Lipid based anti-fibrotic formulations and their impact on extracellular vesicles

Liver fibrosis is a major global health concern: its evolution into liver cirrhosis is followed by the death of over a million of people every year worldwide. The transdifferentiation of hepatic stellate cells (HSCs, the main collagen-producing cells) from a quiescent into an activated, pro-fibrotic status plays a key role in liver fibrogenesis, making these cells interesting from both a therapeutic and a diagnostic perspective. Can membrane-bound nanovesicles shuttled between HSCs become a diagnostic tool for liver fibrosis? The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers that might become a valid alternative to highly invasive liver biopsies. We developed robust sets of methods to isolate and characterize EVs from differently treated LX-2 (human HSCs cell line) in vitro, and we investigated the biological effect they exert onto naïve cells, proving that EVs do play an active role in affecting HSCs’ status. Different purification methods (size exclusion chromatography, SEC, and asymmetrical flow field-flow fractionation, AF4) revealed EV subpopulations with different physicochemical behaviors. Proteomic data from our samples was mined for EV-associated proteins whose expression correlated with HSCs treatment. Consequently, the secreted protein acidic and cysteine rich (SPARC) emerged as a candidate protein to explore the feasibility of using fluorescence nanoparticle tracking analysis (f-NTA) as a non-destructive tool for the determination of HSCs’ physiological state based on EVs. We could thus use EVs to directly evaluate the efficacy of treatments with polyenylphosphatidylcholines (PPC)-rich lipid S80, with and without experimental drugs elafibranor and obeticholic acid, proving that S80 (but neither of the drugs) greatly reduces relative SPARC-positive EVs’ abundance, while pro-fibrotic treatment with transforming growth factor β1 (TGF) does the opposite. Here, for the first time, we could measurably correlate the cellular response to PPC-treatment to the relative presence of SPARC on the generated EVs. Our results pave the way for more precise and less invasive ex vivo analyses: markers related to diseased and healthy states, as well as proteins that are tissue specific or preferentially expressed by specific cells could all be conceivably checked within one EV sample if appropriately selected

Tracking matricellular protein SPARC in extracellular vesicles as a non-destructive method to evaluate lipid-based antifibrotic treatments.

Uncovering the complex cellular mechanisms underlying hepatic fibrogenesis could expedite the development of effective treatments and noninvasive diagnosis for liver fibrosis. The biochemical complexity of extracellular vesicles (EVs) and their role in intercellular communication make them an attractive tool to look for biomarkers as potential alternative to liver biopsies. We developed a solid set of methods to isolate and characterize EVs from differently treated human hepatic stellate cell (HSC) line LX-2, and we investigated their biological effect onto naïve LX-2, proving that EVs do play an active role in fibrogenesis. We mined our proteomic data for EV-associated proteins whose expression correlated with HSC treatment, choosing the matricellular protein SPARC as proof-of-concept for the feasibility of fluorescence nanoparticle-tracking analysis to determine an EV-based HSCs' fibrogenic phenotype. We thus used EVs to directly evaluate the efficacy of treatment with S80, a polyenylphosphatidylcholines-rich lipid, finding that S80 reduces the relative presence of SPARC-positive EVs. Here we correlated the cellular response to lipid-based antifibrotic treatment to the relative presence of a candidate protein marker associated with the released EVs. Along with providing insights into polyenylphosphatidylcholines treatments, our findings pave the way for precise and less invasive diagnostic analyses of hepatic fibrogenesis

Formulating elafibranor and obeticholic acid with phospholipids decreases drug-induced association of SPARC to extracellular vesicles from LX-2 human hepatic stellate cells.

