Periportal Hepatocytes Research Articles

Nodular regenerative hyperplasia: The role of the CK7 immunohistochemistry pattern of expression in diagnosis.

Nodular regenerative hyperplasia (NRH) is a rare vascular disorder of the liver. Clinically, patients present with portal hypertension with or without a cholestatic pattern of injury. Histologically, the liver parenchyma is composed of small nodules of hypertrophic hepatocytes surrounded by atrophic hepatocytes without significant fibrosis. Nodular regenerative hyperplasia is a difficult diagnosis on biopsy specimens, but biopsy remains the gold standard for diagnosis. In this retrospective review, cytokeratin 7 (CK7) immunohistochemistry (IHC) was used to aid in the diagnosis and further characterization of NRH and NRH-like changes. The H&E-stained slides, reticulin, and CK IHC were reviewed for 22 cases. The percentage of hepatocytes staining for CK7 (0%-100%), the location of staining (centrilobular hepatic progenitor cells vs periportal/bile ductular reaction), and the pattern of staining distribution (patchy or diffuse) were recorded for comparison. Of the 22 cases, 9 were CK7 positive. Cases of NRH, however, expressed various degrees of CK7 positivity in centrilobular hepatic progenitor cells, unlike NRH-like changes, which were either CK7 negative or CK7 positive in periportal hepatocytes or in areas of bile ductular reaction. In cases with the appropriate clinical history and histology, CK7 immunohistochemistrycan be performed to distinguish nodular regenerativehyperplasia (primary) and NRH-like changes (secondary). In difficult cases, CK7 positivity in centrilobular hepatic progenitor cells can help confirm the diagnosis of NRH.These data support NRH as a true entity with a distinct pathophysiology from NRH-like changes.

Single-Cell Transcriptomic Analysis Revealed the Cell Population Changes and Cell–Cell Communication in the Liver of a Carnivorous Fish in Response to High-Carbohydrate Diet

BackgroundCarnivorous fish have a low carbohydrate utilization ability, and the physiologic and molecular basis of glucose intolerance has not been fully illustrated. ObjectivesThis study aimed to use largemouth bass as a model to investigate the possible mechanism of glucose intolerance in carnivorous fish with the help of single-nuclei RNA sequencing (snRNA-seq). MethodsTwo diets were formulated, a low-carbohydrate (LC) diet and a high-carbohydrate (HC) diet. The feeding trial lasted for 6 wk, and then, growth performance, biochemical parameters, liver histology, and snRNA-seq were performed. ResultsGrowth performance of fish was not affected by the HC diet, while liver glucolipid metabolism disorder and liver injury were observed. A total of 13,247 and 12,848 cells from the liver derived from 2 groups were isolated and sequenced, and 7 major liver cell types were annotated by the marker genes. Hepatocytes and cholangiocytes were lower and hepatic stellate cells (HSCs) and immune cells were higher in the HC group than those in the LC group. Reclustering analysis identified 7 subtypes of hepatocytes and immune cells, respectively. The HSCs showed more cell communication with other cell types, and periportal hepatocytes showed more cell communication with other hepatocyte subtypes. Cell–cell communication mainly focused on cell junction–related signaling pathways. Uncovered by the pseudotime analysis, midzonal hepatocytes were differentiated into 2 major branches—biliary epithelial hepatocytes and hepatobiliary hybrid progenitor. Cell junction and liver fibrosis–related genes were highly expressed in the HC group. HC diet induced the activation of HSCs and, therefore, led to the liver fibrosis of largemouth bass. ConclusionsHC diet induces liver glucolipid metabolism disorder and liver injury of largemouth bass. The increase and activation of HSCs might be the main reason for the liver injury. In adaption to HC diet, midzonal hepatocytes differentiates into 2 major branches—biliary epithelial hepatocytes and hepatobiliary hybrid progenitors.

Deconvolution analysis identified altered hepatic cell landscape in primary sclerosing cholangitis and primary biliary cholangitis.

Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are characterized by ductular reaction, hepatic inflammation, and liver fibrosis. Hepatic cells are heterogeneous, and functional roles of different hepatic cell phenotypes are still not defined in the pathophysiology of cholangiopathies. Cell deconvolution analysis estimates cell fractions of different cell phenotypes in bulk transcriptome data, and CIBERSORTx is a powerful deconvolution method to estimate cell composition in microarray data. CIBERSORTx performs estimation based on the reference file, which is referred to as signature matrix, and allows users to create custom signature matrix to identify specific phenotypes. In the current study, we created two custom signature matrices using two single cell RNA sequencing data of hepatic cells and performed deconvolution for bulk microarray data of liver tissues including PSC and PBC patients. Custom signature matrix files were created using single-cell RNA sequencing data downloaded from GSE185477 and GSE115469. Custom signature matrices were validated for their deconvolution performance using validation data sets. Cell composition of each hepatic cell phenotype in the liver, which was identified in custom signature matrices, was calculated by CIBERSORTx and bulk RNA sequencing data of GSE159676. Deconvolution results were validated by analyzing marker expression for the cell phenotype in GSE159676 data. CIBERSORTx and custom signature matrices showed comprehensive performance in estimation of population of various hepatic cell phenotypes. We identified increased population of large cholangiocytes in PSC and PBC livers, which is in agreement with previous studies referred to as ductular reaction, supporting the effectiveness and reliability of deconvolution analysis in this study. Interestingly, we identified decreased population of small cholangiocytes, periportal hepatocytes, and interzonal hepatocytes in PSC and PBC liver tissues compared to healthy livers. Although further studies are required to elucidate the roles of these hepatic cell phenotypes in cholestatic liver injury, our approach provides important implications that cell functions may differ depending on phenotypes, even in the same cell type during liver injury. Deconvolution analysis using CIBERSORTx could provide a novel approach for studies of specific hepatic cell phenotypes in liver diseases.

Spatial mapping of hepatic ER and mitochondria architecture reveals zonated remodeling in fasting and obesity

The hepatocytes within the liver present an immense capacity to adapt to changes in nutrient availability. Here, by using high resolution volume electron microscopy, we map how hepatic subcellular spatial organization is regulated during nutritional fluctuations and as a function of liver zonation. We identify that fasting leads to remodeling of endoplasmic reticulum (ER) architecture in hepatocytes, characterized by the induction of single rough ER sheet around the mitochondria, which becomes larger and flatter. These alterations are enriched in periportal and mid-lobular hepatocytes but not in pericentral hepatocytes. Gain- and loss-of-function in vivo models demonstrate that the Ribosome receptor binding protein1 (RRBP1) is required to enable fasting-induced ER sheet-mitochondria interactions and to regulate hepatic fatty acid oxidation. Endogenous RRBP1 is enriched around periportal and mid-lobular regions of the liver. In obesity, ER-mitochondria interactions are distinct and fasting fails to induce rough ER sheet-mitochondrion interactions. These findings illustrate the importance of a regulated molecular architecture for hepatocyte metabolic flexibility.

Spatial transcriptomics unravels palmitoylation and zonation-dependent gene regulation by AEG-1 in mouse liver

Obesity-induced metabolic dysfunction-associated steatohepatitis (MASH) leads to hepatocellular carcinoma (HCC). Astrocyte-elevated gene-1/Metadherin (AEG-1/MTDH) plays a key role in promoting MASH and HCC. AEG-1 is palmitoylated at residue cysteine 75 (Cys75) and a knock-in mouse representing mutated Cys75 to serine (AEG-1-C75S) showed activation of MASH- and HCC-promoting gene signature when compared to wild-type littermates (AEG-1-WT). The liver consists of 3 zones, periportal, midlobular and pericentral, and zone-specific dysregulated gene expression impairs metabolic homeostasis in the liver, contributing to MASH and HCC. Here, to elucidate how palmitoylation influences AEG-1-mediated gene regulation in regards to hepatic zonation, we performed spatial transcriptomics (ST) in the livers of AEG-1-WT and AEG-1-C75S littermates. ST identified six different clusters in livers and using zone- and cell type-specific markers we attributed specific zones and cell types to specific clusters. Ingenuity Pathway Analysis (IPA) of differentially expressed genes in each cluster unraveled activation of pro-inflammatory and MASH- and HCC-promoting pathways, mainly in periportal and pericentral hepatocytes, in AEG-1-C75S liver compared to AEG-1-WT. Interestingly, in AEG-1-C75S liver, the midlobular zone exhibited widespread inhibition of xenobiotic metabolism pathways and inhibition of PXR/RXR and LXR/RXR activation, vs AEG-1-WT. In conclusion, AEG-1-C75S mutant exhibited zone-specific differential gene expression, which might contribute to metabolic dysfunction and dysregulated drug metabolism leading to MASH and HCC.

A spatiotemporal atlas of cholestatic injury and repair in mice.

