Pereyra, David

Background and Aims: Liver fibrosis and cirrhosis are major global health burdens with limited treatments. Activated HSCs drive fibrosis through increased proliferation, migration, collagen deposition and glycolysis. The aim of this study is to elucidate the signaling events that lead to enhanced glycolysis, subsequent HSC activation, and liver fibrosis. Approach and Results: Utilizing a phospho-kinase array, we identified with no lysine kinase 1 (WNK1) as a new kinase in primary human HSCs activated by the profibrotic PDGF-B. PDGF-mediated glycolysis in HSCs was attenuated by the WNK1 selective inhibitor, WNK-IN-11, as measured by live-cell metabolic assay, Glifon-300 fluorescence, Glucose-Glo assay, and glucose transporter 1 immunofluorescence. To understand how WNK1 promotes glycolysis, we employed chemical genetics where an engineered WNK1 carrying threonine 301 to glycine mutation (WNK1 T301G ) mutant with a modified ATP pocket accommodated a specific ATP-γ-S analogue to thio-phosphorylate its substrates. Thio-phosphorylated proteins were analyzed by mass spectrometry, which identified the glycolytic protein, triosephosphate isomerase 1 (TPI1), as a novel WNK1 direct substrate. TPI1 interacted with WNK1 in condensates, membrane-less phase separation structures. Condensate disruption and knockdown of TPI1 or WNK1 reduced HSC proliferation and migration in vitro. In vivo, pharmacological or genetic WNK1 inhibition significantly attenuated liver fibrosis in carbon tetrachloride and high-fat, choline-deficient, L -amino acid diet mouse models. Finally, WNK1 inhibition reduced fibrogenesis ex vivo in human precision-cut liver slices. Conclusions: These findings identify WNK1 as a previously unrecognized kinase that promotes HSC activation and liver fibrosis via TPI1-mediated glycolysis, highlighting the WNK1-TPI1 axis as a potential therapeutic target.

Hepatic resection (HR) and liver transplantation (LT) are curative-intent treatments for hepatocellular carcinoma (HCC). Patients without cirrhosis are typically analyzed as a single group, obscuring potentially important differences between those with histologically normal liver and those with non-cirrhotic parenchymal disease. We aimed to characterize long-term outcomes following HR stratified by underlying parenchymal quality, with a contemporary LT cohort as an external benchmark. Retrospective cohort study of 662 adults undergoing curative-intent surgery for HCC at 2 tertiary centers (2010–2024). Resection patients (n=380) were stratified by non-tumor parenchymal histology into HR-Normal (n=84), HR-Diseased non-cirrhotic (n=159), and HR-Cirrhotic (n=137). A total of 282 LT recipients served as an external reference but were not entered into comparative models. Overall survival (OS) was analyzed by Kaplan–Meier and multivariable Cox regression, and recurrence by Fine–Gray competing-risks regression. Ninety-day mortality was low across resection strata (1.2%–3.2%). OS differed significantly across parenchymal groups (log-rank p =0.01), driven by superior outcomes in HR-Normal patients compared with both HR-Cirrhotic ( p =0.008) and HR-Diseased ( p =0.005). HR-Cirrhotic and HR-Diseased did not differ ( p =0.97). Five-year OS was 74.4%, 59.8% and 57.2%, respectively. On multivariable Cox regression, both cirrhosis (HR 2.03, 95% CI 1.16–3.54, p =0.01) and diseased non-cirrhotic parenchyma (HR 2.04, 95% CI 1.21–3.43, p =0.008) independently predicted death versus normal liver. Fine–Gray analysis confirmed higher recurrence risk in cirrhotic (sHR 2.09, p =0.01) and diseased non-cirrhotic (sHR 1.80, p =0.04) groups. The LT benchmark cohort achieved a 5-year OS of 82.4%. Histologically normal liver identifies a distinct HR subgroup with favorable oncologic outcomes. The conventional cirrhotic/non-cirrhotic dichotomy underestimates the prognostic impact of underlying parenchymal disease.

Abstract The human pancreas contains diverse endocrine and exocrine cell populations whose spatial organization is essential for tissue physiology. While single-cell and spatial transcriptomics revealed molecular heterogeneity across pancreatic cell types, linking these states to physiological activity in situ has remained challenging. Here, we combined single-nucleus RNA sequencing, spatial transcriptomics, and functional calcium imaging across pancreatic samples in health and metabolic disease. We identified heterogeneous endocrine and exocrine cell states associated with obesity and diabetes, including inflammatory remodeling of acinar and ductal populations. To directly couple tissue physiology with molecular state, we developed Slice-seq, which integrates calcium imaging with spatial transcriptomics in acute pancreatic slices. Slice-seq linked local endocrine composition and transcriptional programs with β cell activity and identified extra-islet β cells with reduced glucose responsiveness and mitochondrial oxidative metabolism. Together, our study provides a framework for linking pancreatic cell states to tissue organization and physiological activity in health and disease.

