Journal: Scientific Reports

Article Title: Human Bone Marrow Endothelial Progenitor Cell Transplantation into Symptomatic ALS Mice Delays Disease Progression and Increases Motor Neuron Survival by Repairing Blood-Spinal Cord Barrier

doi: 10.1038/s41598-019-41747-4

Figure Lengend Snippet: Characteristics of disease outcomes in G93A mice receiving hBMEPCs at symptomatic stage. Transplanted ALS mice with cell dose of 1 × 10 6 at 4 weeks post-treatment ( A ) significantly maintained body weight, ( B ) better extended hindlimbs, ( C ) delayed loss in muscle strength, and ( D ) stayed longer on rotarod vs. media-injected mice. Notable beneficial effects on motor function were determined in G93A mice 2–3 weeks after cell transplantation. * p < 0.05, ** p < 0.01.

Article Snippet: Cryopreserved human bone marrow-derived endothelial progenitor cells (hBMEPCs) were purchased from CELPROGEN (Torrance, CA, USA).

Techniques: Injection, Transplantation Assay

Journal: Scientific Reports

Article Title: Human Bone Marrow Endothelial Progenitor Cell Transplantation into Symptomatic ALS Mice Delays Disease Progression and Increases Motor Neuron Survival by Repairing Blood-Spinal Cord Barrier

doi: 10.1038/s41598-019-41747-4

Figure Lengend Snippet: Immunocytochemical analysis of transplanted hBMEPCs in blood smears. For identification of transplanted hBMEPCs within blood circulation of treated mice, immunofluorescent staining with the human anti-vWF was performed in blood smears. ( A ) hBMEPCs were positive for vWF immunoexpression (red, arrowheads). A few cells did not express vWF (asterisk) likely due to damaging during smear preparation. ( B ) There was no detection of human cells by vWF marker in blood smears from media-treated mice (asterisks). ( C , C’) In blood smears from cell-treated ALS mice, some cells were identified with human vWF (red, arrowheads). Cells negative for vWF are noted by asterisks. The nuclei in all images are shown with DAPI. Scale bar in A is 20 µm and B-C’ is 50 µm.

Article Snippet: Cryopreserved human bone marrow-derived endothelial progenitor cells (hBMEPCs) were purchased from CELPROGEN (Torrance, CA, USA).

Techniques: Staining, Marker

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Basic Fibroblast Growth Factor Reduces Permeability and Apoptosis of Human Brain Microvascular Endothelial Cells in Response to Oxygen and Glucose Deprivation Followed by Reoxygenation via the Fibroblast Growth Factor Receptor 1 (FGFR1)/ERK Pathway

doi: 10.12659/MSM.918626

Figure Lengend Snippet: Oxygen-glucose deprivation/reperfusion (OGD/R) resulted in increased human brain microvascular endothelial cell (HBMEC) permeability and apoptosis. ( A ) Fluorescein isothiocyanate (FITC) conjugated with dextran quantified endothelial permeability by analyzing the integrity of the cell monolayer. The human brain microvascular endothelial cells (HBMECs) were examined at 6, 12, 18, and 24 h after OGD/R treatment. ( B ) Quantification of apoptotic cells at 6, 12, 18, and 24 h following OGD/R, detected by the TUNEL assay. ( C ) Endothelial cell viability was measured by the cell counting kit-8 (CCK-8) assay at 6, 12, 18, and 24 h after OGD/R treatment. Control group vs. the OGD/R group (* P<0.05, ** P<0.01). N=6 in each group at each timepoint. N=3 independent experiments.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) were obtained from Angio-Proteomie (Boston, MA, USA).

Techniques: Permeability, TUNEL Assay, Cell Counting, CCK-8 Assay, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Basic Fibroblast Growth Factor Reduces Permeability and Apoptosis of Human Brain Microvascular Endothelial Cells in Response to Oxygen and Glucose Deprivation Followed by Reoxygenation via the Fibroblast Growth Factor Receptor 1 (FGFR1)/ERK Pathway

