Journal: Journal of Extracellular Vesicles

Article Title: Endothelial cell‐derived extracellular vesicles expressing surface VCAM1 promote sepsis‐related acute lung injury by targeting and reprogramming monocytes

doi: 10.1002/jev2.12423

Figure Lengend Snippet: VCAM1 on L‐EC‐EVs contributes to monocyte differentiation and ALI. (a) Volcano plot showing differentially expressed proteins between N‐EC‐EVs and L‐EC‐EVs. Statistical significance was defined as a p value < 0.05 and |log2FC| > 1. (b) Venn diagram showing the KEGG pathways enriched by upregulated EV proteins and the top 100 upregulated monocyte genes. (c) Western blot analysis of VCAM1 in EC‐EVs. (d) The level of VCAM1 on EC‐EVs (10 11 particles/mL) quantified by ELISA. (e) Western blot analysis validating the expression of VCAM1 in EVs. (f) Western blot assay to measure the phosphorylation of p65 (p‐p65) and IκBα (p‐IκBα) in monocytes incubated with different L‐EC‐EVs, and the secreted levels of inflammatory factors (IL‐6 and TNF‐α) were measured by ELISA. (g) Representative histological images of lung sections with H&E staining. Scale bar: 50 μm. (h) The total lung injury score of lung sections from each group. (i) The mRNA levels of Il6 , Tnf , Ccl2 and Cxcl2 measured by qRT‐PCR. (j, k) Quantification of the expression of CD80 (M1 marker) and CD163 (M2 marker) on Mo‐Mφs and TR‐Mφs in lung tissues by flow cytometry. (l) Flow cytometry analysis of neutrophils in lung tissues. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The amount of VCAM1 on the surface of EVs was determined by the mouse VCAM1 Quantikine ELISA Kit (Proteintech, Wuhan, China) or western blotting, and the percentage of VCAM1 + EVs was determined by nanoflow cytometry.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Phospho-proteomics, Incubation, Staining, Quantitative RT-PCR, Marker, Flow Cytometry

Journal: Journal of Extracellular Vesicles

Article Title: Endothelial cell‐derived extracellular vesicles expressing surface VCAM1 promote sepsis‐related acute lung injury by targeting and reprogramming monocytes

doi: 10.1002/jev2.12423

Figure Lengend Snippet: VCAM1 on L‐EC‐EVs regulates monocyte but not TR‐Mφ differentiation by acting on ITGA4. (a) The expression of M1 markers (CD80 and CD86) on monocytes and TR‐Mφs detected by flow cytometry. (b) UMAP plot of single‐cell RNAseq datasets of healthy human lungs showing the expression of ITGA4 in different cell types. The analysis was conducted using the Lung Endothelial Cell Atlas data mining website ( http://www.LungEndothelialCellAtlas.com ). (c) Quantification of the expression of ITGA4 on monocytes and TR‐Mφs by flow cytometry. (d) Immunofluorescence images showing the expression of ITGA4 (red) on monocytes and TR‐Mφs cells. Scale bar: 10 μm. (e–g) Monocytes were pretreated with anti‐ITGA4 blocking antibody or IgG isotype control for 1 h, and then incubated with PBS/L‐EC‐EVs for 24 h. The phosphorylation of p65 (p‐p65) in monocytes was detected by western blotting (e), the expression of M1 markers (CD80 and CD86) on cells was measured by flow cytometry (f), and the levels of inflammatory cytokines (IL‐6 and TNF‐α) were quantified by ELISA (g). (h) Mice were pretreated with either anti‐ITGA4 antibody or IgG isotype 4 h before EV treatment. The expression of M1 markers and M2 markers on Mo‐Mφs in lung tissues was detected by flow cytometry. ns: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The amount of VCAM1 on the surface of EVs was determined by the mouse VCAM1 Quantikine ELISA Kit (Proteintech, Wuhan, China) or western blotting, and the percentage of VCAM1 + EVs was determined by nanoflow cytometry.

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Blocking Assay, Control, Incubation, Phospho-proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Extracellular Vesicles

Article Title: Endothelial cell‐derived extracellular vesicles expressing surface VCAM1 promote sepsis‐related acute lung injury by targeting and reprogramming monocytes

doi: 10.1002/jev2.12423

Figure Lengend Snippet: Endothelial cell‐derived VCAM1 + EVs are increased in the plasma of patients with sepsis‐related ARDS. (a) Western blot analysis of VCAM1 expression in plasma EVs. (b) Representative dot plot, detected by nanoflow cytometry, showing the percentage of VCAM1 + EC‐EVs in plasma EVs of healthy volunteers, non‐ARDS patient and sepsis‐related ARDS patients. (c, d) Quantification of the percentage of EC‐EVs (c) and VCAM1 + EC‐EVs (d) in plasma EVs. (e) Quantification of EVs isolated from the plasma of sepsis‐related ARDS patients and controls. ns: not significant, ** p < 0.01, **** p < 0.0001.

