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 recently shown that TIMP-1 deficiency leads to massive MMP-9 mediated leukocyte recruitment and lethal liver IRI. This study tests the hypothesis that AAV-mediated TIMP-1 overexpression protects livers against IRI. Methods and results: rAAV8-TIMP-1 (5X10¹² gc/Kg) and control rAAV8-Luciferase or saline were administered i.v. to C57/Bl6 mice 7 days prior to 90 min of partial liver ischemia followed by 6h of reperfusion. ELISA studies confirmed an absence of serum anti-AAV8 antibodies in mice prior to rAAV8-TIMP-1 injection. Administration of rAAV8-TIMP-1 increased the liver expression of TIMP-1 mRNA by ∼4 fold (p<0.05) prior to surgery, without significant histological features of hepatic inflammation or cell death, as compared to rAAV8-luciferase or saline treated controls. rAAV8-mediated TIMP-1 expression was sustained after surgery; TIMP-1 mRNA was increased by ∼2 fold (p<0.05) in the rAAV8-TIMP-1 treated livers at 6h of IRI, which correlated with a ∼2 fold (p<0.05) depression in MMP-9 enzymatic activity, when compared to control mice. Importantly, rAAV8-mediated TIMP-1 gene therapy improved liver function, as assessed by significantly decreased AST (4827±1635 vs. 9768±3592; p<0.05) and ALT (7602±3338 vs. 12199±3773; p<0.05) levels at 6h post IRI, when compared to controls. Overall, rAAV8-TIMP-1 treated livers showed notably reduced levels of necrosis and improved hepatic histological architecture after IRI. Furthermore, the rAAV8-TIMP-1 mediated inhibition of MMP-9 activity led to a significant reduction in Ly-6G+ (p<0.05), Mac-1+ (p<0.05) and MMP-9+ (p<0.05) leukocyte infiltration. Pro-inflammatory cytokines, such as TNF-α (p<0.05), IFN-γ (p<0.05), and IL-6 (p<0.05) were also markedly depressed in rAAV-8 TIMP-1 treated livers, relative to controls. Finally, restoring TIMP-1 expression in TIMP-1 -/- knock-out mice via i.v. administration of rAAV8-TIMP-1 led to significantly improved liver histology and reduced serum AST (p<0.05) and ALT (p<0.05) levels in these mice post-IRI. In conclusion: We have demonstrated for the first time that AAV8-mediated TIMP-1 expression is effective in reducing MMP-9 activity and in protecting against liver IRI. Our novel results suggest that TIMP-1 gene therapy is applicable to hepatic IRI.

« Back to 2015 American Transplant Congress