Journal: Function

Article Title: Molecular and Functional Characterization of the Peritoneal Mesothelium, a Barrier for Solute Transport

doi: 10.1093/function/zqae051

Figure Lengend Snippet: Mesothelial and endothelial cell type specific expression of transport related genes, and of tight junction proteins CLDN1 and CLDN5, (A) Expression map of cell junctions, transmembrane channels and transporters, and of transcytotic carriers in human primary peritoneal mesothelial cells (HPMC), immortalized pleural mesothelial cells (MeT-5A), human umbilical vein endothelial cells (HUVEC) and human primary cardiac microvascular endothelial cells (HCMEC). Sealing tight junction claudin1 (CLDN1) is only expressed in mesothelial cells and CLDN5 only in endothelial cells. (B) Immunocytochemical staining of CLDN1 protein in HPMC and MeT-5A, and CLDN5 in HUVEC and HCMEC, together with anchoring protein ZO-1. Pearson correlation analysis of the green and red channel colocalization and RGB spectra at the cell membrane. Scale bar = 10 µm. (C) Quantification of CLDN1 and -5 relative to ZO-1 immunofluorescence in the four cell lines at the cell membrane area (z-stack spacing 0.25 µm). (D) Representative Western blot analysis of CLDN1 and CLDN5 proteins (total protein extraction).

Article Snippet: Human cardiac microvascular endothelial cells (HCMEC), human umbilical vein endothelial cells (HUVEC), and immortalized mesothelial cell line (MeT-5A) were commercially obtained (HUVEC and HCMEC from PromoCell, Heidelberg, Germany; MeT-5A from LGC Standards, Wesel, Germany).

Techniques: Expressing, Staining, Membrane, Immunofluorescence, Western Blot, Protein Extraction

Journal: Function

Article Title: Molecular and Functional Characterization of the Peritoneal Mesothelium, a Barrier for Solute Transport

doi: 10.1093/function/zqae051

Figure Lengend Snippet: Transepithelial resistance, creatinine transport (0.11 kDa), and 4-, 10-, and 70-kDa dextran permeability of polarized mesothelial and endothelial cell monolayers. (A) TER of the four cell lines with increasing cell monolayer density in Transwells, with stable TER being reached with confluence. Confluent HCMEC have a 2.5-fold lower TER, reflecting higher ionic conductance ( n = 5 experiments, 4-5 replicates, P < 0.0001 for HCMEC versus all). (B) The decline in creatinine concentrations in the apical Transwell compartment relative to the initial concentration (10 mg/dL) is given on the left graph. Volume of the basolateral compartment is five times higher (1 mL). The right graph gives the creatinine appearance relative to the creatinine added to the apical compartment and corrected for the higher basolateral volume. The dashed lines indicate the expected equilibration levels between compartments. Transport of creatinine is highest across confluent HCMEC cell monolayers ( P < 0.0001/0.0001 for changes in the apical and basolateral compartment with 4 cell lines, and P < 0.0001/0.0001 for HPMC versus HCMEC only; two-way repeated measure ANOVA; n = 4 experiments, 3 replicates per experiment). (C) 4, 10 and 70 kDa dextran permeabilities of mesothelial and endothelial cell monolayers were calculated from 2 h and 4 h dextran transport kinetics. Paracellular permeability is higher for the smaller macromolecular dextrans across human microvascular endothelial cells. two-way repeated measures ANOVA P < 0.0001 for dextran size, P < 0.0001 for cell type; n = 4 experiments, 3 replicates per experiment, data area mean ± SD, ** P < 0.01, **** P < 0.0001). HCMEC = human cardiac microvascular endothelial cells, HUVEC = human umbilical vein endothelial cells, HPMC = human peritoneal mesothelial cells, MeT-5A = immortalized pleural mesothelial cells.

Article Snippet: Human cardiac microvascular endothelial cells (HCMEC), human umbilical vein endothelial cells (HUVEC), and immortalized mesothelial cell line (MeT-5A) were commercially obtained (HUVEC and HCMEC from PromoCell, Heidelberg, Germany; MeT-5A from LGC Standards, Wesel, Germany).

Techniques: Permeability, Concentration Assay

Journal: bioRxiv

Article Title: The proton-sensing GPR4 receptor regulates paracellular gap formation and permeability of vascular endothelial cells

doi: 10.1101/601088

Figure Lengend Snippet: Plasma membrane staining and paracellular gap area quantitation of ECs treated for up to 5 hours under physiological or acidic pH. Acidosis increases EC gap formation when compared to physiological pH treatment conditions. (A) Representative pictures of plasma membrane staining in Human Umbilical Vein Endothelial Cells (HUVECs) at 0, 3, and 5 hours treated under physiological or acidic pH. Quantitative analysis of gap formation in (B) HUVECs, (C) Human Pulmonary Artery Endothelial Cells (HPAECs), (D) Human Colon Microvascular Endothelial Cells (HMVEC-Colon), and Human Lung Microvascular Endothelial Cells (HMVEC-Lung) over 5 hours. All experiments were performed in triplicate and are representative of four experiments. Data at each time point are presented as mean ± SEM and analyzed for statistical significance between the pH 7.4 group and the pH 6.4 group using the unpaired t -test where **p<0.01 and ***p<0.001. White arrows point to paracellular gaps. Scale bar = 100µm.

