The central goal of this proposal is to establish and characterize novel models of acute and chronic pancreatitis by direct intra-acinar cell trypsinogen activation. Recent discoveries of trypsinogen and trypsin inhibitor mutations in patients with hereditary pancreatitis support the hypothesis that an inappropriate activation of pancreatic zymogens to active enzymes within the pancreas starts the inflammatory process. Thus premature activation of trypsin is believed to be the initial step in the development of pancreatitis. However, current animal models of acute pancreatitis involve treatments that have many non-specific effects and trigger numerous signaling pathways in addition to activating trypsin. The situation for chronic pancreatitis is even less clear, as there are no reliable animal models of this disease.
Specific aim #1 is to induce acute pancreatitis by direct trypsin activation within pancreatic acinar cells in rats. We will initially use adenoviral mediated gene transfer into the rat pancreas of a mutant trypsinogen (AdPACE-TRY) which we developed and have shown to become activated within the acinar cells. We hypothesize that delivery of this virus to the pancreas in vivo will active trypsin and thereby initiate pancreatitis.
Specific aim #2 is to determine whether intracellular trypsin induces NF-KB activation and the role of NF-KB in trypsin induced acute pancreatitis. We have previously shown that NF-KB activation does not cause trypsinogen activation. Here we will test if trypsin activity within the acinar cell can activate NF-KB using AdPACE-TRY to activate trypsin intracellularly and investigate the mechanisms involved. We will further examine the importance of trypsin activation of NF-KB in the initiation of the inflammatory cascade by co-administering an adenovirus expressing kB-cc which will block NF-KB activation.
Specific aim #3 is to establish a chronic pancreatitis model by directly regulating intra-acinar trypsin activity. We will create a chronic pancreatitis model by directly regulating intra-acinar trypsin activity. For this purpose, an elastase promoter driven tamoxifen regulatable Cre-Er transgenic mouse will be crossed with a loxP- GFP flanked PACE-TRY transgenic mouse. Tamoxifen activation of Cre will then remove a stop sequence allowing the expression of the active trypsin. We expect that a high dose tamoxifen will induce high trypsin activity and thus cause severe acute pancreatitis and that a low dose tamoxifen will induce partially pancreatic injury and prolonged pancreatic damage that will result in chronic pancreatitis. ? ? ?
