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The pathogenesis of necrotizing enterocolitis (NEC), a common gastrointestinal disease affecting premature infants, remains poorly understood. We previously found that intestinal VEGF-A expression is decreased in human NEC samples and in a neonatal mouse NEC model prior to detectable histological injury. Therefore, we hypothesized that lack of VEGF receptor 2 (VEGFR2) signaling facilitates neonatal intestinal injury by impairing intestinal microvasculature development. Here, we found that intestinal VEGF-A and its receptor, VEGFR2, were highly expressed at the end of fetal life and significantly decreased after birth in mice. Furthermore, selective inhibition of VEGFR2 kinase activity and exposure to a neonatal NEC protocol significantly decreased the density of the intestinal microvascular network, which was further reduced when both interventions were provided together. Furthermore, VEGFR2 inhibition resulted in greater mortality and incidence of severe injury in pups submitted to the NEC model. The percentage of lamina propria endothelial cells was decreased during NEC induction, and further decreased when VEGFR2 signaling was inhibited. This was associated with decreased endothelial cell proliferation rather than apoptosis. In conclusion, we found that VEGF-A and VEGFR2 proteins are highly expressed in the intestine before birth, and are significantly downregulated in the immediate neonatal period. Furthermore, VEGFR2 signaling is necessary to maintain the integrity of the intestinal mucosal microvasculature during the postnatal period and lack of VEGFR2 signaling predisposes to NEC in neonatal mice. [ABSTRACT FROM AUTHOR]

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-κB p50-p50 with upregulation of the chemokine CXCL2 in the intestine. In this study, we investigate the mechanism of NF-κB activation and the role of the NF-κB p50 subunit in PAF-induced shock and acute bowel injury. NF-κB 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 μg/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 μg/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-κB inhibitory proteins IκBα and β. NF-κB 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-κB p105 into p50, with upregulation of proinfiammatory cytokines, which leads to PAF-induced systemic inflammatory response and acute bowel injury. [ABSTRACT FROM AUTHOR]

BACKGROUND: 8-(N,N'-diethylamino)-n-octyl-3,4,5-trimethoxybenzoate (TMB-8) is a potent Ca(2+)-antagonist that can prevent/treat ischemic stroke and inhibit the contractility of smooth, skeletal, and cardiac muscles. Further studies are warranted to elucidate the efficacy of TMB-8 on rabbit basilar artery preparation and its action mechanisms on vascular smooth muscle cell cultures. METHODS AND RESULTS: Effects of TMB-8 on the contractility of rabbit's basilar artery in vitro and those on intracellular free Ca(2+) concentrations, [Ca(2+)](i), were studies with isolated organ bath and Fura-2 methods. Histamine-induced concentration-response curves were shifted by TMB-8 in a mixed manner whereas those of norepinephrine and KCl were shifted in a non-competitive manner. In the presence of nifedipine or in a Ca(2+)-free medium, 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ) (10 µM) induced an immediate transient contraction in rabbit basilar artery, whereas ryanodine showed a slow, weak, sustained contraction, followed by a weak, sustained relaxation. TMB-8 (30 µM) significantly inhibited these contractions of BHQ and ryanodine. Further, aminophylline enhanced the inhibitory action of TMB-8 on vasocontractions, suggesting that TMB-8's inhibitory actions may be related to the increase of cAMP level. The muscle contraction induced by BHQ was enhanced by pretreatment of the artery ring with TMB-8 for 15 minutes and then TMB-8 was rinsed out. These results indicate that TMB-8 pretreatment can increase Ca(2+) sequestration into sarcoplasmic reticulum, which leads to a larger subsequent Ca(2+) release by BHQ. KCl-induced increase of [Ca(2+)](i) in vascular smooth muscle cells was reduced when the cells were bathed in the medium containing nifedipine. TMB-8 made further reduction on KCl-induced [Ca(2+)](i) increase in nifedipine-containing solution, which had already blocked the voltage-operated Ca(2+) entry. CONCLUSION: These results indicate that (a) TMB-8 can enhance Ca(2+) sequestration into sarcoplasmic reticulum, which leads to a larger amount of Ca(2+) that can be released by BHQ; (b) TMB-8 can inhibit KCl-induced muscle contraction caused by the reduction of [Ca(2+)](i) through saturation of Ca(2+) inside the sarcoplasmic reticulum rather than a direct blockade of Ca(2+)-influx at cell membrane site; and (c) TMB-8 increases cAMP, which enhances Ca(2+) uptake into the sarcoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Necrotizing enterocolitis (NEC) is a devastating disease affecting premature infants with intestinal inflammation and necrosis. The neonatal intestinal inflammatory response is rich in macrophages, and blood monocyte count is low in human NEC. We previously found that NF-κB mediates the intestinal injury in experimental NEC. However, the role of NF-κB in myeloid cells during NEC remains unclear. Herein, inhibitor of kappaB kinase β (IKKβ), a critical kinase mediating NF-κB activation, was deleted in lysozyme M (Lysm)-expressing cells, which were found to be Cd11b + Ly6c + monocytes but not Cd11b + Ly6c - macrophages in the dam-fed neonatal mouse intestine. NEC induced differentiation of monocytes into intestinal macrophages and up-regulation of monocyte recruitment genes (eg, L-selectin) in the macrophage compartment in wild-type mice, but not in pups with IKKβ deletion in Lysm + cells. Thus, NF-κB is required for NEC-induced monocyte activation, recruitment, and differentiation in neonatal intestines. Furthermore, pups with Lysm-IKKβ deletion had improved survival and decreased incidence of severe NEC compared with littermate controls. Decreased NEC severity was not associated with an improved intestinal barrier. In contrast, NEC was unabated in mice with IKKβ deletion in intestinal epithelial cells. Together, these data suggest that recruitment of Ly6c + monocytes into the intestine, NF-κB activation in these cells, and differentiation of Ly6c + monocytes into macrophages are critical cellular and molecular events in NEC development to promote disease., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