doi: 10.12659/MSM.918626

Figure Lengend Snippet: Changes in phospho-fibroblast growth factor receptor 1 (p-FGFR1) and p-ERK expression in human brain microvascular endothelial cells (HBMECs). ( A ) Representative Western blot images for FGFR1 and p-FGFR1. The expression of p-FGFR1 was affected by the use of the agonist, bFGF, and inhibition of FGFR1 with PD173074. ( B ) Representative Western blot images for FGFR2 and p-FGFR2. The expression of the FGFR2 and p-FGFR2 proteins did not change after oxygen-glucose deprivation/reperfusion (OGD/R) treatment. ( C ) Representative Western blot images for ERK and p-ERK. The expression of p-FGFR1 was affected by an agonist, bFGF, and inhibition of ERK with PD98059. ( D ) Immunofluorescence staining of p-FGFR1 and p-ERK in HBMECs shows that the expression levels were changed after OGD/R treatment. ( E ) Quantification of p-FGFR1/2 and p-ERK protein levels. The control group or the bFGF group vs. the OGD/R group (* P<0.05, ** P<0.01). PD173074 or PD98059 treatment vs. the bFGF group ( # P<0.05, ## P<0.01). N=6 in each group. N=3 independent experiments.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) were obtained from Angio-Proteomie (Boston, MA, USA).

Techniques: Expressing, Western Blot, Inhibition, Immunofluorescence, Staining, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Basic Fibroblast Growth Factor Reduces Permeability and Apoptosis of Human Brain Microvascular Endothelial Cells in Response to Oxygen and Glucose Deprivation Followed by Reoxygenation via the Fibroblast Growth Factor Receptor 1 (FGFR1)/ERK Pathway

doi: 10.12659/MSM.918626

Figure Lengend Snippet: Basic fibroblast growth factor (bFGF) reduced the increase in endothelial monolayer permeability and apoptosis induced by oxygen-glucose deprivation/reperfusion (OGD/R). ( A ) Treatment with bFGF significantly decreased the permeability of HBMECs 12 h after OGD/R treatment. ( B ) Quantification and ( C ) immunostaining of apoptotic cells 12 h post-OGD/R, as detected by the TUNEL assay. The number of immunoreactive cells was reduced by bFGF, indicating that it suppressed OGD/R-induced HBMEC apoptosis. The control or bFGF group vs. the OGD/R group (** P<0.01). Inhibition of FGFR1 with PD173074 or inhibition of ERK with PD98059 compared with the bFGF group (## P<0.01). N=6 in each group. N=3 independent experiments.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) were obtained from Angio-Proteomie (Boston, MA, USA).

Techniques: Permeability, Immunostaining, TUNEL Assay, Control, Inhibition

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Basic Fibroblast Growth Factor Reduces Permeability and Apoptosis of Human Brain Microvascular Endothelial Cells in Response to Oxygen and Glucose Deprivation Followed by Reoxygenation via the Fibroblast Growth Factor Receptor 1 (FGFR1)/ERK Pathway

doi: 10.12659/MSM.918626

Figure Lengend Snippet: Following oxygen-glucose deprivation/reperfusion (OGD/R) of endothelial cells, basic fibroblast growth factor (bFGF) inhibited the decrease in tight junction and adherens junction proteins. ( A–D ) Treatment with bFGF reversed or reduced zonula occludens-1 (ZO-1), occludin, claudin-5, and β-catenin expression in HBMECs 24 h after OGD/R treatment. Inhibition of fibroblast growth factor receptor 1 (FGFR1) with PD173074 and inhibition of ERK with PD98059 reversed the protective effect of bFGF.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) were obtained from Angio-Proteomie (Boston, MA, USA).

Techniques: Expressing, Inhibition

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Basic Fibroblast Growth Factor Reduces Permeability and Apoptosis of Human Brain Microvascular Endothelial Cells in Response to Oxygen and Glucose Deprivation Followed by Reoxygenation via the Fibroblast Growth Factor Receptor 1 (FGFR1)/ERK Pathway

doi: 10.12659/MSM.918626

Figure Lengend Snippet: bFGF ameliorated the OGD/R-induced adverse effect of endothelial tight junction, adherens junction, MMPs and apoptosis proteins. ( A ) Representative Western blot image for tight junction and adherens junction proteins, zonula occludens-1 (ZO-1), occludin, claudin-5, and β-catenin. ( B ) Representative Western blot image for matrix metalloproteinases (MMPs) and apoptosis proteins, MMP2, MMP9, caspase-3, Bcl-2, and BAX. ( C–F ) Quantitative data for expression of tight junction and adherens junction proteins, zonula occludens-1 (ZO-1), occludin, claudin-5, and β-catenin in HBMECs 24 h following OGD/R. Note that bFGF promoted the expression of tight junction and adherens junction proteins, suggesting that it reversed or reduced the increased permeability after OGD/R treatment. ( G–K ) Quantitative data for expression of MMPs and apoptosis-related proteins, MMP2, MMP9, caspase-3, Bcl-2, and BAX in HBMECs 24 h following OGD/R. Note that bFGF promoted the expression of Bcl-2 and inhibited the expression of MMP2, MMP9, caspase-3, and BAX, suggesting that it reversed or reduced cellular apoptosis and integrity after OGD/R treatment. The control group or the bFGF group vs. the OGD/R group (** P<0.01). Inhibition of fibroblast growth factor receptor 1 (FGFR1) with PD173074 or inhibition of ERK with PD98059 vs. the bFGF group ( # P<0.05, ## P<0.01). N=6 in each group. N=3 independent experiments.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) were obtained from Angio-Proteomie (Boston, MA, USA).

