Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells

doi: 10.1161/01.ATV.0000222017.05085.8e

Figure Lengend Snippet: PTN downregulates expression of monocytic cell markers. A, Total RNA was extracted from THP-1 cells grown in 10% serum (lane 2), induced to differentiate into macrophage-like cells by addition of 25 ng/mL phorbol 12-myristate 13-acetate (PMA; lane 3), transduced with retroviral bicistronic vector expressing: GFP (lane 4), PTN sense strand (lane 5), PTN antisense strand (lane 6) followed by treatment with PMA. The exponentially growing human coronary artery endothelial cells (lane 7) were used as a negative control. Analyzed monocytic cell markers were c-fms and CD-68 with primers predicted to amplify 97- and 132-bp DNA fragments, respectively. GAPDH primers were used as control a for the RT-PCR. Lane 1 is a DNA ladder marker. B, Flow cytometry analysis was performed by incubating 5×105 THP-1 cells expressing PTN or GFP with phycoerythrin-labeled anti-CD14 antibody from PharMingen. Human coronary artery endothelial cells were used as a negative control. Uninfected THP-1 cells were used as positive control, and human coronary endothelial cells (endothelial) were used as a negative control. FACS analysis was performed at the Cedars-Sinai Research Institute Core Facility. Each experiment was repeated 3 times, and each bar graph represents mean±SEM of 3 experiments.

Article Snippet: Human aortic endothelial cells and smooth muscle cells were obtained from Cell Applications, INC. and cultured according to their recommendation.

Techniques: Expressing, Transduction, Retroviral, Plasmid Preparation, Negative Control, Control, Reverse Transcription Polymerase Chain Reaction, Marker, Flow Cytometry, Labeling, Positive Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells

doi: 10.1161/01.ATV.0000222017.05085.8e

Figure Lengend Snippet: PTN upregulates expression of endothelial cell markers in the monocytic cells. A, RT-PCR analysis of endothelial cell markers. Total RNA isolated from untransduced and transduced cells were used for RT-PCR analysis, using specific primers for endothelial cell markers (see online supplement). To ensure semiquantitative results of the RT-PCR analysis, the number of PCR cycles for each set of primers was checked to be in the linear range of the amplification. In addition, all RNA samples were adjusted to yield equal amplification of GAPDH as an internal standard. The amplified products were separated on 1.2% agarose gels and stained with ethidium bromide. B, A representative flow cytometry analysis of αvβ3 integrin expression by the monocytic cells. GFP (top left panel) or PTN-expressing THP-1 cells (bottom left panel) were incubated with a 1:100 dilution of anti-human αvβ3 mouse antibody (Chemicon Co). After washing, cells were incubated with 1:500 dilution of phycoerythrin-labeled anti-mouse antibody (Sigma), fixed, and analyzed by FACS, as described above. In addition, human coronary artery endothelial cells in the absence (top right panel) or presence of anti-αvβ3 antibody (bottom right panel) were used as a positive control. C, RT-PCR analysis of transcription factors. The PCR was performed as described in A, and the primers and PCR condition are described in the online supplement.

Article Snippet: Human aortic endothelial cells and smooth muscle cells were obtained from Cell Applications, INC. and cultured according to their recommendation.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Staining, Flow Cytometry, Incubation, Labeling, Positive Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells

doi: 10.1161/01.ATV.0000222017.05085.8e

Figure Lengend Snippet: Amplification plot for the expression of CD68 expression in the monocytic cells. Oligonucleotide primer pairs for CD68 gene and an oligonucleotide probe labeled with a reporter fluorescent dye at the 5′ end and quencher dye at the 3′ end were designed using Oligo 4.0 software (National Bioscience, Plymouth, MN). Total RNA with DNase I treatment was used to synthesize first-stand cDNA with RT (GIBCOBRL) and oligo(dT) 15 Primer (Promega). Total RNA (50 ng) was added to a 50 μl reverse transcriptase-polymerase chain reaction (RT-PCR) reaction mixture according to the manufacturer’s protocol (Roche Molecular Systems). The products of the RT reactions was used to seed real-time PCR by using an ABI Prism 7700 Sequence Detector by comparing with GAPDH (internal control) and individual standard curve performed in triplicate. The thermal cycling conditions included one cycle at 48°C for 30 minutes, one cycle at 95°C for 10 minutes, 40 cycles at 95°C for 15 s, annealing at 60°C for 1 minute, and a final hold at 25°C for 2 minutes. Standard curves for the expression of each gene were generated by serial dilution of a standard preparation of total RNA isolated from cultured monocytic or endothelial cells.

