Experimental venous thrombus resolution is driven by IL-6 mediated monocyte actions.

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. Ly6Clo 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.

Pre-Clinical Model to Study Recurrent Venous Thrombosis in the Inferior Vena Cava.

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-ß (TGF-ß), 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.

In Vivo Role of Neutrophil Extracellular Traps in Antiphospholipid Antibody-Mediated Venous Thrombosis.

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.

Alterations in macrophage phenotypes in experimental venous thrombosis.

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]-1ß, 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-1ß, 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 intrathrombus inflammatory (M1) cytokines (IL-1ß, P = .03; IL-12, P = .17; n = 4) and diminished inflammatory response of bone marrow-derived macrophages to lipopolysaccharide (IL-1ß, 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. 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.

Matrix metalloproteinase-9 deletion is associated with decreased mid-term vein wall fibrosis in experimental stasis DVT.

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 21days. 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.

The effect of matrix metalloproteinase 2 and matrix metalloproteinase 2/9 deletion in experimental post-thrombotic vein wall remodeling.

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 ∼ threefold reduced TNFα and IL-1ß 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.

The role of urokinase plasminogen activator and plasmin activator inhibitor-1 on vein wall remodeling in experimental deep vein thrombosis.

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.

Toll-like receptor 9 signaling is critical for early experimental deep vein thrombosis resolution.

OBJECTIVE: Toll-like receptors (TLR) bridge innate immunity and host responses, including inflammation. Sterile inflammation such as a venous thrombus (Vt) may involve TLR signaling, including TLR9. METHODS AND RESULTS: TLR9 signaling on thrombus resolution was investigated using a mouse model of stasis Vt. Vt 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 Vt resolution similar to that of WT. However, inhibition of the NOTCH ligand δ-like 4 was associated with larger Vt. Finally, stimulation with a TLR9 agonist was associated with smaller Vt. CONCLUSIONS: TLR9 signaling is integral for early and mid-Vt resolution through modulation of sterile inflammation, maintaining a TH1 milieu, and effects on the thrombosis pathway.