In metabolic dysfunction–associated steatohepatitis (MASH), liver sinusoidal endothelial cells (LSECs) acquire a proinflammatory phenotype termed lipotoxic endotheliopathy. We previously identified glycogen synthase kinase 3β (GSK3β) as a central signaling hub in LSECs during MASH. To elucidate the molecular mechanisms and functional outcome of lipotoxicity-induced GSK3β activation in LSECs, we utilized endothelial cell–specific Gsk3 β -KO ( Gsk3 β ΔEnd ) mice fed MASH-inducing diets. Endothelial Gsk3 β deletion significantly reduced markers of lipotoxic endotheliopathy, including adhesion molecules and chemokines, alongside liver injury, inflammation, and fibrosis. Immune profiling via flow cytometry and mass cytometry by time of flight (CyTOF) identified decreased hepatic infiltration of proinflammatory myeloid populations, particularly mature DCs in Gsk3 β ΔEnd mice. In a coculture system, GSK3β in lipotoxic LSECs promoted DCs maturation. Mechanistically, GSK3 inhibition restored lipotoxicity-induced alterations in LSEC mitochondrial morphology and respiration by regulating AMP-activated protein kinase and dynamin-related protein 1. This rescue suppressed chemokine and adhesion molecule expression, thereby limiting immune cell recruitment. Collectively, under lipotoxic stress, GSK3β amplifies mitochondrial dysfunction and inflammatory signaling in LSECs, enhancing myeloid cell homing and DC maturation. Targeting LSEC GSK3β may, therefore, represent a promising therapeutic strategy to mitigate LSEC-driven fibroinflammatory response in human MASH.

Background and Aims: Hypothermic oxygenated machine perfusion (HOPE) improves outcomes in orthotopic liver transplantation (OLT), but reliance on University of Wisconsin machine perfusion solution (UW-MPS) increases costs and logistical burden. Histidine-tryptophan-ketoglutarate (HTK) has potential as a single-solution alternative for HOPE. This study evaluated the safety and efficacy of HTK versus UW-MPS during HOPE. Methods: A retrospective, propensity score-matched cohort study including 46 patients who received donation after brain death (DBD) grafts that were preserved with HOPE at the Medical University of Vienna between May 2018 and October 2024 was conducted. A total of 23 patients received grafts perfused with HTK; another 23 patients transplanted with organs perfused with UW-MPS were matched based on recipient age and sodium model of end-stage liver disease score, donor age and sex, cold ischemia time, and perfusion time. Postoperative outcomes, perfusion parameters, and cost differences were assessed. Results: The HTK and UW-MPS cohorts demonstrated comparable perfusion dynamics and vascular resistance. While arterial pressure and flow were higher in the UW-MPS group, clinical outcomes—including early allograft dysfunction (47.8% each), ICU stay, and comprehensive complication index—were statistically similar. A trend toward fewer biliary complications (13.0% vs. 30.4%) and reduced hemodialysis requirement (17.4% vs. 30.4%) was observed in the HTK group. Use of HTK reduced perfusion-related costs by approximately EUR 560 per procedure. Conclusion: HTK is a viable alternative to UW-MPS during HOPE in OLT of DBD grafts, offering comparable short-term outcomes and relevant cost savings. Prospective studies are warranted to validate these findings and explore broader applications of single-solution perfusion strategies.

Portal hypertension is a key factor in posthepatectomy liver failure (PHLF). While preoperative liver function tests like APRI+ALBI assess liver function, they only partially reflect portal hypertension severity. Elevated von Willebrand factor antigen (vWF-Ag) indicates endothelial dysregulation and correlates with portal hypertension. Combining vWF-Ag with APRI+ALBI may enhance PHLF prediction. A total of 534 patients who underwent liver resection at Mayo Clinic Rochester (2020–2024) were analyzed for PHLF incidence, postoperative morbidity, and 90-day mortality. Predictive probability for PHLF was assessed using receiver operating characteristic analysis and validated in an external Austrian cohort of 283 patients (2008–2017). vWF-Ag was then integrated into the existing APRI+ALBI multivariable model. PHLF grade B/C was reported in 56 patients (10.5%). The 90-day mortality rate was 0.9% (n=5). Using 2 cutoffs (182%, 240%), vWF-Ag was found to be an independent predictive factor for PHLF grade B/C that remained statistically significant upon multivariable analysis. The combination of preoperative APRI+ALBI and vWF-Ag increased the positive predictive value to 27.9% for PHLF grade B/C in the APRI+ALBI high and vWF-Ag high group. Incorporation of vWF-Ag into the established APRI+ALBI-based multivariable model revealed a superior AUC of 0.772 for PHLF risk stratification and was successfully validated in an independent cohort (AUC=0.834). The combination of vWF-Ag with APRI+ALBI demonstrates a favorable predictive potential for PHLF risk assessment. We provide 2 pathways for clinical assessment: (1) a cutoff-based system and (2) a more complex multivariable model that can be calculated in a specifically designed smartphone application.

