Therapeutic effects of serum extracellular vesicles in liver fibrosis

Abstract

The lack of approved therapies for hepatic fibrosis seriously limits medical management of patients with chronic liver disease. Since extracellular vesicles (EVs) function as conduits for intercellular molecular transfer, we investigated if EVs from healthy individuals have anti-fibrotic properties. Hepatic fibrogenesis or fibrosis in carbon tetrachloride (CCl4)- or thioacetic acid-induced liver injury models in male or female mice were suppressed by serum EVs from normal mice (EVN) but not from fibrotic mice (EVF). CCl4-treated mice undergoing EVN therapy also exhibited reduced levels of hepatocyte death, inflammatory infiltration, circulating AST/ALT levels and hepatic or circulating pro-inflammatory cytokines. Hepatic histology, liver function tests or circulating proinflammatory cytokine levels were unaltered in control mice receiving EVN. As determined using PKH26-labelled EVN, principal target cells included hepatic stellate cells (HSC; a normally quiescent fibroblastic cell that undergoes injury-induced activation and produces fibrosis during chronic injury) or hepatocytes which showed increased EVN binding after, respectively, activation or exposure to CCl4. In vitro, EVN decreased proliferation and fibrosis-associated molecule expression in activated HSC, while reversing the inhibitory effects of CCl4 or ethanol on hepatocyte proliferation. In mice, microRNA-34c, -151-3p, -483-5p, -532-5p and -687 were more highly expressed in EVN than EVF and mimics of these microRNAs (miRs) individually suppressed fibrogenic gene expression in activated HSC. A role for these miRs in contributing to EVN actions was shown by the ability of their corresponding antagomirs to individually and/or collectively block the therapeutic effects of EVN on activated HSC or injured hepatocytes. Similarly, the activated phenotype of human LX-2 HSC was attenuated by serum EVs from healthy human subjects and contained higher miR-34c, -151-3p, -483-5p or -532-5p than EVs from hepatic fibrosis patients. In conclusion, serum EVs from normal healthy individuals are inherently anti-fibrogenic and anti-fibrotic, and contain microRNAs that have therapeutic actions in activated HSC or injured hepatocytes.Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; CCl4: carbon tetrachloride; CCN2: connective tissue growth factor; E: eosin; EGFP: enhanced green fluorescent protein; EVs: extracellular vesicles; EVF: serum EVs from mice with experimental hepatic fibrosis; EVN: serum EVs from normal mice; H: hematoxylin; HSC: hepatic stellate cell; IHC: immunohistochemistry; IL: interleukin; MCP-1: monocyte chemotactic protein-1; miR: microRNA; mRNA: messenger RNA; NTA: nanoparticle tracking analysis; PCNA: proliferating cell nuclear antigen; qRT-PCR: quantitative real-time polymerase chain reaction; SDS-PAGE: sodium dodecyl sulphate – polyacrylamide gel electrophoresis; αSMA: alpha smooth muscle actin; TAA: thioacetic acid; TG: transgenic; TGF-β: transforming growth factor beta; TEM: transmission electron microscopy; TNFα...

Human Breast Milk-Derived Extracellular Vesicles in the Protection Against Experimental Necrotizing Enterocolitis

“…Western blotting confirmed the presence of the established EV membrane markers CD63, CD81, Alix, and Flotillin (Fig. 1B) [9][10][11]. TEM illustrated the EV with the characteristic cup shaped morphology and size of~100 nm (Fig.…”

“…We have shown in the current study that human breast milk-derived EVs significantly decrease the incidence and severity of NEC. EVs have a vital role in cellular communication and many other biological processes because of their ability to deliver components of their molecular payload (comprising DNA, RNA, micro-RNA and proteins) into recipient cells, which can thereby become functionally reprogrammed [8,9]. Whereas previous studies showed that enteral administration of bovine or rat breast milk-derived EVs protected the intestines from NEC in in vitro and in vivo models [5,7], our study of EVs from human breast milk and their protective role in NEC in vivo provides an important advance in the field that has clear clinical implications.…”

Dynamic Changes in Function and Proteomic Composition of Extracellular Vesicles from Hepatic Stellate Cells during Cellular Activation

“…HSC fibrogenesis is stimulated by these types of hepatocyte-derived EVs [29,32,41], while other phenotypic features of activated HSC such as migration and AKT phosphorylation have been shown to be enhanced by EVs from liver sinusoidal endothelial cells [42]. On the other hand, EVs from healthy hepatocytes [43], various stem cells [44][45][46][47][48][49][50][51] or the serum of healthy mice [52] have the ability to inhibit experimental liver fibrosis, largely by suppressing inflammatory responses and/or pathways of activation or fibrogenesis in HSC. The recognition that HSC are EV targets has highlighted an important new mechanism by which fibrogenic pathways in the liver are modulated and has given a new lead for novel anti-fibrotic therapies based on suppressing the action of pro-fibrotic EVs or harnessing the actions of EVs that are intrinsically anti-fibrotic.…”

Extracellular Vesicles in NAFLD/ALD: From Pathobiology to Therapy

“…Proteomic analysis of EV-HLSCs showed that their cargo included various anti-inflammatory proteins such as Interleukin-10, which may have contributed to the observed beneficial effects. In addition, although yet not explored in the field of NAFLD, the use of MSC-derived EVs to arrest fibrogenesis [53,83] holds promise to treat this condition, as fibrosis is one of the most important determinants of survival. These results underscore the concept that EVs can be exploited for therapy in NAFLD/NASH.…”