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Elevated tissue inhibitor of metalloproteinase 1 (TIMP-1) expression contributes to excess production of extracellular matrix in liver fibrosis. Herein, we constructed a recombinant adeno-associated virus (rAAV) carrying siRNA of the TIMP-1 gene (rAAV/siRNA-TIMP-1) and investigated its effects on liver fibrosis in rats. Two models of rat liver fibrosis, the carbon tetrachloride and bile duct ligation models, were treated with rAAV/siRNA-TIMP-1. In the carbon tetrachloride model, rAAV/siRNA-TIMP-1 administration attenuated fibrosis severity, as determined by histologic analysis of hepatic collagen accumulation, hydroxyproline content, and concentrations of types I and III collagen in livers and sera. Levels of mRNA and active matrix metalloproteinase (MMP) 13 were elevated, whereas levels of mRNA and active MMP-2 were decreased. Moreover, a marked decrease was noted in the expression of α-smooth muscle actin, a biomarker of activated hepatic stellate cells (HSCs), and transforming growth factor-β1, critical for the development of liver fibrosis. Similarly, rAAV/siRNA-TIMP-1 treatment significantly alleviated bile duct ligation-induced liver fibrosis. Furthermore, this treatment dramatically suppressed TIMP-1 expression in HSCs from both model rats. These data indicate that the administration of rAAV/siRNA-TIMP-1 attenuated liver fibrosis by directly elevating the function of MMP-13 and diminishing activated HSCs. It also resulted in indirect decreased expression of type I collagen, MMP-2, and transforming growth factor-β1. In conclusion, rAAV/siRNA-TIMP-1 may be an effective antifibrotic gene therapy agent., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Elevated tissue inhibitor of metalloproteinase (TIMP)-1 expression contributes to excess production of extracellular matrix in liver fibrosis. However, there are few studies on sustained suppression of TIMP-1. We aimed to construct a recombinant adeno-associated virus (AAV) carrying small interfering RNAs (siRNAs) of TIMP-1 and investigate the long-term effects of RNA interference upon the TIMP-1 gene in rat hepatic stellate cells (HSCs). Five siRNA oligomers targeting rat TIMP-1 were designed and transfected into HSCs. A U6 promoter followed by the siRNA which had the strongest suppression effect was cloned into the AAV vector and packed into 293 cells to construct the recombinant AAV/siRNA-TIMP-1/neo. After infecting HSCs with this recombinant AAV, the transcription and expression levels of the TIMP-1 and matrix metalloproteinase-13 (MMP-13) genes were detected at 4 and 12 weeks. Three of the five designed siRNA oligomers had a suppressing effect on TIMP-1 expression in rat HSCs within 72 h. The transcription and expression levels of TIMP-1 were suppressed significantly (P<0.05) following recombinant AAV/siRNA1-TIMP-1/neo infection and lasted 12 weeks. TIMP-1 expression in rAAV/siRNA1-TIMP-1/neo-infected HSCs was suppressed by 60% after four weeks and 90% after twelve weeks when compared to the control recombinant AAV/neo and uninfected HSCs. Furthermore, the transcription and protein expression levels of MMP-13, the main substrate of TIMP-1, were elevated by approximately 40% at twelve weeks in rAAV/siRNA-TIMP-1/neo-infected HSCs. RNA interference exerts suppressive effect on the TIMP-1 gene in cultured HSCs for a longer time when a recombinant AAV is utilized as the gene delivery vector.

Objective: To observe the in vitro effect of compound 861 (Cpd 861) on tissue inhibitor of metalloprotenase 1 (TIMP1) mRNA levels of HSC-T6 cell., Methods: HSC-T6 cells were exposed in different concentrations of Cpd 861 (0.25~1.0 mg/ml) for 48 hours. The TIMP1 level was measured by the quantitative reverse-transcription polymerase chain reaction (RT-PCR)., Results: The TIMP1 mRNA levels of HSC-T6 cells at different concentrations of Cpd 861 were lower (2.50 0.71, 0.50 0.01, 0.11 0.03) than those of the normal control (3.78 0.67, P<0.05 or P<0.01)., Conclusions: The antifibrotic mechanism of Cpd 861 is partly due to its downregulation on TIMP1 mRNA levels of HSC-T6 cells.