Matrix metalloproteinase-9 (MMP-9) is a key mediator of leukocyte migration across vascular barriers in hepatic ischemia/reperfusion injury (IRI). Tissue inhibitor of metalloproteinase-1 (TIMP-1) is the major endogenous regulator of MMP-9 activity. We have shown that TIMP-1 deficiency leads to massive leukocyte recruitment and lethal liver IRI. This study tests the hepatoprotective activity of rAAV8-TIMP-1 mediated TIMP-1 overexpression in liver IRI. Methods and results: rAAV8-TIMP-1 (5X10¹²gc/Kg) and control rAAV8 were administered i.v. to C57/Bl6 mice (negative for anti-AAV8 antibodies) 7 days prior to 90 min of partial liver ischemia followed by 6 and 24h of reperfusion. Biodistribution studies confirmed selective rAAV8-TIMP-1 mediated hepatic TIMP-1 expression (~4fold increase; p<0.05) prior to surgery, without histological features of hepatic inflammation or cell death. TIMP-1 overexpression resulted in the upregulation of anti-apoptotic genes (XIAP, BCL2, BAD) before and after hepatic IRI, relative to their respective controls. Moreover, the sustained overexpression of TIMP-1 markedly reduced the activation levels of caspase-3, -7 and -8 (p<0.05) after liver IRI. Further, high levels of TIMP-1 profoundly depressed MMP-9 activity (p<0.05), inhibited MMP-9 dependent Ly6G+ (p<0.05) and Mac1+ (p<0.05) leukocyte infiltration, and up-regulated the expression of the anti-inflammatory IL-10 post-reperfusion. Reduced apoptosis and inflammation in the rAAV8-TIMP-1 treated mice correlated with a major improvement in hepatic histological architecture and liver function, assessed by lower AST (p<0.05) and ALT (p<0.05) levels. TIMP-1 overexpression also protected primary hepatocytes against H₂O₂ mediated oxidative injury in vitro; rAAV8-TIMP-1 gene transfer significantly increased anti-apoptotic BCL2, BCLxl and BAD expression, reduced PARP1 cleavage, and decreased hepatocyte AST, ALT and LDH release (p<0.05), when compared to rAAV8-GFP treated hepatocytes. Finally, TIMP-1 gene therapy in TIMP-1-/- KO mice significantly reduced their AST and ALT levels (p<0.05) and increased their survival rate (p<0.05) after liver IRI. In conclusion: Here we establish that liver specific AAV8 mediated TIMP-1 overexpression effectively reduces hepatocyte death and MMP-9 mediated inflammation in liver IRI. Our results strongly support the development of therapeutic strategies aimed at selectively increasing the levels of TIMP-1 to treat hepatic IRI.

CITATION INFORMATION: Duarte S, Matian P, Miller M, Busuttil R, Coito A. AAV8 Delivery of TIMP-1 Prevents Hepatocyte Apoptosis After Liver Ischemia/Reperfusion Injury. Am J Transplant. 2017;17 (suppl 3).

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