Additional file 1 of Hepatic small extracellular vesicles promote microvascular endothelial hyperpermeability during NAFLD via novel-miRNA-7
Creators: name: Zuo, Rui
name: Ye, Li-Feng
name: Huang, Yi
name: Song, Zi-Qing
name: Wang, Lei
name: Zhi, Hui
name: Zhang, Min-Yi
name: Li, Jie-Yi
name: Zhu, Li
name: Xiao, Wen-Jing
name: Shang, Hong-Cai
name: Zhang, Yang
name: He, Rong-Rong
name: Chen, Yang
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer

description: Additional file 1: Figure S1. Body weight, liver weight and circulating lipid content in MCD and HFD-induced NAFLD mice. Mice were fed a MCD diet for 4 weeks or HFD for 16 weeks to induce NAFLD. (A) Body weight of NAFLD mice, n = 6 per group. (B) Representative images of liver and the liver weight of NAFLD mice, n = 6 per group. Scale bar:5 mm. (C-E) Relative plasma total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) content, n = 6 per group. Data are expressed as the mean ± SEM. Statistics: Student t-test, *P &lt; 0.05, **P &lt; 0.01 vs. the MCS group; &amp;&amp;P &lt; 0.01 vs. ND group. Figure S2. Changes of microvascular endothelial permeability in lung, liver and spleen induced by hepatic sEVs. NAFLD or control hepatic sEVs were isolated by ultracentrifugation, identified, and administered to naive NLRP3+/+ and NLRP3-/- mice via caudal vein injection. (A-C) In vivo optical imaging system-obtained fluorescence imaging of lung, liver and spleen, n = 6 per group. The pellet derived from the ultracentrifugation of DiR alone was used as a vehicle control. (D-F) Representative images and the summarized data of Evans blue concentrations in lung, liver and spleen, n = 5-6 per group. Data are expressed as the mean ±SEM. Statistics: One-way ANOVA, *P &lt; 0.05 vs. NLRP3+/+ mice injected with MCS hepatic sEVs; &amp;&amp;P &lt; 0.01 vs. NLRP3+/+ mice injected with ND hepatic sEVs; ## P &lt; 0.01 vs. NLRP3+/+ mice injected with MCD hepatic sEVs; $$ P &lt; 0.01 vs. NLRP3+/+ mice injected with HFD hepatic sEVs. Figure S3. Cell viability, LDH leakage and lipid accumulation in steatotic hepatocytes. Human hepatocyte cell line HepG2, HuH7 and L02 were treated with 100 μmol/L palmitic acid (PA) or vehicle for 18 h. (A) Cell viability were measured using cell counting kit (CCK)-8, n = 6 per group. (B) Lactic dehydrogenase (LDH) leakage was estimated by measuring LDH activities in the cell culture supernatants, n = 6 per group. (C) Intracellular triglyceride (TG) contents were measured by chromometry, n = 4 per group. (D) Hepatocytes were stained with Oil Red O and the intracellular lipid accumulation was measured by chromometry, n = 4 per group. Data are expressed as the mean ±SEM. Statistics: Student t-test, Ns, no significance, **P &lt; 0.01 vs. vehicle group. Figure S4. Cytokine contents in the cardiac tissue of NAFLD mice.NLRP3+/+ and NLRP3-/- Mice were fed a MCD diet for 4 weeks or HFD for 16 weeks to induce NAFLD. The contents of cytokines including IL-1β and HMGB1 in the cardiac tissues were measured by ELISA kits. The results were normalized to protein concentration. (A-B) Cardiac IL-1β and HMGB1 content in NLRP3+/+ and NLRP3-/- mice fed a MCS or MCD diet, n = 5-6 per group. (C-D) Cardiac IL-1β and HMGB1 content in NLRP3+/+ and NLRP3-/- Mice fed a ND or HFD, n = 5-6 per group. Data are expressed as the mean ±SEM. Statistics: One-way ANOVA, **P &lt; 0.01 vs. NLRP3+/+ mice fed with a HFD diet. Figure S5. Cytokine contents in the cardiac tissue of hepatic. sEV-treated mice NAFLD or control hepatic sEVs were isolated by ultracentrifugation, identified, and administered to naive NLRP3+/+ and