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: Development (Cambridge, England)

Article Title: Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells

doi: 10.1242/dev.119271

Figure Lengend Snippet: HESC-derived epicardial cells survive and differentiate in vivo . (A-C) Fluorescent (GFP + and mStrawb + ) human epicardial cells detected in the subepicardial region at the base of the chicken heart (arrowheads). (B) Human cells in A are identifiable by bright and distinct Hoechst 33342 staining. (D-F) Fluorescent human epicardial cells detected in the subepicardial region in the middle part of the heart (arrowheads). (F) Human cells in E are identifiable by bright and distinct Hoechst 33342 staining. (G,H) Epicardial cells (GFP + ) that localised at the apex of the heart (G) expressed WT1 (H; indicated by arrowheads). (I) Co-expression of WT1 + and GFP + human cells. (J,K) A few engrafted mStrawb + epicardial cells (J) expressed ACTA2 (K), suggesting differentiation to SMCs in vivo . (L) Cells co-expressing mStrawb and ACTA2 (indicated by arrowheads). (M,N) GFP + and mStrawb + epicardial cells detected within lectin-stained (in cyan and red) coronary vessels. (O) Subepicardial region in a chicken embryo heart injected with mStrawb + human neural crest cells. Scale bars: 100 μm. Myo, myocardium; Epi, epicardium; Subepi, subepicardium.

Article Snippet: Low-passage rat aortic SMCs (RASMCs), primary human coronary artery SMCs (HCASMCs, Promocell), HeLa cells and EPI-SMCs were preloaded with the calcium-sensitive fluorophore Fluo-4 AM (2.5 μM, Molecular Probes) for 1 h at 37°C.

Techniques: Derivative Assay, In Vivo, Staining, Expressing, Injection

Journal: Development (Cambridge, England)

Article Title: Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells

doi: 10.1242/dev.119271

Figure Lengend Snippet: Epicardium-derived SMC differentiation in vitro . (A) Epithelial and mesenchymal marker expression in day 10 epicardial cells (EPI D10) and epicardium-derived SMCs (EPI-SMCs) after 3 (D3) and 6 (D6) days of differentiation with PDGF-BB and TGF-β1 (PT). *** P <0.001, ** P <0.01. (B) SMC marker expression by qRT-PCR in EPI-SMCs differentiated with PT in the presence and absence of p160 Rho-kinase inhibitor (iROCK) after 3, 6 and 12 days of differentiation. Significant differences between PT and iROCK+PT are indicated in black. *** P <0.001, ** P <0.01, * P <0.05. (C) EPI-SMCs after 12 days of PT treatment expressed mesenchymal (VIM) and SMC (ACTA2, CNN1 and TAGLN) markers, similar to human coronary artery SMCs (HCASMCs). SMC marker expression was absent in HUVECs. Scale bars: 100 μm. (D) Percentage of ACTA2 + and CNN1 + cells in H9 and BHX-derived EPI-SMCs. Mouse IgG isotypes served as negative controls.

Article Snippet: Low-passage rat aortic SMCs (RASMCs), primary human coronary artery SMCs (HCASMCs, Promocell), HeLa cells and EPI-SMCs were preloaded with the calcium-sensitive fluorophore Fluo-4 AM (2.5 μM, Molecular Probes) for 1 h at 37°C.

Techniques: Derivative Assay, In Vitro, Marker, Expressing, Quantitative RT-PCR

Journal: Development (Cambridge, England)