Article Snippet: Primary human umbilical vein endothelial cells (HUVEC), human pulmonary artery endothelial cells (HPAEC), and human lung microvascular endothelial cells (HMVEC-Lung) (Lonza, Walkersville, MD, USA), and primary human colon microvascular endothelial cells (HMVEC-Colon) (Cell Biologics Inc., Chicago, IL, USA) were cultured at 37°C and 5% CO 2 in a humidified incubator for experimentation.

Techniques: Staining, Quantitation Assay

Journal: Nanotheranostics

Article Title: Enhanced blood-brain-barrier penetrability and tumor-targeting efficiency by peptide-functionalized poly(amidoamine) dendrimer for the therapy of gliomas

doi: 10.7150/ntno.38954

Figure Lengend Snippet: In vitro evaluation of biocompatibility and antitumor efficacy of the dual-targeting drug delivery system. (a, b) Cytotoxicity of peptides (a) Ang2 and (b) EP-1 to HBMEC and U87-MG cells. (c, d) Cytotoxicity of blank dendrimer-based carriers (P4, P4P and P4PEA), free DOX, P4PD and P4PEAD to (c) HBMEC and (d) U87-MG cells. The cells viability was checked by MTS assay after incubating the cells with different concentrations of peptides, blank carriers and DOX-loaded dendrimer carriers for 48 h. Error bars represent standard deviation (n = 5). (e) IC 50 values of different DOX-loaded dendrimers fitted by Origin 8.1 software. Error bars represent standard deviation (n = 5). (f) Short-term cytotoxicity of different DOX formulations to HBMEC cells after incubating the cells with the DOX formulations for 3 h at the DOX concentration of 20 μΜ. Error bars represent standard deviation (n=5). *p < 0.1, **p < 0.01, ***p < 0.001 (Student's t-test).

Article Snippet: Human brain microvascular endothelial HBMEC cells was purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: In Vitro, MTS Assay, Standard Deviation, Software, Concentration Assay

Journal: Nanotheranostics

Article Title: Enhanced blood-brain-barrier penetrability and tumor-targeting efficiency by peptide-functionalized poly(amidoamine) dendrimer for the therapy of gliomas

doi: 10.7150/ntno.38954

Figure Lengend Snippet: (a, b) Intracellular uptake of different DOX-loaded dendrimers by (a) HBMEC and (b) U87-MG cells detected by flow cytometry. Dendrimers were incubated with the cells for 2 h before flow cytometry measurement. Cells without treatment were used as control. Error bars represent standard deviation (n=5). **p < 0.01, ***p < 0.001, ****p < 0.0001 (Student's t-test). (c, d) Subcellular trafficking of different DOX formulations in (c) HBMEC and (d) U87-MG cells detected by LSCM. Cells were incubated with Cy5.5-labeled different dendrimers for 2 h at the dendrimer concentration of 1 μM. LysoTracker Green DND-26 was used to stain lysosomes at the concentration of 0.1 μM. Hoechst was used to stain nucleus. Scale bar: 50 μm.

Article Snippet: Human brain microvascular endothelial HBMEC cells was purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: Flow Cytometry, Incubation, Control, Standard Deviation, Labeling, Concentration Assay, Staining

Journal: Nanotheranostics

Article Title: Enhanced blood-brain-barrier penetrability and tumor-targeting efficiency by peptide-functionalized poly(amidoamine) dendrimer for the therapy of gliomas

doi: 10.7150/ntno.38954

Figure Lengend Snippet: Evaluation of the BBB penetration and the dual-targeting efficacy of the DOX-loaded dual-targeting dendrimers. (a, b) Schematic illustration of the in vitro BBB model. (a) A monolayer of HBMEC cells were cultured on the transwell inserts, and (b) U87-MG cells were co-cultured. (c) The transport ratio of DOX across the BBB within 3 h. Error bars represent standard deviation (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 (Student's t-test). (d) The cell viability of U87-MG cells in the co-cultured BBB model. Error bars represent standard deviation (n = 3). *p < 0.05, ***p < 0.001 (Student's t-test). (2) The intracellular uptake of DOX by U87-MG cells after crossing BBB by flow cytometry.

Article Snippet: Human brain microvascular endothelial HBMEC cells was purchased from iCell Bioscience Inc. (Shanghai, China).

Techniques: In Vitro, Cell Culture, Standard Deviation, Flow Cytometry