Article Snippet: Human aortic endothelial cells and smooth muscle cells were obtained from Cell Applications, INC. and cultured according to their recommendation.

Techniques: Amplification, Expressing, Labeling, Software, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Control, Generated, Serial Dilution, Isolation, Cell Culture

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells

doi: 10.1161/01.ATV.0000222017.05085.8e

Figure Lengend Snippet: Quail chorioallantotic membrane (CAM) assay. Fertilized eggs of Japanese quail were cultured at 37 °C under ambient atmosphere, for 56 hours, and then opened at embryonic day 3 (panel A) after incubation of the eggs and cultured further at 37°C in 6-well plates. At E7 (Panel B), 1×106 RAW cells in prewarmed PBS (200 μl) were applied in drops to the surface of each CAM. The embryos were incubated further at 37°C for 72 hours, at which time they were harvested (panel C), fixed in 4% paraformaldehyde/2% glutaraldehyde/PBS. Panel D and E show fluorescent Image of CAM implanted with RAW cells expressing PTN/GFP or mouse endothelial cells expressing GFP, respectively.

Article Snippet: Human aortic endothelial cells and smooth muscle cells were obtained from Cell Applications, INC. and cultured according to their recommendation.

Techniques: Membrane, Chick Chorioallantoic Membrane Assay, Cell Culture, Incubation, Expressing

Journal: Scientific Reports

Article Title: Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

doi: 10.1038/s41598-018-23420-4

Figure Lengend Snippet: Canagliflozin stimulates AMPK in human endothelial cells. ( a ) HUVECs were stimulated with A769662 (100 μmol/l, 30 min) or the indicated concentrations of canagliflozin, dapagliflozin or empagliflozin for 15 min and lysates prepared. ( b ) HAECs were stimulated with the indicated concentrations of canagliflozin, empagliflozin or A769662 for 30 min and lysates prepared. AMPK was immunoprecipitated from lysates and assayed for AMPK activity. Data shown represents AMPK activity (% vehicle) from three independent experiments. **p < 0.01; ***p < 0.001 vs vehicle (two-tail t-test).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously .

Techniques: Immunoprecipitation, Activity Assay

Journal: Scientific Reports

Article Title: Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

doi: 10.1038/s41598-018-23420-4

Figure Lengend Snippet: Canagliflozin inhibits 2-deoxyglucose uptake in HUVECs without influencing cell proliferation. ( a ) HUVECs were preincubated in the presence or absence of canagliflozin (10 μmol/l) for the times indicated or dapagliflozin (10 μmol/l, 30 min) and 2-deoxyglucose uptake assessed. Data shown represents the mean 2-deoxyglucose uptake relative to vehicle from three independent experiments. **p < 0.01, ***p < 0.001 relative to vehicle (one-way ANOVA). ( b ) Cell lysates from HUVECs (2 independent lysates), HAECs (4 independent lysates) and HEK-293 cells and mouse kidney membranes were resolved by SDS-PAGE and immunoblotted with anti-SGLT2 antibodies. The immunoblot shown has been cropped, with the full-length immunoblot shown in Supplementary Figure . ( c , d ) HUVECs were incubated in the presence or absence of canagliflozin, dapagliflozin or empagliflozin and ( c ) proliferation assessed in 2.5% (v/v) or 5% (v/v) MV2 Supplement mix (serum) by BrdU incorporation or ( d ) viability assessed by MTS assay. Data shown represents the mean proliferation or viability relative to vehicle (in 5% MV2 supplement mix) from three independent experiments in each cae. ## p < 0.01 relative to vehicle (two-tailed t-test).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously .

