Abstract
MIRNAS EMBEDDED IN HEPATOCYTE DERIVED EXTRACELLULAR VESICLES PROMOTE ENDOTHELIAL CAPILLARIZATION IN CHRONIC LIVER DISEASE
Background:
Liver cells paracrinally affect neighboring cells through the release of extracellular vesicles (EVs), which may contain microRNAs (miRNAs). Our aim was to investigate the role of miRNAs enclosed in hepatocyte-derived EVs as modulators of endothelial dedifferentiation in chronic liver disease (CLD).
Methods:
EVs were purified from the supernatant of healthy (CT) or cirrhotic (CH) primary hepatocytes (hepEVs) from human and rat livers.
In vivo: Healthy rats were intravenously treated with fluorescence-labelled hepEVs-CT, hepEVs-CH or vehicle (n=10) to evaluate their biodistribution, sinusoidal endothelial uptake, and LSEC phenotype through transcriptomic analysis and vWF and eNOS protein expression (n=6).
In vitro: Human hepEVs miRNAs profile was analyzed and those significantly deregulated in CH were validated in rat hepEVs by qPCR (n=3 & 9, respectively). Exogenous over-expression of deregulated miRNAs was performed in CT-LSECs, followed by RNAseq and data analysis using Ingenuity Pathway Analysis (IPA) (n=5). Endothelial deregulations due to miRNAs overexpression was compared to primary cirrhotic rat LSECs (n=6) and human cirrhotic liver tissue (ethanol etiology, n=12).
Results:
Upon in vivo administration, hepEVs-CH mostly accumulated in the liver, inducing endothelial dysfunction as suggested by the deregulation of 144 genes involved in pro-fibrogenic and inflammatory pathways, together with vWF upregulation and eNOS reduction. Characterization of human hepEVs-CH showed 37 miRNAs significantly deregulated in comparison to hepEVs-CT, being miR-A and miR-B validated in rat hepEVs-CH. Interestingly, miR-B target genes were significantly downregulated in CH-LSECs. Transcriptome analysis of LSECs transfected with miR-A or miR-B revealed 868 and 771 genes significantly deregulated vs miR-control. Moreover, the transcriptome of miR-B transfected cells showed 51% homology with CH-LSECs supporting the key role of miR-B in LSEC dedifferentiation. Indeed, IPA confirmed miR-B detrimental effects promoting deregulation in molecular processes involved in fibrogenesis, inflammation and cell death. Differential expression of specific genes of such pathways was confirmed in CH-LSECs and in endothelial population of human liver tissues, suggesting new potential targets for LSEC amelioration in CLD.
Conclusion: miRNAs embedded in hepatocytes’ EVs actively contribute to endothelial dedifferentiation in CLD. We propose this paracrine route as a novel therapeutic approach for endothelial dysfunction and portal hypertension in cirrhosis.