Multi-omics analysis reveals a crosstalk between ferroptosis and peroxisomes on steatotic graft failure after liver transplantation.

To identify the mechanism underlying macrosteatosis (MaS)-related graft failure (GF) in liver transplantation (LT) by multi-omics network analysis. The transcriptome and metabolome were assayed in graft and recipient plasma in discovery (n = 68) and validation (n = 89) cohorts. Differentially expressed molecules were identified by MaS and GF status. Transcriptional regulatory networks were generated to explore the mechanism for MaS-related inferior post-transplant prognosis. The differentially expressed molecules associated with MaS and GF were enriched in ferroptosis and peroxisome-related pathways. Core features of MaS-related GF were presented on decreased transferrin and impaired anti-oxidative capacity dependent upon dysregulation of transcription factors hepatocyte nuclear factor 4A (HNF4A) and hypoxia-inducible factor 1A (HIF1A). Furthermore, miR-362-3p and miR-299-5p inhibited transferrin and HIF1A expression, respectively. Lower M2 macrophages but higher memory CD4 T cells were observed in MaS-related GF cases. These results were validated in clinical specimens and cellular models. Systemic analysis of multi-omics data depicted a panorama of biological pathways deregulated in MaS-related GF. Transcriptional regulatory networks centered on transferrin and anti-oxidant responses were associated with poor MaS graft quality, qualifying as potential targets to improve prognosis of patients after LT.

Multi-omics network analysis on samples from sequential biopsies reveals vital role of proliferation arrest for Macrosteatosis related graft failure in rats after liver transplantation.

To investigate the molecular impact of graft MaS on post-transplant prognosis, based on multi-omics integrative analysis. Rats were fed by methionine-choline deficient diet (MCD) for MaS grafts. Samples were collected from grafts by sequential biopsies. Transcriptomic and metabolomic profilings were assayed. Post-transplant MaS status showed a close association with graft failure. Differentially expressed genes (DEGs) for in-vivo MaS were mainly enriched on pathways of cell cycle and DNA replication. Post-transplant MaS caused arrests of graft regeneration via inhibiting the E2F1 centered network, which was confirmed by an in vitro experiment. Data from metabolomics assays found insufficient serine/creatine which is located on one­carbon metabolism was responsible for MaS-related GF. Pre-transplant MaS caused severe fibrosis in long-term survivors. DEGs for grafts from long-term survivors with pre-transplant MaS were mainly enriched in pathways of ECM-receptor interaction and focal adhesion. Transcriptional regulatory network analysis confirmed SOX9 as a key transcription factor (TF) for MaS-related fibrosis. Metabolomic assays found elevation of aromatic amino acid (AAA) was a major feature of fibrosis in long-term survivors. Graft MaS in vivo increased post-transplant GF via negative regulations on graft regeneration. Pre-transplant MaS induced severe fibrosis in long-term survivors via activations on ECM-receptor interaction and AAA metabolism.

Recent Progress and Future Direction for the Application of Multiomics Data in Clinical Liver Transplantation.

Omics data address key issues in liver transplantation (LT) as the most effective therapeutic means for end-stage liver disease. The purpose of this study was to review the current application and future direction for omics in LT. We reviewed the use of multiomics to elucidate the pathogenesis leading to LT and prognostication. Future directions with respect to the use of omics in LT are also described based on perspectives of surgeons with experience in omics. Significant molecules were identified and summarized based on omics, with a focus on post-transplant liver fibrosis, early allograft dysfunction, tumor recurrence, and graft failure. We emphasized the importance omics for clinicians who perform LTs and prioritized the directions that should be established. We also outlined the ideal workflow for omics in LT. In step with advances in technology, the quality of omics data can be guaranteed using an improved algorithm at a lower price. Concerns should be addressed on the translational value of omics for better therapeutic effects in patients undergoing LT.

Graft-to-recipient weight ratio exerts nonlinear effects on prognosis by interacting with donor liver macrosteatosis.

