Journal: PLoS ONE

Article Title: CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells

doi: 10.1371/journal.pone.0144133

Figure Lengend Snippet: CD40 ligation upregulates membrane CX3CL1 in HAEC and HUVEC. (A–D) HAEC were transduced with MIEG3-based retroviral vector that encode EGFP or EGFP plus wt CD40. CX3CL1 expression was analyzed on transduced (EGFP + ) cells. (A) Histograms show CX3CL1 expression on HAEC transduced with wt CD40-encoding retroviral vector at 24 h post-incubation with or without multimeric CD154. (B) Expression of CX3CL1 (cMFI) on gated EGFP + cells after incubation with or without multimeric CD154 or TNF-α. (C) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with either anti-CD154 or isotype control mAb. (D) Transduced HAEC that express wt CD40 were incubated with recombinant CD154 with or without enhancer used to crosslink CD154. (E) HUVEC were incubated with or without multimeric CD154. Results are shown as mean ± SEM and are representative of 3–5 experiments. ** p

Article Snippet: Cells and in vitro stimulation Primary HAEC and HUVEC obtained from Lonza (Allendale, NJ) and A.T.C.C. (Manassas, VA) were cultured following supplier’s recommendations.

Techniques: Ligation, Transduction, Plasmid Preparation, Expressing, Incubation, Recombinant

Journal: PLoS ONE

Article Title: CD40-TRAF Signaling Upregulates CX3CL1 and TNF-α in Human Aortic Endothelial Cells but Not in Retinal Endothelial Cells

doi: 10.1371/journal.pone.0144133

Figure Lengend Snippet: CD40 ligation stimulates CX3CL1 secretion by HAEC and HUVEC. (A) HAEC transduced with control or wt CD40 retroviral vector were incubated with or without multimeric CD154 or TNF-α for 24 h. CX3CL1 concentrations in supernatants were determined by ELISA. (B) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with either anti-CD154 or isotype control mAb. (C) HAEC transduced with control or wt CD40 retroviral vector were incubated with recombinant CD154 with or without enhancer. (D) HUVEC were incubated with or without multimeric CD154. (E) Transduced HAEC that express wt CD40 were incubated with multimeric CD154 with or without the MMP inhibitor GM 6001. CX3CL1 concentrations in supernatants were determined by ELISA. Results are shown as mean ± SEM and are representative of 3–5 experiments. *** p

Article Snippet: Cells and in vitro stimulation Primary HAEC and HUVEC obtained from Lonza (Allendale, NJ) and A.T.C.C. (Manassas, VA) were cultured following supplier’s recommendations.

Techniques: Ligation, Transduction, Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Stem Cells Translational Medicine

Article Title: Development of a Three‐Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling

doi: 10.5966/sctm.2016-0192

Figure Lengend Snippet: Immunostaining of 3D, multicellular organoids compared with adult human distal lung. (A): Confocal micrograph of cross sections of 3D multicellular lung organoids with immunofluorescence for CD31 (HUVECs), vimentin (FLFs), and pro‐SPB and pro‐SPC (type II alveolar epithelial cells) and T1a (type I alveolar epithelial cells; scale bar = 100 μm). (B): Confocal micrograph of multicellular 3D lung organoids with immunofluorescence for CD31 (HUVECs) and PanCK (SAECs). FLFs were also seeded. (C): Confocal micrograph of a cross‐section of normal adult human lung with immunofluorescence for CD31 (HUVECs) and PanCK (SAECs; scale bar = 100 μm). Abbreviations: 3D, three‐dimensional; DAPI, 4′,6‐diamidino‐2‐phenylindole; FLFs, fetal lung fibroblasts; HUVECs, human umbilical vein endothelial cells; SAECs, small airway epithelial cells; SPB, Surfactant Protein B; SPC, Surfactant Protein C.

Article Snippet: Human umbilical vein endothelial cells (HUVECs) and small airway epithelial cells (SAECs) were maintained according to the manufacturer's recommendations (Lonza, Basel, Switzerland, http://www.lonza.com ) in endothelial growth medium (EGM)‐2 (Thermo Fisher) and small airway growth medium (SAGM) (Lonza), respectively.

Techniques: Immunostaining, Immunofluorescence

Journal: Angiogenesis

Article Title: Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation

doi: 10.1007/s10456-020-09722-0

Figure Lengend Snippet: Clonal populations of CLVM LEC or VEC form vascular or lymphatic channels in mouse. a DNA Sanger sequencing chromatogram for 3 LEC and 3 VEC clones derived from CLVM D EC. b Immunoblot analysis of LEC and VEC clones for PI3K effector AKT (phospho-AKT (Ser473) and total AKT), pan-EC (VEGFR-2) and LEC-specific (PROX1) markers. c Densitometric quantification of phospho-AKT(Ser473) western blot bands relative to total AKT. Horizontal bar shows mean, data is normalized to CLVM D EC mean, n = 2 independent experiments. d Proliferation of LEC and VEC clones at 72 h in EBM-2 no FBS, normalized to time 0 (12 h after seeding). 3 LEC and 3 VEC clones were used. HUVEC and HDLEC served as normal controls. Horizontal bar shows mean ( n = 5 technical repeats). e Resistance of CLVM D EC and clones to cell death induced by growth factor withdrawal at 72 h. HUVEC and HDLEC served as normal controls. Horizontal bar shows mean ( n = 5 technical repeats). The cell death was measured as: [(number of cytotox+cells at 72 h)/ (total number of cells at 72 h) × 100]. f Representative images of 3 LEC and 3 VEC clones injected in vivo. Lesions were analyzed at 9 days for gross morphology and sections stained for H E. n = 4 mice per each LEC or VEC clone. Arrowheads point to lymph fluid material inside the lymphatic channels and arrows point to red blood cells within the vascular channels. Scale bars: 1 cm (plug) and 100 μm (H E)

Article Snippet: Human dermal lymphatic endothelial cells (HDLEC), human umbilical vein endothelial cells (HUVEC), foreskin EC, and human lung fibroblasts served as healthy controls and were obtained from Lonza.

Techniques: Sequencing, Clone Assay, Derivative Assay, Western Blot, Injection, In Vivo, Staining, Mouse Assay

Journal: Angiogenesis

Article Title: Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation

doi: 10.1007/s10456-020-09722-0

Figure Lengend Snippet: Characterization of patient-derived CLVM EC. a Western blot analysis for pan-EC marker VEGFR-2 and LEC-specific markers VEGFR-3 and PROX1 in patient-derived CLVM EC, human umbilical vein endothelial cells (HUVEC), and human dermal lymphatic endothelial cells (HDLEC). β-Actin serves as loading control. b Contrast phase and immunofluorescence images of CLVM EC at 80–90% confluency stained for pan-EC markers CD31 and VE-Cadherin, and LEC-specific markers D2-40 and PROX1. EC and LEC markers (green/red), nuclei (DAPI, blue). Scale bar: phase 100 µm; immunofluorescence 50 µm. c Immunofluorescence images of lung fibroblasts, HUVEC, HDLEC and CLVM EC at 80–90% confluency stained for the stem cell/fibroblast marker CD90 (green) and DAPI for nuclei (blue). Scale bar: 50 µm

Article Snippet: Human dermal lymphatic endothelial cells (HDLEC), human umbilical vein endothelial cells (HUVEC), foreskin EC, and human lung fibroblasts served as healthy controls and were obtained from Lonza.

