Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial to Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction

doi: 10.1161/ATVBAHA.119.312826

Figure Lengend Snippet: A: Averaged curves and quantitation of trans- endothelial electrical resistance (TER) of untreated or PIC treated HAMVECs for 24 hours. TER was monitored in confluent cell monolayers for 24 hours using an electric cell substrate impedance sensor instrument (ECSIS). Data was quantitated using area under curve (AUC) and the results from three different experiments performed in triplicate were presented as average±SD. B: Averaged curves showing the “wound closure” kinetics of untreated and PIC treated cells for 24 hours prior to application of the wound. Wound closure was expressed as speed of closure (μm/hr). Data represent average from three independent experiments performed in duplicate. C: Representative micrographs and quantitation of HAMVEC proliferation using BrDU incorporation assay. Untreated or PIC treated cells for either 1 or 6 days were plated on fibronectin-coated chamber slides and incubated with BrDU for 24 hours. Cell nuclei are stained blue with DAPI. Nuclei of the proliferating cells are stained green using a FITC-labeled BrDU antibody. Overlapping images of the same field show both proliferating cells (light blue nuclei) and non-proliferating cells (dark blue, DAPI only). Magnification is 200x (Scale Bar=50-μm). Quantitation was done by calculation of % of BrDU stained cells to total cells. Data is expressed as % proliferation of PIC treated cells compared to control cells for each of the 1hr and 6 hr time points ± SD. For each experimental condition, three independent experiments with cells from three independent donors were performed in duplicate and 3 different areas for each replicate were imaged and counted. Cells between passages 4–7 were used in all experiments. Statistical analysis was performed using paired Student’s t-test between PIC and control samples.

Article Snippet: Endothelial cells Human adipose microvascular EC (HAMVEC) were purchased from ScienCell Research Laboratories (cat#: 7200, Lot #: 5439).

Techniques: Quantitation Assay, BrdU Incorporation Assay, Incubation, Staining, Labeling, Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial to Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction

doi: 10.1161/ATVBAHA.119.312826

Figure Lengend Snippet: HAMVEC with or without PIC treatment for 1day (A) or 6 days (B) were labeled with calcein, seeded in growth factor reduced Matrigel coated plates and incubated for 6 hours before imaging. Representative micrographs (top panels) show reduced formation of branches and reduced tubule length in PIC treated cells, which were totally blunted after 6 days of treatment; Magnification 40x (Scale Bar=200-μm). Angiogenesis was quantified as average numbers of branches and average tubule lengths in three independent experiments performed in triplicate. Three different pictures for each replicate were analyzed using the Image J Angiogenesis software and data was represented as average ±SD. ND = not detectable. Data represents average of three independent experiments performed in duplicate using EC from 3 individual donors on passages 4–7. Data is expressed as the mean+/−SD. Comparisons were performed using paired Student’s t-test.

Article Snippet: Endothelial cells Human adipose microvascular EC (HAMVEC) were purchased from ScienCell Research Laboratories (cat#: 7200, Lot #: 5439).

Techniques: Labeling, Incubation, Imaging, Software

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial to Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction

doi: 10.1161/ATVBAHA.119.312826

Figure Lengend Snippet: A: Oxygen consumption rate (OCR) was utilized to assess endothelial metabolism in HAMVECs stimulated with PICs for 6 days using a Mitochondrial Stress Test. Cells were seeded into Seahorse XFe24 plates at 50,000 cells/well and allowed to attach and grow for 24 hours until they reached 70–80%confluence. Respiration data was normalized to total cellular protein. B: Proton efflux rate due to glycolysis (GlycoPER) was measured using the glycolysis stress kit in cells seeded in similar conditions as above. Data is normalized to total protein. C: Fatty acid oxidation was measured using palmitate-BSA as a substrate according to a protocol detailed in Methods section. For this assays cells were allowed to reach 100% confluence. Etomoxir (4uM) was used as a positive control for inhibition of beta-oxidation D: Phenogram showing extracellular acidification and oxygen consumption; E: Gene expression of Cpt1, Cpt2 and Acly was measured in control and PIC-treated HAMVEC for 6 days by real-time PCR; F: Western blotting and semi-quantitation of CPT1a expression in HAMVEC treated with PIC for 6 days and untreated controls.G: miR-155–5p measured by real-time PCR in PIC-treated cells for 6 days compared to controls. H: Diagram showing proposed scenario involving down regulation of CPT1, and ACLY via miR-155–5p leading to an overall reduction in HAMVEC metabolism in response to PIC treatment. Data represents average of three independent experiments performed in duplicate using EC from 3 individual donors on passages 4–8. Data is expressed as the mean+/−SD.

