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 American Journal of Tropical Medicine and Hygiene

Article Title: Dengue Virus Infection of Primary Human Smooth Muscle Cells

doi: 10.4269/ajtmh.18-0175

Figure Lengend Snippet: Human umbilical artery smooth muscle cells (HUASMC) are permissive to dengue virus (DENV) infection. Virion production and release was quantified by plaque assays of culture supernatants at different time points post-infection (p.i.) of HUASMC, human umbilical vein endothelial cells (HUVEC), and LLC-MK2 (macaque kidney cells) infected with each of the four DENV serotypes (multiplicity of infection:1). (A) Infection kinetics of HUASMC cells (24–72 hours p.i.) with DENV 1–4 measured by plaque assays (plaque-forming units [PFU]/mL). (B) DENV 1–4 titers in culture supernatants of HUASMC, HUVEC, and LLC-MK2 cells at 72 hours p.i. Data are expressed as the mean ± standard deviation of three independent experiments. ***P < 0.001 compared with its HUVEC and LLC-MK2 cells counterparts. (D) l. = assay detection limit.

Article Snippet: Human umbilical artery smooth muscle cells and HUVEC were purchased and maintained in smooth muscle cell growth medium and endothelial cell growth medium, respectively, according to the manufacturer’s instructions (Cell Applications, San Diego, CA).

Techniques: Virus, Infection, Standard Deviation

Journal: The American Journal of Tropical Medicine and Hygiene

Article Title: Dengue Virus Infection of Primary Human Smooth Muscle Cells

doi: 10.4269/ajtmh.18-0175

Figure Lengend Snippet: Human umbilical artery smooth muscle cells (HUASMC) release dengue virus (DENV) genomes on infection. Dengue virus genomic RNA was quantified by real-time qRT-PCR from cell culture supernatants of HUASMC, human umbilical vein endothelial cells (HUVEC), and LLC-MK2 (macaque kidney cells) infected with DENV (multiplicity of infection:1). (A) Dengue virus 1–4 infection kinetics (24–72 hours post-infection [p.i.]) of HUASMC cells measured by real-time genomic qRT-PCR (copies/mL). (B) Genome copies present in culture supernatants of HUASMC, HUVEC, and LLC-MK2 cells at 72 hours p.i. with the four DENV serotypes. (C) Calculated genome-to-plaque-forming unit (PFU) ratios of HUASMC, HUVEC, and LLC-MK2 cells supernatants at 72 hours p.i. with each DENV serotype. Data are expressed as the mean ± standard deviation of three independent experiments. *P < 0.05, **P < 0.005, and ***P < 0.001 calculated to its HUASMC counterpart.

Article Snippet: Human umbilical artery smooth muscle cells and HUVEC were purchased and maintained in smooth muscle cell growth medium and endothelial cell growth medium, respectively, according to the manufacturer’s instructions (Cell Applications, San Diego, CA).

Techniques: Virus, Infection, Quantitative RT-PCR, Cell Culture, Standard Deviation

Journal: The American Journal of Tropical Medicine and Hygiene

Article Title: Dengue Virus Infection of Primary Human Smooth Muscle Cells

doi: 10.4269/ajtmh.18-0175

Figure Lengend Snippet: Dengue virus (DENV) antigens are detected in human umbilical artery smooth muscle cells (HUASMC). Epifluorescence images of immunostained cells with an anti-DENV 1-4 envelope protein-specific monoclonal antibody (green), and a cytoplasmic (red) and nuclei (blue) counterstains. The images were captured at 72 hours post-infection (p.i.) with each DENV serotype at a multiplicity of infection (MOI) of 1. (A) Representative image of mock-infected HUASMC cells at ×100 magnification (scale bar = 50 μm). (B and C) Representative images of DENV-infected HUASMC (arrows) at ×400 (scale bar = 20 μm) and ×100 (scale bar = 50 μm) magnification, respectively. (D) Image analysis for quantifying infected cells (green outline) vs total cell nuclei (white outlines) with the software CellProfiler 2.0. (E) Percentages of infected cells in HUASMC, human umbilical vein endothelial cells (HUVEC), and LLC-MK2 (macaque kidney cells) cell lines. (F) Comparison of DENV labeling by immunostaining with an anti-DENV 1-4 envelope protein-specific monoclonal antibody (green) and an anti-NS3 polyclonal antibody (red) in HUASMC cells at 72 hours p.i. with DENV-2 and DENV-3 at a MOI of 1. Magnification of ×400 (scale bar = 20 μm). Data are expressed as mean ± standard deviation of three independent experiments. **P < 0.005, ***P < 0.001 calculated to its HUASMC cells counterpart. This figure appears in color at www.ajtmh.org.

