Journal: Nature Aging

Article Title: Gut microbiota-dependent increase in phenylacetic acid induces endothelial cell senescence during aging

doi: 10.1038/s43587-025-00864-8

Figure Lengend Snippet: a , Schematic of quantification of plasma PAA and PAGln in young (3-month-old) and aged (>24-month-old) C57BL/6J male and female mice. b , c , Plasma PAA ( b ) and PAGln ( c ) levels were measured by LC–MS/MS in mice ( n = 6). d , Correlation between plasma PAA (left) and PAGln (right) concentrations and chronological age in the TwinsUK cohort ( n = 7,303, male and female). e , Schema for PAA production from dietary phenylalanine (Phe) via the bacterial VOR/PPFOR system. f , Shotgun metagenomics workflow for analyzing mouse fecal microbiomes. g , Distribution of VOR and PPFOR gene homologs (%) in microbiomes of aged and young mice ( n = 5–6). KOO169, VOR; KOO179, PPFOR). h , Bar plot depicting age-associated abundance of fecal microbiota profiles at strain level (dark colors represent taxa harboring VOR or PPFOR homologs). The leftmost bar plot demonstrates the proportion of VOR, PPFOR or VOR + PPFOR detected in taxa enriched in aged ( n = 44) versus young ( n = 37) mice. i , Heatmap shows correlations between plasma PAA or PAGln levels and gut bacteria among the top enriched taxa in aged versus young mice ( n = 5–6). j , Correlation between plasma PAA (left) and PAGln (right) concentrations and abundance (%) of Clostridium taxa in human participants ( n = 900, TwinsUK; male and female). k , PAA (top) and PAGln (below) concentrations in the supernatants of anaerobic cultures from Clostridium sp. ASF356 ( n = 8). l , Schematic of ex vivo force tension myography. Aortic rings exhibit vasorelaxation responses (%) to acetylcholine (Ach) ( n = 10). m , SA-β-gal staining of ECs from the ascending aortas of mice ( n = 5–6) and quantification of SA-β-gal + cells (%). n , o , Immunoblots and immunofluorescence represent the expression of p16 INK4a ( n ) and VCAM1 ( o ) in aortic ECs ( n = 5–6). p , γ-H2A.X immunostaining in CD31 + ECs ( n = 5–6). Scale bars, 20, 50 and 100 μm. Error bars represent s.d. ( b , c , m , n ) or s.e.m. ( g , k , l ) or 95% confidence intervals ( d ). Statistical analysis was performed using a two-tailed unpaired Student’s t -test ( b , c , l – n ), two-tailed Pearson correlation analysis ( d ), two-tailed Mann–Whitney U -test ( g , k ), ANCOM method for microbial abundance analysis ( h ), two-sided Spearman’s rank correlation test ( i ) and linear mixed model CLR transformation ( j ). Data are shown as median with min–max; boxes represent interquartile range (IQR); center lines represent the median; whiskers extend from the min to max values ( g , k ). Images created with BioRender.com ( a , d – f , l ). * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Primary human aortic ECs (HAECs; Lonza, CC-2535) were cultured in EBM-2 medium (Lonza, 00190860) supplemented with EGM-2 (10% FBS, 2 mM l -glutamine, 100 μg ml −1 penicillin–streptomycin, Lonza, CC-3162/6) at 37 °C, 5% CO 2 .

Techniques: Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Bacteria, Ex Vivo, Staining, Western Blot, Immunofluorescence, Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY, Transformation Assay

Journal: ImmunoHorizons

Article Title: Antibody ligation of HLA class II induces YAP nuclear localization and formation of cytoplasmic YAP condensates in human endothelial cells

doi: 10.1093/immhor/vlae008

Figure Lengend Snippet: Upregulation of HLA II gene expression in primary cultures of human aortic ECs. (A) Human aortic ECs were pretreated with 200 U of TNFα and 500 U of IFNγ in 35 mm dishes coated with 0.1% gelatin for 48 h. The cultures were then stained with an antibody that detects total YAP and with Hoechst 33342 to visualize the cell nuclei. (B) Bars represent the ratio of nuclear/cytoplasm (50–75 cells) mean ± SE with similar results obtained in 2 independent experiments. (C) Ad-CIITA upregulates HLA II expression in endothelial cells. Primary cultures of human aortic ECs were infected with Ad-CIITA for 2 d. Expression of HLA II antigens on ECs was determined by flow cytometric analysis using anti-human HLA II antibody. (D) Quantification of flow cytometric analysis mean ± SE. n = 2. (E) YAP localizes to the cytoplasm following serum starvation. ECs were infected with Ad-CIITA for 2 d and then serum starved for 2 and 4 h. (F) Quantification of ratio of nuclear/cytoplasm (50–75 cells) and mean ± SE with similar results obtained in 3 independent experiments. ** P < 0.01.

Article Snippet: Primary human aortic ECs were isolated from the aortic rings of explanted donor hearts as described previously or commercial (lot no. EC5555) from Lonza/Clonetics (Walkersville, MD).

Techniques: Gene Expression, Staining, Expressing, Infection

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