Journal: The Journal of Biological Chemistry

Article Title: Identification and analyses of inhibitors targeting apolipoprotein(a) kringle domains KIV-7, KIV-10, and KV provide insight into kringle domain function

doi: 10.1074/jbc.RA119.011251

Figure Lengend Snippet: Inhibition of apo(a)-induced primary human coronary artery smooth muscle cell proliferation. A, human coronary artery smooth muscle cells (SMC) were incubated with or without 1 μm fl_apo(a), and proliferation was quantified as outlined under “Experimental procedures”. Apo(a)-induced proliferation could be completely reversed by co-administration of 6 mm TX. Results from a representative experiment and the mean ± standard deviation within the experiment are shown. B, data shown are the IC50 concentrations of compounds AZ-05KIV-10 and AZ-06 KIV-7 that inhibited the increase in SMC proliferation induced by 1 μm recombinant apo(a) compared with cells with medium only by 50%. For TX, the estimated IC50 value was >180 μm. Shown is the mean of three experiments ± S.D.

Article Snippet: Primary human coronary artery smooth muscle cells were from Lonza.

Techniques: Inhibition, Incubation, Standard Deviation, Recombinant

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: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Impaired BK-β1-mediated channel activation, reduced BK-β1 expression, and increased MuRF1 expression in diabetic vessels and in HG-cultured human coronary SMCs. A, inside-out single-channel BK currents were elicited at +60 mV in freshly isolated coronary SMCs from control and diabetic mice before and after exposure to DHS-1 (0.1 μm). DHS-1 robustly increased BK channel NPo in control SMCs, but its effects were diminished in diabetic SMCs. Dashed lines represent the channel closed state (c). B, left panel, the cell lysates of control and diabetic mouse aortas were blotted against anti-ubiquitin antibody. Right panel, the immunoprecipitates (IPs) of anti-BK-β1 antibodies were blotted against anti-ubiquitin (Ub) antibody, showing a significant increase of ubiquitinated BK-β1 protein in diabetic mice. Ctrl, control. C and D, a marked reduction of BK-β1 expression in the aortas of diabetic mice compared with those of control mice and in human coronary SMCs after a 2-week culture with 22 mm glucose (HG) compared with that with 5 mm glucose (NG) culture. Group data with statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Activation Assay, Expressing, Cell Culture, Isolation, Control, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Regulation of BK-β1 expression by MuRF1. A, after a 2-week culture in NG or HG, BK-β1 protein expression in human coronary SMCs was determined under the following conditions: NG culture alone, HG culture after a 48-h transfection with control siRNA (0.1 μm), and HG culture after a 48-h transfection with MuRF1 siRNA (0.1 μm). MuRF1 siRNA suppressed MuRF1 expression under HG culture conditions to that of NG culture conditions. Ctrl, control. B, FLAG-BK-β1 or FLAG-BK-α stably expressed in HEK293 cells 48 h after cotransfection with Myc-MuRF1 WT cDNA or Myc-MuRF1ΔR mutant cDNA. The BK-β1 protein level was halved in cells with MuRF1 WT transfection, but BK-α expression remained unchanged. N.S., not significant. C and D, coimmunoprecipitation experiment shows attenuated BK-β1 protein ubiquitination in the pulldown complexes with anti-HA-ubiquitin (anti-HA-Ub) antibodies or with anti-FLAG antibodies in HEK293 cells after a 24-h transfection with MuRF1ΔR compared with those with MuRF1 WT. Group data with statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Transfection, Control, Stable Transfection, Cotransfection, Mutagenesis, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Reduced BK-β1 expression and BK-β1-mediated channel activation in vascular SMCs by MuRF1 expression. A, fluorescence microscopy images illustrate GPF expression in endothelially denudated mouse coronary artery 12 h after transduction with Ad-GFP but not in a negative control artery. Immunoblot analyses show increased MuRF1 and decreased BK-β1 expression in the aortas of control (Ctrl) mice after a 12-h transduction with Ad-MuRF1 compared with controls with Ad-GFP transduction. B, inside-out single BK currents were elicited from +60 mV at baseline, after exposure to 0.1 μm DHS-1, and after washout (W/O). DHS-1 had no effect on mouse coronary SMCs 12 h after transduction with Ad-MuRF1. Dashed lines represent the channel closed state (c). Group data with statistical analysis are shown in the bar graphs. N.S., not significant. C, effects of Ad-MuRF1 transduction on dose-dependent, NS1619-mediated (10−9-10−5 m) dilation of endothelium-denuded coronary arteries from non-diabetic mice. Transduction with Ad-MuRF1 (12 h) attenuated the NS1619-induced vasodilation, but the NS1619 response was preserved in Ad-MuRF1-treated coronaries by incubation with MG132 (10 μm, 12 h). Control vessels received Ad-GFP transduction. *, p < 0.05, Ad-MuRF1 versus Ad-GFP; +, p < 0.05 Ad-MuRF1+MG132 versus Ad-GFP.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Activation Assay, Fluorescence, Microscopy, Transduction, Negative Control, Western Blot, Control, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Enhanced BK-β1 expression and BK-β1-mediated channel activation by MG132. A, protein expression of BK-β1 in STZ-induced diabetic aortas with or without 24-h treatment of MG132 (10 μm). Inhibition of proteasomal activity by MG132 increased BK-β1 protein expression in diabetic vessels. B, robust BK channel openings by DHS-1 (0.1 μm) were observed in freshly isolated diabetic coronary SMCs after 12-h incubation with MG132. Group data with statistical analysis are shown in the bar graphs. Dashed lines represent the channel closed state (c). W/O, washout.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Activation Assay, Inhibition, Activity Assay, Isolation, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Regulation of Large Conductance Ca 2+ -activated K + (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels *

doi: 10.1074/jbc.M113.520940

Figure Lengend Snippet: Regulation of MuRF1 and BK-β1 expression by NF-κB. A, protein expression of NF-κB1/p105, NF-κB1/p50, and RelA/p-65 in the aortas of control and STZ-induced diabetic mice. The levels of NF-κB/p105, NF-κB1/p50, and p-65 protein expression were significantly increased in diabetic mice compared with controls (Ctrl). B, 24-h incubation with 0.5 μm TPCA-1 suppressed the levels of NF-κB1/p105, NF-κB1/p50, and MuRF1 expression but markedly increased that of IκB-β in both NG- and HG-cultured human coronary SMCs. TPCA-1 treatment did not alter BK-β1 expression in NG-cultured cells but significantly enhanced that in HG-cultured cells. Group data and statistical analysis are shown in the bar graphs.

Article Snippet: Primary human coronary SMCs were purchased from Lonza Walkersville, Inc. and were cultured with Clonetics SmBM (Lonza Walkersville, Inc.) containing 5 m m glucose (normal glucose, NG) or 22 m m glucose (high glucose, HG).

Techniques: Expressing, Control, Incubation, Cell Culture