be a unique strategy to prevent sepsis-associated VT.Supplementary Material to this article is available online at www.thrombosis-online.com.

SummaryDeep-vein thrombosis (DVT) resolves via a sterile inflammatory response. Defining the inflammatory response of DVT may allow for new therapies that do not involve anticoagulation. Previously, we have shown that Toll-like receptor 9 (Tlr9) gene deleted mice had impaired venous thrombosis (VT) resolution. Here, we further characterise the role of Tlr9 signalling and sterile inflammation in chronic VT and vein wall responses. First, we found a human precedent exists with Tlr9 + cells present in chronic post thrombotic intraluminal tissue. Second, in a stasis VT mouse model, endogenous danger signal mediators of uric acid, HMGB-1, and neutrophil extracellular traps marker of citrullinated histone-3 (and extracellular DNA) were greater in Tlr9-/- thrombi as compared with wild-type (WT), corresponding with larger VT at 8 and 21 days. Fewer M1 type (CCR2+) monocyte/macrophages (MØ) were present in Tlr9-/- thrombi than WT controls at 8 days, suggesting an impaired inflammatory cell influx. Using bone marrow-derived monocyte (BMMØ) cell culture, we found decreased fibrinolytic gene expression with exposure to several endogenous danger signals. Next, adoptive transfer of cultured Tlr9+/+ BMMØ to Tlr9-/- mice normalised VT resolution at 8 days. Lastly, although the VT size was larger at 21 days in Tlr9-/- mice and correlated with decreased endothelial antigen markers, no difference in fibrosis was found. These data suggest that Tlr9 signalling in MØ is critical for later VT resolution, is associated with necrosis clearance, but does not affect later vein wall fibrosis. These findings provide insight into the Tlr9 MØ mechanisms of sterile inflammation in this disease process.

Objective— Deep vein thrombosis (VT) can result in vein wall injury, which clinically manifests as post-thrombotic syndrome. Postinjury fibrosis may be modulated in part through cellular cysteine-cysteine receptor 7 (CCR7)–mediated events. We tested the hypothesis that late vein wall fibrotic remodeling is dependent on CCR7. Approach and Results— CCR7 −/− and C57BL/6 wild-type mice had inferior vena cava VT induced by nonstasis or stasis mechanisms. In both models, VT size was largest at day 1 and trended down by day 21, and CCR7 + cells peaked at day 8 in wild-type mice. No significant differences in VT resolution were found in CCR7 −/− as compared with wild type in either model. In the nonstasis VT model, vein wall changes consistent with fibrotic injury were evidenced by significant increases in collagen I, III, matrix metalloproteinase 2, and transforming growth factor-β gene expression, increases in α-smooth muscle actin and fibroblast specific protein-1 antigen, and total collagen at 8 days. Correspondingly, SM22α and fibroblast specific protein-1, but not DDR2 + cells, were increased at 8 days. Early wild-type thrombus exposure inhibited profibrotic gene expression in CCR7 −/− in ex vivo vein wall culture. Bone marrow chimera experiments further showed that circulating CCR7 + leukocytes partially rescued midterm profibrotic changes in CCR7 −/− mice. In human histological sections of chronic thrombosed femoral veins, CCR7 + cells were present in the fibrotic areas. Conclusions— Post-thrombotic vein wall remodeling is impaired in CCR7 −/− mice, with a profibrotic phenotype, is dependent on the thrombotic mechanism, and is mediated by circulating CCR7 + cells. Unlike other postinjury fibrotic responses, CCR7 + signaling may be important for positive vein wall remodeling after VT.

Post thrombotic syndrome therapy is primarily palliative, and the associated vein wall inflammatory mechanisms are unclear. Vein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP9 directly contributes to vein wall remodeling after VT is unknown.WT and MMP9 -/- mice underwent stasis VT by ligation of the inferior vena cava (IVC) and tissue was harvested at 2, 8, and 21days. Assessment of thrombus size, and gene, protein and structural vein wall determinations were done.VT resolution was increased in MMP9-/- mice as compared with controls at 21d only. The primary phenotypic fibrotic vein wall differences occurred at 8d post VT, with significantly less vein wall collagen content as assessed by Picosirius red staining in MMP9 -/- mice as compared with WT. Increased monocytic vein wall influx with less IL-1b and TGFb was found in MMP9 -/- vein walls as compared with WT. Corresponding levels of PAI-1 were increased in MMP9 -/- compared with WT, and no difference in FSP-1+cells as compared with controls.In stasis VT, MMP9 modulates midterm vein wall collagen content, with an altered local inflammatory and profibrotic environment, likely directed by monocytes. Thus, MMP9 plays a role in both vein wall responses as well as late thrombus resolution.

