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: International Journal of Molecular Sciences

Article Title: High-Content Imaging and Machine Learning Classify Phenotypical Change in Coronary Artery Endothelial Cells Caused by BPS

doi: 10.3390/ijms27073259

Figure Lengend Snippet: Representative high-content microscopy images of human coronary artery endothelial cells (HCAEC) exposed to vehicle control (CTRL) or 0.1 µM Bisphenol S (BPS) for 96 h and stained using the PhenoVue Cell Painting assay. For each condition, a representative field acquired at 40× magnification and a higher-magnification inset are shown. Rows correspond to the individual fluorescence channels: Hoechst 33342 (nuclei), PhenoVue Fluor 488 Concanavalin A (endoplasmic reticulum and intracellular membranes), PhenoVue 512 nucleic acid stain (RNA/nucleoli), PhenoVue Fluor 555 wheat germ agglutinin (plasma membrane), PhenoVue 641 mitochondrial stain (mitochondria), and the merged image. White boxes represent the part of the image used for the related inset. Scale bar: 50 µm, 40× objective.

Article Snippet: Primary human coronary artery endothelial cells (HCAEC; ATCC ® PCS-100-020TM, Innovation, VA, USA) were cultured according to the supplier’s recommendations.

Techniques: Microscopy, Control, Staining, Fluorescence, Clinical Proteomics, Membrane

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture restores gene expression of skin barrier molecules filaggrin and loricrin in the 2D psoriasis model. CaCl 2 -differentiated NHEKs were left unstimulated (control) or stimulated with a psoriasis-like cytokine mixture (IL-1β, IL-17A, IL-22 and TNF-alpha, each 10 ng/mL) either in the absence (Psoriasis) or in the presence of the plant extract (Psoriasis + Extract) for 21 h. Gene expression levels of ( a ) FLG , ( b ) LOR and ( c ) IVL were determined by real-time PCR. Statistical significance was tested by a, c) one-way ANOVA with subsequent Sidak’s multiple comparison test or b) Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12 * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Control, Real-time Polymerase Chain Reaction, Comparison

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture downregulates inflammatory markers in the 2D psoriasis model. CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . Gene expression levels of ( a ) IL1A , ( b ) IL1B, ( c ) IL1RN , ( d ) CXCL8 , ( e ) TNFA , ( f ) IL17C , ( g ) IL36G , ( h ) CSF2 , ( i ) VEGFA were measured. Statistical significance was tested by a, b, c, e, h) one-way ANOVA with subsequent Sidak’s multiple comparison test or d, f, g) Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12, * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant). Protein expression levels of ( j ) IL-8 and k) TNFα were measured in the supernatants of the cells by ELISA. Values for unstimulated control and plant extract-treated cells were below the detection limit of 31,3 pg/mL (IL-8) or 62,5 pg/mL (TNFα) and not detectable; therefore, no statistical analysis was performed. n.d.= non-detectable.

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Comparison, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture lowers upregulated antimicrobial peptide expression in the 2D psoriasis model. CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . Gene expression levels of ( a ) S100A7 , ( c ) DEFB4A and ( e ) DEFB103A were measured. Statistical significance was tested by ( a , c ) one-way ANOVA with subsequent Sidak’s multiple comparison test or ( e ) Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12). Protein expression levels of ( b ) psoriasin and ( d ) hBD2 were measured in the supernatants of the cells by ELISA. Statistical significance was tested by b) Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12) or d) one-way ANOVA with subsequent Sidak’s multiple comparison test ( n = 6, * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Expressing, Gene Expression, Comparison, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture reduces NFKBIZ and NFKBIA gene expression and IκBζ protein levels. CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . Gene expression levels of ( a ) NFKBIZ and ( c ) NFKBIA were measured and statistical significance was tested by one-way ANOVA with subsequent Sidak’s multiple comparisons test ( n = 12; * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant). ( b ) Protein expression levels of IκBζ were assessed by western blot using an IκBζ antibody. Detection of pan-actin serves as a loading control. Uncropped blots are shown in Supplementary Figure .