Article Snippet: The amount of VCAM1 on the surface of EVs was determined by the mouse VCAM1 Quantikine ELISA Kit (Proteintech, Wuhan, China) or western blotting, and the percentage of VCAM1 + EVs was determined by nanoflow cytometry.

Techniques: Derivative Assay, Clinical Proteomics, Western Blot, Expressing, Cytometry, Isolation

Journal: Journal of Extracellular Vesicles

Article Title: Endothelial cell‐derived extracellular vesicles expressing surface VCAM1 promote sepsis‐related acute lung injury by targeting and reprogramming monocytes

doi: 10.1002/jev2.12423

Figure Lengend Snippet: Schematic illustration of the effects of EC‐EVs in sepsis‐related ALI/ARDS. In extra‐pulmonary sepsis, the number of VCAM1 + EVs released by endothelial cells was increased, and they were preferentially taken up by monocytes in the lung. VCAM1, highly expressed on these EVs, activates the NF‐κB pathway by acting on ITGA4, thus promoting the differentiation of monocytes into M1‐type macrophages. The latter secreted much proinflammatory cytokines and chemokines, recruiting more inflammatory cells to amplify the inflammatory response and further exacerbate ALI. EC: endothelial cell; Mo: monocyte; PMN: polymorphonuclear neutrophil; RBC: red blood cell; Mo‐Mφ: monocyte‐derived macrophage; TR‐Mφ: tissue resident macrophages; MVB: multivesicular body.

Article Snippet: The amount of VCAM1 on the surface of EVs was determined by the mouse VCAM1 Quantikine ELISA Kit (Proteintech, Wuhan, China) or western blotting, and the percentage of VCAM1 + EVs was determined by nanoflow cytometry.

Techniques: Derivative Assay

Journal: Frontiers in pharmacology

Article Title: Nuciferine induces autophagy to relieve vascular cell adhesion molecule 1 activation via repressing the Akt/mTOR/AP1 signal pathway in the vascular endothelium.

doi: 10.3389/fphar.2023.1264324

Figure Lengend Snippet: FIGURE 1 Nuciferine represses VCAM1 activation both in vitro and in vivo. (A) Chemical structure of nuciferine. (B) Cell viability test in EA.hy926 cells under 24 h treatment of nuciferine (0, 1, 2.5, 5, 10, 20, and 40 μmol/L). Relative mRNA level of VCAM1 verified by qPCR (C), protein level of VCAM1 verified by Western blot assay (D) (left panel), and the relative quantification data (D) (right panel), while EA.hy926 cells were pretreated with nuciferine (0, 1, 2.5, and 5 μmol/L) for 2 h, followed by 12 h incubation with TNFα (10 ng/mL) or vehicle. (E) Plasma VCAM1 level examined by the ELISA method from the indicated mice. (F) VCAM1 immunofluorescence staining of arterial endothelia tissues from the indicated mice; nuclei were visualized by DAPI staining (blue), and VCAM1 was visualized by an FITC-conjugated secondary antibody (green); the final data were captured under a fluorescence digital pathology scanner (magnification ×200; scale bar, 100 μm). All values are denoted as the means ± SD from five animal samples or at least three independent batches of cells. Each group contains the same amount of solvent. ud, undetectable. pp < 0.05 and ppp < 0.01 indicate statistically significant differences.

Article Snippet: VCAM1 content in mice plasma was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit obtained from Cusabio (CSB-E04754m, Wuhan, China).

Techniques: Activation Assay, In Vitro, In Vivo, Western Blot, Incubation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining, Solvent

Journal: International Journal of Molecular Sciences

Article Title: Modulation of Endothelial Function by TMAO, a Gut Microbiota-Derived Metabolite

doi: 10.3390/ijms24065806

Figure Lengend Snippet: Schematic overview of the NF-kB pathway and angiocrine factors activated in endothelial dysfunction. NF-kB activation induces the upregulation of pro-inflammatory factors (ICAM-1, VCAM-1, selectins, and cytokines) and an increase in intracellular ROS. All these angiocrine factors, in combination with reduced eNOS phosphorylation, contribute to the exacerbation of endothelial dysfunction and smooth muscle cell activation, enhancing the atherosclerotic burden. ICAM-1: intracellular adhesion molecule 1; VCAM-1: vascular cell adhesion molecule 1; IL: interleukin; TGF-β: transforming growth factor β; ROS: reactive oxygen species; eNOS: endothelial nitric oxide synthase. Image created with Biorender.com.

Article Snippet: Among different angiocrine factors activated by NF-kB and involved in a positive feedback loop with ROS as previously mentioned [ , ], vascular cell adhesion molecule 1 (VCAM-1) [ , ], ICAM-1 [ , ], selectins, and cytokines are primarily involved in the development of endothelial dysfunction by starting the recruitment of inflammatory cells, like monocytes, neutrophils, leukocytes, and macrophages, and exacerbating the inflammatory response [ ].

Techniques: Activation Assay, Phospho-proteomics