ObjectiveDeep vein thrombosis (DVT) resolution instigates an inflammatory response, resulting in vessel wall damage and scarring. Urokinase-plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), are integral components of the fibrinolytic system, essential for venous thrombosis (VT) resolution. This study determined the vein wall response when exposed to increased and decreased plasmin activity.MethodsA mouse inferior vena cava (IVC) ligation model in uPA −/− or PAI-1 −/− and their genetic wild types (B6/SvEv and C57/BL6, respectively) was used to create stasis thrombi, with tissue harvest at either 8 or 21 days. Tissue analysis included gene expression of vascular smooth muscle cells (alpha smooth muscle actin [αSMA], SM22) and endothelial marker (CD31), by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, matrix metalloproteinase (MMP)-2 and -9 activity by zymography, and vein wall collagen by picro-Sirius red histologic analysis. A P < .05 was considered significant.ResultsThrombi were significantly larger in both 8-day and 21-day uPA −/− as compared with wild type (WT) and were significantly smaller in both 8-day and 21-day PAI-1 −/− as compared with WT. Correspondingly, 8-day plasmin levels were reduced in half in uPA −/− and increased three-fold in PAI-1 −/− when compared with respective WT thrombi (P < .05; n = 5-6). The endothelial marker CD31 was elevated two-fold in PAI-1 −/− mice at 8 days, but reduced 2.5-fold at 21 days in uPA −/− as compared with WT (P = .02; n = 5-6), suggesting less endothelial preservation. Vein wall vascular smooth muscle cell (VSMC) gene expression showed that 8-day and 21-day PAI-1 −/− mice had 2.3- and 3.8-fold more SM22 and 1.8- and 2.3-fold more αSMA expression than respective WT (P < .05; n = 5-7), as well as 1.8-fold increased αSMA (+) cells (P ≤ .05; n = 3-5). No significant difference in MMP-2 or -9 activity was found in the PAI-1 −/− mice compared with WT, while 5.4-fold more MMP-9 was present in 21-day WT than 21-day uPA −/− (P = .03; n = 5). Lastly, collagen was ∼two-fold greater at 8 days in PAI-1 −/− IVC as compared with WT (P = .03; n = 6) with no differences observed in uPA −/− mice.ConclusionsIn stasis DVT, plasmin activity is critical for thrombus resolution. Divergent vein wall responses occur with gain or loss of plasmin activity, and despite smaller VT, greater vein wall fibrosis was associated with lack of PAI-1.Clinical RelevanceDVT resolution is dependent on many factors. Clinical studies suggest that slowly resolving extensive DVTs are associated with greater likelihood of post-thrombotic syndrome, and this is experimentally modeled by vein wall fibrosis and inflammation. The current study suggests a more complex relationship in that the plasmin-mediated events are central to thrombus resolution as well as to how the vessel wall responds. Surprisingly, a larger thrombus from a stasis model is not associated with greater vein wall damage. Thus, novel modalities such as PAI-1 inhibition may require supplemental anticoagulants for full efficacy.

