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. 2024 Jun 3;8(6):e0448.
doi: 10.1097/HC9.0000000000000448. eCollection 2024 Jun 1.

Proteomic analysis of serum extracellular vesicles reveals Fibulin-3 as a new marker predicting liver-related events in MASLD

Affiliations

Proteomic analysis of serum extracellular vesicles reveals Fibulin-3 as a new marker predicting liver-related events in MASLD

Sadatsugu Sakane et al. Hepatol Commun. .

Abstract

Background: There is a need for novel noninvasive markers for metabolic dysfunction-associated steatotic liver disease (MASLD) to stratify patients at high risk for liver-related events including liver cancer and decompensation. In the present study, we used proteomic analysis of proteins in extracellular vesicles (EVs) to identify new biomarkers that change with fibrosis progression and can predict the development of liver-related events.

Methods: We analyzed serum EVs from 50 patients with MASLD assessed for liver fibrosis by biopsy and identified proteins that altered with advanced fibrosis. A further evaluation was conducted on another cohort of 463 patients with MASLD with biopsy.

Results: Eight candidate proteins were identified by proteomic analysis of serum EVs. Among them, serum levels of Fibulin-3, Fibulin-1, and Ficolin 1 correlated with their EV levels. In addition, serum Fibulin-3 and serum Fibulin-1 levels changed significantly with advanced fibrosis. Using another cohort with biopsy, we found that the serum Fibulin-3 concentration was significantly greater in those with advanced fibrosis but that the serum Fibulin-1 concentration was not significantly different. Multivariate Cox proportional hazards analysis revealed that a higher Fibrosis-4 (FIB-4) index and higher serum Fibulin-3 concentration were independent risk factors for liver-related events. When the cutoff value for the serum Fibulin-3 concentration was 6.0 µg/mL according to the Youden index of AUROCs, patients with high serum Fibulin-3 significantly more frequently developed liver-related events than did other patients. Validation using another cohort of 226 patients with clinically diagnosed MASLD confirmed that high serum Fibulin-3 levels are associated with a greater frequency of liver-related events.

Conclusions: Serum Fibulin-3 was identified as a biomarker for predicting liver-related events in patients with MASLD.

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Conflict of interest statement

Hidenori Toyoda is on the speakers’ bureau for Gilead Sciences, AbbVie, Eisai, Terumo, Fujifilm WAKO, Takeda, Kowa, and Astra Zeneca. Naoya Sakamoto is on the speakers’ bureau and received grants from Otsuka Pharm, Gilead, Eisai, and Chugai Pharm. He is on the speakers’ bureau for Abbvie. He received grants from Sumitomo Pharma, Nihon Kayaku, Takeda, and Astellas. The remaining authors have no conflicts to report.

Figures

FIGURE 1
FIGURE 1
Proteins that change with advanced fibrosis according to MASLD criteria were identified through proteomic analysis of serum extracellular vesicles. (A) Relative levels of 8 proteins in serum EVs according to Brunt stage in 50 patients with MASLD; levels were significantly different between F3-F4 and F0-F2. (B) Correlations between the candidate proteins in serum EVs and those in serum. (C) Relative serum Fibulin-3, serum Fibulin-1, and serum Ficolin 1 levels in patients according to fibrosis stage. The dot plot shows individual values. Data are expressed as the mean ± SD. The difference between the 2 groups was assessed by the Mann-Whitney U test. Correlations were assessed by the Pearson product-moment correlation coefficient. Abbreviations: EV, extracellular vesicle; MASLD, metabolic dysfunction–associated steatotic liver disease.
FIGURE 2
FIGURE 2
The serum Fibulin-3 concentration increased in patients with advanced fibrosis according to the MASLD criteria. (A) Relative quantitative values of serum Fibulin-3 and serum Fibulin-1 according to fibrosis stage in 476 patients with MASLD. (B) Correlations between the serum Fibulin-3 concentration and fibrosis markers. (C) Relative quantitative values of serum Fibulin-3 according to steatosis, inflammation, ballooning, and NAS score in 476 patients with MASLD. The dot plot shows individual values. Data are expressed as the mean ± SD. The difference between the 2 groups was assessed by the Mann-Whitney U test. Correlations were assessed by the Pearson product-moment correlation coefficient. Abbreviations: FIB-4, Fibrosis-4; MASLD, metabolic dysfunction–associated steatotic liver disease; NAS, nonalcoholic fatty liver disease activity score.
FIGURE 3
FIGURE 3
The serum Fibulin-3 concentration can predict liver-related events in patients with MASLD. (A) K-M curves for liver-related events in 473 patients with MASLD. (B) ROC curves of the ability of the serum Fibulin-3 concentration and FIB-4 index to predict liver cancer occurrence at 3, 5, and 7 years. (C) K-M curves for liver-related events in 473 patients with MASLD divided into 2 groups: serum Fibulin-3 >6.0 µg/mL and Fibulin-3 <6.0 µg/mL. (D) K-M curves for liver cancer occurrence and decompensation event occurrence in 471 patients with MASLD divided into 2 groups: FIB-4 index >1.3 and <1.3. (E) K-M curves for liver-related events occurring in 304 patients with MASLD with an FIB-4 index >1.3 according to the following 2 groups: serum Fibulin-3 >6.0 µg/mL and Fibulin-3 <6.0 µg/mL. Cutoff values were determined by the Youden index. The difference in cumulative event rates between the 2 groups was assessed by the log-rank test. Abbreviations: FIB-4, Fibrosis-4; K-M curve, Kaplan-Meier curve; MASLD, metabolic dysfunction–associated steatotic liver disease.
FIGURE 4
FIGURE 4
The serum Fibulin-3 concentration was validated to be a predictor of liver-related events in patients with MASLD. (A) K-M curves for liver-related events in 226 patients with MASLD divided into 2 groups: serum Fibulin-3 >6.0 µg/mL and Fibulin-3 <6.0 µg/mL. (B) K-M curves for liver cancer occurrence in 131 patients with MASLD syndrome with an FIB-4 index >1.3 according to the following 2 groups: serum Fibulin-3 >6.0 µg/mL and Fibulin-3 <6.0 µg/mL. The difference in cumulative event rates between the 2 groups was assessed by the log-rank test. Abbreviations: FIB-4, Fibrosis-4; K-M curve, Kaplan-Meier curve; MASLD, metabolic dysfunction–associated steatotic liver disease.

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