Journal: PLoS Pathogens

Article Title: Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions

doi: 10.1371/journal.ppat.1005011

Figure Lengend Snippet: Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml IL-1β or 0.025% DMSO (solvent control, equivalent to that in 125ng/ml MYC) for 24 hours (or other times as indicated). Cells were harvested and subjected to flow cytometry for TM and EPCR. A. Forward and side scatter plots. B. Histogram plots for TM and EPCR. Untreated unstained cells; filled grey, untreated cells with isotype control; dotted black line, untreated cells with TM or EPCR-PE; black line; MYC treated cells (7.8ng/ml) with TM or EPCR-PE, red line; IL-1β treated cells with TM or EPCR-PE, green line. C. Quantitation of TM and EPCR surface expression. MFIs are expressed as % of untreated cells with TM or EPCR-PE (mean±SEM, n = 3). Unstained and isotype bars are for untreated cells. D Quantitation of TM and EPCR surface expression over time in the presence of 7.8ng/ml mycolactone. MFIs are expressed as % of untreated cells with TM or EPCR-PE. DMSO control (0.08%) is after 48hrs exposure (mean±SEM, n = 3). *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: Primary human dermal microvascular endothelial cells (HDMVEC; LONZA) from two donors were used and cultured according to manufacturer’s recommendation in EGM-2MV (LONZA).

Techniques: Solvent, Control, Flow Cytometry, Quantitation Assay, Expressing

Journal: PLoS Pathogens

Article Title: Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions

doi: 10.1371/journal.ppat.1005011

Figure Lengend Snippet: Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml IL-1β or 0.08% DMSO (solvent control) for 24 hours. A. Cells were lysed and subject to Western blot analysis. B. Cells were treated as above in the absence or presence of wiskostatin (1μM), harvested and subject to Western blot analysis. C. Cells were treated as above or with 5μg/ml N-elastase. Supernatants of cells were collected, clarified by centrifugation and sTM was quantified by ELISA (mean±SEM, n = 3, except for elastase where n = 1). D. Cells were treated as above in the absence or presence of protease inhibitor I (PSI, 5μM), harvested and subject to Western blot analysis. E. HeLa cells were stably transfected with a plasmid encoding either GFP alone (expressed in the cytosol) or TM-GFP (a C-terminal fusion of human TM and GFP, therefore expressed on the membrane in an ER-dependent manner). These cell lines were subsequently exposed to 125ng/ml mycolactone or 0.025% DMSO over 21 hours and fluorescence was captured by time-lapse microscopy using a Nikon A1 confocal laser scanning unit attached to an Eclipse Ti microscope. Whole-field fluorescence is expressed as a percentage of starting fluorescence for each of triplicate fields and is expressed as the mean (corrected for background fluorescence).

Article Snippet: Primary human dermal microvascular endothelial cells (HDMVEC; LONZA) from two donors were used and cultured according to manufacturer’s recommendation in EGM-2MV (LONZA).

Techniques: Solvent, Control, Western Blot, Centrifugation, Enzyme-linked Immunosorbent Assay, Protease Inhibitor, Stable Transfection, Transfection, Plasmid Preparation, Membrane, Fluorescence, Time-lapse Microscopy, Microscopy

Journal: PLoS Pathogens

Article Title: Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions

doi: 10.1371/journal.ppat.1005011

Figure Lengend Snippet: A. Thrombin generation was measured by calibrated automated thrombography. Thrombin generation was quantified in human pooled plasma containing various concentrations of mycolactone or 0.013% DMSO as a control. The experiment was initiated with 4pM tissue factor, 4μM phospholipid vesicles, and 16.6mM CaCl 2 . Thrombin generation was monitored using 0.42mM of the fluorogenic substrate Z-GlyArg-AMC-HCl as described in the text. B. Platelet aggregation was assessed using an optical platelet aggregometer by the addition of 1μg/ml collagen to washed human platelets that had been treated with various concentrations of mycolactone or 0.1% DMSO as a control, and are expressed relative to an untreated control. Mean±SEM n = 3 (except for resting platelets where n = 1). C. Platelet activation was determined by quantifying fibrinogen binding to, and P-selectin exposure on, human platelets by flow cytometry. Washed human platelets were treated with various concentrations of mycolactone or 0.1% DMSO as a control then stimulation with 1μg/ml CRP-XL, and are expressed relative to an untreated control. Mean±SEM n = 3. D and E. Protein C activation over human dermal microvascular endothelial cells. Protein C was added to cells in the presence of Ca 2+ and Mg 2+ . Activation was initiated by the addition of 13.5nM thrombin and proceeded for 30 mins at which point the reaction was stopped 5μg antithrombin and 3U heparin. Activated protein C was quantified by assessing the rate of chromogenic substrate S2366 cleavage compared to an APC standard curve. D. Michaelis-Menton curve of protein C activation. E. Cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml IL-1β or 0.025% DMSO (solvent control) for 24 hours. In one case, untreated cells were exposed to a function-blocking antibody (CTM) for 1hr prior to the assay. Protein C (5nM) activation was assessed as before and is expressed as % of untreated cells. Mean±SEM n = 3.

Article Snippet: Primary human dermal microvascular endothelial cells (HDMVEC; LONZA) from two donors were used and cultured according to manufacturer’s recommendation in EGM-2MV (LONZA).