Aim: To investigate the interactions between the graft-to-recipient weight ratio (GWRWR) and other risk factors responsible for inferior allograft outcomes. Methods: A total of 362 patients who received liver transplantation (LT) were enrolled. Indicators such as graft/recipient weight and other prognostic factors were collected. Comparisons of indicators and survival analysis were performed in groups categorized by the GWRWR. Interactions of large-for-size grafts (LFSGs) with graft macrosteatosis (MaS) were evaluated in terms of relative excess risk caused by interaction (RERI) and attributable proportion (AP). Cytoscape visualized the role of LFSGs in the risk profile for poor prognosis. Results: Based on the GWRWR, LT cases can be categorized into three subgroups, standard (1%-2.5%), optimal (2.5%-3.0%), and inferior prognosis (>3.0%). Survival analysis confirmed clear separations in cases categorized by the above-defined limits on the GWRWR (P < 0.05). LFSGs caused inferior prognosis by initiating positive interactions with MaS severity. Conclusion: The GWRWR exerted nonlinear effects on prognosis in deceased donor LT cases. LFSGs (GWRWR > 3.0%) caused inferior outcomes, while grafts sized within (2.5%-3.0%) had optimal post-transplant prognosis. MaS increased the risk of poor prognosis by exerting positive synergistic effects on LFSGs.

Integrative Network Analysis Revealed Genetic Impact of Pyruvate Kinase L/R on Hepatocyte Proliferation and Graft Survival after Liver Transplantation.

BACKGROUND: Pyruvate kinase L/R (PKLR) has been suggested to affect the proliferation of hepatocytes via regulation of the cell cycle and lipid metabolism. However, its impact on the global metabolome and its clinical implications remain unclear. AIMS: We aimed to clarify the genetic impact of PKLR on the metabolomic profiles of hepatoma cells and its potential effects on grafts for liver transplantation (LT). METHODS: Nontargeted and targeted metabolomic assays were performed in human hepatoma cells transfected with lentiviral vectors causing PKLR overexpression and silencing, respectively. We then constructed a molecular network based on integrative analysis of transcriptomic and metabolomic data. We also assessed the biological functions of PKLR in the global metabolome in LT grafts in patients via a weighted correlation network model. RESULTS: Multiomic analysis revealed that PKLR perturbations significantly affected the pyruvate, citrate, and glycerophospholipid metabolism pathways, as crucial steps in de novo lipogenesis (DNL). We also confirmed the importance of phosphatidylcholines (PC) and its derivative lyso-PC supply on improved survival of LT grafts in patients. Coexpression analysis revealed beneficial effects of PKLR overexpression on posttransplant prognosis by alleviating arachidonic acid metabolism of the grafts, independent of operational risk factors. CONCLUSION: This systems-level analysis indicated that PKLR affected hepatoma cell viability via impacts on the whole process of DNL, from glycolysis to final PC synthesis. PKLR also improved prognosis after LT, possibly via its impact on the increased genesis of beneficial glycerophospholipids.

Systematic Assessment of Safety Threshold for Donor Age in Cadaveric Liver Transplantation.

Background: Donor age affects allograft quality and the prognosis of recipients after liver transplantation (LT). Clinicians have assessed the quality of grafts from older donors based on their appearance and texture, with no reliable quantitative evidence. Our study aimed to assess the quantitative impact of donor age on post-transplant outcomes and its safety threshold for LT, based on the published literature. Methods: Relevant studies were retrieved from the Embase, PubMed, and ISI Web of Science databases. Pooled dichotomous relative risks (RRs) were calculated using metan. Continuous RRs were calculated using a two-stage random-effects model. Results: Eleven studies including 30,691 LT cases were included for further analysis. For categorical comparison, the RR of death within the first post-transplant year was significantly higher among patients who received grafts from older donors. Similarly, the RR of graft failure (GF) was increased within the 3 years after transplantation. For continuous comparison, advanced donor age affected transplant outcomes in a linear manner (P > 0.05). A 10-year increment in donor age was associated with RRs 1.10, 1.12, 1.15, 1.10, and 1.08 for 90-day, 180-day, 1-year, 3-year, and 5-year patient mortality and 1.08, 1.06, 1.10, 1.11, and 1.12, for 90-day, 180-day, 1-year, 2-year, and 3-year GF, respectively (all P < 0.05). A spline model showed that transplants using grafts from donors <43 years old were not associated with age-related risks (P > 0.05). The risk of GF was increased in subgroups with fewer LT cases, longer cold ischemic time, fewer male donors, and recipients with viral hepatitis (P < 0.05). Conclusion: Donor age might affect post-LT outcomes in a dose-dependent manner. The safety threshold for donor age in terms of GF should be lowered to 43 years as an early warning for the guarantee of satisfactory outcomes. Clinicians should weigh the benefits against the risks carefully for patients receiving grafts from older donors. Further studies are warranted to investigate the mechanisms responsible for the relationship between donor age and graft quality.

