Impact of liver failure on the circulating extracellular vesicle miRNA repertoire.

BACKGROUND & AIMS: Cell-derived small extracellular vesicles (sEVs) participate in cell-cell communication via the transfer of molecular cargo including selectively enriched microRNAs (miRNAs). Utilizing advances in sEV isolation and characterization, this study investigates the impact of liver injury and dysfunction on the circulating EV-miRNA profile. METHODS: High-throughput screening of 799 sEV-miRNAs isolated from plasma was performed in patients across a spectrum of liver disorders including compensated and decompensated chronic liver disease, acute-on-chronic liver failure (ACLF), and acute liver failure, in addition to healthy controls and those with severe sepsis. miRNA levels were compared with clinical and biochemical parameters, composite scores of liver disease, and patient outcomes. RESULTS: miRNA screening revealed the degree of hepatic dysfunction to be the main determinant of changes in circulating sEV-miRNA profile, with liver-specific miRNA-122 being among the most highly dysregulated in severe injury. Principal components analyses of the 215 differentially expressed miRNAs showed differing profiles, particularly among those with acute liver injury and ACLF. A distinct profile of dysregulated miRNA, but not circulating cytokines, was shown to characterize ACLF, with four consensus miRNAs identified-miR-320e, miR-374-5p, miR-202-3p, and miR-1910-5p. High miR-320e was associated with poorer 90-day survival (p = 0.014) and regulated the functional gene targets IK, RPS5, MANBAL, and PEBP1. CONCLUSIONS: This first comprehensive analysis to the best of our knowledge of patients with varying degrees and stages of liver failure demonstrates miRNA profiles specifically within the sEV compartment to be significantly altered in progressive liver disease and highlights the diagnostic and prognostic potential of sEV-miRNA in ACLF while also establishing downstream gene targets.

A novel microRNA-based prognostic model outperforms standard prognostic models in patients with acetaminophen-induced acute liver failure.

BACKGROUND & AIMS: Acetaminophen (APAP)-induced acute liver failure (ALF) remains the most common cause of ALF in the Western world. Conventional prognostic models, utilising markers of liver injury and organ failure, lack sensitivity for mortality prediction. We previously identified a microRNA signature that is associated with successful regeneration post-auxiliary liver transplant and with recovery from APAP-ALF. Herein, we aimed to use this microRNA signature to develop outcome prediction models for APAP-ALF. METHODS: We undertook a nested, case-control study using serum samples from 194 patients with APAP-ALF enrolled in the US ALF Study Group registry (1998-2014) at early (day 1-2) and late (day 3-5) time-points. A microRNA qPCR panel of 22 microRNAs was utilised to assess microRNA expression at both time-points. Multiple logistic regression was used to develop models which were compared to conventional prognostic models using the DeLong method. RESULTS: Individual microRNAs confer limited prognostic value when utilised in isolation. However, incorporating them within microRNA-based outcome prediction models increases their clinical utility. Our early time-point model (AUC = 0.78, 95% CI 0.71-0.84) contained a microRNA signature associated with liver regeneration and our late time-point model (AUC = 0.83, 95% CI 0.76-0.89) contained a microRNA signature associated with cell-death. Both models were enhanced when combined with model for end-stage liver disease (MELD) score and vasopressor use and both outperformed the King's College criteria. The early time-point model combined with clinical parameters outperformed the ALF Study Group prognostic index and the MELD score. CONCLUSIONS: Our findings demonstrate that a regeneration-linked microRNA signature combined with readily available clinical parameters can outperform existing prognostic models for ALF in identifying patients with poor prognosis who may benefit from transplantation. LAY SUMMARY: While acute liver failure can be reversible, some patients will die without a liver transplant. We show that blood test markers that measure the potential for liver recovery may help improve identification of patients unlikely to survive acute liver failure who may benefit from a liver transplant.

Serum MicroRNA Signatures in Recovery From Acute and Chronic Liver Injury and Selection for Liver Transplantation.

