Here, we compared bile EVs in ESLD patients who received LT with those of donors with normal liver for identification of potential biomarkers of ESLD. First, we found that the levels of EVs secreted into the bile increased in ESLD patients, particularly in patients with HCC. Second, there were no major changes in the overall diversity of miRNA in the bile EVs between the two study groups, but the total amount of miRNA was higher and the types of miRNA were changed dramatically in the bile EVs of ESLD patients. Third, we found that altered miRNA expression in bile EVs was not reflected in serum miRNA in ESLD patients. Furthermore, to our best knowledge, this study is the first analysis of bile EVs extracted from normal liver (healthy control).
EVs, which are secreted by a large variety of cells, vary in count and content depending on the type or health of the EV-producing cells. Thus, they have become an area of intense research as potential diagnostic biomarkers for various diseases (6, 7). A majority of these studies have focused on EVs extracted from blood samples. The relative ease of the extraction process has made EVs a promising clinically effective biomarker. However, EVs extracted from blood include EVs secreted from various organs, and therefore blood EVs are only an indirect reflection of changes in EV secreted from a certain type of cell or tissue. Importantly, they are not completely reflective of changes in the EV secretion profiles of hepatocytes in the state of liver disease (14). In contrast, bile EVs are secreted into the bile canaliculi, which do not interact with the sinusoids. Thus, the only cells that secrete EVs into bile canaliculi are hepatocytes and bile duct cells (10). Therefore, bile does not contain EVs secreted from other organs and from nonparenchymal liver cells, such as hepatic stellate cells, Kupffer cells, and sinusoidal endothelial cells. This means that changes in bile EVs more accurately reflect the changes in the EVs secreted from hepatocytes, which are exposed to the liver microenvironment, than do blood EVs. Indeed, many studies have reported the altered expression of various miRNAs in blood and liver tissue of LC patients (15–17). However, the miRNAs in bile EVs identified in this study differed greatly from their results, suggesting that bile EVs contain a unique miRNA profile independent of the blood and liver tissues samples.
Bile EV concentration was significantly higher in ESLD liver than in normal liver as revealed by NTA. Furthermore, bile EV concentration decreased promptly after transplantation with normal liver, reaching levels similar to those in NL donors. This suggests that EV secretion by hepatocytes increased following exposure to the liver microenvironment during ESLD. We found no associations between bile EV concentration and underlying disease, alanine aminotransferase (ALT) levels, or hepatic reserve (data not shown). Several previous reports have stated that blood EV concentration increases with liver injury (18, 19). Changes in blood EV concentrations have been correlated with ALT, hepatic fibrosis, cell death, and pathological angiogenesis. In mouse models of nonalcoholic fatty liver disease, blood EV concentration increased early on after liver injury and subsequently displayed time-dependent changes (18). These studies showed that EV secretion increased several folds, but these likely reflect not only the EVs secreted by hepatocytes, but also systemic changes, including EV secretion by other organs that accompany fibrosis progression.
Patients with ESLD complicated with HCC had significantly higher EV concentrations than those without HCC. A recent study has reported the superiority of bile EVs for diagnosing malignant bile duct stenosis compared to tumor markers, such as serum Ca19-9 (11); thus, bile EV concentration may also be effective for diagnosing HCC. EV secretion is more elevated in cancer cells than in normal cells, and EVs from cancer cells are involved in angiogenesis, inflammation, cancer-related fibroblast differentiation, and epithelial-mesenchymal transition in the tumor microenvironment (20, 21). Increase in EV secretion by HCC itself may result in higher bile EV concentrations, but the pathway of EV secretion into the bile duct by HCC cells that have lost polarity and normal function is unknown, thus warranting further investigations.
Here, we found that the expression of five bile EV miRNAs, viz., miR-17, miR-92a, miR-25, miR-423, and miR-451a, significantly increased in the ESLD liver relative to the normal liver. Furthermore, a comparison of underlying disease or presence of HCC showed that miR-17 expression significantly increased in alcohol-induced ESLD, which is consistent with the report of markedly higher miR-17 and miR-18a levels in the liver tissue of a rodent model of alcohol-induced liver injury (22). miR-17 and miR-92a are components of the miR-17-92 cluster, and miR-25 composes the miR-106b-25 cluster; both clusters are inhibited by the transcription factor E2F and are known as oncogenic miRNAs (23, 24). It is possible that these EVs secreted by hepatocytes in the microenvironment of liver injury with fibrosis progression contribute to HCC progression and could be promising targets of new liver cancer treatments. In terms of the function of EVs released into the bile, an analysis of a rat model has shown that EV uptake occurs via cholangiocyte cilia to modulate bile duct cell growth (10), but how target cells are determined or how they functionally transmit their content is still unknown. The present study did not elucidate how EVs released by hepatocytes in the liver injury microenvironment act on bile duct cells, or whether they also act on the gastrointestinal tract after secretion. Future studies should investigate these aspects in more detail.
These results must be interpreted with caution, and some limitations should be borne in mind. First, since the study participants were recipients undergoing living donor LT, the sample size was small. Therefore, statistical analysis on the background factors of liver failure (presence of background liver disease and hepatocellular carcinoma) was insufficient. More cases are needed to clarify the effects of hepatocellular carcinoma progression and other background liver diseases on bile EVs. Second, in the analysis of miRNA expression level, exogenous cel-miR-39 spiked-in was used for normalization. However, the deviation of miR-39 levels in bile EVs was large between individual cases (a few samples showed outliers in the scatter plot). Li et al. have similarly reported that the level of the same amount of miR-39 added as an internal control in bile varied upon qPCR quantification, suggesting that the efficiency of RNA extraction from bile was not constant among the samples (12). Therefore, further verification on the relationship between miRNA levels and disease state is needed. Additionally, the establishment of a reliable internal control for bile samples will help improve future studies. Furthermore, collecting bile samples from chronic liver disease patients is invasive and difficult; thus, it may have limited utility as a clinical biomarker. In contrast, bile samples can be obtained easily through the drainage tubes placed in patients after liver transplant. Future studies should uncover the benefits of bile EVs as a diagnostic tool of post-transplant complications, such as acute rejection, blood flow obstruction, and bile duct stricture.
In conclusion, EVs secreted from the liver into the bile change to reflect liver disease state. Furthermore, miRNA in bile EVs change as they do in blood, but the changes are independent of changes in serum miRNA. Various studies have already focused on EVs in blood samples, but we found that EVs in bile could also serve as new biomarkers that provide direct insights into the liver microenvironment in liver disease.