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: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Activin A–Endothelin-1 Axis Governs Pulmonary Vascular Remodeling: Mechanistic Basis for Emerging Therapies in PAH

doi: 10.1161/ATVBAHA.125.323681

Figure Lengend Snippet: Activin A induces excessive ET-1 (endothelin-1) production in pulmonary artery endothelial cells (PAECs), reversible by follistatin or bosentan. A and B , Quantitative real-time polymerase chain reaction analysis of ET-1 mRNA expression ( A , n=6 biologically independent samples per group) and ELISA measurement of ET-1 concentration in culture medium ( B , n=3–4) from PAECs treated for 6 hours with recombinant activin A (100 ng/mL) or vehicle. C and D , ET-1 mRNA expression ( C , n=6) and secreted ET-1 concentration ( D , n=4-5) in PAECs 48 hours after INHBA (inhibin β-A) overexpression (OE) or GFP (green fluorescent protein) control retroviral transfection. E and F , INHBA mRNA expression ( E , n=6) and activin A concentration in culture medium ( F , n=4) in PAECs treated for 6 hours with recombinant ET-1 (100 nmol/L) or vehicle. G , ET-1 mRNA expression in PAECs treated for 6 hours with vehicle or recombinant activin A in the presence of vehicle (VEH), FST (follistatin; 100 ng/mL), bosentan (BOS; 10 μM), or both (FST+BOS; n=4). H , ET-1 mRNA expression in PAECs 48 hours after INHBA OE or GFP transfection, followed by 24 hours of treatment with VEH, FST (100 ng/mL), BOS (10 μM), or FST+BOS (n=4). I , ET-1 mRNA expression in PAECs treated for 6 hours with vehicle or recombinant activin A in the presence of VEH or ACTRIIA-Fc (activin receptor type IIa fusion protein; 2500 ng/mL; n=3-4). J , ET-1 mRNA expression in PAECs 48 hours after INHBA OE or GFP transfection, followed by 24 hours of treatment with VEH or ACTRIIA-Fc (2500 ng/mL; n=4). Data are mean±SEM. P <0.05 is deemed statistically significant. Statistical tests: 2-sided Student t test for A through F ; 1-way ANOVA with Tukey post hoc test for G and H .

Article Snippet: Human pulmonary artery ECs (PAECs; no. C-12241; PromoCell) and pulmonary artery smooth muscle cells (PASMCs; no. C-12521; PromoCell) were cultured in HuMedia-EG2 (Kurabo) and smooth muscle cells growth medium 2 (PromoCell), respectively, at 37 °C and 5% CO 2 .

Techniques: Real-time Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Recombinant, Over Expression, Control, Retroviral, Transfection

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Activin A–Endothelin-1 Axis Governs Pulmonary Vascular Remodeling: Mechanistic Basis for Emerging Therapies in PAH

doi: 10.1161/ATVBAHA.125.323681

Figure Lengend Snippet: Activin A–driven ET-1 (endothelin-1) contributes to pulmonary artery endothelial cell (PAEC) dysfunction. A and B , Representative images ( A ) and quantification ( B ) of chord length and number of branching points in a Matrigel tube formation assay (n=3–4) using PAECs transfected with GFP (green fluorescent protein) or INHBA (inhibin β-A) overexpression (OE), treated with vehicle (VEH), FST (follistatin; 100 ng/mL), bosentan (BOS; 10 μM), or both (FST+BOS). C and D , Representative images ( C ) and quantification ( D ) of apoptotic cells assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining (n=3–4) under serum starvation. Apoptotic cells are indicated by white arrows. E , Cell proliferation measured by WST-1 (water-soluble tetrazolium-1) assay in GFP- or INHBA OE–transfected PAECs treated with VEH, FST, BOS, or FST+BOS (n=3). Data are mean±SEM. P <0.05 is deemed statistically significant. Statistical test: 1-way ANOVA with Tukey post hoc test for B , D , and E . AU indicates a bsorbance units; and DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: Human pulmonary artery ECs (PAECs; no. C-12241; PromoCell) and pulmonary artery smooth muscle cells (PASMCs; no. C-12521; PromoCell) were cultured in HuMedia-EG2 (Kurabo) and smooth muscle cells growth medium 2 (PromoCell), respectively, at 37 °C and 5% CO 2 .

