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

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.

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Kallistatin Inhibits Endothelial Cell Proliferation, Migration and Adhesion in Vitro and Angiogenesis in the Ischemic Hindlimb of Rats

Hypertension ◽

10.1161/hyp.36.suppl_1.706-e ◽

2000 ◽

Vol 36 (suppl_1) ◽

pp. 706-707

Author(s):

Robert Q Miao ◽

Jun Agata ◽

Lee Chao ◽

Julie Chao

Keyword(s):

Cell Proliferation ◽

Endothelial Cell ◽

Gene Delivery ◽

Fluorescent Protein ◽

Regional Blood Flow ◽

Endothelial Cell Proliferation ◽

Hek 293

P76 Kallistatin is a serine proteinase inhibitor (serpin) which has multifunctions including regulation of tissue kallikrein activity, blood pressure, inflammation and neointima hyperplasia. In this study, we investigated the potential role of kallistatin in vascular biology by studying its effects on the proliferation, migration and adhesion of cultured primary human endothelial cells in vitro, and angiogenesis in the ischemic hindlimb of rats. Purified kallistatin significantly inhibits cultured endothelial cell proliferation, migration and adhesion induced by VEGF or bFGF. To further investigate the role of kallistatin in vascular growth in vivo, we prepared adenovirus carrying the human kallistatin gene under the control of the cytomegalovirus promoter/enhancer (Ad.CMV-cHKBP). Expression of recombinant human kallistatin in HEK 293 cells transfected with Ad.CMV-cHKBP was identified by a specific ELISA. The effect of adenovirus-mediated kallistatin gene delivery on angiogenesis was evaluated in a rat model of hindlimb ischemia. Adenovirus carrying the human kallistatin or green fluorescent protein (GFP) gene were injected locally into the ischemic adductor at the time of surgery. Histological and morphometric analysis at 14 days post injection showed that adenovirus-mediated kallistatin gene delivery significantly reduced capillary density in the ischemic muscle as compared to that of control rats injected with GFP. The anti-angiogenic effect of kallistatin was associated with reduced regional blood flow in the ischemic hindlimb measured by microsphere assays. Expression of human kallistatin was identified in the injected muscle and immunoreactive human kallistatin levels were measured in the muscle and in the circulation of rats following kallistatin gene delivery. These results demonstrate a novel role of kallistatin in the inhibition of angiogenesis and in vascular remodeling.

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The Mitogenic Effect of 17β-Estradiol on in vitro Endothelial Cell Proliferation and on in vivo Reendothelialization Are Both Dependent on Vascular Endothelial Growth Factor

Journal of Vascular Research ◽

10.1159/000025732 ◽

2000 ◽

Vol 37 (3) ◽

pp. 202-208 ◽

Cited By ~ 23

Author(s):

P. Concina ◽

S. Sordello ◽

M.A. Barbacanne ◽

R. Elhage ◽

M.T. Pieraggi ◽

...

Keyword(s):

Vascular Endothelial Growth Factor ◽

Cell Proliferation ◽

Endothelial Cell ◽

Endothelial Cell Proliferation ◽

Vascular Endothelial ◽

Mitogenic Effect ◽

17Β Estradiol ◽

Endothelial Growth Factor

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Potential role of leptin in angiogenesis: leptin induces endothelial cell proliferation and expression of matrix metalloproteinases in vivo and in vitro

Experimental & Molecular Medicine ◽

10.1038/emm.2001.17 ◽

2001 ◽

Vol 33 (2) ◽

pp. 95-102 ◽

Cited By ~ 284

Author(s):

Hyun-Young Park ◽

Hyuck Moon Kwon ◽

Hyun Joung Lim ◽

Bum Kee Hong ◽

Ju Yong Lee ◽

...

Keyword(s):

Cell Proliferation ◽

Endothelial Cell ◽

Matrix Metalloproteinases ◽

Potential Role ◽

Endothelial Cell Proliferation

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Carcinoembryonic Antigen (CEA) Regulates Tumor-Angiogenesis in Colon Carcinomas.

