Hepatic Epithelial Cells Research Articles

Cellular Plasticity in Gut and Liver Regeneration.

The intestine and liver share a unique regenerative property that sets them apart from other mammalian visceral organs. The intestinal epithelium exhibits rapid renewal, making it one of the fastest renewing tissues in humans. Under physiological conditions, intestinal stem cells within each intestinal crypt continuously differentiate into the different types of intestinal epithelial cells to maintain intestinal homeostasis. However, when exposed to tissue damage or stressful conditions such as inflammation, intestinal epithelial cells in the gastrointestinal tract exhibit plasticity, allowing fully differentiated cells to regain their stem cell properties. Likewise, hepatic epithelial cells possess a remarkable regenerative capacity to restore lost liver mass through proliferation-mediated liver regeneration. When the proliferation-mediated regenerative capacity is impaired, hepatocytes and biliary epithelial cells (BECs) can undergo plasticity-mediated regeneration and replenish each other. The transition of mammalian liver progenitor cells to hepatocytes/BECs can be observed under tightly controlled experimental conditions such as severe hepatocyte injury accompanied by the loss of regenerative capacity. In this review, we will discuss the mechanism by which cellular plasticity contributes to the regeneration process and the potential therapeutic implications of understanding and harnessing cellular plasticity in the gut and liver.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin.

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell–cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.

Enterocytozoon hepatopenaei (EHP) Infection Alters the Metabolic Processes and Induces Oxidative Stress in Penaeus vannamei

Enterocytozoon hepatopenaei (EHP) is highly contagious and can cause hepatopancreatic microsporidiosis (HPM), which is typically characterized by the slow growth of shrimp. In this study, the differences in histology, metabolism, oxidative stress and growth between healthy and EHP-infected Penaeus vannamei were analyzed using an EHP challenge experiment. Histology showed that EHP caused lesions in the hepatic tubules of P. vannamei, such as hepatic tubular atrophy and epithelial cell shedding, with mature spores. Meanwhile, white feces may appear when the infection is severe. Furthermore, the content of total protein, glycogen, ATP and glucose in the EHP challenge group was significantly reduced. The qPCR results showed that EHP infection changed the expression of key genes in glucose metabolism, among which hexokinase (HK), phosphofructokinase (PFK), pyruvatekinase (PK), citrate synthase (CS) and isocitric dehydrogenase (IDH) were significantly down-regulated, while phosphoenolpyruvate carboxykinase (PEPCK), fructose bisphosphatase (FBP) and glucose-6-phosphatase (G6P) were significantly up-regulated. Obviously, the expression of growth-related genes was disordered. Simultaneously, the antioxidant genes manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPX), glutathione-S-transferases (GST) and nuclear factor E2-related factor2 (Nrf2) were up-regulated to varying degrees in the EHP challenge group, and EHP infection induced significant increases in the oxidative damage products lipid peroxide (LPO) and malondialdehyde (MDA). Ultimately, the shrimp weight of the challenge group was 6.85 ± 0.86 g, which was significantly lower than that of the control group (8.95 ± 0.75 g). Taken together, we speculate that EHP changes the substance metabolism and growth process by causing oxidative damage to the hepatopancreas, which may lead to the growth retardation of P. vannamei.

Microfluidic Chip-Based Modeling of Three-Dimensional Intestine-Vessel-Liver Interactions in Fluorotelomer Alcohol Biotransformation.