Background: Venous thrombosis (VT) and the vein wall responses may be related to interleukin 6 (IL-6) signaling in humans and mice. Inflammatory and fibrotic effects of IL-6 largely signal through the trans pathway: IL-6/soluble IL-6Rα complex associates with ubiquitously expressed signal transducing protein gp130. We hypothesized that IL-6 signaling would differ depending upon thrombosis mechanism (complete v. partial stasis), and disruption of IL-6/IL-6Rα/gp130 axis would alter vein wall response to VT. Methods: Wild type C57BL/6 or IL-6-/- mice underwent induction of VT via inferior vena cava (IVC) stenosis (partial stasis) or ligation (complete stasis). Select WT mice received sgp130Fc chimera to inhibit trans pathway signaling. Vein wall, thrombus, and plasma were harvested at various time points and analyzed by PCR, western blot, ELISA or immunohistochemistry. Results: Compared to sham animals, those undergoing VT (complete or partial stasis) exhibited elevated vein wall IL-6 on post-VT day 4 (n=6-9, p Conclusions: Experimental VT stimulates IL-6 and downstream signaling processes and is dependent on the mechanism of thrombosis. Genetic deletion of IL-6 and selective disruption of trans-signaling pathway by sgp130 decreases vein wall inflammation and influences vein wall remodeling. Targeting of IL-6 trans-signaling pathway may represent a therapeutic option to treat vein wall inflammation post thrombosis.

Background/Aims: Pneumonia is a significant risk factor for the development of venous thrombosis (VT). Cell-adhesion molecules (CAMs) are linked to the pathogenesis of both pneumonia and VT. We hypothesized that remote infection would confer a prothrombogenic milieu via systemic elevation of CAMs. Methods: Lung injury was induced in wild-type (C57BL/6) mice by lung contusion or intratracheal inoculation with Klebsiella pneumoniae or saline controls. K. pneumoniae-treated mice and controls additionally underwent inferior vena cava (IVC) ligation to generate VT. Results: Lung-contusion mice demonstrated no increase in E-selectin or P-selectin whereas mice infected with K. pneumoniae demonstrated increased circulating P-selectin, ICAM-1, VCAM-1 and thrombin-antithrombin (TAT) complexes. Mice with pneumonia formed VT 3 times larger than controls, demonstrated significantly more upregulation of vein-wall and systemic CAMs, and formed erythrocyte-rich thrombi. Conclusion: Elevated CAM expression was identified in mice with pneumonia, but not lung contusion, indicating that the type of inflammatory stimulus and the presence of infection drive the vein-wall response. Elevation of CAMs was associated with amplified VT and may represent an alternate mechanism by which to target the prevention of VT.

Macrophages are involved in venous thrombus (VT) resolution and vein wall remodeling. This study was undertaken to identify variations in macrophage phenotypes in thrombi and vein wall in multiple models of VT to clarify the natural history of macrophage polarization in clearance of VT. We also sought to demonstrate the feasibility of macrophage phenotyping in human VT.Established murine models of VT were used to mimic the clinical spectrum of human VT (stasis and nonstasis models). Vein wall and thrombi were isolated at acute (2 days) or chronic (6-21 days) time points and analyzed by Bio-Plex assay (Bio-Rad, Carlsbad, Calif) for cytokines (interleukin [IL]-1β, IL-6, IL-10, IL-12), by immunohistochemistry for "M1-like" (IL-12) or "M2-like" (arginase 1 [Arg-1]) markers, and by histology for intimal thickness and collagen content (Sirius red staining). Bone marrow was harvested from animals 2 days after undergoing sham, stasis, or nonstasis surgery. Macrophages were skewed toward M1 using lipopolysaccharide, and RNA analysis was done for inflammatory cytokine genes (IL-1β, IL-12). Human blood samples were similarly analyzed with reverse transcription polymerase chain reaction for macrophage polarization markers (CD206, inducible nitric oxide synthase, CCR2) and thrombi with immunohistochemistry (inducible nitric oxide synthase, Arg-1).Stasis (chronic) and nonstasis (acute and chronic) thrombi were characterized by a predominance in anti-inflammatory (M2) macrophages (n = 4-5/group; P .05). Larger thrombi were found in the stasis model at both time points (n = 3; P .01), correlating with decreased intrathrombus inflammatory (M1) cytokines (IL-1β, P = .03; IL-12, P = .17; n = 4) and diminished inflammatory response of bone marrow-derived macrophages to lipopolysaccharide (IL-1β, P = .03; IL-12, P = .04; n = 4) compared with nonstasis model. Anti-inflammatory (M2 [Arg-1]) macrophage cell counts were elevated in the post-thrombotic vein wall of stasis mice compared with nonstasis mice (acute: n = 4, P .05; chronic: n = 5, P .01), consistent with increased intimal thickness (P .01; n = 4-6) and collagen deposition chronically (P = .005; n = 12). M2-like thrombi (Arg-1, P .05; n = 4-7) and circulating markers (CD206, P .05; n = 9-17) decreased over time in human VT.Experimental VT is characterized by an anti-inflammatory predominant macrophage phenotype, possibly impairing thrombus resolution, and is model dependent. Altering the M1/M2 macrophage balance may accelerate thrombus resolution and allow the development of translatable novel therapies to treat VT and to prevent post-thrombotic syndrome.

