Citations

Epoxyeicosatrienoic acids (EETs) potently and stereospecifically activate the cardiac ATP‐sensitive K + (Kir6.2) channels by reducing channel sensitivity to ATP. In this study, we determined the effects of EETs on the binding of ATP to Kir6.2 by using 2,4,6‐trinitrophenyl (TNP)‐ATP, which increases fluorescence emission >8‐fold with binding to protein, and by using GST fusion protein containing Kir6.2. TNP‐ATP binds to the GST fusion protein containing the Kir6.2C‐terminus but not to that with the Kir6.2N‐terminus. Binding of TNP‐ATP to GST‐Kir6.2C (K D 5.0 μ M) was unchanged by 5 μ M 11,12‐EET (K D 4.4 μ M) but B max was reduced by half, suggesting non‐competitive inhibition of ATP binding. The effect of 11,12‐EET was dose‐dependent and 8,9‐ and 14,15‐EETs were equipotent inhibitors of TNP‐ATP binding to GST‐Kir6.2C. Arachidonic acid and 11,12‐DHET were not effective inhibitors, whereas the methyl ester of 11,12‐EET was as effective as 11,12‐EET in inhibiting TNP‐ATP binding to GST‐Kir6.2C. R195A mutation in GST‐Kir6.2C abolished the EET effects on TNP‐ATP binding, while the K185A mutation, which reduced ATP binding to Kir6.2, had no effect. These findings are consistent with previous patch clamp results that showed the epoxide group in EET and R195 in Kir6.2 are critical for K ATP channel activation by EET. We conclude that EETs bind to the C‐terminus of the K ATP channel and allosterically alter the ATP binding site, reducing the channel sensitivity to ATP.

Despite the importance of the vasculature during tumorigenesis and during antiangiogenesis treatment, there are few methods available to visualize conveniently the three‐dimensional microvascular structure of tumors. Micro‐computed tomography provides a means to visualize conveniently and quantitatively the three‐dimensional tumor microvascular architecture during tumorigenesis and treatment. In the present study, micro‐computed tomography was utilized to visualize the vasculature of hepatic tumors in SV‐40 transgenic rats. The rats were anesthetized and the vasculature of the livers was infused via an aortic cannula, first with heparinized 0.9% saline, then with 10% buffered formalin and finally with radiopaque silicone rubber (Microfil 122). The livers were first scanned by micro‐CT to visualize the microvasculature and then prepared for histology to visualize the parenchyma. At 10 months of age, all transgenic rats developed gross tumors in their livers. Histological examination revealed that the malignant hepatic lesions in the livers displayed mixed structures resembling hepatoblastoma, cholangiocellular or hepatocellular differentiation in human. Various sized vessels were visible extending to the tumors. Three‐dimensional micro‐computed tomography showed that those vessels were disorganized and tortuous with atypical branching patterns. Often vascular “lakes” were found in the mass of the large tumors. These results show the utility of micro‐computed tomography for studying tumor vasculogenesis. Supported in part by NIH grant EB 000305.

