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: EMBO Molecular Medicine

Article Title: Characterization and therapy of fertilization failure in murine and human models with HNRNPR mutations

doi: 10.1038/s44321-026-00374-z

Figure Lengend Snippet: ( A ) MII oocyte and zygote fluorescence imaging reflecting fertilization after ICSI of Hnrnpr -mutated sperm. Formation of male and female pronuclei indicates successful fertilization. Tubulin labels the spindle, showing that the oocyte is at metaphase of meiosis II, and P1 marks the first polar body. The white dotted line defines the boundary of the zygotes. Scale bar = 10 μm. ( B ) Quantification of the percentage of zygotes from ( A ). Data are presented as mean ± s.d., and P values were calculated using a two-sided Student’s t test. Data in ( A , B ) represent results from six independent experiments (two technical and three biological replicates, n = 6).

Article Snippet: Rabbit anti-alpha Tubulin , MedChemExpress , Cat# HY- P86200.

Techniques: Fluorescence, Imaging

Journal: Physiological Reports

Article Title: The effect of mitoTEMPO on the development of hypoxia‐induced pulmonary hypertension in male mice

doi: 10.14814/phy2.70804

Figure Lengend Snippet: Effect of mitoTEMPO treatment on HIF‐1α stabilization in vitro. HIF‐1α protein levels were assessed by western blot, and steady‐state mRNA levels for lactate dehydrogenase A ( Ldha ) and pyruvate dehydrogenase kinase 1 ( Pdk1 ) by quantitative real‐time PCR in (a) CMT167 cells, (b) hPASMCs, and (c) mPASMCs, exposed to normoxia (21% O 2 ), severe hypoxia (1% O 2 ), or mild hypoxia (10% O 2 ) for 24 h and treated with triphenylphosphonium (TPP + ) (blue dots) or mitoTEMPO (MT) (red dots). Immunoblots shown are representative of three independent experiments. CMT167: mouse lung carcinoma epithelial cells; hPASMCs: human pulmonary artery smooth muscle cells; mPASMCs: mouse pulmonary artery smooth muscle cells. Densitometric analysis of HIF‐1α bands normalized to β‐Actin. Data are presented as mean ± SD. Statistical comparisons were made using two‐way ANOVA with Tukey's post hoc test ( n = 3 per group). (ns: no signal). Quantitative real‐time PCR data reflect mean ΔCt ± SD ( n = 3 per experimental group).

Article Snippet: Mouse lung carcinoma epithelial (CMT167) cells (10032302, Merck, Germany) and human PASMCs (hPASMCs) (C‐12521, PromoCell, Germany) were purchased.

Techniques: In Vitro, Western Blot, Real-time Polymerase Chain Reaction

Journal: JVS-Vascular Science

Article Title: Comparison between vascular smooth muscle cells isolated from descending thoracic and abdominal aortas of mice in cell function, phenotype features, and gene expression

doi: 10.1016/j.jvssci.2026.100411

Figure Lengend Snippet: (A) Schematic flowchart of vascular smooth muscle cell (VSMC) isolation from the thoracic and abdominal aortas of mice (figure created with BioRender.com). (B) Representative images of α-smooth muscle actin ( α-SMA ) staining (original magnification ×20) of thoracic aortic smooth muscle cell ( Th-SMC ) and abdominal aortic smooth muscle cell ( Ab-SMC ) at passages 3 and 7, respectively. Green indicates α-SMA, and blue means nuclei. (C) The ratio of α-SMA-positive cells was assessed. There was no significant difference in α-SMA expression between passages in each cell type (n = 4 per condition, two images per well, two wells analyzed). nd , not significant; P , passage.

Article Snippet: Wells were washed with phosphate-buffered saline and cultured in fetal bovine serum-free Smooth Muscle Cell Growth Medium (Basal medium, Cell Applications).

Techniques: Isolation, Staining, Expressing

Journal: JVS-Vascular Science

Article Title: Comparison between vascular smooth muscle cells isolated from descending thoracic and abdominal aortas of mice in cell function, phenotype features, and gene expression

doi: 10.1016/j.jvssci.2026.100411

Figure Lengend Snippet: Cell migration and proliferation were assessed to investigate the cell function between cell types. (A) Representative images of the scratch assay for thoracic aortic smooth muscle cells ( Th-SMCs ) and abdominal aortic smooth muscle cells ( Ab-SMCs ) at 0, 12, 24, and 72 hours. (B) Th-SMCs migrated into the scratched wound area (between the dotted lines ) significantly faster than Ab-SMCs at 24, 48, and 72 hours, as evaluated by the reduced wound region (n = 6 per group). (C) Representative immunofluorescence images of Ki67 ( red ) and 4′,6-diamidino-2-phenylindole (DAPI) ( blue ) staining during the scratch migration assay (0, 12, and 72 hours) under serum-free conditions. Scale bar , 100 μm. (D) Quantification of proliferative activity expressed as the Ki67/DAPI intensity ratio during the migration assay. Ki67/DAPI intensity was minimal and not significantly different between Th-SMCs and Ab-SMCs at any time point, indicating negligible proliferation under serum-free conditions. (E) Representative Ki67 staining images of Th-SMCs and Ab-SMCs under growth conditions at day 0, day 1, and day 4 corresponding with the proliferation assay. (F) Quantification of Ki67/DAPI intensity ratio under growth conditions, demonstrating increased proliferative activity over time and a significant difference between Th-SMCs and Ab-SMCs at day 4. ns , not significant. ∗∗∗ P ≤ .001; ∗∗∗∗ P ≤ .0001.

Article Snippet: Wells were washed with phosphate-buffered saline and cultured in fetal bovine serum-free Smooth Muscle Cell Growth Medium (Basal medium, Cell Applications).

Techniques: Migration, Cell Function Assay, Wound Healing Assay, Immunofluorescence, Staining, Activity Assay, Proliferation Assay

Journal: JVS-Vascular Science

Article Title: Comparison between vascular smooth muscle cells isolated from descending thoracic and abdominal aortas of mice in cell function, phenotype features, and gene expression

doi: 10.1016/j.jvssci.2026.100411

Figure Lengend Snippet: Oxidative stress assay on vascular smooth muscle cells (VSMCs) and expression of antioxidant markers in the oxidative stressed VSMCs. (A) VSMCs were treated with different concentrations of H 2 O 2 (0-750 μM) for 12 hours, followed by evaluating the cell viability using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Viability in thoracic aortic smooth muscle cells ( Th-SMCs ) was significantly higher than in abdominal aortic smooth muscle cells ( Ab-SMCs ) at 100 μM and 750 μM H 2 O 2 (n = 4 per group). To evaluate the expression of antioxidant markers in VSMCs treated with hydrogen peroxide ( H 2 O 2 ) at the same concentration used in the oxidative stress assay, reverse transcription-polymerase chain reaction was performed. (B) Nrf2 expression was significantly higher in Th-SMCs than in Ab-SMCs at 100 μM and 750 μM. (C, D) Similarly, Catalase and Sod1 expression were significantly greater in Th-SMCs at each concentration than in Ab-SMCs.

Article Snippet: Wells were washed with phosphate-buffered saline and cultured in fetal bovine serum-free Smooth Muscle Cell Growth Medium (Basal medium, Cell Applications).

Techniques: Expressing, Concentration Assay, Reverse Transcription, Polymerase Chain Reaction