Deep venous thrombosis and residual thrombus burden correlates with circulating IL-6 levels in humans. To investigate the cellular source and role of IL-6 in thrombus resolution, Wild type C57BL/6J (WT), and IL-6 −/− mice underwent induction of VT via inferior vena cava (IVC) stenosis or stasis. Vein wall (VW) and thrombus were analyzed by western blot, immunohistochemistry, and flow cytometry. Adoptive transfer of WT bone marrow derived monocytes was performed into IL6 -/- mice to assess for rescue. Cultured BMDMs from WT and IL-6 −/− mice underwent quantitative real time PCR and immunoblotting for fibrinolytic factors and matrix metalloproteinase activity. No differences in baseline coagulation function or platelet function were found between WT and IL-6 −/− mice. VW and thrombus IL-6 and IL-6 leukocyte-specific receptor CD126 were elevated in a time-dependent fashion in both VT models. Ly6C lo Mo/MØ were the predominant leukocyte source of IL-6. IL-6 −/− mice demonstrated larger, non-resolving stasis thrombi with less neovascularization, despite a similar number of monocytes/macrophages (Mo/MØ). Adoptive transfer of WT BMDM into IL-6 −/− mice undergoing stasis VT resulted in phenotype rescue. Human specimens of endophlebectomized tissue showed co-staining of Monocyte and IL-6 receptor. Thrombosis matrix analysis revealed significantly increased thrombus fibronectin and collagen in IL-6 −/− mice. MMP9 activity in vitro depended on endogenous IL-6 expression in Mo/MØ, and IL-6 −/− mice exhibited stunted matrix metalloproteinase activity. Lack of IL-6 signaling impairs thrombus resolution potentially via dysregulation of MMP-9 leading to impaired thrombus recanalization and resolution. Restoring or augmenting monocyte-mediated IL-6 signaling in IL-6 deficient or normal subjects, respectively, may represent a non-anticoagulant target to improve thrombus resolution.

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. Although antiphospholipid antibodies have been shown to promote thrombosis in mice, the role of neutrophils has not been explicitly studied. The aim of this study was to characterize neutrophils in the context of a new model of antiphospholipid antibody-mediated venous thrombosis.Methods. Mice were administered fractions of IgG obtained from patients with APS. At the same time, blood flow through the inferior vena cava was reduced by induction of 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 (APS IgG) demonstrated exaggerated thrombosis as compared with control IgG-treated mice. Thrombi in mice treated with APS IgG were enriched for citrullinated histone H3 (a marker of neutrophil extracellular traps [NETs]). APS IgG-treated 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 IgG-treated mice and control mice. Treatment with either DNase (which dissolves NETs) or a neutrophil-depleting antibody reduced thrombosis in APS IgG-treated mice to the level in control mice.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.

Summary Venous 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.

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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.

Objective: 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.Methods: 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]-1 beta, 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-1 beta, 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).Results: 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 intra-thrombus inflammatory (M1) cytokines (IL-1 beta, P =.03; IL-12, P =.17; n = 4) and diminished inflammatory response of bone marrow-derived macrophages to lipopolysaccharide (IL-1 beta, 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 = 917) decreased over time in human VT.Conclusions: 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.

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<0.0001) but only complete stasis also had significantly elevated IL-6Rα (n=6-9, p<0.0001) and downstream intracellular signaling pathway molecule pSTAT3 (n=6-9, p=0.0002). Complete stasis VT in IL-6-/- mice was characterized by decreased circulating gp130 (n=4-6 p=0.0257), similar size thrombi to WT controls (0.020±0.005 v. 0.023±0.006, p=ns), and decreased CD68+ macrophage recruitment to the vein wall (n=3-5, p=0.0279). The IL-6-/- mice were protected from endothelial to mesenchymal-like transformation, as reflected by decreased FSP-1 (n=3-6, p<0.0001) and increased VE cadherin (n=3-6, p=0.0421) as compared with WT. Disruption of trans-signaling pathway via gp130Fc chimera resulted in less vein IL-6 (n=6-7, p=0.0027), TGF-β (n=6-7, p=0.0094), and decreased CD68 cell recruitment (n=3, p=0.0299). 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: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 < .05). In contrast, the sizes of nonstasis thrombi were not significantly different in 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 < .05). Last, platelet depletion (>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.

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.

Recent work has shown that activated PMNs release prothrombotic neutrophil extracellular traps (NETs) and elastase. We have previously shown that stasis venous thrombosis (VT) resolution is toll like receptor (TLR)-9 dependent but the mechanisms are not fully elucidated. We hypothesize that TLR9 signaling directly mediates PMN and possibly platelet related thrombotic activity.

Patients who suffer deep venous thrombosis (DVT) have at least a 30% recurrence rate during 10 years. Current murine models of inferior vena cava (IVC) DVT all model primary thrombosis and not recurrent thrombosis. The aim of this project was to create and characterize the first murine model to study recurrent DVT in the IVC.

