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Platelet-activating factor (PAF), an endogenous proinflammatory phospholipid, when injected intravascularly to rats and mice, causes shock, acute bowel injury, and a rapid activation of NF-[kappa]B p50-p50 with upregulation of the chemokine CXCL2 in the intestine. In this study, we investigate the mechanism of NF-[kappa]B activation and the role of the NF-[kappa]B p50 subunit in PAF-induced shock and acute bowel injury. NF-[kappa]B p50-deficient mice and wild-type mice were anesthetized and tracheotomized, and their carotid artery was cannulated for blood pressure monitoring, blood sampling, and PAF administration. For determination of bowel injury, shock, and survival, PAF (2.2 [micro]g/kg, intra-arterially, i.a.) was injected. Two hours later, animals were euthanized, and their small intestines were removed for histological examination. For biochemical studies, PAF (1.5 [micro]g/kg i.a.) was administered and the small intestine removed after 15-60 min. We found that PAF induced an increase in p105 processing within 30 min, but there were no changes in the levels of the NF-[kappa]B inhibitory proteins I[kappa]B[alpha] and [beta]. NF-[kappa]B p50-deficient mice were protected against PAF-induced mortality, shock, intestinal hypoperfusion, and injury compared with wild-type animals. We also found that p50-deficient mice had decreased gene expression of CXCL2 and TNF and a decrease in CXCL2 protein production compared with wild-type mice. Our study suggests that PAF increases the processing of NF-[kappa]B p105 into p50, with upregulation of proinflammatory cytokines, which leads to PAF-induced systemic inflammatory response and acute bowel injury. intestine; necrotizing enterocolitis

Han, Xin-Bing, Xueli Liu, Wei Hsueh, and Isabelle G. De Plaen. Macrophage inflammatory protein-2 mediates the bowel injury induced by platelet-activating factor. Am J Physiol Gastrointest Liver Physiol 287: G1220-G1226, 2004. First published August 19, 2004; doi: 10.1152/ajpgi.00231.2004.--Platelet-activating factor (PAF) is a potent endogenous mediator of bowel inflammation. It activates neutrophils that are needed to initiate the inflammatory response. Macrophage inflammatory protein-2 (MIP-2), a critical C-X-C chemokine secreted by macrophages and epithelial cells, is a potent chemoattractant for neutrophils. Whereas MIP-2 has been previously shown to mediate the injury in various organs, its role in acute intestinal injury has never been assessed. In this study, we first investigated the effect of PAF on MIP-2 expression in the intestine. Anesthetized young adult male Sprague-Dawley rats were injected intravenously with either PAF (1.5 [micro]g/kg) or saline. Sixty minutes later, ileal MIP-2 gene expression was determined by semiquantitative RT-PCR, and plasma and ileal MIP-2 protein was determined by ELISA. In a second step, we assessed the role of MIP-2 in PAF-induced bowel injury. Rats were pretreated with rabbit anti-rat MIP-2 antibodies or control IgG for 90 min and then injected intravenously with PAF (2.5 [micro]/kg) for 90 min. We found that, in the rat intestine, 1) MIP-2 mRNA was only minimally expressed constitutively in sham-operated animals; 2) MIP-2 mRNA was significantly upregulated in response to PAF; 3) MIP-2 protein plasma levels and local production of MIP-2 in the ileum were markedly induced by PAF; 4) the administration of anti-rat MIP-2 IgG, but not control rabbit IgG, markedly reduced PAF-induced bowel injury (injury scores of 0.19 [+ or -] 0.09 vs. 1.12 [+ or -] 0.43, P < 0.05), hypotension, and leukopenia but did not reduce PAF-induced hemoconcentration. Thus we conclude that MIP-2 mediates PAF-induced intestinal injury. chemokines; polynuclear neutrophils

Sirtuin 6 (Sirt6) is predominantly expressed in epithelial cells in intestinal crypts. It plays an important role in protecting intestinal epithelial cells against inflammatory injury. Previously, we found that colitis is associated with the downregulation of Sirt6 protein in the intestines. Here, we report that murine interferon-γ (Ifnγ) inhibits Sirt6 protein but not mRNA expression in young adult mouse colonocytes (YAMC, a mouse colonic epithelial cell line) in a dose- and time-dependent manner. Using microRNA array analysis, we showed that Ifnγ induces expression of miR-92b in YAMC cells. With in silico analysis, we found that the Sirt6 3=-untranslated region (UTR) contains a putative binding site for miR-92b. Luciferase assay showed that Ifnγ inhibited Sirt6 3=-UTR activity and this effect was mimicked by miR-92b via directly targeting the miR-92b seed site in the 3=-UTR of Sirt6 mRNA. Furthermore, Western blot demonstrated that miR-92b downregulated Sirt6 protein expression in YAMC cells. Blocking miR-92b with a specific inhibitor attenuated the inhibitory effect of Ifnγ on Sirt6 protein expression in the cells. Collectively, our data suggest that Ifnγ inhibits Sirt6 protein expression in intestinal epithelial cells via a miR-92b-mediated mechanism. miR-92b may be a novel therapeutic target for rescuing Sirt6 protein levels in intestinal epithelial cells, thereby protecting against intestinal mucosal injury caused by inflammation. [ABSTRACT FROM AUTHOR]

Prenatal inflammation is a risk factor for necrotizing enterocolitis (NEC), and it increases intestinal injury in a rat NEC model. We previously showed that maldevelopment of the intestinal microvasculature and lack of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) signaling play a role in experimental NEC. However, whether prenatal inflammation affects the intestinal microvasculature remains unknown. In this study, mouse dams were injected intraperitoneally with lipopolysaccharide (LPS) or saline at embryonic day 17. Neonatal intestinal microvasculature density, endothelial cell proliferation, and intestinal VEGF-A and VEGFR2 proteins were assessed in vivo. Maternal and fetal serum TNF concentrations were measured by ELISA. The impact of TNF on the neonatal intestinal microvasculature was examined in vitro and in vivo, and we determined whether prenatal LPS injection exacerbates experimental NEC via TNF. Here we found that prenatal LPS injection significantly decreased intestinal microvascular density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression in neonatal mice. Prenatal LPS injection increased maternal and fetal serum levels of TNF. TNF decreased VEGFR2 protein in vitro in neonatal endothelial cells. Postnatal TNF administration in vivo decreased intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression and increased the incidence of severe NEC. These effects were ameliorated by stabilizing hypoxia-inducible factor-1α, the master regulator of VEGF. Furthermore, prenatal LPS injection significantly increased the incidence of severe NEC in our model, and the effect was dependent on endogenous TNF. Our study suggests that prenatal inflammation increases the susceptibility to NEC, downregulates intestinal VEGFR2 signaling, and affects perinatal intestinal microvascular development via a TNF mechanism. NEW & NOTEWORTHY This report provides new evidence that maternal inflammation decreases neonatal intestinal VEGF receptor 2 signaling and endothelial cell proliferation, impairs intestinal microvascular development, and predisposes neonatal mouse pups to necrotizing enterocolitis (NEC) through inflammatory cytokines such as TNF. Our data suggest that alteration of intestinal microvascular development may be a key mechanism by which premature infants exposed to prenatal inflammation are at risk for NEC and preserving the VEGF/VEGF receptor 2 signaling pathway may help prevent NEC development. [ABSTRACT FROM AUTHOR]