Article Snippet: Human umbilical artery smooth muscle cells and HUVEC were purchased and maintained in smooth muscle cell growth medium and endothelial cell growth medium, respectively, according to the manufacturer’s instructions (Cell Applications, San Diego, CA).

Techniques: Virus, Infection, Software, Comparison, Labeling, Immunostaining, Standard Deviation

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: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Bone morphogenetic protein (BMP)-4, transforming growth factor (TGF)-β1, serotonin (or 5-hydroxytryptamine; 5-HT), endothelin (ET)-1, and glycogen synthase kinase (GSK)-3β inhibitors increase pulmonary smooth muscle cell size and protein synthesis. A: change in forward scatter in human pulmonary artery smooth muscle cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, SB-216763, and EGF. B: overall protein synthesis of cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763, as assessed by [3H]leucine incorporation (cpm/well). C: Overall DNA synthesis of cells treated with PBS, BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763, as assessed by [3H]thymidine incorporation (cpm/well); n = 3, means ± SE; *P < 0.05, ANOVA.

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: DNA Synthesis

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Phosphorylation of GSK-3β is required for BMP-4-, TGF-β1-, 5-HT-, and ET-1-induced hypertrophy. A: representative immunoblots for phospho-GSK-3β and total GSK-3β in human pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763. B: GSK-3β-A9 was expressed in A7R5 cells via retroviral gene transfer. Expression of GSK-3β-A9 acts as a “dominant-negative,” decreasing the binding of upstream kinases and scaffolding proteins to native GSK-3β. This leads to a relative reduction of phosphorylated, inactive GSK-3β, and an increase in GSK-3β activity. C: effect of GSK-3β-A9 overexpression on the size of cells treated with BMP-4, TGF-β1, 5-HT, ET-1, LiCl, or SB-216763 (*different from MSCV-transduced cells, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Expressing, Dominant Negative Mutation, Binding Assay, Scaffolding, Activity Assay, Over Expression

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Mechanism of GSK-3β-mediated cell hypertrophy. A: representative immunoblots for phospho- and total eIF2B in pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, ET-1, and GSK-3β inhibitors. B: effect of BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763 on serum response factor (SRF) reporter activity. A7R5 cells were transiently transfected with SV40 Renilla luciferase vector and SRF-luc. Forty-eight hours after treatment, cells were lysed and luciferase activity determined. Each stimulus increased SRF activity (n = 8, means ± SE; *different from control cells, P < 0.05, ANOVA). C: effect of BMP-4, TGF-β1, 5-HT, ET-1, LiCl, and SB-216763 on α-actin mRNA in human pulmonary artery cells. Cells were treated for 4 days and processed for qPCR analysis of α-actin mRNA levels relative to GAPDH mRNA. Each stimulus increased α-actin mRNA (n = 3, means ± SE, *different from control cells, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Activity Assay, Transfection, Luciferase, Plasmid Preparation

