Interleaved trinuclear MRS for single-session investigation of carbohydrate and lipid metabolism in human liver at 7T.

The liver plays a central role in metabolic homeostasis, as exemplified by a variety of clinical disorders with hepatic and systemic metabolic disarrays. Of particular interest are the complex interactions between lipid and carbohydrate metabolism in highly prevalent conditions such as obesity, diabetes, and fatty liver disease. Limited accessibility and the need for invasive procedures challenge direct investigations in humans. Hence, noninvasive dynamic evaluations of glycolytic flux and steady-state assessments of lipid levels and composition are crucial for basic understanding and may open new avenues toward novel therapeutic targets. Here, three different MR spectroscopy (MRS) techniques that have been combined in a single interleaved examination in a 7T MR scanner are evaluated. 1 H-MRS and 13 C-MRS probe endogenous metabolites, while deuterium metabolic imaging (DMI) relies on administration of deuterated tracers, currently 2 H-labelled glucose, to map the spatial and temporal evolution of their metabolic fate. All three techniques have been optimized for a robust single-session clinical investigation and applied in a preliminary study of healthy subjects. The use of a triple-channel 1 H/2 H/13 C RF coil enables interleaved examinations with no need for repositioning. Short-echo-time STEAM spectroscopy provides well resolved spectra to quantify lipid content and composition. The relative benefits of using water saturation versus metabolite cycling and types of respiratory synchronization were evaluated. 2 H-MR spectroscopic imaging allowed for registration of time- and space-resolved glucose levels following oral ingestion of 2 H-glucose, while natural abundance 13 C-MRS of glycogen provides a dynamic measure of hepatic glucose storage. For DMI and 13 C-MRS, the measurement precision of the method was estimated to be about 0.2 and about 16 mM, respectively, for 5 min scanning periods. Excellent results were shown for the determination of dynamic uptake of glucose with DMI and lipid profiles with 1 H-MRS, while the determination of changes in glycogen levels by 13 C-MRS is also feasible but somewhat more limited by signal-to-noise ratio.

Risk of liver-related events in metabolic dysfunction-associated steatohepatitis (MASH) patients with fibrosis: A comparative analysis of various risk stratification criteria.

BACKGROUND AND AIMS: International regulatory agencies recommend testing drug therapy for patients with noncirrhotic high-risk metabolic dysfunction-associated steatohepatitis (MASH) because they are at risk of liver-related events (LRE). We aimed to compare the risk of LRE in patients with MASLD stratified for F2-F4 fibrosis and MASH. APPROACH AND RESULTS: Overall, 1938 consecutive patients with biopsy-proven MASLD were enrolled. High-risk MASH was defined as MASH with F2-F4 fibrosis. LSM was measured by transient elastography. LRE were recorded during follow-up. Cox multivariate models were used to assess the association between high-risk MASH or F2-F4 fibrosis without MASH, of LSM (≥8 or ≥10 Kpa), and of AGILE 3+ with LRE. The diagnostic performance for the prediction of LRE was assessed using the area under the receiver operating characteristic curves. The observed 5-year actuarial rate of LRE was 0.4%, 0.2%, 5.1%, and 6.6% in patients with F0-F1 fibrosis without MASH, F0-F1 fibrosis with MASH, F2-F4 fibrosis without MASH, and high-risk MASH, respectively. At multivariate Cox regression analysis using F0-F1 fibrosis without MASH as a reference, both F2-F4 fibrosis without MASH [adjusted HR (aHR) 9.96] and high-risk MASH (aHR 10.14) were associated with LRE. In the 1074 patients with available LSM, LSM ≥ 10 kPa (aHR 6.31) or AGILE 3+ > 0.67 (aHR 27.45) independently predicted the development of LRE and had similarly acceptable 5-year area under the receiver operating characteristic to high-risk MASH and F2-F4 fibrosis (0.772, 0.818, 0.739, and 0.780, respectively). CONCLUSIONS: The risk of LRE is similar in patients with high-risk MASH and with F2-F4 fibrosis without MASH. The use of LSM ≥ 10 kPa or AGILE 3+ > 0.67 could be an accurate option to identify patients with MASLD worthy to be included in clinical trials.

Validation of the Blood Test MACK-3 for the Noninvasive Diagnosis of Fibrotic Nonalcoholic Steatohepatitis: An International Study With 1924 Patients.