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Expressing, Western Blot, Control

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Plant extract mixture activates the AhR in the 2D psoriasis model. ( a ) CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . Gene expression levels of CYP1A1 were measured and statistical significance was tested by Kruskal-Wallis test with subsequent Dunn’s multiple comparisons test ( n = 12). ( b ) To determine AhR activation, NHEKs were transfected with the pGudLuc6.1 plasmid containing firefly luciferase which expression depends on AhR activation and the pGL4.74 [ hRLuc /TK] reference plasmid containing renilla luciferase. One day after transfection, cells were stimulated as described in ( a ). After cell lysis, activation of AhR was determined by measurement of relative luciferase activities. Statistical significance was tested by one-way ANOVA with subsequent Sidak’s multiple comparison test ( n = 12; ** p < 0.01; *** p < 0.001; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Expressing, Lysis, Comparison

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: Downregulation of AhR inhibits the filaggrin-inducing effects but not the anti-inflammatory effects of the plant extract in the 2D psoriasis model. NHEKs were transfected with control or AhR siRNA and stimulated as described in Fig. . Gene expression levels of ( a ) CYP1A1 , ( b ) FLG , ( c ) NFKBIZ , ( d ) TNFA , ( e ) IL36G ( f ) CXCL8 and ( g ) DEFB4A were measured. Statistical significance was determined by ( a – f ) one-way ANOVA with subsequent Sidak’s multiple comparison test or ( c ) Kruskal-Wallis with subsequent Dunn’s multiple comparison test (n = 9; * p < 0.05; ** p < 0.01; *** p 0.001; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Transfection, Control, Gene Expression, Comparison

Journal: Scientific Reports

Article Title: Plant extract mixture shows anti-inflammatory and barrier-strengthening effects and activates aryl hydrocarbon receptor in a 2D psoriasis model

doi: 10.1038/s41598-026-50000-8

Figure Lengend Snippet: The plant extract mixture exhibits antioxidant effects in the 2D psoriasis model. CaCl 2 -differentiated NHEKs were stimulated as described in Fig. . ( a ) Gene expression levels of NQO1 were measured and statistical significance was tested by one-way ANOVA with subsequent Sidak’s multiple comparisons test ( n = 12). ( b ) Intracellular reactive oxygen species were measured by DCFDA-based assay. The mean fluorescence value of the control cells was set to 100%, and the relative intracellular ROS levels of the other samples were calculated as percentages of the control. Statistical significance was tested by Kruskal-Wallis test with subsequent Dunn’s multiple comparison test ( n = 12, * p < 0.05; ** p < 0.01; *** p < 0.001; ns = not significant). ( c ) NHEKs were transfected and stimulated as described in Fig. . Gene expression levels of NQO1 were measured and statistical significance was determined by one-way ANOVA with subsequent Sidak’s multiple comparison test ( n = 9; * p < 0.05; ns = not significant).

Article Snippet: For the 2D psoriasis model, primary normal human epidermal keratinocytes (NHEKs, pooled from four donors, PromoCell, Heidelberg Germany), used at passage 4, were seeded in a 24-well plate in Derma Life Complete medium (CellSystems, Troisdorf, Germany).

Techniques: Plant Extract, Gene Expression, Fluorescence, Control, Comparison, Transfection

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Spatial ratiometric images and histograms of all pixels within each field of view are shown for dimeric amine/cys Fn-DA in 0 and 1 M GdnHCl and monomeric amine/cys Fn-DA in 1 and 4 M GdnHCl (A). Amine/cys Fn-DA was added to the culture medium of fibroblasts for 24 h, and excess Fn-u was added to suppress intermolecular energy transfer. Confocal microscopic images of acceptor and donor peak intensities taken 1 μm above the glass–cell interface were background subtracted, averaged, and thresholded, and the I A / I D ratiometric image of acceptor to donor was color-coded within the range of 0.05 to 1.0. A histogram (B) for all pixels of amine/cys Fn-DA–containing ECM (C) and an overlay of I A / I D on the DIC image (D) are shown in a region in which the matrix showed a transition from low to intermediate I A / I D within a single Fn fiber. Histograms are overlaid in (B) for regions of extended (E; purple) and unfolded Fn (F; pink). Histograms were generated with 0.01-ratio-unit bin widths. Scale bars = 25 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Generated

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Amine/cys Fn-DA and excess Fn-u were added to the culture medium of fibroblasts for 24 h. Color-coded I A / I D ratiometric images are shown for control cells (A), extracted cell-free matrix (B), and fibroblast cells after 60 min exposure to the ROCK inhibitor Y-27632 (C). Histograms with 0.01-ratio-unit bin widths for all pixels of control (black), cell-free (purple), and ROCK-inhibited matrix (pink) were derived from three random fields of view each from three separate experiments in each group (D). Solution denaturation values for dimeric Fn-DA in 0 M GdnHCl and monomeric Fn-DA in 1 and 4 M GdnHCl are shown as red, green, and blue lines, respectively. Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Control, Derivative Assay

