Journal: PLoS Biology

Article Title: Blood–brain barrier genetic disruption leads to protective barrier formation at the Glia Limitans

doi: 10.1371/journal.pbio.3000946

Figure Lengend Snippet: (A) Primary BMECs were isolated from 12-week-old C57Bl/6 mice, and Dhh , Ihh , Shh , Pecam1 , Sma , Ng2 , Cd45 , and Gfap expressions were quantified by qRT-PCR (cycle threshold mean values). β-actin is used as a reference. (B) Human cortical sections from healthy donors were obtain from the NeuroCEB biobank and immunostained with anti-CDH5 (in red), anti-PECAM1 (in red), and anti-DHH (in green) antibodies. Nuclei were stained with DAPI (in blue). (C–E) HBMECs were cultured until confluency and starved for 24 h. HBMECs were then treated with PBS (control condition) or IL-1β 10 ng/mL for 24 h and (C) DHH , (D) ICAM1 , and (E) VCAM1 expression were quantified by qRT-PCR. (F–H) Twelve-week-old C57Bl/6 females (6 animals per group) were induced with MOG 35-55 EAE versus placebo. At day 13 post induction, mice were humanely killed, and spinal cord microvascular endothelial cells were isolated. (F) Dhh , (G) Icam1 , (H) Vcam1 , (I) Cldn5 , and (J) Zo1 expression were measured via qRT-PCR in both groups (MOG 35-55 versus placebo). ** P ≤ 0.01, **** P ≤ 0.0001 Mann – Whitney U test. The underlying data for Fig 1 can be found in S1 Data ( https://doi.org/10.6084/m9.figshare.12625034.v6 ). *It is important to note that CNS endothelial cells are from a pooled source including both brain and spinal cord tissues. Therefore, the resulting cell cultures/lysates may be heterogeneous in their use of DHH. This remark applies to Figs – . BMECs, brain microvascular endothelial cells; CD45, cluster of differentiation 45; CDH5, cadherin5; Cldn5, claudin5; CNS, central nervous system; Ctrl, control; DHH desert hedgehog; EAE, experimental autoimmune encephalomyelitis; GFAP, glial fibrillary acidic protein; HBMECs, human brain microvascular endothelial cells; IHH, Indian hedgehog; cam1, intercellular adhesion molecule 1; IL-1β, interleukin 1 beta; MEC, microvascular endothelial cell; MOG 35-55 , myelin oligodendrocyte glycoprotein-35-55; NS, non-significant; NG2, neural/glia antigen 2; qRT-PCR, quantitative reverse transcription polymerase chain reaction; PECAM1, platelet/endothelial cell adhesion molecule 1; SMA, smooth muscle actin; SHH, Sonic hedgehog; Vcam1, vascular cell adhesion molecule 1; ZO1, zonula occludens 1.

Article Snippet: HBMECs (Alphabioregen–CliniSciences, Nanterre, France) were cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM-2 BulletKits (Lonza).

Techniques: Isolation, Quantitative RT-PCR, Staining, Cell Culture, Control, Expressing, MANN-WHITNEY, Reverse Transcription, Polymerase Chain Reaction

Journal: PLoS Biology

Article Title: Blood–brain barrier genetic disruption leads to protective barrier formation at the Glia Limitans

doi: 10.1371/journal.pbio.3000946

Figure Lengend Snippet: (A–K) NHA were cultured until confluency and starved for 24 h. NHA were then treated for 24 h with HBMEC medium from untreated cells (control condition), conditioned media from HBMECs treated with VEGFA, conditioned media from HBMECs treated with Mannitol, or HBMEC medium with 20% plasma from healthy donors (Mannitol and VEGFA were washed out of the HBMEC cultures before the medium was used to treat the NHA cultures). (A) Gfap , (B) Aldh1l1 , (C) Vim , and (D) Cldn4 mRNA expression was quantified by qRT-PCR. (Post-VEGFA n = 5, Mannitol n = 8 to 9, Plasma n = 7 to 8, Vehicle control n = 14 to 15). (E–H) GFAP (in green) and (E) CLDN4 (in red) localizations were evaluated by immunofluorescent staining of a confluent NHA monolayer. Nuclei were stained with DAPI (in blue). The experiment was repeated 3 times. (I, J) GFAP and CLDN4 positive areas were then quantified. (Post-VEGFA n = 7 to 8, Mannitol n = 7 to 8, Plasma n = 8, Vehicle control n = 7 to 8). (K–O) Cerebral cortices of 10-week-old C57BL/6 mice were harvested 24 h following stereotactic microinjection of murine VEGFA (60 ng in 3 μL PBS), healthy C57BL/6 mouse plasma (3 μL), or vehicle control (3 μL PBS). (K–M) Cortical lesions were immunostained with anti-GFAP and anti-CLDN4 antibodies. (N) GFAP positive areas and (O) CLDN4 positive areas were quantified (VEGFA n = 4, healthy C57BL/6 mouse plasma n = 4, Vehicle control n = 4). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 Kruskal–Wallis test. The underlying data for Fig 5 can be found in S1 Data ( https://doi.org/10.6084/m9.figshare.12625034.v6 ). Aldh1l1, aldehyde dehydrogenase 1 family, member l1; AU, arbitrary units; CLDN4, Claudin4; Ctrl, control; GFAP, glial fibrillary acidic protein; HBMEC, human brain microvascular endothelial cells; NHA, normal human astrocytes; NS, non-significant; qRT-PCR, quantitative reverse transcription polymerase chain reaction; VEGFA, vascular endothelial growth factor A; Vim, vimentin.

Article Snippet: HBMECs (Alphabioregen–CliniSciences, Nanterre, France) were cultured in endothelial basal medium-2 (EBM-2) supplemented with EGM-2 BulletKits (Lonza).

Techniques: Cell Culture, Control, Clinical Proteomics, Expressing, Quantitative RT-PCR, Staining, Microinjection, Reverse Transcription, Polymerase Chain Reaction

Journal: Brain

Article Title: Gene-mapping study of extremes of cerebral small vessel disease reveals TRIM47 as a strong candidate

doi: 10.1093/brain/awab432

Figure Lengend Snippet: TRIM47 depletion in human brain endothelial cells increases endothelial permeability. ( A ) Efficient reduction of TRIM47 expression in endothelial cells was achieved by siTRIM47 #1 and 2 versus siControl. Ve-cadherin expression, a marker of endothelial cell junction was followed and α tubulin was used as a loading control. ( B ) Endothelial transwell permeability assay. Quantification of dextran leakage expressed as fold change compared to HBMEC and ( C ) hCMEC/D3 ( D ) cells treated with control siRNA TRIM47 #1 and #2 versus siControl. Fluorescent intensity was normalized to siControl knock down. Two-way ANOVA with a Dunnett’s multiple comparisons test was performed. Data are presented as mean ± SD. ** P -value < 0.01, *** P < 0.0001, *** P > 0.00001.

Article Snippet: HBMEC (human brain microvascular endothelial cells; Alphabioregen-Clinisciences, #ALHE02) were grown in endothelial basal medium-2 (EBM-2) supplemented with EGM-2 BulletKits (Lonza #CC-3162) with 0.1% gelatin coating.

Techniques: Permeability, Expressing, Marker, Control, Knockdown