Interpreting elevated liver blood test results through a genetic lens: A genome-wide association study.

BACKGROUND AND AIMS: Individuals with genetic polymorphisms in UGT1A1 exhibit bilirubin levels that belie their risk of liver disease (Gilbert's syndrome) but it is not known if this phenomenon extends to other common liver blood tests (LBTs). METHODS: A genome-wide association analysis of 10 LBTs was conducted using the UK biobank. Polygenic scores (PGS) were created from discordant loci (e.g. loci associated with the LBT but not associated with cirrhosis morbidity risk). Participants were assigned to a low, intermediate or high PGS for each LBT. A high PGS approximates Gilbert's syndrome (i.e. elevated LBT without an analogous increase in disease risk). The prognostic significance of an 'elevated' LBT-and how this differs by PGS-was assessed through competing risk survival analysis. RESULTS: This study included 157 005 and 166 871 participants for the discovery and validation phases, respectively. Elevated LBTs were more prevalent in the high versus low PGS group, yet the 10-year risk of cirrhosis morbidity was comparable. For example, in the low PGS group, 4.3% had an elevated gamma-glutamyltransferase (GGT) and the 10-year risk of cirrhosis morbidity was .45%. Conversely, in the high PGS group, 21.2% had an elevated GGT and the 10-year risk was .38%. Accordingly, the 10-year risk of cirrhosis morbidity for individuals with an elevated GGT was markedly different in the low vs. high group (4.2% vs. 1.2%; p < .001). Similar results were apparent for Fibrosis-4 index, total bilirubin, and platelet count. CONCLUSION: Variability in LBTs is influenced by genetic polymorphisms that have a neutral effect on disease risk. These findings have implications for interpreting elevated LBTs in clinical practice.

Outcome prediction in metabolic dysfunction-associated steatotic liver disease using stain-free digital pathological assessment.

Computational quantification reduces observer-related variability in histological assessment of metabolic dysfunction-associated steatotic liver disease (MASLD). We undertook stain-free imaging using the SteatoSITE resource to generate tools directly predictive of clinical outcomes. Unstained liver biopsy sections (n = 452) were imaged using second-harmonic generation/two-photon excitation fluorescence (TPEF) microscopy, and all-cause mortality and hepatic decompensation indices constructed. The mortality index had greater predictive power for all-cause mortality (index >.14 vs. .31 vs.

Hepatic angiotensin-converting enzyme 2 expression in metabolic dysfunction-associated steatotic liver disease and in patients with fatal COVID-19.

BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD), characterised by hepatic lipid accumulation, causes inflammation and oxidative stress accompanied by cell damage and fibrosis. Liver injury (LI) is also frequently reported in patients hospitalised with coronavirus disease 2019 (COVID-19), while pre-existing MASLD increases the risk of LI and the development of COVID-19-associated cholangiopathy. Mechanisms of injury at the cellular level remain unclear, but it may be significant that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes COVID-19, uses angiotensin-converting expression enzyme 2 (ACE2), a key regulator of the 'anti-inflammatory' arm of the renin-angiotensin system, for viral attachment and host cell invasion. AIM: To determine if hepatic ACE2 levels are altered during progression of MASLD and in patients who died with severe COVID-19. METHODS: ACE2 protein levels and localisation, and histological fibrosis and lipid droplet accumulation as markers of MASLD were determined in formalin-fixed liver tissue sections across the MASLD pathological spectrum (isolated hepatocellular steatosis, metabolic dysfunction-associated steatohepatitis (MASH) +/- fibrosis, end-stage cirrhosis) and in post-mortem tissues from patients who had died with severe COVID-19, using ACE2 immunohistochemistry and haematoxylin and eosin and picrosirius red staining of total collagen and lipid droplet areas, followed by quantification using machine learning-based image pixel classifiers. RESULTS: ACE2 staining is primarily intracellular and concentrated in the cytoplasm of centrilobular hepatocytes and apical membranes of bile duct cholangiocytes. Strikingly, ACE2 protein levels are elevated in non-fibrotic MASH compared to healthy controls but not in the progression to MASH with fibrosis and in cirrhosis. ACE2 protein levels and histological fibrosis are not associated, but ACE2 and liver lipid droplet content are significantly correlated across the MASLD spectrum. Hepatic ACE2 levels are also increased in COVID-19 patients, especially those showing evidence of LI, but are not correlated with the presence of SARS-CoV-2 virus in the liver. However, there is a clear association between the hepatic lipid droplet content and the presence of the virus, suggesting a possible functional link. CONCLUSION: Hepatic ACE2 levels were elevated in nonfibrotic MASH and COVID-19 patients with LI, while lipid accumulation may promote intra-hepatic SARS-CoV-2 replication, accelerating MASLD progression and COVID-19-mediated liver damage.

