331 Validating the Role of Necroptosis in Inflammatory Bowel Disease (IBD) Pathogenesis

activation of AMPK inhibits cell proliferation, reduces protein synthesis, induces autophagy and activates both FAO and glycolysis. Using explant cultures, we show that FAO is fivefold greater in the colon than small intestine and is activated equally in germ-free and conventionally-housed mice. Interestingly, by contrast to both Caco-2 and LS174T cells, the activation of AMPK by AICAR inhibits FAO both in vivo and ex vivo, a response that is reversed when explant cultures are deprived of glucose showing that the Randle cycle is operative in primary intestinal epithelial cells but not in transformed cell lines. Finally, small intestinal FAO is dramatically inhibited in two different Th2-mediated models of inflammation where crypt hypertrophy is associated with dramatic expansion of TA cells and a more modest increase in LGR5-positive stem cells. Conclusions: Cellular metabolism of the intestinal epithelium can be modulated by the activation of AMPK whereby the induction of glycolysis secondarily inhibits FAO, a response that is distinctly different from transformed cells. The reduction in FAO associated with intestinal inflammation and crypt hypertrophy may, therefore, be due to the induction of glycolytic metabolism in rapidly proliferating TA cells. The modulation of intestinal epithelial metabolism by AMPK may be particularly relevant in IBD where pharmacologic activators have been shown to reduce injury in animal models of colitis as well as the inhibit development of intestinal neoplasia. 331 Validating the Role of Necroptosis in Inflammatory Bowel Disease (IBD) Pathogenesis Maria Pierdomenico, Anna Negroni, Roberta Vitali, Enrica Prete, Francesca Palone, Vincenzo Cesi, Marina Aloi, Anna Dilillo, Giovanni Di Nardo, Salvatore Cucchiara, Laura Stronati Background: Excessive intestinal epithelial cell death has been associated with chronic inflammation as occurs in IBD. Recently, an apoptosis and caspase-independent form of programmed necrotic cell death, termed necroptosis, has been described in the intestinal epithelium and considered as a possible trigger of inflammation. In necroptosis, dying cells show the morphological features of necrosis but cell death is highly regulated by an intracellular protein platform. Receptor-interacting kinase 3 (RIP3), a RIP family member, has been recently identified as a key regulator of caspase-independent cell death. Upon activation of Tumor Necrosis Factor Receptor (TNFR) and related receptors, RIP1 and RIP3 assemble in a complex through their homotypic interaction motif (RHIM) with Fas-Associated Death Domain (FADD) and caspase-8. When the activity of caspase-8 is blocked, mutual direct and indirect phosphorylation of RIP1 and RIP3 in the necrosome activates necroptotic signaling. Aims: We performed an in vitro study to demonstrate the role of RIP3 in necroptosis; furthermore, we investigated ex vivo the expression levels of RIP3 in inflamed tissues of IBD patients to suggest its possible role as a necroptosis inducer. Methods: The HT29 colon adenocarcinoma cell line was cultured and treated with: INF-gamma, caspase inhibitor z-VAD-fmk (z-VAD) and apoptosis inducer doxorubicin (Dox). After 48 hrs, cell viability was analyzed by MTT test. Expression levels of RIP3 and Poly (ADP-ribose) polymerase 1 (PARP1), as a necrosis marker, were also analysed. 60 pediatric IBD patients (range age: 2-18 years) (30 Crohn's disease [CD] and 30 ulcerative colitis [UC]) and 24 controls entered into the study. Biopsies were snap frozen immediately after endoscopy and RIP3 and caspase-8 protein expression levels were analysed by Western blot. Results: A significant decrease of cell viability was shown in HT29 cells treated with Interferon(INF)- γ+z-VAD and with INF-γ +z-VAD+Dox compared to cells treated with INF- γ alone (P,0.05). In addition, RIP3 was highly up-regulated when apoptosis was inhibited, while caspase-8 was down-regulated (P,0.05). The strong increase of necrotic band expression of PARP1 excluded apoptosis in these experimental conditions. Ex vivo analysis showed that RIP3 was significantly up-regulated in the inflamed ileal and colonic mucosa of CD and in UC patients (P,0.001). Expression analysis on caspase-8 is in progress. Conclusion: We demonstrated that RIP3 is strongly implicated in necroptosis, as recently suggested. Moreover, we showed for the first time that RIP3 is strongly up-regulated in IBD patients, suggesting that necroptosis could be involved in the disease pathogenesis