BACKGROUND & AIMS: Drug development in nonalcoholic steatohepatitis (NASH) is hampered by a high screening failure rate that reaches 60% to 80% in therapeutic trials, mainly because of the absence of fibrotic NASH on baseline liver histology. MACK-3, a blood test including 3 biomarkers (aspartate aminotransferase, homeostasis model assessment, and cytokeratin 18), recently was developed for the noninvasive diagnosis of fibrotic NASH. We aimed to validate the diagnostic accuracy of this noninvasive test in an international multicenter study. METHODS: A total of 1924 patients with biopsy-proven nonalcoholic fatty liver disease from 10 centers in Asia, Australia, and Europe were included. The blood test MACK-3 was calculated for all patients. FibroScan-aspartate aminotransferase score (FAST), an elastography-based test for fibrotic NASH, also was available in a subset of 655 patients. Fibrotic NASH was defined as the presence of NASH on liver biopsy with a Nonalcoholic Fatty Liver Disease Activity Score of 4 or higher and fibrosis stage of F2 or higher according to the NASH Clinical Research Network scoring system. RESULTS: The area under the receiver operating characteristic of MACK-3 for fibrotic NASH was 0.791 (95% CI 0.768-0.814). Sensitivity at the previously published MACK-3 threshold of less than 0.135 was 91% and specificity at a greater than 0.549 threshold was 85%. The MACK-3 area under the receiver operating characteristic was not affected by age, sex, diabetes, or body mass index. MACK-3 and FAST results were well correlated (Spearman correlation coefficient, 0.781; P < .001). Except for an 8% higher rate of patients included in the grey zone, MACK-3 provided similar accuracy to that of FAST. Both tests included 27% of patients in their rule-in zone, with 85% specificity and 35% false positives (screen failure rate). CONCLUSIONS: The blood test MACK-3 is an accurate tool to improve patient selection in NASH therapeutic trials.

Hepatocyte apoptosis fragment product cytokeratin-18 M30 level and non-alcoholic steatohepatitis risk diagnosis: an international registry study.

BACKGROUND: Liver biopsy for the diagnosis of non-alcoholic steatohepatitis (NASH) is limited by its inherent invasiveness and possible sampling errors. Some studies have shown that cytokeratin-18 (CK-18) concentrations may be useful in diagnosing NASH, but results across studies have been inconsistent. We aimed to identify the utility of CK-18 M30 concentrations as an alternative to liver biopsy for non-invasive identification of NASH. METHODS: Individual data were collected from 14 registry centers on patients with biopsy-proven non-alcoholic fatty liver disease (NAFLD), and in all patients, circulating CK-18 M30 levels were measured. Individuals with a NAFLD activity score (NAS) ≥5 with a score of ≥1 for each of steatosis, ballooning, and lobular inflammation were diagnosed as having definite NASH; individuals with a NAS ≤2 and no fibrosis were diagnosed as having non-alcoholic fatty liver (NAFL). RESULTS: A total of 2571 participants were screened, and 1008 (153 with NAFL and 855 with NASH) were finally enrolled. Median CK-18 M30 levels were higher in patients with NASH than in those with NAFL (mean difference 177 U/L; standardized mean difference [SMD]: 0.87 [0.69-1.04]). There was an interaction between CK-18 M30 levels and serum alanine aminotransferase, body mass index (BMI), and hypertension ( P  < 0.001, P  = 0.026 and P  = 0.049, respectively). CK-18 M30 levels were positively associated with histological NAS in most centers. The area under the receiver operating characteristics (AUROC) for NASH was 0.750 (95% confidence intervals: 0.714-0.787), and CK-18 M30 at Youden's index maximum was 275.7 U/L. Both sensitivity (55% [52%-59%]) and positive predictive value (59%) were not ideal. CONCLUSION: This large multicenter registry study shows that CK-18 M30 measurement in isolation is of limited value for non-invasively diagnosing NASH.

2D shear wave elastography of the rectus femoris muscle in patients with cirrhosis: Feasibility and clinical findings. A pilot study.

