Abstract 651 Neonatal Disease Oriented Research: Intestinal Development Platform, Saturday, 5/1

The histopathology of necrotizing enterocolitis (NEC) is characterized by destruction of the mucosal layer in initial stages and by transmural necrosis of the intestinal wall in advanced stages of the disease. Many features of the human disease can be reproduced in rodent models acutely, by intravascular injection of inflammatory mediators such as platelet activating factor (PAF), lipopolysaccharide (LPS) or tumor necrosis factor α, or chronically, by formula feeding and cold/asphyxia stressing of premature neonatal rats. Histological NEC elicited in any of these models is accompanied by widespread apoptosis in the mucosal layer. In the present study we analyzed the mechanisms by which PAF and LPS cause apoptosis in intestinal epithelial cells in vitro, using the IEC-6, rat small intestinal epithelial cell tissue culture model. Apoptosis was quantified by an ELISA assay based on the capture of cytoplasmic histone-bound DNA fragments, or was visualized by fluorescence TUNEL staining. Caspase 3 enzyme activity from cell lysates was measured using the DEVD-AMC fluorogenic substrate. Assessment of mRNA expression was performed by semi-quantitative RT-PCR, using GAPDH and/or actin as internal control. Quantitative data were normalized to untreated controls and presented below as percent control; mean ± S.E.M. Statistical analysis was performed using Student's t test; * indicates p less than 0.05 and ** indicates p less than 0.005. Both PAF and LPS increased the rate of apoptosis (241±32% and 281±54%; **, n=24), albeit, by different mechanisms. PAF-elicited apoptosis was accompanied by significantly enhanced caspase 3 activation (274±57%; *, n=4) and was completely blocked by the caspase 3 inhibitory peptide DEVD-CHO. The known pro-apoptotic mediator and protein kinase C (PKC) inhibitor sphingosine greatly potentiated PAF-induced apoptosis (654±105%; **, n=12) and PAF-induced caspase 3 activity (851±137%; *, n=4). Consistent with these findings, phorbol myristate acetate (PMA), a direct activator of PKC inhibited apoptosis in IEC-6 cells (55±2.5%; *, n=4). Unlike PAF-induced apoptosis, LPS-induced apoptosis was not blocked by the caspase 3 inhibitor DEVD-CHO and was accompanied by an increase of iNOS mRNA expression in IEC-6 cells. These data indicate that inflammatory mediators can elicit apoptosis in small intestinal epithelial cells via multiple mechanisms. PAF stimulates apoptosis in IEC-6 cells via the activation of caspase 3 and a signaling mechanism that is under inhibitory control by PKC, while LPS stimulates apoptosis in these cells independently of caspase 3, but maybe involving the upregulation of iNOS. It is yet to be determined whether the specific blockade of apoptosis could prevent the development of NEC in our in vivo models.