Background and Aims We conducted a comprehensive serum transcriptomic analysis to explore the roles of microRNAs (miRNAs) in alcohol‐associated hepatitis (AH) pathogenesis and their prognostic significance. Approach and Results Serum miRNA profiling was performed in 15 controls, 20 heavy drinkers without liver disease, and 65 patients with AH and compared to publicly available hepatic miRNA profiling in AH patients. Among the top 26 miRNAs, expression of miR‐30b‐5p, miR‐20a‐5p, miR‐146a‐5p, and miR‐26b‐5p were significantly reduced in both serum and liver of AH patients. Pathway analysis of the potential targets of these miRNAs uncovered the genes related to DNA synthesis and cell‐cycle progression pathways, including ribonucleotide reductase regulatory subunit M2 (RRM2), cyclin D1 (CCND1), cyclin D2 (CCND2), MYC proto‐oncogene (MYC), and phorbol‐12‐myristate‐13‐acetate‐induced protein 1 (PMAIP1). We found a significant increase in the protein expression of RRM2, CCND1, and CCND2, but not MYC and PMAIP1, in AH patients who underwent liver transplantation; miR‐26b‐5p and miR‐30b‐5p inhibited the 3′‐UTR (untranslated region) luciferase activity of RRM2 and CCND2, and miR‐20a‐5p reduced the 3′‐UTR luciferase activity of CCND1 and CCND2. During a median follow‐up of 346 days, 21% of AH patients died; these patients had higher body mass index (BMI), Model for End‐Stage Liver Disease (MELD), and serum miR‐30b‐5p, miR‐20a‐5p, miR‐146a‐5p, and miR‐26b‐5p than those who survived. Cox regression analysis showed that BMI, MELD score, miR‐20a‐5p, miR‐146a‐5p, and miR‐26b‐5p predicted mortality. Conclusions Patients with AH attempt to deal with hepatocyte injury by down‐regulating specific miRNAs and up‐regulating genes responsible for DNA synthesis and cell‐cycle progression. Higher expression of these miRNAs, suggestive of a diminished capacity in liver regeneration, predicts short‐term mortality in AH patients.

ATF6α is a critical driver of hepatic stellate cell (HSC) activation in vitro. HSC-specific deletion of Atf6a limits fibrogenesis in vivo despite increased IRE1α signaling. Conditional deletion of Ire1α from HSCs limits fibrogenic gene transcription without impacting overall fibrosis. This could be due in part to observed upregulation of the ATF6α pathway. Dual loss of Atf6a and Ire1a from HSCs worsens fibrosis in vivo through enhanced HSC activation.

AbstractBackgroundAlcoholic hepatitis (AH) is a severe and life‐threatening alcohol‐associated liver disease. Only a minority of heavy drinkers acquires AH and severity varies among affected individuals, suggesting a genetic basis for the susceptibility to and severity of AH.MethodsA cohort consisting of 211 patients with AH and 176 heavy drinking controls was genotyped for five variants in five candidate genes that have been associated with chronic liver diseases: rs738409 in patatin‐like phospholipase domain‐containing protein 3 (PNPLA3), rs72613567 in hydroxysteroid 17‐beta dehydrogenase 13 (HSD17B13), rs58542926 in transmembrane 6 superfamily member 2 (TM6SF2), rs641738 in membrane bound O‐acyltransferase domain containing 7 (MBOAT7), and a copy number variant in the haptoglobin (HP) gene. We tested the effects of individual variants and the combined/interacting effects of variants on AH risk and severity.ResultsWe found significant associations between AH risk and the risk alleles of rs738409 (p = 0.0081) and HP (p = 0.0371), but not rs72613567 (p = 0.3132), rs58542926 (p = 0.2180), or rs641738 (p = 0.7630), after adjusting for patient's age and sex. A multiple regression model indicated that PNPLA3 rs738409:G [OR = 1.59 (95% CI: 1.15–2.22), p = 0.0055] and HP*2 [OR = 1.38 (95% CI: 1.04–1.82), p = 0.0245], when combined and adjusted for age and sex also had a large influence on AH risk among heavy drinkers. In the entire cohort, variants in PNPLA3 and HP were associated with increased total bilirubin and Model for End‐stage Liver Disease (MELD) score, both measures of AH severity. The HSD17B13 rs72613567:AA allele was not found to reduce risk of AH in patients carrying the G allele of PNPLA3 rs738409 (p = 0.0921).ConclusionPNPLA3 and HP genetic variants increase AH risk and are associated with total bilirubin and MELD score, surrogates of AH severity.

