Circulating extracellular vesicles are a biomarker for NAFLD resolution and response to weight loss surgery

Abstract

There is increasing interest in the development of minimally invasive biomarkers for the diagnosis and prognosis of NAFLD via extracellular vesicles (EV). Plasma EVs were isolated by differential ultracentrifugation and quantified by nanoparticle tracking analysis from pre (n = 28) and post (n = 28) weight loss patients. In the pre weight loss group 22 had NAFLD. Nanoplasmon enhanced scattering (nPES) of gold nanoparticles conjugated to hepatocyte-specific antibodies was employed to identify hepatocyte-specific EVs. Complex lipid panel and targeted sphingolipids were performed. Logistic regression analysis was used to identify predictors of NAFLD. Plasma levels of EVs and hepatocyte-derived EVs are dynamic and decrease following NAFLD resolution due to weight loss surgery. Hepatocyte-derived EVs correlate with steatosis in NAFLD patients and steatosis and inflammation in NASH patients. Plasma levels of small EVs correlate with EV sphingolipids in patients with NASH. Hepatocyte-derived EVs measured by the nPES assay could serve as a point-of-care test for NAFLD.

Keywords: Bariatric surgery; Exosome; Fatty liver; Sphingolipid.

Figure 1.. Circulating EVs decrease following weight loss surgery.

(A) Small EV and (B) large EV concentrations in obese patients, N = 56 (pre 28, post 28). (C) Small EV and (D) large EV concentrations in NAFLD patients, N = 44 (pre 22 NAFLD vs post 22 no-NAFLD). (E) Small EV and (F) large EV concentrations in 14 paired pre- and post-weight loss NAFL patient samples. (G) Paired small EV and (H) large EV concentrations from 7 NASH patients. Two-tailed Student's t test was used for statistical analyses. P values of <0.05, 0.01, and 0.001 were denoted as *, **, and ***, respectively.

Figure 2.. Hepatocyte-derived EVs decrease after bariatric surgery.

(A) A graphical representation of nanoplasmonic enhanced scattering (nPES) assay. (B) Optical response with ASGR2 antibody and (C) CYP2E1 antibody is lower in post bariatric surgery patients than in pre bariatric surgery patient, N = 44 (pre 22 NAFLD vs post 22 no-NAFLD). (D) Optical response with ASGR2 antibody and (E) CYP2E1 antibody is lower in post bariatric surgery patients than in pre bariatric surgery patients, N = 14 NAFLD paired samples. (F) Optical response with ASGR2 antibody and (G) CYP2E1 antibody is lower in post bariatric surgery patients than in pre bariatric surgery patients, n = 7 NASH patients. P values of <0.05 and 0.01 were denoted as * and **, respectively.

Figure 3.. Small EVs are hepatocyte-derived EVs.

Graph showing linear regression between (A) small EV concentration and ASGR2 nPES signal; (B) small EV concentration and CYP2E1 nPES signal; (C) large EVs concentration and ASGR2 nPES signal; and (D) large EVs concentration and CYP2E1nPES signal. N = 8 NASH patients.

Figure 4.. Targeted sphingolipid measurement of small EVs.

(A) Sphingolipid concentration of small EVs, N = 56 (pre 28 vs post 28) according to NAS; two-tailed Student's t test was used for statistical analyses. P values of <0.05 were denoted as *. (B) Sphingolipid concentration of small EVs, N = 44 (pre 22 NAFLD vs post 22 no-NAFLD); two-tailed Student's t test was used for statistical analyses; P values of <0.05 and 0.01 were denoted as * and ** respectively. (C) Heatmap of correlations between small EV sphingolipids and clinical parameters (pre 22 NAFLD vs post 22 no-NAFLD). (D) Graph showing linear regression between small EV concentration and C22:0 CER (pre 22 NAFLD vs post 22 no-NAFLD). (E) Sphingolipid concentration of small EVs, N = 7 NASH paired patients (pre vs post); one-tailed Student's t test was used for statistical analyses. P values of <0.05 and 0.01 were denoted as # and * respectively. (F) Heatmap of correlations between EV sphingolipids and clinical parameters, N = 7 NASH paired patients (pre vs post). (G) Graph showing linear regression between ALT and C16:0 CER 7 NASH paired patients (pre vs post).

Figure 5.. The complex lipid panel analysis of small EVs.

(A) Sphingolipid concentration of small EVs. N = 56 (pre 28 vs post 28); two-tailed Student's t test was used for statistical analyses; P values of <0.05, 0.01, and 0.001 were denoted as *, **, and *** respectively. (B) Volcano plot of lipid changes in small EVs, N = 56, according to NAS score (0 vs 1, 1 vs 2, 2 vs ≧3, respectively); x axis is log2 fold change; y axis is −log10P values; labeled dot indicates upregulated or downregulated lipids (log2FC > 0.5 or log2FC < −0.5), which passed threshold for t test (P < 0.05); two-tailed Student's t test was used for statistical analyses.

Figure 6.. Metabolic pathways reflected in small EV sphingolipid enrichment.

The schematic representation of selected sphingolipid metabolic reactions from Kyoto Encyclopedia of Genes and Genomes (KEGG) with indications of quantified average lipid classes and acyl chains. (A) High in healthy liver (NAS =0), (B) high in NAS = 1, (C) high in NAS =2, and (D) high in NAS ≥ 3. (E) Change of small EV sphingolipid content expressed as the ratio of each sphingolipid normalized to mean of the total ceramide.

Figure 7.. Changes in EV sphingolipids with each NAS score.

Nonlinear regression model was applied to depict lipid parameters according to their respective evolution with NAS. The 95% confidence interval is included in the display. All data were normalized by mean of each lipids class (NAS = 0).