Genetic alteration of endothelial heparan sulfate selectively inhibits tumor angiogenesis

To examine the role of endothelial heparan sulfate during angiogenesis, we generated mice bearing an endothelial-targeted deletion in the biosynthetic enzyme N-acetylglucosamine N-deacetylase/N-sulfotransferase 1 (Ndst1). Physiological angiogenesis during cutaneous wound repair was unaffected, as was growth and reproductive capacity of the mice. In contrast, pathological angiogenesis in experimental tumors was altered, resulting in smaller tumors and reduced microvascular density and branching. To simulate the angiogenic environment of the tumor, endothelial cells were isolated and propagated in vitro with proangiogenic growth factors. Binding of FGF-2 and VEGF164 to cells and to purified heparan sulfate was dramatically reduced. Mutant endothelial cells also exhibited altered sprouting responses to FGF-2 and VEGF164, reduced Erk phosphorylation, and an increase in apoptosis in branching assays. Corresponding changes in growth factor binding to tumor endothelium and apoptosis were also observed in vivo. These findings demonstrate a cell-autonomous effect of heparan sulfate on endothelial cell growth in the context of tumor angiogenesis.

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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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Members of the microRNA-17-92 cluster exhibit a cell-intrinsic antiangiogenic function in endothelial cells

Blood ◽

10.1182/blood-2010-01-264812 ◽

2010 ◽

Vol 115 (23) ◽

pp. 4944-4950 ◽

Cited By ~ 255

Author(s):

Carmen Doebele ◽

Angelika Bonauer ◽

Ariane Fischer ◽

Alexander Scholz ◽

Yvonne Reiss ◽

...

Keyword(s):

Endothelial Cells ◽

Tumor Angiogenesis ◽

Janus Kinase ◽

Negative Regulator ◽

3 Dimensional ◽

Small Noncoding Rnas ◽

A Cell ◽

Sprout Formation

Abstract MicroRNAs are endogenously expressed small noncoding RNAs that regulate gene expression on the posttranscriptional level. The miR-17-92 cluster (encoding miR-17, -18a, -19a/b, -20a, and miR-92a) is highly expressed in tumor cells and is up-regulated by ischemia. Whereas miR-92a was recently identified as negative regulator of angiogenesis, the specific functions of the other members of the cluster are less clear. Here we demonstrate that overexpression of miR-17, -18a, -19a, and -20a significantly inhibited 3-dimensional spheroid sprouting in vitro, whereas inhibition of miR-17, -18a, and -20a augmented endothelial cell sprout formation. Inhibition of miR-17 and miR-20a in vivo using antagomirs significantly increased the number of perfused vessels in Matrigel plugs, whereas antagomirs that specifically target miR-18a and miR-19a were less effective. However, systemic inhibition of miR-17/20 did not affect tumor angiogenesis. Further mechanistic studies showed that miR-17/20 targets several proangiogenic genes. Specifically, Janus kinase 1 was shown to be a direct target of miR-17. In summary, we show that miR-17/20 exhibit a cell-intrinsic antiangiogenic activity in endothelial cells. Inhibition of miR-17/20 specifically augmented neovascularization of Matrigel plugs but did not affect tumor angiogenesis indicating a context-dependent regulation of angiogenesis by miR-17/20 in vivo.

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The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen

Blood ◽

10.1182/blood-2011-08-376038 ◽

2012 ◽

Vol 119 (5) ◽

pp. 1302-1313 ◽

Cited By ~ 31

Author(s):

Cheng-Hsiang Kuo ◽

Po-Ku Chen ◽

Bi-Ing Chang ◽

Meng-Chen Sung ◽

Chung-Sheng Shi ◽

...

Keyword(s):

Endothelial Cells ◽

Egf Receptor ◽

Tube Formation ◽

Endothelial Tube Formation ◽

A Cell ◽

Lewis Y ◽

Tm Domain

AbstractLewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM—TM domain 1 (rTMD1)—in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.