Plyfluoroalkyl substance (PFAS), featured with incredible persistence and chronic toxicity, poses an emerging ecological and environmental crisis. Although significant progress has been made in PFAS metabolism in vivo, the underlying mechanism of metabolically active organ interactions in PFAS bioaccumulation remains largely unknown. We developed a microfluidic-based assay to recreate the intestine-vessel-liver interface in three dimensions, allowing for high-resolution, real-time images and precise quantification of intestine-vessel-liver interactions in PFAS biotransformation. In contrast to the scattered arrangement of vascular endothelium on the traditional d-polylysine-modified two-dimensional (2D) plate, the microtubules in our three-dimensional (3D) platform formed a dense honeycomb network through the ECM, with longer tubular structures. Additionally, the slope culture of epithelial cells in our platform exhibited a closely arranged and thicker cell layer than the planar culture. To dynamically monitor the metabolic crosstalk in the intestinal-vascular endothelium-liver interaction under exposure to fluorotelomer alcohols (FTOHs), we combined the chip with a solid-phase extraction-mass spectrometry (SPE-MS) system. Our findings revealed that endothelial cells were involved in the metabolic process of FTOHs. The transformation of intestinal epithelial and hepatic epithelial cells produces toxic metabolite fluorotelomer carboxylic acids (FTCAs), which circulate to endothelial cells and affect angiogenesis. This system shows promise as an enhanced surrogate model and platform for studying pollutant exposure as well as for biomedical and pharmaceutical research.

Comprehensive analysis of ASB3 as a prognostic biomarker in hepatocellular carcinoma

BackgroundSome reports have indicated a high expression level of ASB3 in various cancers, but its role in hepatocellular carcinoma (HCC) remains elusive. MethodsASB3 levels and clinical features were obtained from the TCGA database. Meanwhile, the expression levels of ASB3 in tumor and paraneoplastic tissues were further verified by qRT-PCR and Imunohistochemistry (IHC). ASB3-related downstream molecular analysis was carried out with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Pathways linked to ASB3 expression were identified by means of gene set enrichment analysis (GSEA). Single-sample GSEA (ssGSEA) aided in conducting a correlation analysis of ASB3 with immune infiltration. Functional experiments were performed in HepG2 cells by using the small interfering RNA. ResultsASB3 expression was remarkably higher in HCC tissues. Its remarkable precision in forecasting cancer suggests that ASB3 might serve as an unidentified diagnostic and prognostic indicator of HCC. Higher ASB3 expression led to worse overall survival (OS), particularly in various clinical subgroups of HCC. GO/KEGG analysis indicated that critical biological activities, such as the activation of complement systems and humoral immune response, could potentially underlie the progression of HCC. Furthermore, GSEA demonstrated enrichment of certain pathways, including the MAPK, IL17, and fibrinolysis pathways, in samples with elevated ASB3 levels. ASB3 exhibited a substantial association with T helper cells, dendritic cells (DCs), and central memory T (Tcm) cell infiltration level. Cell function experiments confirmed elevated ASB3 levels in HCC cell lines as opposed to hepatic epithelial cell lines. Moreover, the ability of HCC cells to proliferate and invade was remarkably reduced by ASB3 knockdown. ConclusionSummarize briefly, we found that ASB3 can be a promising biomarker in HCC.

Antioxidant and Anti-inflammatory Activities Mediate the Radioprotective Effect of Trianthema portulacastrum L. Extracts

Background: Ionizing radiation (IR) generates reactive oxygen species (ROS), which leads to oxidative stress that often leads to inflammatory responses in organisms. Objective: Trianthema portulacastrum L., a plant commonly growing in India, is rich in antioxidant phytochemicals. This is responsible for scavenging free radicals and may provide radioprotective and anti-inflammatory effects in response to ionizing radiation. Methods: The effect of T. portulacastrum extracts was studied in hepatic cells, which are susceptible to radiation-induced damage and in macrophages, which are the primary inflammatory cells of the body. Results: T. portulacastrum stem extracts showed efficient free radical scavenging activity in hepatocytes and decreased radiation-induced lipid peroxidation in cell and mitochondrial membranes. Treatment of irradiated cells with T. portulacastrum stem extracts enhanced cell viability at lower concentration and reduced cell viability at higher concentration. Treatment with low concentration of T. portulacastrum stem extract also reduced cellular ROS generation and increased the concentration of cellular anti-oxidant, glutathione. T. portulacastrum extracts also showed remarkable anti-inflammatory properties in macrophages activated by the inflammatory agonist bacterial lipopolysaccharide (LPS). The extract reduced nitric oxide (NO) production and suppressed the expression of inflammatory genes. Conclusion: Together, these observations demonstrated a potential radioprotective role of T. portulacastrum extract mediated by both its antioxidant activity on hepatic epithelial cells and its anti-inflammatory activity on immune cells.

