RESUMO
INTRODUCTION: Hypoxia-inducible factor (HIF) 1α is essential for the pathogenesis of necrotizing enterocolitis (NEC). HIF-1α is stabilized by glutaredoxin-1 (Grx1) deletion. The precise role of HIF-1α in the intestinal microcirculation in NEC is not well defined. We aimed to determine the role of HIF-1α in the regulation of the intestinal microcirculation during the development of NEC. METHODS: Experimental NEC was induced in full-term C57BL/6 mice and Grx1-/- pups through the formula gavage and hypoxia technique. HIF-1α signaling was blocked using the HIF-1α inhibitor, YC-1 [3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole]. Intestinal tissues were collected at predetermined time points for the assessment of the intestinal microcirculation and HIF-1α activity and signaling. RESULTS: We found that NEC induction impaired the intestinal microcirculation, but the impairment of the intestinal blood flow and capillary density was ameliorated in Grx1-/- mice. This amelioration was associated with tripeptide glutathione-protein adducts in the intestinal tissue. Grx1 ablation also promoted vascular endothelial growth factor A production in the intestinal tissue. This intestinal microvascular improvement was not found in HIF-1α-inhibited mice, suggesting that HIF-1α was involved in the intestinal microcirculatory perfusion. CONCLUSIONS: The current data demonstrated that HIF-1α signaling is involved in the intestinal microvascular modification during the pathogenesis of NEC, suggesting that targeting HIF-1α might be a promising strategy for NEC treatment.
Assuntos
Enterocolite Necrosante , Animais , Camundongos , Enterocolite Necrosante/metabolismo , Glutarredoxinas/metabolismo , Glutationa , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Indazóis , Camundongos Endogâmicos C57BL , Microcirculação , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Enterocolite Necrosante/metabolismo , Inflamação/metabolismo , Intestino Delgado/irrigação sanguínea , Microvasos/metabolismo , Neovascularização Fisiológica , Efeitos Tardios da Exposição Pré-Natal , Fator de Necrose Tumoral alfa/metabolismo , Animais , Permeabilidade Capilar , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Enterocolite Necrosante/etiologia , Enterocolite Necrosante/patologia , Enterocolite Necrosante/fisiopatologia , Feminino , Idade Gestacional , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/complicações , Inflamação/patologia , Inflamação/fisiopatologia , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Microvasos/patologia , Microvasos/fisiopatologia , Gravidez , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The intestinal microvasculature (iMV) plays multiple pathogenic roles during chronic inflammatory bowel disease (IBD). The iMV acts as a second line of defense and is, among other factors, crucial for the innate immunity in the gut. It is also the therapeutic location in IBD targeting aggravated leukocyte adhesion processes involving ICAM-1 and E-selectin. Specific targeting is stressed via nanoparticulate drug vehicles. Evaluating the iMV in enterocyte barrier models in vitro could shed light on inflammation and barrier-integrity processes during IBD. Therefore, we generated a barrier model by combining the enterocyte cell line Caco-2 with the microvascular endothelial cell line ISO-HAS-1 on opposite sides of a transwell filter-membrane under culture conditions which mimicked the physiological and inflamed conditions of IBD. The IBD model achieved a significant barrier-disruption, demonstrated via transepithelial-electrical resistance (TER), permeability-coefficient (Papp) and increase of sICAM sE-selectin and IL-8. In addition, the impact of a prospective model drug-vehicle (silica nanoparticles, aSNP) on ongoing inflammation was examined. A decrease of sICAM/sE-selectin was observed after aSNP-exposure to the inflamed endothelium. These findings correlated with a decreased secretion of ICAM/E-selectin bearing exosomes/microvesicles, as evaluated via ELISA. Our findings indicate that aSNP treatment of the inflamed endothelium during IBD may hamper exosomal/microvesicular systemic communication.
Assuntos
Exossomos/metabolismo , Inflamação/patologia , Nanopartículas/toxicidade , Dióxido de Silício/toxicidade , Células CACO-2 , Selectina E/metabolismo , Impedância Elétrica , Exossomos/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/metabolismoRESUMO
Blood supply to enterocytes dictates intestinal health and nutrient absorption. These two aspects are impaired in low birthweight (LBW) piglets, but whether the perfusion to intestinal tissues is implicated as well is still unknown. Thus, structural changes in the microvasculature of LBW and normal birthweight (NBW) piglets were investigated during early postnatal development. Additionally, the presence of endothelial nitric oxide synthase (eNOS) in the intestinal mucosa was assessed given its important role to assure perfusion. A total of 22 pigs (11 LBW and 11 NBW) were sacrificed at days 0, 3, 8 and 19 of life. Body weight and intestinal length were recorded and a piece of the small intestine was sampled for immunohistochemical analysis of von Willebrand Factor (vWF, an endothelial cell marker) and eNOS. LBW piglets had a relatively (to body weight) longer intestine than their NBW counterparts. Age did not affect microvasculature, which was more abundant (85% larger vWF-positive area) in NBW than LBW pigs. However, an interaction age*BW was observed for eNOS-IR, showing that eNOS presence peaked in NBW piglets on the first day of life and subsequently decreased. This pattern was not observed in LBW piglets. The less abundant intestinal endothelial mass and the different pattern of eNOS expression observed in LBW piglets suggests microcirculation as a contributing factor in the impaired digestive functioning and gut health of LBW pigs. However, revealing whether the origin of this alteration is prenatal or postnatal, for example due to a lower milk intake, needs further study.
