Journal: American Journal of Physiology - Cell Physiology

Article Title: Shear stress induces Gα q/11 activation independently of G protein-coupled receptor activation in endothelial cells

doi: 10.1152/ajpcell.00148.2016

Figure Lengend Snippet: Ligand- and shear stress-induced activation of the sphingosine-1-phosphate (S1P3)-Gαq/11 complex. In situ proximity ligation assay (PLA) was performed using antibodies directed against S1P3 and Gαq/11 (A and B), serotonin (5-HT)2A and Gαq/11 (C and D), S1P3 and G protein-coupled receptor kinases (GRK2; E), and S1P3 and β-arrestin-1/2 (F) on human coronary artery endothelial cells (HCAECs) treated with vehicle alone (Control), S1P; 2 μM for 30 s) (A, E, and F), or 5-HT (100 nM for 30 s) (C) and on HCAECs that were either unstimulated (Sham) or subjected to a step change in shear stress (Flow) for the indicated times (B, D, E, and F). Representative confocal images in a single z-plane of cells probed with the S1P3/Gαq/11 antibody pair are shown (A and B). Scale bar, 20 μm. Each bar graph shows quantification of at least 3 independent experiments as PLA signal (red dots) per cell (blue nuclei) relative to the control condition, with error bars indicating SE; n = 6 for C, n = 5 for A, B, E (Flow), and F (Flow), n = 3 for D, E (S1P), and F (S1P). *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Human coronary artery endothelial cells (HCAECs) from male donors were obtained from either Cell Applications (San Diego, CA) or Lonza (Walkersville, MD) and maintained in complete endothelial growth medium (EGM-2; Lonza) supplemented with 10% heat-inactivated FBS and penicillin-streptomycin.

Techniques: Activation Assay, In Situ, Proximity Ligation Assay

Journal: bioRxiv

Article Title: miR-10b Deficiency Affords Atherosclerosis Resistance

doi: 10.1101/248641

Figure Lengend Snippet: A. Nucleotide sequences of miR-10b and those of its target in 3’ UTR in LTBP1. B. Western blotting for LTBP1 and β-tubulin (TUBB) using various human ECs that showed Type-I phenotypes (HUVEC, HAEC, HCAEC, HMVEC and ESdEC[P6]) and those with Type-II phenotypes (iPS(BJ)EC, iPS(HU)EC, ESdEC[P0] and ESdEC[P1]) as reported previously 1 . C . Type-II ECs that were transfected with an empty vector (CMV-vector (+), mock) or an miR-10b expression vector (CMV-vector (+), miR-10b), and Type-II ECs without transfection (CMV-vector (-)) were subjected to Western blotting for using an anti-LTBP1 body or an anti-β-tubulin (TUBB) antibody. D . Western blotting for LTBP1 and β-tubulin (TUBB) proteins using Type-I ECs that were transfected with a control HIV vector (control) or an miR-10b inhibitor-expressing HIV vector (miR-10bi) together with Western blotting using Type-II ECs without transfection were shown as indicated. E. Proliferation indexes of SMCs that were co-cultured with Type-II ECs in the absence or the presence of increasing concentrations of LY2157299, a TGF-β signaling inhibitor, as indicated. N=3. F and G . SMCs were co-cultured with Type-I or Type-II ECs and the percentages of phosphorylated SMAD2/3-positive cells in were calculated by flow cytometry (F) and nuclear localization of SMAD3 in PKH-26 (red)-stained SMCs was estimated by immunostaining studies with an anti-smad3 antibody (green) along with nuclear counterstaining with DAPI (blue) (G). Full-length blots are presented in Supplementary information. Abbreviations: ESdEC[P6], human ES cell-derived ECs at passage 6; ESdEC[P0], human ES cell-derived ECs at passage 0; ESdEC[P1], human ES cell-derived ECs at passage 1.