Techniques: Derivative Assay, Western Blot, Marker, Immunofluorescence, Staining

Journal: Angiogenesis

Article Title: Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation

doi: 10.1007/s10456-020-09722-0

Figure Lengend Snippet: Proliferative capacity of CLVM EC and response to PI3K-AKT inhibition. a Proliferation rates of CLVM EC, HDLEC and HUVEC in growth medium with 20% fetal bovine serum (FBS) (left) and in basal medium without FBS (right); ( n = 3–4 independent experiments), two-way ANOVA. * P

Article Snippet: Human dermal lymphatic endothelial cells (HDLEC), human umbilical vein endothelial cells (HUVEC), foreskin EC, and human lung fibroblasts served as healthy controls and were obtained from Lonza.

Techniques: Inhibition

Journal: Angiogenesis

Article Title: Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation

doi: 10.1007/s10456-020-09722-0

Figure Lengend Snippet: Increased PI3K-AKT signaling in CLVM EC. a Western blot analysis of AKT (Ser473), AKT (Thr308), and ERK1/2 phosphorylation in CLVM EC compared to normal HUVEC and HDLEC. β-Actin serves as loading control. b Densitometric quantification of phospho-AKT(Ser473), phospho-AKT(Thr308), phospho-ERK1/2 western blot bands relative to total AKT and ERK1/2. Horizontal bar shows mean, data is normalized to control EC mean, Welch’s t -test

Article Snippet: Human dermal lymphatic endothelial cells (HDLEC), human umbilical vein endothelial cells (HUVEC), foreskin EC, and human lung fibroblasts served as healthy controls and were obtained from Lonza.

Techniques: Western Blot

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Activated protein C inhibits LPS-mediated acetylation and secretion of HMGB1 in endothelial cells

doi: 10.1111/jth.14425

Figure Lengend Snippet: APC inhibits LPS-induced HMGB1 translocation from nucleus to cytoplasm in endothelial cells. (A) EA.hy926 endothelial cells were pretreated with APC (20 nM for 3h) followed by stimulation with LPS (1 µg/mL for 1h). Cytoplasmic and nuclear fractions were isolated and HMGB1 levels were analyzed by immunoblotting. β-actin and PCNA were used as loading controls for cytoplasmic and nuclear fractions, respectively. (B) The same as panel A except that following treatment of cells with APC and LPS, cells were fixed and permeabilized followed by staining HMGB1 with rabbit anti-HMGB1 antibody and Alexa Fluor 488-conjugated goat anti-rabbit IgG. Nucleus was stained with DAPI. Immunofluorescence images were taken by confocal microscopy. Arrows indicate cytoplasmic translocation of HMGB1. The magnified insets correspond to the cells marked with yellow dashed boxes. (C) The same as panel B except that primary HUVECs, instead of EA.hy926 cells, were used for the HMGB1 staining. (D and E) The quantification of LPS-mediated translocated cells from the nucleus to the cytoplasm for HUVECs (panel C) and EA.hy926 cells (panel B), respectively. Scale bar: 50 μm. Results are shown as means ± SE. *p

Article Snippet: Cell lines and culturePrimary human umbilical vein endothelial cells (HUVECs, Invitrogen), transformed HUVECs (EA.hy926 cells, ATCC) and murine macrophage cell line J774A.1 (ATCC® TIB67™) was maintained in complete Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FBS, 100 µg/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine, and cultured at 37°C in a humidified incubator with 5% CO2 as described ( , ).

Techniques: Translocation Assay, Isolation, Staining, Immunofluorescence, Confocal Microscopy

Journal: Tissue Engineering. Part A

Article Title: The Dominant Role of IL-8 as an Angiogenic Driver in a Three-Dimensional Physiological Tumor Construct for Drug Testing

doi: 10.1089/ten.tea.2013.0245

Figure Lengend Snippet: HUVEC migration through Transwell ® inserts. (a) HUVEC migration through the Transwell inserts for untreated 3D monoculture (left), 3D monoculture treated with 1 μg/mL anti-VEGF-A monoclonal antibody (center), and 3D monoculture treated with 1 μg/mL anti-IL-8 monoclonal antibody (right); (b) 2D monoculture (left), 2D cocultured with fibroblasts (center), and 2D cocultured with fibroblasts treated with 1 μg/mL anti-IL-8 monoclonal antibody (right); and (c)

Article Snippet: For the experiments involving transfected U2OS cells cocultured with fibroblasts or HUVECs in 2D monolayers, the cells first underwent fluorescence-activated cell sorting (FACS) using an FACSVantage SE flow cytometer (Becton Dickinson), and those found positive for GFP were collected for RNA extraction.

Techniques: Migration

Journal: Tissue Engineering. Part A

Article Title: The Dominant Role of IL-8 as an Angiogenic Driver in a Three-Dimensional Physiological Tumor Construct for Drug Testing

doi: 10.1089/ten.tea.2013.0245

Figure Lengend Snippet: (a) Effect of coculture with human umbilical vein endothelial cells (HUVECs) on angiogenic factor secretion by 2D U2OS monoculture, 2D GFP-sorted U2OS cocultured with HUVECs, and 2D unsorted U2OS cocultured with HUVECs. **Statistically similar at p > 0.05. (b) Upregulation of IL-8 and VEGF-A in 3D coculture with HUVECs. *Statistically different at p

Article Snippet: For the experiments involving transfected U2OS cells cocultured with fibroblasts or HUVECs in 2D monolayers, the cells first underwent fluorescence-activated cell sorting (FACS) using an FACSVantage SE flow cytometer (Becton Dickinson), and those found positive for GFP were collected for RNA extraction.

Techniques:

Journal:

Article Title: Placenta Growth Factor in Diabetic Wound Healing

doi: 10.2353/ajpath.2006.051314

Figure Lengend Snippet: Expression of VEGFR-1 by cultured normal human dermal fibroblasts: proliferative and chemotactic responses to PlGF. A: RT-PCR analysis of VEGFR-1 and VEGFR-2 transcription by cultured dermal fibroblasts. Human umbilical vein endothelial cells have been

Article Snippet: Human umbilical vein endothelial cells were isolated from freshly delivered umbilical cords as previously described and cultured in Endothelial Cell Growth Medium-2 kit from Clonetics (BioWhittaker Inc., Walkersville, MD).

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: In Vivo Targeting of Inflammation-Associated Myeloid-Related Protein 8/14 Via Gadolinium Immunonanoparticles

doi: 10.1161/ATVBAHA.111.244509

Figure Lengend Snippet: Anti-Mrp14 nanoprobes (aMrp-NPs) bind to endothelial cells displaying exogenous Mrps on the surface. Human umbilical vein endothelial cells (HUVECs) were pretreated with either rMrp8/14 or sham (phosphate-buffered saline), or cultured in the presence

Article Snippet: Human umbilical vein endothelial cells (HUVECs, Invitrogen) were maintained in medium 199 (Invitrogen) supplemented with a brain bovine extract (Lonza) and 20% FBS according to vendor's directions.