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Heparanase Modulation by Wingless/INT (Wnt)

10.21203/rs.3.rs-256563/v1 ◽

2021 ◽

Author(s):

Carina Mucciolo Melo ◽

Helena Bonciani Nader ◽

Giselle Zenker Justo ◽

Maria Aparecida Silva Pinhal

Keyword(s):

Heparan Sulfate ◽

Chinese Hamster ◽

Beta Catenin ◽

Ovary Cells ◽

Protein Levels ◽

Coated Plate ◽

A Cell ◽

Chloride Treatment

Abstract Heparanase is an endo-beta-glucuronidase, the only enzyme in mammals capable of cleaving heparan sulfate/heparin chains from proteoglycans. The oligosaccharides generated by heparanase present extensive biological functions since such oligosaccharides interact with adhesion molecules, growth factors, angiogenic factors and cytokines, modulating cell proliferation, migration, inflammation, and carcinogenesis. However, the regulation of heparanase activity is not fully understood. It is known that heparanase is synthesized as an inactive 65 kDa isoform and that post-translation processing forms an active 50 kDa enzyme. In the present study, we are interested in investigating whether heparinase is regulated by its own substrate as observed with many other enzymes. Wild-type Chinese hamster (Cricetulus griséus) ovary cells (CHO-K1) were treated with different doses of heparin. Heparanase expression was analyzed by Real-time PCR and flow cytometry. Also, heparanase activity was measured. The heparanase activity assay was performed using a coated plate with biotinylated heparan sulfate. In the present assay, a competitive heparin inhibition scenario was set aside. Exogenous heparin trigged a cell signaling pathway that increased heparanase mRNA and protein levels. The Wnt/beta-catenin pathway, judged by TCF-driven luciferase activity, seems to be involved to enhance heparanase profile during treatment with exogenous heparin. Lithium chloride treatment, an activator of the Wnt/beta-catenin pathway, confirmed such mechanism of transduction in vivo using zebrafish embryos and in vitro using CHO-K1 cells. Taken together the results suggest that heparin modulates heparanase expression by Wnt/beta-catenin.

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Tie2 Activation Promotes Protection and Reconstitution of the Endothelial Glycocalyx in Human Sepsis

Thrombosis and Haemostasis ◽

10.1055/s-0039-1695768 ◽

2019 ◽

Vol 119 (11) ◽

pp. 1827-1838 ◽

Cited By ~ 5

Author(s):

Carolin Christina Drost ◽

Alexandros Rovas ◽

Kristina Kusche-Vihrog ◽

Paul Van Slyke ◽

Harold Kim ◽

...

Keyword(s):

Heparan Sulfate ◽

Boundary Region ◽

Endothelial Glycocalyx ◽

Human Sepsis ◽

Force Microscopy ◽

Control Serum ◽

A Cell ◽

Angiopoietin 2

AbstractThe endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal surface of the endothelium, provides a first vasoprotective barrier against vascular leakage in sepsis. We hypothesized that angiopoietin-2 (Angpt-2), antagonist of the endothelium-stabilizing receptor Tie2, induces a rapid loss of the eGC in human sepsis. Using intravital microscopy, we measured the perfused boundary region (PBR), an inverse parameter of eGC dimensions in sublingual microvessels, in patients with sepsis and age-matched nonseptic subjects. Median PBR values were significantly higher in patients compared with controls and correlated with serum Angpt-2 levels. To transfer and further explore these findings in a cell culture system, we exposed endothelial cells (ECs) to serum (5%) from a subgroup of septic patients and nonseptic controls. Confocal and atomic force microscopy revealed that sepsis serum, but not control serum, induced thinning of the eGC on human ECs in vitro, which correlated with paired PBR values obtained in vivo (r = 0.96, p < 0.01). Inhibition of Angpt-2 or Tie2 activation completely abolished eGC damage. Mechanistically, sepsis-induced eGC breakdown required the loss of its main constituent heparan sulfate; a result of heparan sulfate-specific enzyme heparanase, which was suppressed by Tie2 activation. Finally, Tie2 activation, but not Angpt-2 inhibition, initiated after septic or enzymatic damage provoked rapid refurbishment of the eGC. Our data indicate that eGC breakdown in human sepsis is mediated via Tie2 deactivation by Angpt-2. Activation of Tie2 seems to accelerate recovery of the eGC and might hold promise as a therapeutic target in human sepsis.

