Journal: Cells

Article Title: Loss of Endothelial Barrier Function in the Inflammatory Setting: Indication for a Cytokine-Mediated Post-Transcriptional Mechanism by Virtue of Upregulation of miRNAs miR-29a-3p, miR-29b-3p, and miR-155-5p

doi: 10.3390/cells10112843

Figure Lengend Snippet: Human endothelial cells (cell line TIME, ATCC ® number CRL-4025) were evaluated following stimulation with 5 ng/mL each of IL-1β, TNF-α, and IFN-γ for 24 h and compared to untreated controls. Three biological replicates were tested in each group. Transcriptome data obtained by next generation sequencing (NGS) were gathered into a gene set enrichment analysis (GSEA). Shown are 5 enrichment plots, which correspond to defined gene sets. ( A ) GO-Cellular-Response-To-Cytokine-Stimulation, ( B ) GO-Inflammatory-Response, ( C ) GO-Regulation-Of-Cytokine-Production, ( D ) GO-Homophilic-Cell-Adhesion-Via-Plasma-Membrane-Adhesion-Molecules, ( E ) GO-Cell-Adhesion-Via-Plasma-Membrane-Adhesion-Molecules. Each plot shows the running Enrichment score (ES) for the gene set, which reflects the degree to which a gene set is over-represented at the top or bottom of a ranked list of genes. A positive ES indicates gene set enrichment at the top of the ranked list (=upregulation); a negative ES indicates gene set enrichment at the bottom of the ranked list (=downregulation).

Article Snippet: The human cell line TIME (ATCC ® number CRL-4025) cultured at 37 °C and 5% CO 2 in a humid atmosphere served as an in vitro model for microvascular endothelial cells.

Techniques: Next-Generation Sequencing

Journal: Cells

Article Title: Loss of Endothelial Barrier Function in the Inflammatory Setting: Indication for a Cytokine-Mediated Post-Transcriptional Mechanism by Virtue of Upregulation of miRNAs miR-29a-3p, miR-29b-3p, and miR-155-5p

doi: 10.3390/cells10112843

Figure Lengend Snippet: The cellular impedance of human endothelial cells (cell line TIME, ATCC ® number CRL-4025) was investigated in real-time using the xCELLigence ® RTCA DP instrument (ACEA Bioscience Incorporation). Changes in impedance are reported by the dimensionless parameter “cell index”. The blue and red data points symbolize cells cultured in basal cell culture medium versus medium containing 5 ng/mL each of IL-1β, TNF-α, and IFN-γ for a defined time, respectively. The arrows indicate the time points of the performed medium changes. Three biological replicates and two technical replicates were tested in each group. Data are shown as means ± standard deviation.

Article Snippet: The human cell line TIME (ATCC ® number CRL-4025) cultured at 37 °C and 5% CO 2 in a humid atmosphere served as an in vitro model for microvascular endothelial cells.

Techniques: Cell Culture, Standard Deviation

Journal: Cells

Article Title: Loss of Endothelial Barrier Function in the Inflammatory Setting: Indication for a Cytokine-Mediated Post-Transcriptional Mechanism by Virtue of Upregulation of miRNAs miR-29a-3p, miR-29b-3p, and miR-155-5p

doi: 10.3390/cells10112843

Figure Lengend Snippet: Human endothelial cells (cell line TIME, ATCC ® number CRL-4025) were evaluated following stimulation with 5 ng/mL each of IL-1β, TNF-α, and IFN-γ for 24 h, and compared to unstimulated controls. Absolute quantification of miRNAs from isolated total RNA was performed using ddPCR analysis. The QX 200 Droplet Digital PCR System (Bio-Rad) and specific LNA PCR primers (Qiagen) were used according to the manufacturer’s instructions. Based on the number of measured positive droplets, the system calculates the copy number of the target RNA in the total mixture, assuming a Poisson distribution. Since a defined amount of RNA sample is used in each ddPCR assay, the copy number per ng RNA can be determined. The box plots depict the median of determined miRNA expression levels, the lower and upper quantile, and the two extreme values. Each group included six biological replicates and two technical replicates. An unpaired t-test was used to identify significant differences. Statistical analysis was performed utilizing GraphPad Prism 9 (GaphPad Software, La Jolla, CA, USA). In all cases, p < 0.05 was assumed to indicate significant differences.

Article Snippet: The human cell line TIME (ATCC ® number CRL-4025) cultured at 37 °C and 5% CO 2 in a humid atmosphere served as an in vitro model for microvascular endothelial cells.