Techniques: Tube Formation Assay, Transfection, Over Expression, TUNEL Assay, Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Activin A–Endothelin-1 Axis Governs Pulmonary Vascular Remodeling: Mechanistic Basis for Emerging Therapies in PAH

doi: 10.1161/ATVBAHA.125.323681

Figure Lengend Snippet: Endothelial cell (EC)–derived activin A–induced ET-1 (endothelin-1) alters vasoconstrictive properties of the pulmonary vasculature. A , eNOS (endothelial NO synthase) mRNA expression in pulmonary artery endothelial cells (PAECs) 48 hours after GFP (green fluorescent protein) or INHBA (inhibin β-A) overexpression (OE), treated with vehicle (VEH), FST (follistatin; 100 ng/mL), bosentan (BOS; 10 μM), or FST+BOS for 24 hours (n=3). B , Representative immunoblots and quantification of p-eNOS (phosphorylated eNOS) and total eNOS under the same conditions (n=3–4). C , ET-1 concentration in pulmonary artery smooth muscle cell (PASMC) culture medium after coculture with PAECs treated with recombinant activin A for 24 or 48 hours (n=3). D , ET-1 and INHBA mRNA expression in PASMCs cocultured for 48 hours with GFP- or INHBA OE–PAECs, with VEH, FST, BOS, or FST+BOS added for the last 24 hours (n=3–4). E , PASMC mRNA expression of PCNA (proliferating cell nuclear antigen), fibronectin, SM22α (smooth muscle protein 22-α), and α-SMA (α-smooth muscle actin) under the same conditions (n=3–4). F , Representative immunoblots and quantification of MYH11 (myosin heavy chain 11), SM22α, and MMP2 (matrix metalloproteinase-2) in PASMCs cocultured with GFP- or INHBA OE–PAECs, treated as in E (n=3). Data are mean±SEM. P <0.05 is deemed statistically significant. Tests: 2-sided Student t test for C ; 1-way ANOVA with Tukey post hoc test for A , B , D through F .

Article Snippet: Human pulmonary artery ECs (PAECs; no. C-12241; PromoCell) and pulmonary artery smooth muscle cells (PASMCs; no. C-12521; PromoCell) were cultured in HuMedia-EG2 (Kurabo) and smooth muscle cells growth medium 2 (PromoCell), respectively, at 37 °C and 5% CO 2 .

Techniques: Derivative Assay, Expressing, Over Expression, Western Blot, Concentration Assay, Recombinant

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Activin A–Endothelin-1 Axis Governs Pulmonary Vascular Remodeling: Mechanistic Basis for Emerging Therapies in PAH

doi: 10.1161/ATVBAHA.125.323681

Figure Lengend Snippet: Activin A–derived ET-1 (endothelin-1) drives multiple proremodeling pathways in pulmonary artery endothelial cells (PAECs). A , mRNA expression of BMP4 (bone morphogenetic protein 4), SLUG (snail family transcriptional repressor 2), SNAIL (snail family transcriptional repressor 1), VE-cadherin (vascular endothelial cadherin), fibronectin, and SOD2 (superoxide dismutase 2) in GFP (green fluorescent protein)- or INHBA (inhibin β-A) overexpression (OE)–PAECs treated with vehicle (VEH), FST (follistatin), bosentan (BOS), or FST+BOS for 24 hours (n=3–4). B , Representative immunoblots and quantification of vimentin, SOD2, and NRF2 (nuclear factor, erythroid 2-related factor 2) under the same conditions (n=3). C , Reactive oxygen species (ROS) production in GFP- or INHBA OE–PAECs treated as in A , measured at 4, 6, 16, and 24 hours (n=3–4). Data are mean±SEM. P <0.05 is deemed statistically significant. Tests: 1-way ANOVA with Tukey post hoc test for A and B ; 2-way ANOVA with Tukey post hoc test for C .

