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: 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: 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

Journal: Heliyon

Article Title: Human serum albumin and oxidative stress in preeclamptic women and the mechanism of albumin for stress reduction

doi: 10.1016/j.heliyon.2017.e00369

Figure Lengend Snippet: (A) Representative images of in situ superoxide production. Blue or red fluorescence indicates nuclei and superoxide in the human coronary arterial smooth muscle cells, respectively. (B-D) Relative superoxide production in the human coronary arterial smooth muscle cells treated with the addition of L-glucose, D-glucose, D-glucose in combination with Tiron, gp91ds-tat, sgp91ds-tat, human serum albumin (BENESIS, 0.05 to 0.5 g/dL), methyl-β-cyclodextrin, gp60 antibody (20 μg/mL) or the combination for 60 min. (B) *: P < 0.05 vs. control. (C) *: P < 0.05 vs. D-glucose, and #: P < 0.05 vs. albumin (0.5 g/dL). (D) *: P < 0.05 vs. D-glucose.

Article Snippet: Human coronary arterial smooth muscle cells (Cat No. KS-4209, Kurabo Industries Ltd., Osaka, Japan) were grown in HuMedia-SG2 medium (Kurabo Industries Ltd., Osaka, Japan) at 37 °C in the humid air with 5% carbon dioxide.

Techniques: In Situ, Fluorescence, Control

Journal: Heliyon

Article Title: Human serum albumin and oxidative stress in preeclamptic women and the mechanism of albumin for stress reduction

doi: 10.1016/j.heliyon.2017.e00369

Figure Lengend Snippet: (A) Relative protein expressions of NADPH oxidase subunits, p47phox, rac-1, NOX2, and Na + /K + ATPase in the membrane fraction from human coronary arterial smooth muscle cells 60 min after addition of L-, D-glucose (20 mmol/L) or D-glucose in combination with human serum albumin (BENESIS, 0.5 g/dL). *: P < 0.05 vs. L-glucose. Note that p47phox images split were taken from the same picture in the Supplementary Figure, where the full, non-adjusted images are available. (B) Immunofluorescent image analysis in the human coronary arterial smooth muscle cells 60 min after addition of L-, D-Glucose or D-Glucose in combination with human serum albumin (BENESIS, 0.5 g/dL) from five separate experiments. P47phox (green) was localized to the cytoplasmic region whereas it migrates to the cell periphery after incubation with D-glucose.

Article Snippet: Human coronary arterial smooth muscle cells (Cat No. KS-4209, Kurabo Industries Ltd., Osaka, Japan) were grown in HuMedia-SG2 medium (Kurabo Industries Ltd., Osaka, Japan) at 37 °C in the humid air with 5% carbon dioxide.

Techniques: Membrane, Incubation

Journal: bioRxiv

Article Title: A Molecular Pathway for Arterial-Specific Association of Vascular Smooth Muscle Cells

doi: 10.1101/2019.12.27.889782

Figure Lengend Snippet: A,B, Schematic diagrams illustrating the experimental design for using the mrc1a promoter to drive ectopic mosaic expression of cxcl12b in veins. A, A Tol2(mrc1a:cxcl12b-2a-mCherry) DNA construct co-translationally expressing cxcl12b and mCherry under the control of the mrc1a promoter is injected into Tg(tagln:eGFP) transgenic zebrafish embryos at the 1 cell stage. B, At 4 dpf tol2(mrc1a:cxcl12b-2a-mCherry) -injected zebrafish larvae are analyzed for vSMC (eGFP) association at sites of mCherry (i.e. cxcl12b ) expression in the dorsal aorta and cardinal vein. C,D , Representative confocal images of the mid-trunk of 4 dpf Tg(tagln:eGFP) transgenic larvae injected with either control Tol2(mrc1a) “empty vector” (C) or Tol2(mrc1a:cxcl12b-2a-mCherry) (D). eGFP-expressing vSMCs are shown in green, cxcl12b-2a-mCherry expression in dorsal aorta (DA) or cardinal vein (CV) endothelium is shown in magenta. E , Quantification of eGFP-positive vSMC associated with the dorsal aorta (DA) or cardinal vein (CV) in 4 dpf Tg(tagln:eGFP) transgenic zebrafish injected with either control Tol2(mrc1a) “empty vector” (black columns) or Tol2(mrc1a:cxcl12b-2a-mCherry) (green columns), showing strongly increased association of vSMCs with the cardinal vein. F , Schematic diagrams showing potential models for direct (left) versus indirect (right) mechanisms for promoting arterial recruitment of vSMC via CXCL12. G , Schematic diagram illustrating the 3D pulmonary artery smooth muscle cell (PASMC) motility assay. CXCL12, PDGFB, or nothing (control) is placed within the collagen gel to determine if PASMCs migrate towards these potential chemoattractants. H, Representative lateral images of 3D collagen gels showing PASMCs within the collagen matrix for each gel condition. I , Quantification of the relative number of PASMCs invading the collagen gel. The control is set to 100% and the CXCL12 and PDGFB conditions normalized to this level of invasion. Scale bars = 75 µm (panels C,D), 200 µm (panel H). Box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box respectively). The whiskers demonstrate the spread of data within 1.5x above and below the interquartile range. All data points are shown as individual dots, with outliers shown above or below the whiskers. P-values are indicated above statistically significant datasets.