We previously demonstrated a distinct hepatic microRNA (miRNA) signature (down-regulation of miRNA-23a, -150, - 200b, -503, and -663 and up-regulation of miRNA-20a) is associated with successful regeneration in auxiliary liver transplantation (ALT). This study aimed to evaluate whether the serum expression of this regeneration-linked miRNA signature is associated with clinical outcomes in acute and chronic liver disease. These were represented by patients with acetaminophen-induced acute liver failure (ALF; n = 18) and patients with hepatitis C virus (HCV) undergoing treatment with direct-acting antivirals (n = 56), respectively. Patients were grouped depending on their clinical outcome. Global serum miRNA expression was analyzed using polymerase chain reaction (PCR) arrays and selected miRNA expression using targeted PCR. We demonstrate that specific regeneration-linked miRNAs discriminate outcomes in both clinical scenarios. We further show that miRNA-20a, -23a, -150, -200b, -503, and -663 undergo concordant changes in expression in 3 distinct clinical settings: liver regeneration accompanying successful ALT, clinical recovery after ALF, and clinical recompensation after cure of HCV. This miRNA signature represents a potentially novel biomarker to predict outcome and optimize patient selection for liver transplantation in both acute and chronic liver disease.

The challenges and potential in developing microRNA associated with regeneration as biomarkers to improve prognostication for liver failure syndromes and hepatocellular carcinoma.

INTRODUCTION: Determining the need for liver transplantation remains critical in the management of hepatocellular carcinoma (HCC) and liver failure syndromes (including acute liver failure and decompensated cirrhosis states). Conventional prognostic models utilize biomarkers of liver and non-liver failure and have limitations in their application. Novel biomarkers which predict regeneration may fulfil this niche. microRNA are implicated in health and disease and are present in abundance in the circulation. Despite this, they have not translated into mainstream clinical biomarkers. AREAS COVERED: We will discuss current challenges in the prognostication of patients with liver failure syndromes as well as for patients with HCC. We will discuss biomarkers implicated with liver regeneration. We then provide an overview of the challenges in developing microRNA into clinically tractable biomarkers. Finally, we will provide a scoping review of microRNA which may have potential as prognostic biomarkers in liver failure syndromes and HCC. EXPERT OPINION: Novel biomarkers are needed to improve prognostic models in liver failure syndromes and HCC. Biomarkers associated with liver regeneration are currently lacking and may fulfil this niche. microRNA have the potential to be developed into clinically tractable biomarkers but a consensus on standardizing methodology and reporting is required prior to large-scale studies.

The challenges and potential of microRNA-based therapy for patients with liver failure syndromes and hepatocellular carcinoma.

INTRODUCTION: Morbidity and mortality from liver disease continues to rise worldwide. There are currently limited curative treatments for patients with liver failure syndromes, encompassing acute liver failure and decompensated cirrhosis states, outside of transplantation. Whilst there have been improvements in therapeutic options for patients with hepatocellular carcinoma (HCC), there remain challenges necessitating novel therapeutic agents. microRNA have long been seen as potential therapeutic targets but there has been limited clinical translation. AREAS COVERED: We will discuss the limitations of conventional non-transplant management of patients with liver failure syndromes and HCC. We will provide an overview of microRNA and the challenges in developing and delivering microRNA-based therapeutic agents. We will finally provide an overview of microRNA-based therapeutic agents which have progressed to clinical trials. EXPERT OPINION: microRNA have great potential to be developed into therapeutic agents due to their association with critical biological processes which govern health and disease. Utilizing microRNA sponges to target multiple microRNA associated with specific biological processes may improve their therapeutic efficacy. However, there needs to be significant improvements in delivery systems to ensure the safe delivery of microRNA to target sites and minimize systemic distribution. This currently significantly impacts the clinical translation of microRNA-based therapeutic agents.

Distinct microRNA profiles are associated with the severity of hepatitis C virus recurrence and acute cellular rejection after liver transplantation.

