Introduction Pancreatic fibrosis is a characteristic feature of chronic pancreatic injury and is thought to result from a change in the balance between synthesis and degradation of extracellular matrix proteins. Pancreatic stellate cells (PSC) have the capacity to synthesise both matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitor of metalloproteinase (TIMPs). These may play a key role in pancreatic fibrogenesis by protecting extracellular matrix from degradation and supporting the survival of fibrogenic PSC. Therefore it was hypothesised that TIMP-1 has proproliferative and anti-apoptotic effects on culture activated PSC.
Methods Culture activated rat PSC were used between passages 2 and 4 and serum deprived prior to all experiments. Apoptosis was induced with cycloheximide (50 mm/l) in the presence or absence of recombinant rat TIMP-1 (1–200 ng/ml). Apoptosis was quantified using acridine orange and counting of apoptotic bodies. PSC proliferation was determined by [3H]-thymidine incorporation. Culture-activated rat PSC were transfected with TIMP-1 and a negative control siRNA by electroporation. Cytotoxicity of siRNA was assessed by lactate dehydrogenase activity assay. Culture supernatants were collected to quantitate TIMP-1 protein knockdown following electroporation.
Results TIMP-1 showed a dose dependent inhibition of cycloheximide induced PSC apoptosis. TIMP-1 at 100 ng/ml and 200 ng/ml significantly reduced apoptosis by up to 61% at 6 h, and up to 74% at 24 h (see table). Although there was a trend towards TIMP-1 stimulation of increased proliferation, this did not reach statistical significance. TIMP-1 electroporation was an effective means of silencing PSC TIMP-1 expression. Peak knockdown of target TIMP-1 protein was 77% at 24 h for TIMP-1 compared to negative control siRNA (p<0.05). There was no increase in cell toxicity in TIMP-1 siRNA treated cells compared with electroporation alone.
Conclusion These data suggest that TIMP-1 reduces PSC apoptosis in the presence of cycloheximide, with no significant effect on proliferation. These data also suggest that siRNA are an effective means of silencing TIMP-1 in PSC. This may represent an important therapeutic target in the treatment of pancreatic fibrosis.