Objective— Toll-like receptors (TLR) bridge innate immunity and host responses, including inflammation. Sterile inflammation such as a venous thrombus (V t ) may involve TLR signaling, including TLR9. Methods and Results— TLR9 signaling on thrombus resolution was investigated using a mouse model of stasis V t . V t were significantly larger in TLR9−/− mice compared with wild-type (WT) at 2 and 8 days, despite a 2-fold increase in thrombus polymorphonucleic neutrophils at 2 days and monocytes at 8 days, whereas thrombus collagen and neovascularization was 55% and 37% less, respectively, at 8 days. Coincidently, decreased fibrinogen and increased thrombin-antithrombin complex were observed in TLR9−/− mouse thrombi. Vein wall interferon-α, interleukin-1α, and interleukin-2 were significantly reduced in TLR9−/− mice compared with WT. Thrombus cell death pathway markers were not significantly altered at 2 days, but caspase-1 was reduced in TLR9−/− thrombi at 8 days. MyD88 confers TLR9 intracellular signaling, but MyD88−/− mice had V t resolution similar to that of WT. However, inhibition of the NOTCH ligand δ-like 4 was associated with larger V t . Finally, stimulation with a TLR9 agonist was associated with smaller V t . Conclusion— TLR9 signaling is integral for early and mid-V t resolution through modulation of sterile inflammation, maintaining a TH1 milieu, and effects on the thrombosis pathway.

Several epigenetic phenomena occur at ribosomal DNA loci in eukaryotic cells, including the silencing of Pol I and Pol II transcribed genes, silencing of replication origins and repression of recombination. In Saccharomyces cerevisiae, studies focusing on the silencing of Pol II transcription and genetic recombination at the ribosomal DNA locus (rDNA) have provided insight into the mechanisms through which chromatin and chromatin-associated factors regulate gene expression and chromosome stability. The core histones, H2A, H2B, H3 and H4, the fundamental building blocks of chromatin, have been shown to regulate silent chromatin at the rDNA; however, the function of the linker histone H1 has not been well characterized. Here, we show that S. cerevisiae histone H1 represses recombination at the rDNA without affecting Pol II gene silencing. The most highly studied repressor of recombination at the rDNA is the Silent information regulator protein Sir2. We find that cells lacking histone H1 do not exhibit a premature-ageing phenotype nor do they accumulate the rDNA recombination intermediates and products that are found in cells lacking Sir2. These results suggest that histone H1 represses recombination at the rDNA by a mechanism that is independent of the recombination pathways regulated by Sir2.

Objective: Sepsis is a strong risk factor for the development of deep venous thrombosis (DVT). However, little is understood about the underlying pathobiology responsible for this phenomenon. This study was undertaken to evaluate the endothelial response of cellular adhesion molecules (CAM) involved in early thrombogenesis in a mouse model of sepsis. Methods: Wild-type mice underwent treatment with vehicle or LPS (10mg/kg), or followed by inferior vena cava (IVC) ligation to generate a venous thrombus (VT). Tissue and plasma were harvested at 2 hours post-intervention and were assessed for CAMs, thrombus size, and markers of extracellular DNA (NETs). Pre-treatment (12 hours prior and at time of thrombosis) with P/E selectin (sel) and ICAM exogenous inhibition was undertaken. Antibody depletion was accomplished in selected mice with anti-Ly6G antibody. To understand leukocyte trafficking to the vein wall under sepsis conditions, a separate group of animals underwent LPS or vehicle injection followed by intravital fluorescence microscopy of rhodamine-6G-labeled leukocytes. Results: Pre-thrombosis, IVC CAMs (P-sel p=0.15, E-sel, p Conclusions: In a mouse model of endotoxemia, CAM elevation and leukocyte trafficking occurs in the serum and local vein wall; and with stasis insult, larger VT form. Inhibitor experiments suggest that ICAM may be central to the sepsis related early VT, while PMNs and P/E sel may be less important. CAM inhibition may be a unique strategy to prevent sepsis-associated DVT.