Techniques: Cell Culture

Journal: Journal of Virology

Article Title: Dengue Virus Induces Novel Changes in Gene Expression of Human Umbilical Vein Endothelial Cells

doi: 10.1128/JVI.77.21.11822-11832.2003

Figure Lengend Snippet: Multiplex PCR for the expression of TLR1 to -5 in uninfected and D2V-infected HUVECs. cDNA template was obtained from an RT step performed on total RNA isolated from control and D2V-infected cultures 48 h postinfection. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is given as a control for equal loading. In the PCR step, two different quantities of cDNA (3 and 6 μl) were used. GAPDH is shown at 680 bp.

Article Snippet: To analyze the virus-host interaction, we studied the effect of DV infection on gene expression in human umbilical vein endothelial cells (HUVECs) by using differential display reverse transcription-PCR (DD-RTPCR), quantitative RT-PCR, and Affymetrix oligonucleotide microarrays.

Techniques: Multiplex Assay, Polymerase Chain Reaction, Expressing, Infection, Isolation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Angiogenesis Potential of Human Limbal Stromal Niche Cells

doi: 10.1167/iovs.11-9414

Figure Lengend Snippet: Epithelial sphere growth in 3D Matrigel. LEPCs derived from dispase-isolated epithelial sheets alone or mixed with fluorescence prelabeled ( red ) HUVEC or P4/3D cells to generate sphere growth from Day 2 to Day 10 in 3D Matrigel ( A ). Compared with those formed by LEPC alone, spheres formed by LEPC+HUVEC and by LEPC+P4/3D expressed significantly more ΔNp63α, CK15, and CEBPδ transcripts ( B, n = 3, * P

Article Snippet: Single cells obtained from P4/3D aggregates or human umbilical vein endothelial cells (HUVEC) were prelabeled with red fluorescent nanocrystals (Qtracker cell labeling kits; Invitrogen, Carlsbad, CA), mixed with single cells derived from dispase-isolated limbal epithelial sheets at a ratio of 1:4, and seeded at the density of 5 × 104 per cm2 to generate sphere growth.

Techniques: Derivative Assay, Isolation, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Angiogenesis Potential of Human Limbal Stromal Niche Cells

doi: 10.1167/iovs.11-9414

Figure Lengend Snippet: Support of HUVEC vascular tube network. Fluorescence pre-labeled ( red ) HUVEC and P4/3D cells were seeded alone or together on the surface of 100% Matrigel in EGM2. Although vascular tube-like network was noted in all three conditions at Day 1 ( A–C ), such network in P4/3D cells ( A ) or HUVEC ( B ) alone was disintegrated by Day 2 ( E, F ). In contrast, the network formed by cocultured P4/3D cells and HUVEC ( C ) was maintained at Day 2 ( G ) and Day 5 (not shown). High magnification of insets ( C, G ) revealed close association between P4/3D cells and HUVEC ( red ) in the network at Day 1 ( D ) and Day 2 ( H ). Scale bar = 200 μm for A–C and E–G , and 50 μm for D and H .

Article Snippet: Single cells obtained from P4/3D aggregates or human umbilical vein endothelial cells (HUVEC) were prelabeled with red fluorescent nanocrystals (Qtracker cell labeling kits; Invitrogen, Carlsbad, CA), mixed with single cells derived from dispase-isolated limbal epithelial sheets at a ratio of 1:4, and seeded at the density of 5 × 104 per cm2 to generate sphere growth.

Techniques: Fluorescence, Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Angiogenesis Potential of Human Limbal Stromal Niche Cells

doi: 10.1167/iovs.11-9414

Figure Lengend Snippet: Differentiation into vascular endothelial cells. Single cells of P4/3D aggregates were cultured on plastic in EGM2 with VEGF for 3 days, yielding spindle cells similar to HUVEC. They uniformly expressed Flk-1, CD31, and von Willebrand factor, and took up Dil-Ac-LDL ( top ) in a similar fashion to the positive control of HUVEC cultured in the same condition ( bottom ). Nuclei were counterstained by Hoechst 33342 ( blue ). Scale bar = 100 μm.

Article Snippet: Single cells obtained from P4/3D aggregates or human umbilical vein endothelial cells (HUVEC) were prelabeled with red fluorescent nanocrystals (Qtracker cell labeling kits; Invitrogen, Carlsbad, CA), mixed with single cells derived from dispase-isolated limbal epithelial sheets at a ratio of 1:4, and seeded at the density of 5 × 104 per cm2 to generate sphere growth.

Techniques: Cell Culture, Positive Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Epidermal Growth Factor Reverses the Inhibitory Effects of the Bisphosphonate, Zoledronic Acid, on Human Oral Keratinocytes and Human Vascular Endothelial Cells In Vitro via the Epidermal Growth Factor Receptor (EGFR)/Akt/Phosphoinositide 3-Kinase (PI3K) Signaling Pathway

doi: 10.12659/MSM.911579

Figure Lengend Snippet: The effects of zoledronic acid (ZA) and epidermal growth factor (EGF) on the cell viability of human umbilical vein endothelial cells (HUVECs) and human oral keratinocytes (HOKs) measured by the cell counting kit-8 (CCK-8) assay. The results are presented as the average ± standard deviation (SD) of three independent experiments. a: Zoledronic acid treatment showed significant effects on human umbilical vein endothelial cells (HUVECs) after 48 h at the concentrations of 5, 50, and 100 μmol/L. Zoledronic acid significantly affected HOKs after 72 h at these concentrations (P

Article Snippet: Cell culture and treatment of human umbilical vein endothelial cells (HUVECs) and human oral keratinocytes (HOKs) and the four treatment groups Human umbilical vein endothelial cells (HUVECs) and human oral keratinocytes (HOKs) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GE Healthcare, Hyclone, South Logan, UT, USA) with 10% fetal bovine serum (FBS) (GE Healthcare, Hyclone, South Logan, UT, USA) and 1% penicillin and streptomycin (Sigma-Aldrich, St Louis, MO, USA) in a 37°C humidified atmosphere containing 95% air and 5% CO2 .

Techniques: Cell Counting, CCK-8 Assay, Standard Deviation

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Epidermal Growth Factor Reverses the Inhibitory Effects of the Bisphosphonate, Zoledronic Acid, on Human Oral Keratinocytes and Human Vascular Endothelial Cells In Vitro via the Epidermal Growth Factor Receptor (EGFR)/Akt/Phosphoinositide 3-Kinase (PI3K) Signaling Pathway

doi: 10.12659/MSM.911579

Figure Lengend Snippet: The effects of zoledronic acid on apoptosis in human umbilical vein endothelial cells (HUVECs) and human oral keratinocytes (HOKs) was measured by an Annexin-V conjugated to fluorescein isothiocyanate (FITC) apoptosis assay. The results are presented as the average ± standard deviation (SD) of three independent experiments. There was a significant difference between the control group and the human umbilical vein endothelial cells (HUVECs) treated with 100 μmol/L of zoledronic acid, and a significant difference between the control group and the human oral keratinocytes (HOKs) treated with 50 and 100 μmol/L of zoledronic acid (P

Article Snippet: Cell culture and treatment of human umbilical vein endothelial cells (HUVECs) and human oral keratinocytes (HOKs) and the four treatment groups Human umbilical vein endothelial cells (HUVECs) and human oral keratinocytes (HOKs) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GE Healthcare, Hyclone, South Logan, UT, USA) with 10% fetal bovine serum (FBS) (GE Healthcare, Hyclone, South Logan, UT, USA) and 1% penicillin and streptomycin (Sigma-Aldrich, St Louis, MO, USA) in a 37°C humidified atmosphere containing 95% air and 5% CO2 .