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Protease-activated receptors 1 and 4 mediate thrombin signaling in endothelial cells

Blood ◽

10.1182/blood-2003-04-1130 ◽

2003 ◽

Vol 102 (9) ◽

pp. 3224-3231 ◽

Cited By ~ 128

Author(s):

Hiroshi Kataoka ◽

Justin R. Hamilton ◽

David D. McKemy ◽

Eric Camerer ◽

Yao-Wu Zheng ◽

...

Keyword(s):

Endothelial Cells ◽

Wild Type ◽

Protease Activated Receptors ◽

Erk Phosphorylation ◽

Low Concentrations ◽

Extracellular Signal ◽

High Concentrations ◽

Mouse Aorta

AbstractDefining the relative importance of protease-activated receptors (PARs) for thrombin signaling in mouse endothelial cells is critical for a basic understanding of thrombin signaling in these cells and for the rational use of knockout mice to probe the roles of thrombin's actions on endothelial cells in vivo. We examined thrombin- and PAR agonist–induced increases in cytoplasmic calcium, phosphoinositide hydrolysis, extracellular signal-regulated kinase (ERK) phosphorylation, and gene expression in endothelial cells from wild-type and PAR-deficient mice. PAR1 and PAR4 agonists triggered responses in wild-type but not in Par1–/– and Par4–/– endothelial cells, respectively. Calcium imaging confirmed that a substantial fraction of individual endothelial cells responded to both agonists. Compared with wild-type cells, Par1–/– endothelial cells showed markedly decreased responses to low concentrations of thrombin, and cells that lacked both PAR1 and PAR4 showed no responses to even high concentrations of thrombin. Similar results were obtained when endothelial-dependent vasorelaxation of freshly isolated mouse aorta was used as an index of signaling in native endothelial cells. Thus PAR1 is the major thrombin receptor in mouse endothelial cells, but PAR4 also contributes. These receptors serve at least partially redundant roles in endothelial cells in vitro and in vivo and together are necessary for the thrombin responses measured.

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Either exogenous or endogenous fibronectin can promote adherence of human endothelial cells

Journal of Cell Science ◽

10.1242/jcs.82.1.263 ◽

1986 ◽

Vol 82 (1) ◽

pp. 263-280

Author(s):

R.A. Clark ◽

J.M. Folkvord ◽

L.D. Nielsen

Keyword(s):