Cholestatic liver injuries, characterized by regional damage around the bile ductular region, lack curative therapies and cause considerable mortality. Here we generated a high-definition spatiotemporal atlas of gene expression during cholestatic injury and repair in mice by integrating spatial enhanced resolution omics sequencing and single-cell transcriptomics. Spatiotemporal analyses revealed a key role of cholangiocyte-driven signaling correlating with the periportal damage-repair response. Cholangiocytes express genes related to recruitment and differentiation of lipid-associated macrophages, which generate feedback signals enhancing ductular reaction. Moreover, cholangiocytes express high TGFβ in association with the conversion of liver progenitor-like cells into cholangiocytes during injury and the dampened proliferation of periportal hepatocytes during recovery. Notably, Atoh8 restricts hepatocyte proliferation during 3,5-diethoxycarbonyl-1,4-dihydro-collidin damage and is quickly downregulated after injury withdrawal, allowing hepatocytes to respond to growth signals. Our findings lay a keystone for in-depth studies of cellular dynamics and molecular mechanisms of cholestatic injuries, which may further develop into therapies for cholangiopathies.

Region-specific cellular and molecular basis of liver regeneration after acute pericentral injury

Liver injuries often occur in a zonated manner. However, detailed regenerative responses to such zonal injuries at cellular and molecular levels remain largely elusive. By using a fate-mapping strain, Cyp2e1-DreER, to elucidate liver regeneration after acute pericentral injury, we found that pericentral regeneration is primarily compensated by the expansion of remaining pericentral hepatocytes, and secondarily by expansion of periportal hepatocytes. Employing single-cell RNA sequencing, spatial transcriptomics, immunostaining, and invivo functional assays, we demonstrated that the upregulated expression of the mTOR/4E-BP1 axis and lactate dehydrogenase A in hepatocytes contributes to pericentral regeneration, while activation of transforming growth factor β (TGF-β1) signaling in the damaged area mediates fibrotic responses and inhibits hepatocyte proliferation. Inhibiting the pericentral accumulation of monocytes and monocyte-derived macrophages through an Arg-Gly-Asp (RGD) peptide-based strategy attenuates these cell-derived TGF-β1 signalings, thus improving pericentral regeneration. Our study provides integrated and high-resolution spatiotemporal insights into the cellular and molecular basis of pericentral regeneration.

Citrus limon (L.) Osbeck Fruit Peel Extract Attenuates Carbon Tetrachloride-Induced Hepatocarcinogenesis in Sprague-Dawley Rats.

Traditional herbal medicine practitioners in the Ashanti region of Ghana use the fruit peels of Citrus limon (L.) Osbeck (C. limon) in preventive and curative treatment of many cancers including liver cancer. This ethnobotanical claim remains to be verified scientifically. Aim of the Study. This study investigated prophylactic hepatoprotective and anti-HCC effects of C. limon peel extract (LPE) in CCl4/olive oil-induced HCC-like rats. After preparation of LPE, it was subjected to phytochemical screening using standard phytochemical methods. A total of 30 healthy adult male Sprague-Dawley rats (weighing 150-200 g) were randomly assigned into six groups of 5 rats each. Rats in the control group received olive oil (5 mL/kg ip) twice weekly for 16 weeks. Rats in the model group received CCl4/olive oil (2 mL/kg, ip) twice weekly for 16 weeks. Rats in capecitabine (10 mg/kg po) and LPE (50, 100, and 200 mg/kg po) groups received CCl4/olive oil (2 mL/kg, i.p) in the morning and their respective treatments in the afternoon twice a week for 16 weeks. Rats in all groups had free access to food and water ad libitum. Body weight and survival rates were monitored. Rats were sacrificed under deep anesthesia, blood was collected, and liver and other organs were isolated. Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), prothrombin time, bilirubin, C-reactive protein (CRP), alpha- (α-) fetoprotein (AFP), and liver histology were assessed. Alkaloids, tannins, flavonoids, terpenoids, and saponins were detected in LPE. Model rats demonstrated increased serum levels of AFP, CRP, ALP, GGT, ALT, and AST, prothrombin time, total bilirubin, direct bilirubin, blood lymphocyte, and monocyte counts, but decreased serum albumin and total protein compared to control rats. Unlike the control, model rats demonstrated fat accumulation in periportal and centrilobular hepatocytes and neoplastic transformation. Semiquantitation of periodic acid Schiff- (PAS-) stained liver sections showed decreased glycogen storage in hepatocytes of model rats compared to control rats. Compared to the model, LPE treatment protected against CCl4-induced hepatocarcinogenesis, which was evidenced by decreased AFP, CRP, liver enzymes, total and direct bilirubin, prothrombin time, and blood lymphocyte and monocyte counts; attenuation of fat accumulation; and increased glycogen storage, albumin, and total protein. LPE abates CCl4-induced hepatocarcinogenesis by attenuating liver inflammation and improving metabolic, biosynthetic, and detoxification functions of the liver. The prophylactic hepatoprotective and anti-hepatocarcinogenic effects of LPE are attributable to its phytochemical composition raising hopes of finding potential anticancer bioactive compounds from C. limon fruit peels.