Background: Corroborating evidence for the use of hypothermic oxygenated machine perfusion (HOPE) prior to orthotopic liver transplantation (OLT) suggests a beneficial effect in regard to biliary complications. Here, the authors aim to evaluate whether perfusion via portal vein alone (sHOPE) or via additional perfusion of the hepatic artery (dHOPE) have diverging impact on outcomes after OLT when compared to the use of static cold storage (SCS). Methods: Consecutive patients undergoing OLT at the Medical University of Vienna (2018–2023) were retrospectively analyzed. Donor organs were procured using SCS, or subjected to end-ischemic sHOPE or dHOPE. The severity of biliary complications was classified according to the degree of therapeutic intervention (endoscopic retrograde cholangiopancreatography or surgical revision). Results: Two hundred forty-seven patients were included (69 SCS, 76 sHOPE, and 102 dHOPE). Hospitalization was shorter for patients after HOPE (median in days: SCS=25 vs HOPE=20, P=0.019). Biliary complications were less frequent in patients after HOPE (SCS=37.7% vs HOPE=22.5%, P=0.015). A significantly lower incidence of surgical revisions for biliary complications was observed in the HOPE cohort (24.6% vs 11.8%, P=0.012). When evaluating outcome according to HOPE-modality, a significant reduction in biliary complications (P=0.006) and surgical revisions (P=0.002) was only observed in dHOPE patients in comparison to SCS. Further, only dHOPE was significantly associated with a reduced need for surgical revision for biliary complications upon univariable and multivariable logistic regression (odds ratio=0.336, P=0.011). Conclusion: HOPE leads to a reduction of biliary complications and associated surgical revisions. This effect seems to be primarily associated with use of dHOPE, while both methods appear as feasible options for preconditioning of donor grafts prior to OLT.

Post-hepatectomy liver failure (PHLF) remains a significant risk for patients undergoing partial hepatectomy (PHx). Reliable prognostic markers and treatments to enhance liver regeneration are lacking. Plasma nanoparticles, including lipoproteins, exosomes, and extracellular vesicles (EVs), can reflect systemic and tissue-wide proteostasis and stress, potentially aiding liver regeneration. However, their role in PHLF is still unknown. Methods: Our study included nine patients with hepatocellular carcinoma (HCC) undergoing PHx: three patients with PHLF, three patients undergoing the associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure, and three matched controls without complications after PHx. Patient plasma was collected before PHx as well as 1 and 5 days after. EVs were isolated by ultracentrifugation, and extracted proteins were subjected to quantitative mass spectrometry using a super-SILAC mix prepared from primary and cancer cell lines. Results: We identified 2625 and quantified 2570 proteins in the EVs of PHx patients. Among these, 53 proteins were significantly upregulated and 32 were downregulated in patients with PHLF compared to those without PHLF. Furthermore, 110 proteins were upregulated and 78 were downregulated in PHLF patients compared to those undergoing ALPPS. The EV proteomic signature in PHLF indicates significant disruptions in protein translation, proteostasis, and intracellular vesicle biogenesis, as well as alterations in proteins involved in extracellular matrix (ECM) remodelling and the metabolic and cell cycle pathways, already present before PHx. Conclusions: Longitudinal proteomic analysis of the EVs circulating in the plasma of human patients undergoing PHx uncovers proteomic signatures associated with PHLF, which reflect dying hepatocytes and endothelial cells and were already present before PHx.

Liver resection (LR) is the primary treatment for hepatic tumors, yet posthepatectomy liver failure (PHLF) remains a significant concern. While the precise etiology of PHLF remains elusive, dysregulated inflammatory processes are pivotal. Therefore, we explored the theragnostic potential of extracellular high-mobility-group-box protein 1 (HMGB1), a key damage-associated molecular pattern (DAMP) released by hepatocytes, in liver recovery post LR in patients and animal models. Plasma from 96 LR patients and liver tissues from a subset of 24 LR patients were analyzed for HMGB1 levels, and associations with PHLF and liver injury markers were assessed. In a murine LR model, the HMGB1 inhibitor glycyrrhizin, was administered to assess its impact on liver regeneration. Furthermore, plasma levels of keratin-18 (K18) and cleaved cytokeratin-18 (ccK18) were quantified to assess suitability as predictive biomarkers for PHLF. Patients experiencing PHLF exhibited elevated levels of intrahepatic and circulating HMGB1, correlating with markers of liver injury. In a murine LR model, inhibition of HMGB1 improved liver function, reduced steatosis, enhanced regeneration and decreased hepatic cell death. Elevated levels of hepatic cell death markers K18 and ccK18 were detected in patients with PHLF and correlations with levels of circulating HMGB1 was observed. Our study underscores the therapeutic and predictive potential of HMGB1 in PHLF mitigation. Elevated HMGB1, K18, and ccK18 levels correlate with patient outcomes, highlighting their predictive significance. Targeting HMGB1 enhances liver regeneration in murine LR models, emphasizing its role in potential intervention and prediction strategies for liver surgery.