Endothelial Cells ◽

Wound Repair ◽

Cell Types ◽

Culture Conditions ◽

Collagen Type Iv ◽

Human Endothelial Cells ◽

Small Vessels ◽

Microvascular Cells

Recently, we have presented evidence that proliferating blood vessels produce and deposit fibronectin in situ during the angiogenesis of wound repair. This report extends these observations by demonstrating that human endothelial cells from both large and small vessels depend on fibronectin for their adherence in vitro. Endothelial cells were grown from human umbilical veins (HUVEC) by the method of Gimbrone and from the microvasculature of human omentum by the method of Kern, Knedler and Eckel. Second-passage cells were plated into microtitre wells that had been coated with 100 micrograms ml-1 of fibronectin, types I and III collagen, type IV collagen or laminin. After a 3-h incubation, adherent cells were solubilized with Zap-Isoton and quantified on a Coulter Counter. Under normal culture conditions HUVEC showed no preference for fibronectin substrates while microvascular cells always demonstrated a striking preference for fibronectin substrates. However, when HUVEC were exposed to 2.5 or 25 micrograms ml-1 of cycloheximide for 4 h before and during the adherence assays, the adherence to fibronectin was 50–200% greater than to types I and III collagen. Immunofluorescence studies showed that while HUVEC expressed a large quantity of surface fibronectin, microvascular cells expressed very little. Metabolic labelling studies confirmed that HUVEC cultures had substantial quantities of fibronectin in their cell layer while microvascular cells did not. In antibody blocking experiments, preincubation of fibronectin-coated surfaces with anti-fibronectin antibodies totally blocked microvascular cell adhesion but only abrogated HUVEC adherence by 50%, presumably since these latter cells were able to deposit additional fibronectin onto the surface during the 3 h assay period. In the presence of cycloheximide anti-fibronectin antibodies totally blocked HUVEC adherence. These results demonstrate that both endothelial cell types rely, at least in part, on fibronectin for adherence in vitro. HUVEC can synthesize, secrete and deposit enough fibronectin for their adherence in vitro, while microvascular cells rely on an exogenous source of fibronectin under these culture conditions. Thus, the increased blood vessel fibronectin observed during angiogenesis in vivo may mediate adherence of the proliferating and migrating endothelial cells.

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Abstract 3377: Modulation of Vessel Stability by the ELR-negative Chemokine IP-10

Circulation ◽

10.1161/circ.118.suppl_18.s_415 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Richard J Bodnar ◽

Cecelia C Yates ◽

Xiaoping Du ◽

Alan Wells

Keyword(s):

Endothelial Cells ◽

Wound Repair ◽

Critical Role ◽

Tissue Development ◽

Platelet Factor ◽

Signaling Mechanism ◽

Extracellular Signaling ◽

Vessel Stability

Angiogenesis plays a critical role in tissue development and wound repair. Regulation of the vascular tree is critical to organogenesis and neo-organogenesis. The initial vigorous vessel proliferation and arborization that occurs during tissue development and wound repair must be pruned to form mature tissue. The signals that trigger this vascular regression are still ill-defined. Mitotic endothelial cells express the CXC receptor 3 (CXCR3), which binds the ELR-negative chemokines IP-10 (CXCL10), platelet factor 4 (PF4, CXCL4), IP-9/I-TAC (CXCL11), and MIG (CXCL9), with IP-9 and IP-10 being expressed during the later remodeling phases of wound repair when vascular involution occurs. We hypothesized that not only does this signaling system limit angiogenesis, but that CXCR3 signaling also compromises endothelial cell tube integrity contributing to regression. Treatment of newly formed tubes/cords on Matrigel (in vitro) or vessels in a subcutaneous Matrigel plug (in vivo) with IP-10 in either presence or absence of VEGF caused a dissociation of the tubes and subsequent vascular involution. This followed CXCR3 induced cleavage of β3 integrin in endothelial cells. CXCR3 triggering of mu-calpain activity caused cleavage of the cytoplasmic tail of human β3 integrins at the calpain cleavage sites NPLY747 and TSTF754. IP-10 stimulation was also found to activate caspase 3, blockage of which prevented cell death but not tube dissociation. Thus, CXCR3 activation in newly formed vessels results in endothelial dissociation from matrix, followed by anoikis. This is the first direct evidence for an extracellular signaling mechanism through CXCR3 causing the dissociation of newly formed blood vessels. This study was supported by funds from VA Medical Research Program and National Institute of General Medical Sciences (NIH, USA).

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Angiomotin

The Journal of Cell Biology ◽

10.1083/jcb.152.6.1247 ◽

2001 ◽

Vol 152 (6) ◽

pp. 1247-1254 ◽

Cited By ~ 254

Author(s):

Boris Troyanovsky ◽

Tetyana Levchenko ◽

Göran Månsson ◽

Olga Matvijenko ◽

Lars Holmgren

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Leading Edge ◽

Tube Formation ◽

Endothelial Cell Migration ◽

Kringle Domains ◽

A Cell ◽

Cell Type Specific

Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type–specific manner. We have used a construct encoding the kringle domains 1–4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

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