Blood ◽

10.1182/blood.v112.11.1897.1897 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1897-1897

Author(s):

Kira Braemswig ◽

Marina Poettler ◽

Wazlawa Kalinowska ◽

Christoph Zielinski ◽

Gerald W Prager

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Tumor Angiogenesis ◽

Endothelial Cell Proliferation ◽

Erk Phosphorylation ◽

Dose Dependent Increase ◽

Dose Dependent

Abstract Human carcinoembryonic antigen (CEA) is a cell surface adhesion molecule member of the Immunoglobulin Superfamily (IgSF). Aberrant upregulation and secretion of soluble CEA is a common feature found in a wide variety of human cancers such as colon, breast and lung. Previous in vitro and in vivo results have demonstrated that CEA can affect tumor cell behavior including the inhibition of cell differentiation and apoptosis. However, any functional effects on angiogenic endothelial cell behavior are so far unknown. In the present work we found that in endothelial cells exogenous CEA led to a time and dose dependent increase in ERK phosphorylation, which was inhibited by the specific MEK inhibitor U0126. Thereby, the observed CEA effect was comparable in time and intense with the canonical angiogenic growth factor VEGF. The CEA-induced ERK phosphorylation was not affected by the blockage of VEGFR-2 / flk-1 using a specific inhibiting peptide (CBO-P11), which indicates a VEGF-independent mechanism. Furthermore, co-stimulation of endothelial cells with VEGF and CEA shows synergistic effects on ERK phosphorylation. While in endothelial cells no endogenous expression of CEA is detected, its putative receptor, the CEA receptor (CEAR), is highly expressed as shown by immunohistochemical staining of paraffin-embedded colon carcinoma sections as well as in biochemical analyses. When an activating antibody against CEAR was used, CEA-induced ERK phosphorylation was mimicked, while downregulation of CEAR by siRNA diminished CEA-induced signal transduction, significantly. To test a biological relevance of our findings, we first measured endothelial cell proliferation: CEA led to a dose dependent increase in endothelial cell proliferation in vitro, which again revealed a synergistic effect with VEGF. Thereby, CEA-induced endothelial cell proliferation was again independent of VEGFR-2 / flk-1. A biological role of CEA in tumor-angiogenesis was reflected by an in vivo model using CEA Mimotope immunized BALB/c mice, which were transplanted with MethA/CEA overexpressing tumor cells. Immunohistological analyses of these tumors revealed a significantly reduced vascular density, which was accompanied with diminished tumor growth. Our data provide first evidence of CEA as a novel pro-angiogenic activator of endothelial cells, which results in an increase in endothelial cell proliferation, independent of VEGFR-2. Furthermore, by targeting CEA in an in vivo mouse model, tumor-angiogenesis was markley reduced, indicating a potential therapeutic target in cancer.

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Canstatin-N fragment inhibits in vitro endothelial cell proliferation and suppresses in vivo tumor growth

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2003.11.003 ◽

2003 ◽

Vol 312 (3) ◽

pp. 801-805 ◽

Cited By ~ 39

Author(s):

Guo-An He ◽

Jin-Xian Luo ◽

Tian-Yuan Zhang ◽

Fang-Yu Wang ◽

Rui-Fang Li

Keyword(s):

Cell Proliferation ◽

Endothelial Cell ◽

Tumor Growth ◽

Endothelial Cell Proliferation

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Calreticulin and Calreticulin Fragments Are Endothelial Cell Inhibitors That Suppress Tumor Growth

Blood ◽

10.1182/blood.v94.7.2461.419a26_2461_2468 ◽

1999 ◽

Vol 94 (7) ◽

pp. 2461-2468 ◽

Cited By ~ 129

Author(s):

Sandra E. Pike ◽

Lei Yao ◽

Joyce Setsuda ◽

Karen D. Jones ◽

Barry Cherney ◽

...

