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: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Regulatory role of HSPB1 in endothelial cell EndoMT (A) Western blot shows HSPB1 expression in HUVECs following lentiviral-mediated overexpression (LV-HSPB1) or knockdown (LV-HSPB1-RNAi); β-actin served as a loading control. (B) Quantification of HSPB1/β-actin ratio shows significant differences between groups. (C) Representative images of Transwell migration assays evaluating the effect of HSPB1 on TGF-β1–induced endothelial migration (scale bars, 100 μm). (D) Quantification of migrated cells per field. (E) Representative tube formation images showing the effect of HSPB1 modulation on TGF-β1–induced angiogenic activity (scale bars, 200 μm). (F–H) Quantitative analysis of tube formation parameters, including the number of branches (F), loops (G), and total tube length (H), measured using ImageJ software. Data are presented as mean ± SD ( n ≥ 6). Exact p values are indicated in the graphs. Statistical analyses were performed using one-way ANOVA followed by a Bonferroni post hoc test.

Article Snippet: Human umbilical vein endothelial cells (HUVECs; primary, pooled donors; ATCC) were purchased through an authorized distributor in China and originally sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Western Blot, Expressing, Over Expression, Knockdown, Control, Migration, Activity Assay, Software

Journal: iScience

Article Title: Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis

doi: 10.1016/j.isci.2026.115028

Figure Lengend Snippet: Proposed model of HSPB1-mediated redox regulation of TGF-β1 maturation during post-MI fibrosis. During myocardial fibrosis following myocardial infarction, the expression of HSPB1 is markedly upregulated in the peri-infarct region. Upon activation, HSPB1 exposes its reactive cysteine residue (Cys137), which may interact with critical cysteine sites within pre-pro-TGF-β1, thereby influencing its redox-dependent folding and disulfide bond formation. This interaction potentially interferes with the maturation and secretion of active TGF-β1 into the extracellular space. Reduced secretion of mature TGF-β1 limits Smad2/3 phosphorylation and endothelial-to-mesenchymal transition, ultimately alleviating myocardial fibrosis. The red dashed box highlights the hypothesized redox regulatory interaction between HSPB1 and pre-pro-TGF-β1, which requires further biochemical validation.

Article Snippet: Human umbilical vein endothelial cells (HUVECs; primary, pooled donors; ATCC) were purchased through an authorized distributor in China and originally sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Activation Assay, Residue, Phospho-proteomics, Biomarker Discovery

Journal: bioRxiv

Article Title: Kinetic proofreading as a mechanism for transcriptional specificity in living human cells

doi: 10.64898/2026.03.17.711757

Figure Lengend Snippet: (A) Schematic for transcriptional regulation by ligand-inducible nuclear receptors. (B) Experimental designs for either 2 or 4h activation of GR, RXR, RARa, and PPARy nuclear receptors using synthetic ligands in human bronchial epithelial cells. (C) Volcano plots showing differentially expressed genes by RNA-seq after activation of nuclear receptors 2h post-treatment (dashed lines indicate adj p-val threshold of 0.05). (D-E) RNA-seq genome browser tracks (D) and read count values (E) at the ERRFI1 and MYH9 loci at the indicated ligand treatments. (F) GR-ChIP and H3K27ac-ChIP genome browser tracks at ERRFI1 in the absence and presence of Dex. (G) Summary of GR transcription factor acting as a specific transcription factor for ERRFI1 and non-specific transcription factor for MYH9 gene.

Article Snippet: Cells were cultured in Airway Epithelial Cell Basal Medium (ATCC, PCS-300-030) supplemented with 1% penicillin/streptomycin and Bronchial Epithelial Cell Growth Kit (ATCC, PCS-300-040) containing HLL Supplement, L-glutamine, Extract P, and Airway Epithelial Cell Supplement (“complete medium”).

Techniques: Activation Assay, RNA Sequencing