primary human aortic ecs  (PromoCell)

Journal: Redox Biology

Article Title: Big data analytics for MerTK genomics reveals its double-edged sword functions in human diseases

doi: 10.1016/j.redox.2024.103061

Figure Lengend Snippet: Proteomics in HAECs with MerTK gene knockout or control. ( A ) MerTK expression in HAECs incubated with apoptotic Jurkat cells for 1 h. ( B ) Immunochemical staining for MerTK expression in the aortic arch from WT mice. ( C ) Efferocytosis of apoptotic Jurkat cells by HAECs after 1 h of co-incubation. P < Apoptotic Jurkat cells were labeled with green PKH67 (Sigma) and HAECs were labeled with red PKH26 (Sigma). Green cells are apoptotic Jurkat cells that were not engulfed by HAECs. Green/red small round cells are apoptotic Jurkat cells that were engulfed by HAECs. Large red cells are HAECs. (D) Volcano plot illustration in MerTK KO vs. control. Relative protein abundance (log2) plotted against significance level (-log10 P-value), showing significantly (p < 0.05) downregulated (blue), upregulated (red) or non-differentially expressed proteins (grey). (E) Graphic summarization for pathways in MerTK KO vs. control. (F) MerTK KO activates apoptosis signaling. (G) Canonical pathway analysis in MerTK KO vs. control. Color depends on z-score. Blue signifies negative value; orange signifies positive value; and grey signifies no activity pattern. Size is proportional to the number of genes that overlap the pathway. (H) Machine learning analysis for activated or inhibited disease pathways. (I–K) IPA prediction shows that MerTK KO activates premature aging, kidney failure and heart failure. Proteomics data were analyzed by IPA. Data were analyzed with GraphPad Prism 9.4.1 and shown as the mean ± SD (n = 3–5). P < 0.05 was considered statistically significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Primary human aortic ECs (HAECs) and the human Jurkat cell line were purchased from ATCC (Manassas, VA, USA).

Techniques: Gene Knockout, Control, Expressing, Incubation, Staining, Labeling, Quantitative Proteomics, Activity Assay

Journal: iScience

Article Title: Deciphering the role of endothelial granulocyte macrophage-CSF in chronic inflammation associated with HIV

doi: 10.1016/j.isci.2024.110909

Figure Lengend Snippet: Dual gene expression analysis and GM-CSF protein quantification in the vascular endothelium of PWH (A) Representative combined FISH and immunofluorescence staining in the vascular endothelium and subendothelial tissues obtained an HIV negative (top) and an PWH (bottom) donors for CSF2 mRNA (red) and GM-CSF protein (green); the endothelial lining is shown by the space between dashed lines; each circle represents the average fluorescence intensity data from at least 5 images acquired from a tissue sample. Right panel: Violin plots show the quantitative assessment of CSF2 mRNA (upper: MFI-red) and GM-CSF protein (lower: MFI-green) expression in HIV Negative donors ( n = 12), and PWH ( n = 10), (B) Positive correlations of CSF2 mRNA and GM-CSF protein expression through combined FISH and immunofluorescence staining in the vascular endothelium obtained from negative controls ( n = 12) and PWH ( n = 10), (A: ∗ p < 0.05). (C) Measurement of GM-CSF levels in plasma samples obtained from HIV Neg. ( n = 17) and PWH ( n = 17) using ELISA., (∗ p < 0.05). All data are represented as mean ± SEM.

Article Snippet: The primary human aortic endothelial cell (EC) was purchased from PromoCell and cultured in glass slide chambers (Lab-Tek) with EGM-MV medium (PromoCell), according to the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Deciphering the role of endothelial granulocyte macrophage-CSF in chronic inflammation associated with HIV

doi: 10.1016/j.isci.2024.110909

Figure Lengend Snippet: LPS-induced GM-CSF expression and release by human aortic endothelial cells (A) Immunofluorescence microscopy image shows intracellular staining of GM-CSF (red), eNOS (green), and DAPI (blue) in primary human aortic endothelial cells (ECs). Time-dependent changes in intracellular GM-CSF and eNOS levels are quantified using the intracellular GM-CSF-to-DAPI MFI ratio and intracellular eNOS-to-DAPI MFI ratio. The image represents endothelial responses to LPS at indicated time points from one of the three identical experiments. (B) (Top image) Detection of GM-CFS by Western Blot (see Figure S3 C for the complete WB data) in EC supernatant and in purified, lysed EVs from cells stimulated overnight with LPS (100 ng/mL) or incubated without stimulation (supernatant 2). (Bottom image) ELISA data ( n = 3) illustrating a dose-dependent increase in GM-CSF concentration in cell culture supernatants or in purified and lysed EVs, derived from culture supernatants following overnight incubation of ECs with or without exposure to LPS at varying concentrations. Data shown here (A, B) are from one of the 3 independent experiments, each circle represents average MFI values from one image with ∼10 ECs, n = ∼100 cells/experiment (∗ p < 0.05). (C) GM-CSF detection in endothelial cell culture supernatants after overnight incubation with the indicated TLR agonists ( n = 3, ∗ p < 0.05). All data are represented as mean ± SEM.