Article Title: Robust derivation of epicardium and its differentiated smooth muscle cell progeny from human pluripotent stem cells

doi: 10.1242/dev.119271

Figure Lengend Snippet: Functional characterisation of epicardium-derived SMCs. (A) Change in the relative fluorescence unit (ΔRFU) of Fluo-4 AM-loaded HeLa cells, rat aortic SMCs (RASMC), human coronary artery (HCASMC) and epicardium-derived SMCs (EPI-SMC) by flow cytometry over 10 min after the addition of carbachol. (B) Peak ΔRFU 1 min after carbachol addition. Differences in Fluo-4 intensity compared with HeLa cells. *** P <0.001. (C) Fluo-4 AM-loaded cells displayed a change in cell surface area following carbachol stimulation. (D) EPI-SMCs, RASMCs and HCASMCs displayed 10-30% decreases in cell surface area with negligible change in HeLa cells. (E) Uptake of Alexa Fluor 594-conjugated acetylated low density lipoprotein (Ac-LDL) in EPI-SMCs and HCASMCs appears as red droplets after 2 and 3 h of incubation. Nuclei counterstained with DAPI (blue). Effective lowering of LDL uptake observed after treatment with atorvastatin (Ac-LDL+statin) in both cell types. (F) Quantification of Alexa Fluor 594 intensity in the absence (solid line) and presence (dashed line) of atorvastatin. ** P <0.01. Scale bars: 100 μm.

Article Snippet: Low-passage rat aortic SMCs (RASMCs), primary human coronary artery SMCs (HCASMCs, Promocell), HeLa cells and EPI-SMCs were preloaded with the calcium-sensitive fluorophore Fluo-4 AM (2.5 μM, Molecular Probes) for 1 h at 37°C.

Techniques: Functional Assay, Derivative Assay, Fluorescence, Flow Cytometry, Incubation

Journal: Vascular Biology

Article Title: OxLDL induces the release of IL-1β from primed human endothelial and smooth muscle cells via different caspase -1-dependent mechanisms

doi: 10.1530/VB-22-0009

Figure Lengend Snippet: (A, B, C and D) Representative bright-field microscope images of stimulated, untreated HCAECs (A) and HCASMCs (C) compared with cytokine-stimulated (10 ng/mL of TNF-α and IL-1α for 48 h) HCAECs (B) and HCASMCs (D) treated with 50 µg/mL oxLDL for 6 h (HCAECs) or 2h (HCASMCs) and stained with Oil red O. Droplets stained with Oil red O were observed within both cell types upon stimulation and treatment. Images were taken using a Leica© bright-field microscope and are representative of 3 independent experiments, scale bar = 100 µm. (E and F) ELISA analysis of intracellular IL-1β in HCAECs (E) and HCASMCs (F) showed a significant increase following cytokine priming but no effect with oxLDL treatment when compared to the cytokine-only control. Data are presented as mean ± s.e.m. and analysed using a one-way ANOVA followed by a Bonferroni post-hoc test, **** P < 0.0001, *** P < 0.001, n = 3 independent donors.

Article Snippet: Human coronary artery endothelial (HCAECs) and smooth muscle cells (HCASMCs) were purchased from PromoCell (Heidelberg, Germany) and maintained according to the manufacturer’s instructions.

Techniques: Microscopy, Staining, Enzyme-linked Immunosorbent Assay

Journal: Vascular Biology

Article Title: OxLDL induces the release of IL-1β from primed human endothelial and smooth muscle cells via different caspase -1-dependent mechanisms

doi: 10.1530/VB-22-0009

Figure Lengend Snippet: (A and B) Cytokine stimulation and treatment with higher concentrations of oxLDL for 6 h (HCAECs) or 2 h (HCASMCs) induced significant IL-1β release from both HCAECs (A) and HCASMCs (B) compared to the cytokine-only control. (C and D) Increased release of IL-1β following cytokine-priming and treatment with 50 µg/mL oxLDL is associated with elevated LDH release in HCAECs (C) but not in HCASMCs (D). Samples are compared to the cytokine-only controls. Data are presented as mean ± s.e.m. and analysed using a one-way ANOVA followed by a Bonferroni post-hoc test, **** P < 0.0001, *** P < 0.001, n = 3 independent donors.

Article Snippet: Human coronary artery endothelial (HCAECs) and smooth muscle cells (HCASMCs) were purchased from PromoCell (Heidelberg, Germany) and maintained according to the manufacturer’s instructions.

