Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 is repressed in endothelial cells under inflammatory conditions in vivo. A, left panel, in situ hybridization detection of Egfl7 transcripts in endothelial cell nuclei of adult mouse lungs (blue staining, arrows). Right panel and inset, CD31 immunostaining (brown, arrows) and hematoxylin counterstaining of a parallel section of the same area. Bar, 25 μm. B, expression levels of CD31 and Egfl7 transcripts in CD31− cells (white bars) and CD31+ cells (black bars) isolated from mouse lungs using immunoaffinity and measured by duplex RT-qPCR using a mouse CD31-FAM or a mouse Egfl7-FAM TaqMan probe mixed with a mouse β-actin-VIC probe (see “Experimental Procedures”). The results are plotted as quantities relative to CD31− controls values set to 1. RQ, relative quantities. C, LPS (5 mg/kg, +) or LPS-free PBS (−) was instilled in mice nostrils, and animals were sacrificed at the onset of treatment (0 h) or after 10 or 24 h; the lungs were dissected and processed for total RNA isolation. Expression levels of ICAM-1, VCAM-1, E-selectin, and Egfl7 were measured by duplex RT-qPCR using the indicated FAM-labeled TaqMan probe for the mouse transcript of interest and a mouse β-actin-VIC-labeled TaqMan probe and expressed as 2−ΔΔCT quantities relative to t = 0 h values set to 1. *, p < 0.05; **, p < 0.01; ***, p < 0,001. The results are representative of three experiments performed in triplicate. RQ, relative quantities. D, left panel, HUVEC were treated with increasing doses of LPS for 4 h and Egfl7 transcript levels assessed by duplex RT-qPCR. Right panel, expression levels of Egfl7 transcripts in primary HUVEC cells treated with 0.1 μg/ml of LPS for the indicated length of time and assessed by duplex RT-qPCR. E, TNFα (0.25 mg/kg, +) or LPS-free PBS (−) was instilled in mice nostrils and lungs processed as in C for the analysis of expression levels of ICAM-1, VCAM-1, E-selectin, and Egfl7 expressed as relative to t = 0 values set to 1. RQ, relative quantities. **, p < 0.01; ***, p < 0,001.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: In Vivo, In Situ Hybridization, Staining, Immunostaining, Expressing, Isolation, Quantitative RT-PCR, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Expression of Egfl7 in endothelial cells is repressed by pro-inflammatory cytokines. A and B, expression levels of Egfl7 measured by duplex RT-qPCR in HUVEC treated with PBS (□) or with 10 ng/ml TNFα (●) (A) for the indicated length of time or with PBS (□), 10 ng/ml IL1β (●), or 10 ng/ml IL6 (▵) (B). The results are representative of three experiments performed in triplicate. C, Western blotting analysis of endogenous Egfl7 or of actin in HUVEC treated with TNFα as in A for the indicated length of time. The numbers below indicate the TNFα treated/non-treated ratio of Egfl7 protein levels normalized to actin levels taken at the same time points and assessed by densitometry. The results are representative of two experiments. D, immunostaining of Egfl7 in confluent HUVEC monolayers treated for 4 h with or without TNFα. Bar, 25 μm. E, expression levels of Egfl7 measured by duplex RT-qPCR in HUVEC treated for 24 h with complete medium (EGM-2) or in basal medium supplemented with the indicated amounts of FGF-2 or VEGF-A165. The results are representative of two experiments performed in triplicate.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 expression in endothelial cells is regulated by TNFα at the transcriptional level. A, Egfl7 transcript levels measured by duplex RT-qPCR in confluent HUVEC treated with 10 ng/ml TNFα (●) or with PBS (□) for 1 h and then with 10 μg/ml actinomycin D (ActD, t = 0) and assessed during the next 6 h. B, Egfl7 transcripts levels measured by duplex RT-qPCR in confluent HUVEC treated with DMSO or 10 μg/ml actinomycin D for 1 h before stimulation with or without 10 ng/ml TNFα for 6 h. *, p < 0.05; **, ns, non-significant. C, luciferase activities measured in HUVEC transfected with pGL3basic (Ctrl) or with the indicated reporter constructs containing fragments of the human egfl7 gene promoter and with the pCMV-β-Gal normalizing vector. The cells were then treated with 10 ng/ml TNFα (black bars) or with PBS (white bars) and lysed 18 h later. The letters correspond to conserved promoter regions (16). The numbers indicate the base position relative to the exon 1b transcription initiation site (2). Activities were normalized with β-galactosidase values, folds of induction were calculated using pGL3basic values as reference; the results are representative of three experiments performed in triplicate. **, p < 0.01; ***, p < 0.001; ns, non-significant.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Expressing, Quantitative RT-PCR, Luciferase, Transfection, Construct, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 repression by TNFα is mediated via the NF-κB pathway. A, confluent HUVEC were cultured in the presence or not of 5 ng/ml TNFα and of 10 μm of the NF-κB inhibitor BAY117085 (BAY) for 6 h, after which RNA were isolated and Egfl7 transcripts were quantified by duplex RT-qPCR. *, p < 0.05. B, HUVEC were transfected with pGL3basic (Ctrl) or with the −7585/+50Luc construct in the absence (NT) or presence of pRC-CMV (Ctrl) or of pRC-CMV-IκBα S32/36A vector and with pCMV-β-Gal. 24 h later, the cells were treated with 10 ng/ml TNFα (+, black bars) or with PBS (−, white bars) and lysed after 18 h, and luciferase and β-galactosidase activities were measured in cell extracts. The results are representative of two experiments performed in triplicate. *, p < 0.05; **, p < 0.01; ns, non-significant.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Cell Culture, Isolation, Quantitative RT-PCR, Transfection, Construct, Plasmid Preparation, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Specific targeting and rescue of egfl7 do not affect expression of the miR-126 locus. A, HUVEC were transfected with siCtrl (white) or with two different siRNA targeting Egfl7: siEgfl7 #9 (black) or siEgfl7 #10 (gray) and cultured for 4 days. Left panel, total RNA were prepared and analyzed by duplex RT-qPCR for Egfl7 expression. ***, p < 0.001. Middle panel, protein extracts were prepared and analyzed for the presence of Egfl7 or actin by 12% SDS-PAGE and Western blotting. Right panel, HUVEC were treated as in A, and analysis of miR126-3p and miR126-5p expression was performed by RT-qPCR, using the U6 snRNA as normalizer. B, HUVEC were transfected with siCtrl or siEgfl7#10 as in A and then with pcDNA3 (pCtrl) or a pcDNA3-human Egfl7 expression plasmid (pEgfl7). Left panel, RT-qPCR analysis of Egfl7 expression on triplicate experimental samples. Right panel, RT-qPCR analysis of expression of miR126-3p and of miR126-5p in pCtrl- or in pEgfl7-transfected HUVEC on triplicate experimental samples. ***, p < 0.001; ns, non-significant.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Expressing, Transfection, Cell Culture, Quantitative RT-PCR, SDS Page, Western Blot, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 represses the activation of endothelial cells by TNFα. A, expression levels of ICAM-1 (left panel), VCAM-1 (middle panel), and E-selectin (right panel) measured using duplex RT-qPCR in HUVEC transfected with siCtrl (□) or siEgfl7 (●) and stimulated 3 days later with TNFα. The results are representative of two experiments performed in triplicate. At time 0, the levels of Egfl7, ICAM-1, VCAM-1, and E-selectin in the siEgfl7 condition relative to siCtrl condition were 0.31 ± 0.01, 3.18 ± 0.23, 1.00 ± 0.10, and 2.14 ± 0.29, respectively. B, left panel, HUVEC were transfected with pCtrl or pEgfl7 and treated or not for 6 h with TNFα and expression levels of Egfl7 assessed by RT-qPCR on triplicate experimental samples. The numbers indicate the pEgfl7/pCtrl ratio of Egfl7 expression. ***, p < 0.001. Right three panels, HUVEC were transfected with siCtrl or siEgfl7, rescued with pCtrl or pEgfl7, and stimulated or not 2 days later with TNFα for 6 h. Expression levels of ICAM-1, VCAM-1, and E-selectin were assessed by duplex RT-qPCR on triplicate experimental samples. *, p < 0.05; ***, p < 0.001. C, left panel, adhesion of fluorescently labeled Jurkat T-lymphocytes plated onto monolayers of HUVEC transfected 2 days earlier with pCtrl or pEgfl7 and stimulated or not with TNFα. The results are representative of two experiments performed in triplicate. Right panel, mean numbers of adherent fluorescent cells counted over 10 microscopic fields in each condition. *, p < 0.05; ns, non-significant

