Impact of FASN-enriched EVs on endothelial cell function in obstructive sleep apnea hypopnea syndrome

Figures

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  Fig. 1. The impact of serum-derived extracellular vesicles (s-EVs) on lung endothelial function in patients with obstructive sleep apnea hypopnea syndrome (OSAHS). (A) Distribution of s-EVs labeled with PKH26 in lung tissue. (B) Observation of lung tissue morphology and lung injury scoring statistics with hematoxylin and eosin (H&E) staining. (C) Determination of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positivity rate in lung tissue using TUNEL staining. (D) Measurement of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) levels using enzyme-linked immunosorbent assay (ELISA) assay. (E–H) Assessment of reactive oxygen species (ROS) levels (E), myeloperoxidase (MPO) activity (F), malondialdehyde (MDA) levels (G), and superoxide dismutase (SOD) activity (H) in lung tissue. (I) Estimation of endothelial integrity in vascular cross-section using cluster of differentiation 31 (CD31) fluorescence intensity. Each group consisted of 6 mice, and values are presented as mean ± standard deviation (SD), where ∗P < 0.05 denotes significance, ∗∗P < 0.01 denotes high significance, ∗∗∗P < 0.001, and ns…

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  Fig. 2. Joint analysis of differentially expressed proteins (DEPs) and metabolites. (A) Correlation heat map of the results between DEPs and differentially expressed metabolites (DEMs), where orange to red indicates a positive correlation and blue indicates negative correlation. (B) Significant association network between DEPs and DEMs, where circles represent DEPs, squares represent DEMs, and darker colors indicate higher correlation. (C) Joint Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEPs-DEMs lipid metabolism network, where red indicates upregulated proteins or metabolites, blue indicates downregulated proteins or metabolites, and gray indicates metabolic pathways. (D) Quantitative polymerase chain reaction (qPCR) shows changes in arnitine palmitoyltransferase 1A (Cpt1a) and fatty acid synthase (Fasn) expression in obstructive sleep apnea hypopnea syndrome (OSAHS) mouse group (n = 20). (E) Protein correlation analysis involved in lipid metabolism. (F) Western blot detection of FASN protein expression in Ctrl s-EVs and OSHAS s-EVs (n = 3). ∗P < 0.05 indicates significance, ∗∗P < 0.001 indicates high significance, ∗∗∗P < 0.001 indicates very high…

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  Fig. 3. The influence of serum-derived extracellular vesicles (s-EVs) from chronic intermittent hypoxia (CIH) mice on endothelial function. (A) Confocal microscopy images of uptake of PKH67-labeled s-EVs across mouse aortic endothelial cells (MAECs). (B) Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay detecting endothelial cell viability in different treatment groups; (C, D) Flow cytometry assessment of cell apoptosis in different treatment groups. (E, F) Scratch assay measuring cell migration in different treatment groups (E) and the statistic plot (F). (G, H) Matrigel tube formation assay determining the tube-forming ability of MAECs in different treatment groups (G) and the statistic plot (H). (I) Immunofluorescence images of cluster of differentiation 31 (CD31) (green fluorescence) and intercellular adhesion molecule-1 (ICAM-1) (red fluorescence) staining in MAECs. (J, K) Western blot experiments measuring levels of endothelial dysfunction markers CD31, von Willebrand factor (vWF), ICAM-1, and vascular cell adhesion molecule-1 (VCAM-1) in different treatment groups (J) and the statistic plot (K). ∗∗P < 0.001 denotes high significance, ∗∗∗P < 0.001 denotes very high…

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  Fig. 4. Fatty acid synthase (FASN) activation mediates mitochondrial dysfunction-induced endothelial cell apoptosis. (A) Evaluation of endothelial cell mitochondrial membrane potential (MMP) using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) showing aggregates (red) and monomers (green). (B) The statistic plot of monomers (green). (C) Mitochondrial permeability transition pore (mPTP) opening rate in different treatment groups. (D) Fluorescence microscopy displaying total reactive oxygen species (ROS) levels and mitochondrial ROS (mtROS) levels, with ROS (green) and mtROS (red). (E) Fluorescence microplate showing ROS and mtROS. (F) Immunofluorescence detecting leakage of mitochondrial Cytochrome c (Cyt c) into the cytoplasm and nucleus. (G, H) Western blot analysis of changes in mitochondrial apoptosis proteins (G) and the statistical plot (H). (I, J) Observation of cell permeability in different treatment groups (I) and the statistic plot (J). (K) Changes in cell resistance in different treatment groups. ∗∗P < 0.01 denotes high significance, and ∗∗∗P < 0.001denotes very high significance, with cell experiments repeated three times. NO: normoxia;…

