Journal: Scientific reports

Article Title: Soluble adenylyl cyclase links Ca 2+ entry to Ca 2+ /cAMP-response element binding protein (CREB) activation in vascular smooth muscle.

doi: 10.1038/s41598-019-43821-3

Figure Lengend Snippet: Figure 1. SOCE increases intracellular cyclic AMP and activates PKA in human coronary artery smooth muscle cells (hCASMCs). (a) Intracellular Ca2+ measurements recorded in fura-2-AM-loaded hCASMCs. Cells were initially bathed in nominally Ca2+-free solution for 10 minutes before treatment with thapsigargin (TG, 2 μM). Ca2+ (1.8 mM) was then introduced into the extracellular solution and was present hereafter (n = 54 from 4 experimental repeats). (b) Intracellular cyclic AMP levels measured in hCASMCs transduced with adenoviruses encoding the cyclic AMP biosensor H187 (upper panel, see Methods for details) and subjected to the same experimental scheme as described in (a). At the end of the experiment saturating concentrations of forskolin (20 μM) and IBMX (500 μM) were added to generate maximal cyclic AMP responses (n = 20 cells from 4 experimental repeats). (c) PKA activity measured in hCASMCs transduced with adenoviruses encoding the PKA reporter, AKAR4-NES (upper panel, see Methods for details) in response to the experimental scheme described in a (n = 19 cells from 4 experimental repeats). All extracellular solutions contain 1% serum. Error bars represent the standard error of the mean. (d) Western blot analyses of hCASMC homogenates immunoblotted with anti-VASP (upper panel) and anti-phospho-PKA substrates (lower panel). Before being lysed, cells were exposed to either: vehicle control (0.1% DMSO); thapsigargin (TG, 2 μM) for 5 min in zero extracellular Ca2+, TG for 5 min in zero extracellular Ca2+ followed by introduction of 1.8 mM extracellular Ca2+ for a further 5 min; or forskolin (20 μM) plus IBMX (500 μM) for 5 min. In lysates immunoblotted with anti-VASP, phosphorylated VASP appears as a slower-migrating band. Maximal phosphorylation is seen following exposure to saturating concentrations of forskolin and IBMX. Blots shown representative of 3 experimental repeats. (e) Densiometric analysis; the density of the phospho-VASP immunoreactive band relative to the lower (unphosphorylated) VASP band (n = 3, Control vs TG/0Ca2+: P = 0.024; Control vs TG/1.8Ca2+: P = 0.026; Control vs Fsk/IBMX: P = 0.001; one-way ANOVA with Student Newman Keuls post- hoc test).

Article Snippet: Human coronary artery smooth muscle cells (hCASMCs; Promocell, Heidelberg, Germany) were cultured in smooth muscle cell growth medium 2 containing 5% (v/v) foetal calf serum supplemented with basic fibroblast growth factor (2 ng mL−1), epidermal growth factor (0.5 ng mL−1) and insulin (5 μg mL−1; all Promocell).

Techniques: Transduction, Activity Assay, Western Blot, Control, Phospho-proteomics

Journal: PLOS One

Article Title: β-Nicotinamide adenine dinucleotide (β-NAD) acts as a bronchodilator

doi: 10.1371/journal.pone.0334491

Figure Lengend Snippet: (A, B) Force recordings in organ bath, β-NAD (A) and salbutamol (B) concentration-dependently relax precontracted human bronchioli. n refers to the number of bronchioli, followed by the number of lungs from which they were taken, presented as bronchioli/lungs. (C) Recording of [Ca 2+ ] i in HBSMC with Fura-2 AM. Both β-NAD and ATP cause rise in [Ca 2+ ] i . n refers to the number of cells, taken from 4 independent experiments.

Article Snippet: HBSMC (C-12561, PromoCell, Heidelberg, Germany) were cultured in Smooth Muscle Cell Growth Medium 2 for at least 24 h at 37°C.