Multiomics analyses decipher intricate changes in the cellular and metabolic landscape of steatotic livers upon dietary restriction and sleeve gastrectomy.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a chronic, progressive liver disease that encompasses a spectrum of steatosis, steatohepatitis (or MASH), and fibrosis. Evidence suggests that dietary restriction (DR) and sleeve gastrectomy (SG) can lead to remission of hepatic steatosis and inflammation through weight loss, but it is unclear whether these procedures induce distinct metabolic or immunological changes in MASLD livers. This study aims to elucidate the intricate hepatic changes following DR, SG or sham surgery in rats fed a high-fat diet as a model of obesity-related MASLD, in comparison to a clinical cohort of patients undergoing SG. Single-cell and single-nuclei transcriptome analysis, spatial metabolomics, and immunohistochemistry revealed the liver landscape, while circulating biomarkers were measured in serum samples. Artificial intelligence (AI)-assisted image analysis characterized the spatial distribution of hepatocytes, myeloid cells and lymphocytes. In patients and experimental MASLD rats, SG improved body mass index, circulating liver injury biomarkers and triglyceride levels. Both DR and SG attenuated liver steatosis and fibrosis in rats. Metabolism-related genes (Ppara, Cyp2e1 and Cyp7a1) were upregulated in hepatocytes upon DR and SG, while SG broadly upregulated lipid metabolism on cholangiocytes, monocytes, macrophages, and neutrophils. Furthermore, SG promoted restorative myeloid cell accumulation in the liver not only ameliorating inflammation but activating liver repair processes. Regions with potent myeloid infiltration were marked with enhanced metabolic capacities upon SG. Additionally, a disruption of periportal hepatocyte functions was observed upon DR. In conclusion, this study indicates a dynamic cellular crosstalk in steatotic livers of patients undergoing SG. Notably, PPARα- and gut-liver axis-related processes, and metabolically active myeloid cell infiltration indicate intervention-related mechanisms supporting the indication of SG for the treatment of MASLD.

Periportal hepatocyte proliferation at midgestation governs maternal glucose homeostasis in mice

The maternal liver is challenged by metabolic demands throughout pregnancy. However, hepatocyte dynamics and their physiological significance in pregnancy remain unclear. Here, we show in mice that hepatocyte proliferation is spatiotemporally regulated in each liver lobular zone during pregnancy, with transient proliferation of periportal and pericentral hepatocytes during mid and late gestation, respectively. Using adeno-associated virus (AAV)−8-mediated expression of the cell cycle inhibitor p21 in hepatocytes, we show that inhibition of hepatocyte proliferation during mid, but not late, gestation impairs liver growth. Transcriptionally, genes involved in glucose/glycogen metabolism are downregulated in late pregnancy when midgestational hepatocyte proliferation is attenuated. In addition, hepatic glycogen storage is abolished, with concomitant elevated blood glucose concentrations, glucose intolerance, placental glycogen deposition, and fetal overgrowth. Laser capture microdissection and RNA-seq analysis of each liver lobular zone show zone-specific changes in the transcriptome during pregnancy and identify genes that are periportally expressed at midgestation, including the hyaluronan-mediated motility receptor (Hmmr). Knockdown of Hmmr in hepatocytes by AAV8-shHmmr suppresses periportal hepatocyte proliferation at midgestation and induces impaired hepatic glycogen storage, glucose intolerance, placental glycogen deposition and fetal overgrowth. Our results suggest that periportal hepatocyte proliferation during midgestation is critical for maternal glycogen metabolism and fetal size.

Spatial and single-cell profiling of the metabolome, transcriptome and epigenome of the aging mouse liver

Tissues within an organism and even cell types within a tissue can age with different velocities. However, it is unclear whether cells of one type experience different aging trajectories within a tissue depending on their spatial location. Here, we used spatial transcriptomics in combination with single-cell ATAC-seq and RNA-seq, lipidomics and functional assays to address how cells in the male murine liver are affected by age-related changes in the microenvironment. Integration of the datasets revealed zonation-specific and age-related changes in metabolic states, the epigenome and transcriptome. The epigenome changed in a zonation-dependent manner and functionally, periportal hepatocytes were characterized by decreased mitochondrial fitness, whereas pericentral hepatocytes accumulated large lipid droplets. Together, we provide evidence that changing microenvironments within a tissue exert strong influences on their resident cells that can shape epigenetic, metabolic and phenotypic outputs.

