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: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: Analysis of miR-155 binding sites on kras 3’UTR and Gli1/2 binding sites on mir155hg promotor by dual-luciferase reporter assays. A Conservation of the miR-155 sequence among different species (upper panel), and conservation of the miR-155 target sequence in KRAS among different species (lower panel). Human, Homo sapiens ; Mouse, Mus musculus ; Rat, Rattus norvegicus . B The miRNA response elements (MREs) of miR-155 were shown on the sequence of kras 3′-UTR, and mutations were introduced on these MREs. Both wild-type and mutated sequences were cloned into the psiCHECK-2 plasmid. C dual-luciferase reporter assay testing miR-155 binding with kras 3’UTR. HEK-293 T cells were co-transfected with has-miR-155 mimics or mimics NC as control oligonucleotide (final concentration at 50 nM) together with the wild-type (KRAS-WT-3’UTR) or mutated (KRAS-Mut-3’UTR) kras 3′-UTR luciferase reporter plasmids. The renilla luciferase activity was measured and normalized to firefly luciferase activity after 36 h. ** p < 0.01. The assays were performed in triplicates, and results are presented as mean ± SEM. D Schematic of the three predicted Gli1/2 binding sites on mir155 hg promotor (upper panel) and their binding sequences accordingly (lower panel). The binding sites were located at − 1088 to − 1077 (site 1), − 461 to − 447 (site 2), and − 407 to − 393 (site 3) of the mir155hg promotor. The gli2 luciferase activities were tested by applying a series of truncations ( E ) as well as site-targeted mutations ( F ) on the mir155hg promoter, along with pcDNA3.1-Gli1 and pcDNA3.1-Gli2 and pRL-TK plasmids. The specific constructs used in the truncation assays ( E ) included pGL3-basic vector, pGL3-mir155hg-promo-WT (containing promotor region from − 1440 to + 226), pGL3-mir155hg-promo-truncation1 (from − 913 to + 226) and pGL3-mir155hg-promo-truncation2 (from − 339 to + 226). The specific constructs used in the site-mutation assays ( F ) included pGL3-basic vector, pGL3- mir155hg-promo-WT (containing all 3 sites), pGL3-mir155hg-promo-mut1 (lack of site 1), pGL3-mir155hg-promo-mut2 (lack of site 2) and pGL3-mir155hg-promo-mut3 (lack of site 3). The luciferase activities were determined and presented as the ratio of firefly and renilla luciferase activity. The assays were performed with three replicates and data are presented as mean ± SEM. ** p < 0.01. G Schematic of the Gli1 and Gli2 binding to the mir155hg promotor at around − 1088 in hBMECs

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Binding Assay, Luciferase, Sequencing, Clone Assay, Plasmid Preparation, Reporter Assay, Transfection, Control, Concentration Assay, Activity Assay, Construct, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: rTGFβ1 inhibited ERK1/2 signaling and immune reaction of RS218 infected hBMECs relying on inducted noncanonical HH signaling. A Detecting expression alterations of IL-6, MIP-2, and E-selectin in hBMECs with qPCR or Western blot. The cells were infected with RS218 with or without rTGFβ1 pre-treatment (at 50 ng/mL). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. B qPCR detecting IL-6, MIP-2, and E-selectin transcription upon RS218 infection in wild-type hBMECs or Gli-KO hBMECs. The wild-type hBMECs were pretreated with rTGFβ1 (at 50 ng/mL) or together with GANT61 (at 10 μM). The Gli-KO hBMECs were pretreated with rTGFβ1 (at 50 ng/mL). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. C Western blot detecting phosphorylation of p65, JNK, ERK1/2, and p38 in RS218 infected hBMECs. The cells were pretreated with or without rTGFβ1 at 50 ng/mL. D Western blot detecting expression of E-selectin and ERK1/2 phosphorylation in RS218 infected hBMECs. The cells were pretreated with rTGFβ1 (at 50 ng/mL) or GANT61 (at 10 μM). E Detecting expression alterations of IL-6, MIP-2, and E-selectin in hBMECs with qPCR or Western blot. The cells were infected with RS218 with or without U0126 treatment (at 5 μM). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Infection, Expressing, Western Blot, Phospho-proteomics