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  Fig. 5. Regulation of energy metabolism by fatty acid synthase (FASN) under chronic intermittent hypoxia (CIH) conditions. (A) Seahorse XFe96 was used to measure oxygen consumption rate (OCR). Respective components of OCR via calculation as follows: Basal OCR = pre oligomycin − post antimycin/rotenone, adenosine triphosphate (ATP)-linked = pre oligomycin − post oligomycin, Max respiration = post carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) − post antimycin/rotenone, and Reserve OCR = post FCCP − pre FCCP. (B) Seahorse XFe96 was used to measure extracellular acidification rate (ECAR). Respective ECAR components with calculations as follows: Basal ECAR = pre glucose − post 2-deoxyglucose (2-DG), Glycolysis = post glucose − pre glucose, Max glycolysis = post oligomycin − post 2-DG, and Reserve ECAR = post oligomycin − pre oligomycin. n = 11–12 individual replicates per group. (C) Mitochondrial ATP quantification analysis. (D–F) Measurement of metabolite levels related to fatty acid β-oxidation (D), tricarboxylic acid (TCA) (E), and glycolysis (F) in mitochondria. (G, H) Analysis of enzyme levels related to fatty acid β-oxidation, TCA cycle, and glycolysis in mitochondria…

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  Fig. 6. Fatty acid synthase (FASN) modulates cell adhesion behavior to promote inflammatory factor aggregation in endothelial cells. (A) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of FASN in THP-P cells. (B) Western blot was applied to determine the protein expression of FASN in THP-P cells. (C, D) Assessment of cell adhesion in different treatment groups (C) and the statistical data (D). (E, F) Measurement of vascular cell adhesion molecule-1 (VCAM-1) expression in cells through RT-qPCR (E) and Western blot (F). (G) Observation of changes in cell adhesion molecules using immunofluorescence. (H–J) Evaluation of the expression of inflammatory factors tumor necrosis factor-alpha (TNF-α) (H), interleukin-6 (IL-6) (I), monocyte chemoattractant protein-1 (MCP-1) (J) in endothelial cells via Western blot. ∗P < 0.05 indicates a significant difference between the two groups, ∗∗P < 0.01 indicates a significant difference, and ∗∗∗P < 0.001 indicates a very highly significant difference between the two groups, with the cell experiments repeated three times. NO: normoxia; s-EVs: serum-derived extracellular vesicles; CIH: chronic…

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  Fig. 7. The intervention of fatty acid synthase (FASN) improves endothelial permeability by inhibiting apoptosis of endothelial cells. (A) enzyme-linked immunosorbent assay (ELISA) assay was performed to measure the levels of endothelial function markers normoxia (NO), inorganic phosphate (Pi), and endothelin-1 (ET-1) in the peripheral blood of mice. (B, C) Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)/ cluster of differentiation 31 (CD31) double immunofluorescence staining in the dysfunctional area (B) and the statistical data (C). (D, E) Estimation of endothelial integrity in vascular cross-sections based on CD31 fluorescence intensity, with lower intensity indicating greater endothelial injury (D) and the statistical data (E). (F, G) Triple immunofluorescence staining of F4/80/α-smooth muscle actin (α-SMA)/ interleukin-6 (IL-6) to observe endothelial cells in vascular cross-sections (F) and the statistical data (G). (H, I) Lung blue dye perfusion images (H) and Evans blue leakage index (I). Tissue samples shown in each group represent typical samples, reflecting the overall trend. Each group consisted of 6 mice, and data are presented as mean ± standard…

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  Fig. 8. Mechanism of endothelial dysfunction induced by high expression of fatty acid synthase (FASN) in serum-derived extracellular vesicles (s-EVs) from obstructive sleep apnea hypopnea syndrome (OSAHS) patients under intermittent hypoxia conditions. TNF-α: tumor necrosis factor α; IL-6: interleukin 6; MCP-1: monocyte chemoattractant protein 1; ICAM-1: intercellular cell adhesion molecule 1; VCAM-1: vascular cell adhesion molecule 1; LDHA: lactate dehydrogenase A; CPT1b: carnitine palmitoyltransferase 1b; SDHB: succinate dehydrogenase subunit B; CS: citrate synthase; CD31: cluster of differentiation 31; vWF: von Willebrand factor; MMP: matrix metalloproteinase; Cyt-c: cytochrome c; Bax: Bcl-2-associated X protein; Bcl2: B-cell lymphoma 2 (Created by Biorender).