BackgroundHeavy alcohol consumption disrupts gut epithelial integrity, leading to increased permeability of the gastrointestinal tract and subsequent translocation of microbes. Regenerating islet‐derived protein 3α (REG3α) and Trefoil factor 3 (TFF3) are mainly secreted to the gut lumen by Paneth and Goblet cells, respectively, and are functionally linked to gut barrier integrity. Circulating levels of REG3α and TFF3 have been identified as biomarkers for gut damage in several human diseases. We examined whether plasma levels of REG3α and TFF3 were dysregulated and correlated with conventional markers of microbial translocation (MT) and pro‐inflammatory mediators in heavy drinkers with and without alcoholic hepatitis (AH).MethodsCross‐sectional and longitudinal studies were performed to monitor plasma levels of REG3α and TFF3 in 79 AH patients, 66 heavy drinkers without liver disease (HDC), and 46 healthy controls (HC) at enrollment and at 6‐ and 12‐month follow‐ups. Spearman correlation was used to measure the relationships of REG3α and TFF3 levels with MT, disease severity, inflammation, and effects of abstinence from alcohol.ResultsAt enrollment, AH patients had significantly higher levels of REG3α and TFF3 than HDC and HC. The elevated REG3α levels were positively correlated with the 30‐day fatality rate. Plasma levels of REG3α and TFF3 in AH patients differentially correlated with conventional MT markers (sCD14, sCD163, and LBP) and several highly up‐regulated inflammatory cytokines/chemokines/growth factors. At follow‐ups, although REG3α and TFF3 levels were decreased in AH patients with alcohol abstinence, they did not fully return to baseline levels.ConclusionsCirculating levels of REG3α and TFF3 were highly elevated in AH patients and differentially correlated with AH disease severity, MT, and inflammation, thereby serving as potential biomarkers of MT and gut epithelial damage in AH patients.

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

Modern medical care produces large volumes of multimodal patient data, which many clinicians struggle to process and synthesize into actionable knowledge. In recent years, artificial intelligence (AI) has emerged as an effective tool in this regard. The field of hepatology is no exception, with a growing number of studies published that apply AI techniques to the diagnosis and treatment of liver diseases. These have included machine‐learning algorithms (such as regression models, Bayesian networks, and support vector machines) to predict disease progression, the presence of complications, and mortality; deep‐learning algorithms to enable rapid, automated interpretation of radiologic and pathologic images; and natural‐language processing to extract clinically meaningful concepts from vast quantities of unstructured data in electronic health records. This review article will provide a comprehensive overview of hepatology‐focused AI research, discuss some of the barriers to clinical implementation and adoption, and suggest future directions for the field.

Alcohol‐associated liver disease (ALD) is a major driver of global liver related morbidity and mortality. There are 2.4 billion drinkers (950 million heavy drinkers) and the lifetime prevalence of any alcohol use disorder (AUD) is 5.1%‐8.6%. In 2017, global prevalence of alcohol‐associated compensated and decompensated cirrhosis was 23.6 million and 2.5 million, respectively. Combined, alcohol‐associated cirrhosis and liver cancer account for 1% of all deaths worldwide with this burden expected to increase. Solutions for this growing epidemic must be multi‐faceted and focused on both population and patient‐level interventions. Reductions in ALD‐related morbidity and mortality require solutions that focus on early identification and intervention, reducing alcohol consumption at the population level (taxation, reduced availability and restricted promotion), and solutions tailored to local socioeconomic realities (unrecorded alcohol consumption, focused youth education). Simple screening tools and algorithms can be applied at the population level to identify alcohol misuse, diagnose ALD using non‐invasive serum and imaging markers, and risk‐stratify higher‐risk ALD/AUD patients. Novel methods of healthcare delivery and platforms are needed (telehealth, outreach, use of non‐healthcare providers, partnerships between primary and specialty care/tertiary hospitals) to proactively mitigate the global burden of ALD. An integrated approach that combines medical and AUD treatment is needed at the individual level to have the highest impact. Future needs include (1) improving quality of ALD data and standardizing care, (2) supporting innovative healthcare delivery platforms that can treat both ALD and AUD, (3) stronger and concerted advocacy by professional hepatology organizations, and (4) advancing implementation of digital interventions.