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Diagnosing plasma conditions can give great advantages in optimizing plasma wakefield accelerator experiments. One possible method is that of photon acceleration. By propagating a laser probe pulse through a plasma wakefield and extracting the imposed frequency modulation, one can obtain an image of the density modulation of the wakefield. In order to diagnose the wakefield parameters at a chosen point in the plasma, the probe pulse crosses the plasma at oblique angles relative to the wakefield. In this paper, mathematical expressions relating the frequency modulation of the laser pulse and the wakefield density profile of the plasma for oblique crossing angles are derived. Multidimensional particle-in-cell simulation results presented in this paper confirm that the frequency modulation profiles and the density modulation profiles agree to within 10%. Limitations to the accuracy of the measurement are discussed in this paper. This technique opens new possibilities to quantitatively diagnose the plasma wakefield density at known positions within the plasma column.

One obstacle in plasma accelerator development is the limitation of techniques to diagnose and measure plasma wakefield parameters. In this paper, we present a novel concept for the density measurement of a plasma wakefield using photon acceleration, supported by extensive particle in cell simulations of a laser pulse that copropagates with a wakefield. The technique can provide the perturbed electron density profile in the laser’s reference frame, averaged over the propagation length, to be accurate within 10%. We discuss the limitations that affect the measurement: small frequency changes, photon trapping, laser displacement, stimulated Raman scattering, and laser beam divergence. By considering these processes, one can determine the optimal parameters of the laser pulse and its propagation length. This new technique allows a characterization of the density perturbation within a plasma wakefield accelerator.

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.

Current treatments to prevent thrombosis, namely anticoagulants and platelets antagonists, remain complicated by the persistent risk of bleeding. Improved therapeutic strategies that diminish this risk would have a huge clinical impact. Antithrombotic agents that neutralize and inhibit polyphosphate (polyP) can be a powerful approach towards such a goal. Here, we report a design concept towards polyP inhibition, termed macromolecular polyanion inhibitors (MPI), with high binding affinity and specificity. Lead antithrombotic candidates are identified through a library screening of molecules which possess low charge density at physiological pH but which increase their charge upon binding to polyP, providing a smart way to enhance their activity and selectivity. The lead MPI candidates demonstrates antithrombotic activity in mouse models of thrombosis, does not give rise to bleeding, and is well tolerated in mice even at very high doses. The developed inhibitor is anticipated to open avenues in thrombosis prevention without bleeding risk, a challenge not addressed by current therapies., (© 2023. The Author(s).)

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., (© 2023. The Author(s).)

Objective: Deep vein thrombosis (DVT) and its sequela, post-thrombotic syndrome (PTS), remain a clinically significant problem. Interleukin-6 (IL-6) is a proinflammatory cytokine that is elevated in patients who develop PTS. We hypothesized that genetic deletion of IL-6 and the use of anti-IL-6 pharmacologic agents would be associated with decreased late vein wall injury., Methods: Wild-type C57BL/6J (WT) and IL-6 -/- mice underwent induction of stasis venous thrombosis by ligation of the infrarenal IVC. Vein wall inferior vena cava and thrombus were harvested at 21 days after ligation and analyzed by Western blot and immunohistochemistry of the vein wall using monocyte markers CCR2 and arginase 1, the endothelial marker CD31, and fibroblast markers DDR2 and FSP-1. Two anti-IL-6 pharmacologic agents (gp130 [glycoprotein 130] and tocilizumab) were tested and compared with low-molecular-weight heparin (LMWH) as the reference standard in WT mice. Plasma was collected at 4 and 48 hours to confirm the pharmacologic agents' effects., Results: Less fibrosis but no increase in luminal endothelialization was found in IL-6 -/- mice compared with WT mice at 21 days. The IL-6 -/- mice had fewer DDR2- and arginase 1-positive cells in the vein wall compared with the WT mice. However, no difference was found in the CCR2 + cells. Despite documented in vivo activity, exogenous gp130 and tocilizumab were not associated with decreased vein wall fibrosis or increased endothelial luminal coverage at 21 days. LMWH therapy, both before and after treatment, was not associated with decreased vein wall fibrosis at 21 days., Conclusions: IL-6 genetic deletion was associated with less fibrotic vein wall injury at a late time point, consistent with the PTS timeframe. However, neither the standard of care LMWH nor two available anti-IL-6 agents showed antifibrotic biologic effects in this model., (© 2022 by the Society for Vascular Surgery. Published by Elsevier Inc.)

