Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jul 1;317(1):G57-G66.
doi: 10.1152/ajpgi.00332.2018. Epub 2019 May 24.

Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis

Xiaocai Yan  1   2 , Elizabeth Managlia  1   2 , Xiao-Di Tan  3   2 , Isabelle G De Plaen  1   2
Affiliations

Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis

Xiaocai Yan et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

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.

Keywords: endothelial cells; inflammation; intestinal microvasculature; necrotizing enterocolitis; neonate.

PubMed Disclaimer

Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

Fig. 1.
Fig. 1.
Prenatal lipopolysaccharide (LPS) affects the perinatal development of the intestinal microvasculature. Pregnant dams were injected with lipopolysaccharide (LPS) or normal saline (controls) at embryonic day 17, and pups were allowed to be delivered naturally. Alexa Fluor 647-labeled wheat germ agglutinin was injected by intracardiac perfusion at day 1 of life. The intestinal submucosal (A) and villous (C) microvasculature was imaged at ×10 magnification on formalin-fixed, whole mount tissues. Representative images are presented. A: ×10 images (top); locally magnified images (bottom). Scale bars = 200 μm. B: intestinal submucosal microvascular area density was assessed using Photoshop software and vessel area percentage is presented (***P < 0.001, analyzed using two-sided Student’s t-test). Pups were examined (2–3 per litter) and 3 litters were studied in each group. C: luminal dye leakage noted in pups prenatally treated with LPS (arrowheads).
Fig. 2.
Fig. 2.
Prenatal lipopolysaccharide (LPS) increases neonatal intestinal microvascular permeability. To measure intestinal microvascular permeability, pups were administered 10 μl of Evans blue albumin (EBA) solution (0.5% Evans blue/40 mg/ml BSA) retro-orbitally, and EBA that had leaked into the small intestinal tissue was quantified 1 h later (n = 11 in pups prenatally treated with LPS; n = 8 in the control group). *P < 0.05, analyzed with two-sided Student’s t-test.
Fig. 3.
Fig. 3.
Prenatal lipopolysaccharide (LPS) decreases intestinal villous endothelial cell proliferation. Pregnant dams were intraperitoneally injected with LPS or normal saline at embryonic day 17. Pups were allowed to deliver naturally. Pups that were 24 h old were injected intraperitoneally with BrdU 4 h before euthanasia and tissue collection. Small intestine tissue sections were co-stained with antibodies against BrdU (red-purple) and CD31 (green) to identify proliferating endothelial cells. A: images from pups whose dams were administered prenatal LPS (right) vs. saline (left) (scale bars = 50 μm). B: proliferating endothelial cells of the villi were counted at ×20 magnification in 3–4 fields per sample (n = 15 in each group). ****P < 0.0001. Data represent three independent experiments and were analyzed using two-sided Student’s t-test.
Fig. 4.
Fig. 4.
Prenatal lipopolysaccharide (LPS) decreases VEGF and VEGF receptor 2 (VEGFR2) protein expression in the small intestine of neonatal mice. Pregnant dams were intraperitoneally injected with LPS or normal saline at embryonic day 17. Pups were allowed to deliver naturally, and small intestines were collected at 24 h. A: intestinal VEGF-164 and VEGFR2 proteins were accessed by Western blot. B: VEGF and VEGFR2 band intensities were quantified by densitometry and normalized to endogenous β-actin. ****P < 0.0001. Data represent three independent experiments and were analyzed using two-sided Student’s t-test.
Fig. 5.
Fig. 5.
Prenatal administration of lipopolysaccharide (LPS) increases maternal and fetal serum TNF. TNF decreases VEGF receptor 2 (VEGFR2) protein expression in neonatal intestinal endothelial cells in vitro. A: cesarean section was performed at indicated time points after LPS injection to the dams at embryonic day 17, and serum was immediately obtained from dams and fetuses. Serum levels of TNF from dams (left) and fetuses (right) were measured by ELISA; ****P < 0.0001. Data were analyzed with one-way ANOVA test and represent three experiments with a total of 6–8 dams per group and 16–30 pups in each group (serum was pooled from 2 to 3 pups). B: endothelial cells isolated from small intestines were cultured until 90% confluence and treated with indicated concentrations of TNF for 12 h. VEGFR2 protein was assessed by Western blot (left). Band densities for each sample were normalized to β-actin and a ratio to the mean control value from endothelial cells treated with culture medium only was calculated (right). *P < 0.05, **P < 0.01. Data represent three independent experiments combined and were analyzed by one-way ANOVA.