Techniques: SDS Page, Western Blot, Incubation, BrdU Incorporation Assay, MTS Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

doi: 10.1038/s41598-018-23420-4

Figure Lengend Snippet: Expression of SGLT2 and SGLT1 mRNA in human endothelial cells. Expression of SGLT2, SGLT1 relative to TBP was assessed by qPCR in cDNA prepared from three independent cultures of HUVECs, HAECs and HEK-293 cells, using cDNA from human kidney cortex as a positive control. Data shown are the mean ± SEM Ct values and 2 −ΔΔCt in each case, from three independent samples. ND = Not detected.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously .

Techniques: Expressing, Positive Control

Journal: Scientific Reports

Article Title: Canagliflozin inhibits interleukin-1β-stimulated cytokine and chemokine secretion in vascular endothelial cells by AMP-activated protein kinase-dependent and -independent mechanisms

doi: 10.1038/s41598-018-23420-4

Figure Lengend Snippet: Canagliflozin inhibits IL-1β-stimulated MCP-1 and IL-6 secretion in human endothelial cells. ( a , c ) HUVECs were stimulated with IL-1β (10 ng/ml) for ( a ) 6 h or ( c ) 24 h following preincubation in the presence or absence of canagliflozin (10 μmol/l, 15 min), dapagliflozin (1 μmol/l, 15 min), empagliflozin (1 μmol/l, 15 min) or A769662 (100 μmol/l, 30 min) and conditioned medium collected. ( b ) HAECs were infected with 100 pfu/cell Ad.null or Ad.AMPK-DN for 24 h and preincubated in the presence or absence of canagliflozin (10 μmol/l, 15 min) or A769662 (100 μmol/l, 30 min) prior to stimulation with IL-1β (10 ng/ml) for 6 h and conditioned medium collected. ( a , b ) MCP-1, ( a ) IL-6 or ( c ) endothelin-1 levels were assayed in conditioned medium by ELISA. Data shown represents the % IL-1β-stimulated MCP-1, IL-6 or endothelin-1 secretion from ( a , c ) three ( b ) four (Ad.null) or five (Ad.AMPK-DN) independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 relative to IL-1β alone (ANOVA).

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously .

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Inflammasome Signaling and Impaired Vascular Health in Psoriasis

doi: 10.1161/ATVBAHA.118.312246

Figure Lengend Snippet: (A) Differential transcript expression following in vitro human aortic endothelial cells stimulation with IL-17 versus PBS, IL-17+TNF-α versus PBS, IL-17+IFN-γ versus PBS (p<0.05 for all changes). (B) Direct analysis of venous endothelial cells harvested from psoriasis patients compared to controls show transcript upregulation in intracellular adhesion molecules (VCAM-1, ICAM-1), inflammation (COX-2) as well as chemokines (CXCL10, CXCL1, CX3CL1, CCL3, MCP-1) interleukins and tumor necrosis factors (IL-1β, IL-8, Lymphotoxin beta). (C) Differential endothelial transcript expression in psoriasis over controls stratified by psoriasis severity (control, PASI: ≤ 10, > 10). (D) Endothelial inflammatory activation in psoriatic patients, as assessed by nuclear p65 NFκB localization in the vascular endothelium of brachial vein endothelial cells. Direct immunofluorescence staining of harvested venous endothelial cells (VE-cadherin, green) of psoriasis patients compared to healthy controls show increased p65 NFκB (red) nuclear (DAPI - blue) co-localization (n=6). p<0.05*, p<0.01**. DAPI, 4’,6-diamidino-2-phenylinodle; IL, interleukin; NFκB, Nuclear factor kappa-light-chain-enhancer of activated B cells; PASI, psoriasis area and severity index; PSB, phosphate buffered-saline; TNF, tumor necrosis factor; VEGFA, vascular endothelial growth factor A; vWF, von willebrand factor.

Article Snippet: In vitro human aortic endothelial methods and analysis: Human aortic endothelial cells (HAECs) were cultured in Endothelial Cell Growth Medium MV 2 (Promocell® GmbH) at passage three.

Techniques: Expressing, In Vitro, Activation Assay, Immunofluorescence, Staining