Techniques: Concentration Assay

Journal: PLOS One

Article Title: β-Nicotinamide adenine dinucleotide (β-NAD) acts as a bronchodilator

doi: 10.1371/journal.pone.0334491

Figure Lengend Snippet: (A, B) Recording of intracellular cAMP concentration in HBSMC via FRET, with low FRET ratio indicating high cAMP concentration. β-NAD and isoproterenol cause a decrease in FRET ratio, reflecting rise in intracellular cAMP concentration. In the presence of KH7 (30 µM), a soluble adenylyl cyclase antagonist, the cAMP response to β-NAD was blocked, while the isoproterenol-induced cAMP increase remained unaffected. (A) FRET ratio over time and (B) ΔFRET ratio (%) represents the change in response to β-NAD and isoproterenol, measured in the presence and absence of KH-7. Within-group comparisons include the ΔFRET ratio before agonist addition versus after the addition of β-NAD or isoproterenol. n represents the number of cells analyzed, derived from three independent experiments. Error bars indicate mean ± SEM throughout. Statistical analysis was performed using the Wilcoxon signed-rank test (two-tailed) with Bonferroni correction for multiple comparisons. (C-J) Force recording from trachea in organ bath (C-F) and videomorphometric recording of luminal bronchial area in PCLS (G-J) in specimens taken from mice lacking the C1 (C, E, G, I) or C2 domain of soluble adenylyl cyclase (D, F, H, J). In both assays, β-NAD-induced relaxation of muscarine-precontracted airways was not significantly reduced in knockout mice compared to their respective wildtype controls. (G-J) In PCLS, KH7 (30 µM) has no significant effect upon muscarine-induced contraction and β-NAD-induced relaxation, both in knockout and in wild-type mice. (E, F, I, J) Scatterplots depict changes induced by β-NAD related to the preceding response to muscarine. (E, F) Scatterplots show the β-NAD-induced relaxation effect (%) relative to the muscarine response. (I, J) Scatterplot showing the maximum peak responses of the second stimulation (first response set as 100%) in the presence of KH7 within C1 and C2 knockout groups and their corresponding wild-type controls. The corresponding controls with the application of vehicle (DMSO) instead of KH7 are depicted in . Statistical analysis was performed using the Mann-Whitney test. Data are expressed as mean ± SEM. n refers to the number of animals.

Article Snippet: HBSMC (C-12561, PromoCell, Heidelberg, Germany) were cultured in Smooth Muscle Cell Growth Medium 2 for at least 24 h at 37°C.

Techniques: Concentration Assay, Derivative Assay, Two Tailed Test, Knock-Out, MANN-WHITNEY

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Endothelial cell-derived exosomes influence vascular smooth muscle cell phenotype and calcification-related gene expression. HAVSMCs were incubated for 8 days with 10 µg/mL exosomes derived from endothelial cells (ECs) in ECM (control), TNFα, TGFβ, or varying concentrations of TMAO (1–100 μM). ( A – D ) qPCR analysis of osteogenic markers RUNX2 and OPN, confirming transcriptional reprogramming toward an osteoblast-like phenotype. ( E ) TNAP (Tissue Non-Specific Alkaline Phosphatase) involved in vascular calcification and osteogenic transformation of VSMCs. Data are presented as mean ± SD from four independent biological replicates. Statistical significance was determined by one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. SMCM control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Derivative Assay, Gene Expression, Incubation, Control, Transformation Assay

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Differential effects of endothelial cell-derived exosomes on calcification of HAVSMCs, assessed by Alizarin Red staining. ( A – G ) Representative images of Alizarin Red staining in HAVSMCs after 8 days of culture with 10 µg/mL endothelial cell-derived exosomes (EC-EXOs) obtained from endothelial cell maintenance medium (ECM EC EXO), TNFα-stimulated EC exosomes (TNFα EC EXO), TGFβ-stimulated EC exosomes (TGFβ EC EXO), TMAO-treated EC exosomes (1 µM, 10 µM, and 50 µM TMAO EC EXO), and control smooth muscle cell medium (SMCM). ( H ) Quantification of Alizarin Red stain intensity was normalized to total protein concentration. Data are presented as mean ± SD from four independent biological replicates. Statistical significance was determined by one-way ANOVA, followed by Tukey’s post hoc test. * p < 0.05 vs. SMCM control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Derivative Assay, Staining, Control, Protein Concentration