Efficacy of Autologous Skeletal Myoblast Cell Sheet Transplantation for Liver Regeneration in Liver Failure.

No effective therapies have yet been established for liver regeneration in liver failure. Autologous skeletal myoblast cell sheet transplantation has been proven to improve cardiac function in patients with heart failure, and one of the mechanisms has been reported to be a paracrine effect by various growth factors associated with liver regeneration. Therefore, the present study focused on the effect of myoblast cells on liver regeneration in vitro and in vivo. We assessed the effect of myoblast cells on the cells comprising the liver in vitro in association with liver regeneration. In addition, we examined in vivo effect of skeletal myoblast cell sheet transplantation in C57/BL/6 mouse models of liver failure, such as liver fibrosis induced by thioacetamide and hepatectomy. In vitro, the myoblast cells exhibited a capacity to promote the proliferation of hepatic epithelial cells and the angiogenesis of liver sinusoidal endothelial cells, and suppress the activation of hepatic stellate cells. In vivo, sheet transplantation significantly suppressed liver fibrosis in the induced liver fibrosis model and accelerated liver regeneration in the hepatectomy model. Autologous skeletal myoblast cell sheet transplantation significantly improved the liver failure in the in vitro and in vivo models. Sheet transplantation is expected to have the potential to be a clinically therapeutic option for liver regeneration in liver failure.

A solitary intracranial rich focus with early meningeal involvement: A correlative image

•Rich foci are caseating brain tuberculous (TB) granulomas surrounding the immigrated TB bacilli.•Rich foci are hypothesized as an underlying pathological mechanism of TB meningitis.•The hypothesis is based on necropsy studies and has rarely been depicted in vivo.•Ruptured foci discharge their contents into the neighboring subarachnoid space.•Discharged contents trigger a T cell response, leading to inflammatory meningitis. A 24-year-old man presented to the emergency department with a 2-day history of fever and headache, not responsive to over-the-counter medications. He reported a previous history of latent tuberculosis, for which he received antitubercular therapy a couple of years ago. Positive results were obtained from interferon-γ release and adenosine deaminase (ADA) assays, but chest computed tomography (CT) was unremarkable. The patient had unremarkable general and neurologic examinations. Brain CT demonstrated a left-sided focal lesion that delineates perfectly post-contrast administration, besides an adjacent area of meningeal enhancement at the Sylvian sulcus (Figure 1). Cerebrospinal fluid analysis revealed monocytic pleocytosis, an elevated protein level, marked hypoglycorrhachia, and positive ADA activity. Based on the data obtained thus far, we concluded that the patient had an early focal tuberculous meningitis (TBM) caused by a rupturing rich focus, consistent with the natural history of TBM that has rarely been confirmed in vivo (Supplement 1) [1Rich AR McCordock HA. Evidence for the presence of ribonucleic acid in the cytoplasmic bodies that appear in the hepatic and adrenal epithelial cells of man in acute infection.Bull Johns Hopkins Hosp. 1933; 52: 5-37Google Scholar, 2Donald PR Schaaf HS Schoeman JF. Tuberculous meningitis and miliary tuberculosis: the Rich focus revisited.J Infect. 2005; 50: 193-195https://doi.org/10.1016/j.jinf.2004.02.010Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 3Be NA Kim KS Bishai WR Jain SK. Pathogenesis of central nervous system tuberculosis.Curr Mol Med. 2009; 9: 94-99https://doi.org/10.2174/156652409787581655Crossref PubMed Scopus (100) Google Scholar]. Subsequently, the patient was started on antitubercular treatment using INH, RIF, PZA, and EMB for 2 months. After which, PZA and EMB were discontinued, while INH and RIF were continued for an additional nine months. The focus with the associated TBM resolved substantially following treatment with no evidence of recurrence over a 2-year follow-up.

