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: Pharmaceuticals

Article Title: Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ph17121567

Figure Lengend Snippet: The cytotoxicity analysis of the NPs. Our results indicate that low (L: 15.62 µg/mL), normal (N: 31.25 µg/mL), and high (H: 62.5 µg/mL) doses of NP formulations including PLGA, BSA, BSA-Tf, HSA, HSA-Tf, and NLC did not cause any toxic effects on hBMECs ( a ), hBVPs ( b ), or hASTROs ( c ) compared to the control group after 3 h of incubation.

Article Snippet: Primary human microvascular endothelial cells (hBMECs) (Cat# cAP-0002, Angio-Proteomie, Boston, MA, USA), primary human brain vascular pericytes (hBVPs) (Cat# SC-1200, Caltag Medsystems, Buckingham, UK), and primary human astrocytes (hASTROs) (Cat# SC-1800, Caltag Medsystems, Buckingham, UK) were cultured in their respective growth media: endothelial cell growth medium-2 (EGM-2; Cat# CC-3162, Lonza Group, CH, Basel, Switzerland), pericyte growth medium (Cat# SC-1201, Caltag Medsystems, UK), and astrocyte medium (Cat# 1801, ScienceCell Research Laboratories, Carlsbad, CA, USA).

Techniques: Control, Incubation

Journal: Pharmaceuticals

Article Title: Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ph17121567

Figure Lengend Snippet: Micrographs demonstrating Tf receptor expression in hBMECs, while no Tf receptor expression was observed in hASTROs or hBVPs. TfR: transferrin receptor (×400 maginification).

Article Snippet: Primary human microvascular endothelial cells (hBMECs) (Cat# cAP-0002, Angio-Proteomie, Boston, MA, USA), primary human brain vascular pericytes (hBVPs) (Cat# SC-1200, Caltag Medsystems, Buckingham, UK), and primary human astrocytes (hASTROs) (Cat# SC-1800, Caltag Medsystems, Buckingham, UK) were cultured in their respective growth media: endothelial cell growth medium-2 (EGM-2; Cat# CC-3162, Lonza Group, CH, Basel, Switzerland), pericyte growth medium (Cat# SC-1201, Caltag Medsystems, UK), and astrocyte medium (Cat# 1801, ScienceCell Research Laboratories, Carlsbad, CA, USA).

Techniques: Expressing

Journal: Pharmaceuticals

Article Title: Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ph17121567

Figure Lengend Snippet: Semi-thin and ultra-thin sections of hBMECs, hBVPs, and hASTROs from control groups displaying healthy and active cellular profiles. N: nucleus, arrowhead: mitochondria, *: autophagic figures, <: extracellular vesicles, red cycles: small transport vesicles, blue cycle: cell-to-cell contact.

Article Snippet: Primary human microvascular endothelial cells (hBMECs) (Cat# cAP-0002, Angio-Proteomie, Boston, MA, USA), primary human brain vascular pericytes (hBVPs) (Cat# SC-1200, Caltag Medsystems, Buckingham, UK), and primary human astrocytes (hASTROs) (Cat# SC-1800, Caltag Medsystems, Buckingham, UK) were cultured in their respective growth media: endothelial cell growth medium-2 (EGM-2; Cat# CC-3162, Lonza Group, CH, Basel, Switzerland), pericyte growth medium (Cat# SC-1201, Caltag Medsystems, UK), and astrocyte medium (Cat# 1801, ScienceCell Research Laboratories, Carlsbad, CA, USA).

Techniques: Control

Journal: Pharmaceuticals

Article Title: Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ph17121567

Figure Lengend Snippet: Ultrastructural observations of the application of PLGA, BSA, BSA-Tf, HSA, HSA-Tf, and NLC NP formulations on hBMECs, hBVPs, and hASTROs at 62.5 µg/mL doses after 3 h of exposure.

Article Snippet: Primary human microvascular endothelial cells (hBMECs) (Cat# cAP-0002, Angio-Proteomie, Boston, MA, USA), primary human brain vascular pericytes (hBVPs) (Cat# SC-1200, Caltag Medsystems, Buckingham, UK), and primary human astrocytes (hASTROs) (Cat# SC-1800, Caltag Medsystems, Buckingham, UK) were cultured in their respective growth media: endothelial cell growth medium-2 (EGM-2; Cat# CC-3162, Lonza Group, CH, Basel, Switzerland), pericyte growth medium (Cat# SC-1201, Caltag Medsystems, UK), and astrocyte medium (Cat# 1801, ScienceCell Research Laboratories, Carlsbad, CA, USA).