Techniques: Apoptosis Assay, Standard Deviation

Journal: Nature Communications

Article Title: The critical role of AMPK in driving Akt activation under stress, tumorigenesis and drug resistance

doi: 10.1038/s41467-018-07188-9

Figure Lengend Snippet: AMPK-Skp2-Akt axis is critical for survival under stress and glucose deprivation induced VEGF secretion and EGF-induced glycolysis and migration. a , c Cell survival analysis of MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown, AMPKα1 knockdown along with Myr-Akt, Skp2 WT, or Skp2 S256A or Skp2 S256D restoration under hypoxia (1% O 2 ) for 72 h. b , d Cell survival analysis of MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown, AMPKα1 knockdown along with Myr-Akt, Skp2 WT, or Skp2 S256A or Skp2 S256D restoration under glucose deprivation for 16 h. e MDA-MB-231 cells with control (shLuc), AMPKα1 knockdown and AMPKα1 knockdown with vector control, Skp2 WT and S256A or Skp2 S256D restoration were treated with a glucose-free medium for 8 h, and supernatant was collected for HUVEC tube formation assay. Each value represents the mean ± SEM in three independent experiments. ** P

Article Snippet: HEK293T, HEK293, MDA-MB-231, HUVEC cells were purchased from American Type Culture Collection.

Techniques: Migration, Multiple Displacement Amplification, Plasmid Preparation, HUVEC Tube Formation Assay

Journal: Journal of Bacteriology

Article Title: A Novel Protein, RafX, Is Important for Common Cell Wall Polysaccharide Biosynthesis in Streptococcus pneumoniae: Implications for Bacterial Virulence

doi: 10.1128/JB.01696-14

Figure Lengend Snippet: Decreased adherence of Δ rafX mutant R6 to MLE12 cells and HUVEC. Adherence of bacterial strains to MLE12 cells (A) or HUVEC (B). ΔEL4, H106A, H306A, and WL denote the Δ rafX mutant complemented with the RafX ΔEL4, RafX H106A,

Article Snippet: Briefly, MLE12 cells and human umbilical vein endothelial cells (HUVEC) (1 × 105 ; American Type Culture Collection) were incubated with 107 CFU of mid-exponential-phase bacteria, respectively, at 37°C for 1 h. After washes and lysis, the cell suspensions were properly diluted and plated onto blood agar plates to obtain colonies with easily recognizable sizes and the numbers of bacteria were determined.

Techniques: Mutagenesis

Journal:

Article Title: Inhibition of IFN-? signaling by a PKC- and protein tyrosine phosphatase SHP-2-dependent pathway

doi: 10.1073/pnas.0408854102

Figure Lengend Snippet: Inhibition of IFN-α-induced Jak–STAT signaling by FcγRs and R848 is mediated by PKC. ( A )FcγRs on primary human macrophages were crosslinked by using plate-bound IgG for 1 h with or without GF109203X pretreatment and stimulated

Article Snippet: GF109203X, LY 294002, SB203580, PD98059, and actinomycin D were purchased from Calbiochem.

Techniques: Inhibition

Journal: The Journal of Clinical Investigation

Article Title: microRNA-21-5p dysregulation in exosomes derived from heart failure patients impairs regenerative potential

doi: 10.1172/JCI123135

Figure Lengend Snippet: Effects of exosome treatment on cardiomyocytes, endothelial cells, and cardiac fibroblasts in vitro. ( A ) Representative fluorescent micrographs showing uptake of DiI-labeled NEXO and FEXO by NRCMs. Endocytosed exosomes (red) can be seen within the cytoplasm of cardiomyocytes (green). Scale bar: 10 μm. ( B ) Quantitation of exosomes uptake ( n = 10). Two-tailed t test. ( C ) NRCM proliferation in response to NEXO, FEXO, or PBS treatment. White arrows indicate Ki-67 + /α-SA + cells. Scale bar: 20 μm. ( D ) Quantitation of proliferating cardiomyocytes ( n = 6). ( E ) Apoptotic NRCMs in response to NEXO, FEXO, or PBS treatment. White arrows indicate TUNEL + /α-SA + cells. Scale bar: 20 μm. ( F ) Quantitation of apoptotic cardiomyocytes ( n = 6). ( G ) Measurement of tube formation in HUVECs co-cultured with NEXO, FEXO, or PBS. Scale bar: 100 μm. ( H ) Quantitation of average HUVEC tube length ( n = 20). ( I ) Neonatal rat fibroblasts underwent phenotypic transition to myofibroblasts in response to NEXO, FEXO, or PBS treatment. Scale bar: 20 μm. ( J ) Quantitation of myofibroblasts ( n = 12). * P

Article Snippet: HUVECs were co-incubated with 1.5 × 108 exosomes for 24 hours, then plated on growth factor–deprived Matrigel (356230, Corning) to evaluate angiogenesis ( ).

Techniques: In Vitro, Labeling, Quantitation Assay, Two Tailed Test, TUNEL Assay, Cell Culture

Journal: Oncotarget

Article Title: The protective role of sphingosine-1-phosphate against the action of the vascular disrupting agent combretastatin A-4 3-O-phosphate

doi: 10.18632/oncotarget.21172

Figure Lengend Snippet: CA4P treatment does not alter the quantity of VE-cadherin expressed on HUVEC cell surfaces FACS analysis of S1P/CA4P treated HUVEC labelled for VE-cadherin reveals that there is no statistical significance in level of expression of extracellular VE-cadherin on treated HUVEC monolayers (A) . Bars represent means ± SEM for 3 repeated experiments. Fluorescence immunohistochemistry using an antibody against VE-cadherin on treated HUVEC monolayers shows that extracellular VE-cadherin expression is ‘re-arranged’ rather than downregulated (B) . In untreated or S1P-treated cells, VE-cadherin expression is localized to the outer edges of the cells, whereas 10 min after CA4P treatment the VE-cadherin is rearranged into a zigzag pattern (arrowheads), an effect which is decreased by pretreatment with S1P. The change in expression is more prominent after 60 min CA4P treatment but again the effect is somewhat curtailed by S1P pre-treatment. Scalebars = 5 μm.

Article Snippet: Immunofluorescence and immunohistochemistry To stain VE-cadherin or tubulin on HUVEC, HUVEC (Promocell, Heidelberg, Germany) up to passage 5 were grown on gelatin coated 4-chamber slides (Lab-Tek, Nunc, Rochester, New York) to confluence.