NLRP3-/- mice via caudal vein injection. The contents of cytokines including IL-1β and HMGB1 in the cardiac tissues were measured by ELISA kits. The results were normalized to protein concentration. (A-B) Cardiac IL-1β and HMGB1 content in NLRP3+/+ and NLRP3-/- mice injected with MCS-sEVs or MCD-sEVs, n = 5-6 per group. (C-D) Cardiac IL-1β and HMGB1 content in NLRP3+/+ and NLRP3-/- mice injected with ND-sEVs or HFD-sEVs, n = 5-6 per group. Data are expressed as the mean ±SEM. Statistics: One-way ANOVA, **P &lt; 0.01 vs. NLRP3+/+ mice injected with MCS-sEVs; ##P &lt; 0.01 vs. NLRP3+/+ mice injected with MCD-sEVs; &amp;&amp;P &lt; 0.01 vs. NLRP3+/+ mice injected with ND-sEVs; $$P &lt; 0.01 vs. NLRP3+/+ mice injected with HFD-sEVs. Figure S6. Novel-miR-7 antagomir does not alter the changes of body weight, circulating lipid content and aminotransferase activities of NAFLD mice. Mice were fed with a MCD diet for 2 weeks or a HFD for 6 weeks, followed by administration of 1 μmol/kg novel miR-7 antagomir or negative control (NC) lipoprotein cholesterol (LDL-C) content, n = 6 per group. (E-F) Plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), n = 6 per group. Data are expressed as the mean ± SEM. Statistics: One-way ANOVA, **P &lt; 0.01 vs. the MCS plus NC antagomir group; &amp;P &lt; 0.05, &amp;&amp;P &lt; 0.01 vs. the ND plus NC antagomir group; Ns: no significance, the MCD plus novel-miR-7 antagomir group were compared with the MCD plus NC antagomir group, and the HFD plus novel-miR-7 antagomir group were compared with the HFD plus NC antagomir group. Figure S7 Novel-miR-7 antagomir reduces the cardiac cytokine contents and ameliorates coronary microvascular hyperpermeability of NAFLD mice. Mice were fed with a MCD diet for 2 weeks or a HFD for 6 weeks, followed by administration of 1 μmol/kg novel miR-7 antagomir or negative control (NC) antagomir via the caudal vein once a week. (A-D) Cardiac IL-1β and HMGB1 contents were measured by ELISA kits, n = 5-6 per group. (E-F) Representative images and the summarized data of Evans blue concentrations in heart, n = 6 per group. Data are expressed as the mean ± SEM. Statistics: One-way ANOVA, **P &lt; 0.01 vs. the MCS plus NC antagomir group; &amp;P &lt; 0.05, &amp;&amp;P &lt; 0.01 vs. the ND plus NC antagomir group; #P &lt; 0.05, ##P &lt; 0.01 vs. the MCD plus NC antagomir group; $$P Figure S8. NAFLD hepatic sEVs promote pulmonary microvascular hyperpermeability via novel-miR-7. Mouse lung microvascular endothelial cells (MLVECs) were transfected with 100 nmol/L novel-miR-7 inhibitor or negative control (NC) inhibitor and incubated with or without 120 μg/mL NAFLD hepatic sEVs for 24 h. Mice were fed with a MCD diet for 2 weeks or a HFD for 6 weeks, followed by administration of 1 μmol/kg novel miR-7 antagomir or negative control (NC) antagomir via the caudal vein once a week. (A-B) Representative flow cytometry images of caspase-1 FLICA staining and the summarized data of the positive cells, n = 4 per group. The fold changes were obtained by calculating the ratio of the positive cells of the treated groups to the NC inhibitor group. (C) Relative permeability of the endothelial monolayer to FITC-dextran, n = 4 per group. (D-E) Representative images and the summarized data of Evans blue concentrations in lung, n = 6 per group. Data are expressed as the mean ± SEM. Statistics: One-way ANOVA, *P &lt; 0.05, **P &lt; 0.01 vs. the NC inhibitor group or the MCS plus NC antagomir group; &amp;&amp;P &lt; 0.01 vs. the NC inhibitor group or the ND plus NC antagomir group; #P &lt; 0.05, ##P &lt; 0.01 vs. the NC inhibitor