BACKGROUND AND AIMS: Frailty is frequent in cirrhosis and associated with skeletal muscle abnormalities and worse prognosis. 2D shear-wave elastography (2D-SWE) could mirror biomechanical properties of skeletal muscle reflecting muscle quality. However, there is no data on 2D-SWE on skeletal muscle stiffness assessment in cirrhosis and on frailty. METHODS: Outpatients with cirrhosis were prospectively included in a single center. Skeletal muscle stiffness was studied at the rectus femoris by 2D-SWE. Ileo-psoas area and index (area/height2), and antero-posterior diameter of rectus femoris (RF) was measured on ultrasound. RESULTS: We included 44 patients (24 male, age 59 [IQR 49-66]) with a median liver frailty index (LFI) of 3.7 (IQR 3.2-4.0). Measurement of RF muscle stiffness (RFMS) was feasible in all with high inter-measurement reproducibility. RFMS did not correlate with LFI, liver function and skeletal muscle diameters. Ileo-psoas index was lower in frail patients (1.7 vs 1.0 cm2/m2, p = 0.024). RF antero-posterior diameter inversely correlated with LFI (r -0.578: p<0.001). CONCLUSION: RFMS by 2D-SWE is feasible and reproducible in cirrhosis and is independent of liver function and LFI, and warrants further studies in this setting. RF antero-posterior diameter could be reported as an objective parameter mirroring sarcopenia and frailty.

Non-invasive tools for compensated advanced chronic liver disease and portal hypertension after Baveno VII - an update.

Non-invasive tests (NITs) and liver stiffness measurement (LSM) in particular, have entered clinical practice over 20 years ago as point-of-care tests to diagnose liver fibrosis in patients with compensated chronic liver disease. Since then, NITs use has evolved thanks to a large number of studies in all major etiologies of liver disease, and they have become important tools to stratify the risk of portal hypertension and liver-related events. The Baveno VII consensus workshop provided several novel recommendations regarding the use of well-established and novel NITs in the specific setting of portal hypertension screening, diagnosis and follow-up. The Baveno VII expert panels paid special attention to summarizing the existing data into simple clinical rules able to guide clinicians in their practice. The "rule of five" for LSM is a tool to stratify the risk of liver-related events, and LSM alone or in combination with platelet count, can be used now to rule-in and rule-out compensated advanced chronic liver disease (cACLD) and clinically significant portal hypertension, as well as to rule-out high-risk varices. Use of NITs in obese subjects with non-alcoholic fatty liver disease (NAFLD) and patients with viral hepatitis C that has been successfully treated, require specific knowledge. This review will update the reader on these aspects.

Endoplasmic Reticulum and Mitochondria Contacts Correlate with the Presence and Severity of NASH in Humans.

The interaction between the mitochondria and the endoplasmic reticulum (ER) is essential for hepatocyte function. An increase in ER-mitochondria contacts (ERMCs) is associated with various metabolic diseases. Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and type 2 diabetes, and its progressive form non-alcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma. However, the role of ERMCs in the progression of NAFL to NASH is still unclear. We assessed whether ERMCs could correlate with NAFLD severity. We used a proximity ligation assay to measure the abundance of ERMCs in liver biopsies from patients with biopsy-proven NAFLD (n = 48) and correlated the results with histological and metabolic syndrome (MetS) features. NAFLD patients were included according to inclusion and exclusion criteria, and then assigned to NAFL (n = 9) and NASH (n = 39) groups. ERMCs density could discriminate NASH from NAFL (sensitivity 61.5%, specificity 100%). ERMCs abundance correlated with hepatocellular ballooning. Moreover, the density of ERMCs increased with an increase in the number of MetS features. In conclusion, ERMCs increased from NAFL to NASH, in parallel with the number of MetS features, supporting a role for this interaction in the pathophysiology of NASH.

PPAR-Targeted Therapies in the Treatment of Non-Alcoholic Fatty Liver Disease in Diabetic Patients.

Peroxisome proliferator-activated receptors (PPAR), ligand-activated transcription factors of the nuclear hormone receptor superfamily, have been identified as key metabolic regulators in the liver, skeletal muscle, and adipose tissue, among others. As a leading cause of liver disease worldwide, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) cause a significant burden worldwide and therapeutic strategies are needed. This review provides an overview of the evidence on PPAR-targeted treatment of NAFLD and NASH in individuals with type 2 diabetes mellitus. We considered current evidence from clinical trials and observational studies as well as the impact of treatment on comorbid metabolic conditions such as obesity, dyslipidemia, and cardiovascular disease. Future areas of research, such as possible sexually dimorphic effects of PPAR-targeted therapies, are briefly reviewed.

