Journal:

Article Title: The orphan nuclear receptor ROR? is a negative regulator of the inflammatory response

doi: 10.1093/embo-reports/kve007

Figure Lengend Snippet: Fig. 1. RORα is expressed in different vascular SMC types. (A and B) RT–PCR (35 cycles) analysis of RORα and GAPDH mRNA. C-PCR and C-RT are negative controls for PCR and RT, respectively; VSMC, smooth muscle cells from saphenous veins; CASMC, human coronary artery smooth muscle cells; HASMC, human aortic smooth muscle cells. (C) RT–PCR (30 cycles) analysis of RORα mRNA in SMC infected with Ad-RORα1 or Ad-GFP for 24 h. (D) Analysis of RORα protein expression in SMC infected for 24 h with or without Ad-RORα1. Immunocytochemistry experiments were performed as previously described (Chinetti et al., 1998) using a rabbit polyclonal RORα antibody raised against aa 163–225.

Article Snippet: Primary human aortic (HA) and coronary artery (CA) SMC (PromoCell, Heidelberg, Germany) and primary SMC from saphenous veins (VSMC: a kind gift of Dr Walsh, Boston, MA) were cultured under standard conditions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Expressing, Immunocytochemistry

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: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Effects of inhibition of AGEs on coronary artery tensions and BK channel densities and protein expression (a) Representative tracings for 60 mmol/L KCl and 100 nmol/L IBTX induced vascular tension alterations of coronary arterial rings from C+V, DM+V, C+A and DM+A groups. (b) Graph data showing the vascular tension alterations induced by KCl. (c) Graph data showing the vascular tension alterations (IBTX/KCl). (d and e) Whole-cell potassium currents before and after application of 100 nmol/L IBTX, and the I-V relationship of IBTX-sensitive currents of control and AGEs-cultured freshly isolated rat coronary arterial SMCs ( n = 3∼6 per group). (f) The representative tracings of baseline potassium currents and potassium currents after application of 100 nM IBTX in rat coronary arterial SMCs of the C+V, DM+V, C+A and DM+A groups, respectively ( n = 3∼5 per group). (g) Graph data showing IBTX-sensitive current densities at the testing potential of +100 mV in rat coronary arterial SMCs of the four groups. (h–j) The protein expressions of BK-α and BK-β1 in human coronary arterial SMCs in the BSA and BSA-AGEs groups ( n = 6∼9 per group). Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels. (k-l) The mRNA expression of BK-α and BK-β1 in rat coronary arteries of the C+V, DM+V, C+A and DM+A groups. β-actin was used as an internal control to normalize differences in the amount of total RNA in each rat sample ( n = 4 per group). (m and n) The mRNA expression of BK-α and BK-β1 in human coronary arterial SMCs of the NG, HG, NG+A, HG+A groups. GAPDH was used as an internal control to normalize differences in the amount of total RNA in each cell sample ( n = 4∼5 per group). (o–q) Protein expressions of BK-α and BK-β1 in rat coronary arteries of the C+V, DM+V, C+A and DM+A groups. Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels ( n = 5 per group). (r–t) Protein expressions of BK-α and BK-β1 in human coronary arterial SMCs of the NG, HG, NG+A, HG+A groups. Quantitative analysis of BK-α and BK-β1 were normalized to GAPDH protein expression levels ( n = 5∼9 per group). (C+V: Control + Vehicle; C+A: Control + aminoguanidine; DM+V: DM + Vehicle; DM+A: DM + aminoguanidine. NG: normal glucose; HG: high glucose; NG+A: normal glucose + aminoguanidine; HG+A: high glucose + aminoguanidine).

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Inhibition, Expressing, Control, Cell Culture, Isolation

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Regulation of Akt in AGEs-mediated FBXO32-induced BK-β1 degradation (a and b) Protein expression of FBXO32 in rat coronary arteries of four groups ( n = 5 per group). (c and d) Protein expression of FBXO32 in human coronary arterial SMCs of four cell groups. Quantitative analysis of FBXO32 was normalized to GAPDH protein expression levels. (e–g) Phosphorylation levels of Akt and total Akt in rat coronary arteries of four groups ( n = 8 per group). (h–j) Phosphorylation levels of Akt and total Akt in human coronary arterial SMCs of four groups ( n = 3 per group). The phosphorylation level of Akt (k and n) and the protein expressions of FBXO32 (l and o) and BK-β1 (m and p) were measured after human coronary arterial SMCs were incubated for 96 h in DMEM containing 25.5 mmol/L glucose, or 25.5 mmol/L glucose with aminoguanidine in the absence or presence of MK2206 (0.3 μM) ( n = 5∼10 per group). MK2206 was added at the beginning and remained for 6 h (C+V: Control + Vehicle; C+A: Control + aminoguanidine; DM+V: DM + Vehicle; DM+A: DM + aminoguanidine. NG: normal glucose; HG: high glucose; NG+A: normal glucose + aminoguanidine; HG+A: high glucose + aminoguanidine.)