The essential role of O-GlcNAcylation in hepatic differentiation.

O-GlcNAcylation is a post-translational modification catalyzed by the enzyme O-GlcNAc transferase, which transfers a single N-acetylglucosamine sugar from UDP-GlcNAc to the protein on serine and threonine residues on proteins. Another enzyme, O-GlcNAcase (OGA), removes this modification. O-GlcNAcylation plays an important role in pathophysiology. Here, we report that O-GlcNAcylation is essential for hepatocyte differentiation, and chronic loss results in fibrosis and HCC. Single-cell RNA-sequencing (RNA-seq) was used to investigate hepatocyte differentiation in hepatocyte-specific O-GlcNAc transferase-knockout (OGT-KO) mice with decreased hepatic O-GlcNAcylation and in O-GlcNAcase-KO mice with increased O-GlcNAcylation in hepatocytes. Patients HCC samples and the diethylnitrosamine-induced HCC model were used to investigate the effect of modulation of O-GlcNAcylation on the development of liver cancer. Loss of hepatic O-GlcNAcylation resulted in disruption of liver zonation. Periportal hepatocytes were the most affected by loss of differentiation, characterized by dysregulation of glycogen storage and glucose production. O-GlcNAc transferase-KO mice exacerbated diethylnitrosamine-induced HCC development with increased inflammation, fibrosis, and YAP signaling. Consistently, O-GlcNAcase -KO mice with increased hepatic O-GlcNAcylation inhibited diethylnitrosamine-induced HCC. A progressive loss of O-GlcNAcylation was observed in patients with HCC. Our study shows that O-GlcNAcylation is a critical regulator of hepatic differentiation, and loss of O-GlcNAcylation promotes hepatocarcinogenesis. These data highlight increasing O-GlcNAcylation as a potential therapy in chronic liver diseases, including HCC.

Effect of Lauric acid against ethanol-induced hepatotoxicity by modulating oxidative stress/apoptosis signalling and HNF4α in Wistar albino rats

Ethanol (EtOH) is most widely used in alcoholic beverages to prepare alcohol. As EtOH is mainly metabolised in the liver, the excessive consumption of EtOH forms a primary toxic metabolic product called acetaldehyde, as the gradual increase in acetaldehyde leads to liver injury, as reported. Lauric acid (LA) is rich in antioxidant, antifungal, antibacterial, anticancer, and antiviral properties. LA is an edible component highly present in coconut oil. However, no report on LA protective effects against the EtOH-instigated hepatotoxicity exists. Therefore, the experiment is carried out to investigate the potency effects of LA on EtOH-instigated hepatotoxicity in thirty male albino rats. Rats were divided into five groups (n-6): control DMSO alone, EtOH -intoxicated, EtOH + LA 180 mg/kg, EtOH + LA 360 mg/kg, and LA alone were administered orally using oral gavage. The study measured body weight every weekend in all rat groups. The rats were sacrificed and assessed for serum markers (alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase), antioxidant activity (superoxide dismutase, reduced glutathione, glutathione peroxidase), lipid peroxidation (malondialdehyde), histopathological, cytokine levels (TNF-α, IL-1β and IL-6), protein expression (caspase 3 and caspase 8 and Bcl-2 and HNF4α) were evaluated after the 56-days study period. The impact of EtOH intoxication reduces the rat's body weight by 90 g, upregulates the liver enzyme markers, depletes the antioxidant levels, produces malondialdehyde, changes the histoarchitecture (periportal inflammation and hepatocyte damage), downregulates the Bcl-2 expressions and HNF4α, and elevates the expression of cytokines and apoptotic markers. LA alleviated EtOH-induced liver toxicity by significant (p < 0.05) modulation of biochemical levels, caspase-8/3 signalling, reducing pro-inflammatory cytokines, and restoring the normal histoarchitecture, upregulating the Bcl-2 and HNF4α Expressions. In conclusion, LA treatment can protect the liver against EtOH-induced hepatotoxicity, evidenced by alleviating Oxidative stress, lipid peroxidation, inflammation, apoptosis, and upregulation of HNF4α.

Spatiotemporally quantitative in vivo imaging of mitochondrial fatty acid β-oxidation at cellular-level resolution in mice.