Keyword(s):

Amino Acids ◽

Cell Proliferation ◽

Endothelial Cell ◽

Tumor Growth ◽

Angiogenesis Inhibitors ◽

Full Length ◽

Endothelial Cell Proliferation ◽

Proliferation In Vitro

Several angiogenesis inhibitors are fragments of larger proteins that are themselves not active as angiogenesis inhibitors. Vasostatin, the N-terminal domain of calreticulin inclusive of amino acids 1-180, is an angiogenesis inhibitor that exerts antitumor effects in vivo. In the present study, we examined whether the full-length calreticulin molecule shares the antiangiogenic and antitumor activities of vasostatin. Similar to vasostatin, calreticulin selectively inhibited endothelial cell proliferation in vitro, but not cells of other lineages, and suppressed angiogenesis in vivo. When inoculated into athymic mice, calreticulin inhibited Burkitt tumor growth comparably with vasostatin. Calreticulin lacking the N-terminal 1-120 amino acids inhibited endothelial cell proliferation in vitro and Burkitt tumor growth in vivo comparably with vasostatin. An internal calreticulin fragment encompassing amino acids 120-180 also inhibited endothelial cell proliferation in vitro and angiogenesis in vivo comparably with calreticulin and vasostatin. These results suggest that the antiangiogenic activities of vasostatin reside in a domain that is accessible from the full-length calreticulin molecule and localize to calreticulin N-terminal amino acids 120-180. Thus, calreticulin and calreticulin fragments are inhibitors of angiogenesis that directly target endothelial cells, inhibit angiogenesis, and suppress tumor growth. This information may be critical in designing targeted inhibitors of pathological angiogenesis that underlies cancer and other diseases.

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Quantifying endothelial cell proliferation in the zebrafish embryo

F1000Research ◽

10.12688/f1000research.73130.1 ◽

2021 ◽

Vol 10 ◽

pp. 1032

Author(s):

George Bowley ◽

Timothy JA Chico ◽

Jovana Serbanovic-Canic ◽

Paul C Evans

Keyword(s):

Cell Proliferation ◽

Endothelial Cell ◽

Zebrafish Embryo ◽

Time Lapse ◽

Endothelial Cell Proliferation ◽

Small Scale ◽

Zebrafish Embryos ◽

Time Lapse Imaging

Introduction: Endothelial cell (EC) proliferation is a fundamental determinant of vascular development and homeostasis, and contributes to cardiovascular disease by increasing vascular permeability to blood-borne lipoproteins. Rodents have been traditionally used to analyse EC proliferation mechanisms in vascular health and disease; however, alternative models such as the zebrafish embryo allow researchers to conduct small scale screening studies in a physiologically relevant vasculature whilst reducing the use of mammals in biomedical research. In vitro models of EC proliferation are valuable but do not fully recapitulate the complexity of the in vivo situation. Several groups have used zebrafish embryos for vascular biology research because they offer the advantages of an in vivo model in terms of complexity but are also genetically manipulable and optically transparent. Methods: Here we investigated whether zebrafish embryos can provide a suitable model for the study of EC proliferation. We explored the use of antibody, DNA labelling, and time-lapse imaging approaches. Results: Antibody and DNA labelling approaches were of limited use in zebrafish due to the low rate of EC proliferation combined with the relatively narrow window of time in which they can label proliferating nuclei. By contrast, time-lapse imaging of fluorescent proteins localised to endothelial nuclei was a sensitive method to quantify EC proliferation in zebrafish embryos. Discussion: We conclude that time-lapse imaging is suitable for analysis of endothelial cell proliferation in zebrafish, and that this method is capable of capturing more instances of EC proliferation than immunostaining or cell labelling alternatives. This approach is relevant to anyone studying endothelial cell proliferation for screening genes or small molecules involved in EC proliferation. It offers greater biological relevance than existing in vitro models such as HUVECs culture, whilst reducing the overall number of animals used for this type of research.

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