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Expressing, Phospho-proteomics, Incubation, Control

Journal: Diabetes & Vascular Disease Research

Article Title: Advanced glycation end products impair coronary artery BK channels via AMPK/Akt/FBXO32 signaling pathway

doi: 10.1177/14791641231197107

Figure Lengend Snippet: Regulation of AMPK in Akt-mediated FBXO32-induced BK-β1 degradation by AGEs (a–c) Protein expression of p-AMPK and AMPK in rat coronary arteries from the four groups ( n = 8 per group). (d–f) Protein expression of p-AMPK and AMPK in human coronary arterial SMCs from the four groups ( n = 9 per group). Quantitative analysis of p-AMPK and AMPK was normalized to GAPDH protein expression levels. (g) Human coronary arterial SMCs were incubated for 96 h in DMEM containing 25.5 mmol/L glucose, or 25.5 mmol/L glucose and aminoguanidine in the absence or presence of Compound C (CC, 1 μM). Subsequently, the phosphorylation level of AMPK (h and i), AKT (j and k), and the protein expressions of FBXO32 (l) and BK-β1 (m) were measured ( n = 8 and 9 per group). Quantitative analysis of FBXO32 and BK-β1 was normalized to GAPDH protein expression levels.

Article Snippet: Human coronary arterial SMCs (ATCC, #PCS-100-021) and the culture medium (ATCC, #PCS-100-042 and #PCS-100-030) were purchased from ATCC.

Techniques: Expressing, Incubation, Phospho-proteomics

Journal: Cellular signalling

Article Title: PAI-1 contributes to homocysteine-induced cellular senescence

doi: 10.1016/j.cellsig.2019.109394

Figure Lengend Snippet: Cultures of EA.hy926 endothelial cells (A) and HCAEC (B, C) were pretreated with TM5441 (10 μM) (A, B) or TM5A15 (10 μM) (C) in triplicate followed by Homocysteine (Hcy) treatment for 4–5 days. Whole cell extracts were prepared and equal amount of pooled proteins from three wells were subjected to Western blot analysis for senescence markers and regulators using specific antibodies as indicated (A–C). Bar represents mean ± sem. Quantitative data are shown on the right (A’-C’). The levels of at least 2–3 senescence markers were determined in repeat experiments. D, E. Whole cell extracts (HCAEC) were prepared from two separate experiments and equal amount of pooled proteins from three wells were subjected to Western blot analysis for senescence markers and regulators p53 and pERK1/2 (D), integrin β3 and PAI-1 (E) using specific antibodies. Quantitative data in the lower panel showing the levels of each regulator relative to loading control Actin (D’, E’).

Article Snippet: Endothelial cell culture: treatment with Hcy and small molecule inhibitors of PAI-1 Primary cultures of Human Coronary Artery Endothelial Cells (HCAEC) (Cell Applications; Cat # 300–05a) and EA.hy926 (ATCC cat #CRL-2922) were grown in MesoEndo Cell Growth Media and Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin and streptomycin respectively and maintained at 37 °C in a 5% CO 2 incubator.

Techniques: Western Blot, Control

Journal: Frontiers in Physiology

Article Title: Myoendothelial Junctions of Mature Coronary Vessels Express Notch Signaling Proteins

doi: 10.3389/fphys.2020.00029

Figure Lengend Snippet: Notch receptors and ligand are located within and active at the cellular extensions of the human in vitro MEJ. (A) Representative images showing localization of PAI-1, Jagged1, Notch1, and Notch2 in human coronary ECs and human coronary VSCMs cultured on the top and bottom of transwell inserts, respectively. Note that PAI-1 expression is limited to ECs while Jagged1, Notch1, and Notch2 are expressed in both ECs and VSMCs and are also expressed at the MEJs (white arrows). Negative controls were incubated with appropriate secondary antibodies only. Since both Pai-1 and Notch2 required the same secondary antibody, the same negative control was used for those images only. * indicates endothelial side of transwell. n = 3–4 per group. Scale bar = 10 μm. (B) Human coronary VSMC (hcVSMC) Notch was activated at the in vitro MEJ (across transwell inserts) by co-culture with human coronary ECs (hcECs). This induction was completely blocked by the γ-secretase inhibitor, DAPT, which prevents Notch signaling activation. n = 5 per group. ** p < 0.01 and *** p < 0.001.

Article Snippet: Vascular cell co-cultures (VCCC) were assembled as previously described ( ; , ) using human coronary ECs (hcECs) and human coronary VSMCs (hcVSMCs) (Lonza, Morristown, NJ, United States and ATCC, Manassas, VA, United States).

Techniques: In Vitro, Cell Culture, Expressing, Incubation, Negative Control, Co-Culture Assay, Activation Assay