Recurrent hepatitis C virus (HCV) infection is associated with accelerated fibrosis rates after liver transplantation (LT) and is the leading cause of graft failure. Furthermore, distinguishing recurrent HCV from acute cellular rejection (ACR) can be problematic, and this can lead to inappropriate treatments and adverse outcomes. We hypothesized that intragraft microRNA (miRNA) expression profiles could distinguish the severity of recurrent HCV and differentiate recurrent HCV from ACR. We established meticulously matched post-LT patient cohorts in order to derive robust global miRNA expression profiles and minimize the impact of variables known to influence HCV recurrence. These cohorts consisted of patients with slow HCV fibrosis progression (Ishak stage < F2), fast HCV fibrosis progression (Ishak stage ≥ F2), ACR, and nonviral etiologies. We found increased intragraft expression of miRNA-146a, miRNA-19a, miRNA-20a, and miRNA-let7e in slow progressors versus fast progressors, and we validated these findings with quantitative PCR. This miRNA network regulates the expression of cardinal genes implicated in promoting antifibrogenic, antiangiogenic, and anti-inflammatory pathways. miRNA-19a and miRNA-20a were also specifically detected in the serum of slow progressors. Furthermore, intragraft miRNA expression distinguished fast HCV progression from ACR. Here, changes in the expression of key miRNAs regulating fibrogenic and angiogenic pathways were associated with fast HCV progression. We demonstrate specific miRNA expression signatures that discriminate the rates of fibrosis progression in patients with recurrent HCV, and we distinguish recurrent HCV from ACR after LT. A pathway analysis indicates that specific miRNAs may play a regulatory role in these processes. Selected miRNAs may serve as intragraft and serum biomarkers for recurrent HCV after LT and help to distinguish between ACR and recurrent HCV.

Regeneration linked miRNA modify tumor phenotype and can enforce multi-lineage growth arrest in vivo.

Regulated cell proliferation is an effector mechanism of regeneration, whilst dysregulated cell proliferation is a feature of cancer. We have previously identified microRNA (miRNA) that regulate successful and failed human liver regeneration. We hypothesized that these regulators may directly modify tumor behavior. Here we show that inhibition of miRNAs -503 and -23a, alone or in combination, enhances tumor proliferation in hepatocyte and non-hepatocyte derived cancers in vitro, driving more aggressive tumor behavior in vivo. Inhibition of miRNA-152 caused induction of DNMT1, site-specific methylation with associated changes in gene expression and in vitro and in vivo growth inhibition. Enforced changes in expression of two miRNA recapitulating changes observed in failed regeneration led to complete growth inhibition of multi-lineage cancers in vivo. Our results indicate that regulation of regeneration and tumor aggressiveness are concordant and that miRNA-based inhibitors of regeneration may constitute a novel treatment strategy for human cancers.

Gjb3 Gene Mutations in Non-Syndromic Hearing Loss of Bloch, Kurd, and Turkmen Ethnicities in Iran.

BACKGROUND: Hearing loss (HL) is one of the most common heterogeneous congenital disabilities worldwide. Gap junction protein ß-3 (GJB3) gene encodes Connexin31 protein (Cx31). The hereditary type of hearing impairment in this gene are known to cause both autosomal recessive and autosomal dominant form. In addition, GJB3 mutations have been involved in sensorineural deafness, erythrokeratodermia variabilis (EKV), and neuropathy diseases. We aimed to investigate GJB3 mutations in people suffering from HL among three different ethnicities of Iranian population (Baloch, Kurd, and Turkmen). METHODS: In this descriptive study, 50 GJB2-negative non-syndromic hearing loss (NSHL) Iranian individuals from 3 ethnic groups of Baloch (n=17), Kurd (n =15) and Turkmen (n=18) were enrolled. DNA extractions, PCR, and mutation detection was carried out for the two large deletions of the GJB6, del (GJB6 -D13S1830,) and del (GJB6 -D13S1854) followed by direct DNA sequencing method for the GJB3. RESULTS: DNA sequencing of GJB3 was shown a missense heterozygous mutation rs199689484 (NM_024009.3) GJB3: c.340G>A (p.Ala114Thr) in a Baloch patient, and a polymorphism rs35983826 (NM_024009.3) GJB3: c.798C>T (p.Asn266=) in a Turkman patient, in coding region of the GJB3. We did not detect del (GJB6 -D13S1830) and del (GJB6 -D13S1854) among these three ethnicities in Iran. CONCLUSION: Deafness is a heterogeneous disorder. Specific genes and mutations contribute to hearing loss that varies from locus to locus as well as from population to population.