Article Snippet: Human umbilical vein endothelial cells (HUVEC), human aortic endothelial cells (HAEC), human microvascular endothelial cells (HMVEC) and human coronary arterial endothelial cells (HCAEC) were purchased from Dainippon Sumitomo Pharma Co., Ltd. (Osaka Japan).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Cell Culture, Flow Cytometry, Staining, Immunostaining, Derivative Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling reduced TNF-α-induced monocyte-adhesion. Cultured endothelial cells were stimulated with 10 ng/mL recombinant human tumor necrosis factor-α (TNF-α) and supplemented with either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) for 18 h. Calcein-labeled THP-1 cells were allowed to adhere to human umbilical vein endothelial cells (HUVECs) (A) or HCAECs (B) for 30 min, then adherent cells were quantified and expressed as a fold change of control ( n = 5 each). In HUVECs, VCAM-1 (C) , and ICAM-1 (D) from whole cell lysates were quantified by Western blotting, normalized to stain-free loading controls and expressed as a fold change of TNF-α ( n = 6 and 4, respectively). Representative Western blots shown. In HCAECs, VCAM-1 (E) , and ICAM-1 (F) from whole cell lysates were quantified by Western blotting, normalized to stain-free loading controls and expressed as a fold change of TNF-α ( n = 3 and 5, respectively). Representative Western blots and stain-free loading controls are shown. *Indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS denotes not significant.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Cell Culture, Recombinant, Control, Labeling, Western Blot, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling restored barrier function in TNF-α-stimulated endothelial cells. Cultured endothelial cells were stimulated with 10 ng/mL recombinant human TNF-α in the presence of either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) for 18 h. Human umbilical vein endothelial cells (HUVECs) (A) or HCAECs (B) were seeded in Transwell inserts and streptavidin-HRP leakage across the endothelial monolayers was quantified. Data are expressed as a fold change of control ( n = 6 and 3, respectively) (C) . In HUVECs, following immunofluorescence for phospho-paxillin (Tyr118), total phospho-paxillin (Tyr118) levels were quantified using a Fiji-based macro, and normalized to cell count ( n = 6). Representative images of HUVECs immunostained (green) for VE-cadherin (D) , ZO-1 (E) or phospho-paxillin (Tyr118) (F) . Nuclei were stained with DAPI (blue). Scale bars represent 10 μm and apply to all panels. *Indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS denotes not significant.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Cell Culture, Recombinant, Control, Immunofluorescence, Cell Counting, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Inhibition of Wnt/β-catenin signaling enhanced platelet binding to TNF-α-stimulated endothelial cells. Cells were treated with either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) in the presence or absence of 10 ng/mL recombinant human TNF-α stimulus for 18 h. BCECF-AM-labeled, thrombin-activated platelets were co-cultured with human umbilical vein endothelial cells (HUVECs) (A) and HCAECs (B) for 10 min, bound platelets lysed, and the fluorescent signal quantified. Data are expressed as optical density ( n = 8 and 4, respectively). (C) Representative Western blots for integrins α v and β 3 in whole cell lysates, and vWF and ADAMTS13 in conditioned media, and stain-free controls in HUVECs. (D) Segments of human saphenous vein were co-cultured with BCECF-AM-labeled, thrombin-activated platelets for 10 min, and the number of bound platelets quantified ( n = 4). (E) In HUVECs, integrin α v from whole cell lysates was quantified by Western blotting, normalized to stain-free controls and expressed as a fold change from control ( n = 4). Quantification and representative images of immunofluorescence (green) for vWF in permeabilised (F,G) and non-permeabilised (H,I) HUVECs, to detect intracellular and membrane-tethered vWF, respectively ( n = 5 and 4, respectively). Nuclei were stained with DAPI (blue). Scale bars represent 10 μm and apply to all panels. From cultured HUVECs, ADAMTS13 (J) , and soluble vWF (K) in conditioned culture medium were quantified by Western blotting, normalized to stain-free controls and expressed as a fold change from control ( n = 7 and 3, respectively). *Indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS denotes not significant.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Binding Assay, Control, Recombinant, Labeling, Cell Culture, Western Blot, Staining, Immunofluorescence, Membrane

