Targeted Deletion of MMP-2 Inhibits Post-Thrombotic Vein Wall Fibrosis

      Objective: Post-thrombotic syndrome (PTS) is clinically characterized by a fibrotic vein wall injury following deep vein thrombosis (DVT). Previous studies have demonstrated exacerbated injury with loss of both matrix metalloproteinase (MMP)-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1, each associated with an increase in MMP-2 activity. We hypothesized that genetic deletion of MMP-2 would attenuate fibrotic vein wall injury. Methods: DVT was produced in the mouse by ligation of the infrarenal inferior vena cava (IVC) or sham operation in MMP-2 -/- (KO) and c57 (WT) animals, and tissue was harvested at 2, 8, 21 and 42 days. The vein wall tissue was processed for real-time reverse transcriptase-polymerase chain reaction, gelatin zymography (5 per time point), reverse zymography, ELISA and immunohistochemistry. Measurement of collagen deposition and scarring was performed using picrosirius red staining in polarized light. ANOVA was used for multiple comparisons, Student's test was used for direct pair comparisons, and a P < .05 was significant. Results: Thrombus resolution was documented by a decrease in the thrombosed IVC weight from 2 days to 42 days in both groups. At baseline and 2d following DVT formation, vein wall thickness and collagen scores were similar. At 8 days following DVT formation, collagen content and thickness of the vein wall was 32% less in the MMP-2 KO vs WT mice (P= .017). At 21d, the collagen content and thickness of the vein wall was less in the MMP-2 KO, but this difference did not attain significance (P=0.12). Collagen III gene expression was 5.8-fold lower at 8d (P=.009), and collagen I gene expression was 25.7-fold lower at 21d (P=0.034) in MMP-2 KO than in WT. Tropoelastin gene expression was 6.7-fold lower in MMP-2 KO than in WT at 21d (P=.008). There were no significant alterations in expression of MMP-9, although by gelatin zymography, 3 fold more MMP-9 activity was present in MMP-2 KO than WT at 8d (P=.006). TGF-β expression was similar at baseline and at 2d and 8d after DVT creation, but was 4.4 fold higher in MMP -2 KO at 21 days (P=.009). There were no differences in TNFα or MCP-1 levels by ELISA, nor were differences found in vein wall monocyte influx. There were no significant differences in the levels of either TIMP-1 or TIMP-2 at 8, 21, or 42 days in MMP-2 KO as compared with WT. Lastly, no significant differences in cellular proliferation (Ki-67 positive staining) or in apoptosis (TUNEL assay) at 8d were found between groups. Conclusions: In an experimental DVT injury model, loss of MMP-2 activity attenuates the post thrombotic vein wall injury with diminished expression and accumulation of collagen following DVT. These changes are not dependent on alterations in cellular proliferation or apoptosis. Further study will be required to determine if this is a suitable target for reducing PTS.
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