Background Deletion of Toll-like receptor 9 (Tlr9) signaling, which is important for sterile inflammatory processes, results in impaired resolution of venous thrombosis (VT) in mice. The purpose of this study was to determine if deletion of Tlr9 affected sterile necrosis, apoptosis, and neutrophil extracellular trap (NET) production in VT. Methods Stasis and nonstasis murine models of VT were used in wild-type (WT) and Tlr9 −/− mice, with assessment of thrombus size and determination of NETs, necrosis, and apoptosis markers. Anti-polymorphonuclear neutrophil (PMN) and antiplatelet antibody strategies were used to determine the cellular roles and their roles in WT and Tlr9 −/− mice. Results At 2 days, stasis thrombi in Tlr9 −/− mice were 62% larger (n = 6-10), with 1.4-fold increased uric acid levels, 1.7-fold more apoptotic cells, 2-fold increased citrullinated histones, 2-fold increased peptidylarginine deiminase 4 (PAD4), and 1.5-fold increased elastase and a 2.4-fold reduction in tissue factor pathway inhibitor compared with WT mice (all n = 4-7; P Tlr9 −/− mice (n = 4-6), and they did not have elevated necrosis or NET markers. Stasis thrombus size was not reduced at the 2-day time point in WT or Tlr9 −/− mice that received treatment with deoxyribonuclease I or in PAD4 −/− mice, which are incapable of forming NETs. In Tlr9 −/− mice undergoing PMN depletion (n = 8-10), stasis thrombus size was reduced 18% and was associated with 29-fold decreased citrullinated histones, 1.3-fold decreased elastase, and 1.5-fold increased tissue factor pathway inhibitor (all n = 6; P 90% reduction) did not significantly reduce stasis thrombus size in Tlr9 −/− mice. Conclusions These data suggest that the thrombogenic model affects Tlr9 thrombogenic mechanisms and that functional Tlr9 signaling in PMNs, but not in platelets or NETs, is an important mechanism in early stasis experimental venous thrombogenesis.

Citation Elfline M, Clark A, Petty HR, Romero R. Bi-directional calcium signaling between adjacent leukocytes and trophoblast-like cells. Am J Reprod Immunol 2010 Problem Trophoblasts are believed to play an important role in mitigating immunological responses against the fetus. To better understand the nature of trophoblast–leukocyte interactions, we have studied signal transduction during intercellular interactions. Method of study Using a highly sensitive microfluorometric ratioing method and Ca2+-sensitive dyes, we measured Ca2+ signals in trophoblast-like cell lines (JEG-3 and JAR) or in leukocytes (neutrophils and monocytes) during intercellular contact. Results Trophoblast cell lines exhibit Ca2+ signals during leukocyte contact. In contrast, leukocytes cannot elicit Ca2+ signals in non-opsonized tumour cells, suggesting that Ca2+ signaling is not a general feature of cell–cell encounters. Similarly, leukocytes demonstrate Ca2+ signals during contact with trophoblast cell lines. Ca2+ signals were confirmed using three dyes and with the Ca2+ buffer BAPTA. Conclusion We suggest that leukocyte-to-trophoblast interactions lead to mutual Ca2+ signaling events in both cell types, which may contribute to immunoregulation at the materno–fetal interface.