BackgroundNecrotizing enterocolitis (NEC) is a devastating neonatal disease characterized by intestinal necrosis. Hypoxia-inducible factor-1α (HIF-1α) has a critical role in cellular oxygen homeostasis. Here, we hypothesized that prolyl hydroxylase (PHD) inhibition, which stabilizes HIF-1α, protects against NEC by promoting intestinal endothelial cell proliferation and improving intestinal microvascular integrity via vascular endothelial growth factor (VEGF) signaling.MethodsTo assess the role of PHD inhibition in a neonatal mouse NEC model, we administered dimethyloxalylglycine (DMOG) or vehicle to pups before or during the NEC protocol, and determined mortality and incidence of severe intestinal injury. We assessed intestinal VEGF by western blot analysis and quantified endothelial cell and epithelial cell proliferation following immunofluorescence.ResultsDMOG decreased mortality and incidence of severe NEC, increased intestinal VEGF expression, and increased intestinal villus endothelial and epithelial cell proliferation in experimental NEC. Inhibiting VEGFR2 signaling eliminated DMOG's protective effect on intestinal injury severity, survival, and endothelial cell proliferation while sparing DMOG's protective effect on intestinal epithelial cell proliferation.ConclusionDMOG upregulates intestinal VEGF, promotes endothelial cell proliferation, and protects against intestinal injury and mortality in experimental NEC in a VEGFR2 dependent manner. DMOG's protective effect on the neonatal intestinal mucosa may be mediated via VEGFR2 dependent improvement of the intestinal microvasculature.

Intestinal adaptation to small-bowel resection (SBR) after necrotizing enterocolitis expands absorptive surface areas and promotes enteral autonomy. Survivin increases proliferation and blunts apoptosis. The current study examines survivin in intestinal epithelial cells after ileocecal resection. Wild-type and epithelial Pik3r1 (p85α)-deficient mice underwent sham surgery or 30% resection. RNA and protein were isolated from small bowel to determine levels of β-catenin target gene expression, activated caspase-3, survivin, p85α, and Trp53. Healthy and post-resection human infant small-bowel sections were analyzed for survivin, Ki-67, and TP53 by immunohistochemistry. Five days after ileocecal resection, epithelial levels of survivin increased relative to sham-operated on mice, which correlated with reduced cleaved caspase-3, p85α, and Trp53. At baseline, p85α-deficient intestinal epithelial cells had less Trp53 and more survivin, and relative responses to resection were blunted compared with wild-type. In infant small bowel, survivin in transit amplifying cells increased 71% after SBR. Resection increased proliferation and decreased numbers of TP53-positive epithelial cells. Data suggest that ileocecal resection reduces p85α, which lowers TP53 activation and releases survivin promoter repression. The subsequent increase in survivin among transit amplifying cells promotes epithelial cell proliferation and lengthens crypts. These findings suggest that SBR reduces p85α and TP53, which increases survivin and intestinal epithelial cell expansion during therapeutic adaptation in patients with short bowel syndrome., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Milk fat globule-EGF factor 8 (MFG-E8) is expressed by macrophages and plays an important role in attenuating inflammation and maintaining tissue homeostasis. Previously, we and others found that lipopolysaccharide (LPS) inhibits MFG-E8 gene expression in macrophages. Here, we characterized the 5'-flanking region of the mouse MFG-E8 gene. To functionally analyze the upstream regulatory region of the MFG-E8 gene, a series of luciferase reporter gene constructs containing deleted or mutated regulatory elements were prepared. Using the luciferase assay, we revealed that Sp1 binding motifs within the proximal promoter region were necessary for full activity of the MFG-E8 promoter, whereas AP-1 like binding sequence at -372 played a role in governing the promoter activity at a homeostatic level. With chromatin immunoprecipitation assay, we showed that Sp1 and c-Jun physically interact with the MFG-E8 promoter region in vivo. In addition, Sp1 was found to regulate the MFG-E8 promoter activity positively and c-Jun negatively. Furthermore, we demonstrated that LPS inhibited MFG-E8 promoter activity via targeting Sp1 and AP-1-like motifs in the 5'-flanking region. Collectively, our data indicate that Sp1 and AP-1-related factors are involved in the regulation of MFG-E8 gene transcription by targeting their binding sites in the 5'-flanking region under physiological and inflammatory states., (© 2015 Wiley Periodicals, Inc.)

