Extent of Differentiated Gene Expression in the Human Endothelium-Derived EA.hy926 Cell Line

Rieber, Alison J; Marr, Henry S.; Comer, Mary B.; Edgell, Cora‐Jean S.

doi:10.1055/s-0038-1651636

Cited by 39 publications

(22 citation statements)

References 36 publications

(21 reference statements)

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“…For the experiments, confluent cultures were used at the second passage. Human endothelial cell line EA.hy 926, derived by fusion of HUVECs with continuous human lung carcinoma cell line A549 were grown as monolayers in 25-cm 2 tissue culture flasks under standard conditions in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with HAT medium (hypoxanthine/aminopterin/thymidine), streptomycin (100 g/ ml), penicillin (100 units/ml), and 10% fetal bovine serum at 37°C in a humidified 5% CO 2 atmosphere (24,25). For the experiments, cells were transferred to 6-well dishes and used at 70% confluence in the DMEM supplemented with the components mentioned above.…”

Section: Methodsmentioning

confidence: 99%

DNAzymes to β1 and β3 mRNA Down-regulate Expression of the Targeted Integrins and Inhibit Endothelial Cell Capillary Tube Formation in Fibrin and Matrigel

Cieślak¹,

Niewiarowska²,

Nawrot³

et al. 2002

Journal of Biological Chemistry

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A novel approach based on DNA-cleaving deoxyribozymes (DNAzymes) was developed to control expression of ␤ 1 and ␤ 3 integrins in endothelial cells. To engineer a specific cleavage site in mRNA, the flanking domains of DNAzymes were derived from oligodeoxynucleotides complementary to sequences corresponding to 1053-1070 and 1243-1267 in ␤ 1 and ␤ 3 mRNA, respectively. Phosphorothioate analogues of these antisense oligodeoxynucleotides, designated ␤1-1053 and ␤3-1243, significantly inhibited expression of ␤ 1 and ␤ 3 integrin subunits in endothelial and K562 cells at the level of mRNA and protein synthesis. They also specifically decreased the cell surface expression of corresponding subunits in endothelial cells and K562 cells, as measured by flow cytometry. In functional tests, ␤1-1053 and ␤3-1243 markedly reduced adhesion of cells to fibronectin and vitronectin, respectively. We designed DNAzymes to ␤ 1 and ␤ 3 mRNAs containing a 15-deoxynucleotide catalytic domain that was flanked by two substrate recognition segments of 8 and 10 deoxynucleotides for ␤ 1 and ␤ 3 DNAzymes, respectively. Both DNAzymes in the presence of Mg 2؉ specifically cleaved their substrates, synthetic ␤ 1 and ␤ 3 mRNA fragments. Although DNAzymes were partially modified with phosphorothioate and with 2-O-methyl groups at both the 5 and 3 ends indicated similar kinetic parameters, they were significantly more potent than the unmodified DNAzymes because of their much higher resistance to nuclease degradation. Similar to the antisense oligonucleotides, DNAzymes abolished microvascular endothelial cell capillary tube formation in fibrin and Matrigel. In conclusion, DNAzymes to ␤ 1 and ␤ 3 mRNAs with 2-O-methyl modifications are potentially useful as gene-inactivating agents and may ultimately provide a therapeutic means to inhibit angiogenesis in vivo.Angiogenesis is a multistep sequence of cellular reactions beginning with degradation of extracellular matrix and then proliferation and migration of endothelial cells followed by lumen formation and maturation (1). The formation of new blood vessels is critical to the development of normal tissues as well as the growth of solid tumors and to a large extent depends on specific molecular interactions between vascular cells and extracellular matrix (2, 3). Aberrant angiogenesis is also a key process for progress of many disorders including atherosclerosis (4), diabetic retinopathy (5) and restenosis (6). Currently, two groups of integrin receptors are known to regulate adhesive interactions during angiogenesis. Integrins in the ␤ 1 subfamily, particularly ␣ 5 ␤ 1 and ␣ 2 ␤ 1 , which are substantially expressed in human angiogenic blood vessels, promote endothelial cell morphogenesis, migration, and tube formation (7,8). The second group is composed of vitronectin receptors ␣ V ␤ 3 and ␣ V ␤ 5 , which are poorly expressed in quiescent vasculature (9, 10) but can be substantially expressed by neoplastic vasculature and cells (11,12). Expression of ␣ V ␤ 3 and ␣ V ␤ 5 can be differentially up-regulate...

