Journal: Pharmacological research

Article Title: Modulation of cellular bioenergetics by CO-releasing molecules and NO-donors inhibits the interaction of cancer cells with human lung microvascular endothelial cells.

doi: 10.1016/j.phrs.2018.09.005

Figure Lengend Snippet: Fig. 4. Effect of CORM-401 and PAPA NONOate on MDA-MB-231-luc2-tdTomato cell transendothelial migration and adhesion to lung microvascular endothelial cells.

Article Snippet: Human lung microvascular endothelial cell line (hLMVEC) was purchased from the European Cell Culture Collection (Cell Applications, San Diego, CA, USA), human breast adenocarcinoma MDA-MB-231-luc2-tdTomato cell line stably expressing the firefly luciferase gene and tdTomato fluorescent protein was kindly provided by Prof. Joanna Wietrzyk (Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences). hLMVEC cells were maintained in Microvascular Endothelial Cell Growth Medium (Cell Applications), MDA-MB-231-luc2-tdTomato cells were cultured in RPMI1640 GlutaMAX Medium (Gibco) with 10% FBS (Gibco), AAS (Sigma-Aldrich) containing 20 units of penicillin, 20 mg streptomycin and 0.05 mg amphotericin B.

Techniques: Migration

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: 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: 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