Techniques: Isolation, Digital PCR, Expressing, Software

Journal: bioRxiv

Article Title: TAK1 blockade as a therapy for retinal neovascularization

doi: 10.1101/2021.01.29.428701

Figure Lengend Snippet: TAK1 deletion reduced the expression of TAK1, ICAM-1, CXCL8 and VEGF genes in human microvascular endothelial cells. qPCR results revealed that TAK1 deletion suppressed the expression of selected cytokines ( CXCL8 and VEGFA) and adhesion molecules ( ICAM-1) in TIMEs stimulated with IL1 β for 24 hours (n = 4; technical duplicate from each biological repeat was shown as separated dot plot). Group data are shown as means ± SEM. Statistical analysis was undertaken with one-way ANOVA and Tukey’s multiple comparison test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Telomerase-immortalized human microvascular endothelial cell (TIME; CRL-4025™) and primary human retinal microvascular endothelial cell (HRMEC; ACBRI 181V) were purchased from American Type Culture Collection (ATCC; Manassas, VA) and Cell Systems (Kirkland, WA).

Techniques: Expressing

Journal: bioRxiv

Article Title: TAK1 blockade as a therapy for retinal neovascularization

doi: 10.1101/2021.01.29.428701

Figure Lengend Snippet: TAK1 knockdown by siRNA inhibited the expression of TAK1, ICAM-1, CXCL8 and VEGF genes in human primary retinal microvascular endothelial cells (HRMECs). qPCR results revealed that TAK1 knockdown by siRNA targeting TAK1 (siTAK1) suppressed the expression of selected cytokines ( CXCL8 and VEGFA) and adhesion molecules ( ICAM-1) in HRMECs stimulated with TNFα for 24 hours (n = 3). Group data are shown as means ± SEM. Statistical analysis was undertaken with one-way ANOVA and Tukey’s multiple comparison test; ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Telomerase-immortalized human microvascular endothelial cell (TIME; CRL-4025™) and primary human retinal microvascular endothelial cell (HRMEC; ACBRI 181V) were purchased from American Type Culture Collection (ATCC; Manassas, VA) and Cell Systems (Kirkland, WA).

Techniques: Expressing

Journal: bioRxiv

Article Title: TAK1 blockade as a therapy for retinal neovascularization

doi: 10.1101/2021.01.29.428701

Figure Lengend Snippet: TAK1 inhibition by 5Z-7-oxozeaenol inhibited the expression of TAK1, ICAM-1, CXCL8 and VEGF genes in primary human retinal microvascular endothelial cells (HRMECs). qPCR results revealed that pharmacological inhibition of TAK1 by 5Z-7-oxozeaenol (Oxo) suppressed ICAM-1 , PTGS2 , CXCL8 and VEGFA in HRMECs stimulated with TNFα in a dose-dependent manner (n = 3; technical duplicate from each biological repeat was shown as separated dot plot). Group data are shown as means ± SEM. Statistical analysis was undertaken with one-way ANOVA and Tukey’s multiple comparison test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Telomerase-immortalized human microvascular endothelial cell (TIME; CRL-4025™) and primary human retinal microvascular endothelial cell (HRMEC; ACBRI 181V) were purchased from American Type Culture Collection (ATCC; Manassas, VA) and Cell Systems (Kirkland, WA).

Techniques: Inhibition, Expressing

Journal: bioRxiv

Article Title: TAK1 blockade as a therapy for retinal neovascularization

doi: 10.1101/2021.01.29.428701

Figure Lengend Snippet: (A) Experimental protocol for 5Z-7-oxozeaenol treatment in the OIR rat model. A single intravitreal injection of vehicle, low (18 ng) or high (90 ng) dose of 5Z-7-oxozeaenol was given to Sprague-Dawley OIR rats at P14, and retinae were collected for qPCR and immunostaining at P16 and P18. (B) Intravitreal injection of 5Z-7-oxozeaenol in OIR rats at P14 inhibited TNFα , VEGFA and ICAM-1 gene expression assessed using qPCR (n = 5-7). (C) Representative images of microglial adhesion to the vascular surface in a retina from a control rat and OIR rats that had a single intravitreal injection of vehicle, low or high dose of 5Z-7-oxozeaenol (Oxo). Vessels were visualized with isolectin GS-IB4 staining (green), and distribution of microglia was immunolabeled with Iba-1 (red). Scale bar: 50 µm. (D) Quantitative analysis of microglial number relative to retinal vessel area in vehicle or 5Z-7-oxozeaenol treated OIR rats (n = 7-10; 10 images from each retina were shown as separated dot plot). (E) Schematic diagram for the role of TAK1 signaling in microvascular endothelial cells. Proinflammatory cytokines such as TNFα can activate TAK1 in microvascular endothelial cells, and TAK1/TAB1/TAB2-3 complex can lead to activation of NFκB and MAPKs/AP-1 cascades. NFκB and MAPKs/AP-1 induce expression of “cytokines” and “adhesion proteins”, thereby triggering the “inflammatory cycle” and “leukostasis”, and leading to retinal neovascularization. Group data are shown as means ± SEM. Statistical analysis was undertaken with one-way ANOVA and Tukey’s multiple comparison test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Telomerase-immortalized human microvascular endothelial cell (TIME; CRL-4025™) and primary human retinal microvascular endothelial cell (HRMEC; ACBRI 181V) were purchased from American Type Culture Collection (ATCC; Manassas, VA) and Cell Systems (Kirkland, WA).

Techniques: Injection, Immunostaining, Expressing, Staining, Immunolabeling, Activation Assay