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: A schematic of the strain device is shown in the relaxed configuration with length L before (A) and length L + DL after (B) application of strain. PDMS sheets were covalently modified with Fn-u as described in Materials and Methods, and fibroblast cells were cultured for 24 h in the presence of amine/cys Fn-DA and excess Fn-u. Cells were extracted in mild detergent. Color-coded I A / I D ratiometric images are shown for a field of view without application of stretch (C) and after application of 70% elongation strain with 28% transverse compression (D). Region of interest analysis on individual fibrils was used to determine the impact of elongation on I A / I D on a per fibril basis (circles, mean ± standard deviation), and binned averages were calculated for fibrils between −37% and −20%, −20% and −10%, −10% and 10%, 10% and 40%, and 40% and 73% strain (red squares, mean ± standard deviations) (E). Abscissa is also plotted as relative length change. Solution values for dimeric Fn-DA in 0 M GdnHCl and monomeric Fn-DA in 1 and 4 M GdnHCl are shown as horizontal red, green, and blue lines, respectively. Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Modification, Cell Culture, Standard Deviation

Journal: PLoS Biology

Article Title: Force-Induced Unfolding of Fibronectin in the Extracellular Matrix of Living Cells

doi: 10.1371/journal.pbio.0050268

Figure Lengend Snippet: Cys/cys Fn-DA (A–C) or amine/cys Fn-DA (D–G) was incorporated into fibroblast matrix on Fn-u that was adsorbed to plasma cleaned PDMS, and after cell extraction the substrate was relaxed to 4/5 (A and B; 3.7% transverse stretch) or 3/5 the starting length (D–F; 10% transverse stretch). I A / I D ratiometric images of cys/cys Fn-DA–containing matrix are shown at the PDMS–ECM interface (A), where a portion of the cell-free fibers are still attached to the substrate, and from the same field of view but acquired 3 μm above the PDMS surface (B), where the strain-free Fn mat randomly diffused around its points of attachment to the underlying ECM. Histograms are shown for all pixels within the field of view at the substrate (C; black) and from the upper, strain-free confocal slice (C; pink). An I A / I D ratiometric image of amine/cys Fn-DA is shown with both detached (E) and still-attached (F) regions of matrix within the same confocal slice. Region of interest analysis was used to generate histograms (G0 for all pixels within the detached (E and G; purple) and attached (F and G; pink) regions of matrix, which were overlaid on a histogram of all pixels in the field of view (black). Scale bars = 50 μm.

Article Snippet: Primary human dermal fibroblast cells derived from foreskins (PromoCell, http://www.promocell.com/ ) were maintained for less than eight passages in Fibroblast Growth Medium plus Supplement (PromoCell).

Techniques: Clinical Proteomics, Extraction

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 is expressed in the kidney as assessed by western blot performed on total kidney extracts. ( b ) Gata5 is essentially expressed in the glomeruli as assessed by qPCR on isolated glomeruli (Glo) and microdissected tubules (Tub) from Wt mice kidneys. ( n =3–5 per group). The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice (Mann–Whitney test). ( c , d ) Specific deletion of Gata5 in endothelial cells (e Gata5-null mice) virtually abolished renal and glomerular expression of Gata5 ( n =3 per group). The results are reported as mean±s.e.m. * P <0.05 versus eGata5 +/+ mice (Mann–Whitney test). ( e ) The expression of the glomerular genes Nphs1 (nephrin) and Nphs2 (podocin) as measured by qPCR ( n =6 per group) was increased in Gata5 -null mice. The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice; *** P <0.005 versus Gata5 +/+ mice ( t -test for nephrin; Mann–Whitney test for podocin). ( f – i ) Absence of Gata5 induces glomerular lesions (sections are stained with periodic acid Schiff; scale bar, 30 μm; n =5 per group) and renal inflammation as assessed by leucocytes CD45 immunostaining (scale bar, 200 μm; n =4 per group). The results are reported as mean±s.e.m. *** P <0.005 versus Gata5 +/+ mice (Mann–Whitney test). ( j – n ) Deletion of Gata5 from endothelial cells reproduces the renal phenotype of global Gata5 deletion: both Nephs1 and Nphs2 transcript levels were increased ( n =4–6 per group) as well as glomerular lesion score (scale bar, 30 μm; n =4–6 per group) and renal leucocytes infiltration (scale bar, 200 μm; n =4–5 per group) in e Gata5 -null mice in comparison with their controls. The results are reported as mean±s.e.m. * P <0.05 versus e Gata5 +/+ mice; *** P <0.005 versus e Gata5 +/+ mice (Mann–Whitney test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Western Blot, Isolation, MANN-WHITNEY, Expressing, Staining, Immunostaining, Comparison