Techniques: Western Blot, Expressing, Permeability, Control, Inhibition

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Basic Fibroblast Growth Factor Reduces Permeability and Apoptosis of Human Brain Microvascular Endothelial Cells in Response to Oxygen and Glucose Deprivation Followed by Reoxygenation via the Fibroblast Growth Factor Receptor 1 (FGFR1)/ERK Pathway

doi: 10.12659/MSM.918626

Figure Lengend Snippet: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) showed that bFGF enhanced the expression of tight junction, adherens junction and apoptosis-related genes in HBMECs, while inhibition of fibroblast growth factor receptor 1 (FGFR1) with PD173074 and inhibition of ERK with PD98059 removed the protective effect of bFGF. ( A–F ) Expression of genes in HBMECs, 24 h following OGD/R, after treatment with bFGF, inhibition of FGFR1 with PD173074, or inhibition of ERK with PD98059. In the bFGF group, the relative expression levels of zonula occludens-1 (ZO-1), occludin, claudin-5, β-catenin, and Bcl-2 were significantly increased. The expression level of BAX was decreased in the bFGF group, but the protective effects of bFGF were reversed after PD173074 or PD98059 treatment. The control group or the bFGF group vs. the OGD/R group (* P<0.05, ** P<0.01). Inhibition of fibroblast growth factor receptor 1 (FGFR1) with PD173074 or inhibition of ERK with PD98059 vs. the bFGF group ( # P<0.05, ## P<0.01). N=6 in each group. N=3 independent experiments.

Article Snippet: Primary human brain microvascular endothelial cells (HBMECs) were obtained from Angio-Proteomie (Boston, MA, USA).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Inhibition, Control

Journal: Stem Cell Reviews and Reports

Article Title: Outgrowth Endothelial Cell Conditioned Medium Negates TNF-α-Evoked Cerebral Barrier Damage: A Reverse Translational Research to Explore Mechanisms

doi: 10.1007/s12015-022-10439-4

Figure Lengend Snippet: Schematic diagram of an in vitro model of human BBB and the effect of OEC-CM on BBB integrity and function and actin cytoskeleton organization in HBMECs and OECs. (A) In vitro models of human BBB consisting of astrocytes, pericytes, and HBMECs alone or mixed with OECs. (B, C) TNF-α significantly disrupted BBB integrity and function, as shown by decreases in TEER and concomitant increases in paracellular flux of sodium fluorescein, which were prevented by OEC-CM treatment. (D) Co-treatment with OEC-CM prevented the effects of TNF-α on cytoskeletal reorganization in HBMECs and OECs and decreased stress fiber formation (white arrows). (E) Quantification of stress fiber formation in both cells. Scale bar: 25 μm. * P < 0.05 versus BBB formed by HBMECs or control, # P < 0.05 versus BBB formed by HBMECs exposed to TNF-α, † P < 0.05 versus BBB formed by HBMECs exposed to TNF-α and OEC-CM, φ P < 0.05 versus BBB formed by HBMECs and OECs, ψ P < 0.05 versus BBB formed by HBMECs and OECs exposed to TNF-α (one-way ANOVA followed by Tukey's post-hoc analysis). BBB, blood–brain barrier; HBMECs, human brain microvascular endothelial cells; OEC-CM, outgrowth endothelial cell-derived conditioned medium; OECs, outgrowth endothelial cells; TNF-α, tumor necrosis factor-α

Article Snippet: Human brain microvascular endothelial cells (HBMECs), pericytes, and astrocytes were purchased from TCS CellWorks Ltd. (Buckingham, UK) and cultured at 37 °C in a humidified atmosphere (75% N 2 , 20% O 2 , 5% CO 2 ) with their respective media (Sciencell Research Laboratories, San Diego, USA).

