Journal: PLoS ONE

Article Title: CXCL12 Mediates Trophic Interactions between Endothelial and Tumor Cells in Glioblastoma

doi: 10.1371/journal.pone.0033005

Figure Lengend Snippet: ( A ) Human glioblastoma specimen immunostained for CXCL12 (brown) demonstrates expression in vascular endothelial cells. t = tumor cells, e = cross-section through tumor-associated capillary, and Scale bar = 25 µm. ( B ) HBMECs, in co-culture on Matrigel, express CXCL12 (red). Nuclei are counterstained blue with DAPI. Scale bar equals 25 µm. ( C ) Human brain micro-vascular endothelial cells cultured on Matrigel (Mat HBMEC) secrete CXCL12 into the media as determined by ELISA. Mat alone indicates results from Matrigel alone-conditioned media. N = 3. ** = p<0.005 as determined by two-tailed t -test. ( D ) Single cell suspensions from two different adult GBM patients (GBM1, GBM2) and cultured U87 cells express CXCR4 by western blotting. CXCR4 appears red and the actin loading control appears green.

Article Snippet: Primary human brain microvascular endothelial cells (HBMEC) were obtained from ScienCell, Carlsbad, CA).

Techniques: Expressing, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot, Control

Journal: PLoS ONE

Article Title: CXCL12 Mediates Trophic Interactions between Endothelial and Tumor Cells in Glioblastoma

doi: 10.1371/journal.pone.0033005

Figure Lengend Snippet: ( A ) Twenty-four hours after establishing a capillary-like network of mcherry-expressing HBMECs in Matrigel, eGFP-expressing U87 cells were added to the culture. Within 24 hours U87 cells were seen in physical contact with HBMECs. Scale bar = 50 microns. ( B ) The mean distances between U87 cells (500 to 800 cells) and HBMECs were calculated at different time points after the addition of the tumor cells to the HBMEC networks. There was a significant increase in co-localization (reduction in mean distance) within 24 hrs, which was maintained over a 72 hr period. * = p<0.05 as determined by one way ANOVA for the means of three separate experiments involving 500–800 measurements per experiment. ( C ) The distance between approximately 1000 eGFP-expressing U87 cells and mCherry fluorescent protein-expressing HBMECs in co-culture (24 hrs) was measured and the distribution was plotted as the percentage of total cells in 20 micron increments (black triangles). More than 50% of the total U87 cells in the culture were within 40 microns of an endothelial cell. A theoretical plot of a random distribution of cells is shown (open circles). ( D ) The distance between GFAP positive GBM cells and mCherry fluorescent protein-expressing HBMECs in co-culture were measured and the distribution was plotted as the percentage of total cells in 20 micron increments (black triangles). Error bars represent SEM from three independent experiments involving three different GBM isolates. Approximately 500 GBM (GFAP positive) cells were counted. Nearly 80% of the GFAP positive GBM cells in the culture were within 20 microns of an endothelial cell. A theoretical plot of a random distribution of cells is shown (open circles).

Article Snippet: Primary human brain microvascular endothelial cells (HBMEC) were obtained from ScienCell, Carlsbad, CA).

Techniques: Expressing, Co-Culture 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: 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: BMC Microbiology

Article Title: Role of Mycobacterium tuberculosis pknD in the Pathogenesis of central nervous system tuberculosis

doi: 10.1186/1471-2180-12-7

Figure Lengend Snippet: Invasion and survival of M. tuberculosis pknD mutant in host-derived cells . A . BALB/c mice were infected with M. tuberculosis CDC1551 or pknD mutant, and sacrificed at days 1 and 49 after infection. The mutant for M. tuberculosis pknD was significantly attenuated (P = 0.004) in mouse brain, but not lung tissue, 49 days after infection. No defect was observed in the lungs at either time point. Bacterial burden is represented as log 10 CFU/organ for all animal experiments. B . Invasion of host-cell monolayers by wild-type CDC1551, wild-type intergenic transposon control, pknD transposon mutant (pknD:Tn), and pknD genetic complement (pknD:Comp) was examined and normalized to the wild-type control. Invasion assays were performed in brain microvascular endothelial cells (HBMEC), epithelial A549 cells, and umbilical vein endothelia (HUVEC). No difference in invasion was observed in A549 cells (P = 0.31) or HUVEC (P = 0.41). A significant reduction in invasive capacity, however, was observed in the CNS-derived HBMEC (P = 0.02). This defect was restored by genetic complementation with the native pknD/pstS2 operon. N.S. = not significantly different. C . Intracellular survival of each of the above M. tuberculosis strains was examined in HBMEC at days 1, 3, 5, and 7 after infection. The pknD:Tn mutant demonstrated an invasion and intracellular survival defect in HBMEC relative to wild-type over the course of the seven day infection. D . Survival was also examined by infection of activated J774 macrophages. No corresponding survival defect for the pknD:Tn mutant was observed in these cells during the seven day infection. A mutant for the gene Rv0442c , known to be attenuated in the macrophage model, is included as a control. All CFU counts are represented as mean ± standard deviation.

Article Snippet: Primary human brain microvascular endothelial cells and HUVEC were kind gifts from Dr. Kwang Sik Kim, Department of Pediatrics, Johns Hopkins University School of Medicine.

Techniques: Mutagenesis, Derivative Assay, Infection, Control, Standard Deviation

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