Accurate prediction of all-cause mortality in patients with metabolic dysfunction-associated steatotic liver disease using electronic health records.

INTRODUCTION AND OBJECTIVES: Despite the huge clinical burden of MASLD, validated tools for early risk stratification are lacking, and heterogeneous disease expression and a highly variable rate of progression to clinical outcomes result in prognostic uncertainty. We aimed to investigate longitudinal electronic health record-based outcome prediction in MASLD using a state-of-the-art machine learning model. PATIENTS AND METHODS: n = 940 patients with histologically-defined MASLD were used to develop a deep-learning model for all-cause mortality prediction. Patient timelines, spanning 12 years, were fully-annotated with demographic/clinical characteristics, ICD-9 and -10 codes, blood test results, prescribing data, and secondary care activity. A Transformer neural network (TNN) was trained to output concomitant probabilities of 12-, 24-, and 36-month all-cause mortality. In-sample performance was assessed using 5-fold cross-validation. Out-of-sample performance was assessed in an independent set of n = 528 MASLD patients. RESULTS: In-sample model performance achieved AUROC curve 0.74-0.90 (95 % CI: 0.72-0.94), sensitivity 64 %-82 %, specificity 75 %-92 % and Positive Predictive Value (PPV) 94 %-98 %. Out-of-sample model validation had AUROC 0.70-0.86 (95 % CI: 0.67-0.90), sensitivity 69 %-70 %, specificity 96 %-97 % and PPV 75 %-77 %. Key predictive factors, identified using coefficients of determination, were age, presence of type 2 diabetes, and history of hospital admissions with length of stay >14 days. CONCLUSIONS: A TNN, applied to routinely-collected longitudinal electronic health records, achieved good performance in prediction of 12-, 24-, and 36-month all-cause mortality in patients with MASLD. Extrapolation of our technique to population-level data will enable scalable and accurate risk stratification to identify people most likely to benefit from anticipatory health care and personalized interventions.

Practical considerations for pathological diagnosis and molecular profiling of cholangiocarcinoma: an expert review for best practices.

INTRODUCTION: Advances in precision medicine have expanded access to targeted therapies and demand for molecular profiling of cholangiocarcinoma (CCA) patients in routine clinical practice. However, pathologists face challenges in establishing a definitive intrahepatic CCA (iCCA) diagnosis while preserving sufficient tissue for molecular profiling. Additionally, they frequently face challenges in optimal tissue handling to preserve nucleic acid integrity. AREAS COVERED: This article first identifies the challenges in establishing a definitive diagnosis of iCCA in a lesional liver biopsy while preserving sufficient tissue for molecular profiling. Then, the authors explore the clinical value of molecular profiling, the basic principles of single gene and next-generation sequencing (NGS) techniques, and the challenges in tissue sampling for genomic testing. They also propose an algorithm for best practice in tissue management for molecular profiling of CCA. EXPERT OPINION: Several practical challenges face pathologists during tissue sampling and processing for molecular profiling. Optimized tissue processing, careful tissue handling, and selection of appropriate approaches to molecular testing are essential to ensure that the highest possible quality of diagnostic information is provided in the greatest proportion of cases.

Recommendations on maximising the clinical value of tissue in the management of patients with intrahepatic cholangiocarcinoma.

Background & Aims: Patients with intrahepatic cholangiocarcinoma can now be managed with targeted therapies directed against specific molecular alterations. Consequently, tissue samples submitted to the pathology department must produce molecular information in addition to a diagnosis or, for resection specimens, staging information. The pathologist's role when evaluating these specimens has therefore changed to accommodate such personalised approaches. Methods: We developed recommendations and guidance for pathologists by conducting a systematic review of existing guidance to generate candidate statements followed by an international Delphi process. Fifty-nine pathologists from 28 countries in six continents rated statements mapped to all elements of the specimen pathway from receipt in the pathology department to authorisation of the final written report. A separate survey of 'end-users' of the report including surgeons, oncologists, and gastroenterologists was undertaken to evaluate what information should be included in the written report to enable appropriate patient management. Results: Forty-eight statements reached consensus for inclusion in the guidance including 10 statements about the content of the written report that also reached consensus by end-user participants. A reporting proforma to allow easy inclusion of the recommended data points was developed. Conclusions: These guiding principles and recommendations provide a framework to allow pathologists reporting on patients with intrahepatic cholangiocarcinoma to maximise the informational yield of specimens required for personalised patient management. Impact and Implications: Biopsy or resection lesional tissue from intrahepatic cholangiocarcinoma must yield information about the molecular abnormalities within the tumour that define suitability for personalised therapies in addition to a diagnosis and staging information. Here, we have developed international consensus guidance for pathologists that report such cases using a Delphi process that sought the views of both pathologists and 'end-users of pathology reports. The guide highlights the need to report cases in a way that preserves tissue for molecular testing and emphasises that reporting requires interpretation of histological characteristics within the broader clinical and radiological context. The guide will allow pathologists to report cases of intrahepatic cholangiocarcinoma in a uniform manner that maximises the value of the tissue received to facilitate optimal multidisciplinary patient management.

