miRNA-ome plasma analysis unveils changes in blood-brain barrier integrity associated with acute liver failure in rats.

BACKGROUND: Hepatic encephalopathy (HE) symptoms associated with liver insufficiency are linked to the neurotoxic effects of ammonia and other toxic metabolites reaching the brain via the blood-brain barrier (BBB), further aggravated by the inflammatory response. Cumulative evidence documents that the non-coding single-stranded RNAs, micro RNAs (miRs) control the BBB functioning. However, miRs' involvement in BBB breakdown in HE is still underexplored. Here, we hypothesized that in rats with acute liver failure (ALF) or rats subjected to hyperammonemia, altered circulating miRs affect BBB composing proteins. METHODS: Transmission electron microscopy was employed to delineate structural alterations of the BBB in rats with ALF (thioacetamide (TAA) intraperitoneal (ip.) administration) or hyperammonemia (ammonium acetate (OA) ip. administration). The BBB permeability was determined with Evans blue dye and sodium fluorescein assay. Plasma MiRs were profiled by Next Generation Sequencing (NGS), followed by in silico analysis. Selected miRs, verified by qRT-PCR, were examined in cultured rat brain endothelial cells. Targeted protein alterations were elucidated with immunofluorescence, western blotting, and, after selected miR mimics transfection, through an in vitro resistance measurement. RESULTS: Changes in BBB structure and increased permeability were observed in the prefrontal cortex of TAA rats but not in the brains of OA rats. The NGS results revealed divergently changed miRNA-ome in the plasma of both rat models. The in silico analysis led to the selection of miR-122-5p and miR-183-5p with their target genes occludin and integrin ß1, respectively, as potential contributors to BBB alterations. Both proteins were reduced in isolated brain vessels and cortical homogenates in TAA rats. We documented in cultured primary brain endothelial cells that ammonia alone and, in combination with TNFα increases the relative expression of NGS-selected miRs with a less pronounced effect of TNFα when added alone. The in vitro study also confirmed miR-122-5p-dependent decrease in occludin and miR-183-5p-related reduction in integrin ß1 expression. CONCLUSION: This work identified, to our knowledge for the first time, potential functional links between alterations in miRs residing in brain endothelium and BBB dysfunction in ALF.

Mitochondrial Changes in Rat Brain Endothelial Cells Associated with Hepatic Encephalopathy: Relation to the Blood-Brain Barrier Dysfunction.

The mechanisms underlying cerebral vascular dysfunction and edema during hepatic encephalopathy (HE) are unclear. Blood-brain barrier (BBB) impairment, resulting from increased vascular permeability, has been reported in acute and chronic HE. Mitochondrial dysfunction is a well-documented result of HE mainly affecting astrocytes, but much less so in the BBB-forming endothelial cells. Here we review literature reports and own experimental data obtained in HE models emphasizing alterations in mitochondrial dynamics and function as a possible contributor to the status of brain endothelial cell mitochondria in HE. Own studies on the expression of the mitochondrial fusion-fission controlling genes rendered HE animal model-dependent effects: increase of mitochondrial fusion controlling genes opa1, mfn1 in cerebral vessels in ammonium acetate-induced hyperammonemia, but a decrease of the two former genes and increase of fis1 in vessels in thioacetamide-induced HE. In endothelial cell line (RBE4) after 24 h ammonia and/or TNFα treatment, conditions mimicking crucial aspects of HE in vivo, we observed altered expression of mitochondrial fission/fusion genes: a decrease of opa1, mfn1, and, increase of the fission related fis1 gene. The effect in vitro was paralleled by the generation of reactive oxygen species, decreased total antioxidant capacity, decreased mitochondrial membrane potential, as well as increased permeability of RBE4 cell monolayer to fluorescein isothiocyanate dextran. Electron microscopy documented enlarged mitochondria in the brain endothelial cells of rats in both in vivo models. Collectively, the here observed alterations of cerebral endothelial mitochondria are indicative of their fission, and decreased potential of endothelial mitochondria are likely to contribute to BBB dysfunction in HE.

The Effect of TGF-ß1 Reduced Functionality on the Expression of Selected Synaptic Proteins and Electrophysiological Parameters: Implications of Changes Observed in Acute Hepatic Encephalopathy.

Decreased platelet count represents a feature of acute liver failure (ALF) pathogenesis. Platelets are the reservoir of transforming growth factor 1 (TGF-ß1), a multipotent cytokine involved in the maintenance of, i.a., central nervous system homeostasis. Here, we analyzed the effect of a decrease in TGF-ß1 active form on synaptic proteins levels, and brain electrophysiology, in mice after intraperitoneal (ip) administration of TGF-ß1 antibody (anti-TGF-ß1; 1 mg/mL). Next, we correlated it with a thrombocytopenia-induced TGF-ß1 decrease, documented in an azoxymethane-induced (AOM; 100 mM ip) model of ALF, and clarified the impact of TGF-ß1 decrease on blood-brain barrier functionality. The increase of both synaptophysin and synaptotagmin in the cytosolic fraction, and its reduction in a membrane fraction, were confirmed in the AOM mice brains. Both proteins' decrease in analyzed fractions occurred in anti-TGF-ß1 mice. In turn, an increase in postsynaptic (NR1 subunit of N-methyl-D-aspartate receptor, postsynaptic density protein 95, gephyrin) proteins in the AOM brain cortex, but a selective compensatory increase of NR1 subunit in anti-TGF-ß mice, was observed. The alterations of synaptic proteins levels were not translated on electrophysiological parameters in the anti-TGF-ß1 model. The results suggest the impairment of synaptic vesicles docking to the postsynaptic membrane in the AOM model. Nevertheless, changes in synaptic protein level in the anti-TGF-ß1 mice do not affect neurotransmission and may not contribute to neurologic deficits in AOM mice.

Insight into microRNAs-Mediated Communication between Liver and Brain: A Possible Approach for Understanding Acute Liver Failure?

Acute liver failure (ALF) is a life-threatening consequence of hepatic function rapid loss without preexisting liver disease. ALF may result in a spectrum of neuropsychiatric symptoms that encompasses cognitive impairment, coma, and often death, collectively defined as acute hepatic encephalopathy. Micro RNAs are small non-coding RNAs that modulate gene expression and are extensively verified as biomarker candidates in various diseases. Our systematic literature review based on the last decade's reports involving a total of 852 ALF patients, determined 205 altered circulating miRNAs, of which 25 miRNAs were altered in the blood, regardless of study design and methodology. Selected 25 miRNAs, emerging predominantly from the analyses of samples obtained from acetaminophen overdosed patients, represent the most promising biomarker candidates for a diagnostic panel for symptomatic ALF. We discussed the role of selected miRNAs in the context of tissue-specific origin and its possible regulatory role for molecular pathways involved in blood-brain barrier function. The defined several common pathways for 15 differently altered miRNAs were relevant to cellular community processes, indicating loss of intercellular, structural, and functional components, which may result in blood-brain barrier impairment and brain dysfunction. However, a causational relationship between circulating miRNAs differential expression, and particular clinical features of ALF, has to be demonstrated in a further study.