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 is expressed in human cardiac microvascular (CM), coronary artery (CA), dermal microvascular (DM) and pulmonary microvascular (PM) endothelial cells. ( b , c ) The vasoconstrictor response of Gata5 -null mice mesenteric arteries to norepinephrine is unaltered ( n =7 per group), while the vasodilatory response to acetylcholine is decreased ( n =10–11 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (comparison of best-fit values—effector concentration for half-maximum response (EC 50 ) and Hill slope—using an F-test). ( d – f ) Deletion of Gata5 in endothelial (e Gata5 -null mice; n =4–5 per group) but not smooth muscle cells (sm Gata5 -null mice; n =5 per group) decreases mesenteric arteries sensitivity to acetylcholine and increases BP (e Gata5 -null mice n =6–10 per group; sm Gata5 -null mice n =6–9 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (two-factor ANOVA). ( g ) The vasodilatory response of Gata5 -null mice to diethylamine NONOate, an NO donor, is unaltered ( n =7 per group). The results are reported as mean±s.e.m. (comparison of best-fit values—EC 50 and Hill slope—using an F-test). ( h , i ) NOS3 and Akt phosphorylation are decreased in Gata5 -null mice mesenteric arteries. PTEN and PDK1 phosphorylation and expression are unaltered ( n =5–7 per group). Phosphorylated proteins are normalized to total proteins. Total proteins are normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice ( t -test). ( j ) Quantification of protein nitrotyrosination in mesenteric arteries of Gata5 -null mice and their controls as measured by ELISA. 3-Nitrotyrosine content is expressed as picomole of nitrotyrosine per milligram of protein ( n =5–7 per group). The results are reported as mean±s.e.m. ( t -test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Comparison, Concentration Assay, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 expression is significantly decreased in human dermal microvascular endothelial cells infected with a lentiviral vector containing an anti- GATA5 shRNA (HDMEC-GATA5-KD). Control cells were infected with a vector containing a control shRNA (targets no known mammalian gene) (HDMEC-pLKO-Ctrl, referred here as Ctrl). ( b ) Heatmap representation of the differentially regulated genes between HDMEC-GATA5-KD cells and their controls as identified by transcriptomic analysis. Colour is function of Log2 RMA (Affymetrix microarray, n =3 per group). ( c ) Functional analysis of the differentially regulated genes between HDMEC-GATA5-KD cells and their controls. Protein kinase A pathway is the most significantly enriched pathway. Fisher's exact test P value. ( d ) Validation by qPCR (upper panel) of genes predicted by microarray (lower panel) to be up- and downregulated in HDMEC-GATA5-KD endothelial cells. ( n =5 wells per condition). Downregulated genes: PRKACB codes for the PKA catalytic subunit β, PRKAR2B for the PKA regulator subunit 2β and PRKAA2 for the AMPK catalytic subunit α2. Upregulated genes: ICAM1 codes for the intercellular adhesion molecule 1, BMP4 for the bone morphogenetic protein 4 and IL6 for the interleukin 6. The results are reported as mean±s.e.m. ** P <0.01 versus Ctrl ( t -test). ( e ) Western blot representation of phospho-NOS3, NOS3 (Ser1177) and phospho-(Ser/Thr) PKA substrate motif in HDMEC-GATA5-KD cells and their controls. ( f ) Phosphorylation of NOS3 on Ser1177 is decreased in HDMEC-GATA5-KD cells (performed twice, 2–3 wells per condition). Phospho-NOS3 is normalized to total NOS3. NOS3 is normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Ctrl (Mann–Whitney test). ( g ) Phosphorylation of (Ser/Thr) PKA substrate motif is decreased in HDMEC-GATA5-KD cells (performed twice, 2–3 wells per condition). Phospho-(Ser/Thr) PKA substrate motif (between 25 and 250 kDa) is normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Ctrl (Mann–Whitney test). ( h ) Western blot representation of phospho-(Ser/Thr) PKA substrate motif in mesenteric arteries of Gata5 -null mice and their controls. ( i ) In mesenteric arteries of Gata5 -null mice, there is a trend to decrease in the (Ser/Thr) PKA substrate motif phosphorylation ( n =4–5 per group). Phospho-(Ser/Thr) PKA substrate motif (between 25 and 250 kDa) is normalized to actin. The results are reported as mean±s.e.m. (Mann–Whitney test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Expressing, Infection, Plasmid Preparation, shRNA, Control, Microarray, Functional Assay, Biomarker Discovery, Western Blot, Phospho-proteomics, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) Administration of hydralazine (a smooth muscle cell relaxant) for 4 weeks decreased blood pressure similarly in both Gata5 -null mice and their controls ( n =5–7 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons). ( b ) Hydralazine had no effect on Gata5 -null mice endothelial dysfunction ( n =5–7 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (comparison of best-fit values—EC 50 and Hill slope—using an F-test). ( c ) Hydralazine decreased partially glomerular injuries in Gata5 -null mice (sections are stained with periodic acid Schiff; scale bar, 30 μm; n =5–7 per group). * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons). ( d ) Hydralazine decreased completely renal CD45+ cells infiltration in Gata5 -null mice and also their controls (scale bar, 200 μm; n =5–7 per group). * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Comparison, Staining