Techniques: In Vitro, Control, Derivative Assay

Journal: Stem Cell Reviews and Reports

Article Title: Outgrowth Endothelial Cell Conditioned Medium Negates TNF-α-Evoked Cerebral Barrier Damage: A Reverse Translational Research to Explore Mechanisms

doi: 10.1007/s12015-022-10439-4

Figure Lengend Snippet: The effect of OEC-CM on HBMEC and OEC functional characteristics and analysis of angiogenesis-related proteins in HBMEC and OEC secretomes and OEC-CM. (A, B) OEC-CM accelerated wound closure in both HBMEC and OECs. (C-E) OEC-CM negated the impact of TNF-α on HBMEC and OEC tubule network. (F, G) Treatments with OEC-CM neutralized the inhibitory effect of TNF-α on HBMEC and OEC adhesion to fibronectin, an extracellular matrix protein. (H, I) Proteome profiling of OEC-CM along with HBMEC and OEC secretomes revealed significant variations in various pro- and anti-angiogenic factors e.g. endothelin-1, MCP-1 and endostatin in OEC-CM. Scale bars = 100 μm. * P < 0.05 versus control, # P < 0.05 versus TNF-α (one-way ANOVA followed by Tukey's post-hoc analysis). HBMECs, human brain microvascular endothelial cells; IL-8, interleukin-8; MCP-1, monocyte chemoattractant protein-1; OEC-CM, outgrowth endothelial cell-derived conditioned medium; OECs, outgrowth endothelial cells; TIMP-1, tissue inhibitors of metalloproteinase-1; TNF-α, tumor necrosis factor-α; uPA, urokinase plasminogen activator

Article Snippet: Human brain microvascular endothelial cells (HBMECs), pericytes, and astrocytes were purchased from TCS CellWorks Ltd. (Buckingham, UK) and cultured at 37 °C in a humidified atmosphere (75% N 2 , 20% O 2 , 5% CO 2 ) with their respective media (Sciencell Research Laboratories, San Diego, USA).

Techniques: Functional Assay, Control, Derivative Assay

Journal: Basic Research in Cardiology

Article Title: Acid sphingomyelinase deactivation post-ischemia promotes brain angiogenesis and remodeling by small extracellular vesicles

doi: 10.1007/s00395-022-00950-7

Figure Lengend Snippet: Amitriptyline inhibits ASM activity in vivo and promotes angiogenesis after I/R in an Asm dependent way. A Asm activity in the reperfused ischemic striatum (labeled I/R) and contralateral non-ischemic striatum (labeled C), measured using BODIPY-labeled sphingomyelin in wildtype mice exposed to transient MCAO, which were intraperitoneally treated with vehicle or amitriptyline (2 or 12 mg/kg b.w., b.i.d.) immediately after MCAO or with 24 h delay, followed by animal sacrifice after 24 h or after 14 days. B Ceramide content, measured by LC–MS/MS in I/R and C of wildtype MCAO mice treated with vehicle or amitriptyline for 14 days as above. C Total microvascular length, D branching point density and E mean microvascular branch length, evaluated by LSM in I/R and C of wildtype MCAO mice treated with vehicle or amitriptyline for 14 days. F Microvascular length, G branching point density and ( H ) mean branch length in C and I/R of Smpd1 + / + (wildtype) and Smpd1 −/− (that is, ASM-deficient) MCAO mice treated with vehicle or amitriptyline for 14 days. Note that amitriptyline increases angiogenesis in wildtype but not Smpd1 −/− mice. Representative 3D stacks post-I/R are shown in ( I ), ROIs for the evaluation of microvascular networks in ( J ), and maximum projection images inside these ROIs in ( K ). Data are means ± SD values. * p ≤ 0.05/** p ≤ 0.01/*** p ≤ 0.001 compared with non-ischemic C; † p ≤ 0.05/ †† p ≤ 0.01 compared with corresponding vehicle; ‡ p ≤ 0.05/ ‡‡ p ≤ 0.01 compared with corresponding Smpd1 + / + (n = 4–7 animals/group [in ( A )]; n = 7–9 animals/group [in ( B )]; n = 7–8 animals/group [in ( C – E )]; n = 5–7 animals/group [in ( F – H )]; analyzed by one-way ANOVA followed by LSD tests). Scale bars in 3D reconstructions in ( I ), 500 µm; in horizontal sections in ( I ), 1000 µm

Article Snippet: For comparative studies on sphingomyelinase expression and activities, primary human brain microvascular endothelial cells (HBMEC; catalog #1000; ScienCell™ Research Laboratories, Carlsbad, CA, U.S.A.) cultured in endothelial cell growth medium MV (ECGM-MV, PromoCell, Heidelberg, Germany) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium MV supplement mix (PromoCell) and human umbilical vein endothelial cells (HUVEC) cultured in endothelial cell growth medium (ECGM, PromoCell) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium supplement mix (PromoCell) were used.