Article Snippet: Human pulmonary artery ECs (PAECs; no. C-12241; PromoCell) and pulmonary artery smooth muscle cells (PASMCs; no. C-12521; PromoCell) were cultured in HuMedia-EG2 (Kurabo) and smooth muscle cells growth medium 2 (PromoCell), respectively, at 37 °C and 5% CO 2 .

Techniques: Derivative Assay, Expressing, Over Expression, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Activin A–Endothelin-1 Axis Governs Pulmonary Vascular Remodeling: Mechanistic Basis for Emerging Therapies in PAH

doi: 10.1161/ATVBAHA.125.323681

Figure Lengend Snippet: Canonical SMAD2/3 signaling mediates activin A–induced ET-1 (endothelin-1) expression. A , Representative immunoblots and quantification of p-SMAD2/3 (phosphorylated SMAD2/3; small mother against decapentaplegic family member 2/3) and total SMAD2/3 in GFP (green fluorescent protein)- or INHBA (inhibin β-A) overexpression (OE)–pulmonary artery endothelial cells (PAECs) treated with vehicle (VEH), FST (follistatin), bosentan (BOS), or FST+BOS for 24 hours (n=3). B , Immunoblots showing p-SMAD2/3 and total SMAD2/3 in GFP- or INHBA OE–PAECs treated with SB505124 (5 μM) or vehicle for 24 hours. C , ET-1 mRNA expression in GFP- or INHBA OE–PAECs treated with SB505124 or vehicle for 24 hours ( left , n=3) and in PAECs treated for 6 hours with activin A±SB505124 ( right , n=3–4). D , Immunoblots showing SMAD2/3 and β-actin in PAECs pretreated with SMAD2 siRNA (small interfering RNA; siSMAD2), SMAD3 siRNA (siSMAD3), dual SMAD2-SMAD3 siRNA (dual siSMAD), or control siRNA (siNC [siRNA negative control]). E , ET-1 mRNA expression in GFP- or INHBA OE–PAECs pretreated with siSMAD2, siSMAD3, dual siSMAD, or siNC (n=3–4). F , INHBA and ET-1 mRNA expression in PAECs exposed to normoxia or hypoxia (0.1% O 2 , 24 hours; n=6). G and H , ET-1 mRNA expression in PAECs under hypoxia treated with FST ( G ) or SB505124 ( H ; n=3). Data are mean±SEM. P <0.05 is deemed statistically significant. Tests: 1-way ANOVA with Tukey post hoc test for A , C , E , G , and H ; 2-sided Student t test for F .

Article Snippet: Human pulmonary artery ECs (PAECs; no. C-12241; PromoCell) and pulmonary artery smooth muscle cells (PASMCs; no. C-12521; PromoCell) were cultured in HuMedia-EG2 (Kurabo) and smooth muscle cells growth medium 2 (PromoCell), respectively, at 37 °C and 5% CO 2 .

Techniques: Expressing, Western Blot, Over Expression, Small Interfering RNA, Control, Negative Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Activin A–Endothelin-1 Axis Governs Pulmonary Vascular Remodeling: Mechanistic Basis for Emerging Therapies in PAH