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: TGF-HH axis suppressed ERK1/2 signaling in RS218 infected hBMECs via modulating miR155 and KRAS. A Western blot and qPCR detecting expression of KRAS in RS218 infected hBMECs. The cells were pretreated with rTGFβ1 (at 50 ng/mL) or GANT61 (at 10 μM). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. B IF assays showing the KRAS expression in BMECs of mice challenged by RS218 with or without rTGFβ1 pre-treatment (at 1 μg/kg). The KRAS was stained in red. CD31 was specifically applied for labeling the micro-vessels in green. The cell nucleus was stained in blue with DAPI. Scale bar = 50 μm. C qPCR detecting MIR155HG and miR-155 expression upon RS218 infection in hBMECs. The cells were pretreated with rTGFβ1 (at 50 ng/mL) or GANT61 (at 10 μM). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. D Western blot detecting expression of E-selectin, KRAS and ERK1/2 phosphorylation in RS218 infected hBMECs with or without rTGFβ1 pre-treatment (at 50 ng/mL). The cells were transfected with has-miR-155 mimics, mimics NC, has-miR-155 inhibitors, or inhibitors NC at 50 nM as indicated. E qPCR detecting expression alterations of IL-6, MIP-2, and E-selectin in RS218 infected hBMECs with or without rTGFβ1 pre-treatment (at 50 ng/mL). The cells were transfected with has-miR-155 mimics, mimics NC, has-miR-155 inhibitors, or inhibitors NC at 50 nM as indicated. ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Infection, Western Blot, Expressing, Staining, Labeling, Phospho-proteomics, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: SAG repressed BMECs immune reaction and neuroinflammation of infected mice in vivo and in vitro. A Western blot detecting expression of E-selectin, KRAS and ERK1/2 phosphorylation in RS218 infected hBMECs with or without SAG pre-treatment (at 10 μM). B qPCR detecting expression alterations of IL-6, MIP-2, and E-selectin in RS218 infected hBMECs with or without SAG pre-treatment (at 10 μM). ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. C qPCR detecting MIR155HG, miR-155, and KRAS expression upon RS218 infection in hBMECs. The cells were pretreated with or without SAG pre-treatment (at 10 μM). * p < 0.05, ** p < 0.01. The qPCR assays were performed in triplicates, and results are presented as mean ± SEM. D Effects of the SAG pre-treatment at 10 mg/kg (for 12 h) on the survival of the mice after the challenge of RS218 ( n = 10). ** p < 0.01. E ELISA analysis of IL-6 and MIP-2 in brain lysates from RS218 challenged mice with or without SAG pre-treatment at 10 mg/kg. Data are presented as mean ± SEM from five individual mice in each group. F IF assays showing the E-selectin and KRAS expression in brains of mice challenged by RS218 with or without SAG pre-treatment (at 10 mg/kg). The E-selection and KRAS were stained in red. CD31 was specifically applied for labeling the micro-vessels in green. The cell nucleus was stained in blue with DAPI. Scale bar = 50 μm

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Infection, In Vivo, In Vitro, Western Blot, Expressing, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Selection, Staining, Labeling

Journal: Cell Communication and Signaling : CCS

Article Title: TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

doi: 10.1186/s12964-023-01383-y

Figure Lengend Snippet: Schematic representation of the TGFβ1 or SAG immunosuppression effects on BMECs through upregulating miR-155 and negative regulating KRAS as well as downstream ERK1/2 signaling. The exogenous TGFβ1 or SAG triggered the HH signaling in E. coli -infected BMECs and upregulated intracellular miR-155. Further, the promoted miR-155 suppressed the ERK1/2 activation by negatively regulating KRAS, thus decreasing IL-6, MIP-2, and E-selectin expression

Article Snippet: The human BMECs (hBMECs) were kindly gifted from Prof. Kwang Sik Kim at Johns Hopkins University School of Medicine, and the Gli-KO hBMECs cell lines were constructed and tested in a previous study [ ].