Clear mortality gap caused by graft macrosteatosis in Chinese patients after cadaveric liver transplantation.

BACKGROUND: Liver transplantation (LT) is one of the most effective surgical treatment for patients with end-stage liver disease. Steatosis is a contributor for inferior graft quality. But its impact and safety on transplantation was less assessed in Chinese patients. METHODS: Graft steatosis and related information involved in recipients, donors and surgical procedures were retrospectively collected from 239 patients. RESULTS: Donor macrosteatosis (MaS) caused about 2.14 and 2.80 folds of increment on patient and graft mortality. Dose-response analysis revealed prominent risk of grafts on overall patient/organ mortality when MaS content exceeded 10% (P<0.05). Noteworthy, deaths were only observed in MaS group when concurrent with extremely higher post-transplant alanine aminotransferase (ALT, 64%). However, microsteatosis (MiS) grafts didn't affect outcomes after LT. In a cohort of Chinese patients, MaS had comprehensive effects on post-transplant outcomes with relatively lower safety threshold at 10%. Mortality gap caused by MaS grafts was observed in patients with severer ischemia reperfusion injury. CONCLUSIONS: Our study revealled the graft MaS affected the post-transplant outcomes in lower risk cutoff in Chinese patients. Further study is worthy to validate these results and investigate inner mechanism under the phenomenon.

Metabonomic Profile of Macrosteatotic Allografts for Orthotopic Liver Transplantation in Patients With Initial Poor Function: Mechanistic Investigation and Prognostic Prediction.

BACKGROUND: Our previous study revealled amplified hazardous effects of macrosteatosis (MaS) on graft failure (GF) in recipients with severe liver damage in short post-operative days, with vague mechanism inside. AIM: We aimed to uncover the molecular mechanism of donor MaS on GF, and construct the predictive model to monitor post-transplant prognosis based on "omics" perspective. METHODS: Ultra-performance liquid chromatography coupled to mass spectrometry metabolomic analysis was performed in allograft tissues from 82 patients with initial poor function (IPF) from multi-liver transplant (LT) centers. Pathway analysis was performed by on-line toolkit Metaboanalyst (v 3.0). Predictive model was constructed based on combinative metabonomic and clinical data extracted by stepwised cox proportional analysis. RESULTS: Principle component analysis (PCA) analysis revealled stratification on metabolic feature in organs classified by MaS status. Differential metabolits both associated with MaS and GF were significantly enriched on pathway of glycerophospholipid metabolism (P < 0.05). Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) involved in glycerophospholipid metabolism was significantly decreased in cases with MaS donors and GF (P < 0.05). Better prediction was observed on graft survival by combinative model (area under the curve = 0.91) and confirmed by internal validation. CONCLUSION: Metabonomic features of allografts can be clearly distinguished by MaS status in patients with IPF. Dysfunction on glycerophospholipid metabolism was culprit to link donor MaS and final GF. Decrement on PC and PE exerted the fatal effects of MaS on organ failure. Metabonomic data might help for monitoring long-term graft survival after LT.

