Matrix metalloproteinase-9 (MMP-9) is a key mediator of leukocyte migration across vascular barriers in hepatic ischemia/reperfusion injury (IRI). Tissue inhibitor of metalloproteinase1 (TIMP-1) is the major endogenous regulator of MMP-9 activity. We have shown that TIMP-1 deficiency leads to massive MMP9 mediated leukocyte recruitment and lethal liver IRI. This study evaluates the effect of rAAV8-TIMP-1 mediated TIMP-1 overexpression on the hepatic inflammatory response in IRI. Methods and results: rAAV8-TIMP-1 (5X10¹²gc/Kg) and control rAAV8 were administered i.v. to C57/Bl6 mice 7 days prior to 90 min of partial liver ischemia followed by reperfusion. ELISA studies confirmed an absence of serum anti-AAV8 antibodies in mice prior to rAAV8-TIMP-1 injection. TIMP-1 expression was increased (~4-fold; p<0.05) by rAAV8-TIMP-1 administration prior to surgery, without histological features of hepatic inflammation or cell death, compared to controls. TIMP-1 overexpression was sustained after surgery; TIMP-1 mRNA was increased in rAAV8-TIMP1 treated livers at 6h (~2 fold, p<0.05) and 24h (~2.6 fold, p<0.05) of IRI. Elevated rAAV8-TIMP-1 expression depressed MMP-9 activity at both 6h (706±341 vs. 3820±1915, p<0.05) and 24h (1139±972 vs. 2132±647, p<0.05) of IRI. Furthermore, rAAV8-TIMP-1 mediated MMP-9 inhibition significantly reduced inflammatory Ly6G⁺ (p<0.05), Mac1⁺ (p<0.05) and MMP-9⁺ (p<0.05) leukocyte infiltration at both 6h and 24h post-IRI. Proinflammatory cytokines, such as TNFα (p<0.05), IFNγ (p<0.05), and IL6 (p<0.05) were also markedly depressed in rAAV8-TIMP-1 treated livers, relative to controls. Reduced inflammation correlated with improved liver function in the rAAV8-TIMP-1 treaded mice, as assessed by lower AST (6h: 4827±1635 vs. 9768±3592, 24h: 1077±859 vs. 3974± 2566; p<0.05) and ALT (6h: 7602±3338 vs. 12199±3773, 24h: 3018±2933 vs. 7735±4284; p<0.05) levels; rAAV8-TIMP-1 treated livers had notably reduced levels of necrosis and improved hepatic histological architecture post-IRI. Finally, TIMP-1 gene transfer to TIMP-1-/- mice significantly improved their liver function and reduced liver injury (AST: p<0.05; ALT p<0.05) at 6h and 24h of IRI. In conclusion: Here we establish that AAV8 mediated TIMP-1 overexpression effectively reduces MMP-9 activity and inflammation in liver IRI. Our results strongly support that therapeutic approaches aimed at selectively increasing the levels of TIMP-1 are applicable to treat hepatic IRI.

CITATION INFORMATION: Duarte S, Fujii T, Lipshutz G, Busuttil R, Coito A. Anti-Inflammatory Effects of AAV-Mediated TIMP-1 Gene Transfer in Hepatic Ischemia-Reperfusion Injury. Am J Transplant. 2016;16 (suppl 3).

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