A prognostic molecular signature of hepatic steatosis is spatially heterogeneous and dynamic in human liver.

Metabolic dysfunction-associated steatotic liver disease (MASLD) prevalence is increasing in parallel with an obesity pandemic, calling for novel strategies for prevention and treatment. We defined a circulating proteome of human MASLD across ≈7000 proteins in ≈5000 individuals from diverse, at-risk populations across the metabolic health spectrum, demonstrating reproducible diagnostic performance and specifying both known and novel metabolic pathways relevant to MASLD (central carbon and amino acid metabolism, hepatocyte regeneration, inflammation, fibrosis, insulin sensitivity). A parsimonious proteomic signature of MASLD was associated with a protection from MASLD and its related multi-system metabolic consequences in >26000 free-living individuals, with an additive effect to polygenic risk. The MASLD proteome was encoded by genes that demonstrated transcriptional enrichment in liver, with spatial transcriptional activity in areas of steatosis in human liver biopsy and dynamicity for select targets in human liver across stages of steatosis. We replicated several top relations from proteomics and spatial tissue transcriptomics in a humanized "liver-on-a-chip" model of MASLD, highlighting the power of a full translational approach to discovery in MASLD. Collectively, these results underscore utility of blood-based proteomics as a dynamic "liquid biopsy" of human liver relevant to clinical biomarker and mechanistic applications.

A data-driven approach to decode metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD), defined by the presence of liver steatosis together with at least one out of five cardiometabolic factors, is the most common cause of chronic liver disease worldwide, affecting around one in three people. Yet the clinical presentation of MASLD and the risk of progression to cirrhosis and adverse clinical outcomes is highly variable. It, therefore, represents both a global public health threat and a precision medicine challenge. Artificial intelligence (AI) is being investigated in MASLD to develop reproducible, quantitative, and automated methods to enhance patient stratification and to discover new biomarkers and therapeutic targets in MASLD. This review details the different applications of AI and machine learning algorithms in MASLD, particularly in analyzing electronic health record, digital pathology, and imaging data. Additionally, it also describes how specific MASLD consortia are leveraging multimodal data sources to spark research breakthroughs in the field. Using a new national-level 'data commons' (SteatoSITE) as an exemplar, the opportunities, as well as the technical challenges of large-scale databases in MASLD research, are highlighted.

An integrated gene-to-outcome multimodal database for metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the commonest cause of chronic liver disease worldwide and represents an unmet precision medicine challenge. We established a retrospective national cohort of 940 histologically defined patients (55.4% men, 44.6% women; median body mass index 31.3; 32% with type 2 diabetes) covering the complete MASLD severity spectrum, and created a secure, searchable, open resource (SteatoSITE). In 668 cases and 39 controls, we generated hepatic bulk RNA sequencing data and performed differential gene expression and pathway analysis, including exploration of gender-specific differences. A web-based gene browser was also developed. We integrated histopathological assessments, transcriptomic data and 5.67 million days of time-stamped longitudinal electronic health record data to define disease-stage-specific gene expression signatures, pathogenic hepatic cell subpopulations and master regulator networks associated with adverse outcomes in MASLD. We constructed a 15-gene transcriptional risk score to predict future hepatic decompensation events (area under the receiver operating characteristic curve 0.86, 0.81 and 0.83 for 1-, 3- and 5-year risk, respectively). Additionally, thyroid hormone receptor beta regulon activity was identified as a critical suppressor of disease progression. SteatoSITE supports rational biomarker and drug development and facilitates precision medicine approaches for patients with MASLD.

British Society of Gastroenterology guidelines for the diagnosis and management of cholangiocarcinoma.