MiR-9 and miR-181a Target Gab2 to Inhibit the Proliferation and Migration of Hepatocellular Carcinoma HepG2 Cells.

The incidence of liver cancer ranks seventh globally, with nearly half of all cases occurring in East Asia, but currently, there are very few drugs to treat it. Our previous studies demonstrated that the signal integration protein Gab2 is a potential drug target for the prevention and therapy of liver cancer. Here, we screened for and identified two miRNAs that target Gab2 to suppress the proliferation and migration of hepatocellular carcinoma (HCC) cells. First, we predicted Gab2-targeting miRNAs through biological websites, and we selected nine miRNAs that were reported in the literature as being abnormally expressed in liver cancer and fatty liver tissue. Then, we measured the expression of these miRNAs in the hepatic epithelial cell line HL-7702 and the HCC cell line HepG2. The expression levels of miR-9, miR-181a, miR-181c, miR-34a, and miR-134 were high in HL-7702 cells but low in HepG2 cells, and their expression patterns were the opposite of Gab2 in these cells. Furthermore, we transfected miR-9, miR-34a, miR-181a, and miR-181c mimics into HepG2 cells and found that only miR-9 and miR-181a reduced the level of Gab2 proteins. miR-9 also reduced the Gab2 mRNA level, but miR-181a did not affect the Gab2 mRNA levels. Using a miRNA-Gab2 3'UTR binding reporter, we confirmed that miR-9 and miR-181a bind to the Gab2 3'UTR region. Finally, we introduced miR-9 and miR-181a mimics into HepG2 cells and found that cell proliferation and migration were significantly inhibited. In conclusion, we identified two novel miRNAs targeting Gab2 and provided potential drug targets for the prevention and treatment of liver cancer.

Distinct vulnerability to oxidative stress determines the ammonia sensitivity of crayfish (Procambarus clarkii) at different developmental stages

Red swamp crayfish (Procambarus clarkii) has increasingly become a high-value freshwater product in China. During the intensive cultivation, excessive ammonia exposure is an important lethal factor of crayfish. We investigated the toxic effects and mechanisms of ammonia on crayfish at two different developmental stages. A preliminary ammonia stress test showed a 96-h LC50 of 135.10 mg/L and 299.61 mg/L for Stage_1 crayfish (8.47 ± 1.68 g) and Stage_2 crayfish (18.33 ± 2.41 g), respectively. During a prolonged ammonia exposure (up to 96 h), serum acid phosphatase and alkaline phosphatase showed a time-dependent decrease. Histological assessment indicated the degree of hepatopancreatic injury, which was mainly characterized as tubule lumen dilatation, degenerated tubule, vacuolization and dissolved hepatic epithelial cell, increased with exposure time. Enhanced malondialdehyde level and reduced antioxidant capacity of hepatopancreas were also observed. The mRNA expression and activity of catalase and superoxide dismutase showed an initial up-regulation within 24 h, and then gradually down-regulated with the exposure time. In the post-treatment recovery period, the Stage_2 crayfish exerted a stronger antioxidant and detoxification capacity than that of the Stage_1 crayfish, and thus quickly recovered from the ammonia exposure. Our findings provide a further understanding of the adverse effects of ammonia stress and suggest guidelines for water quality management during crayfish farming.

The Effect of Microcirculation Disturbance of Peribiliary Vascular Plexus on Hepatic Bile Duct Epithelial Cells in Rats Undergoing Liver Transplantation and Postoperative Rejection Reduction by Nanocarrier Mediation.