Citation Xu Y, Madsen-Bouterse SA, Romero R, Hassan S, Mittal P, Elfline M, Zhu A, Petty HR. Leukocyte pyruvate kinase expression is reduced in normal human pregnancy but not in pre-eclampsia. Am J Reprod Immunol 2010; 64: 137–151 Problem Emerging evidence suggests that metabolism influences immune cell signaling and immunoregulation. To examine the immunoregulatory role of glycolysis in pregnancy, we evaluated the properties of pyruvate kinase in leukocytes from non-pregnant women and those with normal pregnancy and pre-eclampsia. Method of study We evaluated pyruvate kinase expression in lymphocytes and neutrophils from non-pregnant, pregnant, and pre-eclampsia patients using fluorescence microscopy and flow cytometry. Leukocyte pyruvate kinase activity and pyruvate concentrations were also evaluated. To study pyruvate’s effect on signaling, we labeled Jurkat T cells with Ca2+ dyes and measured cell responses in the presence of agents influencing intracellular pyruvate. Results The expression of pyruvate kinase is reduced in lymphocytes and neutrophils from normal pregnant women in comparison with those of non-pregnant women and pre-eclampsia patients. Similarly, the activity of pyruvate kinase and the intracellular pyruvate concentration are reduced in leukocytes of normal pregnant women in comparison with non-pregnant women and women with pre-eclampsia. Using Jurkat cells as a model of leukocyte signaling, we have shown that perturbations of intracellular pyruvate influence Ca2+ signals. Conclusion Normal pregnancy is characterized by reduced pyruvate kinase expression within lymphocytes and neutrophils. We speculate that reduced pyruvate kinase expression modifies immune cell responses due to reduced pyruvate concentrations.

SummaryDeep-vein thrombosis (DVT) resolution is thought to be primarily a urokinase plasminogen activator (uPA) -dependent mechanism, although observations suggest other non-fibrinolytic mechanisms may exist. We explored the role of matrix metalloproteinase (MMP) -2 and –9 in early DVT resolution in uPA-deficient mice. Male B6/SVEV (WT) and genetically matched uPA -/- mice underwent inferior vena cava (IVC) ligation to create stasis venous thrombi, with IVC and thrombus harvest. Thrombus size was similar between WT and uPA -/- mice at day 4, suggesting early non uPA-dependent resolution. Intrathrombus neutrophils and monocytes were reduced 3- and 3.5-fold in uPA -/- mice as compared with WT. By ELISA, tumour necrosis factor α and interleukin 1β were not altered, while interferon (IFN)γ was significantly elevated in uPA -/- mice. A compensatory increase in thrombus tPA was not observed, plasmin activity was reduced and PAI-1 was elevated 2.5-fold in uPA -/- mice. Active MMP2, but not MMP9, was elevated 3-fold in uPA-/- mice as compared with WT as well as MMP-14, an MMP2 activator. Collagen type IV and fibrinogen were reduced in uPA -/- mice thrombi as compared with WT. IFNγ induces MMP2, and blockade of IFNγ was associated with larger venous thrombi and reduced active MMP2, as compared with WT. Consistently, MMP2 -/- mice had larger VT as compared with WT controls, despite normal thrombus plasmin levels. Taken together, early experimental venous thrombus resolution is independent of uPA, and, in part, inflammatory cell influx. MMP2-dependent thrombolysis is an important compensatory mechanism of venous thrombus resolution, possibly by collagen type IV metabolism, and may represent an exploitable therapeutic avenue.

Ca(2+) messages are broadly important in cellular signal transduction. In immune cells, Ca(2+) signaling is an essential step in many forms of activation. Neutrophil-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is one form of leukocyte activation that plays an important role in tumor cell killing in vitro and in patient care. Using fluorescence methodologies, we found that neutrophils exhibit Ca(2+) signals during ADCC directed against breast fibrosarcoma cells. Importantly, these signals were localized to Ca(2+) microdomains at the neutrophil-to-tumor cell interface where they display dynamic features such as movement, fusion, and fission. These signals were blocked by the intracellular Ca(2+) buffer BAPTA. At the neutrophil-tumor cell synapse, the neutrophil's cytoplasm was enriched in STIM1, a crucial mediator of Ca(2+) signaling, whereas the Ca(2+)-binding proteins calbindin and parvalbumin were not affected. Our findings suggest that Ca(2+) microdomains are due to an active signaling process. As Ca(2+) signals within neutrophils were necessary for specific tumor cell apoptosis, a central role of microdomains in leukocyte-mediated tumor cell destruction is indicated.