These guidelines for the diagnosis and management of cholangiocarcinoma (CCA) were commissioned by the British Society of Gastroenterology liver section. The guideline writing committee included a multidisciplinary team of experts from various specialties involved in the management of CCA, as well as patient/public representatives from AMMF (the Cholangiocarcinoma Charity) and PSC Support. Quality of evidence is presented using the Appraisal of Guidelines for Research and Evaluation (AGREE II) format. The recommendations arising are to be used as guidance rather than as a strict protocol-based reference, as the management of patients with CCA is often complex and always requires individual patient-centred considerations.

A TGFß-ECM-integrin signaling axis drives structural reconfiguration of the bile duct to promote polycystic liver disease.

The formation of multiple cysts in the liver occurs in a number of isolated monogenic diseases or multisystemic syndromes, during which bile ducts develop into fluid-filled biliary cysts. For patients with polycystic liver disease (PCLD), nonsurgical treatments are limited, and managing life-long abdominal swelling, pain, and increasing risk of cyst rupture and infection is common. We demonstrate here that loss of the primary cilium on postnatal biliary epithelial cells (via the deletion of the cilia gene Wdr35) drives ongoing pathological remodeling of the biliary tree, resulting in progressive cyst formation and growth. The development of cystic tissue requires the activation of transforming growth factor-ß (TGFß) signaling, which promotes the expression of a procystic, fibronectin-rich extracellular matrix and which itself is perceived by a changing profile of integrin receptors on the cystic epithelium. This signaling axis is conserved in liver cysts from patients with either autosomal dominant polycystic kidney disease or autosomal dominant polycystic liver disease, indicating that there are common cellular mechanisms for liver cyst growth regardless of the underlying genetic cause. Cyst number and size can be reduced by inhibiting TGFß signaling or integrin signaling in vivo. We suggest that our findings represent a therapeutic route for patients with polycystic liver disease, most of whom would not be amenable to surgery.

Antifibrotic therapy in nonalcoholic steatohepatitis: time for a human-centric approach.

Nonalcoholic steatohepatitis (NASH) might soon become the leading cause of end-stage liver disease and indication for liver transplantation worldwide. Fibrosis severity is the only histological predictor of liver-related morbidity and mortality in NASH identified to date. Moreover, fibrosis regression is associated with improved clinical outcomes. However, despite numerous clinical trials of plausible drug candidates, an approved antifibrotic therapy remains elusive. Increased understanding of NASH susceptibility and pathogenesis, emerging human multiomics profiling, integration of electronic health record data and modern pharmacology techniques hold enormous promise in delivering a paradigm shift in antifibrotic drug development in NASH. There is a strong rationale for drug combinations to boost efficacy, and precision medicine strategies targeting key genetic modifiers of NASH are emerging. In this Perspective, we discuss why antifibrotic effects observed in NASH pharmacotherapy trials have been underwhelming and outline potential approaches to improve the likelihood of future clinical success.

Single-nucleus RNA sequencing of pre-malignant liver reveals disease-associated hepatocyte state with HCC prognostic potential.

Current approaches to staging chronic liver diseases have limited utility for predicting liver cancer risk. Here, we employed single-nucleus RNA sequencing (snRNA-seq) to characterize the cellular microenvironment of healthy and pre-malignant livers using two distinct mouse models. Downstream analyses unraveled a previously uncharacterized disease-associated hepatocyte (daHep) transcriptional state. These cells were absent in healthy livers but increasingly prevalent as chronic liver disease progressed. Copy number variation (CNV) analysis of microdissected tissue demonstrated that daHep-enriched regions are riddled with structural variants, suggesting these cells represent a pre-malignant intermediary. Integrated analysis of three recent human snRNA-seq datasets confirmed the presence of a similar phenotype in human chronic liver disease and further supported its enhanced mutational burden. Importantly, we show that high daHep levels precede carcinogenesis and predict a higher risk of hepatocellular carcinoma development. These findings may change the way chronic liver disease patients are staged, surveilled, and risk stratified.

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling.

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.

Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

Large scale phenotype imputation and in vivo functional validation implicate ADAMTS14 as an adiposity gene.

Obesity remains an unmet global health burden. Detrimental anatomical distribution of body fat is a major driver of obesity-mediated mortality risk and is demonstrably heritable. However, our understanding of the full genetic contribution to human adiposity is incomplete, as few studies measure adiposity directly. To address this, we impute whole-body imaging adiposity phenotypes in UK Biobank from the 4,366 directly measured participants onto the rest of the cohort, greatly increasing our discovery power. Using these imputed phenotypes in 392,535 participants yielded hundreds of genome-wide significant associations, six of which replicate in independent cohorts. The leading causal gene candidate, ADAMTS14, is further investigated in a mouse knockout model. Concordant with the human association data, the Adamts14-/- mice exhibit reduced adiposity and weight-gain under obesogenic conditions, alongside an improved metabolic rate and health. Thus, we show that phenotypic imputation at scale offers deeper biological insights into the genetics of human adiposity that could lead to therapeutic targets.