This study was to investigate the effect of microcirculation disturbance of PVP on HBD epithelial cells in rats undergoing liver transplantation and to explore the postoperative rejection reduction by nanocarriers mediation. For this aim, adult male rats weighing 210-250 g, were fed cleanly and were subjected to liver transplantation. 3 days after the surgery, the rats were randomly divided into three groups based on different intervention factors: group A (HAL), group B (HAMI combined with HAL), and group C (control). The three groups of rats were divided into three subgroups according to the duration of the University of Wisconsin (UW) solution (UW time) used to preserve the donor organs for transplantation, which were 2h, 8h, and 16h, respectively. In addition, the RNA sequence of rat class-II transactivator (CIITA) the rat was searched, and the target interference sequence was designed concerning the RNA. Results showed that the carrier nanoparticles were spherical without obvious oxygen vacancies, the distribution was relatively tight and concentrated, and the main particle size was 50-140 nm. As the mass ratio of HGPAE to DNA increased, the mobility speed of the nanocarrier/shRNA plasmid complex decreased due to the decrease in surface charge. When the mass ratio reached 90:1, the mobility of the complex was completely blocked, suggesting that the DNA was completely compounded. The counts of PCNA, CK-19, F-VIII-Ag, VEGFA, VEGFB, and VEGFC in the 3 groups all showed a downward trend with the increase of UW time; the count in group B was lower than that of groups A and C. In the PCNA count statistics, there was no obvious difference between group A and group B at UW8h, but there were differences in contrast to group C (p<0.05). It was concluded that the blood supply of the microcirculation of the PVP was extremely important for the transplanted liver tissue. When the blood vessels around the HBD of the rat were completely ischemic, the HBD epithelial cells became the most important target of damage, and the proliferation and changes of the HBD epithelial cells can be directly observed. In addition, the nanocarrier-mediated genes were applied to discuss postoperative rejection. The expression of class-II MHC-II gene in nanocarrier CIITA-shRNA was inhibited, which interfered with the recipient's immune recognition of the graft, thereby reducing the intensity of the rejection reaction and relieving the rejection reaction.

Plasmolipin regulates basolateral-to-apical transcytosis of ICAM-1 and leukocyte adhesion in polarized hepatic epithelial cells

Apical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory response.

DEP domain containing 1B (DEPDC1B) exerts the tumor promoter in hepatocellular carcinoma through activating p53 signaling pathway via kinesin family member 23 (KIF23)

ABSTRACT Hepatocellular carcinoma (HCC) is closely associated with chronic liver disease and possesses a high incidence. DEP domain containing 1B (DEPDC1B) expression has been found to be upregulated in HCC according to bioinformatics analysis. This paper sought to study the specific role of DEPDC1B in HCC. The data of DEPDC1B expression and individual overall survival in HCC and normal liver tissues were acquired from UALCAN database. The association between DEPDC1B and the downstream signal, kinesin family member 23 (KIF23), was determined using LinkedOmics and STRING database, and subsequently confirmed by co-immunoprecipitation assay. The expression levels of DEPDC1B and KIF23 in normal hepatic epithelial cells and HCC cell lines were assessed by RT-qPCR and Western blotting, respectively. Following transfection with small interference RNA-DEPDC1B, the influences of DEPDC1B knockdown on cell proliferation, colony formation, cell cycle, cell invasion, migration, and KIF23 expression were evaluated. In addition, the effects of KIF23 overexpression on the above aspects of HCC cells were also determined, as well as the expression level of p53 signaling-related proteins. The results indicated that DEPDC1B was highly expressed in HCC cells. DEPDC1B knockdown inhibited the proliferation, migration, invasion, cycle, and KIF23 expression in HCC cells. Moreover, KIF23 overexpression reversed the inhibitory effect of DEPDC1B knockdown in HCC cells and the activation of the p53 signaling. In conclusion, DEPDC1B knockdown exerts anti-cancer role in HCC by activating the p53 signaling through KIF23.