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: β-catenin inhibition attenuates endothelial exosome-induced β-catenin activation in HAVSMCs. ( A , C ) Representative Western blot images showing non-phosphorylated (active) β-catenin protein expression in human aortic vascular smooth muscle cells (HAVSMCs) treated with endothelial cell-derived exosomes (EC-EXOs) obtained from TNFα-, TGFβ-, or TMAO-stimulated endothelial cells, in the presence or absence of the β-catenin transcriptional inhibitor ICG-001 for 8 days. β-actin was used as a loading control. ( B , D ) Quantitative densitometric analysis demonstrates a significant increase in β-catenin protein levels following EC-EXO treatment, which was markedly reduced upon β-catenin inhibition with ICG-001. Protein expression levels were normalized to β-actin and expressed as fold change relative to vehicle-treated controls. Data are presented as mean ± standard deviation (SD) from three independent biological replicates. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test to assess differences between EC-EXO treatment groups and the effect of β-catenin inhibition. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Derivative Assay, Control, Standard Deviation

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: β-catenin inhibition suppresses endothelial exosome-induced osteogenic gene expression in HAVSMCs. ( A – E ) Quantitative real-time PCR analysis of osteogenic gene expression in HAVSMCs treated with endothelial cell-derived exosomes (EC-EXOs) from TNFα-, TGFβ-, or TMAO-stimulated endothelial cells, in the presence of the β-catenin inhibitor ICG-001. Relative mRNA expression levels of ( A ) SM22A, ( B ) αSMA, ( C ) RUNX2, ( D ) osteopontin (OPN), and ( E ) tissue-nonspecific alkaline phosphatase (TNAP) were normalized to housekeeping genes and expressed relative to vehicle-treated control cells (0.1% v / v DMSO). EC-EXO co-treatment with ICG-001 significantly attenuated the expression of RUNX2, OPN, and TNAP, indicating that β-catenin signaling is required for endothelial exosome-induced osteogenic reprogramming of HAVSMCs. Data are presented as mean ± SD from three independent biological replicates. Statistical significance was assessed using one-way ANOVA, followed by post-hoc analysis. * p < 0.05, ** p < 0.01, *** p < 0.001, vs. CTL vehicle.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Inhibition, Gene Expression, Real-time Polymerase Chain Reaction, Derivative Assay, Expressing, Control

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: Uptake kinetics of MemBright-labeled endothelial cell-derived exosomes by HAVSMC. Representative confocal microscopy images showing the time-dependent uptake of MemBright-labeled endothelial cell-derived exosomes by human aortic vascular smooth muscle cells (HAVSMCs). ( A ) HAVSMCs treated with control endothelial cell-derived exosomes (CTL EC EXO). ( B ) HAVSMCs treated with exosomes derived from endothelial cells exposed to 50 µM TMAO (TMAO EC EXO). Exosomes were labeled with MemBright (green), and cell nuclei were counterstained with Hoechst (blue). Images were acquired immediately after exosome addition (T = 0 h) and after 1, 3, and 4 h of incubation. Merged images illustrate progressive internalization and intracellular accumulation of exosomes over time, with 20× objective. All images were captured using a Leica confocal laser scanning microscope under identical acquisition settings. Scale bar: 194 µm.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Labeling, Derivative Assay, Confocal Microscopy, Control, Incubation, Laser-Scanning Microscopy

Journal: Cells

Article Title: TMAO-Triggered Endothelial–Mesenchymal Transition and Microvesicle Release as Mediators of Vascular Smooth Muscle Cell Osteogenic Differentiation and Vascular Calcification

doi: 10.3390/cells15050466

Figure Lengend Snippet: miR-222-3p overexpression promotes osteogenic signaling in HAVSMCs through activation of β-catenin pathway. ( A ) Quantitative PCR analysis confirming successful transfection of HAVSMCs with miR-222-3p mimic compared with the results for scrambled mimic control. Relative miR-222-3p expression levels were normalized to miR5S and expressed as fold change. ( B – F ) Quantitative PCR analysis of gene expression levels of RUNX2, OPN and TNAP in HAVSMCs after miR-222-3p mimic transfection for 48 h. ( G ) Representative Western blot images showing β-catenin protein expression in HAVSMCs following transfection with scrambled mimic or miR-222-3p mimic. ( H ) Quantitative densitometric analysis of protein expression levels of β-catenin protein expression levels were normalized to housekeeping protein and expressed relative to scrambled control. Data are presented as mean ± SD from independent biological replicates. Statistical significance was determined using unpaired two-tailed Student’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. scrambled mimic control.

