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 TLR9Ϫ/Ϫ 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 TLR9Ϫ/Ϫ mouse thrombi. Vein wall interferon-␣, interleukin-1␣, and interleukin-2 were significantly reduced in TLR9Ϫ/Ϫ mice compared with WT. Thrombus cell death pathway markers were not significantly altered at 2 days, but caspase-1 was reduced in TLR9Ϫ/Ϫ thrombi at 8 days. MyD88 confers TLR9 intracellular signaling, but MyD88Ϫ/Ϫ mice had VT resolution similar to that of WT. However, inhibition of the NOTCH ligand ␦-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.
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 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 21d. Tissue analysis included gene expression of vascular smooth muscle cells (alpha SMA [αSMA], SM22) and endothelial marker (CD31), by real time PCR, ELISA, matrix metalloproteinase (MMP) -2 and 9 activity by zymography and vein wall collagen by picrosirius red histological analysis. A P < .05 was considered significant. RESULTS Thrombi were significantly larger in both 8d and 21d uPA −/− as compared to WT, and were significantly smaller in both 8 and 21d PAI-1 −/− as compared to WT. Correspondingly, 8d plasmin levels were reduced in half in uPA −/− and increased 3 fold in PAI-1 −/− when compared to respective WT thrombi (P < .05, N = 5 – 6). The endothelial marker CD31 was elevated 2 fold in PAI-1 −/− mice at 8d, but reduced 2.5 fold at 21d in uPA −/− as compared with WT (P = .02, N = 5 – 6), suggesting less endothelial preservation. Vein wall VSMC gene expression showed that 8d and 21d 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 (N = 3 – 5, P ≤ .05). No significant difference in MMP2 or 9 activity was found in the PAI-1 −/− mice compared with WT, while 5.4 fold more MMP9 was present in 21d WT than 21d uPA −/− (P = .03, N = 5). Lastly, collagen was ~2 fold greater at 8d in PAI-1 −/− IVC as compared to 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 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 (IVC) and tissue was harvested at 2, 8, and 21 days in MMP2 −/− and genetic wild type (WT) mice. Tissue analysis by immunohistochemistry, ELISA, real time PCR, and zymography was performed. Results Thrombus resolution was impaired at 8d in MMP2 −/− as compared with WT, evidenced by a 51% increase in VT size (p < .01), and 3 fold fewer vWF positive channels (p<.05). In MMP2 −/− mice, the main phenotypic fibrotic differences occurred at 8d post VT, with significantly less vein wall collagen content (p=.013), 4 fold lower procollagen III gene expression (p < .01) but no difference in procollagen I as compared to WT. Decreased inflammation in MMP2−/− vein walls was suggested by ~ 3 fold reduced TNFα and IL1β at 2d and 8d post VT (p < .05). A 4 fold increase in vein wall monocytes (p = .03) with 3 fold decreased apoptosis (p < .05), but no difference in cellular proliferation at 8d was found in MMP2−/− as 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 8d. Consistently, 2 fold larger VT, a 3 fold decrease in vein wall collagen, and a 3 fold increase in monocytes was found (all p < .05). Similar findings were observed in MMP9 −/− mice administered an exogenous MMP2 inhibitor. Conclusion 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.
How the vein wall endothelium responds after deep vein thrombosis (DVT) has not been well documented owing to limited human specimens. This report shows that low-molecular-weight heparin accelerates or protects the endothelium and preserves medial smooth muscle cell integrity after DVT, but that this effect is limited to a relatively early time period. Although most DVT prophylaxis is pharmacologic (a heparin agent), use of nonpharmacologic measures is also common. The use of heparin prophylaxis, compared with after DVT treatment, and the acceleration of post-DVT re-endothelialization require clinical correlation.
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.
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