Fig. 6.
Fig. 6.
TNF decreases small intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGF receptor 2 (VEGFR2) protein expression in vivo, which are prevented by dimethyloxalylglycine (DMOG) administration. DMOG attenuates TNF detrimental effects on intestinal injury and mortality in experimental necrotizing enterocolitis (NEC). Pups were administered DMOG or vehicle only (control) intraperitoneally 16 h before intraperitoneal injection of TNF on day 1. A: dye was injected and intestinal tissues were collected 24 h later. Small intestinal submucosal microvasculature images (left) and quantification of microvascular density (right) are shown. Data represent 3 independent experiments from 3 litters (n = 11: control, n = 18: TNF, and n = 12: DMOG+TNF group). Data were analyzed by one-way ANOVA. Scale bar = 200 μm. Intestinal tissues were collected at day 2 (24 h after TNF treatment) and examined by immunofluorescence for endothelial cell proliferation (Ki-67+ endomucin+ cells) (B) or Western blot analysis for VEGF and VEGFR2 expression (C). Data represent 3 separated experiments from 3 litters and were analyzed by one-way ANOVA. D: pups were injected intraperitoneally with TNF or vehicle control and submitted 2 hr later to the NEC model. Animal survival (left) and tissue injury severity score (right) were assessed. Data represent three independent experiments (n = 38: TNF group, n = 31: DMOG+TNF group, n = 32: control group. Log-rank test was used for survival curve and χ2 test for histological score analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. n.s., not significant.
Fig. 7.
Fig. 7.
Prenatal administration of lipopolysaccharide (LPS) to dams decreases survival and increases the incidence of severe intestinal injury in neonatal mice submitted to a necrotizing enterocolitis (NEC) model, which are improved by neutralizing TNF. Dams injected with 50 μg/kg LPS or saline at embryonic day 17 were allowed to deliver naturally. Pups were submitted to a NEC protocol and euthanized when showing signs of distress. Mortality was recorded and intestinal tissues were collected for histology. A: animal survival curves (left) and histological injury scores (right) (n = 39 in the prenatal LPS group and n = 24 in the control group). Data were obtained from three separate experiments. (B: representative histological images from each group are presented (Scale bar = 100 μm). C: pregnant dams were injected with anti-TNF neutralizing antibody or control antibody at E17 2 h before LPS injection. Pups were submitted to the experimental NEC protocol, and survival (left) and histological tissue injury (right) were assessed (n = 36: anti-TNF antibodies and n = 34: control antibodies group). Data were obtained from three separate experiments. *P < 0.05, **P < 0.01, ****P < 0.0001. Log-rank test was used for survival curve and χ2 test for histological score analysis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Aaltonen R, Heikkinen T, Hakala K, Laine K, Alanen A. Transfer of proinflammatory cytokines across term placenta. Obstet Gynecol 106: 802–807, 2005. doi:10.1097/01.AOG.0000178750.84837.ed. - DOI - PubMed
    1. Ashdown H, Dumont Y, Ng M, Poole S, Boksa P, Luheshi GN. The role of cytokines in mediating effects of prenatal infection on the fetus: implications for schizophrenia. Mol Psychiatry 11: 47–55, 2006. doi:10.1038/sj.mp.4001748. - DOI - PubMed
    1. Ballance WA, Dahms BB, Shenker N, Kliegman RM. Pathology of neonatal necrotizing enterocolitis: a ten-year experience. J Pediatr 117: S6–S13, 1990. doi:10.1016/S0022-3476(05)81124-2. - DOI - PubMed
    1. Been JV, Lievense S, Zimmermann LJ, Kramer BW, Wolfs TG. Chorioamnionitis as a risk factor for necrotizing enterocolitis: a systematic review and meta-analysis. J Pediatr 162: 236–42.e2, 2013. doi:10.1016/j.jpeds.2012.07.012. - DOI - PubMed
    1. Bein A, Zilbershtein A, Golosovsky M, Davidov D, Schwartz B. LPS induces hyper-permeability of intestinal epithelial cells. J Cell Physiol 232: 381–390, 2017. doi:10.1002/jcp.25435. - DOI - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources