Necrotizing enterocolitis (NEC) is a life-threatening inflammatory bowel disorder of unknown cause that affects approximately 10% of premature infants born <1500gm. Platelet activating factor (PAF) is a phospholipid inter and intra-cellular mediator which has been shown in clinical studies to rise several days before the onset of clinical symptoms in some patients, suggesting that it is a critical factor in the development of the disease. Thus inhibition of the early effects of PAF could limit the clinical morbidity of NEC. However, the exact mechanisms of PAF-induced intestinal injury are poorly understood. The objective of this proposal is to investigate the early effects of PAF directly on intestinal epithelial cells in order to identify a therapeutic intervention to prevent development of NEC. Preliminary data for this proposal demonstrate four potentially linked, early effects of PAF directly on intestinal epithelial cells -PAF-induced intracellular acidification, along with PAF-induced Cl- channel activation, increased free intracellular [Ca2+]I, and apoptosis. I hypothesize that PAF induces intracellular acidosis via a Ca2+ dependent Cl- channel and that PAF induced intracellular acidosis results in apoptosis of intestinal epithelial cells leading to compromised intestinal barrier function. Various Cl- channels have been shown to conduct bicarbonate in addition to Cl-, thereby altering intracellular pH. Intracellular acidosis has been linked to apoptosis in other cell types. Apoptosis of intestinal epithelial cells may lead to increased intestinal permeability, bacterial translocation across the intestinal epithelium, inflammation, and NEC. Thus, PAF-induced activation of an intestinal mucosal Cl- channel and resultant acidosis may be an important early trigger of epithelial cell apoptosis resulting in compromise of intestinal barrier function. This proposal is designed to 1. Determine the mechanism of PAF induced acidosis in intestinal epithelial cells 2. Investigate the specific relationship between PAF induced acidosis and apoptosis. 3. Examine the early effects of PAF on intestinal barrier function. The experimental design uses the well described rat small intestinal epithelial cell line IEC-6, the human fetal small intestinal cell line H4 which is a model of the human immature small intestine uniquely susceptible to NEC, and pre-weaned rat intestinal explants. These studies will begin to answer the critical questions "What begins the cycle of intestinal injury in NEC?" and "What is the role of PAF in initial intestinal epithelial cellular injury?" Since apoptosis of intestinal epithelial cells associated with Cl- channel activation has not been previously described, understanding this mechanism may provide a novel approach to treating or preventing NEC in at risk infants. PUBLIC HEALTH RELEVANCE Necrotizing enterocolitis is a poorly understood, life threatening inflammatory bowel disease of premature infants in which 20-30% of patients die and survivors are at risk for significant intestinal and neurodevelopmental consequences. Platelet activating factor (PAF) is believed to play a critical role in the development of this disease. This proposal will investigate how PAF causes intestinal injury, in order to develop a treatment to limit necrotizing enterocolitis in vulnerable infants.

R21HD055237

2008-04-01

2010-03-31