Assuntos
Peso ao Nascer/fisiologia , Intestinos/irrigação sanguínea , Microvasos/crescimento & desenvolvimento , Óxido Nítrico Sintase Tipo III/metabolismo , Suínos/anatomia & histologia , Animais , Feminino , Imuno-Histoquímica/veterinária , Intestinos/anatomia & histologia , Intestinos/crescimento & desenvolvimento , Suínos/crescimento & desenvolvimentoRESUMO
PURPOSE: Necrotizing enterocolitis is associated with decreased intestinal perfusion and ischemia. Paneth cells, specialized epithelial cells, have been shown to regulate the intestinal vasculature and disruption of these cells has been associated with NEC. We hypothesized that Paneth cell disruption in immature mice intestine would decrease the perfusion of the intestinal microvasculature. METHODS: Paneth cells were disrupted in P14-16 mice using chemical (dithizone) and transgenic (diphtheria toxin) methodology. Six hours after Paneth cell disruption, Dylight 488 was injected directly into the left ventricle and allowed to perfuse for 5 minutes prior to intestinal harvesting. Tissue samples were evaluated with confocal fluorescence microscopy to quantify intestinal perfusion and samples were quantified by real time RT-PCR for gene expression. RESULTS: Dithizone treatment significantly decreased intestinal perfusion compared to controls (pâ¯<â¯0.01). However, diphtheria toxin treatment demonstrated no significant difference in perfusion (pâ¯>â¯0.21). Intestines from all treatment groups had similar PECAM staining, but intestines treated with dithizone had significantly decreased nNOS and iNOS gene expression compared to controls (pâ¯<â¯0.007). CONCLUSIONS: Paneth cell disruption significantly decreases the perfusion of the small intestinal microvasculature in a dithizone-specific manner. Dithizone has no effect on the amount of microvasculature, but does impact genes critical to nitric oxide signaling likely contributing to mesenteric vasoconstriction.
Assuntos
Ditizona/farmacologia , Intestino Delgado/irrigação sanguínea , Microcirculação/efeitos dos fármacos , Celulas de Paneth/efeitos dos fármacos , Animais , Toxina Diftérica/farmacologia , Modelos Animais de Doenças , Enterocolite Necrosante/etiologia , Enterocolite Necrosante/metabolismo , Enterocolite Necrosante/patologia , Isquemia/induzido quimicamente , Camundongos , Óxido Nítrico/metabolismo , Celulas de Paneth/metabolismo , Celulas de Paneth/fisiologia , Transdução de SinaisRESUMO
Necrotizing enterocolitis (NEC), a leading cause of morbidity and mortality in preterm neonates, is a devastating disease characterized by intestinal tissue inflammation and necrosis. NEC pathogenesis is multifactorial but remains unclear. Translocation of bacteria and/or bacterial products across a weak intestinal barrier in the setting of impaired mucosal immunity leads to an exaggerated inflammatory response and secondary mucosal epithelial injury. In addition to prematurity, other risk factors for NEC include congenital heart disease, maternal pre-eclampsia with placental vascular insufficiency, severe anemia and blood transfusion - all conditions that predispose the intestine to ischemia. We recently found that maldevelopment of the intestinal microvasculature plays an important role in NEC pathogenesis. Here we review the evidence supporting a role for defective development of the intestinal mucosal microvasculature and perturbations of intestinal blood flow in NEC, emphasizing the importance of vascular endothelial growth factor (VEGF) and the VEGF receptor-2 signaling pathway.
Assuntos
Enterocolite Necrosante/metabolismo , Mucosa Intestinal/irrigação sanguínea , Intestinos/irrigação sanguínea , Microcirculação/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Humanos , Recém-Nascido , Recém-Nascido PrematuroRESUMO
The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-ß) and a nanoparticle (Ba/Gd: coprecipitated BaSO4 and Gd(OH)3), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (Papp) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the Papp decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-ß, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the Papp, the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying endothelial cells. In summary, this coculture model exhibits in vivo-like features, which cannot be observed in conventional monocultures, making the former more suitable to study interactions with external stimuli.