Background: Decreased intestinal perfusion may contribute to the development of necrotizing enterocolitis (NEC). Vascular endothelial growth factor (VEGF) is an angiogenic protein necessary for the development and maintenance of capillary networks. Whether VEGF is dysregulated in NEC remains unknown., Objectives: The objective of this study was to determine whether intestinal VEGF expression is altered in a neonatal mouse model of NEC and in human NEC patients., Methods: We first assessed changes of intestinal VEGF mRNA and protein in a neonatal mouse NEC model before significant injury occurs. We then examined whether exposure to formula feeding, bacterial inoculation, cold stress and/or intermittent hypoxia affected intestinal VEGF expression. Last, we visualized VEGF protein in intestinal tissues of murine and human NEC and control cases by immunohistochemistry., Results: Intestinal VEGF protein and mRNA were significantly decreased in pups exposed to the NEC protocol compared to controls. Hypoxia, cold stress and commensal bacteria, when administered together, significantly downregulated intestinal VEGF expression, while they had no significant effect when given alone. VEGF was localized to a few single intestinal epithelial cells and some cells of the lamina propria and myenteric plexus. VEGF staining was decreased in murine and human NEC intestines when compared to control tissues., Conclusion: Intestinal VEGF protein is reduced in human and experimental NEC. Decreased VEGF production might contribute to NEC pathogenesis., (© 2015 S. Karger AG, Basel.)

Whether intestinal barrier disruption precedes or is the consequence of intestinal injury in necrotizing enterocolitis (NEC) remains unknown. Using a neonatal mouse NEC model, we examined the changes in intestinal permeability and specific tight-junction (TJ) proteins preceding NEC and asked whether these changes are prevented by administration of Bifidobacterium infantis, a probiotic known to decrease NEC incidence in humans. Compared with dam-fed controls, pups submitted to the NEC protocol developed i) significantly increased intestinal permeability at 12 and 24 hours (as assessed by 70-kDa fluorescein isothiocyanate-dextran transmucosal flux); ii) occludin and claudin 4 internalization at 12 hours (as assessed by immunofluorescence and low-density membrane fraction immunoblotting); iii) increased claudin 2 expression at 6 hours and decreased claudin 4 and 7 expression at 24 hours; and iv) increased claudin 2 protein at 48 hours. Similar results were seen in human NEC, with claudin 2 protein increased. In mice, administration of B. infantis micro-organisms attenuated increases in intestinal permeability, preserved claudin 4 and occludin localization at TJs, and decreased NEC incidence. Thus, an increase in intestinal permeability precedes NEC and is associated with internalization of claudin 4 and occludin. Administration of B. infantis prevents these changes and reduces NEC incidence. The beneficial effect of B. infantis is, at least in part, due to its TJ and barrier-preserving properties., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Necrotizing enterocolitis (NEC) is a major cause of morbidity and death in premature infants. NEC is associated with increased levels of pro-inflammatory cytokines in plasma and tissues that are regulated by the transcription factor nuclear factor-kappaB (NF-kappaB). It remains unknown, however, whether NF-kappaB mediates injury in neonatal NEC. We therefore examined the activation status of NF-kappaB perinatally in the small intestine and in a neonatal rat model of NEC. We found that intestinal NF-kappaB is strongly activated at birth and, in dam-fed newborn rats, is down-regulated within a day. In contrast, NF-kappaB remains strongly activated at both d 1 and d 2 in stressed animals, and this is accompanied by a significant decrease in the levels of the endogenous NF-kappaB inhibitor protein IkappaBalpha and IkappaBbeta at d 2. To determine the importance of elevated NF-kappaB activity in intestinal injury in NEC, we administered the NEMO-binding domain (NBD) peptide that selectively inhibits the critical upstream IkappaB kinase (IKK). NBD but not a control peptide decreased mortality and bowel injury in this model, supporting the hypothesis that bowel injury in NEC results from elevated NF-kappaB activity. Our findings therefore lead us to conclude that selective NF-kappaB inhibition represents a promising therapeutic strategy for NEC.