Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: A . Physiological rationale: Ambient PM2.5 exposure is epidemiologically linked to increased ischemic stroke risk. This in vitro model simulates the real-life scenario of pre-existing PM2.5 exposure followed by ischemic stroke and subsequent reperfusion. B . Primary adult male HBMEC were exposed to 5, 15, 75, or 300 μg/m 3 PM 2.5 for 48h in total. To compare with the effects of physiological ischemic-like injury, some plates were exposed to hypoxia (1% O 2 ) and glucose deprived media (HGD) for 3h after the initial 24h incubation. Following HGD or normoxia, cells were reperfused with nutrient-enriched media and incubated with PM 2.5 at normoxic (21% O 2 ) conditions as a reference for resolution of ischemia. Barrier integrity, cell viability, reactive oxygen species (ROS), inflammation and LOX-1 expression was assessed. Figure created in BioRender.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: In Vitro, Incubation, Expressing

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Adult male HBMEC were exposed to vehicle or PM 2.5 (5, 15, 75, or 300 μg/m 3 ) for 24h and incubated for 3h in normoxia- or hypoxia and glucose deprivation (HGD) followed by 24h reperfusion. A . Live cell count (CyQUANT nuclear stain) decreased when exposed to ≥75 μg/m 3 PM 2.5 compared to vehicle. HGD treatment reduced live cell count compared to normoxia but did not differ between particle treated groups. B . Reactive oxygen species (ROS) signal (DCHF-DA) normalized to live cell count. Relative ROS levels increased dose-dependently with PM 2.5 concentration, with significant increase observed at PM 2.5 ≥75 μg/m 3 , in comparison to normoxia vehicle. ROS levels were uniformly elevated following HGD across all doses in comparison to normoxia vehicle and significantly higher than untreated HBMEC. (n=12 technical replicates for vehicle and 5, n=8 technical replicates for 15, 75 and 300) C . Analysis of crystal violet-stained HBMEC shows a longer maximum cellular length when treated with ≥15 μg/m 3 PM 2.5 . (n=21-37 individual cells) D . Representative images of crystal violet-stained HBMEC visualizing a differentiated morphology in cells treated with higher PM 2.5 concentration, where cells appear more elongated and expanding towards neighbouring cells. Data presented as mean ± SD. Statistical significance assessed through Kruskal-Wallis test within treatment groups (Normoxia/HGD) and Mann-Whitney test between groups with different treatment (300 normoxia/vehicle HGD). *p<0.05. ***p<0.001. ****p<0.0001.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Incubation, Cell Characterization, CyQUANT Assay, Staining, Concentration Assay, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Urban PM 2.5 at Realistic Environmental Concentrations Impairs Blood–Brain Barrier Integrity and Enhances LOX-1 Expression in Human Brain Endothelial Cells

doi: 10.64898/2026.01.29.702473

Figure Lengend Snippet: Western Blot assessment of adult male HBMEC exposed to vehicle, 5, 15, 75, or 300 μg/m 3 PM 2.5 during normoxia or ischemic-like injury with hypoxia, glucose deprivation and reperfusion (HGD). A . Representative Western Blot image of IL-6 and β-actin band migration. B . Signal quantification of 25kDa IL-6 shows no difference between PM 2.5 exposure or HGD treated group. C . Signal quantification of 17kDa IL-6 shows dose-dependency with higher IL-6 expression from higher PM 2.5 exposure, with significant increase ≥75 μg/m 3 and from HGD treatment compared to vehicle. D . Representative Western Blot image of LOX-1 and β-actin. E . Signal quantification of LOX-1 displays a dose-dependent increase in LOX-1 with exposure to ≥15 μg/m 3 PM 2.5 or HGD. (n=4-7 technical replicates). Data presented as mean +-SD. Statistical significance assessed by Kruskal-Wallis test. *p<0.05, **p<0.01.

Article Snippet: Primary adult male HBMEC were purchased from Innoprot (Spain, Catalog number: P10361, Lot number: 111224CS).

Techniques: Western Blot, Migration, Expressing

Journal: Neural Regeneration Research

Article Title: Outgrowth endothelial cells form a functional cerebral barrier and restore its integrity after damage

doi: 10.4103/1673-5374.269029

Figure Lengend Snippet: Assessment of the integrity and function of the BBB constructed with HBMECs or OECs. Triple culture of HBMEC or OECs with human astrocytes and pericytes lead to generation of equally tight and functional BBB as evidenced by assessments of transendothelial electrical resistance (TEER, A) and paracellular flux of sodium fluorescein (NaF), a low molecular weight permeability marker (B). Exposure of both types of BBB models to oxygen-glucose deprivation alone (OGD) or followed by reperfusion (OGD + R) affect the integrity and function of both barriers in a similar fashion. * P < 0.05, *** P < 0.001, vs . control; # P < 0.05, ### P < 0.001, vs . OGD (one-way analysis of variance followed by Tukey’s post hoc analysis). All experiments were performed in triplicate. BBB: Blood-brain barrier; HBMECs: human brain microvascular endothelial cells; OECs: outgrowth endothelial cells.

Article Snippet: Human astrocytes, pericytes and HBMECs were bought from TCS CellWorks Ltd. (Buckingham, UK) and cultured at 37°C in relevant specialised media (Sciencell, Caltag Systems, Buckingham, UK) in a humidified atmosphere consisting of 75% N 2 , 20% O 2 and 5% CO 2 .

Techniques: Construct, Functional Assay, Molecular Weight, Permeability, Marker, Control

Journal: Neural Regeneration Research

Article Title: Outgrowth endothelial cells form a functional cerebral barrier and restore its integrity after damage

doi: 10.4103/1673-5374.269029

Figure Lengend Snippet: Analyses of the vasculoreparative function of OECs on an in vitro model of human BBB. Exogenous OECs repair the wound scratch induced on endothelial layer of a triple culture model of human BBB established by astrocytes, pericytes and human brain microvascular endothelial cells and maintained in serum-free conditions as assessed by measurements of transendothelial electrical resistance (TEER, A) and paracellular flux of low molecular weight permeability marker sodium fluorescein (NaF, B). Data are presented as mean ± SEM. * P < 0.05, *** P < 0.001, vs . control; §§§ P < 0.001, vs . scratch without OECs (one-way analysis of variance followed by Tukey’s post hoc analysis). All experiments were performed in triplicate. BBB: Blood-brain barrier; HBMECs: human brain microvascular endothelial cells; OECs: outgrowth endothelial cells.

Article Snippet: Human astrocytes, pericytes and HBMECs were bought from TCS CellWorks Ltd. (Buckingham, UK) and cultured at 37°C in relevant specialised media (Sciencell, Caltag Systems, Buckingham, UK) in a humidified atmosphere consisting of 75% N 2 , 20% O 2 and 5% CO 2 .

Techniques: In Vitro, Molecular Weight, Permeability, Marker, Control