Techniques:

Journal: Vascular Biology

Article Title: OxLDL induces the release of IL-1β from primed human endothelial and smooth muscle cells via different caspase -1-dependent mechanisms

doi: 10.1530/VB-22-0009

Figure Lengend Snippet: (A and B) ELISA analysis showed a significant reduction in oxLDL-induced IL-1β release from HCAECs following inhibition of both caspase-1 (YVAD) (A) and the NLRP3 inflammasome (MCC950) (B). (C and D) The release of IL-1β from oxLDL-treated HCASMCs was only significantly reduced upon inhibition of caspase-1 (C) and not the NLRP3 inflammasome (D). Data are expressed as mean ± s.e.m. and analysed using a one-way ANOVA and a Bonferroni post-hoc test, **** P < 0.0001, n = 3 independent donors.

Article Snippet: Human coronary artery endothelial (HCAECs) and smooth muscle cells (HCASMCs) were purchased from PromoCell (Heidelberg, Germany) and maintained according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Vascular Biology

Article Title: OxLDL induces the release of IL-1β from primed human endothelial and smooth muscle cells via different caspase -1-dependent mechanisms

doi: 10.1530/VB-22-0009

Figure Lengend Snippet: (A and B) ELISA analysis showed a significant reduction in oxLDL-induced IL-1β release from HCAECs following inhibition of gasdermin D (disulfiram) (A) but disulfiram was without effect on the release of IL-1β from HCASMCs (B). (C and D) The release of LDH was measured in HCAEC after cytokine treatment which was significantly reduced by 1 µM disulfiram treatment (C). There was no LDH release under any condition from HCASMC. Data are expressed as mean ± s.e.m. and analysed using a one-way ANOVA and a Bonferroni post-hoc test, *** P < 0.001, ** P < 0.01, n = 3 independent donors.

Article Snippet: Human coronary artery endothelial (HCAECs) and smooth muscle cells (HCASMCs) were purchased from PromoCell (Heidelberg, Germany) and maintained according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition

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: Physiological Genomics

Article Title: Transcriptomic sequencing reveals diverse adaptive gene expression responses of human vascular smooth muscle cells to nitro-conjugated linoleic acid

doi: 10.1152/physiolgenomics.00090.2017

Figure Lengend Snippet: Transcriptomic profiling of human coronary artery smooth muscle cells (hCASMCs) treated by nitro-conjugated linoleic acid (NO2-CLA). A: Venn diagram of differentially expressed genes (DEGs) identified in NO2-CLA vs. conjugated linoleic acid (CLA) and NO2-CLA vs. control. B: pairwise volcano plot of gene expression differences between conditions. The threshold of DEGs is defined at false discovery rate (FDR) > 0.05, and 1.3 fold up- (red) or down- (green) regulated upon treatment.

Article Snippet: hCASMCs were purchased from PromoCell (Germany) and cultured in Smooth Muscle Cell Growth Medium 2 from same company at 37°C, 5% CO 2 in a standard cell culture incubator.

Techniques: Expressing

Journal: Physiological Genomics

Article Title: Transcriptomic sequencing reveals diverse adaptive gene expression responses of human vascular smooth muscle cells to nitro-conjugated linoleic acid

doi: 10.1152/physiolgenomics.00090.2017

Figure Lengend Snippet: Differentially expressed genes (DEGs) in NO2-CLA-treated hCASMCs. A: heat map of 578 DEGs expression levels. Color bar denotes the row-scaled fragments per kilobase of transcript per million mapped reads (FPKM) value, representing the Z score of gene expression across samples. B: correlation matrix of 12 samples constructed based on DEGs. C: plot of the 1st and 2nd principal component by principal component analysis (PCA) of sample variations.

Article Snippet: hCASMCs were purchased from PromoCell (Germany) and cultured in Smooth Muscle Cell Growth Medium 2 from same company at 37°C, 5% CO 2 in a standard cell culture incubator.

Techniques: Expressing, Construct