329 Reduced Expression of Nerve Growth Factor in Aging Gastric Mucosal Endothelial Cells - A New Key Mechanism for Impaired Angiogenesis in Aging Gastric Mucosa? Amrita Ahluwalia, Michelle Giffin, Michael K. Jones, Andrzej S. Tarnawski Background/Aims: In aging gastric mucosa, the formation of new blood vessels (angiogenesis) is impaired and healing of mucosal injury is delayed. However the mechanisms of impaired angiogenesis in aging gastric mucosa are not fully elucidated. Recent studies indicate that nerve growth factor (NGF) may play a role in blood vessel development and remodeling in some tissues (Circ Res. 106:1275-84, 2010). However, it is not known whether NGF plays a mechanistic role in gastric angiogenesis; whether gastric endothelial cells express NGF and/or its high affinity TrkA receptor; or, how aging impacts the expression of these proteins. The aims of this study were to test the hypotheses that: 1) NGF is expressed in gastric mucosal endothelial cells; and, 2) reduced expression of NGF in aging gastric mucosal microvascular endothelial cells (AGEC) plays a causal role in impaired angiogenesis. Methods: Gastric mucosal microvascular endothelial cells were isolated from Fisher F-344 rats, 3 months of age (young, YGEC) and 24 months of age (aging, AGEC) using anti-PECAM-1 selection and magnetic bead separation. YGEC and AGEC cells were cultured in growth media and were treated with vehicle (PBS), NGF or VEGF. Studies: 1) Expression of NGF and its TrkA receptor by Western blotting and immunohistochemistry, 2) In vitro angiogenesis on matrigel and quantification of tube formation using Metamorph image analysis system, 3) To determine the mechanisms of impaired angiogenesis, we treated AGEC with exogenous NGF and examined whether exogenous NGF induces angiogenesis. Results: YGEC and AGEC expressed NGF and TrkA receptor. NGF protein was reduced ~ 2-fold (P ,0.05) in AGEC vs. YGEC but TrkA levels were similar in AGEC and YGEC. In vitro angiogenesis was reduced 4.1-fold in AGEC (vs. YGEC; P ,0.001). Treatment of AGEC with exogenous NGF significantly increased in vitro angiogenesis by 1.8-fold (P ,0.001), which was similar to treatment with VEGF, a fundamental angiogenic factor. Conclusions: 1) This study demonstrated for the first time that gastric endothelial cells express NGF and its receptor. 2) NGF expression is reduced in gastric endothelial cells isolated from aging rats and is causally associated with impaired angiogenesis. 3) Treatment with exogenous NGF increases angiogenesis in aging gastric mucosal microvascular endothelial cells. 4) These findings demonstrate that reduced NGF expression plays a novel causal role in impaired angiogenesis in aging gastric mucosa.

332 After Isolation From Luminal Nutrition, Human Small Intestine Shows Increased Paneth Cell Numbers and Decreased Proliferation, and Phosphorylation of S6 Protein Christa N. Grant, Mark R. Frey, Tracy Grikscheit Purpose: Adult intestinal stem cells and their niche are responsive to the host's nutritional state. In the mouse, Paneth cells alter intestinal epithelial stem cell fate in response to caloric restriction through inhibition of S6 protein phosphorylation in the mechanistic target of rapamycin complex 1 signaling pathway. Using specimens from patients undergoing ileostomy reversal, in which the distal part of the intestine was removed from luminal flow, we asked whether this response is conserved in humans. Methods: This study was approved and monitored by the CHLA IRB. Human small intestinal tissue samples were collected from adjacent segments of the small intestine of children at a single institution between July and September 2012. These patients had all had previous intestinal division in the ileum without extensive resection, so that compared segments were adjacent. The segment contiguous with enteral feeding was denoted "fed" (n=5) while the segment out of luminal flow because of the intestinal division was denoted "unfed" (n=4). Tissue was processed for immunofluorescence, immunohistochemistry, and western blot. Villus height, crypt depth, and cell type quantifications were made per hemi-crypt and per hemi-villus for all 20x fields included in a 5 micron section of each specimen. Mouse intestine served as a histological control. Results: We observed a 37% decrease in S6 phosphorylation in unfed versus fed human intestinal tissue by quantitative western blot analysis (p=0.03). Paneth cells per hemi crypt were increased in the unfed tissue (1.75 versus 0.76, p ,0.02). Villi in unfed ileum were on average 22% shorter (p ,0.02) and crypts were 31% more shallow (p ,0.008). In contrast, there was no significant difference in crypt width or crypt fission rates (0.97 bifid crypts per 20x field in unfed versus 0.85 in fed). Immunofluorescent staining with proliferating cell nuclear antigen (PCNA) showed decreased rates of proliferation in crypts (2.05 versus 3.38 PCNA+ cells per hemi crypt p=0.008) in unfed tissue. In terms of differentiation, there were more chromogranin A positive enteroendocrine cells per villus in unfed tissue