Techniques: FACS, Expressing, Fluorescence, Immunohistochemistry

Journal: Oncotarget

Article Title: The protective role of sphingosine-1-phosphate against the action of the vascular disrupting agent combretastatin A-4 3-O-phosphate

doi: 10.18632/oncotarget.21172

Figure Lengend Snippet: CA4P disrupts N-cadherin adherens junctions Untreated co-cultured HUVEC and vSMC stained for CD31 (indicating endothelial cells) and N-cadherin (indicating EC/SMC junctions) display mostly intact CD31 and N-cadherin staining at intercellular junctions (A, C, E) , although some cytoplasmic N-cadherin is also evident. Staining for F-actin in (A) and (B) , indicated in red, reveals the cytoskeleton of the cells. (A) shows untreated co-cultures in monolayer stained for N-cadherin (green) and actin (red), whilst (B) shows CA4P treated cells. (C) shows a tubular structure, typically formed from around 40% of the co-cultured cells, with the rest forming flat monolayers as in (E); N-cadherin (red) and CD31 (green). CD31 staining was relatively unaffected by CA4P treatment (green staining in (D) and (F) versus (C) and (E)). However, N-cadherin staining appears considerably more intracellular and punctate following CA4P treatment (green staining in (B) versus (A) and red staining in (F) versus (E). The change in cellular distribution of N-cadherin following CA4P treatment was blocked by pre-treatment with 1μM S1P, allowing N-cadherin to retain its intercellular location, although its appearance was less smooth than in untreated cells (red staining in (G) versus (F)). Cells treated with S1P alone also retained intercellular location of N-cadherin, but with a less smooth appearance than in untreated cells (red staining in (H) versus (E)). Yellow in merged images indicates co-localisation at cell-cell junctions. Arrowheads = EC, asterisks = SMC (revealed by lack of CD31 staining). Scalebars = 15μm.

Article Snippet: Immunofluorescence and immunohistochemistry To stain VE-cadherin or tubulin on HUVEC, HUVEC (Promocell, Heidelberg, Germany) up to passage 5 were grown on gelatin coated 4-chamber slides (Lab-Tek, Nunc, Rochester, New York) to confluence.

Techniques: Cell Culture, Staining

Journal: Oncotarget

Article Title: The protective role of sphingosine-1-phosphate against the action of the vascular disrupting agent combretastatin A-4 3-O-phosphate

doi: 10.18632/oncotarget.21172

Figure Lengend Snippet: CA4P-induced tubulin depolymerisation as well as VE-cadherin redistribution is prevented by S1P HUVEC monolayers were treated with 1 μM CA4P for 4 (A, E) , 8 (B, F) , or 10 (C, G) minutes then fixed and stained for either tubulin (A-D) or VE-cadherin (E-H) . HUVEC were also pretreated with 1 μM S1P prior to CA4P treatment; (D,H) show tubulin and VE-cadherin staining following S1P pretreatment followed by 10 minutes CA4P treatment. Tubulin disruption and VE-cadherin rearrangement are both evident from around 8 minutes post CA4P treatment (arrowheads). Tubulin disruption is pronounced by 10 minutes after starting CA4P treatment ( * ) but tubulin is protected at this timepoint by pretreatment with S1P ( ** ). Scale bars = 5μm.

Article Snippet: Immunofluorescence and immunohistochemistry To stain VE-cadherin or tubulin on HUVEC, HUVEC (Promocell, Heidelberg, Germany) up to passage 5 were grown on gelatin coated 4-chamber slides (Lab-Tek, Nunc, Rochester, New York) to confluence.

Techniques: Staining

Journal: Science Advances

Article Title: Epac1 inhibition ameliorates pathological angiogenesis through coordinated activation of Notch and suppression of VEGF signaling

doi: 10.1126/sciadv.aay3566

Figure Lengend Snippet: Epac1 cross-talks with VEGF signaling and regulates EC functions. ( A and B ) Images of 3D angiogenesis assay of HUVECs, which were cultured alone but treated with vehicle or 2.5 μM ESI-09 (A), or transfected with control siRNA or Epac1 siRNA and then cocultured with fibroblasts (B) for 5 days. Red, isolectin B4 staining to highlight ECs. Scale bars, 50 μm. ( C and D ) 3D images of the same experiments shown in (A) and (B). ( E ) Activation of Epac1 by 007-AM (5 μM, 30 min) alone promoted phospho-Akt (S473) and phospho-eNOS (S1177) and synergized with VEGF (10 ng/ml, 30 min) in activating Akt and eNOS. Values are expressed as relative fold change in the format of means ± SEM ( n = 4 independent experiments). ** P

Article Snippet: For experiments involving RNA interference (RNAi), HUVECs at 70% confluence were transfected with Epac1-specific (1299001) or nontargeting control (12935-300; Thermo Fisher Scientific) Stealth RNAi siRNA oligonucleotides at a final concentration of 50 nM using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Angiogenesis Assay, Cell Culture, Transfection, Staining, Activation Assay

Journal: Science Advances

Article Title: Epac1 inhibition ameliorates pathological angiogenesis through coordinated activation of Notch and suppression of VEGF signaling

doi: 10.1126/sciadv.aay3566

Figure Lengend Snippet: Epac1 regulates Notch signaling in HUVECs. ( A ) Gene silencing of Epac1 by siRNA increased the mRNA expression of Notch target genes Hes1, Hey1, and DLL4. **** P

Article Snippet: For experiments involving RNA interference (RNAi), HUVECs at 70% confluence were transfected with Epac1-specific (1299001) or nontargeting control (12935-300; Thermo Fisher Scientific) Stealth RNAi siRNA oligonucleotides at a final concentration of 50 nM using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Expressing

Journal: Science Advances

Article Title: Epac1 inhibition ameliorates pathological angiogenesis through coordinated activation of Notch and suppression of VEGF signaling

doi: 10.1126/sciadv.aay3566

Figure Lengend Snippet: Epac1 interacts with γ-secretase and inhibits its cellular activity. ( A ) Coimmunoprecipitation of Epac1 and γ-secretase. Affinity pulldown of Epac1 using anti-FLAG M2 resin in HeLa cells ectopically expressing Epac1 -FLAG protein or control empty pOZ vector. Total cell lysates were probed by anti-Epac1 antibody, while immunoprecipitation (IP) was probed by an antibody specific for the catalytic subunit of γ-secretase, PS1. IB, immunoblotting. ( B ) Affinity pulldown of HUVEC or HeLa cell lysate by GST-Epac1 or GST control probed by an antibody specific for PS1. ( C and E ) Epac1 activation reduces γ-secretase activity for Notch cleavage. γ-Secretase activity was assayed using recombinant Notch substrate in HUVECs (C) and HeLa-Epac1 cells (E). The enzymatic activity was measured following the treatment of 007-AM (5 μM) at different time points. Data are represented as AlphaLISA signal in arbitrary units (A.U.) in the format of means ± SEM ( n = 4). Statistical analysis was done with one-way analysis of variance (ANOVA) compared to time point 0. * P

Article Snippet: For experiments involving RNA interference (RNAi), HUVECs at 70% confluence were transfected with Epac1-specific (1299001) or nontargeting control (12935-300; Thermo Fisher Scientific) Stealth RNAi siRNA oligonucleotides at a final concentration of 50 nM using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Activation Assay, Recombinant

Journal: British Journal of Pharmacology

Article Title: Serelaxin-mediated signal transduction in human vascular cells: bell-shaped concentration–response curves reflect differential coupling to G proteins

doi: 10.1111/bph.12964

Figure Lengend Snippet: The expression of RXFP1 and RXFP2 receptor mRNA and RXFP1 receptor protein in human primary umbilical vascular cells and human primary cardiac fibroblasts. qPCR (A) was utilized to show expression levels of RXFP1 and RXFP2 receptor mRNA in HUAECs, HUVECs, HUASMCs, HUVSMCs and HCFs relative to β-actin ( n = 2). RXFP2 receptor mRNA was only measureable in the positive control. Cell surface RXFP1 receptor protein expression was determined by radioligand binding (B) utilizing [ 125 I]-serelaxin and showed specific serelaxin binding in HEK-RXFP1 cells ( n = 6), HUASMCs ( n = 4), HUVECs ( n = 4), HUVSMCs ( n = 3) and HCFs ( n = 3), but not in HUAECs ( n = 2).