Techniques: Clinical Proteomics, Control, Activation Assay, Binding Assay, Flow Cytometry, Solvent, Blocking Assay

Journal: PLoS Pathogens

Article Title: Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions

doi: 10.1371/journal.ppat.1005011

Figure Lengend Snippet: Human dermal microvascular endothelial cells were exposed to various concentrations of mycolactone (MYC), 10ng/ml TNF, 1μM staurosporin or 0.025% DMSO (solvent control) as appropriate. A. Both attached and detached cells were subjected to Calcein/EtBr staining for live/dead cells. The proportion of cells that were either attached or detached and alive (Calcein +/EtBr-), and attached or detached and dead (Calcein-/EtBr +) are expressed as a % of the total population of cells. mean±SEM, n = 3. B. HDMVECs were exposed to 7.8ng/ml mycolactone over a timecourse. Cells were then analysed by confocal microscopy following staining of cells with no-wash reagents. CellEvent caspase-3/7 green detection reagent identified cells undergoing apoptosis, alongside PI and DRAQ5. The number of cells in late apoptosis (positive for both active caspase 3/7 and PI) were counted in 3 fields and expressed as a proportion of total cells (DRAQ5 stained) Mean±SEM (n = 3). C. Cells were harvested and subjected to flow cytometry for VE-Cadherin and CD31 (PECAM-1). MFIs are expressed as % of untreated cells (mean±SEM, n = 4). Unstained and isotype bars are for untreated cells. **, P<0.01; ***, P<0.001.

Article Snippet: Primary human dermal microvascular endothelial cells (HDMVEC; LONZA) from two donors were used and cultured according to manufacturer’s recommendation in EGM-2MV (LONZA).

Techniques: Solvent, Control, Staining, Confocal Microscopy, Flow Cytometry

Journal: Cell reports

Article Title: Aedes aegypti sialokinin facilitates mosquito blood feeding and modulates host immunity and vascular biology

doi: 10.1016/j.celrep.2022.110648

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human Primary Dermal Microvascular Endothelial Cells (HDMVECn) , ATCC , Cat# PCS-110-010.

Techniques: Purification, Blocking Assay, Virus, Titration, Recombinant, Lysis, DNA Purification, Staining, Luminex, Mass Spectrometry, Software

Journal: Journal of Biomedical Optics

Article Title: Functionalized erythrocyte-derived optical nanoparticles to target ephrin-B2 ligands

doi: 10.1117/1.JBO.24.8.085002

Figure Lengend Snippet: Ephrin-B2 Western blot expression. Western blot showing the expression of ephrin-B2 in hDMVECs infected with an ephrin-B2 lentivirus compared to the ephrin-B2 expression level of hDMVECs not infected with the ephrin-B2 lentivirus. Beta-actin levels are shown as the internal loading control.

Article Snippet: Human dermal microvascular endothelial cells (hDMVECs) were cultured in endothelial cell basal medium (ECBM) with growth supplement (Cell Applications, Inc.) as previously described.

Techniques: Western Blot, Expressing, Infection, Control

Journal: Journal of Biomedical Optics

Article Title: Functionalized erythrocyte-derived optical nanoparticles to target ephrin-B2 ligands

doi: 10.1117/1.JBO.24.8.085002

Figure Lengend Snippet: Cellular fluorescence of control and ephrin-B2 hDMVECs incubated with F-NETs at various ρ * values. (a) Fluorescent images of control and ephbin-B2 hDMVECs after 40 min of incubation at 4°C with F-NETs solutions. All images are falsely colored with the blue and red corresponding to DAPI and ICG NIR emission from the NETs, respectively. Scale bars are 30 μ m . (b) Averaged fluorescence intensity ( I ¯ ) [see Eq. (3)] as a function of ρ * . Cells from 3 to 4 images were analyzed, resulting in 16 to 41 measurements for each combination of nanoparticles and cells. Statistical significance of p < 0.001 is denoted by ***. Only statistically significant populations with the same ρ * value of F-NETs are indicated. (c) Sigmoidal fit to the I ¯ values of the ephrin-B2 hDMVECs versus the ρ * value of F-NETs. Error bars in (b) and (c) represent SDs.

Article Snippet: Human dermal microvascular endothelial cells (hDMVECs) were cultured in endothelial cell basal medium (ECBM) with growth supplement (Cell Applications, Inc.) as previously described.

Techniques: Fluorescence, Control, Incubation

Journal: Acta Dermato-Venereologica

Article Title: Novel Detection and Clinical Utility of Serum-Derived Extracellular Vesicle in Angiosarcoma

doi: 10.2340/actadv.v105.40902

Figure Lengend Snippet: miR-3926, miR-5703, miR-3925-5p, and miR-184 were upregulated in angiosarcoma (AS) cell lines. Expression of microRNAs (miRNAs) in an AS cell line (ISO-HAS B) and benign human endothelial cell lines (HUVEC and HDMVECn.) were compared by quantitative reverse transcription PCR. ISO-HAS B showed significant upregulation of miR-5703 ( P adj = 0.0078 and P adj = 0.0261, respectively) and miR-184 ( P adj = 0.0214 and P adj = 0.0053, respectively) in comparison with HUVEC and HDMVECn (F value for each one-way ANOVA analysis was 12.14 and 14.06, respectively). ISO-HAS B also showed a tendency to upregulations in miR-3926 and miR-3925-5p, while the differences were not significant (F value for each one-way ANOVA analysis was 3.154 and 3.237, respectively). The experiment was performed 3 times with triplicate samples for each cell line. HUVEC: human umbilical vein endothelial cell; HDMVECn: human dermal microvascular endothelial cells neonatal. * p < 0.05, ** p < 0.01.

Article Snippet: Commercial human umbilical vein endothelial cells (HUVEC) (ATCC ® , Manassas, VA, USA, PCS-100-010TM) and human dermal microvascular endothelial cells neonatal (HDMVECn) (ATCC ® , PCS-110-010TM) were cultured in vascular cell basal medium (ATCC ® , PCS-110-030TM) supplemented with an Endothelial Cell Growth Kit-VEGF (ATCC ® , PCS-110-041TM).

Techniques: Expressing, Reverse Transcription, Comparison