Inflammatory activity affects the accuracy of liver stiffness measurement by transient elastography but not by two-dimensional shear wave elastography in non-alcoholic fatty liver disease.

BACKGROUND: In patients with non-alcoholic fatty liver disease (NAFLD), the impact of the severity of steatosis and inflammatory activity on the accuracy of liver stiffness measurement (LSM) by transient elastography (TE) and by two-dimensional shear wave elastography (2D-SWE) in staging liver fibrosis is still debated and scarce. We aimed to focus on this aspect. METHODS: We prospectively studied 104 patients requiring biopsy for the assessment of NAFLD. We used ordinary least squares regression to test for differences in the association between fibrosis and LSM by TE and 2D-SWE when other factors (steatosis and inflammatory activity) are considered. RESULTS: Among 104 patients, 102 had reliable LSM by TE, and 88 had valid LSM by 2D-SWE. The association between fibrosis based on histology and LSM was significantly stronger when 2D-SWE assessed LSM compared to TE (Spearman's correlation coefficient of .71; P < .001 vs .51, P < .001; Z = 2.21, P = .027). Inflammatory activity was an independent predictor of LSM by TE but not of LSM by 2D-SWE. After controlling for fibrosis, age, sex and body mass index, the inflammatory activity and the interaction between inflammatory activity and fibrosis independently explained 11% and 13% of variance in LSM by TE respectively. Steatosis did not affect the association of fibrosis and LSM by either method. CONCLUSION: Inflammatory activity on histology significantly affects LSM by TE, but not LSM by 2D-SWE in NAFLD. LSM by 2D-SWE reflects liver fibrosis more accurately than LSM by TE. Furthermore, the severity of steatosis on histology did not influence the association of LSM and fibrosis by either elastography method.

Prevention of NAFLD-associated HCC: Role of lifestyle and chemoprevention.

In many countries worldwide, the burden of hepatocellular carcinoma (HCC) associated with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is increasing. Preventive strategies are needed to counteract this trend. In this review, we provide an overview of the evidence on preventive strategies in NAFLD-associated HCC. We consider the impact of lifestyle factors such as weight loss, physical activity, smoking, dietary patterns and food items, including coffee and alcohol, on both HCC and NAFLD/NASH. Furthermore, evidence on chemopreventive treatments, including aspirin, antidiabetic treatments and statins is summarised. The role of adjuvant therapies for tertiary prevention of HCC is briefly reviewed.

High subcutaneous adipose tissue density correlates negatively with survival in patients with hepatocellular carcinoma.

BACKGROUND & AIMS: Body composition parameters have been reported to add information, which can lead to tailored treatment and prognostication for oncological patients. Data for patients with hepatocellular carcinoma (HCC) are scarce. We assessed the association between different body composition parameters and overall survival (OS) in two different newly diagnosed HCC populations. METHODS: The area (cm2 ) and density (Hounsfield Units [HU]) of skeletal muscle (SM) and adipose tissue (subcutaneous [SAT], visceral [VAT] and intermuscular [IMAT]) were measured on computed tomography (CT) scans at the level of the third lumbar vertebra (L3) in two cohorts of patients diagnosed in different HCC stages (Bern, Switzerland n = 187 and Newcastle, United Kingdom n = 216). Univariate and multivariate Cox regressions analyses were used to assess the crude and adjusted association of body composition parameters with OS. RESULTS: By univariate analysis, in both cohorts, Bern and Newcastle, high SAT density (hazard ratio [HR]: 1.35; 1.12-1.62, P < .001 and 1.44; 1.27-1.63, P < .001, respectively) and high VAT density (HR: 1.38; 1.1-1.72, P = .005 and HR: 1.53; 1.3-1.81, P < .001, respectively) correlated negatively with survival. After model adjustment for potential baseline confounders (gender, age, diabetes, cirrhosis, MELD score, BCLC stage) in a multivariate analysis, SAT density remained associated with mortality in Bern and Newcastle (Bern: HR: 1.27; 1.04-1.57, P = .022; Newcastle: HR: 1.23; 1.03-1.48, P = .022) and VAT remained associated with mortality in Bern (HR: 1.31; 1.05-1.65, P = .019). CONCLUSIONS: Based on two HCC cohorts, our data show that high SAT density correlates negatively with OS in HCC patients.