Article Snippet: Human coronary artery smooth muscle cells (PASMC, Lonza) were cultured in 10% FBS in Advanced DMEM base media (Gibco) on 1mg/ml gelatin coated tissue culture flasks.

Techniques: Expressing, Construct, Control, Injection, Transgenic Assay, Plasmid Preparation, Motility Assay

Journal: bioRxiv

Article Title: A Molecular Pathway for Arterial-Specific Association of Vascular Smooth Muscle Cells

doi: 10.1101/2019.12.27.889782

Figure Lengend Snippet: A,B, PDGFB transcript (A) and protein (B) in HUVEC cells cultured in vitro in a confluent cell monolayer for up to 8 hours with (“+CXCL12”) or without (“CTRL”) added recombinant CXCL12. Relative PDGFB transcript levels (A) and protein levels (B) were measured by qPCR and Western blot, respectively, showing an upregulation of both PDGFB transcript and PDGFB protein levels in response to stimulation by CXCL12. C-E , PDGFB transcript (C) and protein levels (D,E) in HUVEC cells cultured in vitro in a confluent cell monolayer and treated with either control, CXCR4, or CXCL12 siRNAs. Relative PDGFB transcript (C) and protein (E,F) levels were measured by qPCR and Western blot, respectively, showing suppression of both PDGFB transcript and protein in response to either CXCR4 or CXCL12 knockdown. Values in A, C, and E are averaged from three individual experiments and expressed as a percentage of control. Error bars ± s.d. (A,C). F, Confocal images of immunohistochemically stained transverse sections through the dorsal aorta of E12.5 Cxcr4+/- heterozygous sibling (F) and Cxcr4-/- mutant (G) mice, probed for platelet derived growth factor B (PDGFB; green) and for smooth muscle 22 alpha (SM22, aka transgelin) for vascular smooth muscle cells (vSMC, red). G, Quantification of relative PDGFB protein expression in Cxcr4+/- heterozygous embryos versus Cxcr4-/- homozygous mutant embryos. Values are expressed as a percentage of heterozygous control and averaged from five individual mice per condition. H , Schematic diagram of a zebrafish larva with the red box highlighting the area imaged in panels I and J. I,J, Whole mount in situ hybridization of the mid-trunk of 2.5 dpf zebrafish injected with control (I) or cxcl12b (J) RNA, showing upregulation of pdgfb transcript in response to exogenous cxcl12b . Red and blue brackets in panel J indicate the dorsal aorta and cardinal vein, respectively. K, Western blot of whole embryo protein lysate from 2.5 dpf zebrafish injected with either control (left) or cxcl12b (right) RNA, probed for pdgfb (top) or alpha tubulin (bottom), showing upregulation of pdgfb protein levels in response to exogenous cxcl12b . Images are representative of data from three individual experiments. M , Schematic diagram illustrating the proposed model for endothelial-autonomous chemokine signaling driving increased endothelial PDGFB ligand production, thereby indirectly promoting vSMC acquisition by arteries. Putative upstream regulators of CXCL12 and CXCR4 are noted in red. Scale bars = 50 µm (panel F). Box plots are graphed showing the median versus the first and third quartiles of the data (the middle, top, and bottom lines of the box respectively). The whiskers demonstrate the spread of data within 1.5x above and below the interquartile range. All data points are shown as individual dots, with outliers shown above or below the whiskers. P-values are indicated above statistically significant datasets.

Article Snippet: Human coronary artery smooth muscle cells (PASMC, Lonza) were cultured in 10% FBS in Advanced DMEM base media (Gibco) on 1mg/ml gelatin coated tissue culture flasks.

Techniques: Cell Culture, In Vitro, Recombinant, Western Blot, Control, Knockdown, Staining, Mutagenesis, Derivative Assay, Expressing, In Situ Hybridization, Injection

Journal: Cell stem cell

Article Title: Patient hiPSCs identify vascular smooth muscle arylacetamide deacetylase as protective against atherosclerosis

doi: 10.1016/j.stem.2020.04.018

Figure Lengend Snippet: (A and B) Representative immunostaining of PCNA positive cells (Scale bar: 50 μm.) and percentage of PCNA positive cells of CAVSMCs (A; n = 5) and dVSMCs (B; n = 10) infected by GFP- or AADAC- overexpressing lentivirus with or without doxycycline (Dox). one-way ANOVA with Sidak’s multiple comparisons test.

Article Snippet: Human primary coronary artery endothelial cells (CAEC) and CAVSMCs from healthy individuals were purchased from Lonza (CC-2545 and CC-2583).

Techniques: Immunostaining, Infection