Journal: bioRxiv
Article Title: IsoDGR-Induced Endothelial Cytoskeletal Disruption Drives Age-Related Blood-Brain Barrier Breakdown
doi: 10.1101/2025.07.08.663800
Figure Lengend Snippet: (A) Endothelial permeability assay: Confluent monolayers of bEnd.3 brain endothelial cells cultured on Transwell inserts were treated with isoDGR-peptide (50 μg/mL) or vehicle control. Permeability to 60-70 kDa FITC-dextran was measured in the lower chamber at indicated time points. IsoDGR significantly increased paracellular tracer flux, indicating compromised barrier integrity (n = 6 wells per group). Fluorescence was quantified using a fluorometer, and statistical analysis was performed using repeated measures ANOVA. (B) Scratch-wound motility assay: bEnd.3 monolayers were scratched with a pipette tip and cultured in the presence or absence of isoDGR-peptide (50 μg/mL). Wound width was imaged and measured up to 48 hours. IsoDGR treatment significantly delayed wound closure, demonstrating impaired endothelial cell migration (n = 6). Statistical analysis: one-way repeated measures ANOVA. (C) Representative brightfield images of scratched bEnd.3 cultures at 24 hours post-injury. Untreated control cells exhibit narrower wound gaps, whereas isoDGR-treated cells show substantially delayed closure. Red scale bars and yellow outlines demarcate wound width (μm). (D) Integrin αV immunostaining: bEnd.3 cells treated 24 hours with isoDGR-peptide (50 μg/mL) or vehicle control were fixed and stained for Integrin αV (green) and DAPI (blue). Fluorescence imaging revealed enhanced integrin αV signal in isoDGR-treated cells. (E) Quantification of Integrin αV fluorescence intensity across image using Image J. IsoDGR significantly upregulated integrin αV expression (****p < 0.0001, unpaired t-test), suggesting possible involvement of integrin signaling. (F) Densitometric analysis of six independent blots, normalized to control. IsoDGR increases the expression of integrin αV and β3 proteins, consistent with the immunofluorescence results (mean ± SEM; *p < 0.05, unpaired t-test). Scale bars: 100 μm. Error bars represent SEM. These findings support a functional link between isoDGR exposure and endothelial dysfunction via increased permeability, reduced motility, and integrin-mediated signaling.
Article Snippet: Briefly, 24-well permeable cell culture inserts with a 0.4 μm pore size (Corning, or A1048301, ThermoFisher Scientific) were coated with rat tail type I collagen (A1048301, ThermoFisher Scientific).
Techniques: Permeability, Cell Culture, Control, Fluorescence, Motility Assay, Transferring, Migration, Immunostaining, Staining, Imaging, Expressing, Immunofluorescence, Functional Assay
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