Mitochondrial fatty acid β-oxidation (FAO) plays a key role in energy homeostasis. Several FAO evaluation methods are currently available, but they are not necessarily suitable for capturing the dynamics of FAO in vivo at a cellular-level spatial resolution and seconds-level time resolution. FAOBlue is a coumarin-based probe that undergoes β-oxidation to produce a fluorescent substrate, 7-hydroxycoumarin-3-(N-(2-hydroxyethyl))-carboxamide (7-HC). After confirming that 7-HC could be specifically detected using multiphoton microscopy at excitation/emission wavelength = 820/415-485 nm, wild-type C57BL/6 mice were randomly divided into control, pemafibrate, fasting (24 or 72 h), and etomoxir groups. These mice received a single intravenous injection of FAOBlue. FAO activities in the liver of these mice were visualized using multiphoton microscopy at 4.2 s/frame. These approaches could visualize the difference in FAO activities between periportal and pericentral hepatocytes in the control, pemafibrate, and fasting groups. FAO velocity, which was expressed by the maximum slope of the fluorescence intensity curve, was accelerated in the pemafibrate and 72-h fasting groups both in the periportal and the pericentral hepatocytes in comparison with the control group. Our approach revealed differences in the FAO activation mode by the two stimuli, i.e., pemafibrate and fasting, with pemafibrate accelerating the time of first detection of FAO-derived fluorescence. No increase in the fluorescence was observed in etomoxir-pretreated mice, confirming that FAOBlue specifically detected FAO in vivo. Thus, FAOBlue is useful for visualizing in vivo liver FAO dynamics at the single-cell-level spatial resolution and seconds-level time resolution.NEW & NOTEWORTHY Fatty acid β-oxidation (FAO) plays a key role in energy homeostasis. Here, the authors established a strategy for visualizing FAO activity in vivo at the cellular-level spatial resolution and seconds-level time resolution in mice. Quantitative analysis revealed spatiotemporal heterogeneity in hepatic FAO dynamics. Our method is widely applicable because it is simple and uses a multiphoton microscope to observe the FAOBlue-injected mice.

COVID-19 vaccination-related autoimmune hepatitis-a perspective.

Autoimmune hepatitis (AIH) is the inflammation of the liver with clear-cut interface hepatitis and piecemeal necrosis located at the boundary between portal areas and periportal hepatocytes, and characterized by autoimmunity to hepatocytes with an increase in the antinuclear antibody. After the disastrous SARS-CoV-2 pandemic flagellated several countries, several vaccines have been commercialized and have become a ground for social responsibility. The mRNA vaccines, issued by Pfizer-BioNTech (BNT162b2) and Moderna (mRNA-1273), do not use prebuilt viruses to supply the antigen in the subject's body and are not perfect but have been useful in tackling the pandemic. Nevertheless, both myocarditis and AIH have been reported as side effects of the vaccination programs in addition to thromboembolic events. Here, we explore this topic and give a data-based perspective, gathering a comparison between the titin protein of the sarcomere and myocarditis. The isolation of a Drosophila gene using the serum from a patient with autoimmune scleroderma recognized an epitope on chromosomes (condensed mitotic form) in both human cultured cells and early Drosophila embryos. It revealed that this gene encodes a Drosophila homolog of the vertebrate titin (D-Titin). Moreover, anti-titin antibodies have been found in a subset of patients with myasthenia gravis, a neuromuscular junction disease that is mostly associated with autoimmune antibodies, such as the anti-acetylcholine receptor antibody. The co-existence of myasthenia gravis and autoimmune hepatitis is rare, and a cohort of patients with myasthenia gravis anti-titin antibodies seems to be highly relevant. In consideration of these data and the number of patients who may not be symptomatic, we postulated that autoimmune phenomena may not be exceedingly rare, following the administration of mRNA technology-based vaccines, and a balance between pros and cons in administrating boosters is critical.

Zonal expression of StARD1 and oxidative stress in alcoholic-related liver disease

Alcoholic-related liver disease (ALD) is one of the leading causes of chronic liver disease and morbidity. Unfortunately, the pathogenesis of ALD is still incompletely understood. StARD1 has emerged as a key player in other etiologies of chronic liver disease, and alcohol-induced liver injury exhibits zonal distribution. Here, we report that StARD1 is predominantly expressed in perivenous (PV) zone of liver sections from mice-fed chronic and acute-on-chronic ALD models compared to periportal (PP) area and is observed as early as 10 days of alcohol feeding. Ethanol and chemical hypoxia induced the expression of StARD1 in isolated primary mouse hepatocytes. The zonal-dependent expression of StARD1 resulted in the accumulation of cholesterol in mitochondria and increased lipid peroxidation in PV hepatocytes compared to PP hepatocytes, effects that were abrogated in PV hepatocytes upon hepatocyte-specific Stard1 KO mice. Transmission electron microscopy indicated differential glycogen and lipid droplets content between PP and PV areas, and alcohol feeding decreased glycogen content in both areas while increased lipid droplets content preferentially in PV zone. Moreover, transmission electron microscopy revealed that mitochondria from PV zone exhibited reduced length with respect to PP area, and alcohol feeding increased mitochondrial number, particularly, in PV zone. Extracellular flux analysis indicated lower maximal respiration and spared respiratory capacity in control PV hepatocytes that were reversed upon alcohol feeding. These findings reveal a differential morphology and functional activity of mitochondria between PP and PV hepatocytes following alcohol feeding and that StARD1 may play a key role in the zonal-dependent liver injury characteristic of ALD.