Senolytic treatment preserves biliary regenerative capacity lost through cellular senescence during cold storage.

Liver transplantation is the only curative option for patients with end-stage liver disease. Despite improvements in surgical techniques, nonanastomotic strictures (characterized by the progressive loss of biliary tract architecture) continue to occur after liver transplantation, negatively affecting liver function and frequently leading to graft loss and retransplantation. To study the biological effects of organ preservation before liver transplantation, we generated murine models that recapitulate liver procurement and static cold storage. In these models, we explored the response of cholangiocytes and hepatocytes to cold storage, focusing on responses that affect liver regeneration, including DNA damage, apoptosis, and cellular senescence. We show that biliary senescence was induced during organ retrieval and exacerbated during static cold storage, resulting in impaired biliary regeneration. We identified decoy receptor 2 (DCR2)-dependent responses in cholangiocytes and hepatocytes, which differentially affected the outcome of those populations during cold storage. Moreover, CRISPR-mediated DCR2 knockdown in vitro increased cholangiocyte proliferation and decreased cellular senescence but had the opposite effect in hepatocytes. Using the p21KO model to inhibit senescence onset, we showed that biliary tract architecture was better preserved during cold storage. Similar results were achieved by administering senolytic ABT737 to mice before procurement. Last, we perfused senolytics into discarded human donor livers and showed that biliary architecture and regenerative capacities were better preserved. Our results indicate that cholangiocytes are susceptible to senescence and identify the use of senolytics and the combination of senotherapies and machine-perfusion preservation to prevent this phenotype and reduce the incidence of biliary injury after transplantation.

In vivo partial reprogramming by bacteria promotes adult liver organ growth without fibrosis and tumorigenesis.

Ideal therapies for regenerative medicine or healthy aging require healthy organ growth and rejuvenation, but no organ-level approach is currently available. Using Mycobacterium leprae (ML) with natural partial cellular reprogramming capacity and its animal host nine-banded armadillos, we present an evolutionarily refined model of adult liver growth and regeneration. In infected armadillos, ML reprogram the entire liver and significantly increase total liver/body weight ratio by increasing healthy liver lobules, including hepatocyte proliferation and proportionate expansion of vasculature, and biliary systems. ML-infected livers are microarchitecturally and functionally normal without damage, fibrosis, or tumorigenesis. Bacteria-induced reprogramming reactivates liver progenitor/developmental/fetal genes and upregulates growth-, metabolism-, and anti-aging-associated markers with minimal change in senescence and tumorigenic genes, suggesting bacterial hijacking of homeostatic, regeneration pathways to promote de novo organogenesis. This may facilitate the unraveling of endogenous pathways that effectively and safely re-engage liver organ growth, with broad therapeutic implications including organ regeneration and rejuvenation.

Role of Hepatocyte Senescence in the Activation of Hepatic Stellate Cells and Liver Fibrosis Progression.

Hepatocyte senescence is associated with liver fibrosis. However, the possibility of a direct, causal relation between hepatocyte senescence and hepatic stellate cell (HSC) activation was the subject of this study. Liver biopsy specimens obtained from 50 patients with non-alcoholic fatty liver disease and a spectrum of liver fibrosis stages were stained for p16, αSMA, and picrosirius red (PSR). Primary human HSCs were cultured in conditioned media derived from senescent or control HepG2 cells. Expression of inflammatory and fibrogenic genes in HSCs cultured in conditioned media were studied using RT-PCR. ELISAs were undertaken to measure factors known to activate HSCs in the conditioned media from senescent and control HepG2 cells and serum samples from healthy volunteers or patients with biopsy-proven cirrhosis. There was a strong association between proportion of senescent hepatocytes and hepatic stellate cell activation. Both proportion of hepatocyte senescence and hepatic stellate cell activation were closely associated with fibrosis stage. Inflammatory and fibrogenic genes were up-regulated significantly in HSCs cultured in conditioned media from senescent HepG2 cells compared with control HepG2 cells. PDGF levels were significantly higher in the conditioned media from senescent hepatocytes than control HepG2-conditioned media, and in serum samples from patients with cirrhosis than healthy volunteers. In conclusion, this 'proof of concept' study revealed activation of human HSCs by media from senescent HepG2 cells, indicating direct involvement of factors secreted by senescent hepatocytes in liver fibrosis.