Systematic Evaluation of the Safety Threshold for Allograft Macrovesicular Steatosis in Cadaveric Liver Transplantation.

Background: Currently, 30% macrovesicular steatosis (MaS) content is usually assigned empirically as the boundary between "use" and "refuse" a donor liver for liver transplantation (LT); however, this cut-off is questionable due to the lack of systemic evidence of the efficiency relative to prognosis prediction. Clinicians have tried to identify the threshold for optimized utilization of marginal steatotic allografts, but controversy exists among different studies. Aim: Our study aimed to systematically determine an acceptable donor MaS content cut-off without incurring extra risk in liver transplantation, using meta-analysis. Methods: The relevant literature reporting the relationship between MaS content and post-transplant mortality/morbidity was searched and retrieved in Pubmed, Embase, and ISI Web of Science. Results: Nine studies were enrolled into the final analysis. A categorical comparison revealed that patients who received allografts with moderate steatosis (MaS content >30%) had significantly higher risks of graft failure/dysfunction, but not of mortality. Dose-response analysis showed that donor MaS content affected the graft failure/dysfunction in a non-linear relationship. Risks associated with MaS content in terms of poorer outcomes were independent of other risk covariates for liver transplantation. A non-significant increase in risk of inferior post-transplant outcomes was observed in patients who received allografts with a MaS content <35%. The risks of post-transplant graft failure and dysfunction increased with severe donor MaS content infiltration, without a consistent relationship. Conclusions: The threshold of allograft MaS content can be safely extended to 35% without additional risk burden on post-transplant inferior outcomes. Clarification on "the effects of stratification" for MaS content can provide theoretical evidence for further optimal utilization of marginal steatotic allografts in liver transplantation.

Meta-Analysis of Adiponectin as a Biomarker for the Detection of Metabolic Syndrome.

Previous studies revealed the potential significance of circulating adiponectin levels with respect to the diagnosis and prediction of metabolic syndrome, but uncertainty has been noted across different cohorts. Systematic evaluation was performed for diagnostic accuracy and predictivity of adiponectin variation for metabolic syndrome in enrolled studies including 1,248 and 6,020 subjects, respectively. Adiponectin can identify metabolic syndrome with moderate accuracy (area under the curve = 0.81, 95% CI: 0.77-0.84). Heterogeneity analysis revealed that an increasing index of insulin resistance was independently associated with improving the performance of adiponectin upon metabolic syndrome diagnosis (ratio of diagnostic odds ratio = 3.89, 95% CI: 1.13-13.9). In addition, reductions in adiponectin were associated with increasing metabolic syndrome incidence in a linear dose-response manner. The risk of hypoadiponectinemia with metabolic syndrome was especially increased in men (P < 0.05). Further Mendelian randomization analysis identified that the amplified risk could be attributed to increased susceptibility (up to 7%) to insulin resistance compared with women. In conclusion, adiponectin measurement might have potential benefits in the detection of metabolic syndrome. Factors that affect insulin resistance should be considered for adjustment in future assessments.

The effect of the TM6SF2 E167K variant on liver steatosis and fibrosis in patients with chronic hepatitis C: a meta-analysis.

The impact of Transmembrane 6 superfamily member 2 (TM6SF2) E167K variant, which causes hepatocellular fat retention by altering lipoprotein secretion, on liver damage and metabolic traits in chronic hepatitis C patients is still debated. We performed a systematic review and meta-analysis to clarify this relationship. Four studies with a total of 4325 patients were included. The risk of histologically-determined advanced steatosis, fibrosis, and cirrhosis (but not of severe inflammation) were increased in carriers of the TM6SF2 variant (P < 0.05). Unlike the inconsistent association with steatosis severity, due to the confounding effect of infection by the genotype-3 hepatitis C virus, the TM6SF2 variant was robustly associated with advanced fibrosis (OR = 1.07; 95% confidence interval [CI] = 1.01-1.14) and in particular with cirrhosis (OR = 2.05; 95% CI = 1.39-3.02). Regarding metabolic features, individuals positive for the TM6SF2 variant exhibited 5.8-12.0% lower levels of circulating triglycerides and non-HDL cholesterol (P < 0.05). Carriers of the variant were leaner, but there was high heterogeneity across studies (I2 = 97.2%). No significant association was observed between the TM6SF2 variant and insulin resistance or hepatitis C viral load (both P > 0.05). In conclusion, the TM6SF2 E167K variant promotes the development of steatosis, fibrosis and cirrhosis in patients with chronic hepatitis C. Conversely, this variant reduces circulating atherogenic lipid fractions.