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Effects of inhibition of Wnt/β-catenin signaling on wound healing, apoptosis and proliferation in cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with either 0.05% DMSO vehicle control or 25 μM inhibitor of β-catenin-responsive transcription (iCRT) in the presence or absence of 10 ng/mL recombinant human tumor necrosis factor-α (TNF-α) stimulus for 18 h. (A) HUVECs were subjected to scratch wounding and regrowth was quantified; data is expressed in μm ( n = 4 each). (B) HUVECs were subjected to immunofluorescence for cleaved caspase-3, and apoptosis quantified and expressed as the percentage of cleaved caspase-3-positive cells ( n = 3). (C) HUVECs were subjected to fluorescent labeling of incorporated EdU, and proliferation quantified and expressed as the percentage of EdU-positive cells ( n = 4). (D) Representative images of scratch wound assay performed on HUVECs. Dashed line indicates wound edge. Scale bar represents 500 μm. (E) Representative images of HUVECs immunostained (green) for cleaved caspase-3. White arrowhead indicates cleaved caspase-3-positive cell. Nuclei were stained with DAPI (blue). Scale bar represents 10 μm and applies to all panels. *Indicates p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. NS denotes not significant.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Cell Culture, Control, Recombinant, Immunofluorescence, Labeling, Scratch Wound Assay Assay, Staining

Journal: Frontiers in Cardiovascular Medicine

Article Title: Pro-inflammatory role of Wnt/β-catenin signaling in endothelial dysfunction

doi: 10.3389/fcvm.2022.1059124

Figure Lengend Snippet: Schematic diagram illustrating effect of inhibition of Wnt/β-catenin signaling on endothelial-platelet interaction in unchallenged and TNF-α-challenged cultured endothelial cells. Endothelial cells were treated with either 0.05% DMSO vehicle control or 25 μM iCRT in the presence or absence of 10 ng/mL recombinant human TNF-α stimulus for 18 h, then co-cultured with thrombin-activated platelets for 10 min. (A) Control: Unchallenged endothelial cells treated with 0.05% DMSO vehicle control. UL-vWF multimers remain stored in the Weibel-Palade bodies. (B) Control + iCRT-14: Unchallenged endothelial cells treated with 25 μM iCRT-14. UL-vWF multimers remain stored in the Weibel-Palade bodies. (C) TNF-α: TNF-α-challenged endothelial cells treated with 0.05% DMSO vehicle control. TNF-α stimulates release of UL-vWF from Weibel–Palade bodies. TNF-α-driven up-regulation in ADAMTS13 results in proteolytic cleavage of membrane-tethered UL-vWF; hence, TNF-α-stimulation does not promote endothelial-platelet interaction. (D) TNF-α + iCRT-14: TNF-α-challenged endothelial cells treated with 25 μM iCRT-14. TNF-α stimulates release of UL-vWF from Weibel–Palade bodies. Treatment with iCRT-14 blocks TNF-a-mediated up-regulation of ADAMTS13 thereby maintaining high levels of membrane-tethered UL-vWF; hence, platelet recruitment is enhanced. Acronyms: ADAMTS13 - a disintegrin-like and metalloprotease with thrombospondin type-1 repeats-13; TNF-α - tumor necrosis factor-a; UL-vWF - ultra-large von Willebrand factor.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, pooled from up to four different donors per lot) and human coronary artery endothelial cells (HCAECs, single donor per lot) from different donors were purchased from Promocell (C-12203 and C-12221), and were utilized between passages 2 and 6.