Techniques:

Journal: Pharmaceuticals

Article Title: Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ph17121567

Figure Lengend Snippet: Representative TEM micrographs of PLGA application at 62.5 µg/mL in hBMECs, hBVPs, and hASTROs. PLGA, identified by its Zr marker (arrows), was observed within single-layered vesicles distributed throughout the cytoplasm of hBMECs, hBVPs, and hASTROs. Ly: lysosomes, arrowhead: large autophagic vacuoles, *: large vacuoles.

Article Snippet: Primary human microvascular endothelial cells (hBMECs) (Cat# cAP-0002, Angio-Proteomie, Boston, MA, USA), primary human brain vascular pericytes (hBVPs) (Cat# SC-1200, Caltag Medsystems, Buckingham, UK), and primary human astrocytes (hASTROs) (Cat# SC-1800, Caltag Medsystems, Buckingham, UK) were cultured in their respective growth media: endothelial cell growth medium-2 (EGM-2; Cat# CC-3162, Lonza Group, CH, Basel, Switzerland), pericyte growth medium (Cat# SC-1201, Caltag Medsystems, UK), and astrocyte medium (Cat# 1801, ScienceCell Research Laboratories, Carlsbad, CA, USA).

Techniques: Marker

Journal: Pharmaceuticals

Article Title: Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ph17121567

Figure Lengend Snippet: Representative TEM micrographs of BSA and BSA-Tf applications at 62.5 µg/mL in hBMECs, hBVPs, and hASTROs. BSA and BSA-Tf (arrows) were occasionally observed in hBMECs, hBVPs, and hASTROs. Ly: lysosomes, *: autophagic vacuoles, red cycles: Au particles accumulated in the lysosomes or in the mitochondria.

Article Snippet: Primary human microvascular endothelial cells (hBMECs) (Cat# cAP-0002, Angio-Proteomie, Boston, MA, USA), primary human brain vascular pericytes (hBVPs) (Cat# SC-1200, Caltag Medsystems, Buckingham, UK), and primary human astrocytes (hASTROs) (Cat# SC-1800, Caltag Medsystems, Buckingham, UK) were cultured in their respective growth media: endothelial cell growth medium-2 (EGM-2; Cat# CC-3162, Lonza Group, CH, Basel, Switzerland), pericyte growth medium (Cat# SC-1201, Caltag Medsystems, UK), and astrocyte medium (Cat# 1801, ScienceCell Research Laboratories, Carlsbad, CA, USA).

Techniques:

Journal: Pharmaceuticals

Article Title: Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ph17121567

Figure Lengend Snippet: Representative TEM micrographs of HSA and HSA-Tf applications at 62.5 µg/mL in hBMECs, hBVPs, and hASTROs. HSA and HSA-Tf (arrows) were occasionally observed in hBMECs, hBVPs, and hASTROs. Ly: lysosomes, *: autophagic vacuoles, red cycles: Au particle accumulations in autophagic vacuoles, LD: lipid droplets.

Article Snippet: Primary human microvascular endothelial cells (hBMECs) (Cat# cAP-0002, Angio-Proteomie, Boston, MA, USA), primary human brain vascular pericytes (hBVPs) (Cat# SC-1200, Caltag Medsystems, Buckingham, UK), and primary human astrocytes (hASTROs) (Cat# SC-1800, Caltag Medsystems, Buckingham, UK) were cultured in their respective growth media: endothelial cell growth medium-2 (EGM-2; Cat# CC-3162, Lonza Group, CH, Basel, Switzerland), pericyte growth medium (Cat# SC-1201, Caltag Medsystems, UK), and astrocyte medium (Cat# 1801, ScienceCell Research Laboratories, Carlsbad, CA, USA).

Techniques:

Journal: Pharmaceuticals

Article Title: Comparison of Drug Delivery Systems with Different Types of Nanoparticles in Terms of Cellular Uptake and Responses in Human Endothelial Cells, Pericytes, and Astrocytes

doi: 10.3390/ph17121567

Figure Lengend Snippet: Representative TEM micrographs of NLC application at 62.5 µg/mL in hBMECs, hBVPs, and hASTROs. NLC (arrows) was occasionally observed in hBMECs, hBVPs, and hASTROs. Ly: lysosomes, Pr: peroxisomes, *: autophagic vacuoles, red cycles: Au particle accumulations in peroxisomes or autophagic vacuoles.