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Section: Methodsmentioning

confidence: 99%

DNAzymes to β1 and β3 mRNA Down-regulate Expression of the Targeted Integrins and Inhibit Endothelial Cell Capillary Tube Formation in Fibrin and Matrigel

Cieślak¹,

Niewiarowska²,

Nawrot³

et al. 2002

Journal of Biological Chemistry

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“…This cell line was originally established by hybridization of A549 human lung cancer cells and human umbilical vein endothelial cells and retained some characteristics of vein endothelial cells (Rieber et al, 1993). Because lymphatic vessels originated from veins during embryonic development, we thought this cell line might be suitable for the investigation of the biological activity of Prox1.…”

Section: Lys556 (K556) Is the Major Sumoylation Site Of Prox1mentioning

confidence: 99%

Sumoylation of Prox1 controls its ability to induce VEGFR3 expression and lymphatic phenotypes in endothelial cells

Pan

Chang

et al. 2009

Journal of Cell Science

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lymphangiogenic signals, including the enhancement of proliferation, sprouting and tube formation and the inhibition of apoptosis. This effect is SUMO-dependent because ectopic expression of SUMO-specific protease 2 (SENP2) effectively reduced Prox1 sumoylation and Prox1-induced VEGFR3 expression. In addition, K556R mutant Prox1 could not induce lymphatic phenotypes. Taken together, our results indicate that Prox1 is a target for SUMO-1 and suggest that sumoylation of Prox1 controls its ability to induce VEGFR3 expression and lymphatic phenotypes in endothelial cells.

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“…EA.hy 926 cells closely resemble human umbilical vein endothelial cells and maintain the characteristics of differentiated endothelium (21,22). The cells were cultured in growth medium containing DMEM, HAT (100 mM hypoxanthine, 0.4 mM aminopterin, 16 mM thimidine) and 10% FCS in a 90 -95% humidified atmosphere of 5% CO 2 at 37°C.…”

Section: Cell Culturesmentioning

confidence: 99%

Cerivastatin, a HMG-CoA Reductase Inhibitor, Reduces Plasminogen Activator Inhibitor-1 (PAI-1) Expression in Endothelial Cells by Down-Regulation of Cellular Signaling and the Inhibition of PAI-1 Promoter Activity

Świątkowska¹,

Pawłowska²,

Szemraj

et al. 2002

Japanese Journal of Pharmacology

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ABSTRACT-Statins, which competitively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity and reduce mevalonate synthesis, are believed to exert a plethora of pleiotropic effects. In this report, molecular mechanisms of the inhibitory effect on plasminogen activator inhibitor type 1 (PAI-1) expression produced by cerivastatin (CRV), the most active compound in this class, were studied using monocultures of human endothelial cell line (EA.hy 926). CRV similar to another statin, lovastatin (LOV), significantly inhibited PAI-1 expression and its release from endothelial cells, nonstimulated and stimulated with TNF-a. The inhibitory effect of CRV could be detected at the level of PAI-1 promoter in EA.hy 926 cells transfected with plasmid p800 LUC containing PAI-1 promoter fragment (+71 to -800), as well as at the level of PAI-1 mRNA. The PAI-1 promoter activity was markedly suppressed in the nonstimulated cells and almost completely inhibited in TNF-a-stimulated cells. In addition, CRV at low doses (IC50 of 4 -6 mM) significantly inhibited mitogen-activated protein kinases (MAPKs) phosphorylation. The majority of inhibitory effects occurred at significantly lower concentrations for CRV compared to LOV. The mechanism by which CRV inhibits PAI-1 expression appears to be directly associated with geranylgeranylation of some cell proteins, since the inhibitory effect on PAI-1 expression can be reversed by geranylgeranylpyrophosphate but not by farnesyl-pyrophosphate.

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Extent of Differentiated Gene Expression in the Human Endothelium-Derived EA.hy926 Cell Line

Cited by 39 publications

References 36 publications

DNAzymes to β1 and β3 mRNA Down-regulate Expression of the Targeted Integrins and Inhibit Endothelial Cell Capillary Tube Formation in Fibrin and Matrigel

DNAzymes to β1 and β3 mRNA Down-regulate Expression of the Targeted Integrins and Inhibit Endothelial Cell Capillary Tube Formation in Fibrin and Matrigel

Sumoylation of Prox1 controls its ability to induce VEGFR3 expression and lymphatic phenotypes in endothelial cells

Cerivastatin, a HMG-CoA Reductase Inhibitor, Reduces Plasminogen Activator Inhibitor-1 (PAI-1) Expression in Endothelial Cells by Down-Regulation of Cellular Signaling and the Inhibition of PAI-1 Promoter Activity

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