Liver Fibrosis: Underlying Mechanisms and Innovative Therapeutic Approach. A Review Article

Liver fibrosis is considered: “a pathological repairing process in liver injuries leading to extracellular cell matrix (ECM) accumulation evidencing chronic liver diseases”. Chronic viral hepatitis, alcohol consumption, autoimmune diseases as well as non-alcoholic steatohepatitis are from the main causes of liver fibrosis (Lee et al., 2015; Mieli-Vergani et al., 2018). Hepatic stellate cells (HSCs) exist in the sinus space next to the hepatic epithelial cells as well as endothelial cells (Yin et al., 2013). Normally, HSCs are quiescent and mainly participate in fat storage and in the metabolism of vitamin A. HSCs are produced during liver injury and then transformed into myofibroblasts. The activated HSCs resulted in a sequence of events considered as marks fibrosis. The activation of HSCs mostly express alpha smooth muscle actin (α-SMA). Moreover, ECM is synthesized and secreted by HSCs that affects markedly the structure and function of the liver tissue leading to fibrosis (Tsuchida et al., 2017; Han et al., 2020). Hence, activated HSCs are attracting attention as potential targets in liver fibrosis. Many signaling molecules are involved in HSCs activation first and foremost, platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-β) (Tsuchida et al., 2017; Wang et al., 2020c) as interfering the PDGF or TGF-β signaling pathways is a growing field for liver fibrosis treatment.

Serum Proteomics Uncovers Biomarkers of Clinical Portal Hypertension in Children With Biliary Atresia.

Children with biliary atresia (BA) often develop portal hypertension (PHT) and its complications, which are associated with high morbidity and mortality. The goal of this study was to identify serum biomarkers of PHT by using large‐scale proteomics. We applied the slow off‐rate modified aptamer scan (SOMAscan) to measure 1,305 proteins in serum samples of children with BA with and without clinical evidence of PHT in validation and discovery cohorts enrolled in the Biliary Atresia Study of Infants and Children. Serum proteomics data was analyzed using logistic regression to identify protein(s) with an area under the receiver operating characteristic curve (AUROC) ≥ 0.90. Immunostaining was used to characterize the cellular localization of the new biomarker proteins in liver tissues. We identified nine proteins in the discovery cohort (n = 40 subjects) and five proteins in the validation cohort (n = 80 subjects) that individually or in combination predicted clinical PHT with AUROCs ≥ 0.90. Merging the two cohorts, we found that semaphorin 6B (SEMA6B) alone and three other protein combinations (SEMA6B+secreted frizzle protein 3 [SFRP3], SEMA6B+COMM domain containing 7 [COMMD7], and vascular cell adhesion molecule 1 [VCAM1]+BMX nonreceptor tyrosine kinase [BMX]) had AUROCs ≥ 0.90 in both cohorts, with high positive‐ and negative‐predictive values. Immunostaining of the new protein biomarkers showed increased expression in hepatic endothelial cells, cholangiocytes, and immune cells within portal triads in BA livers with clinical PHT compared to healthy livers. Conclusion: Large‐scale proteomics identified SEMA6B, SFRP3, COMMD7, BMX, and VCAM1 as biomarkers highly associated with clinical PHT in BA. The expression of the biomarkers in hepatic epithelial, endothelial, and immune cells support their potential role in the pathophysiology of PHT.

Identification of Key Amino Acids that Impact Organic Solute Transporter α/β (OSTα/β)