Low-volume hydrodynamic gene delivery to the rat liver via an isolated segment of the inferior vena cava: efficiency, cardiovascular response and intrahepatic vascular dynamics.

BACKGROUND: Clinical application of hydrodynamic gene delivery to the liver requires the use of small volumes, an evaluation of the cardiovascular consequences of acute volume overload, and a better understanding of the intrahepatic vascular pressures driving gene delivery. Injection of DNA solution into the isolated segment of inferior vena cava (IVC) draining the hepatic veins is a potentially valuable low-volume approach. METHODS: Various volumes of DNA solution (pGL3 plasmid) were injected at 100 ml/min either systemically or into the isolated IVC segment in the DA rat. Arterial pressure, portal venous pressure, heart rate and electrocardiogram, in addition to reporter gene expression in the liver, were monitored. RESULTS: The 2% volume was > 10 000-fold more effective when delivered via the IVC segment than when given systemically, and as effective as 6% systemically. Isolation of the IVC segment caused profound falls in arterial pressure, with electrocardiogram signs of myocardial ischemia. On release of the IVC ties, without DNA infusion (no volume overload), arterial pressure recovered rapidly. However, with DNA infusion (volume overload) there was a brief recovery of arterial pressure, followed by complete heart block and fall in arterial pressure and pulse for several minutes. Portal venous pressure rose steeply to 30-33 mm Hg during the infusion. CONCLUSIONS: The IVC segment approach enables excellent gene delivery to the whole liver with small volumes, but causes severe cardiovascular disturbances in the rat. Portal venous pressures are slightly higher than in the mouse, and suggest functional outflow obstruction by the capillary bed of the intestines.

An in silico argument for mitochondrial microRNA as a determinant of primary non function in liver transplantation.

Mitochondria have their own genomic, transcriptomic and proteomic machinery but are unable to be autonomous, needing both nuclear and mitochondrial genomes. The aim of this work was to use computational biology to explore the involvement of Mitochondrial microRNAs (MitomiRs) and their interactions with the mitochondrial proteome in a clinical model of primary non function (PNF) of the donor after cardiac death (DCD) liver. Archival array data on the differential expression of miRNA in DCD PNF was re-analyzed using a number of publically available computational algorithms. 10 MitomiRs were identified of importance in DCD PNF, 7 with predicted interaction of their seed sequence with the mitochondrial transcriptome that included both coding, and non coding areas of the hypervariability region 1 (HVR1) and control region. Considering miRNA regulation of the nuclear encoded mitochondrial proteome, 7 hypothetical small proteins were identified with homolog function that ranged from co-factor for formation of ATP Synthase, REDOX balance and an importin/exportin protein. In silico, unconventional seed interactions, both non canonical and alternative seed sites, appear to be of greater importance in MitomiR regulation of the mitochondrial genome. Additionally, a number of novel small proteins of relevance in transplantation have been identified which need further characterization.

The microRNA Expression Profile in Donation after Cardiac Death (DCD) Livers and Its Ability to Identify Primary Non Function.