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: BMP-4, TGF-β1, 5-HT, and ET-1 activate the p70S6K signaling pathway. A: representative immunoblots for phospho-p70S6K, total p70S6K (top), phospho-S6, and total S6 (bottom) in pulmonary artery smooth muscle cells treated with BMP-4, TGF-β1, 5-HT, and ET-1. B: group mean data (n = 3, ± SE, *different from unstimulated cells, P < 0.05, ANOVA). C: specific siRNAs against p70S6K (top) and S6 (bottom) block phosphorylation of these proteins. D: group mean data (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Western Blot, Blocking Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: Pulmonary artery smooth muscle hypertrophy: roles of glycogen synthase kinase-3β and p70 ribosomal S6 kinase

doi: 10.1152/ajplung.00108.2009

Figure Lengend Snippet: Activation of the p70S6K pathway is required for cell hypertrophy. Pulmonary artery smooth muscle cells were transfected with either nontargeting siRNA, specific siRNA against p70S6K (A), or siRNA against S6 (B), and treated with BMP-4, TGF-β1, 5-HT, or ET-1. Cell size was measured by flow cytometry. C: representative immunoblots for α-actin and β-actin from cells transfected with either nontargeting siRNA, p70S6K siRNA, or S6 siRNA. D: group mean data for p70S6K siRNA experiments (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA). E: group mean data for S6 siRNA experiments (n = 3, ± SE, *different from nontargeting siRNA, P < 0.05, ANOVA).

Article Snippet: Human pulmonary artery smooth muscle cells were obtained from Lonza (Conshohocken, PA).

Techniques: Activation Assay, Transfection, Flow Cytometry, Western Blot

Journal: Nature medicine

Article Title: KLF4 Dependent Phenotypic Modulation of SMCs Plays a Key Role in Atherosclerotic Plaque Pathogenesis

doi: 10.1038/nm.3866

Figure Lengend Snippet: ( a ) SMCs within advanced atherosclerotic lesion specimens were identified based on PLA detection of the SMC specific stable epigenetic signature H3K4dime on the MYH11 . MYH11 H3K4dime PLA + cells exhibit a punctate red dot within the nucleus while the non-nuclear amorphous red staining is autofluorescence or non-specific background. ( a ) Samples were also immuno-stained for CD68 (green), and DAPI (blue). Results showed three distinct cell populations highlighted in enlarged panels to the right and indicated with white arrows: (i) MYH11 H3K4dime PLA + SMCs that are CD68 − , (ii) MYH11 H3K4dime PLA − CD68 + (HSC-derived Mϕs), and (iii) H3K4dime MYH11 H3K4dime PLA + CD68 + SMC-derived Mϕ-like cells. Scale bar = 100 μm. ( b ) Shoulder regions within plaques [stained with DAPI (blue), ACTA2 (green), PLA (red), and CD68 (cyan)] exhibited a high incidence of SMC-derived Mϕ-like cells ( MYH11 H3K4dime PLA + CD68 + ) (yellow arrows) and several phenotypically modulated SMCs negative for CD68 ( MYH11 H3K4dime PLA + ACTA2 − CD68 − ) (white arrows). Scale bar = 50 μm. ( c ) Quantitative analysis of SMC-derived Mϕ-like cells within human coronary lesions based on MYH11 H3K4dime ISH-PLA +/− adjustment for the efficiency of PLA . Error bars = S.E.M. for 12 independent samples of human atherosclerosis in the right coronary artery. ( d ) Combined epigenetic SMC and genetic HSC lineage tracing analyses of cross gender human heart transplant samples. Coronary artery specimens from a male patient who received a female heart were processed for MYH11 H3K4dime PLA (red), Y-chromosome FISH (green), and CD68 staining (yellow). Results show cells that were MYH11 H3K4dime PLA + Y-chromosome − and CD68 + (yellow arrows) reflecting a SMC-derived Mϕ-like cell not of hematopoietic origin (top). In contrast, Mϕs of hematopoietic origin are MYH11 H3K4dime PLA − Y-chromosome + CD68 + (red arrows) (bottom). Scale bar = 50 μm.

Article Snippet: Human coronary SMC were purchased from Lonza and cultured in SMC maintaining media (Lonza).

Techniques: Staining, Derivative Assay