Organic solute transporter α/β (OSTα/β) is a bidirectional bile acid transporter localized on the basolateral membrane of hepatic, intestinal, and renal epithelial cells. OSTα/β plays a critical role in intestinal bile acid reabsorption and is upregulated in hepatic diseases characterized by elevated bile acids, whereas genetic variants in SLC51A/B have been associated with clinical cholestasis. OSTα/β also transports and is inhibited by commonly used medications. However, there is currently no high-resolution structure of OSTα/β, and structure-function data for OSTα, the proposed substrate-binding subunit, are lacking. The present study addressed this knowledge gap and identified amino acids in OSTα that are important for bile acid transport. This was accomplished using computational modeling and site-directed mutagenesis of the OSTα subunit to generate OSTα/β mutant cell lines. Out of the 10 OSTα/β mutants investigated, four (S228K, T229S, Q269E, Q269K) exhibited decreased [3H]-taurocholate (TCA) uptake (ratio of geometric means relative to OSTα/β wild type (WT) of 0.76, 0.75, 0.79, and 0.13, respectively). Three OSTα/β mutants (S228K, Q269K, E305A) had reduced [3H]-TCA efflux % (ratio of geometric means relative to OSTα/β WT of 0.86, 0.65, and 0.79, respectively). Additionally, several OSTα/β mutants demonstrated altered expression and cellular localization when compared with OSTα/β WT. In summary, we identified OSTα residues (Ser228, Thr229, Gln269, Glu305) in predicted transmembrane domains that affect expression of OSTα/β and may influence OSTα/β-mediated bile acid transport. These data advance our understanding of OSTα/β structure/function and can inform future studies designed to gain further insight into OSTα/β structure or to identify additional OSTα/β substrates and inhibitors. SIGNIFICANCE STATEMENT: OSTα/β is a clinically important transporter involved in enterohepatic bile acid recycling with currently no high-resolution protein structure and limited structure-function data. This study identified four OSTα amino acids (Ser228, Thr229, Gln269, Glu305) that affect expression of OSTα/β and may influence OSTα/β-mediated bile acid transport. These data can be utilized to inform future investigation of OSTα/β structure and refine molecular modeling approaches to facilitate the identification of substrates and/or inhibitors of OSTα/β.

P-73 HEPATIC CHANGES BY SARS-COV 2 IN PATIENTS OF THE INTENSIVE CARE UNIT OF THE TROPICAL MEDICINE CENTER IN RONDÔNIA

IntroductionCoronavirus (SARS-CoV2) infection occurs through the receptor's angiotensin converting enzyme 2, present in the pulmonary, biliary, and hepatic epithelial cells. Therefore, the liver is a potential target for infection.ObjectivesTo analyze liver changes resulting from Sars-Cov-2 infection in patients admitted to the Intensive Care Unit of the Rondônia Tropical Medicine Center (CEMETRON).MethodsPatients admitted between April and August 2020 in the CEMETRON ICU were included in the study. Project approved by the Research Ethics Committee. For statistical analysis, the SPSS® program was used.Results307 patients were admitted to the CEMETRON ICU. 81 (26.4%) non-COVID and 226 (73.6%) diagnosed with COVID. Among the 226 tested positive for COVID, 52.3% and 54.3% had, respectively, an increase in ALT and AST up to three times the upper limit of normal (40-120U/L). Non-COVID patients showed this increase in 20.8% for ALT and 33.3% for AST, being statistically significant (p: <0.005 for both). Transaminases above 120U/L had no statistically significant difference between the two groups. Regarding liver function assessed through bilirubin, albumin and platelets, there was no statistically significant difference in any of the variables (p: 0.93 p: 0.45 p: 0.599 respectively). The means varied within the normal range, except for both groups there was a tendency towards hypoalbuminemia (3.1 g / dL).ConclusionPatients with COVID evolved in more than 50% of the cases with changes in liver enzymes, showing that despite the inflammation, liver function was not directly affected. We associate hypoalbuminemia more with basal malnutrition than with hepatic impairment.

CircTP63 promotes hepatocellular carcinoma progression by sponging miR-155-5p and upregulating ZBTB18