Objective: Venous thrombosis (VT) damages the vein wall, both physically by prolonged distension and from inflammation. These factors contribute to post-thrombotic syndrome (PTS). Interleukin (IL)-6 might play a role in experimental PTS and vein wall responses. Previous assessments of post-thrombotic vein wall injury used static measures such as histologic examination and immunologic assays. The purpose of the present study was to use myography to quantify the changes in contraction and relaxation of murine vessels exposed to an acute VT., Methods: Wild-type (WT) C57BL/6 mice were used to determine the baseline vein wall passive tension on a DMT 610m myograph (DMT-USA, Inc., Ann Arbor, Mich), including dosing concentrations of phenylephrine (Phe) and acetylcholine (Ach). WT and IL-6 -/- mice underwent VT using inferior vena cava (IVC) ligation (complete stasis) and stenosis (partial stasis), with no-surgery mice used as controls. The mice were harvested at 2 days (2D) and analyzed using a myograph. The vessels were stimulated with Phe and Ach to stimulate a contraction and relaxation response. The endothelial responses to VT were quantified by CD31 immunohistochemistry, Greiss assay, polymerase chain reaction, and Evans blue assay., Results: Optimal passive tension was determined to be 2 mN, with an optimal concentration of Phe and Ach of 7E-3M and 1E-5M, respectively. No significant differences were found in the contractions when exposed to Phe between the WT control, WT 2D ligation, and WT 2D stenosis IVC segments and the IL-6 -/- mice with and without thrombus (P > .05 for all). When treated with Ach, significantly more relaxation was found in the nonthrombosed control IVC segments than in those IVC segments that had had a 2D thrombus from either ligation- or stenosis-derived thrombotic mechanisms in both WT and IL-6 -/- mice. CD31 staining showed ∼20% less luminal endothelium after stasis thrombosis (P ≤ .01) but no loss in the controls (P > .05). Evans blue staining showed a trend toward increased leakiness in post-thrombotic vein walls. No significant difference in the endothelial gene markers or nitric oxide production was found., Conclusions: Compared with the controls, acute thrombosis in the total or partial stasis models did not impair IVC contractile responses, suggesting no effect on the medial vascular smooth muscle response. The relaxation response was significantly reduced in the post-thrombotic groups, likely from direct endothelial injury. These findings suggest, at acute points, that VT impairs the endothelial function of a vein wall while retaining the vascular smooth muscle cell function and might be a mechanism that promotes PTS., (Copyright © 2019 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: J.A.D. is on the Board of Directors of the American Venous Forum, as a Research Council Chair and Member of the ISTH-SSC Board. All the other authors report no conflict of interest., (Schattauer GmbH Stuttgart.)

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., (© 2016, American College of Rheumatology.)

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., (Copyright © 2015 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

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]-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)., 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-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., 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., (Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

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., (© 2016 S. Karger AG, Basel.)

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., Competing Interests: There are no conflicts-of-interest to report for any authors of this work.

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-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., (Copyright © 2013 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

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., (© 2013.)

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., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

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., (Copyright © 2011 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

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.

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.

Introduction: Deep venous thrombosis (DVT) resolution involves the plasmin and the matrix metalloproteinase (MMP) system. This study tested the hypothesis that pharmacological inhibition of the plasmin system would impair DVT resolution and worsen vein wall damage., Methods: A rat model of stasis DVT by inferior vena cava (IVC) ligation was performed with intravenous control saline or aprotinin (AP; 2.8 mg/kg at operation), and harvest of thrombosed IVC at 7 days. After laser Doppler imaging, DVT were separated and weighed, and vein wall stiffness was assessed by tensiometry. Thrombus and vein wall tissue analysis included total collagen by colorimetric assay, cytokines, chemokines, and d-dimer by ELISA, urokinase-plasminogen activator (uPA), and plasminogen activator inhibitor-1 (PAI-1) by immuno-blotting, MMP-2 and -9 by zymography, and neutrophil (PMN) and monocyte (ED-1) leukocytes by immunohistochemistry., Results: DVT weights were 2-fold greater in the AP-treated rats (P < 0.05), but no significant differences in thrombus perfusion, collagen, or d-dimer levels were found. Vein wall stiffness was reduced 50% (P < 0.05), suggesting less biomechanical injury. The total vein wall MMP-9 was increased (P < 0.05) 5-fold in the AP group compared with controls, while MMP-2 was elevated but did not reach significance. No difference was found in vein wall tumor necrosis factor-alpha, tissue growth factor-beta, vein wall or thrombus monocytes, PMN, or uPA/PAI-1 ratio between groups., Discussion: AP inhibition of the plasmin system was associated with larger thrombi but less vein wall injury, but no difference in other measures of resolution, possibly because of increased vein wall MMP-9 activity. These data suggest an important redundant mechanism for DVT resolution.