Dose-response Relationship of Serum Uric Acid with Metabolic Syndrome and Non-alcoholic Fatty Liver Disease Incidence: A Meta-analysis of Prospective Studies.

Emerging evidence has shown that serum uric acid (SUA) elevation might cause metabolic derangements, including metabolic syndrome (MetS) and non-alcoholic fatty liver disease (NAFLD); however, magnitude of the risk has not been quantified. We searched PubMed, EMBASE, and ISI databases for relevant studies through 10 May 2015. Prospective studies reporting the risk of SUA elevation on the incidence of MetS/NAFLD were enrolled. Pooled HR of MetS was 1.55 (95%CI: 1.40-1.70) for the highest versus lowest SUA categories, and 1.05 (95%CI: 1.04-1.07) per incremental increased in SUA of 1 mg/dl. The pooled HR of MetS in younger women was higher than age-matched men and older women (1.17 vs. 1.05 and 1.04, respectively, P < 0.05). Individuals in the highest SUA category had a 40% greater risk of disease NAFLD occurrence. Dose-response increment of NAFLD events was 1.03 (95%CI: 1.02-1.05). A positive relationship with a linear trend for SUA elevation with MetS and NAFLD in different genders was examined by a dose-response meta-analysis (P < 0.001).SUA assay is useful in screening metabolic disorders for linear trend between its elevation and MetS/NAFLD incidence. SUA-lowering therapy is a potential strategy for preventing systemic/hepatic metabolic abnormalities.

Meta-analysis reveals a specific association of the PNPLA3 I148M polymorphism with ALT level in adolescents.

AIM: To evaluate potential link between the PNPLA3 rs738409 polymorphism and alanine aminotransferase (ALT) levels through an evidence-based study. MATERIALS & METHODS: Electronic literature databases, including PubMed, Embase and the Institute for Scientific Information, were searched for relevant studies. Pooling standardized mean differences for quantitative variables and summary odds ratios (OR) were respectively calculated using per-allele comparison. RESULTS: Although a genotype-phenotype association was inconsistent in adults, this genetic effect was stable in adolescents. There was an approximate increase of 23% in ALT value, and 1.99-fold higher ALT elevation per risk allele increase with low heterogeneity. CONCLUSION: The PNPLA3 rs738409 polymorphism can have a differentiated influence on ALT level. Our meta-analysis provides reference data for the adjustment of diverse susceptibility due to the rs738409 polymorphism when evaluating liver injury in various populations.

Alanine aminotransferase-old biomarker and new concept: a review.

Measurement of serum alanine aminotransferase (ALT) is a common, readily available, and inexpensive laboratory assay in clinical practice. ALT activity is not only measured to detect liver disease, but also to monitor overall health. ALT activity is influenced by various factors, including viral hepatitis, alcohol consumption, and medication. Recently, the impact of metabolic abnormalities on ALT variation has raised concern due to the worldwide obesity epidemic. The normal ranges for ALT have been updated and validated considering the metabolic covariates in the various ethnic districts. The interaction between metabolic and demographic factors on ALT variation has also been discussed in previous studies. In addition, an extremely low ALT value might reflect the process of aging, and frailty in older adults has been raised as another clinically significant feature of this enzyme, to be followed with additional epidemiologic investigation. Timely updated, comprehensive, and systematic introduction of ALT activity is necessary to aid clinicians make better use of this enzyme.

Complex association between alanine aminotransferase activity and mortality in general population: a systematic review and meta-analysis of prospective studies.