Article Snippet: The primary human aortic endothelial cell (EC) was purchased from PromoCell and cultured in glass slide chambers (Lab-Tek) with EGM-MV medium (PromoCell), according to the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Microscopy, Staining, Western Blot, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Derivative Assay

Journal: iScience

Article Title: Deciphering the role of endothelial granulocyte macrophage-CSF in chronic inflammation associated with HIV

doi: 10.1016/j.isci.2024.110909

Figure Lengend Snippet: Single-cell RNA sequencing (RNAseq) reveals dynamic gene expression patterns in human aortic endothelial cells (ECs) ECs were cultured in medium alone or medium supplemented with LPS (100 ng/mL) or Dapa (1μg/mL) for 24 h. An additional 7days incubation period was included for estradiol (10 ng/mL) only (D5-E2-D7). (A). Unbiased clustering and UMAP representation of scRNAseq data analyzed with a resolution setting of 0.12, displaying distinct clusters of human aortic endothelial cells on a UMAP plot. (B) HeatMap of treatment-dependent differentially expressed genes illustrates the differential expression of genes in ECs across various treatment conditions, highlighting treatment-dependent clusters. (C and D) Violin plots display the expression profiles of selected genes (KLF2, eNOS, ICAM-1, VCAM-1, CSF-1, CSF2, and CSF3) within clusters from LPS treated cells. Expression levels are represented as probability distributions across clusters on the y axis.

Article Snippet: The primary human aortic endothelial cell (EC) was purchased from PromoCell and cultured in glass slide chambers (Lab-Tek) with EGM-MV medium (PromoCell), according to the manufacturer’s instructions.

Techniques: RNA Sequencing Assay, Expressing, Cell Culture, Incubation

Journal: iScience

Article Title: Deciphering the role of endothelial granulocyte macrophage-CSF in chronic inflammation associated with HIV

doi: 10.1016/j.isci.2024.110909

Figure Lengend Snippet: Modulation of LPS-induced GM-CSF expression by human aortic endothelial cells (EC) through pharmacological interventions Endothelial cells were cultured in medium alone or medium supplemented with LPS (100 ng/mL), LPS + Dapa (1μg/mL) or LPS + TLR-4 Inhibitor (TLR4-C34) (10μg/mL). (A) Representative microscopy and fluorescent images of NBD glucose uptake under the indicated conditions (left) and violin plots show the quantification of NBD glucose uptake (MFI, right) in EC receiving indicated treatments (data from one of the 3 independent experiments, each circle represents MFI value from one EC, n = ∼100 cells/experiment, ∗∗∗ p < 0.0005). (B) Immunofluorescence microscopy images (left) and violin plots showing quantified image data (MFI/DAPI ratio, right) illustrating KLF2 protein expression in indicated treatment groups (data from 100 ECs per condition; each circle represents average MFI values from one image with 5–10 ECs). (C) ELISA data illustrate GM-CSF protein concentrations in supernatants of endothelial cell cultures after 24 h of incubation under specified conditions ( n = 3; ∗ p < 0.05). (D) Violin plots show ELISA data of GM-CSF levels in plasma samples from PWH, with ( n = 7) and without ( n = 10) receiving Dapa treatment (∗∗∗ p < 0.0005). All data are represented as mean ± SEM.

Article Snippet: The primary human aortic endothelial cell (EC) was purchased from PromoCell and cultured in glass slide chambers (Lab-Tek) with EGM-MV medium (PromoCell), according to the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Microscopy, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Incubation

Journal: iScience

Article Title: Deciphering the role of endothelial granulocyte macrophage-CSF in chronic inflammation associated with HIV

doi: 10.1016/j.isci.2024.110909

Figure Lengend Snippet:

Article Snippet: The primary human aortic endothelial cell (EC) was purchased from PromoCell and cultured in glass slide chambers (Lab-Tek) with EGM-MV medium (PromoCell), according to the manufacturer’s instructions.

Techniques: Virus, Microarray, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Fluorescence, Plasmid Preparation, Expressing, Software

Journal: Acta Neuropathologica Communications

Article Title: ER stress induced immunopathology involving complement in CADASIL: implications for therapeutics

doi: 10.1186/s40478-023-01558-1

Figure Lengend Snippet: Markers of Inflammation and Immunoreactivity in Cerebral Arteries and Arterioles in CADASIL

Article Snippet: For the co-cultures with VSMCs, we used human aortic endothelial cells (ECs; Life Technologies) in M 200 medium with low serum growth supplement (ThermoFisher Scientific) [ ].

Techniques: Marker, Ubiquitin Proteomics