Liver ischemia reperfusion injury (IRI) is an inflammatory event that contributes to graft dysfunction in orthotopic liver transplantation (OLT). Matrix Metalloproteinases (MMPs) are responsible for extracellular matrix (ECM) turnover in physiological and inflammatory conditions. We established that matrix metalloproteinase-9 (MMP-9) proteolytic activity facilitates inflammatory leukocyte invasion in murine models of warm and cold liver IRI. Here we characterize MMP-9 expression in biopsies of human OLT recipients. Methods: Liver biopsies were collected from 26 OLT recipients after reperfusion and prior to abdominal closure. In 5 cases, ischemic donor livers were also biopsied prior to transplant. Controls were biopsied from non-lesion liver of patients subject to liver resection. Biopsies were fixed in formalin prior to MMP-9 detection. Results: 15 male and 11 female recipients had a median age of 63, a mean MELD score of 28, with mean CIT and WIT of ~7h and ~1h, respectively. Triple immunofluorescence confirmed CD45⁺ infiltrating inflammatory leukocytes as the major sources of MMP-9 in human OLTs. MMP-9⁺ leukocytes were modestly detected in biopsies of both non-lesion controls and cold ischemic livers, and were significantly increased (~2-fold) in liver biopsies after OLT (MMP-9; OLT vs. CI vs. non-lesion: 32±5 vs. 17±3 vs. 11±4; p<0.002). Leukocyte MMP-9 expression had a positive correlation with liver injury post-OLT; MMP-9⁺ leukocyte numbers correlated with serum AST and ALT levels at post-operative day 5 (POD5) (AST: R=0.457, p<0.04; ALT: R=0.524, p<0.02) and 7 (POD7) (AST: R=0.407, ns; ALT: R=0.570, p<0.01). Moreover, recipients with serum transaminases elevated above the upper limit of normal (AST>48 & ALT>55 IU/L) had increased MMP-9⁺ leukocytes compared to recipients within normal AST and ALT range at POD5 (MMP-9: 37±16 vs. 8±6; p<0.02) and POD7 (MMP-9: 31±9 vs. 15±13; p<0.05). MMP-9⁺ leukocytes were also increased in reperfusion biopsies from steatotic donor grafts (~30% mean macrosteatosis) compared to reperfusion biopsies from non-steatotic donor grafts (MMP-9: 44±26 vs. 19±12; p<0.03). Conclusion: Our results show that MMP-9 is produced by infiltrating inflammatory leukocytes in human OLT biopsies and that its expression associates with liver injury and graft steatosis. Overall, this study correlates with our previous findings in murine models of liver IRI and together support an important role for MMP-9 expression in liver transplantation.

CITATION INFORMATION: Duarte S, Datta N, Baber J, Busuttil R, Zarrinpar A, Coito A. Matrix Metalloproteinase-9 in Human Orthotopic Liver Transplant Biopsies. Am J Transplant. 2016;16 (suppl 3).

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