Changing Epidemiology of HCC: How to Screen and Identify Patients at Risk?

The prognosis of patients with hepatocellular carcinoma is dependent on the stage of tumor at diagnosis. The earlier the tumor is found, the higher the chances to offer a curative treatment. In order to diagnose hepatocellular carcinoma early, patients at risk should be enrolled in a surveillance program. The population at risk is usually defined as patients with cirrhosis. These patients should have twice a year a ultrasonographic examination of the liver. However, more and more patients will develop hepatocellular carcinoma in the context of nonalcoholic fatty liver disease which is tightly linked to obesity and diabetes. In these patients, this approach is jeopardized by the difficulty to perform a sonography of good quality due to the obesity and more importantly by the fact that hepatocellular carcinoma occurs frequently in the context of nonalcoholic fatty liver disease before the cirrhosis. This article reviews the impact of the changing epidemiology of hepatocellular carcinoma on its screening.

Anticoagulation quality and clinical outcomes in multimorbid elderly patients with acute venous thromboembolism.

BACKGROUND: Multimorbid patients with acute venous thromboembolism (VTE) are often excluded from clinical trials and little is known about their prognosis. OBJECTIVES: To examine whether multimorbidity is associated with adverse clinical outcomes and lower anticoagulation quality in older patients with VTE. PATIENTS/METHODS: We studied 991 patients aged ≥65 years with acute VTE in a Swiss prospective multicenter cohort study. A modified Charlson Comorbidity Index was used to measure multimorbidity, which was defined as the presence ≥2 of 17 predefined comorbid conditions. We examined the association between multimorbidity and recurrent VTE and major bleeding, adjusting for confounders and periods of anticoagulation. We assessed whether the percentage of time spent in the therapeutic international normalized ratio (INR) range varied by the number of comorbidities present. RESULTS: Overall, 708 (71%) patients were multimorbid. Multimorbid patients had a higher 3-year cumulative incidence of recurrent VTE (16.8 vs. 10.8%; P = 0.056) and major bleeding (18.7 vs. 9.0%; P = 0.001) than non-multimorbid patients. After adjustment, multimorbid patients had a significantly higher risk of recurrent VTE (sub-hazard ratio [SHR] 1.66, 95% confidence interval [CI] 1.08-2.57) and a higher risk of major bleeding (SHR 1.55, 95% CI 0.96-2.50), although the latter failed to achieve statistical significance. With increasing numbers of comorbid conditions, patients spent less time in and more time above and below the therapeutic INR range. CONCLUSIONS: Multimorbid patients with acute VTE have not only a lower anticoagulation quality but also more complications. Clinical trials should explicitly enroll multimorbid patients to determine the optimal anticoagulation strategy in such patients.

Loss of the alpha7 integrin promotes extracellular signal-regulated kinase activation and altered vascular remodeling.

Vascular smooth muscle cell (VSMC) proliferation and migration are underlying factors in the development and progression of cardiovascular disease. Studies have shown that altered expression of vascular integrins and extracellular matrix proteins may contribute to the vascular remodeling observed after arterial injury and during disease. We have recently shown that loss of the alpha7beta1 integrin results in VSMC hyperplasia. To investigate the cellular mechanisms underlying this phenotype, we have examined changes in cell signaling pathways associated with VSMC proliferation. Several studies have demonstrated the mitogen-activated protein kinase signaling pathway is activated in response to vascular injury and disease. In this study, we show that loss of the alpha7 integrin in VSMCs results in activation of the extracellular signal-regulated kinase and translocation of the activated kinase to the nucleus. Forced expression of the alpha7 integrin or use of the mitogen-activated protein kinase kinase 1 inhibitor U0126 in alpha7 integrin-deficient VSMCs suppressed extracellular signal-regulated kinase activation and restored the differentiated phenotype to alpha7 integrin-null cells in a manner dependent on Ras signaling. Alpha7 integrin-null mice displayed profound vascular remodeling in response to injury with pronounced neointimal formation and reduced vascular compliance. These findings demonstrate that the alpha7beta1 integrin negatively regulates extracellular signal-regulated kinase activation and suggests an important role for this integrin as part of a signaling complex regulating VSMC phenotype switching.