Chronic hepatic diseases often compromise liver function and are directly responsible for up to two million yearly deaths world-wide. There are yet no treatment options to solve this global medical need. Experimental drugs elafibranor (Ela) and obeticholic acid (OA) appeared promising in numerous earlier studies, but they recently struggled to show significant benefits in patients. Little is known on the drugs' impact on hepatic stellate cells (HSCs), key players in liver fibrogenesis. We recently reported a beneficial effect of polyenylphosphatidylcholines (PPCs)-rich formulations in reverting fibrogenic features of HSCs, including differences in their extracellular vesicles (EVs). Here, we newly formulated Ela and OA in PPC liposomes and evaluated their performance on the LX-2 (human HSC) cell line through our rigorous methods of EV-analysis, now expanded to include lipidomics. We show that direct treatments with Ela and OA increase EV-associated secreted protein acidic and cysteine rich (SPARC), a matricellular protein overexpressed in fibrogenesis. However, our results suggest that this potentially damaging drugs' action to HSCs could be mitigated when delivering them with lipid-based formulations, most notably with a PPC-rich phospholipid inducing specific changes in the cellular and EV phospholipid composition. Thus, EV analysis substantially deepens evaluations of drug performances and delivery strategies

Synergy of Phospholipid—Drug Formulations Significantly Deactivates Profibrogenic Human Hepatic Stellate Cells

The pivotal role of hepatic stellate cells (HSCs) in orchestrating the bidirectional process of progression and regression of liver fibrosis makes them an ideal target for exploring new antifibrotic therapies. Essential phospholipids (EPLs), with their polyenylphosphatidylcholine (PPC) fraction, either alone or combined with other hepatoprotective substances such as silymarin, are recommended in hepatic impairment, but a scientific rationale for their use is still lacking. Herein, we compared the ability of EPLs to restore quiescent-like features in HSCs with that of dilinoleoylphosphatidylcholine (DLPC), PPC fraction’s main component. Specifically, we screened at the cellular level the antifibrotic effects of PPC formulations in the presence and absence of silymarin, by using LX-2 cells (pro-fibrogenic HSCs) and by assessing the main biochemical hallmarks of the activated and deactivated states of this cell line. We also proved the formulations’ direct effect on the motional order of cell membranes of adherent cells. LX-2 cells, examined for lipid droplets as a quiescence marker, showed that PPCs led to a more prominent deactivation than DLPC. This result was confirmed by a reduction of collagen and α-SMA expression, and by a profound alteration in the cell membrane fluidity. PPC–silymarin formulations deactivated HSCs with a significant synergistic effect. The remarkable bioactivity of PPCs in deactivating fibrogenic HSCs paves the way for the rational design of new therapeutics aimed at managing hepatic fibrosis

Liver-derived extracellular vesicles: A cell by cell overview to isolation and characterization practices.

BACKGROUND: Extracellular vesicles (EVs) are a diverse group of membrane-bound nanovesicles potentially released by every cell. With the liver's unique ensemble of cells and its fundamental physiological tasks, elucidating the role of EV-mediated hepatic cellular crosstalk and their role in different pathologies has been gaining the attention of many scientists. SCOPE OF REVIEW: The present review shifts the perspective into practice: we aim to critically discuss the methods used to purify and to biochemically analyse EVs from specific liver resident cells, including hepatocytes, hepatic stellate cells, cholangiocytes, liver sinusoidal endothelial cells, Kupffer cells, liver stem cells. The review offers a reference guide to current approaches. MAJOR CONCLUSIONS: Strategies for EV isolation and characterization are as varied as the research groups performing them. We present main advantages and disadvantages for the methods, highlighting common causes for concern, such as FBS handling, reporting of cell viability, EV yield and storage, differences in differential centrifugations, suboptimal method descriptions, and method transferability. We both looked at how adaptable the research between human and rodent cells in vitro is, and also assessed how well either of them translates to ex vivo settings. GENERAL SIGNIFICANCE: We reviewed methodological practices for the isolation and analysis of liver-derived EVs, making a cell type specific user guide that shows where to start, what has worked so far and to what extent. We critically discussed room for improvement, placing a particular focus on working towards a potential standardization of methods