Article Snippet: Primary cultures of human umbilical artery endothelial cells (HUAECs), HUVECs, human umbilical artery smooth muscle cells (HUASMCs), human umbilical vein smooth muscle cells (HUVSMCs) and fetal human cardiac fibroblasts (HCFs: pooled from fetal atria and ventricles) were obtained from ScienCell Research Laboratories (San Diego, CA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Positive Control, Binding Assay

Journal: Molecular Vision

Article Title: The neuroprotective effect of resveratrol on retinal ganglion cells after optic nerve transection

doi:

Figure Lengend Snippet: Expression of sirtuin 1 in retinal ganglion cells three days after optic nerve transection in eyes treated with resveratrol injection ( A ) and in eyes treated with co-injections of sirtinol and resveratrol ( B ). The expression of sirtuin 1 (SIRT1) in retinal ganglion cells (RGCs) decreased in eyes treated with co-injections of sirtinol and resveratrol.

Article Snippet: Sirtinol (Sigma-Aldrich) was dissolved in PBS and diluted to a final concentration of 100 μM, which is a previously established effective concentration [ ].

Techniques: Expressing, Injection

Journal: Molecular Vision

Article Title: The neuroprotective effect of resveratrol on retinal ganglion cells after optic nerve transection

doi:

Figure Lengend Snippet: Neuroprotection of resveratrol blocked by sirtinol, a sirtuin 1 inhibitor. The numbers of retinal ganglion cells (RGCs) in eyes co-injected with resveratrol and sirtinol were smaller than injected with resveratrol at 1 week after treatment with concentrations of 9.4 uM and 31.3 uM of resveratrol (n≥5 for each group, *p

Article Snippet: Sirtinol (Sigma-Aldrich) was dissolved in PBS and diluted to a final concentration of 100 μM, which is a previously established effective concentration [ ].

Techniques: Injection

Journal: Molecular Vision

Article Title: The neuroprotective effect of resveratrol on retinal ganglion cells after optic nerve transection

doi:

Figure Lengend Snippet: Effect of resveratrol and sirtinol on the survival of RGC-5 cells in serum-free media were shown. The control group had cells cultured in 10% fetal bovine serum; the label S(-) indicated a group with cells cultured in serum-free media. The group with resveratrol addition showed more cell viability than the group with PBS addition in cells cultured in serum-free media. Coaddition of resveratrol and sirtinol resulted in lower cell viabilities than the group with resveratrol only at 24 h and 48 h after addition. ( G ; n ≥ 3 for each group, *p

Article Snippet: Sirtinol (Sigma-Aldrich) was dissolved in PBS and diluted to a final concentration of 100 μM, which is a previously established effective concentration [ ].

Techniques: Cell Culture

Journal: Molecular and Cellular Biology

Article Title: miR-31 Functions as a Negative Regulator of Lymphatic Vascular Lineage-Specific Differentiation In Vitro and Vascular Development In Vivo ▿ ▿ †

doi: 10.1128/MCB.00185-10

Figure Lengend Snippet: Direct posttranscriptional regulation of PROX1 by miR-31. (A) Schematic representation of the full-length PROX1 3′ UTR and the consecutive fragments present in the PROX1 3′ UTR-luciferase reporter constructs (PROX1 FL to F6). Candidate miR-31 binding sites identified using standard nucleic acid alignment techniques (gray) and TargetScan 5.0 (black) are indicated. The PROX1 -miR-31 base pairing is shown for the 7mer-m8 TargetScan-predicted binding site. (B to D) LECs or HUVECs were cotransfected with the plasmids containing a miR-31 binding site (miR31BS), the PROX1 3′ UTR (FL to F6), the PROX1 CDS, or PROX1 3′ UTR miR-31 binding site mutant forms (FL-mut or F2-mut) and the pre-miR-31 precursor, anti-miR-31 inhibitor, or negative-control molecules. (B) The activities of luciferase constructs containing miR31BS, PROX1 FL, and PROX1 F2 decreased significantly following miR-31 overexpression (pre-miR-31; n = 3) in LECs compared to those of negative controls (pre-miR-Neg; n = 3). (C) miR31BS and PROX1 FL reporter gene activities increased significantly following miR-31 inhibition (anti-miR-31; n = 3) in HUVECs compared to those of negative controls (anti-miR-Neg; n = 3). miR-31 loss of function also enhanced the activities of PROX1 F1 and PROX1 F2 constructs, but these differences were not statistically significant. (D) miR-31 mutant binding site full-length and F2 PROX1 3′ UTR (PROX1 FL-mut and PROX1 F2-mut) luciferase activities showed no major change following miR-31 overexpression (pre-miR-31, n = 3) or knockdown (anti-miR-31, n = 3). Data were normalized to firefly luciferase activities and are shown as mean relative abundances ± the standard errors. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: LECs and HUVECs were purchased from Cambrex (Verviers, Belgium).

Techniques: Luciferase, Construct, Binding Assay, Mutagenesis, Negative Control, Over Expression, Inhibition

Journal: Scientific Reports

Article Title: Akt2 knock-down reveals its contribution to human lung cancer cell proliferation, growth, motility, invasion and endothelial cell tube formation

doi: 10.1038/srep12759

Figure Lengend Snippet: Analysis of selective Akt1 and AKt2 silencing in LNM35 cells. ( A ) Western blot analysis of the three Akt isoform in the human lung cancer cells LNM35 and A549, breast cancer cells MDA-MB-231 and MCF7, colon cancer cells HT29 and the endothelial cells HUVECs. ( B and C ) Akt1 and Akt2 protein expression in LNM35 cells transiently transfected with siRNA targeting Akt1 and Akt2 transcript ( + ) respectively or with its siControl oligonucleotide ( − ). Western blot analysis was performed at days 2, 3 and 5 following the transfection. D and E ) Akt1 and Akt2 protein expression in LNM35 cells stably transduced with control-shRNA and two different designs of Akt1 and AKT2 shRNA (shRNA1 and shRNA2). Data are representative of three independent experiments.

Article Snippet: The HUVEC cells transduced with Akt1-shRNAs (Akt1-shRNA1 and Akt1-shRNA2), Akt2-shRNAs (Akt2-shRNA1 and Akt2-shRNA2) or control sequences (Control-shRNA) were plated at 40,000 cells per well and incubated for 8 h at 37 °C in 0.1 ml of EndoGROTM-MV-VEGF Complete Media Kit (Millipore, Temecula, CA, USA).

Techniques: Western Blot, Multiple Displacement Amplification, Expressing, Transfection, Stable Transfection, Transduction, shRNA

Journal: Scientific Reports

Article Title: Akt2 knock-down reveals its contribution to human lung cancer cell proliferation, growth, motility, invasion and endothelial cell tube formation

doi: 10.1038/srep12759

Figure Lengend Snippet: Role of Akt1 and −2 in angiogenesis. ( A ) Formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs) transduced with control-shRNA, two different design of AKT1 shRNA (Akt1-shRNA1 and Akt1-shRNA2) and two different design of AKT2 shRNA (Akt2-shRNA1 and Akt2-shRNA2) and cultured on Matrigel matrix in 96-well plates. ( B ) Quantification of tubular morphogenesis induced in HUVEC cells. Tube formation was determined by the length of tube-like structures containing connected cells. ( C ) Viable cells were assayed as described in Materials and Methods. Data are mean ± S.E.M. from three separate experiments. The “ns” indicate no statistical differences. Statistical differences obtained at *P

Article Snippet: The HUVEC cells transduced with Akt1-shRNAs (Akt1-shRNA1 and Akt1-shRNA2), Akt2-shRNAs (Akt2-shRNA1 and Akt2-shRNA2) or control sequences (Control-shRNA) were plated at 40,000 cells per well and incubated for 8 h at 37 °C in 0.1 ml of EndoGROTM-MV-VEGF Complete Media Kit (Millipore, Temecula, CA, USA).

