Journal: Molecular neurobiology

Article Title: A2A adenosine receptor regulates the human blood brain barrier permeability

doi: 10.1007/s12035-014-8879-2

Figure Lengend Snippet: (A) Measurement of trans-endothelial electrical resistance (TEER) in primary human brain endothelial cells (HBMVEC) treated with AR agonists, Lexiscan or NECA or VEGF and SIP as positive and negative controls respectively, and vehicle (control). TEER measurement in primary human brain endothelial cell monolayer (HBMVEC) Data represents mean ± s.e.m. (n=3, + (S1P), ¶ (VEGF), * (Lexiscan), # (NECA) indicate p<0.05 by two-tailed student t-test). (B) TEER measurement in mouse brain endothelial cell monolayer (bEnd 3) after treatment with AR agonists. Data represents mean ± s.e.m. (n=3, + (S1P), ¶ (VEGF), * (Lexiscan), # (NECA) indicate p<0.05 by two-tailed student t-test). (C) FITC-Dextran extravasation through primary human brain endothelial cell monolayer in the presence of Lexiscan, NECA or NECA and SCH58261 at 30, 60, 90 minutes. Data represents mean ± s.e.m. (n=3, * p<0.05, ** p<0.01, *** p<0.001 two-way ANOVA with Bonferroni multiple comparison test). (D) Early time course measurement of FITC extravasation by Lexiscan. Data represents mean ± s.e.m. (n=3, * p<0.05, ** p<0.01, *** p<0.001 by two-way ANOVA with Bonferroni multiple comparison test). (E) 10 kDa FITC-Dextran extravasation in the presence of Lexiscan or NECA in mouse brain endothelial cell monolayer. Data represents mean ± s.e.m. (n=3, * p<0.05, ** p<0.01, two-way ANOVA with Bonferroni multiple comparison test).

Article Snippet: In mouse brain endothelial monolayer (bEnd 3 cells), Lexiscan down-regulated VE-cadherin expression in a time dependent manner, from 30 minutes to 1 hr, after which it increased to baseline levels and was maintained steadily up to 4 hrs ( ).

Techniques: Two Tailed Test

Journal: Molecular neurobiology

Article Title: A2A adenosine receptor regulates the human blood brain barrier permeability

doi: 10.1007/s12035-014-8879-2

Figure Lengend Snippet: (A–D) Western blot result of VE-Cadherin and Claudin 5 in primary human brain endothelial cells with Lexiscan and NECA activation. (A and B) Western blot analysis of Claudin-5 and VE-Cadherin was performed on the HBMVEC cells treated with Lexiscan upto 30 minutes (A). Normalized intensity of band by GAPDH from treated group was divided by that of control group at each time point and plotted as graph (B). (C and D) Western blot on Claudin-5 and VE-Cadherin was performed on the HBMVEC cells treated with NECA upto 120 minutes (C). Normalized intensity of band by GAPDH from treated group was divided by that of control group at each time point and plotted as graph (D). (E–H) Western blot analysis of Claudin-5 and VE-Cadherin levels in mouse brain endothelial cell line (bEnd3). (E and F) Western blot analysis of Claudin-5 and VE-Cadherin was performed on the bEnd 3 cells treated with Lexiscan upto 4 hours (E). Normalized intensity of band by GAPDH from treated group was divided by that of control group at each time point and plotted as graph (F). (G and H) Western blot on Claudin-5 and VE-Cadherin was performed on the bEnd 3 cells treated with NECA upto 4 hours (G). Normalized intensity of band by GAPDH from treated group was divided by that of control group at each time point and plotted as graph (H). In all western blot images M indicates media only control.

Article Snippet: In mouse brain endothelial monolayer (bEnd 3 cells), Lexiscan down-regulated VE-cadherin expression in a time dependent manner, from 30 minutes to 1 hr, after which it increased to baseline levels and was maintained steadily up to 4 hrs ( ).

Techniques: Western Blot, Activation Assay

Journal: Molecular neurobiology

Article Title: A2A adenosine receptor regulates the human blood brain barrier permeability