Techniques: Inhibition, Cell Culture, Control, Recombinant, Membrane

Journal: Journal of Translational Medicine

Article Title: Quinic acid regulated TMA/TMAO-related lipid metabolism and vascular endothelial function through gut microbiota to inhibit atherosclerotic

doi: 10.1186/s12967-024-05120-y

Figure Lengend Snippet: QA improved TMAO-induced inflammatory lesions and endothelial dysfunction in HCAECs. (A) CCK-8 was applied to detect the toxicity of QA on HCAECs. (B) CCK-8 was used to detect HCAECs proliferation. (C) The expression of COX-2, IL-6, E-selectin, ICAM-1, HMGB1 was detected by RT-qPCR. (D) The expression of p-P65, p-MAPK14 protein was detected by western blot. (E) HMGB1 levels were detected by ELISA. (F) The expression of ZO-2, VE-Cadherin and Occludin were detected by western blot. * P < 0.05 vs. Control, # P < 0.05 vs. TMAO

Article Snippet: To investigate the cytotoxicity of QA, human coronary artery endothelial cells (HCAECs, HUM-iCell-c006, iCell) were treated with 1, 2.5, 5, 10 and 20 μM QA.

Techniques: CCK-8 Assay, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Time dependence of the absolute cell numbers of HAAF (A) , HASMC (B) , and HCAEC (C) cells cultured on SilkGraft and on polystyrene. Absolute cell numbers were lower on SilkGraft than on polystyrene because the available surface area was reduced due to the use of the steel ring which kept the silk substrate under water. Total cell growth differences between cells cultured on SilkGraft or on polystyrene are expressed by the areas under the corresponding curves, the statistical levels of significance of which are: for HAAFs, P < 0.001; for HASMCs, P < 0.01; and for HCAECs, P < 0.001.

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Living cells density/mm 2 of apparent surface area after 21 days of in vitro culture.

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: In Vitro

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Cumulative consumption of glucose and glutamine and release of lactate. Results were normalized per 10 3 cells. (A–C) The cumulative glucose consumption was higher for HAAFs ( P < 0.05) and HCAECs ( P < 0.001) seeded on SilkGraft, whereas it showed only marginal differences between the two substrates for HASMCs ( P > 0.05). (D–F) Glutamine consumption was lower for HAAFs ( P < 0.05) seeded on SilkGraft, similar for HASMCs ( P > 0.05) cultured on the two substrates, and significantly larger for HCAECs ( P < 0.001) grown on the silk substrate. (G,H) The cumulative amount of lactate released by HAAFs and HASMCs was the same whichever the substrate ( P > 0.05). Lactate release could not be assessed for HCAECs because the released lactate was re-uptaken and used for metabolic purposes. The statistical analysis of these data is shown in .

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Comparison of metabolic parameters ^* of the different cell types cultured on SilkGraft and polystyrene.

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Comparison, Cell Culture

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests

doi: 10.3389/fbioe.2019.00356

Figure Lengend Snippet: Relevant cytokines and chemokines secreted by each cell type cultured between 18 and 20 days on SilkGraft and polystyrene: HAFFs (A) , HCAECs (B) , and HASMCs (C) . Results of immunofluorescence intensities were normalized to 10 3 cells. IL-6: Interleukin-6; MCP-1: Monocyte chemoattractant protein-1; TIMP-2: Tissue inhibitor of metal proteinases-2; IP-10: Interferon gamma-induced protein-10; MCP-2: Monocyte chemoattractant protein-2; Eotaxin-1; RANTES: Regulated on activation normal T cell expressed and secreted; MIP-1β: Macrophage inflammatory protein-1β; TNF-β: Tumor necrosis factor-β; GM-CSF: Granulocyte-macrophage colony stimulating factor; IL-1α: Interleukin-1α; IL-1β: Interleukin-1β; ICAM-1: Intercellular adhesion molecule-1. The bars are the mean values of three independent experiments corrected for cell numbers. * P < 0.01; ** P < 0.001. SEMs, not shown, ranged between 5 and 10% of corresponding mean values.

Article Snippet: Adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) were provided by ScienCell Research Laboratories (Carlsbad, CA, USA).

Techniques: Cell Culture, Immunofluorescence, Activation Assay