Journal: bioRxiv

Article Title: Temporal AI model predicts drivers of cell state trajectories across human aging

doi: 10.64898/2026.03.30.715396

Figure Lengend Snippet: ( A ) Predicted impact of in silico inhibition vs. slope of expression change across human aging for genes in cardiac fibroblasts. ( B ) Concordant genes whose inhibition was predicted to be rejuvenating were upregulated across aging, and vice versa. ( C ) Predicted impact of in silico inhibition of genes across the indicated cardiac cell types. ( D ) Beta-gal staining and cell counts quantifying senescence of primary human cardiac capillary endothelial cells in response to predicted pro-aging perturbation of ZBTB16 inhibition. *p<0.05, Wilcoxon rank sums, n=4. ( E ) Predicted impact of in silico perturbation and expression change across human aging and in telomere-shortened mice in each cardiac cell type for top predicted cardiomyocyte age-modulating targets. ( F ) PCA plot and ( G ) differential gene expression heatmap of transcriptional response (measured by bulk RNA sequencing) to predicted pro-aging perturbations (AAV overexpression of indicated genes vs. GFP) in human iPSC-derived cardiomyocytes. p<0.05, Wald test with BH correction, n=4. ( H ) Gene set enrichment of genes differentially expressed in response to all predicted pro-aging perturbations in human iPSC-derived cardiomyocytes (hypergeometric test with g:Set Counts and Sizes (g:SCS) correction). ( I ) Slowed calcium cycle kinetics and ( J ) rhythm irregularities in response to predicted pro-aging perturbations (AAV overexpression of indicated genes vs. GFP) in human iPSC-derived cardiomyocytes. *p<0.05, Wilcoxon rank sums with BH correction. Rhythm n=10, time to peak n=180, 111, 139, 37, 237, 150, 191 (bars left to right), decay n=169, 30, 86, 11, 213, 105, 172 (bars left to right). ( K ) Differential gene expression heatmap of transcriptional response (measured by bulk RNA sequencing) to predicted pro-aging perturbations (AAV overexpression of indicated genes vs. GFP) in human iPSC-derived cardiomyocytes or human primary cardiac fibroblasts where dysregulation of the given gene was statistically significant across all four conditions. Statistical significance of differential expression: p<0.05, Wald test with BH correction, n=4. Statistical significance of overlap of shared dysregulation between the four conditions: p<0.05, one-sided binomial test. RASG.=RASGEF1B. ( L ) Beta-gal staining quantifying senescence of primary human cardiac fibroblasts in response to predicted pro-aging perturbations (AAV overexpression of indicated genes). *p<0.05, Wilcoxon rank sums with BH correction, empty n=6, P4HA1 and RASGEF1B n=4. ( M ) Schematic of in vivo validation experiment. o/e=overexpression. ( N ) In vivo systolic function echocardiographic measurements at 6 weeks post-AAV9 injection. EF: GFP n=8, P4ha1 n=10, Rasgef1b n=10; GLS: GFP n=8, P4ha1 n=9, Rasgef1b n=8. *p<0.05 one-way ANOVA with multiple hypothesis correction.

Article Snippet: Human cardiac capillary endothelial cells were purchased from PromoCell (C-12285) and cultured in endothelial basal medium (EBM, CC-3121, Lonza) supplemented with 10% fetal bovine serum (10100147, Gibco).

Techniques: In Silico, Inhibition, Expressing, Staining, Gene Expression, RNA Sequencing, Over Expression, Derivative Assay, Quantitative Proteomics, In Vivo, Biomarker Discovery, Injection