scholarly journals A Simple Method to AssessIn VivoProliferation in Lung Vasculature with EdU: The Case of MMC-Induced PVOD in Rat

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Antigny Fabrice ◽  
Ranchoux Benoît ◽  
Nadeau Valérie ◽  
Edmund Lau ◽  
Bonnet Sébastien ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Pulmonary Veins ◽  
Endothelial Cell Proliferation ◽  
Pulmonary Vasculature ◽  
Microvascular Endothelial Cell ◽  
Proliferating Cells ◽  
Simple Method ◽  
Gold Standard Method

5-Ethynyl-2′-deoxyuridine (EdU) incorporation is becoming the gold standard method forin vitroandin vivovisualization of proliferating cells. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a “terminal” state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD.In vivopulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.

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

Hypertension ◽  
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.

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

2019 ◽  
Vol 317 (1) ◽  
pp. G57-G66 ◽  
Author(s):  
Xiaocai Yan ◽  
Elizabeth Managlia ◽  
Xiao-Di Tan ◽  
Isabelle G. De Plaen
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Necrotizing Enterocolitis ◽  
Endothelial Cell Proliferation ◽  
Vegf Receptor ◽  
Fetal Serum ◽  
Prenatal Inflammation ◽  
Vegfr2 Signaling

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.

scholarly journals ASC and SVF Cells Synergistically Induce Neovascularization in Ischemic Hindlimb Following Cotransplantation

2021 ◽  
Vol 23 (1) ◽  
pp. 185
Author(s):  
Hong Zhe Zhang ◽  
Dong-Sik Chae ◽  
Sung-Whan Kim
Keyword(s):  
Cell Proliferation ◽  
Cell Transplantation ◽  
Stromal Vascular Fraction ◽  
Blood Perfusion ◽  
Immunohistochemical Analysis ◽  
Tube Formation ◽  
Endothelial Cell Proliferation ◽  
Therapeutic Effects

Previously, we reported the angio-vasculogenic properties of human stromal vascular fraction (SVF) and adipose tissue-derived mesenchymal stem cells (ASCs). In this study, we investigated whether the combination of ASCs and SVF cells exhibited synergistic angiogenic properties. We conducted quantitative (q)RT-PCR, Matrigel plug, tube formation assays, and in vivo therapeutic assays using an ischemic hind limb mouse model. Immunohistochemical analysis was also conducted. qRT-PCR results revealed that FGF-2 was highly upregulated in ASCs compared with SVF, while PDGF-b and VEGF-A were highly upregulated in SVF. Conditioned medium from mixed cultures of ASCs and SVF (A+S) cells showed higher Matrigel tube formation and endothelial cell proliferation in vitro. A+S cell transplantation into ischemic mouse hind limbs strongly prevented limb loss and augmented blood perfusion compared with SVF cell transplantation. Transplanted A+S cells also showed high capillary density, cell proliferation, angiogenic cytokines, and anti-apoptotic potential in vivo compared with transplanted SVF. Our data indicate that A+S cell transplantation results in synergistic angiogenic therapeutic effects. Accordingly, A+S cell injection could be an alternative therapeutic strategy for treating ischemic diseases.

scholarly journals Utilizing a Kidney-Targeting Peptide to Improve Renal Deposition of a Pro-Angiogenic Protein Biopolymer

Pharmaceutics ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 542 ◽  
Author(s):  
Fakhri Mahdi ◽  
Alejandro R. Chade ◽  
Gene L. Bidwell
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Endothelial Cell Proliferation ◽  
Microvascular Endothelial Cell ◽  
Similar Distribution ◽  
Target Tissue ◽  
Cell Binding ◽  
Targeting Peptide

Elastin-like polypeptides (ELP) are versatile protein biopolymers used in drug delivery due to their modular nature, allowing fusion of therapeutics and targeting agents. We previously developed an ELP fusion with vascular endothelial growth factor (VEGF) and demonstrated its therapeutic efficacy in translational swine models of renovascular disease and chronic kidney disease. The goal of the current work was to refine renal targeting and reduce off-target tissue deposition of ELP–VEGF. The ELP–VEGF fusion protein was modified by adding a kidney-targeting peptide (KTP) to the N-terminus. All control proteins (ELP, KTP–ELP, ELP–VEGF, and KTP–ELP–VEGF) were also produced to thoroughly assess the effects of each domain on in vitro cell binding and activity and in vivo pharmacokinetics and biodistribution. KTP–ELP–VEGF was equipotent to ELP–VEGF and free VEGF in vitro in the stimulation of primary glomerular microvascular endothelial cell proliferation, tube formation, and extracellular matrix invasion. The contribution of each region of the KTP–ELP–VEGF protein to the cell binding specificity was assayed in primary human renal endothelial cells, tubular epithelial cells, and podocytes, demonstrating that the VEGF domain induced binding to endothelial cells and the KTP domain increased binding to all renal cell types. The pharmacokinetics and biodistribution of KTP–ELP–VEGF and all control proteins were determined in SKH-1 Elite hairless mice. The addition of KTP to ELP slowed its in vivo clearance and increased its renal deposition. Furthermore, addition of KTP redirected ELP–VEGF, which was found at high levels in the liver, to the kidney. Intrarenal histology showed similar distribution of all proteins, with high levels in blood vessels and tubules. The VEGF-containing proteins also accumulated in punctate foci in the glomeruli. These studies provide a thorough characterization of the effects of a kidney-targeting peptide and an active cytokine on the biodistribution of these novel biologics. Furthermore, they demonstrate that renal specificity of a proven therapeutic can be improved using a targeting peptide.

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

Blood ◽  
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.

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

2003 ◽  
Vol 312 (3) ◽  
pp. 801-805 ◽  
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

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

Blood ◽  
1999 ◽  
Vol 94 (7) ◽  
pp. 2461-2468 ◽  
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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