Donation after cardiac death (DCD) livers are marginal organs for transplant and their use is associated with a higher risk of primary non function (PNF) or early graft dysfunction (EGD). The aim was to determine if microRNA (miRNA) was able to discriminate between DCD livers of varying clinical outcome. DCD groups were categorized as PNF retransplanted within a week (n=7), good functional outcome (n=7) peak aspartate transaminase (AST) ≤ 1000 IU/L and EGD (n=9) peak AST ≥ 2500 IU/L. miRNA was extracted from archival formalin fixed post-perfusion tru-cut liver biopsies. High throughput expression analysis was performed using miRNA arrays. Bioinformatics for expression data analysis was performed and validated with real time quantitative PCR (RT-qPCR). The function of miRNA of interest was investigated using computational biology prediction algorithms. From the array analysis 16 miRNAs were identified as significantly different (p<0.05). On RT-qPCR miR-155 and miR-940 had the highest expression across all three DCD clinical groups. Only one miRNA, miR-22, was validated with marginal significance, to have differential expression between the three groups (p=0.049). From computational biology miR-22 was predicted to affect signalling pathways that impact protein turnover, metabolism and apoptosis/cell cycle. In conclusion, microRNA expression patterns have a low diagnostic potential clinically in discriminating DCD liver quality and outcome.

Intestinal lactase as an autologous beta-galactosidase reporter gene for in vivo gene expression studies.

Intestinal lactase has potential as an autologous beta-galactosidase reporter gene for long-term gene expression studies in vivo, using chromogenic, luminescent, and fluorogenic substrates developed for Escherichia coli beta-galactosidase. In normal rat tissues, reactivity with a chromogenic fucopyranoside (X-Fuc, the preferred substrate of lactase) was present only at the lumenal surface of small intestine epithelial cells. Full-length lactase (domains I-IV), mature lactase (domains III and IV), and a cytosolic form of mature lactase (domains III and IV, without the signal sequence or transmembrane region) were evaluated. Transfection of HuH-7 cells in vitro, and hydrodynamic gene delivery to the liver in vivo, resulted in excellent gene expression. The full-length and mature (homodimeric, membrane-bound) forms reacted strongly with X-Fuc but not with the corresponding galactopyranoside (X-Gal). However, the presumptively monomeric cytosolic lactase unexpectedly reacted equally well with both substrates. The fluorogenic substrate fluorescein-di-beta-D-galactopyranoside was cleaved by cytosolic lactase, but not by full-length or mature lactase. Full-length lactase, when expressed ectopically in hepatocytes in vivo, localized exclusively to the bile canalicular membrane. Intestinal lactase is highly homologous in mice, rats, and humans and has considerable potential for evaluating long-term gene expression in experimental animals and the clinic.

Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways.

The thyroid target Ag for disease-inducing autoantibodies in Graves' disease is the receptor for thyroid-stimulating hormone (TSH), but little is known about the molecular basis of this pathogenic Ab response. We describe the characteristics of two high- affinity mAbs developed from an experimental murine model of hyperthyroid Graves' disease that exhibit potent thyroid-stimulating activity. Nanogram concentrations of the IgG mAbs KSAb1 and KSAb2 and their Fab induce full stimulation of the TSH receptor that is matched by the ligand TSH and, thus, act as full agonists for the receptor. However, KSAb1 and KSAb2 display differential activities in their ability to block TSH-mediated stimulation of the receptor, indicating subtle differences in their biological properties. In displacement studies, IgG and Fabs of KSAb1 and KSAb2 compete with Graves' disease autoantibodies as well as thyroid-blocking Abs present in some hypothyroid patients, indicating a close relationship between these autoimmune determinants on the receptor. In passive transfer studies, single injections of microgram quantities of KSAb1 or KSAb2 IgG led to rapid elevation of serum thyroxine and a hyperthyroid state that was maintained for a number of days. The thyroid glands showed evidence of cell necrosis, but there was no accompanying mononuclear cell infiltrate. In studying their receptor activation pathways, both KSAb1 and KSAb2 provoked phosphorylation of the intracellular ERK1/2 pathway in primary thyrocytes, indicating that multiple signaling pathways may participate in the pathogenesis of Graves' disease. In summary, our findings emphasize the similarities of the experimental mouse model in reproducing the human disorder and provide improved means for characterizing the molecular basis of this pathogenic response.