Dysregulated peripheral proteome reveals NASH-specific signatures identifying patient subgroups with distinct liver biology.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. The prognosis may vary from simple steatosis to more severe outcomes such as nonalcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma. The understanding of the biological processes leading to NASH is limited and non-invasive diagnostic tools are lacking. The peripheral immunoproteome in biopsy-proven NAFL (n=35) and NASH patients (n=35) compared to matched, normal-weight healthy controls (n=15) was studied using a proximity extension assay, combined with spatial and single cell hepatic transcriptome analysis. We identified 13 inflammatory serum proteins that, independent of comorbidities and fibrosis stage, distinguished NASH from NAFL. Analysis of co-expression patterns and biological networks further revealed NASH-specific biological perturbations indicative of temporal dysregulation of IL-4/-13, -10, -18, and non-canonical NF-kβ signaling. Of the identified inflammatory serum proteins, IL-18 and EN-RAGE as well as ST1A1 mapped to hepatic macrophages and periportal hepatocytes, respectively, at the single cell level. The signature of inflammatory serum proteins further permitted identification of biologically distinct subgroups of NASH patients. NASH patients have a distinct inflammatory serum protein signature, which can be mapped to the liver parenchyma, disease pathogenesis, and identifies subgroups of NASH patients with altered liver biology.

345 The Role of TCF7L2 in Hepatic Metabolic Zonation

OBJECTIVES/GOALS: Single nucleotide polymorphisms in the transcription factor 7-like 2 (TCF7L2) gene are associated with Type 2 Diabetes (T2D) and nonalcoholic fatty liver disease (NAFLD). The metabolic function of TCF7L2 in the liver remains to be fully elucidated, but we hypothesized that TCF7L2 contributes to NAFLD through regulation of zonal metabolic pathways. METHODS/STUDY POPULATION: Using single nuclei RNA sequencing, we examined Tcf7l2 expression in periportal (PP) hepatocytes around the portal triad and pericentral (PC) hepatocytes surrounding the central vein of the liver. To visualize TCF7L2 transcriptional activity we used a TCF reporter mice, which expresses an H2B-eGFP fusion protein downstream of the conserved TCF DNA binding site. We disrupted Tcf7l2 transcriptional activity in mouse liver by breeding mice with a floxed Tcf7l2 exon 11, which encodes part of the DNA binding domain (DBD), to albumin-Cre mice (Hep-TCF7L2ΔDBD). Eight-week-old mice were fed a choline-deficient amino acid-defined high fat (CDAHFD) diet for 8 weeks. In liver samples harvested from these mice, we examined disruption to several key zonated metabolic pathways, and quantified the development of fibrosis. RESULTS/ANTICIPATED RESULTS: Single nuclei analysis revealed that Tcf7l2 mRNA was expressed primarily in parenchymal cells of the liver but was ubiquitous across the liver lobule. However, in immunofluorescence analysis of TCF reporter mice, the transcriptional activity of TCF7L2 was highly restricted to PC hepatocytes. Classic PC hepatocyte markers, including glutamine synthetase (Glul), were absent in Hep-TCF7L2ΔDBD mice. Following the CDAHFD, Hep-TCF7L2ΔDBD mice developed more severe fibrosis in histological analysis, and expressed elevated levels of genes involved in fibrogenesis, collagen synthesis and TGFβsignaling. Hep-TCF7L2ΔDBD mice also displayed hepatic cholesterol accumulation following the CDAHFD, which was likely the result of impaired pericentral bile acid synthesis. DISCUSSION/SIGNIFICANCE: Our results suggest that TCF7L2 plays an important role in the regulation of zonated metabolic pathways, which may contribute to the development of fibrosis. Ongoing analyses are exploring the mechanisms regulating the zonal transcriptional activity of TCF7L2.