Article Snippet: Primary human microvascular endothelial cells (hBMECs) (Cat# cAP-0002, Angio-Proteomie, Boston, MA, USA), primary human brain vascular pericytes (hBVPs) (Cat# SC-1200, Caltag Medsystems, Buckingham, UK), and primary human astrocytes (hASTROs) (Cat# SC-1800, Caltag Medsystems, Buckingham, UK) were cultured in their respective growth media: endothelial cell growth medium-2 (EGM-2; Cat# CC-3162, Lonza Group, CH, Basel, Switzerland), pericyte growth medium (Cat# SC-1201, Caltag Medsystems, UK), and astrocyte medium (Cat# 1801, ScienceCell Research Laboratories, Carlsbad, CA, USA).

Techniques:

Journal: Biochemical pharmacology

Article Title: Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability

doi: 10.1016/j.bcp.2022.115143

Figure Lengend Snippet: a. Human primary brain microvascular ECs stained for ciliary protein ARL13B (green) and nuclear stain DAPI (blue) showing more than one cilia by immunofluorescence method. b. Human primary umbilical vein endothelial cells. c. Human primary brain choroid plexus endothelial cells. a-c. Scale bar are 20 μm in the merged image and 10 μm in the zoomed image (n=3). White boxed regions are zoomed in the adjacent panel and arrows indicate cilium. d. Human primary umbilical vein endothelial cells were grown for an additional 5 days once confluence was reached prior to cilia staining with acetylated tubulin (red) antibody. Scale bar= 50 μm. Arrows show a cell with multiple cilia. e. Human embryonic stem cell-derived brain microvascular endothelial-like cells staining with ARL13B antibody. Scale bar= 50 μm. (n=4). Arrows show a cell with multiple cilia.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Cell Systems Corporation Cat # ACBRI 376), and primary human brain umbilical vein endothelial cells (HUVECs) (Glyco Tech Inc) were maintained at 37°C in 5% CO 2 incubator in endothelial cell complete medium (Promocell, Cat # C22010 ).

Techniques: Staining, Immunofluorescence, Derivative Assay

Journal: Biochemical pharmacology

Article Title: Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability

doi: 10.1016/j.bcp.2022.115143

Figure Lengend Snippet: a, c & d. HBMECs were knocked down with and without PAK2 siRNA (25 nM) (upper panel, a), with and without overexpression of Arl13b-GFP WT plasmid (middle panel, c), with and without overexpression of Arl13b-GFP Mutant (R78Q) plasmid (lower panel, d) and assessed for ARL13B positive cilia by immunofluorescence. Scale bar= 20 μm in the merged image and 10 μm in the zoomed image. b. Two graphs are depicted. The top graph represents percentage of brain ECs with 1 cilium per cell. Cells with 2-cilia were not included in the quantification. The bottom graph is the ARL13B cilia length measured in each sample set. Number of cilia and its length were assessed by number of nuclei to cilia and its length using ACDC V 0.93 cilia specific software. Each dot represents random field in the slide. Statistical P values were compared to control group; n=3 in each experimental group. Detailed quantification protocol is provided in the methods section. Results were presented as mean ± SEM. SEM, standard error to the mean. ANOVA was used to examine the effects of various conditions on the outcomes.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Cell Systems Corporation Cat # ACBRI 376), and primary human brain umbilical vein endothelial cells (HUVECs) (Glyco Tech Inc) were maintained at 37°C in 5% CO 2 incubator in endothelial cell complete medium (Promocell, Cat # C22010 ).

Techniques: Over Expression, Plasmid Preparation, Mutagenesis, Immunofluorescence, Software, Control

