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: 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: Stem cells and development

Article Title: Efficient Differentiation of TBX18 + /WT1 + Epicardial-Like Cells from Human Pluripotent Stem Cells Using Small Molecular Compounds.

doi: 10.1089/scd.2016.0208

Figure Lengend Snippet: FIG. 5. Functional assay of EPLC-derived SMCs (EPL-SMCs). (A) Time-course of change in relative mean fluorescence intensity of Fluo-4-preloaded EPLC-derived cells, determined by flow cytometry after carbachol addition. Error bars represent SEM; n = 3/group; *p < 0.05, **p < 0.01 versus untreated cultures, analyzed by the Student’s t-test. (B) Total proportion of functional EPLC-SMCs displaying calcium transients after phenylephrine addition in D15 + 8 + 6 NT and TGFb1+ bFGF+TGFb1-induced cultures. Error bars represent SEM; n = 3/group; **p < 0.01 versus untreated cultures, analyzed by the Student’s t-test. (C) Calcium imaging examining the functional response of EPLC-SMCs and HCASMCs to phenylephrine. Average fluorescence intensity normalized to baseline (F/Fo) of five random cells exhibiting calcium transients was measured. Peaks correspond to active calcium cycles in response to 15mM phenylephrine added at 100 s, eliciting different responses. (D) Amplitude of calcium transients after phenylephrine addition. Error bars represent SEM; n = 5 cells; **p < 0.01 versus HCASMCs, analyzed by the Student’s t-test. (E) Kinetic parameters of calcium transients after phenylephrine addition. Error bars represent SEM; n = 5 cells; **p < 0.01 versus HCASMCs, analyzed by the Student’s t-test. (F) Fluo-4 AM-loaded EPLC- SMCs and HCASMCs displayed a change in cell surface area following phenylephrine stimulation. Scale bars, 100 mm. (G) Percentage change in cell surface area. Error bars represent SEM; n = 12 cells; **p < 0.01 versus D15 + 8 + 6 cells that did not exhibit calcium transients, analyzed by the Student’s t-test. HCASMCs, human coronary artery smooth muscle cells; NS, no significant difference. Color images available online at www.liebertpub.com/scd

Article Snippet: Primary human coronary artery SMCs (HCASMCs, ATCC) and EPL-SMCs were preloaded with 2.5 mM Fluo-4 AM in Tyrode’s solution at 37 C for 30 min.

Techniques: Functional Assay, Derivative Assay, Cytometry, Imaging

Journal: International Journal of Molecular Sciences

Article Title: Biodegradable Polymers Influence the Effect of Atorvastatin on Human Coronary Artery Cells

doi: 10.3390/ijms17020148

Figure Lengend Snippet: The viability of vascular cells is influenced by polymers. Human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) were cultured on control and polymer surfaces for six days. Cell viability was determined after incubation with AlamarBlue ® (Biosource, Camarillo, CA, USA). Bars show mean ± SD of six separate experiments normalized to the control with * p < 0.05 compared to the HCAEC control and + p < 0.05 compared to the HCASMC control.

Article Snippet: Primary cultures of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) were purchased from Provitro GmbH, Berlin, Germany.

Techniques: Cell Culture, Control, Polymer, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Biodegradable Polymers Influence the Effect of Atorvastatin on Human Coronary Artery Cells

doi: 10.3390/ijms17020148

Figure Lengend Snippet: Atorvastatin (ATOR) acts cell type-specifically on human coronary artery vascular cells. HCAEC and HCASMC were treated for 48 h with the indicated range of concentrations of ATOR. Proliferation was determined after BrdU incorporation. Dots show mean ± SD of at least three separate experiments normalized to the respective control. IC 50 values and the respective 95% confidence interval (CI) are indicated.

Article Snippet: Primary cultures of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) were purchased from Provitro GmbH, Berlin, Germany.

Techniques: BrdU Incorporation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Biodegradable Polymers Influence the Effect of Atorvastatin on Human Coronary Artery Cells

doi: 10.3390/ijms17020148

Figure Lengend Snippet: Polymers attenuate anti-proliferative actions of ATOR on SMCs. ( a ) HCAEC were cultured on polymer surfaces in the absence of ATOR (−) or presence of 0.1 µM ATOR for 48 h (+); ( b ) HCASMC were cultured on polymer surfaces and treated for 48 h with 0.1 µM ATOR (+). Proliferation was determined after BrdU incorporation. Bars show mean ± SD of three independent experiments with * p < 0.05 compared to the corresponding HCAEC control and +++ p < 0.001 compared to the HCASMC control.

Article Snippet: Primary cultures of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) were purchased from Provitro GmbH, Berlin, Germany.

Techniques: Cell Culture, Polymer, BrdU Incorporation Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Biodegradable Polymers Influence the Effect of Atorvastatin on Human Coronary Artery Cells

doi: 10.3390/ijms17020148

Figure Lengend Snippet: Polymers attenuate cell type-specific anti-proliferative effects of ATOR on vascular cells. HCAEC (EC) and HCASMC (SMC) were cultured on polymer surfaces and treated for 48 h with 0.1 µM ATOR. Proliferation was determined after BrdU incorporation. Bars show mean ± SD of at least five separate experiments with # p < 0.05 and ### p < 0.001.

Article Snippet: Primary cultures of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) were purchased from Provitro GmbH, Berlin, Germany.

Techniques: Cell Culture, Polymer, BrdU Incorporation Assay

Journal: International Journal of Molecular Sciences

Article Title: Biodegradable Polymers Influence the Effect of Atorvastatin on Human Coronary Artery Cells

doi: 10.3390/ijms17020148

Figure Lengend Snippet: IC 50 of ATOR on HCASMC and HCAEC grown on different polymer surfaces. IC 50 values were calculated from sigmoidal dose-response curves as the concentration of ATOR causing 50% reduction in cell growth. The 95% confidence intervals (CI) are indicated.

Article Snippet: Primary cultures of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) were purchased from Provitro GmbH, Berlin, Germany.

Techniques: Polymer, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Biodegradable Polymers Influence the Effect of Atorvastatin on Human Coronary Artery Cells

doi: 10.3390/ijms17020148

Figure Lengend Snippet: Uptake of 3 H-ATOR is cell type-specific and polymer-dependent. ( a ) HCAEC (EC) and HCASMC (SMC) were treated for 20 min with radio-labelled 3 H-ATOR. Bars show mean ± SD of at least 10 separate experiments normalized to HCAEC grown on Thermanox™ (Thermo Fisher Scientific, Darmstadt, Germany) ( ### p < 0.001); ( b ) HCAEC (EC) and HCASMC (SMC) were grown on polymer surfaces prior to 3 H-ATOR treatment. Data are shown as the percentage of the ATOR-uptake in ECs. Each bar shows mean ± SD of at least three separate experiments. In each case, SMC data were normalized to their respective ECs ( ## p < 0.01).

Article Snippet: Primary cultures of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) were purchased from Provitro GmbH, Berlin, Germany.

Techniques: Polymer