Several epigenetic phenomena occur at ribosomal DNA loci in eukaryotic cells, including the silencing of Pol I and Pol II transcribed genes, silencing of replication origins and repression of recombination. In Saccharomyces cerevisiae, studies focusing on the silencing of Pol II transcription and genetic recombination at the ribosomal DNA locus (rDNA) have provided insight into the mechanisms through which chromatin and chromatin-associated factors regulate gene expression and chromosome stability. The core histones, H2A, H2B, H3 and H4, the fundamental building blocks of chromatin, have been shown to regulate silent chromatin at the rDNA; however, the function of the linker histone H1 has not been well characterized. Here, we show that S. cerevisiae histone H1 represses recombination at the rDNA without affecting Pol II gene silencing. The most highly studied repressor of recombination at the rDNA is the Silent information regulator protein Sir2. We find that cells lacking histone H1 do not exhibit a premature-ageing phenotype nor do they accumulate the rDNA recombination intermediates and products that are found in cells lacking Sir2. These results suggest that histone H1 represses recombination at the rDNA by a mechanism that is independent of the recombination pathways regulated by Sir2.

BackgroundVein wall fibrotic injury following deep venous thrombosis (VT) is associated with elevated matrix metalloproteinases (MMPs). Whether and by what mechanism MMP2 contributes to vein wall remodeling after VT is unknown.MethodsStasis VT was produced by ligation of the inferior vena cava and tissue was harvested at 2, 8, and 21 days in MMP2 -/- and genetic wild type (WT) mice. Tissue analysis by immunohistochemistry, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and zymography was performed.ResultsThrombus resolution was less at 8 days in MMP2 -/- compared with WT, evidenced by a 51% increase in VT size (P < .01), and threefold fewer von Willebrand's factor positive channels (P < .05). In MMP2 -/- mice, the main phenotypic fibrotic differences occurred at 8 days post-VT, with significantly less vein wall collagen content (P = .013), fourfold lower procollagen III gene expression (P < .01), but no difference in procollagen I compared with WT. Decreased inflammation in MMP2 -/- vein walls was suggested by ∼ threefold reduced TNFα and IL-1β at 2 days and 8 days post-VT (P < .05). A fourfold increase in vein wall monocytes (P = .03) with threefold decreased apoptosis (P

BackgroundPostthrombotic syndrome is characterized by a fibrotic vein injury following deep vein thrombosis (DVT). We sought to quantify the change in vein wall thickness in patients who fail to resolve DVT by 6 months and whether there were differences in blood or plasma levels of inflammatory proteins associated with venous remodeling.MethodsPatients presenting with confirmed lower extremity DVT were prospectively recruited for this study. Duplex imaging of the lower extremity venous system was performed, and blood was collected at entrance and repeat evaluation with blood draw and ultrasound imaging at 1 and 6 months. DVT resolution and thickness of the vein wall was quantified by ultrasound imaging in each segment affected by thrombus, and a contralateral, unaffected vein wall served as a control. Gene and protein expression of inflammatory markers were examined from leukocytes and serum, respectively. Analysis of variance or Student t-tests were used, and a P < .05 was significant. N = 10 to 12 for all analyses.ResultsThirty-two patients (12 patients with DVT resolution at 6 months, 10 patients with persistent thrombus at 6 months, and 10 healthy controls) were compared. Both resolving and nonresolving DVT were associated with a 1.5- to 1.8-fold increased vein wall thickness at 6 months (P = .008) as compared with nonaffected vein wall segments. However, the thickness of the affected segments was 1.4-fold greater in patients who had total resolution of the DVT by 6 months than in patients who had persistent chronic thrombus 6 months after presentation (P = .01). There was a four- to five-fold increased level of matrix metalloproteinase-9 (MMP-9) antigen in thrombosed patients compared with nonthrombosed patient controls (P < .05), while Toll-like receptor-9 (TLR-9) gene expression was three-fold less than controls (P < .05) at enrollment. D-dimer and P-selectin were higher in thrombosed as compared to controls at diagnosis but not at 6 months. Both TLR-4 (marker of inflammation) and P-selectin gene expression were higher in leukocytes from patients with chronic DVT compared with those who resolved at 1 month after diagnosis (P < .05).ConclusionsThis preliminary study suggests ongoing vein wall remodeling after DVT, measurable by ultrasound and associated with certain biomarkers. At 6 months, the vein wall is markedly thickened and directly correlates with resolution. This suggests that the vein wall response is initiated early following thrombus formation and persists even in the presence of total resolution.