plus MCD-sEVs group or the MCD plus NC antagomir group; $P &lt; 0.05, $$P &lt; 0.01 vs. the NC inhibitor plus HFD-sEVs group or the HFD plus NC antagomir group. Figure S9. Yield and size changes of hepatic sEVs between the two models. NAFLD or control hepatic sEVs were isolated by differential ultracentrifugation. (A) Protein concentration of hepatic sEVs was measured by BCA kit, n = 7–8 per group. (B) Representative images of sEV size distribution analysis by Flow Nanoanlyzer, n = 8 per group. (C) Concentration of hepatic sEVs was measured by Flow Nanoanlyzer, n = 8 per group. (D) Average size of hepatic sEVs was measured by Flow Nanoanlyzer, n = 8 per group. Data are expressed as the mean ±SEM. Statistics: One-way ANOVA, Ns: no significance, the MCD group were compared with the MCS group, and the HFD group were compared with the ND group. Figure S10. Genetic inhibition of NLRP3 suppresses NLRP3 inflammasome-associated microvascular hyperpermeability MVECs were transfected with 100 nmol/L NLRP3 siRNA (siNLRP3) or negative control siRNA (siNC) and incubated with or without 120 μg/mL NAFLD hepatic sEVs for 24 h. (A) Transfection efficiency of 5-FAM labeled siNLRP3. Scale bar, μm. (B) Representative western blot bands and the summarized data determined by densitometric analysis, n = 4 per group. (C-D) Representative flow cytometry images of caspase-1 FLICA staining and the summarized data of the positive cells, n = 4 per group. The fold changes were obtained by calculating the ratio of the positive cells of the treated groups to the siNC group. (E) Relative permeability of the endothelial monolayer to FITC-dextran, n = 4 per group. Data are expressed as the mean ± SEM. Statistics: One-way ANOVA, **P &lt; 0.01 vs. siNC group; # P &lt; 0.05, ## P &lt; 0.01 vs. siNC plus MCD-sEVs group; &amp;&amp;P &lt; 0.01 vs. siNC group; $$ P &lt; 0.01 vs. siNC plus HFD-sEVs group. Figure S11. Effect of novel-miR-705 and novel-miR-1135 inhibitor on NLRP3 inflammasome activation. MVECs were transfected with 100nmol/L novel-miR-705 inhibitor or novel-miR-1135 inhibitor or negative control (NC) inhibitor and incubated with or without 120 μg/mL NAFLD hepatic sEVs for 24 h. (A-B) Representative flow cytometry images of caspase-1 FLICA staining and the summarized data of the positive cells, n = 4 per group. The fold changes were obtained by calculating the ratio of the positive cells of the treated groups to the NC inhibitor group. Data are expressed as the mean ± SEM. Statistics: One-way ANOVA, **P &lt; 0.01 vs. NC inhibitor group; # P &lt; 0.05 vs. NC inhibitor plus MCD-sEVs group; &amp;&amp;P &lt; 0.01 vs. NC inhibitor group; Ns: no significance vs. NC inhibitor plus MCD-sEVs or HFD-sEVs groups. Figure S12. KEGG pathway enrichment analysis of the strong candidate genes. The common genes from cytokine activity and cytokine receptor binding signaling pathway with correlation scores ≥4 were considered as strong candidate genes of microvascular hyperpermeability. KEGG pathway enrichment analysis was performed to reveal the relevant signaling pathway of these strong candidate genes. Figure S13. Three-Dimensional cell model from the Z-stack obtained by stimulated emission depletion (STED) microscopy. MVECs were pretreated with or without 20 μmol/L dynasore for 40 min, followed by incubation of 120 μg/mL DiI-labeled hepatic sEVs for 8 h. Representative fluorescent confocal images of DiI-labeled hepatic sEVs (red), wheat germ agglutinin (WGA, green) with DAPI (blue). The pellet derived from the ultracentrifugation of DiI alone was used as a vehicle control. Scale bar, 10 μm.