Journal: Biochemical pharmacology

Article Title: Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability

doi: 10.1016/j.bcp.2022.115143

Figure Lengend Snippet: a & b. Human brain microvascular endothelial cells (HBMECs) were treated with VEGF-A165 (20 ng/mL) and PDGF-BB ligands (10 ng/mL) for 5 and 60 mins respectively and assessed by immunoblotting for ARL13B and PAK2 proteins and quantified. Panels c & d are HBMECs treated with PDGF-BB (10 ng/mL) ligand for 10, 30 and 60 mins and assessed for ciliary protein ARL13B and vascular stability protein PAK2 by immunoblotting and quantified. Experiment was done in triplicates and the results were presented as mean ± SEM. SEM, standard error to the mean. *P<0.05; and #P<0.001 compared with control group; n=3 in each experimental group. ANOVA was used to examine the effects of various conditions on the outcomes. e-g. PDGF-BB and VEGF-A165 treated HBMECs were assessed for ARL13B positive cilia by immunofluorescence (e) and quantified (f & g). Number of cilia and its length were assessed by number of nuclei to cilia and its length using ACDC V0.93 cilia specific software. The percentage of brain ECs represents 1-cilium per cell. Cells with 2-cilia were excluded in this quantification. Control group n= 65 nuclei; PDGF-BB group n= 57 nuclei; VEGF-A165 group n= 72 nuclei. Results were presented as mean ± SEM. SEM. Scale bar= 20 μm in the merged image and 10 μm in the zoomed image. Each dot represents a random field in the slide. Statistical P values were compared to control group; n=3 in each experimental group. Detailed quantification protocol is provided in the methods section. h & i. HBMECs were knocked down with PAK2 siRNA with and without the EC ligands VEGF-A165 and PDGF-BB for 5 and 60 mins and assessed for PAK2 and ARl13B levels by immunoblotting. (*P<0.05 and #P<0.001 compared with control group; n=3 in each experimental group). Results were presented as mean ± SEM. ANOVA was used to examine the effects of various conditions on the outcomes.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Cell Systems Corporation Cat # ACBRI 376), and primary human brain umbilical vein endothelial cells (HUVECs) (Glyco Tech Inc) were maintained at 37°C in 5% CO 2 incubator in endothelial cell complete medium (Promocell, Cat # C22010 ).

Techniques: Western Blot, Control, Immunofluorescence, Software

Journal: Biochemical pharmacology

Article Title: Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability

doi: 10.1016/j.bcp.2022.115143

Figure Lengend Snippet: a & b. Human brain microvascular endothelial cells (HBMECs) were treated with PDGF-BB (10 ng/mL) ligand for 10, 30 and 60 mins and assessed for total VEGFR2 protein levels and quantified. c & d. HBMECs were treated with VEGF-A165 for 5 mins, VEGFR2 siRNA, VEGF-A165 + VEGFR2 siRNA and PDGF-BB ligand for 60 mins and assessed for phosphorylated VEGFR2 at tyrosine sites Y1175 and Y1059 and total VEGFR2 protein levels and quantified. *P<0.05; **P<0.01 and #P<0.001 compared with control group; n=3 in each experimental group. Results were presented as mean ± SEM. e & f. HBMECs knockdown with VEGFR2 siRNA (25 nM) and treated with PDGF-BB 10, 30 and 60 mins respectively and assessed for VEGFR2, ciliary protein ARL13B and vascular stability protein PAK2 and quantified. g & h. HBMECs treated with VEGF-A165 (20 ng/mL) and knockdown with VEGFR2 siRNA respectively and assessed for ARL13B and PAK2 proteins and quantified. ANOVA was used to examine the effects of various conditions on the outcomes.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Cell Systems Corporation Cat # ACBRI 376), and primary human brain umbilical vein endothelial cells (HUVECs) (Glyco Tech Inc) were maintained at 37°C in 5% CO 2 incubator in endothelial cell complete medium (Promocell, Cat # C22010 ).

Techniques: Control, Knockdown

Journal: Biochemical pharmacology

Article Title: Ciliogenesis mechanisms mediated by PAK2-ARL13B signaling in brain endothelial cells is responsible for vascular stability

doi: 10.1016/j.bcp.2022.115143

Figure Lengend Snippet: Human primary brain microvascular ECs were validated and compared with human primary brain choroid plexus endothelial cells for endothelial specific markers CD31, TIE2, Claudin-5, Ve-cadherin (VE-CAD), CD105 (endoglin) and β-actin by flow cytometry analysis (n=4). Expression of each protein is quantified as median fluorescence intensity (MFI). Results were presented as mean ± SEM. SEM, standard error to the mean. P<0.05 was considered significant. Top panel shows the quantification, while the bottom panel shows representative MFI histogram for respective proteins. For β-actin quantification, secondary antibody was used. Dotted histograms with respective colors mark ‘secondary controls.’

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) (Cell Systems Corporation Cat # ACBRI 376), and primary human brain umbilical vein endothelial cells (HUVECs) (Glyco Tech Inc) were maintained at 37°C in 5% CO 2 incubator in endothelial cell complete medium (Promocell, Cat # C22010 ).

Techniques: Flow Cytometry, Expressing, Fluorescence