doi: 10.1007/s12035-014-8879-2

Figure Lengend Snippet: (A and B) IFA of VE-cadherin (Green) and F-actin (Red) in HBMVEC cells 5 and 30 minutes post-treatment with Lexiscan and NECA. Additionally, NECA was treated concomitantly with SCH58261 which is A2A specific antagonist. Arrows indicate the disrupted junction formation. Nucleus was counterstained with DAPI (Blue). Scale bar indicates 25 um. (C and D) AR agonists increase the permeability to chemotherapeutics, Gemcitabine, in primary human brain endothelial monolayer. Changes in permeability to the chemotherapeutic drug, Gemcitabine, after AR activation was determined using primary human brain endothelial cell monolayer. Donor chamber was treated with Gemcitabine (Gem) (10 ug/ml) for 5, 15, 30, 60 minutes with or without 1uM of Lexiscan or NECA and donor chambers were removed. Receiver chambers on which the YFP-transfected human glioblastoma cells (U251) were cultured and further incubated for 96 hrs and cell viability was measured by the relative intensity of YFP signal compared to untreated YFP-U251 (n=3). Lexiscan or NECA only treatment group was also set as control to test its effect on glioma cell viability (n=3). Data represents mean ± s.e.m. (* indicates where p<0.05, two tailed student t-test). (E) Selective A2A AR agonist Lexiscan increases the permeability of the BBB to 10 kDa FITC-Dextran in mice. Lexiscan (0.05 mg/kg) was intravenously administered concomitantly with 10 kDa FITC-Dextran and perfused with ice-cold PBS at different time point (n=10). Brain was collected and processed for analysis of FITC-Dextran concentration using fluometry (** indicates where p<0.01, two tailed student t-test). Graph from Figure 2D showing the effect of Lexiscan on hBBB permeability to 10 kDa FITC-Dextran was juxtaposed as inset for comparison. Arrows indicate the time point with maximal FITC-Dextran concentration from two different graphs. (F) Adenosine increases the peremeability of the BBB to 10 kDa FITC-Dextran in mice. Adenosine was intravenously administered three times (0.138 mg/kg, 20 seconds apart) concomitantly with 10 kDa Dextran and perfused with ice cold PBS at 1 minute after treatment (n=2). Brain was collected and processed for analysis of FITC-Dextran concentration using fluometry (* indicates where p<0.05, two tailed student t-test).

Article Snippet: In mouse brain endothelial monolayer (bEnd 3 cells), Lexiscan down-regulated VE-cadherin expression in a time dependent manner, from 30 minutes to 1 hr, after which it increased to baseline levels and was maintained steadily up to 4 hrs ( ).

Techniques: Permeability, Activation Assay, Transfection, Cell Culture, Incubation, Two Tailed Test, Concentration Assay

Journal: Nature Communications

Article Title: Japanese encephalitis virus neuropenetrance is driven by mast cell chymase

doi: 10.1038/s41467-019-08641-z

Figure Lengend Snippet: Inhibition of chymase abolishes JEV-induced breakdown of the BBB. a bEND.3 cell monolayers were treated with media alone or media containing JEV or supernatants of MCs only, JEV-stimulated MCs, or JEV-stimulated MCs treated with either TY-51469 (chymase inhibitor) or nafamostat mesylate (tryptase inhibitor). Supernatants from JEV-stimulated BMMCs reduced the TEER, which was reversed by TY-51469 (100 µM), but not nafamostat mesylate (10 µM); analyzed by two-way ANOVA. b JEV-activated BMMC supernatants increased FITC-dextran leakage across transwells 24 h post-exposure. TY-51469, but not nafamostat mesylate, reduced FITC-dextran leakage; n = 3. c Supernatants from JEV-stimulated WT BMMCs reduced TEER of b.END3 monolayers, but control media, JEV, supernatants from WT or MCPT4-KO BMMCs or JEV-stimulated MCPT4-KO BMMCs did not. d JEV-activated WT BMMC supernatants increased FITC-dextran leakage (24 h), but controls or supernatants from JEV-activated MCPT4-KO BMMCs did not; n = 3. e Claudin-5, ZO-1, ZO-2, and occludin levels in bEND.3 cells exposed to JEV-activated MC supernatants were reduced compared to media, JEV, and unstimulated MC supernatant-treated groups, by western blotting, which was inhibited by TY-51469. GAPDH blotting served as loading controls. Quantification is provided (Supplementary Figure ). f TY-51469 reduced EBD leakage into brains of JEV-infected WT mice, to levels similar to JEV-infected Sash mice, 5 days post-i.p. infection with Nakayama (2 × 10 7 PFU); n = 5. g Representative images from f . h IgG (red) of brain sections of JEV-infected WT, Sash, and TY-51469-treated WT mice, 5 days post-i.p. infection with Nakayama. During JEV infection, IgG was detected in the brain parenchyma of WT mice, but not Sash or TY-51469-treated WT mice. Scale bar = 50 μm. i TY-51469 reduced the JEV titers in brains, 6 days post-i.p. Nakayama infection, compared to vehicle-treated WT mice, to levels similar to Sash mice. Chymase injection (30 ng, i.p.) of Sash mice led to increased j EBD leakage ( n = 5) and k brain JEV titers, to levels similar to WT JEV-infected mice (5 days post-infection); n = 8; representative of two experiments. Error bars represent the SEM. Unless indicated, all data are representative of three independent experiments, were analyzed by one-way ANOVA with Holm-Sidak’s multiple comparison test; * denotes P < 0.05 and ** denotes P < 0.01. Chymase inhibition limits JEV penetration of the brain by preventing BBB leakage

Article Snippet: Mouse brain endothelial bEND.3 cells (5 × 10 5 cells in 500 µL DMEM media, passage number 4 or 5) were grown for 1 day on 3 μM transwell inserts (BD Biosciences; Corning, #353492) until confluency and tight junctions were well-established.