doi: 10.1161/ATVBAHA.125.323681

Figure Lengend Snippet: In vivo activin A inhibition improves pulmonary hypertension (PH) phenotype comparably or more than ET-1 (endothelin-1) blockade. A , Experimental design: wild-type (WT) and VE-cadherin (vascular endothelial cadherin)–INHBA (inhibin β-A)-Tg (TG/transgenic) mice were exposed to hypoxia (10% O 2 ) for 3 weeks, with vehicle (VEH), FST (follistatin; 8.5 μg/kg), bosentan (BOS; 30 mg/kg), or FST+BOS administered during the final 2 weeks. B , Right ventricular systolic pressure (RVSP; n=4–9). C , Fulton index (RV/[LV+S] [right ventricle to left ventricle plus septum] ratio; n=4–8). D , Representative hematoxylin and eosin–stained lung sections. Blue arrows indicate vessels. E , Representative immunofluorescent staining of α-SMA (α-smooth muscle actin protein; green, SMC [smooth muscle cell] marker), vWF (von Willebrand Factor; red, endothelial cell [EC] marker), and DAPI (4′,6-diamidino-2-phenylindole; blue, nuclei). White arrows indicate vessels. F , Quantification of pulmonary artery muscularization (non-, partial-, full; n=12–15 fields from 3–4 mice). PA indicates pulmonary artery. G , Lung ET-1 mRNA expression (n=3–4). H , mRNA expression of INHBA, ET-1, eNOS (endothelial NO synthase), SOD2 (superoxide dismutase 2), fibronectin, SLUG (snail family transcriptional repressor 2), CD31 (cluster of differentiation 31), and BMP4 (bone morphogenetic protein 4) in lung ECs isolated from WT and TG mice (n=3–4). Data are mean±SEM. P <0.05 is deemed statistically significant. Tests: 1-way ANOVA with Tukey post hoc test for B , C , and G ; 2-way ANOVA with Tukey post hoc test for F ; 2-sided Student t test for H .

Article Snippet: Human pulmonary artery ECs (PAECs; no. C-12241; PromoCell) and pulmonary artery smooth muscle cells (PASMCs; no. C-12521; PromoCell) were cultured in HuMedia-EG2 (Kurabo) and smooth muscle cells growth medium 2 (PromoCell), respectively, at 37 °C and 5% CO 2 .

Techniques: In Vivo, Inhibition, Transgenic Assay, Staining, Marker, Expressing, Isolation

Journal: Journal of hazardous materials

Article Title: Mechanistic insight into airborne particulate matter PM10 as an environmental hazard for hemorrhagic stroke: Evidence from in vitro and in vivo studies.

doi: 10.1016/j.jhazmat.2024.136319

Figure Lengend Snippet: Fig. 5. PM10 promotes CA development through FasL-mediated necroptosis of VSMC in the cerebrovasculature. Representative immunofluorescence staining images of (a) p-RIPK1 and (b) FasL in 3D vascular spheroids following PM10 exposure. All sections were counterstained with anti-α −SMA antibody for VSMC as well as DAPI for nuclei (x400 magnification). Histograms represent the quantification of fluorescence intensity. The fluorescence intensity profile of FasL expression is shown. (c) Immunofluorescence staining for NF-κB p65 nuclear translocation in the vascular cell culture spheroid model system in response to PM10 (x400 magnification). The fluorescence intensity profile of the p65 localization is shown. (d and e) Assessment of the efficacy of the superoxide-dependent Fenton reaction in the vascular spheroids upon PM10 exposure, evaluated via fluorescent probes corresponding to intracellular labile iron pools (Calcein-AM) and superoxide radicals (HKSOX-1). (f) Immunohistochemical staining for p-MLKL in the internal carotid artery (ICA) (x400 magnification). (g) Immunofluorescence for p-RIPK3 with green fluorescence in ICA, counterstained with α-SMA (red) and DAPI (blue) (x400 magnification). (h) Immunofluorescence staining for NF-κB p65 nuclear translocation in the ICA following intratracheal PM10 instillation in mice (x400 magnification). The fluorescence intensity profile of the p65 localization is shown. (i) Representative immunofluorescence images for FasL upregulation in the ICA in response to PM10 exposure. (j) Serological levels of S100B, a diagnostic marker for hemorrhagic stroke, were assessed by ELISA in serum. (k) Immunofluorescence staining for myeloperoxidase (MPO) in the ICA, indicating increased circulating neutrophil infiltration into the cerebral vasculature following PM10 inhalation (x400 magnification). All data are presented as mean ± standard error of the mean, and *in dicates statistically significant differences between groups at *p < 0.05 (n = 6).

Article Snippet: HITB5 (CLU305), vascular smooth muscle cell (VSMC), was purchased from CEDARLANE (ON, Canada).

Techniques: Immunofluorescence, Staining, Fluorescence, Expressing, Translocation Assay, Cell Culture, Immunohistochemical staining, Diagnostic Assay, Marker, Enzyme-linked Immunosorbent Assay