Characterisation of hepatic lipid signature distributed across the liver zonation using mass spectrometry imaging

Background & AimsLipid metabolism plays an important role in liver pathophysiology. The liver lobule asymmetrically distributes oxygen and nutrition, resulting in heterogeneous metabolic functions. Periportal and pericentral hepatocytes have different metabolic functions, which lead to generating liver zonation. We developed spatial metabolic imaging using desorption electrospray ionisation mass spectrometry to investigate lipid distribution across liver zonation with high reproducibility and accuracy. MethodsFresh frozen livers from healthy mice with control diet were analysed using desorption electrospray ionisation mass spectrometry imaging. Imaging was performed at 50 μm × 50 μm pixel size. Regions of interest (ROIs) were manually created by co-registering with histological data to determine the spatial hepatic lipids across liver zonation. The ROIs were confirmed by double immunofluorescence. The mass list of specific ROIs was automatically created, and univariate and multivariate statistical analysis were performed to identify statistically significant lipids across liver zonation. ResultsA wide range of lipid species was identified, including fatty acids, phospholipids, triacylglycerols, diacylglycerols, ceramides, and sphingolipids. We characterised hepatic lipid signatures in three different liver zones (periportal zone, midzone, and pericentral zone) and validated the reproducibility of our method for measuring a wide range of lipids. Fatty acids were predominantly detected in the periportal region, whereas phospholipids were distributed in both the periportal and pericentral zones. Interestingly, phosphatidylinositols, PI(36:2), PI(36:3), PI(36:4), PI(38:5), and PI(40:6) were located predominantly in the midzone (zone 2). Triacylglycerols and diacylglycerols were detected mainly in the pericentral region. De novo triacylglycerol biosynthesis appeared to be the most influenced pathway across the three zones. ConclusionsThe ability to accurately assess zone-specific hepatic lipid distribution in the liver could lead to a better understanding of lipid metabolism during the progression of liver disease. Impact and ImplicationsZone-specific hepatic lipid metabolism could play an important role in lipid homoeostasis during disease progression. Herein, we defined the zone-specific references of hepatic lipid species in the three liver zones using molecular imaging. The de novo triacylglycerol biosynthesis was highlighted as the most influenced pathway across the three zones.

Self-assembling polymer-based short chain fatty acid prodrugs ameliorate non-alcoholic steatohepatitis and liver fibrosis

With the preponderance of a high-calorie diet and sedentary lifestyle, the prevalence of non-alcoholic steatohepatitis (NASH), a state of abnormally elevated lipid accumulation in the liver with chronic inflammation, is increasing at an alarming rate worldwide. Hence, cost-effective therapeutic interventions are required to manage this disease at an early stage. Numerous reports have suggested a link between gut microbial dysbiosis, particularly a decrease in the abundance of short-chain fatty acids (SCFA)-producing microbiota and NASH pathogenesis. Considering these low molecular weight (LMW) SCFAs such as acetic, propionic, and butyric acids have been used to inhibit hepatic steatosis in mouse models. However, the poor pharmacokinetic (PK) profile of SCFAs, caused due to their LMW, renders them therapeutically ineffective. Thus, to improve the PK characteristic-based therapeutic efficacy of LMW SCFAs, we designed SCFA-based prodrugs that possess self-assembling characteristics in aqueous media. The designed SCFA prodrugs consist of enzyme-metabolizable amphiphilic block copolymers, [poly(ethylene glycol)-b-poly(vinyl ester)s] conjugated to propionic acid (PA) or butyric acid (BA) by an ester linkage, which self-assemble into stable nanosized micelles several tens of nanometers in diameter (NanoPA and NanoBA). Via pharmacological analysis, we confirmed that, after oral administration, LMW BA decreased to a physiological level within 24 h in the liver, whereas BA liberated from NanoBA was observed until 72 h post-administration, implying a sustained release profile. Here, we evaluated the therapeutic efficacy of NanoSCFA in a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD)-induced NASH and liver fibrosis mouse model by ad libitum drinking. NanoSCFA, particularly NanoBA, exhibited the remarkable potential to ameliorate the phenotypic features of fatty liver disease by reducing hepatic lipogenesis and fibrosis, with negligible adverse effects. In contrast, conventional LMW SCFAs failed to prevent the pathogenesis of fatty liver disease, which plausibly can be explained by their rapid clearance and discernible adverse effects. Mechanistic studies revealed that NanoBA restored the nuclear expression of PPARα, a transcriptional factor regulating mitochondrial fatty acid oxidation, in the periportal hepatocytes and decreased the CPT1A expression level in the hepatic tissues, reflecting the therapeutic effects of NanoBA. Taken together, we confirmed that our NanoSCFA potentially improved the PK properties of SCFAs, and it consequently alleviated NASH symptoms and fibrotic liver compared to LMW SCFAs. Our study establishes NanoSCFA as a suitable nano-assembled prodrug for NASH treatment.