330 AMPK-Dependent Metabolic Plasticity of the Intestinal Crypt Compartment Lillian Chau, Mike Sheng, Sue A. Keilbaugh, Laetitia Charrier-Hisamuddin, Ying-Yu Chen, Sarah A. Smith, Gary D. Wu Background: The intestinal epithelial barrier is constantly renewed every 4-5 days making the intestinal epithelium the most rapidly proliferating cell type in adult animals. Intestinal stem cells give rise to transit amplifying (TA) cells that rapidly divide 4 to 5 times before becoming terminally differentiated. The regulation of cellular metabolism is critical to sustain rapidly proliferating cells where glucose metabolism, and to a lesser extent glutamine, is essential to support rapid cellular proliferation in both nontransformed and tumor cells. By contrast, fatty acid oxidation (FAO), principally of short chain fatty acids produced by the gut microbiota, is widely believed to be the primary source of metabolic fuel for the colonic epithelium. Focusing on the master regulator of cellular metabolism, AMP-activated protein kinase (AMPK), we examined the interaction between FAO and glycolysis in the intestinal epithelium. Methods and Results: FAO of 3H-Oleic acid was quantified by the production of tritiated water release and glycolysis was determined by the consumption of glucose and the production of lactate. The LKB1 serine/threonine kinase, a tumor suppressor gene for Putz-Jehgers polyposis, activates AMPK thereby coordinating cell growth with energy metabolism in both Caco-2 and LS174T intestinal cell lines. Specifically, LKB1-depdendent

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AGA Abstracts

AGA Abstracts

of pancreatic innervation has not been possible previously. Confocal laser induced endomicroscopy (CLE) is a novel tool that enables in-vivo histologic imaging by detection of fluorescence. An ultrathin CLE catheter probe that inserts into a 19G FNA needle now allows for Needle-based confocal laser endomicroscopy (nCLE). This study was aimed to determine feasibility of in vivo visualization of the innervation of porcine pancreas using endoscopic ultrasound guided nCLE and a fluorescent neuronal marker; and to determine pancreatic nerve cell expression of: nerve growth factor (NGF - neurotrophic factor that promotes survival of neuronal cells), its high affinity receptor Trk A and melatonin receptor 1 (MR1). Methods. Studies were performed in anesthetized pigs. NeuroTrace (fluorescent neuronal marker) was injected into pancreas tail and body using endoscopic ultrasound guided 19G FNA needle, followed by nCLE in vivo imaging (Cellvizio AQ-Flex-19, Mauna Kea Technologies, Paris, France). Pigs were then euthanized and pancreatic specimens obtained for histology, ex-vivo immunostaining and molecular imaging. Studies: 1) nCLE image analysis, 2) quantitative histology and visualization of neuronal labeling under fluorescence, 3) immunostaining and quantification for NGF, its TrkA receptor and MR1 using specific antibodies. Results: nCLE imaging visualized an extensive network of intrapancreatic ganglia and nerves labeled with NeuroTrace. There was a higher density of neural elements in the body than the tail. On histologic sections examined under fluorescence microscope, in-vivo injected NeuroTrace was localized to the neural structures and nerves in pancreatic stromal tissue with features similar to those seen on nCLE. NGF was strongly expressed in intrapancreatic neuronal cells and nerves and was also expressed in epithelial cells of pancreatic acini and endothelial cells of blood vessels. TrkA was expressed in some neuronal cells and nerves distributed throughout the acini. MR1 was expressed in about 60% of ganglion cells and nerves and also in pancreatic acini. Quantitatively its expression was 2.5fold lower (p,0.01) compared to NGF. Conclusions: 1)This study showed feasibility of in vivo visualization of neuronal network in porcine pancreas nCLE. 2)The pancreatic neural network is extensive; numerous neuronal cells express NGF, Trk A and MTR1. 3)NGF and MR1 expression in epithelial acinar cells and endothelium of blood vessels, in addition to neural structures, indicates local interactions between neural, epithelial and vascular components of pancreas.