Article Snippet: Endothelial cells Human adipose microvascular EC (HAMVEC) were purchased from ScienCell Research Laboratories (cat#: 7200, Lot #: 5439).

Techniques: Positive Control, Inhibition, Gene Expression, Control, Real-time Polymerase Chain Reaction, Western Blot, Quantitation Assay, Expressing

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial to Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction

doi: 10.1161/ATVBAHA.119.312826

Figure Lengend Snippet: A: Nanoparticle size distribution of extracellular vesicles (EV) isolated from untreated control cells (EV-C) and from PIC-treated cells (EV-PIC) shows an average peak size of 140–150-nm for both EV preparations. B: Representative micrographs of negative staining electron microscopy of EV-PIC and EV-C confirms the presence of EVs in the size range calculated by nanoparticle tracking analysis in both preparations; C: Nanosight analysis shows a 3-fold higher number of EV-PIC/cell compared to EV-C. This difference was consistent across 12 different EV preparations from control and PIC treated cells. D: Western blot showing expression of various markers in control and PIC treated cells and in EV-C and EV-PIC extracellular vesicles. Exosomal markers syntenin-1 and CD9 are enriched in both EV-PIC and EV-C compared to parent cells and CD63 and HSP70 were present in both EV preparations; calnexin (microsomal marker) and LAMP1 (lysosomal marker) were not detectable in either one of the EV preparations. E: diagram showing experimental protocol for EV production, isolation and incubation with recipient cells. F: EV-PIC and EV-C were labeled with the fluorescent lipophilic dye Vybrant DiO and incubated with EC overnight; representative micrographs show uptake by HAMVEC at 37°C and show dramatic reduction of uptake at 22°C, suggesting an energy dependent uptake mechanism; magnification 100x (Scale Bar=100-μm); higher magnification inset shows the peri-nuclear distribution of the labeled EVs. G: Quantitative analysis of EV uptake using flow cytometry of the HAMVEC cells after 24 h incubation with labeled EVs. Representative flow cytometry plots show the background control that was subtracted from the uptake data (left panel); negative control (center left) and EV-PIC and EV-C representative uptake plots. Concentration dependent uptake was determined in pilot experiments. Results shown here are from incubation of 105 cells with 1010 EV-C or EV-PIC for 24 hours. Data is expressed as mean of % cell uptake ± SD and shows no difference between uptake of EV-C and EV-PIC. Results are from 3 independent experiments, performed in duplicate, using cells from 3 separate donors, on passages 4–5.

Article Snippet: Endothelial cells Human adipose microvascular EC (HAMVEC) were purchased from ScienCell Research Laboratories (cat#: 7200, Lot #: 5439).

Techniques: Isolation, Control, Negative Staining, Electron Microscopy, Western Blot, Expressing, Marker, Incubation, Labeling, Flow Cytometry, Negative Control, Concentration Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Endothelial to Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction

