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: 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: Respiratory Research

Article Title: Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

doi: 10.1186/1465-9921-12-103

Figure Lengend Snippet: Increased expression of PDGF-BB in pulmonary endothelial cells on treatment with HIV-proteins . Representative western blot showing PDGF-BB expression in cellular extracts from Tat (25 ng/ml), gp-120 LAV (100 ng/ml), gp-120 CM (100 ng/ml), heat-inactivated (HI) Tat or HI-gp-120 treated human pulmonary microvascular endothelial cells. The blots were re-probed with human β-actin antibodies. Histogram represents the average densitometric ratio of PDGF-BB to β-actin of three independent experiments. Statistical significance was calculated using a two-tail, independent t-test. (** p ≤ 0.01 vs. control, #p ≤ 0.05 vs. Tat or gp-120 treatment).

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMVEC) were purchased from ScienCell research laboratories (Carlsbad, CA) and grown in endothelial cell basal media containing 2% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and gentamycin/amphotericin.

Techniques: Expressing, Western Blot, Control

Journal: Respiratory Research

Article Title: Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

doi: 10.1186/1465-9921-12-103

Figure Lengend Snippet: Involvement of oxidative stress in gp-120 mediated PDGF-BB induction in pulmonary endothelial cells . A) Enhanced oxidative stress in pulmonary endothelial cells on Tat and gp120 treatment. Human pulmonary microvascular endothelial cells (HPMVECs) were incubated with carboxy-H2-DCF-DA followed by Tat (25 ng/ml) or gp-120 (100 ng/ml) treatment for 60 min, and assessed for oxidative stress (Mean ± SD., **P ≤ 0.01, ***P < 0.001 vs. control). B) Effect of CCR5 neutralizing antibody on gp-120 CM (100 ng/ml) mediated oxidative stress in HPMVECs. Cells were pretreated with CCR5 antibody (10 μg/ml) or equal amount of IgG isotype control for 30 min, followed by DCF assay (Mean ± SD., ***P < 0.001 treatment versus control; #P < 0.05 vs. gp120 CM treatment). C) Gp-120 CM mediated PDGF-BB expression in the presence of antioxidant cocktail. HPMVECs were pretreated with antioxidant cocktail for 30 min followed by incubation with gp-120 CM (100 ng/ml) for 24 hours. Cells were then used for protein extraction followed by sequential immunobloting with antibodies specifically directed to the PDGF-BB and β-actin. Representative western blot images (upper panel) are shown with histograms (lower panel) showing the average densitometric analysis of the PDGF-BB band normalized to corresponding β-actin band from three independent experiments (*** P < = 0.001 versus control; ###P < = 0.001 versus gp120 CM treatment).

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMVEC) were purchased from ScienCell research laboratories (Carlsbad, CA) and grown in endothelial cell basal media containing 2% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and gentamycin/amphotericin.

Techniques: Incubation, Control, DCF Assay, Expressing, Protein Extraction, Western Blot

Journal: Respiratory Research

Article Title: Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

doi: 10.1186/1465-9921-12-103

Figure Lengend Snippet: Oxidative stress dependent HIF-1α expression is involved in gp-120 CM mediated PDGF-BB induction . A) Western blot analysis of HIF-1α expression in human pulmonary microvascular endothelial cells (HPMVECs) pretreated with or without antioxidant cocktail for 30 min followed by incubation with gp-120 CM (100 ng/ml) for 24 hours. B) Evaluation of HIF-1α knockdown by western blot analysis of whole cell lysates from HPMVECs transfected with siRNA specific to HIF-1α (10nM) or with negative control siRNA in presence of gp120 CM treatment. ( C) Knock down of HIF-1α resulted in inhibition of gp120 CM -mediated induction of PDGF-BB expression in HPMVECs. Blots are representative of three independent experiments with histogram (lower panel) showing the average densitometric analysis normalized to β-actin. All values are mean ± SD. *P < = 0.01,**P < = 0.001 treatment versus control; #P < = 0.01, ##P < = 0.001 treatment versus gp120 CM treated untransfected cells.

Article Snippet: Human primary pulmonary microvascular endothelial cells (HPMVEC) were purchased from ScienCell research laboratories (Carlsbad, CA) and grown in endothelial cell basal media containing 2% fetal bovine serum (FBS), 1 μg/ml hydrocortisone, 10 ng/ml human epidermal growth factor, 3 ng/ml basic fibroblast growth factor, 10 μg/ml heparin, and gentamycin/amphotericin.

Techniques: Expressing, Western Blot, Incubation, Knockdown, Transfection, Negative Control, Inhibition, Control

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