Search

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

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

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

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