BackgroundHepatocellular carcinoma (HCC) is the leading cause of tumor-related death worldwide due to high morbidity and mortality, yet lacking effective biomarkers and therapies. Circular RNAs (circRNAs) are a group of non-coding RNAs that regulate gene expression through interacting with miRNAs, implicating in the tumorigenesis and progression. A novel circRNA, circTP63, was reported to be an oncogene in HCC. However, its role in HCC remains unclear.MethodsqRT-PCR was used to assess the mRNA levels of CircTP63 in 90 pairs of tumor and adjacent normal tissues from HCC patients, one human normal hepatic epithelial cell line and HCC cell lines. CCK-8, colony formation, transwell, and flow cytometry assays were performed to detect the cellular function of circTP63/miR-155-5p/ZBTB18 in HCC cells. HCC xenograft mice models were established to assess the in vivo effect of circTP63. Bioinformatic analysis, RNA pull-down and luciferase assays were used to determine the interaction among circTP63/miR-155-5p/ZBTB18.ResultscircTP63 was significantly upregulated in HCC tissues and cell lines. High circTP63 expression is closely associated with the tumor stages, lymph node metastasis, and poor prognosis of HCC patients. Functionally, knockdown of circTP63 inhibited cell proliferation, migration, invasion, and promoted cell apoptosis of HCC. Meanwhile, overexpression of circTP63 enhanced HCC progression. Mechanically, circTP63 was a sponge of miR-155-5p to facilitate the ZBTB18 expression, and the ZBTB18 expression in HCC tissues was negatively associated with the survival rate of HCC patients. Furthermore, rescued assays revealed that the reduced tumor-promoting effect on HCC cells induced by knockdown of circTP63 can be reversed by miR-155-5p inhibitor or ZBTB18 overexpression.ConclusionOur data highlight a critical circTP63-miR-155-5p-ZBTB18 regulatory network involved in the HCC progression, gaining mechanistic insights into the function of circRNAs in HCC progression, and providing effective biomarkers and therapeutic targets for HCC treatment.

LncRNA CASC9 promotes proliferation, migration and inhibits apoptosis of hepatocellular carcinoma cells by down-regulating miR-424-5p

Introduction and objectivesCASC9 and miR-424-5p are closely related with hepatocellular carcinoma (HCC) progression. This study aimed to evaluate the effect of CASC9 involved with miR-424-5p on the development of HCC. Materials and methodsqRT-PCR was performed to determine the mRNA expressions of CASC9 and miR-424-5p in HCC tissues/cells and adjacent normal tissues/human hepatic epithelial cells, and to analyze the relationship of CASC9 with the clinico-pathological characteristics and prognosis of HCC patients. Then, cell proliferation was measured by CCK-8 and11LncRNAs, long non-coding RNAs; HCC, hepatocellular carcinoma; AFP, alpha-fetoprotein; CASC9, cancer sensitivity 9; ESCC, esophageal squamous cell carcinoma; qRT-PCR, quantitative reverse transcription polymerase chain reaction; ATCC, American Type Culture Collection; DMEM, Dulbecco's Modified Eagle Medium; CCK-8, cell counting kit-8; RIPA, radio-immunoprecipitation assay; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis; PVDF, polyvinylidene fluoride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siNC, small interfering RNA of negative control; M: miR-424-5p mimic; IC, inhibitor control; I, miR-424-5p inhibitor. clone formation assays. Apoptosis of HCC cells was measured by flow cytometry. Besides, cell migration and invasion were determined by scratch wound-healing and Transwell assays, respectively. DIANA-LncBase V2 and dual luciferase reporter gene assay were used to verify the targeted relationship between CASC9 and miR-424-5p. Bcl-2, Bax and cleaved caspase-3 expressions were detected by Western blot. ResultsHigher expression of CASC9 was observed in HCC tissues/ cells than in adjacent normal tissues/ human hepatic epithelial cells, and was closely linked to poor prognosis of HCC, tumor size, TNM stage, differentiation degree, lymph node metastasis and alpha-fetoprotein (AFP). Down-regulation of CASC9 decreased the proliferation, invasion and migration of HCC cells while enhancing apoptosis. Besides, CASC9 was negatively correlated with miR-424-5p. MiR-424-5p inhibitor enhanced cell proliferation, invasion and migration while decreasing apoptosis. Interestingly, siRNA-CASC9 partially offset the effects of miR-424-5p inhibitor on HCC cells. ConclusionCASC9 promoted proliferation, invasion and migration and inhibited apoptosis in HCC cells by inhibiting miR-424-5p.