stimulation human brain microvascular endothelial cell line  (Merck & Co)

Journal: International Journal of Pharmaceutics: X

Article Title: Plant-derived extracellular vesicles as a natural drug delivery platform for glioblastoma therapy: A dual role in preserving endothelial integrity while modulating the tumor microenvironment

doi: 10.1016/j.ijpx.2025.100349

Figure Lengend Snippet: Construction of a 3D BTB model and assessment of the barrier-crossing ability of Citrus limon L. -derived EVs. (A) Schematic representation of the 3D BTB model construction and EVs permeability assessment. (B) TEER measurements assessing endothelial barrier formation in hCMEC/D3 cells over seven days. (C) FITC-dextran permeability in the absence (−) or presence (+) of hCMEC/D3 cells. (D) Immunofluorescence staining of the tight junction protein ZO-1 (green) in hCMEC/D3 cells. Nuclei are stained with DAPI (blue). (E) Nanoparticle tracking analysis (NTA) of size distribution and concentration of EVs. (F) Representative transmission electron microscopy (TEM) image of EVs (Scale bar = 100 nm). (G) Size and (H) zeta potential distribution of Citrus limon L. -derived EVs. (I) Fluorescence images showing the uptake of Calcein-AM-labeled EVs (green) by U87 glioblastoma cells. Nuclei are stained with DAPI (blue). scale bar = 100 μm. The magnified images depict high-magnification views of the white boxed areas. (J) Fluorescence intensities of endothelial barrier-crossed EVs were measured using a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA). Error bars represent the standard deviation (or standard error) of the mean; however, they are shorter than the height of the symbols and therefore not visible in the figure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human brain microvascular endothelial cell line hCMEC/D3 (Catalog #305024) was purchased from Cytion Biosciences.

Techniques: Derivative Assay, Permeability, Immunofluorescence, Staining, Concentration Assay, Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Fluorescence, Labeling, Imaging, Standard Deviation

Journal: International Journal of Pharmaceutics: X

Article Title: Plant-derived extracellular vesicles as a natural drug delivery platform for glioblastoma therapy: A dual role in preserving endothelial integrity while modulating the tumor microenvironment

doi: 10.1016/j.ijpx.2025.100349

Figure Lengend Snippet: Effects of Citrus limon L. -derived EVs on U87 and hCMEC/D3 Cells. (A, D) Representative live/dead staining images of U87 and hCMEC/D3 cells, respectively, following 48-h treatment with EVs (80 μg/mL). Live cells are stained green (Calcein AM), while dead cells are stained red (PI). (B, E) Cell viability of U87 and hCMEC/D3 cells treated with increasing concentrations of EVs (10, 20, 40, and 80 μg/mL). Data are expressed as the percentage of viable cells relative to the control (mean ± SEM, n = 4). (C, F) Intracellular ROS levels in U87 and hCMEC/D3 cells were normalized to the number of viable cells. (G) Wound healing assay showing hCMEC/D3 cell migration at 0-, 4-, and 24-h post-scratch, (+) with or (−) without EVs. (H) Quantification of migrating cells and hCMEC/D3 cells after scratch wounding. Statistical significance was determined using one-way or two-way ANOVA with Tukey's post hoc test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human brain microvascular endothelial cell line hCMEC/D3 (Catalog #305024) was purchased from Cytion Biosciences.

Techniques: Derivative Assay, Staining, Control, Wound Healing Assay, Migration

Journal: International Journal of Pharmaceutics: X

Article Title: Plant-derived extracellular vesicles as a natural drug delivery platform for glioblastoma therapy: A dual role in preserving endothelial integrity while modulating the tumor microenvironment

doi: 10.1016/j.ijpx.2025.100349

Figure Lengend Snippet: Evaluation of EVs@TMZ uptake, cytotoxicity, glioblastoma spheroid progression, and VEGF-A secretion in a 3D BTB model. (A) Schematic illustration of EVs@TMZ uptake by U87 in a 3D BTB co-culture model with hCMEC/D3 cells. (B) Confocal microscopy images of 3D U87 cells incubated with EVs@TMZ at 37 °C for 24 h. EVs@TMZ were labeled with calcein-AM (shown in green), actin filaments were labeled with rhodamine phalloidin, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (scale bar = 10 μm). The magnified images depict high-magnification views of the white boxed areas. (C) 3D Z -stack image of spatial distribution and uptake of EVs@TMZ in U87 cells. (D) Progressive BTB crossing of EVs@TMZ over 24 h. (E) Cytotoxic effects and (F) ROS levels in hCMEC/D3 cells after 48 h of untreated or treatment with TMZ and EVs@TMZ. (G) Representative stitched microscopic images of 3D U87 in the untreated (Ctrl) group at 24 and 48 h, with arrows indicating aggregates and migration. (H) Representative fluorescence images of 3D U87 after 48 h under three conditions: untreated (Ctrl), TMZ-treated, and EVs@TMZ-treated. (I-L) Quantification of 3D U87, including viable cells (I), ROS levels (J), migrated cells (K), and core density (L). (M) VEGF-A levels in U87, U87 co-cultured with hCMEC/D3 (U87 + hCMEC/D3), TMZ-treated, and EVs@TMZ-treated. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human brain microvascular endothelial cell line hCMEC/D3 (Catalog #305024) was purchased from Cytion Biosciences.

