The most common cause of Pancreatitis is excessive alcohol intake which has the potential to induce reactive oxygen species (ROS) leading to DNA damage and apoptosis. In addition, characteristic pathological responses of pancreatitis, including inflammatory chemokines and a stiff, desmoplastic microenvironment may also contribute to this increased oxidative potential in pancreatic ductal cells. ROS can cause a severe inflammatory reaction instigating severe pain, hemorrhagic shock, acute respiratory distress, or death, however, no in vitro human model yet exists to understand this pathology. As such, we have begun to develop a novel, three-dimensional, human pancreatic duct model composed of nestin-positive, immortalized, pancreatic epithelial cells, Matrigel scaffolds, predetermined culture media, and specific growth factors. This model is characterized via confocal microscopy for cellular organization, polarization, and functionality similar to human mammary epithelial acini. Secondly, much progress has been made in understanding pancreatic stellate cell reactions to ethanol, but less is understood of the pancreatic epithelial cell response. Thus, we will analyze the effect of increasing concentrations of ethanol on ROS induction within these human pancreatic ducts via fluorescent staining and lipid peroxidation as well as the subsequent DNA damage response and apoptotic initiation events via the double strand break markers (H2AX and (SP1101 and Caspase-3, Bim, and Annexin V, respectively. Thirdly, pancreatic stellate cells have also been implicated in ROS production via inflammatory mediators, however, little has been advanced in the pancreatic epithelial cell response. As such, we will examine the direct initiation of ROS within the human pancreatic duct model via an inflammatory chemokine regularly found within the pancreatitis environment, IL-8. Through treatment with IL-8, we will determine the human pancreatic epithelial cell duct ROS-induced DNA damage and apoptosis response. Lastly, we will continue to verify the extent of ROS production within human pancreatic epithelial ducts by a stiffening extracellular matrix. By blocking pancreatic-specific integrin (3, (4 and (6 through antibodies/disintegrins and by DNA microarray validation of their expression, we will understand how exogenous elastic stiffness can affect ROS potential internal to pancreatic epithelial cells. We anticipate that the development of our in vitro, 3-D, human pancreatic epithelial duct model along with each of the three independent experiments will uncover parts of the etiologic pathway leading to pancreatitis in a human-relevant model and offer possible targets for future clinical therapy.
No in vitro human model of alcoholic pancreatitis yet exists to examine the major factors contributing to its inflammatory pathogenesis. As such, we aim to create a 3-D human pancreatic epithelial cell duct model capable of recapitulating the pancreas's in vivo organization and function. In addition, we will systematically analyze alcohol-induced and chemokine- initiatied reactive oxygen species as well as their subsequent DNA damage and apoptosis responses within this model. Lastly, we will determine the modulatory effects of the stiff, fibroblastic extracellular matrix on reactive oxygen species induction.
| Botta, Gregory P; Reichert, Maximilian; Reginato, Mauricio J et al. (2013) ERK2-regulated TIMP1 induces hyperproliferation of K-Ras(G12D)-transformed pancreatic ductal cells. Neoplasia 15:359-72 |
| Reichert, Maximilian; Takano, Shigetsugu; Heeg, Steffen et al. (2013) Isolation, culture and genetic manipulation of mouse pancreatic ductal cells. Nat Protoc 8:1354-65 |
| Botta, Gregory P; Reginato, Mauricio J; Reichert, Maximilian et al. (2012) Constitutive K-RasG12D activation of ERK2 specifically regulates 3D invasion of human pancreatic cancer cells via MMP-1. Mol Cancer Res 10:183-96 |
| Frohbergh, Michael E; Katsman, Anna; Botta, Gregory P et al. (2012) Electrospun hydroxyapatite-containing chitosan nanofibers crosslinked with genipin for bone tissue engineering. Biomaterials 33:9167-78 |