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 regulates the activation of endothelial cells through the NF-κB and MEK/Erk pathways. A, left panel, microscopic fields of fluorescent Jurkat T-lymphocytes adhering onto HUVEC transfected 3 days earlier with siCtrl or siEgfl7. DMSO or BAY117085 (BAY, 10 μm) was added 20 h before plating T-lymphocytes. Right panel, mean numbers of adherent Jurkat T-lymphocytes counted in 15 non-overlapping microscope fields. *, p < 0.05. B, expression levels of ICAM-1, VCAM-1, and E-selectin transcripts measured using duplex RT-qPCR in HUVEC transfected with siCtrl or siEgfl7 and treated with DMSO or BAY117085 (10 μm). The results are representative of three experiments performed in triplicate. C, left panel, microscopic fields of fluorescent T-lymphocytes adhering onto HUVEC transfected 3 days earlier with siCtrl or siEgfl7. DMSO or U0126 (10 μm) was added 20 h before plating T-lymphocytes. Right panel, mean numbers of adherent Jurkat T-lymphocytes counted in 15 non-overlapping microscope fields. The results are representative of two experiments performed in triplicate. *, p < 0.05. D, expression levels of ICAM-1, VCAM-1, and E-selectin measured using duplex RT-qPCR in HUVEC transfected with siCtrl or siEgfl7 and treated with DMSO or 10 μm of the MEK1/2 inhibitor U0126 for 16 h. The results are representative of two experiments performed in triplicate. *, p < 0.05.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Activation Assay, Transfection, Microscopy, Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 prevents the activation of the NF-κB pathway in endothelial cells. A, HUVEC were transfected with siCtrl or siEgfl7, treated 4 days later with 10 ng/ml TNFα, and lysed at the indicated time points. The cell extracts were prepared, and proteins (10–20 μg) were analyzed by Western blotting using antibodies against the indicated proteins (left). The results are representative of three experiments. B, membranes were exposed to a Las3000 system, and intensities were quantified using the Multigauge V3.0 software.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Activation Assay, Transfection, Western Blot, Software