Techniques: Activity Assay, In Vivo, Labeling, Liquid Chromatography with Mass Spectroscopy

Journal: Basic Research in Cardiology

Article Title: Acid sphingomyelinase deactivation post-ischemia promotes brain angiogenesis and remodeling by small extracellular vesicles

doi: 10.1007/s00395-022-00950-7

Figure Lengend Snippet: Amitriptyline, fluoxetine and desipramine promote cerebral angiogenesis in vitro in an ASM dependent way. A – C Matrigel-based tube formation evaluated for the number of closed tubes, microvascular length and branching point density, D transwell migration, E , F VEGFR2 abundance examined by Western blot and G , H VEGF concentration in supernatants measured by enzyme-linked immunosorbent assay (ELISA) of hCMEC/D3 exposed to vehicle or amitriptyline (0–50 µM). In ( F , H ), analyses were made after 4 and 24 h amitriptyline exposure, respectively. I Tube formation and J transwell migration of hCMEC/D3 exposed to vehicle or fluoxetine (0–20 µM). K Tube formation and L transwell migration of hCMEC/D3 exposed to vehicle or desipramine (0–50 µM). Note that all three ASM inhibitors increase angiogenesis. M Tube formation, N transwell migration, O VEGFR2 abundance and P VEGF concentration in supernatants of hCMEC/D3 transfected with scrambled siRNA (used as control) or SMPD1 siRNA which were exposed to vehicle or amitriptyline (50 µM). In ( O , P ), measurements were made after 4 and 24 h amitriptyline exposure, respectively. Data are means ± SD values. * p ≤ 0.05/** p ≤ 0.01/*** p ≤ 0.001 compared with corresponding vehicle; ‡ p ≤ 0.05/ ‡‡ p ≤ 0.01/ ‡‡‡ p ≤ 0.001 compared with corresponding scrambled siRNA ( n = 3–7 independent samples/group [in ( A – L )]; n = 5–8 independent samples/group [in ( M,N,O )]; n = 3 independent samples/group [in ( P )]; analyzed by one-way ANOVA [in ( A – D, G–L )] or two-way ANOVA [in ( M , N , P )] followed by LSD tests [in ( A – D , G – N , P )] or paired two-tailed t tests [in ( E , F , O )])

Article Snippet: For comparative studies on sphingomyelinase expression and activities, primary human brain microvascular endothelial cells (HBMEC; catalog #1000; ScienCell™ Research Laboratories, Carlsbad, CA, U.S.A.) cultured in endothelial cell growth medium MV (ECGM-MV, PromoCell, Heidelberg, Germany) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium MV supplement mix (PromoCell) and human umbilical vein endothelial cells (HUVEC) cultured in endothelial cell growth medium (ECGM, PromoCell) containing 100 U/ml penicillin/streptomycin (Life Technologies) and growth medium supplement mix (PromoCell) were used.

Techniques: In Vitro, Migration, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Two Tailed Test

Journal: Molecular Medicine

Article Title: Selective Antihypertensive Dihydropyridines Lower A? Accumulation by Targeting both the Production and the Clearance of A? across the Blood-Brain Barrier

doi: 10.2119/molmed.2010.00180

Figure Lengend Snippet: Effects of amlodipine, nitrendipine and nilvadipine on Aβ transcytosis across an in vitro model of the BBB. Fluorescein labeled human Aβ1–42 was added to the basolateral compartment (“brain” side) whereas different doses of amlodipine, nitrendipine and nilvadipine were added to the apical side (“blood” side) of the in vitro BBB model. The amount of fluorescein-Aβ1–42 was quantified in the apical side over a period of 90 min to calculate the apparent permeability of Aβ1–42 for the different treatments conditions. ANOVA shows a statistically significant effect of nilvadipine (P < 0.05), of nitrendipine (P < 0.001) but not of amlodipine (P = 0.910) on Aβ transcytosis in vitro across the BBB layer of human brain microvascular endothelial cells. Post hoc analysis reveals a significant effect of nilvadipine at 10 μmol/L (P < 0.05) and a significant effect of nitrendipine at 5 μmol/L (P < 0.01) and 10 μmol/L (P < 0.001), showing that nilvadipine and nitrendipine stimulate the transport of Aβ from the brain to the periphery in an in vitro model of the BBB, whereas amlodipine is inefficient.

Article Snippet: Effects of Dihydropyridines on Aβ Transcytosis In Vitro Human brain microvascular endothelial cells (HBMEC), endothelial cell media (ECM), fetal bovine serum, penicillin/streptomycin solution, and endothelial cell growth supplement (ECGS) were purchased from Sciencell Research Laboratories (Carlsbad, CA, USA).

Techniques: In Vitro, Labeling, Permeability