Techniques: Transduction, shRNA, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Endothelial AMP-Activated Kinase α1 Phosphorylates eNOS on Thr495 and Decreases Endothelial NO Formation

doi: 10.3390/ijms19092753

Figure Lengend Snippet: Endothelial nitric-oxide synthase (eNOS) is a substrate of AMPK in vitro. ( A ) Wild-type eNOS, as well as Thr495 and Ser1177 mutants, was overexpressed in HEK293 cells, then immunoprecipitated and used as substrate for AMPKα1 in in vitro kinase assays. The upper panel shows the autoradiograph of eNOS proteins. The lower panel shows the Western blot of the immunoprecipitated (IP) eNOS protein used as input. The graph summarizes the data from four independent experiments. ( B ) Wild-type eNOS (Flag-tagged) overexpressed in human umbilical vein endothelial cells was immunoprecipitated and used as a substrate for AMPKα1. Phosphorylation was assessed using specific antibodies for phosphorylated Ser1177 (p-Ser1177) and Thr945 eNOS (p-Thr495). The graph summarizes the data from five independent kinase reactions. * p

Article Snippet: For lentiviral and adenoviral transduction, human umbilical vein endothelial cells (Promocell, Heidelberg, Germany) were used and cultured up to passage 8 in endothelial growth medium 2 (Promocell, Heidelberg, Germany).

Techniques: In Vitro, Immunoprecipitation, Autoradiography, Western Blot

Journal: Contrast Media & Molecular Imaging

Article Title: Iso-Osmolar Iodixanol Induces Less Increase in Circulating Endothelial Microparticles In Vivo and Less Endothelial Apoptosis In Vitro Compared with Low-Osmolar Iohexol

doi: 10.1155/2018/8303609

Figure Lengend Snippet: The effects of contrast media on apoptosis-related proteins in HUVECs. HUVECs were treated with 20 vol% of iodixanol or iohexol for 4 h and expression of Bcl-2 (a), Bax (b), caspase 3, and cleaved caspase-3 (c) was measured by Western blotting. These experiments were repeated at least 3 times. Data represents mean ± SD. Complete growth medium served as control. ∗ P

Article Snippet: Hoechst Staining To further demonstrate the apoptosis of HUVECs, after treatment with 20 vol% of iodixanol or iohexol for 4 h, the cells were stained with 0.5 mL of Hoechst-33258 (Beyotime Biotechnology, China) for 5 min at room temperature in the dark.

Techniques: Expressing, Western Blot

Journal: Contrast Media & Molecular Imaging

Article Title: Iso-Osmolar Iodixanol Induces Less Increase in Circulating Endothelial Microparticles In Vivo and Less Endothelial Apoptosis In Vitro Compared with Low-Osmolar Iohexol

doi: 10.1155/2018/8303609

Figure Lengend Snippet: The induction of EMP release from HUVECs by iodixanol or iohexol. HUVECs were treated with different concentrations of iodixanol or iohexol for 4 h and CD31 + /CD41a − EMPs (a) and CD62E + EMPs (b) were measured by flow cytometry. The data were expressed as mean ± SD ( n = 9). Complete growth medium served as control. ∗∗∗ P

Article Snippet: Hoechst Staining To further demonstrate the apoptosis of HUVECs, after treatment with 20 vol% of iodixanol or iohexol for 4 h, the cells were stained with 0.5 mL of Hoechst-33258 (Beyotime Biotechnology, China) for 5 min at room temperature in the dark.

Techniques: Flow Cytometry, Cytometry

Journal: Contrast Media & Molecular Imaging

Article Title: Iso-Osmolar Iodixanol Induces Less Increase in Circulating Endothelial Microparticles In Vivo and Less Endothelial Apoptosis In Vitro Compared with Low-Osmolar Iohexol

doi: 10.1155/2018/8303609

Figure Lengend Snippet: The effects of iodixanol and iohexol on viability and apoptosis of HUVECs. (a) HUVECs were stimulated with iodixanol or iohexol at different concentrations and time points and then cell proliferation/cytotoxicity assay was performed using CCK-8 assay. (b) HUVECs were treated with iodixanol or iohexol at different concentrations for 4 h and cell apoptosis was determined by Annexin V and PI staining. (c) HUVECs were treated with 20 vol% of iodixanol or iohexol for 4 h and stained with Hoechst. Reference bar size in 20 μ m. Magnification × 400 folds. The data were expressed as mean ± SD ( n = 9). Complete growth medium served as control. ∗ P

Article Snippet: Hoechst Staining To further demonstrate the apoptosis of HUVECs, after treatment with 20 vol% of iodixanol or iohexol for 4 h, the cells were stained with 0.5 mL of Hoechst-33258 (Beyotime Biotechnology, China) for 5 min at room temperature in the dark.

Techniques: Cytotoxicity Assay, CCK-8 Assay, Staining

Journal: Journal of Cell Science

Article Title: Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells

doi: 10.1242/jcs.187864

Figure Lengend Snippet: Localisation of PI4P, and RNAi of PI4K2A and PI4K2B in endothelial cells. (A) Representative confocal image of a HUVEC transfected with the PI4P probe GFP-SidC, fixed, permeabilised and labelled with DAPI nuclear stain (blue), anti-TGN46 (red), anti-GFP (green) and anti-VWF (cyan). Scale bar: 10 µm. HUVEC were transfected with vehicle (Mock), or siRNA against PI4K2A, PI4K2B or both PI4K2A and PI4K2B (PI4K2A B). (B,C) The efficiency of knockdown was assayed by detecting protein levels (western blotting, B) or mRNA transcript levels (qRT-PCR; C). Means±s.e.m. of six experiments are shown in C.

Article Snippet: VWF string assay Nucleofected HUVECs were seeded onto gelatin-coated µ-slides VI0.4 (ibidi, Munich, Germany).

Techniques: Transfection, Staining, Western Blot, Quantitative RT-PCR

Journal: Journal of Cell Science

Article Title: Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells

doi: 10.1242/jcs.187864

Figure Lengend Snippet: Morphometric analysis of VWF-positive structures in PI4KII-depleted cells. (A–C) The morphology of WPBs in HUVECs transfected with vehicle (Mock) or siRNA against PI4K2A, PI4K2B or both PI4K2A and PI4K2B (PI4K2A B), was analysed by using an unbiased high-throughput method. The Feret diameter (A, B) and maximum fluorescence intensity (C-E) of VWF-positive objects in confocal images of HUVECs labelled with anti-VWF was measured. Graphs are representative of at least three determinations. In A, a cumulative frequency distribution representative of three determinations is shown. In each case, the results for the siRNA-treated groups were considered significant by Wilcoxon rank-sum test ( P

Article Snippet: VWF string assay Nucleofected HUVECs were seeded onto gelatin-coated µ-slides VI0.4 (ibidi, Munich, Germany).

