Megan Elfline

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-&agr;, interleukin-1&agr;, 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 &dgr;-like 4 was associated with larger Vt. Finally, stimulation with a TLR9 agonist was associated with smaller Vt. Conclusion—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.

Postthrombotic vein wall remodeling: preliminary observations.

BACKGROUND Postthrombotic 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. METHODS Patients 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. RESULTS Thirty-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). CONCLUSIONS This 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.

In vivo role of neutrophil extracellular traps in antiphospholipid antibody-mediated venous thrombosis

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.

Urokinase plasminogen activator independent early experimental thrombus resolution: MMP2 as an alternative mechanism

Deep-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.

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.

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 Willebrands 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 < .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.

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.

Leukocyte Pyruvate Kinase Expression is Reduced in Normal Human Pregnancy but not in Pre‐eclampsia

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

Calicum microdomains form within neutrophils at the neutrophil–tumor cell synapse: role in antibody-dependent target cell apoptosis

Ca2+ messages are broadly important in cellular signal transduction. In immune cells, Ca2+ 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 Ca2+ signals during ADCC directed against breast fibrosarcoma cells. Importantly, these signals were localized to Ca2+ 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 Ca2+ buffer BAPTA. At the neutrophil–tumor cell synapse, the neutrophil’s cytoplasm was enriched in STIM1, a crucial mediator of Ca2+ signaling, whereas the Ca2+-binding proteins calbindin and parvalbumin were not affected. Our findings suggest that Ca2+ microdomains are due to an active signaling process. As Ca2+ signals within neutrophils were necessary for specific tumor cell apoptosis, a central role of microdomains in leukocyte-mediated tumor cell destruction is indicated.

Divergent effects of Tlr9 deletion in experimental late venous thrombosis resolution and vein wall injury

Deep-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.