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 international medical research

Article Title: Physical properties of poly(N-isopropylacrylamide) hydrogel promote its effects on cardiac protection after myocardial infarction.

doi: 10.1177/030006051204000615

Figure Lengend Snippet: FIGURE 6: Neovasculature formation in sham-operated and infarct areas of myocardium 90 days after induction of myocardial infarction (MI) in rats, as identified by immunohistochemical staining for α-smooth muscle actin. (A) Sham-operated group; (B) phosphate-buffered saline (PBS)-treated MI group; (C) Gel A-treated MI group; (D) Gel B-treated MI group (scale bars, 100 µm). Immunohistochemical staining was used to quantify (E) blood vessel density per high-power field (HPF) in each group. Black arrows show neovasculature formed in sham-operated or infarcted myocardium. aP < 0.05 compared with sham-operated group; bP < 0.05 compared with MI + PBS group; one-way analysis of variance and unpaired Student’s t-test

Article Snippet: The slides were incubated overnight at 4 °C with anti-αsmooth muscle actin primary antibody (dilution 1 : 50; Boster, Wuhan, China).

Techniques: Immunohistochemical staining, Staining, Saline

Journal: Renal Failure

Article Title: PCSK6 is a novel regulator of venous smooth muscle cell function in arteriovenous fistula remodeling

doi: 10.1080/0886022X.2026.2663246

Figure Lengend Snippet: PCSK6 is increased in smooth muscle cells of stenotic arteriovenous fistula. (A-B) Human stenotic and non-stenotic arteriovenous fistula (AVF) tissues were obtained as described in the Materials and Methods. (A) Representative images of hematoxylin and eosin (HE) staining and immunofluorescence staining for PCSK6, COL1A1, and MYH11 in tissue sections. Immunofluorescence intensity of PCSK6 in the two groups, as well as correlations between PCSK6 and COL1A1 immunofluorescence intensity, neointimal thickness, degree of luminal stenosis, and AVF blood flow were plotted. (B) Total protein and RNA were extracted from tissues. Protein expression of PCSK6 was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (C-D) Primary cultured smooth muscle cells (SMCs) were derived from human stenotic and non-stenotic AVF tissues. (C) Immunofluorescence staining for PCSK6 and the SMC marker MYH11 in primary cultured SMCs. (D) Total protein and RNA were extracted from primary cultured SMCs. Protein expression of PCSK6 was analyzed by Western blot, and mRNA expression was determined by real-time PCR.

Article Snippet: Human venous smooth muscle cells were purchased from ATCC with the Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-04).

Techniques: Staining, Immunofluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Derivative Assay, Marker

Journal: Renal Failure

Article Title: PCSK6 is a novel regulator of venous smooth muscle cell function in arteriovenous fistula remodeling

doi: 10.1080/0886022X.2026.2663246

Figure Lengend Snippet: PCSK6 is increased in smooth muscle cells during venous remodeling after arteriovenous creation. (A-B) Mouse AVF models were generated as described in the Material and Methods. (A) Tissues from the AVF anastomosis were collected at the indicated time points. Representative images of HE staining and immunofluorescence staining for PCSK6 and MYH11 are shown. Neointimal thickness and PCSK6 immunofluorescence intensity across different time points, as well as the correlation between PCSK6 intensity and neointimal thickness were plotted. (B) Total protein and RNA were extracted from the tissues. Protein expression of PCSK6 at different time points was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (C-D) Primary cultured SMCs were derived from the AVF at the indicated time points. (C) Immunofluorescence staining for PCSK6 and the SMC marker MYH11 in primary cultured SMCs. (D) Total protein and RNA were extracted from primary cultured SMCs. Protein expression of PCSK6 was analyzed by Western blot, and mRNA expression was determined by real-time PCR.

Article Snippet: Human venous smooth muscle cells were purchased from ATCC with the Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-04).

Techniques: Generated, Staining, Immunofluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Derivative Assay, Marker

Journal: Renal Failure

Article Title: PCSK6 is a novel regulator of venous smooth muscle cell function in arteriovenous fistula remodeling

doi: 10.1080/0886022X.2026.2663246

Figure Lengend Snippet: PCSK6 promotes smooth muscle cells phenotypic switch and ECM production. (A–G) Venous SMCs were transfected with control or PCSK6 expression vectors. (A) Total protein and RNA were extracted. Protein expression of COL1A1, fibronectin, VIM, and MMP2 was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (B) Cell viability was assessed using CCK-8 assay. (C) Cell proliferation was measured by BrdU assay. (D) Cell migration was evaluated by wound healing assay. (E) Cell contractility was determined by collagen gel contraction assay. (F). Hydroxyproline levels were quantified. (G) MMPs activity was measured using MMPs activity kit as described in the Material and Methods section. (H–N) PrimaryM cultured SMCs were transfected with siRNA targeting either control or PCSK6. (H) Total protein and RNA were extracted. Protein expression of COL1A1, fibronectin, VIM, and MMP2 was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (I) Cell viability was assessed using CCK-8 assay. (J) Cell proliferation was measured by BrdU assay. (K) Cell migration was evaluated by wound healing assay. (L) Cell contractility was determined by collagen gel contraction assay. (M) Hydroxyproline levels were quantified. (N) MMPs activity was measured using MMPs activity kit as described in the Material and Methods section.

Article Snippet: Human venous smooth muscle cells were purchased from ATCC with the Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-04).

Techniques: Transfection, Control, Expressing, Western Blot, Real-time Polymerase Chain Reaction, CCK-8 Assay, BrdU Staining, Migration, Wound Healing Assay, Collagen Gel Contraction Assay, Activity Assay, Cell Culture

Journal: Renal Failure

Article Title: PCSK6 is a novel regulator of venous smooth muscle cell function in arteriovenous fistula remodeling

doi: 10.1080/0886022X.2026.2663246

Figure Lengend Snippet: Silencing of PCSK6 in VSMCs alleviated venous remodeling and AVF stenosis. (A) Smooth muscle cell-specific PCSK6 knockout mice were generated as described in the Material and Methods. The schematic illustrates the experimental timeline after AVF creation in both knockout and control mice. (B) AVF diameter and blood flow were monitored by ultrasound. Quantitative data are presented. (C-D) Functional analysis of harvested IVC segments assessing (C) contraction responses to 40mM KCl and (D) Relaxation responses to the cumulative addition of acetylcholine. (E) Total protein and RNA were extracted. Protein expression of COL1A1, fibronectin, MMP2 and VIM, was analyzed by Western blot, and mRNA expression was determined by real-time PCR. (F) Histological evaluation of IVC sections through HE/EVG/Masson staining and immunofluorescence for PCSK6 and MYH11. Neointimal thickness was quantified in both experimental groups.

Article Snippet: Human venous smooth muscle cells were purchased from ATCC with the Vascular Smooth Muscle Cell Growth Kit (ATCC PCS-100-04).

Techniques: Knock-Out, Generated, Control, Functional Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Staining, Immunofluorescence