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

Abstract

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.

Low-molecular-weight heparin modulates vein wall fibrotic response in a plasminogen activator inhibitor 1-dependent manner

“…These data are congruent with other studies reporting that a smaller thrombus does not correlate with a favorable vein wall response. 13,17,32,33 Our results suggest that the beneficial effect of LMWH on the vein wall fibrotic response is independent of thrombus size but dependent on the presence of PAI-1.…”

“…17 To determine whether elevation of PAI-1 levels affected MMP activity through inhibition of MMP activator plasmin, we measured vein wall MMP-2 during peak activity, at 6 days after thrombosis. Compared with untreated and treated (LMWH) controls, the PAI-1 −/− mice exhibited increased MMP-2 activity by zymography (WT vs PAI-1 −/− : P = .02, n = 10; WT + LMWH vs PAI-1 −/− + LMWH: P < .05, n = 4–10; Fig 3, A ).…”

“…We demonstrate an inverse relationship between PAI-1 levels and MMP activity. The proteolytic activity of MMP-2 17 plays a key role in modulating post-thrombotic vein wall remodeling and fibrosis. Taken together, these data suggest that occlusive thrombi, as many patients have at the time of diagnosis, result in vein wall damage sufficient to increase local MMP activity, which is amplified in the absence of PAI-1.…”

“…10,16,17 MMP activity was visualized as clear bands against a darkly stained background. Optical density was measured with a FOTO/ Analyst CCD camera (Fotodyne, Hartland , Wisc) and EL-Pro Analyzer software version 3.1 (Media Cybernetics, Silver Springs, Md).…”

“…10,17 Samples underwent reverse transcription, and the resultant complementary DNA was amplified by Taq polymerase in the Rotor-Gene quantitative polymerase chain reaction system (Qiagen Inc, Valencia, Calif). DNA levels of each gene of interest were measured with SYBR green intercalating dye (Roche, Indianapolis, Ind).…”