OBJECTIVE: Controversy exists in using alanine aminotransferase (ALT) activity for predicting long-term survival. Therefore, this research study investigated the association between ALT activity and mortality through a systematic review and meta-analysis of previous prospective studies. METHODS: Electronic literature databases, including PubMed, Embase, and the Institute for Scientific Information (ISI), were searched for relevant prospective observational studies (published before Dec 30, 2013) on the association between baseline ALT activity and ensuing all-cause/disease-specific mortality. Information on nationality, sample size, participant characteristics, follow-up duration, comparison, outcome assessment, hazard ratios (HRs) and adjusted covariates was extracted. Pooled HRs and corresponding 95% confidence intervals (CIs) were separately calculated for categorical risk estimates (highest vs. lowest ALT categories) and continuous risk estimates (per 5 U/l of ALT increment) in subgroups separated by age (<70/≥ 70 years). RESULTS: A total of twelve prospective cohort studies, totaling 206,678 participants and 16,249 deaths, were identified and analyzed. In the younger age group, the pooled HR for mortality related to liver-disease was about 1.24 (95% CI: 1.23-1.25) per 5 U/l of ALT increment. The dose-response HRs of all-cause mortality, cardiovascular (CV) disease-related mortality, and cancer-related mortality were 0.91 (0.88-0.94), 0.91 (0.85-0.96), 0.92 (0.86-0.98) respectively per 5 U/l of ALT elevation, with insignificant heterogeneity in the older population. There was an approximate decrease of 4‰ observed on HRs of all-cause, CV-related, and cancer-related mortality followed with one year's increment through meta-regression (all P<0.05). CONCLUSIONS: The ALT-mortality association was inconsistent and seems particularly susceptible to age after synthesizing the previous prospective studies. In terms of the age, ALT activity was more valuable in predicting mortality in the older population; extremely low ALT levels indicated a higher all-cause, CV-related, and cancer-related mortality. ALT activity may therefore be a useful biomarker when predicting the long-term survival of elderly patients.

Elevated alanine aminotransferase is strongly associated with incident metabolic syndrome: a meta-analysis of prospective studies.

BACKGROUND: The incidence of metabolic syndrome (MetS) is rapidly increasing worldwide and associated with alanine aminotransferase (ALT) activity. However, the impact of ALT activity on MetS incidence is inconsistent in published literature. We therefore estimated the association between elevated ALT activity and incident MetS through a meta-analysis of prospective cohort studies. METHODS/PRINCIPAL FINDINGS: All published prospective cohort studies on the association between elevated ALT activity and incident MetS were retrieved from Pubmed, Embase, and the Institute for Scientific Information (ISI). In all, seven prospective cohort studies, with 31545 participants and 2873 cases of incident MetS were recruited. If there was insignificant heterogeneity (P-value>0.05 and I(2)<50%), the fixed-effect model was used to calculate the pooled relative risks (RRs) of incident MetS induced by raised ALT. Otherwise, the random-effect model was used. The calculated RR was 1.81 (95% confidence interval [CI]: 1.49-2.14) when the incidence of MetS was compared between the highest versus the lowest classification of ALT activities. The pooled RR was 1.13 (95% CI: 1.11-1.16) in dose-response analysis with 5 units per liter (U/l) of ALT increment. Subgroup analysis suggested that gender disparity might be the main origin of heterogeneity in overall analysis (P = 0.007 between RRs of gender-specific subgroups evaluated with 5 U/l increments of ALT). Women had a higher dose-response risk of MetS incidence (1.38, 95% CI: 1.20-1.55) than men. Furthermore, sensitivity analysis confirmed the stability of results. No publication bias was found in our meta-analysis. CONCLUSIONS/SIGNIFICANCE: Current evidence from prospective studies supports the association between ALT elevation and increasing MetS incidence. This association is closer and more consistent in female population. Further studies are needed to confirm this association and to investigate the potential mechanism of ALT activity on MetS occurrence.