Techniques: Inhibition, Western Blot, Infection, Injection

Journal: Nature Communications

Article Title: The liver and muscle secreted HFE2-protein maintains central nervous system blood vessel integrity

doi: 10.1038/s41467-024-45303-1

Figure Lengend Snippet: a Knockout of liver Hfe2 reduces pericyte coverage in section from the cerebral cortex (mean ± s.e.m.; unpaired two-tail t-test; AAV8-GFP n = 3, Hfe2 ΔAlb-cre n = 4, and AAV8-AlbCre n = 3). Scale bar, 50μm. b Quantification through quantitative RT-PCR shows a reduction in PDGF-B mRNA levels in liver knock out animals (mean ± s.e.m.; paired two-tail t-test; Hfe2 fl/fl n = 3 and Hfe2 ΔAlb-cre n = 3). c Quantification of PDGF-B mRNA in cultured bEnd.3 cells following indicated treatments. Treatment with RGMa leads to a significant reduction of PDGF-B mRNA levels that is rescued with HFE2 addition (mean ± s.e.m.; paired two-tail t-test; HFE2 n = 3, RGMa n = 4, and RGMa+ HFE2 n = 5; n representing an independent experiment). d Immunocytochemistry of claudin-5 in human primary endothelial cell monolayer after PBS, HFE2, RGMa, and RGMa+HFE2 treatments and quantification (mean ± s.e.m.; unpaired two-tail t-test; replicates n = 3). Scale bar, 100 µm. e Western blotting of Claudin-5 expression in bEnd3 cell lysates after indicated treatment (mean ± s.e.m.; unpaired two-tailed t-test; replicates n = 3). f Transwell permeability leakage assay performed on a monolayer of bEnd3 cells using HRP (mean ± s.e.m.; paired one-tail t-test; replicates n = 3). g Transwell permeability leakage assay performed on a monolayer of human primary endothelial cells using 70 kDa FITC-dextran (mean ± s.e.m.; paired two-tail t-test; replicates n = 3). h In vitro TEER analysis shows that RGMa alters the integrity of a monolayer of bEnd3 cells, this is rescued by HFE2 (mean ± s.e.m.; unpaired two-tail t-test; replicates n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file (includes exact p-values).

Article Snippet: The endothelial monolayer permeability assay was performed as previously described DiStefano et al. Murine Brain Endothelial cells (bEnd3) (ATCC®CRL-2299 TM ) were seeded on 3 μm pore Transwell inserts (Corning Life Sciences) coated with 10 μg/ml human plasma fibronectin (Sigma, FC010).

Techniques: Knock-Out, Quantitative RT-PCR, Cell Culture, Immunocytochemistry, Western Blot, Expressing, Two Tailed Test, Permeability, In Vitro

Journal: Development (Cambridge, England)

Article Title: Hedgehog signaling is essential for endothelial tube formation during vasculogenesis.

doi: 10.1242/dev.01304

Figure Lengend Snippet: Fig. 6. Vascular assembly in cell culture in response to exogenous SHH. (A) RT- PCR analysis detects Ptch1, Smo and Vegfr2 transcripts in bEnd3 cells (lane 1) and Eoma cells (lane 2). (B) Confluent bEnd3 cells were switched to serum-free DMEM or (C) DMEM plus 2 µg/ml of SHH. SHH induces assembly of vascular endothelial cells into tube-like structures. (D) RT-PCR analysis of Vegf transcripts. The cDNA was synthesized from untreated and treated bEND3 cultures 48 hours after the addition of SHH or VEGF. cDNA synthesized from adult mouse heart total RNA was used as a positive control. (E) bEnd3 cells were assayed for proliferation by BrdU labeling, following addition of SHH at concentrations indicated. Data were averaged from three separate experiments. Addition of SHH does not increase endothelial cell proliferation.

Article Snippet: The mouse endothelial cell lines bEnd3 (Montesano et al., 1990) and EOMA (Obeso et al., 1990) were obtained from ATCC and cultured in Dulbecco’s Modified Eagles Medium (DMEM) containing 10% FBS (Hyclone), penicillin and streptomycin.

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Synthesized, Positive Control, Labeling