Techniques: Transfection, High Throughput Screening Assay, Fluorescence

Journal: Journal of Cell Science

Article Title: Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells

doi: 10.1242/jcs.187864

Figure Lengend Snippet: Effect of PI4KII-kinase depletion on secreted VWF protein. (A–C) HUVECs treated with vehicle (Mock) or siRNA against PI4K2A, PI4K2B or both transcripts were either not stimulated (A), stimulated with PMA (B) or with histamine (C). Secreted VWF (A–C) and total VWF levels (D) were measured by ELISA and were typically 3% (±0.6%) (unstimulated), 10% (±1.2%) (histamine) and 24% (±2.9%) (PMA) of total VWF content. Here secreted VWF is presented as a percentage of mock values. Means±s.e.m. of three to six experiments are shown and were not significantly different (analysed using one-way ANOVA). (E) Secreted VWF from unstimulated (60 min) or histamine-stimulated (30 min) HUVECs was analysed for VWF multimer distribution on a non-reducing multimer gel. No differences were observed between samples.

Article Snippet: VWF string assay Nucleofected HUVECs were seeded onto gelatin-coated µ-slides VI0.4 (ibidi, Munich, Germany).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Cell Science

Article Title: Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells

doi: 10.1242/jcs.187864

Figure Lengend Snippet: Effect of PI4KII-kinase depletion on VWF string formation in vitro and bleeding time in vivo . (A) HUVECs treated with vehicle (Mock) or siRNA against PI4K2A, PI4K2B or both transcripts (PI4K2A B) were stimulated with histamine under flow to release VWF strings, then fixed under flow and their surface was labelled with anti-VWF for imaging. Scale bar: 50μm. (B,C) String number per cell (B) and string length (C), were then measured, with whisker plot showing mean, maximum and minimum of more than four experiments. Data were analysed by one-way ANOVA ( P

Article Snippet: VWF string assay Nucleofected HUVECs were seeded onto gelatin-coated µ-slides VI0.4 (ibidi, Munich, Germany).

Techniques: In Vitro, In Vivo, Flow Cytometry, Imaging, Whisker Assay

Journal: Journal of Cell Science

Article Title: Type II PI4-kinases control Weibel-Palade body biogenesis and von Willebrand factor structure in human endothelial cells

doi: 10.1242/jcs.187864

Figure Lengend Snippet: Examination of VWF-positive objects after treatment with monensin. HUVECs were transfected with vehicle (Mock) or siRNA against PI4K2A, PI4K2B or both transcripts, and treated with vehicle or monensin. (A) Opera acquired confocal sections showing VWF immunolabelling. Scale bar: 20 µm. (B) Cumulative frequency distributions of the Feret diameter of VWF-positive structures in cells treated with vehicle (line) or monensin (dotted line), together with results of Wilcoxon rank-sum test ( P value) and Kulback–Leibler distance (KLD). Representative of three determinations. (C) Data from B displayed as a percentage of VWF-positive objects with a Feret diameter > 2.35 µm (Ci) or the difference in mean Feret diameter between vehicle and monensin-treated samples (Cii). Means±95% confidence interval of eight replicate wells are shown. Data shown in Cii were analysed using one-way ANOVA with Dunnett's multiple comparison test compared to the mock sample. ns, non-significant; ** P

Article Snippet: VWF string assay Nucleofected HUVECs were seeded onto gelatin-coated µ-slides VI0.4 (ibidi, Munich, Germany).

Techniques: Transfection

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

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: 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

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: Experimental and Therapeutic Medicine

Article Title: Fenofibrate inhibits the expression of VEGFC and VEGFR-3 in retinal pigmental epithelial cells exposed to hypoxia

doi: 10.3892/etm.2015.2697

Figure Lengend Snippet: Analysis of the migration of human umbilical vein endothelial cells (HUVECs) by scratch-wound assay. (A) Migration of HUVECs cultured for 24 h in the supernatant of control retinal pigment epithelial cells (RPE cells) (left panel) and in the supernatant of RPE cells epxosed to hypoxia (right panel) (x200 magnification). (B) HUVEC migration rate. The scratch width of each group was 200 µm, and the cell migration distance of the cells between the scratch edges was observed after 24 h. Results shown are the means ± SD; n=8. Compared with the control group, cutlure with the supernatant from hypoxic RPE cells significantly increased the HUVEC migration rate. *p

Article Snippet: Cell culture RPE cells (CRL-2302) were obtained from ATCC (Manassas, VA, USA) and human umbilical vein endothelial cells (HUVECs) were from ScienCell.

Techniques: Migration, Scratch Wound Assay Assay, Cell Culture

Journal: Experimental and Therapeutic Medicine

Article Title: Fenofibrate inhibits the expression of VEGFC and VEGFR-3 in retinal pigmental epithelial cells exposed to hypoxia

doi: 10.3892/etm.2015.2697

Figure Lengend Snippet: Effects of culture with the culture supernatant of retinal pigment epithelial cells (RPE cells) on the proliferation of human umbilical vein endothelial cells (HUVECs). By MTT assay, we detected the effects of RPE cell culture medium on the proliferation activity of HUVECs. Control group: HUVECs in the control group were cultured with Dulbeccos modified Eagles medium (DMEM) containing 1% fetal bovine serum (FBS). HUVECs cultured with RPE cell medium: HUVECs were cultured in the culture supernatant of RPE cells exposed to hypoxia. Results shown are the means ± SD; n=8. Compared with the normal control group, culture with the supernatant of RPE cells exposed to hypoxia significantly increased the proliferation of the HUVECs.

Article Snippet: Cell culture RPE cells (CRL-2302) were obtained from ATCC (Manassas, VA, USA) and human umbilical vein endothelial cells (HUVECs) were from ScienCell.

Techniques: MTT Assay, Cell Culture, Activity Assay, Modification

Journal: Experimental and Therapeutic Medicine

Article Title: Fenofibrate inhibits the expression of VEGFC and VEGFR-3 in retinal pigmental epithelial cells exposed to hypoxia

doi: 10.3892/etm.2015.2697

Figure Lengend Snippet: Effects of culture with the supernatant of retinal pigment epithelial cells (RPE cells) on angiogenic activity of human umbilical vein endothelial cells (HUVECs). Cultured HUVEC cells were divided into 2 groups and plated in Matrigel. The control group was treated with Dulbecco's modified Eagle's medium (DMEM) containing 1% fetal bovine serum (FBS). The experimental group was treated with the supernatant of RPE cells exposed to hypoxia. (A) Control group (left panel) and the experimental group (right panel) after 48 h of incubation (x50 magnification). (B) Area covered by cellular extensions linking HUVEC masses and branch points. These calculations were performed on images of standardized fields from at least 5 wells/experimental condition. Results are the means ± SD, with 8 samples in each group. Compared with the control group, culture with the supernatant of RPE cells exposed to hypoxia significantly increased the tube formation ability of the HUVECs. *p

Article Snippet: Cell culture RPE cells (CRL-2302) were obtained from ATCC (Manassas, VA, USA) and human umbilical vein endothelial cells (HUVECs) were from ScienCell.

Techniques: Activity Assay, Cell Culture, Modification, Incubation