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<0.01 and ICAM-1 p=0.11, all n=6) were elevated in mice treated with LPS. Consistently, intravital microscopy of leukocyte behavior revealed increased leukocyte rolling (n=6, p< .0001) and decreased rolling velocity (n=75/group, p<0.0001). Post-thrombosis, LPS treated mice formed larger thrombi (6.4±0.8mg v. 9.9±1.1mg, p=0.03) and exhibited elevations in circulating P-sel, E-sel, and ICAM-1 (all p<0.05, n=10). NETs markers H3 and elastase were not elevated. Pre-treatment with an ICAM-1 inhibitor restored thrombus size (6.6±1.5 mg, n=5) and circulating ICAM-1 levels to that of vehicle treated mice, while P/E sel inhibition resulted in moderately reduced thrombus size (7.3±1.2mg, n=10). Exogenous PMN depletion did not decrease thrombus weight. 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.

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.

Introduction: 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.Methods: WT and MMP9-/- mice underwent stasis VT by ligation of the inferior vena cava (IVC) and tissue was harvested at 2, 8, and 21 days. Assessment of thrombus size, and gene, protein and structural vein wall determinations were done.Results: 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.Conclusions: 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. (C) 2013 Elsevier Ltd. All rights reserved.

Background: Vein 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.Methods: Stasis 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.Results: Thrombus 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 similar to threefold reduced TNF alpha and IL-1 beta at 2 days and 8 days post-VT (P < .05). A fourfold increase in vein wall monocytes (P = .03) with threefold decreased apoptosis (P < .05), but no difference in cellular proliferation at 8 days was found in MMP2 -/- compared with WT. As increased compensatory MMP9 activity was observed in the MMP2 -/- mice, MMP2/9 double null mice had thrombus induced with VT harvest at 8 days. Consistently, twofold larger VT, a threefold decrease in vein wall collagen, and a threefold increase in monocytes were found (all P < .05). Similar findings were observed in MMP9 -/- mice administered an exogenous MMP2 inhibitor.Conclusions: In stasis VT, deletion of MMP2 was associated with less midterm vein wall fibrosis and inflammation, despite an increase in monocytes. Consideration that VT resolution was impaired with MMP2 (and MMP2/9) deletion suggests direct inhibition will likely also require anticoagulant therapy.

OBJECTIVE:Deep 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. METHODS:A 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. RESULTS:Thrombi 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. CONCLUSIONS:In 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.

Introduction: Deep vein thrombosis (DVT) resolution occurs via an inflammatory response. As most DVT are sterile, endogenous danger signals, such as nucleic or uric acids, must incite the inflammatory response. Neutrophils and monocyte/macrophages are important in DVT resolution; the role of dendritic cells is not well defined. Toll-like receptor 9 (TLR9) is expressed on these cells and activation by foreign or endogenous nucleic acids triggers an innate immune response. Previously, we have shown that loss of TLR9 signaling is associated with impaired venous thrombosis (VT) resolution and increased necrotic cell products in mice. in this study, we investigated whether extracellular nucleic acids are present in experimental VT in mice, and further defined the role of TLR9 and sterile inflammation in early VT resolution. Methods: VT were induced in Balb/c mice by inferior vena cava ligation. at 2 days, thrombi were harvested and histological sections were stained for DNA or RNA. to determine the effects of necrotic cell products on VT resolution, bone marrow-derived macrophages (BMMO) or dendritic cells (BMDC) were exposed to TLR9 agonist (CpG DNA), monosodium urate (MSU), necrotic cells, or RNA in vitro with expression analysis of genes important for VT resolution. to further elucidate the role of TLR9 signaling in thrombus-associated macrophages and dendritic cells, Tlr9+/+ BMMO or BMDC were adoptively transferred to Tlr9-/- mice prior to VT induction, with examination of the effect on early VT resolution. Results: We found that extracellular DNA, but not extracellular RNA, is present in experimental VT in mice. the extracellular DNA is located in proximity to neutrophils in the thrombus, suggesting the presence of neutrophil extracellular traps (NETs). in vitro, BMMO and BMDC responded to TLR9 agonist, MSU, and necrotic cells with changes in expression of genes important for VT resolution (e.g. BMMO responded to MSU with an 11-fold increase in IL-1α, 1.7-fold increase in MMP-9, and 60% decrease in uPA compared with control; P<0.05), but did not respond to RNA. Adoptive transfer of Tlr9+/+ macrophages or DC to Tlr9-/- mice did not improve VT resolution (P=0.31 and 0.69, respectively, for thrombus size when compared with appropriate controls). Conclusions: These findings show that extracellular DNA, but not RNA, is present in experimental VT in mice. While monocytes and DCs are activated by sterile inflammatory products in vitro, these cells did not restore normal VT resolution, suggesting that TLR9 signaling in these cells is not important in early VT resolution.