An optimized workflow for analyzing extracellular vesicles as biomarkers in liver diseases

Abstract

Background & Aims: Extracellular vesicles (EVs) play an important role in intercellular communication, serving as vehicles for the exchange of biological materials and being involved in the regulation of physiological processes. EVs and their associated cargoes are considered a promising source of disease-associated biomarkers. The purpose of this study was to establish an easy-to-use, reproducible, and scalable workflow to efficiently analyze EVs in the context of liver disease. Methods: An optimized workflow was established for the pre-analytical processing and isolation of EVs from plasma and serum. Nanoparticle Tracking Analysis (NTA) was used to characterize circulating EVs in the serum of patients with nonalcoholic fatty liver disease (NAFLD), autoimmune liver disease (AIH), and animal models with impaired liver function. EVs were separated from soluble proteins by an optimized, polyethylene glycol (PEG)-based enrichment protocol. Enriched EVs were either labeled and functionally characterized by monitoring cellular uptake or lysed for biomarker identification. Results: Circulating EVs in the serum of patients with NAFLD or AIH and in different animal models have been characterized by NTA. Here we show that both the quantity and size of EVs in the serum of patients/animal models are significantly different from those of healthy individuals. We show that isolated EVs are functional, and their uptake by acceptor cells can be quantified after fluorescence labelling. Enriched EVs were directly used to analyze RNA biomarkers. Several microRNAs, including miR-15b, -16, -21, -122 and -223, were found to be significantly up-regulated in EVs isolated from the sera of patients with NAFLD and AIH. We show that EVs transport cytokines, and that IL-2, IL-6 and IL-8 were significantly up-regulated in EVs enriched from patients with cholangiocarcinoma (CCA) compared to healthy controls. Conclusions: The workflow presented here represents an accessible and easy-to-use approach that enables the analysis and enrichment of EVs from complex biological fluids and their preparation for functional characterization or downstream analysis. In this study, the levels of several miRNAs were found to be significantly increased in EVs isolated from AIH and NAFLD patients compared with healthy controls.

Uncovering Novel Roles of miR-122 in the Pathophysiology of the Liver: Potential Interaction with NRF1 and E2F4 Signaling

“…Nevertheless, the introduction of innovative and potent pharmaceutical agents for hepatitis C treatment [54] has considerably diminished the emphasis on therapeutic strategies involving miR-122 inhibition. The findings presented within this study, coupled with our recently published research [40], propose an intriguing potential physiological role for the reduction in miR-122 in response to inflammation. These insights hold the potential to stimulate a fresh evaluation of therapeutic opportunities associated with targeting miR-122 in CLDs, as inhibiting miR-122 could potentially emerge as a valuable approach for modulating inflammatory responses within the liver, warranting further consideration and exploration.…”

“…Conversely, the downregulation of miR-122 observed in patients with liver diseases, along with the consequent dysregulation of these transcription factors and their associate networks, may contribute to the "chronicization" process associated with liver diseases in these patients, as proposed in Figure 8B. Furthermore, in a recent publication, our research unveiled a significant connection among the bone morphogenetic protein 6 (BMP6), transforming growth factor beta (TGFβ), and regulation of miR-122 transcription [40], shedding light on a potential link between reduced miR-122 levels and hepatic inflammation. This pivotal finding, combined with the insights described in this manuscript, strengthens the hypothesis that the reduction in miR-122 expression is a physiological response triggered (also) by inflammation, leading to the fine-tuning of the signaling cascades downstream of E2F4, YY1, and NRF1.…”