Techniques: Co-Culture Assay, Confocal Microscopy, Incubation, Labeling, Migration, Fluorescence, Cell Culture

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A , Experimental timeline for inflammatory insult with TNF-α and FGF2 co-treatment. Once confluent (4 days), HBEC-5i or bEnd.3 were pretreated with FGF2 or vehicle for one hour and then stimulated with TNF-α or vehicle for up to 7 days. FGF2 alters mouse ( B ) and human ( C ) endothelial transcription in response to acute TNF-α stimulation. FGF2 promotes faster restoration of TNF-α-induced Cldn5 loss in mouse ( D ) and human ( E ) endothelial cells. Chronic stimulation with TNF-α leads to a reduction in endothelial monolayer integrity measured by trans-endothelial electrical resistance (TEER) in mouse ( F ) and human ( G ) endothelial cells. FGF2 co-treatment preserves normal TEER despite TNF-α. H 7 days of TNF-α treatment promotes spikes and discontinuities in Cldn5 tight junction strands in bEnd.3, which is reversed by FGF2 treatment (scalebar = 20 μm). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with two-way ANOVA followed by Bonferroni’s post hoc tests; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques:

Journal: Nature Communications

Article Title: Environmental enrichment and physical exercise prevent stress-induced social avoidance and blood-brain barrier alterations via Fgf2

doi: 10.1038/s41467-025-68058-9

Figure Lengend Snippet: A 1 h pretreatment with Fgf2 increases serine-9 phosphorylation of GSK3β in HBEC-5i when compared to no treatment (CTRL 0 h). TNF-α treatment induces rapid, transient dephosphorylation of GSK3β, but this effect is not reversed by Fgf2 coadministration. Each dot represents a replicate ( n = 3). B 1 h Fgf2 pretreatment diminishes basal β-catenin phosphorylation when compared to no treatment (CTRL 0 h). Further, while TNF-α induces a rapid reduction in phosphorylated β-catenin, Fgf2 reverses this dynamic upon inflammatory activation ( n = 3). C In health control endothelial cells (top), β-catenin interacts with VE-Cadherin at the cell membrane, and this complex inhibits Cldn5 transcriptional suppression by FOXO1. Excess cytosolic β-catenin is phosphorylated by GSK3β, targeting it for degradation. When stimulated with TNFα, unbound β-catenin complexes with FOXO1, leading to suppression of Cldn5 expression (bottom, red arrow), while a small amount is targeted for degradation. Meanwhile, when FGF2 is co-administered with TNF-α (bottom, blue arrow), our results suggest that unbound β-catenin is strongly redirected toward GSK3β-mediated phosphorylation. D 30 min of TNF-α is sufficient to induce β-catenin distribution at tight junctions ( n = 4 replicates) with representative images on the right ( E ) (scalebar = 20 μm). F Fgf2 attenuates TNF-α-induced reductions in the wound healing capacity of HBEC-5i ( n = 4 replicates) (**** p < 0.0001). Data represent mean ± s.e.m., and each experiment was replicated at least twice on independent samples. Group comparisons were evaluated with one or two-way ANOVA followed by Bonferroni’s post hoc tests or two-tailed t-tests with Welch’s correction when appropriate; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human brain microvascular endothelial cell line HBEC-5i (ATCC CRL-3245, male donor according to https://www.cellosaurus.org/CVCL_4D10 ) and the mouse brain endothelial cell line bEnd.3 (ATCC CRL-2299) were subcultured and stored in banks at −150 ° C. Cells were thawed as needed and cultured in DMEM/F12 supplemented with 10% fetal bovine serum, 25 ug/mL gentamicin (Gibco), and 1X endothelial cell growth supplement (ScienCell).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Control, Membrane, Expressing, Two Tailed Test

Journal: Journal of Neuroinflammation

Article Title: Transcriptomic profiling of thrombus-derived extracellular vesicles reveals PECAM-1 as a potential inflammatory marker for cardioembolic stroke patients

doi: 10.1186/s12974-025-03555-8

Figure Lengend Snippet: Effect of oxLDL and thrombo-inflammatory stimuli on the mRNA expression of selected candidates in human brain microvascular endothelial cells (hCMEC/D3). Expression of PECAM-1 (panel A ), IL-8 (panel B ), ITGB1 (panel C ), CD44 (panel D ), and SLC3A2 (panel E) in hCMEC/D3 cell cultures stimulated with oxLDL for 24 h, or with TNFα, IL1β and thrombin for 6 h and 12 h. Data are expressed as fold-change (FC) relative to control. * p < 0.05, ** p < 0.01, *** p < 0.001 vs baseline

Article Snippet: Human brain microvascular endothelial cell line (hCMEC/D3, SC0066, Merck), was seeded on 0.7 mg/mL rat collagen type I in PBS (Sigma-Aldrich, 08–115) coated 75 cm 2 flask and cultured in complete EndoGROTM-MV medium (Merck, SCME004) supplemented with 1% penicillin and streptomycin (PS, 100 U/mL and 100 μg/mL, Sigma-Aldrich).

Techniques: Expressing, Control