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 does not affect the IKK complex. HUVEC were transfected with siCtrl or siEgfl7, treated 4 days later with 10 ng/ml TNFα, and lysed at the indicated time points. Cell extracts were prepared, and proteins (10–20 μg) were analyzed by Western blotting using antibodies against the indicated proteins (left). The results are representative of three experiments.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 regulates the degradation of IκBα by the proteasome. A, HUVEC were transfected with siCtrl or siEgfl7, treated 4 days later with 10 ng/ml TNFα, and lysed at the indicated time points. The cell extracts were prepared, and proteins (10–20 μg) were analyzed by Western blotting using antibodies against the indicated proteins (left). The results are representative of three experiments. B, HUVEC were transfected with siCtrl or siEgfl7 and treated 4 days later with the proteasome inhibitor MG132 for 2 h prior to adding 10 ng/ml TNFα for the indicated time. The cells were lysed, and proteins (5 μg) were analyzed by Western blotting against total IκBα or GAPDH. The results are representative of two experiments.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Transfection, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation *

doi: 10.1074/jbc.M116.731331

Figure Lengend Snippet: Egfl7 participates in a loop of regulation of endothelial activation during inflammation. In normal, unstimulated conditions (left panel), Egfl7 constitutively expressed by endothelial cells represses IκBα phosphorylation and degradation, thus promoting the accumulation of inactive NF-κB dimers complexed to IκBα. Egfl7 contributes to low expression levels of leukocyte adhesion molecules and low cell activation. Upon TNFα stimulation (right panel), activation of the NF-κB pathway leads to the repression of Egfl7, which participates in the destabilization of IκBα and triggers endothelial cell activation.

Article Snippet: Immunoblots The anti-human Egfl7 antibody (AF3638, lot WNK0109011) was from R&D Systems.

Techniques: Activation Assay, Phospho-proteomics, Expressing