Hepatic ischemia/reperfusion injury (IRI) remains a challenge in organ transplantation. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is the major endogenous regulator of matrix metalloproteinase-9 (MMP-9). While MMP-9 inhibition ameliorates liver damage, TIMP-1 deficiency can lead to lethal IRI. Here, we unveil hepatoprotective mechanisms of TIMP-1 in liver IRI. Methods: TIMP-1 knockout (TIMP-1KO), TIMP-1/MMP-9 double knock out (TIMP-1/MMP-9 dKO), MMP-9 knockout (MMP-9KO) mice and respective wild-type (WT) control littermates were subject to 90 min of 70% liver ischemia followed by reperfusion. Results: Serum ALT levels (IU/L) were upregulated in TIMP-1KO mice (∼2-fold increase; p<0.04) compared with WT-controls at 6h post-IRI. WT-control livers had elevated congestion/necrosis; however, TIMP-1 deficient livers showed significant further disruption of lobular architecture post-IRI. The severely impaired liver function/histology observed in the TIMP-1KO mice correlated with their poor survival rate (40% vs. 100%; p<0.05) post-reperfusion. TIMP-1KO livers had significantly higher MMP-9 activity (2.5-fold increase; p<0.05), leukocyte infiltration (Ly6G-neutrophils and Mac-1-macrophages; p<0.001), and pro-inflammatory cytokine expression (TNF-Α and IFN-Γ; p<0.05) than controls at 6h post-IRI. Interestingly, obliteration of TIMP-1 resulted in a significant upregulation of the cell cycle suppressor p53 (∼3-fold increase; p<0.02) and of the p53-regulated inhibitor of cyclin-dependent kinases, p21 (∼2.7-fold increase; p<0.02) at 48h post-IRI. Indeed, BrdU (0.9±0.1 vs. 6.5±0.2; p<0.05), PCNA (2.7±0.3 vs. 27.0±2.7; p<0.05), and Mitotic (1.9±1.7 vs. 10.7±1.8; p<0.05) indexes, markers of cell proliferation, were almost negligible in TIMP-1-/- livers and significantly enhanced in WT-controls. Moreover, the serum ALT levels (IU/L) in TIMP-1KO mice were significantly increased when compared to TIMP-1/MMP-9 dKO (p<0.008), MMP-9KO (p<0.002), and to WT (p<0.002) mice at 48h post-IRI, providing an indication of a fine-tuned TIMP-1/MMP-9 balance in the modulation of hepatic damage and repair post-reperfusion. Conclusion: Our findings support the view of an important hepatoprotective role for TIMP-1 in hepatic IRI. Loss of TIMP-1 resulted in alteration of the cell-cycle dynamics and in impaired liver regeneration after liver IRI. Therefore, this study provides the rationale to identify therapeutic approaches aimed at modulating the TIMP-1/MMP-9 balance in hepatic IRI.

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