Techniques: Infection, Activation Assay, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Felodipine Promotes the Recovery of Mice With Spinal Cord Injury by Activating Macrolipophagy Through the AMPK ‐ mTOR Pathway

doi: 10.1111/jcmm.70543

Figure Lengend Snippet: Felodipine could attenuate lipid accumulation‐induced fibrosis in HBMECs through the AMPK‐mTOR pathway in vitro. (A) The chemical structure formula of felodipine. (B) Adopting CCK‐8 to measure relative cell viability, the optimal concentration of felodipine was 100 nM in HBMECs. (C) Western blot indicates the expression of pAMPK, AMPK, pmTOR and mTOR in HBMECs. (D and E) Quantitative analysis of pAMPK/AMPK and pmTOR/mTOR protein expression. *Represents p < 0.05, **represents p < 0.01 versus the control group and ## represents p < 0.01. The data are the mean ± SD ( n = 3). (F) Immunofluorescence staining of Bodipy (green), collagen I (red) and DAPI (blue) in HBMECs. Scale bar: 100 μm. (G) The mean fluorescence intensity of Bodipy in HBMECs. (H) Quantification of the proportion of Bodipy + and collagen I + cells.

Article Snippet: Human brain microvascular endothelial cells (HBMECs) were obtained from iCell (iCell‐h070, Shanghai, China) and cultured in high‐glucose DMEM supplemented with 100 μg/mL streptomycin, 100 U/mL penicillin and 10% FBS in a 37°C incubator with 5% CO 2 .

Techniques: In Vitro, CCK-8 Assay, Concentration Assay, Western Blot, Expressing, Control, Immunofluorescence, Staining, Fluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Felodipine Promotes the Recovery of Mice With Spinal Cord Injury by Activating Macrolipophagy Through the AMPK ‐ mTOR Pathway

doi: 10.1111/jcmm.70543

Figure Lengend Snippet: Felodipine could induce autophagy in HBMECs through the AMPK‐mTOR pathway in vitro. (A) Immunofluorescence staining of LC3 (green) and DAPI (blue) in HBMECs. Scale bar: 100 μm. (B) The mean fluorescence intensity of LC3 in HBMECs. (C) Western blot indicating the expression of LC3 and P62 in HBMECs. (D and E) Quantitative analysis of LC3II/LC3I and P62 protein expression. (F) Immunofluorescence staining of Bodipy (green) and LC3 (red) in HBMECs. Scale bar: 50 μm. (G and H) The mean fluorescence intensity of Bodipy and LC3 in HBMECs. N.S. (not significant), *represents p < 0.05, **represents p < 0.01 versus the control group and ## represents p < 0.01. The data are the mean ± SD ( n = 3).

Article Snippet: Human brain microvascular endothelial cells (HBMECs) were obtained from iCell (iCell‐h070, Shanghai, China) and cultured in high‐glucose DMEM supplemented with 100 μg/mL streptomycin, 100 U/mL penicillin and 10% FBS in a 37°C incubator with 5% CO 2 .

Techniques: In Vitro, Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing, Control

Journal:

Article Title: Anti-Tumoral Activity of a Short Decapeptide Fragment of the Alzheimer's A? Peptide

doi: 10.1007/s10989-010-9198-8

Figure Lengend Snippet: Effect of 4-hydroxy tamoxifen on angiogenesis in vitro. a Representative pictures depicting the formation of capillary networks in response to a dose range of 4-hydroxy tamoxifen following the plating of human brain microvascular endothelial cells onto a layer of Matrigel. b The graph represents the quantification of capillary network length by image analysis and the amount of LDH activity detected in the culture medium surrounding the capillary like structures following 24 h of treatment with a dose range of 4-hydroxy tamoxifen. ANOVA revealed a significant main effect of 4-hydroxy tamoxifen on LDH released (P < 0.03) but no significant main effect on capillary formation (P = 0.743) and post hoc analysis showed significant differences for LDH released between control conditions and 10 µM of 4-hydroxy tamoxifen (P < 0.009)

Article Snippet: Capillary Morphogenesis Assay Human Brain Microvascular Endothelial Cells (HBMEC) (Sciencell, CA) were cultured in Endothelial Cell Growth Medium (Cell Applications, CA) containing 5% fetal bovine serum, 1% penicillin/streptomycin and 1% Endothelial Cell Growth Supplement (Sigma–Aldrich, MO).

Techniques: In Vitro, Activity Assay, Control