Background: Pulmonary embolism (PE) is a life-threatening condition that is associated with the long-term sequelae of chronic pulmonary hypertension. Prior experimental work has suggested that post-PE inflammation is accompanied by pulmonary artery intimal hyperplasia. This study evaluated the effect of the thrombus and tested the hypothesis that thrombolytic, antiplatelet, and anticoagulant agents would decrease pulmonary injury., Methods: Male Sprague-Dawley rats (n = 267) underwent laparotomy and temporary clip occlusion of the infrarenal inferior vena cava for the formation of endogenous thrombus or placement of an inert silicone "thrombus." Two days later, repeat laparotomy was performed, the clip removed, and the thrombus or silicone plug was embolized to the lungs. The endogenous thrombus group received normal saline, low-molecular-weight heparin (LMWH), tissue plasminogen activator (tPA), or a gIIB/IIIA antagonist (abciximab). Lung tissue was harvested at various times over 21 days and assayed for total collagen, monocyte chemoattractant protein-1 (MCP-1), interleukin-13 (IL-13), and transforming growth factor-beta (TGF-beta). Fixed sections were stained with trichrome for intimal hyperplasia determination and ED-1 monocytes and alpha-actin-positive staining., Results: The overall survival for rats undergoing PE was 90%, was not affected by treatment, and 84% of all PE localized to the right pulmonary artery. The PE significantly reduced Pa(O2) in all groups. Compared with controls, the silicone emboli group had an increased level of IL-13 on day 1, an increased level of MCP-1 on day 4, and an increase in the levels of all inflammatory mediators on day 14 (P < .05). Accompanying these differences were greater pulmonary artery intimal hyperplasia at days 4 and 21 in the silicone group compared with controls (P < .05). LMWH treatment in the thrombus of PE rats significantly decreased IL-13 levels at all time points, whereas treatment with abciximab or tPA significantly increased IL-13 levels compared with controls. TGF-beta levels were significantly increased by LMWH at day 4 and 14, and abciximab was associated with lower TGF-beta at day 14. Only LMWH was associated with less pulmonary artery intimal hyperplasia at day 14 compared with controls and the other treatment groups., Conclusions: Persistent pulmonary artery distention by an inert material is sufficient to invoke significant inflammation and intimal hyperplasia independent of the thrombus itself. Compared with nontreated PE, LMWH is the only therapy associated with a significant reduction in late intimal hyperplasia and, with the exception of TGF-beta, lower profibrotic growth-factor production.

Objective: We tested the hypothesis that a venous thromboembolism to the pulmonary arterial system (pulmonary embolism [PE]) would cause an inflammatory response within the pulmonary arterial (PA) wall marked by elevated cytokines and chemokines and an influx of inflammatory cells., Methods: Experimental PE was induced in 70 rats and confirmed with angiography and O(2) saturation depression, and an additional 70 rats underwent sham operations. PA and lung tissue were removed at 3 hours and at 1, 2, 4, 6, 8, and 14 days (n = 10 per time point), were analyzed for proinflammatory cytokines and chemokines, and underwent histologic analysis. Data were analyzed with analysis of variance and the unpaired Student t test., Results: Average gross PE resolution was 40% at 2 days, 90% at 4 days, and 100% at 6 days. Only monocyte chemoattractant protein-1 levels were greater in affected PAs compared with sham PAs at 3 hours, 1 day, and 2 days (137 +/- 13 pg/mg protein, 285 +/- 40 pg/mg protein, and 249 +/- 36 pg/mg protein versus 101 +/- 6 pg/mg protein, 150 +/- 36 pg/mg protein, and 92 +/- 3 pg/mg protein; P <.01 for all). Keratinocyte-derived chemokine, tissue necrosis factor, interleukin-10, nitric oxide, P-selectin, and E-selectin levels were not elevated. Neutrophils infiltrated the PA wall beginning at 3 hours, peaked at 2 days (69.4 +/- 21.7 per five high-power fields; P <.01), and returned to baseline by 8 days after PE. Macrophages peaked at 1 day after PE (29.3 +/- 6.9; P <.01) and returned to baseline by 4 days after PE. PE also was associated with a significantly increased intima to media ratio (P <.05), apparent at 4 days after PE and persisting through 14 days., Conclusion: PE is associated with an early influx of polymorphonuclears and macrophages and monocyte chemoattractant protein-1 elevation within the PA wall. These are temporally associated with thrombus resolution and intimal hyperplasia. These factors may mediate these two processes after PE. This offers targets for further study with the hopes of minimizing the pathophysiologic response to PE.