Article Snippet: HAVSMCs (ATCC ® PCS-100-012TM) were cultured in Smooth Muscle Cell Growth Medium (SMCM, ScienCell, Carlsbad, CA, USA) supplemented with 2% FBS and 1% Pen-Strep under standard conditions (37 °C, 5% CO 2 ).

Techniques: Over Expression, Activation Assay, Real-time Polymerase Chain Reaction, Transfection, Control, Expressing, Gene Expression, Western Blot, Two Tailed Test

Journal: Advanced Healthcare Materials

Article Title: Nitric Oxide‐Releasing Catheters with Phenol‐Amine Catalytic Coatings for Improved Anti‐Inflammatory Performance

doi: 10.1002/adhm.202500457

Figure Lengend Snippet: a) HCASMCs viability, b) number of cells, and c) endogenous NO generation after incubation with uncoated and coated catheter segments compared to the blank group, measured using the Live/Dead assay, Hoechst staining, and DAF‐FM diacetate, respectively, at i) 48 h and ii) 72 h. Statistical significance relative to control tests was calculated using one‐way ANOVA, ns = not significant, * p < 0.1, ** p < 0.01, **** p < 0.0001. n = 6; error bars represent standard deviation.

Article Snippet: Human coronary artery smooth muscle cells (HCASMCs) and smooth muscle cell growth medium kit were purchased from Cell Applications.

Techniques: Incubation, Live Dead Assay, Staining, Control, Standard Deviation

Journal: The Journal of Physiology

Article Title: Novel identification and modulation of the mechanosensitive Piezo1 channel in human myometrium

doi: 10.1113/jp283299

Figure Lengend Snippet: Figure 3. Piezo1-meidiated Ca2+ influx in CD31+ and CD31−human myometrial cells An intracellular calcium flux assay determined Piezo1 activity in phMEC (CD31+) and phUSMC (CD31−) cells. phMEC, phUSMC and HEK293 Piezo1KO cells were pre-treated with the Ca2+ indicator Calbryte (ex/em 493/515 nm) followed by exposure to Yoda1 (0.3 or 3 μM) ± the Piezo1 antagonist Dooku1 (10 μM) and the change in fluorescence (Cai2+) was measured. A, phMECs treated with 3 μM Yoda1 exhibited 4.09-fold increase in Ca2+ uptake (Cai2+) over 0.3 μM treated cells (P < 0.0001) which decreased by 35.74% when co-treated with Dooku1 (P = 0.0327) at 60 min. B, phUSMC experienced a 2.64-fold increase in fluorescence when challenged with 3 μM Yoda1 (P < 0.0001), with a respective decrease of 22.49% when co-treated with Dooku1 (P = 0.0326). C, Piezo1KO fluorescence did not vary significantly at any dose of Yoda1 or Yoda1 + Dooku1 relative to baseline (Kruskal–Wallis one-way ANOVA, P = 0.2622). D, left, ×10 bright field images of phMEC (top), phUSMC (middle), and Piezo1KO (bottom). Right, Calbryte-induced fluorescence after 3 μM Yoda1 stimulation. ×20 fluorescence images of phMEC (top), phUSMC (middle), and Piezo1KO (bottom). Data presented as ±SD.

Article Snippet: Cells captured by the beads were deemed CD31+ pregnant human myometrial endothelial cells (phMEC) and CD31− pregnant human uterine smooth muscle cells (phUSMC). phMECs cultured in endothelial basal medium 2 (C-22 011, PromoCell, Heidelberg, Germany) containing 10% fetal bovine serum (FBS) and 1% penicillin, while the phUSMC cells were cultured in DMEM with 50 U/ml streptomycin, 50 μg/ml penicillin, and 10% FBS and supplemented with oestrogen (15 ng/ml) and progesterone (200 ng/ml).

Techniques: Calcium Flux Assay, Activity Assay, Fluorescence