doi: 10.1161/ATVBAHA.119.312826

Figure Lengend Snippet: HAMVEC were incubated for 24 hours with either EV-C, EV-PIC or were left untreated. Following treatment, cells were labeled with calcein, seeded in growth factor reduced matrigel coated plates and incubated for 6 hours before imaging. Representative micrographs (top panels) showing reduced formation of branches and reduced tubule length in HAMVEC treated with EV-PIC compared to untreated cells; Magnification 40x (Scale Bar=200-μm). Treatment with EV-PIC severely impaired tube network parameters compared to untreated control cells and EV-C treated cells. Angiogenesis was quantified in three independent experiments performed in triplicate. Three different pictures for each replicate were analyzed using the Image J Angiogenesis Analyzer software and data was represented as average ±SD. B: Oxygen consumption rate (OCR) was utilized to assess endothelial metabolism in cells treated with EV-C or EV-PIC using a Mitochondrial Stress Test. Cells were seeded into Seahorse XFe24 plates and allowed to attach and grow for 24 hours until they reached 70–80% confluence . Respiration data was normalized to total cellular protein. B: Proton efflux rate due to glycolysis (GlycoPER) was measured using the glycolysis stress kit in cells seeded in similar conditions as above. Data is normalized to total protein. C: Fatty acid oxidation was measured using palmitate-BSA as a substrate according to a protocol detailed in Methods section. For this assays cells were allowed to reach 100% confluence. Etomoxir (Eto) (4uM) was used as a positive control for inhibition of beta-oxidation Data represents average of three independent experiments performed in duplicate on cells from 3 individual donors on passages 4–6 and are expressed as the mean+/−SD.

Article Snippet: Endothelial cells Human adipose microvascular EC (HAMVEC) were purchased from ScienCell Research Laboratories (cat#: 7200, Lot #: 5439).

Techniques: Incubation, Labeling, Imaging, Control, Software, Positive Control, Inhibition

Journal: Orphanet Journal of Rare Diseases

Article Title: Impaired osteoblast and osteoclast function characterize the osteoporosis of Snyder - Robinson syndrome

doi: 10.1186/s13023-015-0235-8

Figure Lengend Snippet: Characterization of the bone and osteoblast pathology. A . Photograph of the bone biopsy. B . Steady state SMS mRNA levels relative to GAPDH expression in cultured fibroblasts and osteoblasts. The patient’s cells did not differ significantly from controls. Data were derived by qRT-PCR analysis of 3 independent extractions of total RNA. C . Immunoblot showing steady state SMS protein expression in patient and control osteoblasts. ß-tubulin is shown as a loading control. D . Graph showing steady state SMS protein levels in the patient and control hBMSCs relative to ß-tubulin levels; there was no significant difference. The data are based on 3 independent experiments for each cell line. E - J . Immunofluorescent detection of SMS protein subcellular distribution in unaffected (E-G) and Patient II-1 (H-J) hBMSCs. SMS protein is shown in red and the nucleus is shown in blue. K . Graph quantifying immunoblot detected steady state SMS protein levels in the cytoplasm and nuclei of patient and control hBMSCs. The cytoplasmic expression was normalized to β-tubulin expression and the nuclear expression to p84 expression. L . Polyamine quantification in fibroblasts and osteoblasts. Note that the patient hBMSCs have a more striking imbalance of spermidine and spermine levels than do the patient fibroblasts, * p < 0.05, *** p < 0.005. M . Osteogenic potential of bone marrow stromal cells (hBMSCs) isolated from Patient II-1 sample is markedly lower than that of an unaffected control (cnt). The hBMSCs were seeded in triplicates (6x10 4 /12-well) and either kept untreated (-) or treated (+) with osteogenic differentiation media (see ) for 18 days. After the treatment, cells were fixed and were stained with Alizarin Red S to check for calcium deposition, a marker of osteogenic differentiation.

Article Snippet: We also measured spermine and spermidine levels in cultured human bone marrow stromal cells (hBMSCs) and fibroblasts using the Biochrom 30 polyamine protocol and assessed the osteogenic potential of hBMSCs.

Techniques: Expressing, Cell Culture, Derivative Assay, Quantitative RT-PCR, Western Blot, Control, Isolation, Staining, Marker

Journal: Archives of Gynecology and Obstetrics

Article Title: Glycosylated fibronectin as a first trimester marker for gestational diabetes

doi: 10.1007/s00404-020-05670-8

Figure Lengend Snippet: a Glycosylated fibronectin concentrations in BMI subgroups of 20–25 and 30–35 kg/m 2 in the study and the control group. b Effect of smoking on concentrations of glycosylated fibronectin in the control and GDM groups. c Multiple of median (MoM) values for fibronectin and glycosylated fibronectin between control and GDM groups. d Multiple of median (MoM) values for adiponectin and glycosylated adiponectin between control and GDM groups

Article Snippet: Calibrators for both assays were made from recombinant human adiponectin (1065-AF, R&D Systems, Abingdon, UK).

Techniques: Control