Interleukin-7-based identification of liver lymphatic endothelial cells reveals their unique structural features.

Background & Aims: The lymphatic system plays crucial roles in maintaining fluid balance and immune regulation. Studying the liver lymphatics has been considered challenging, as common lymphatic endothelial cell (LyEC) markers are expressed by other liver cells. Additionally, isolation of sufficient numbers of LyECs has been challenging because of their extremely low abundance (<0.01% of entire liver cell population) in a normal liver. Methods: Potential LyEC markers was identified using our published single-cell RNA sequencing (scRNA-seq) dataset (GSE147581) in mouse livers. Interleukin-7 (IL7) promoter-driven green fluorescent protein knock-in heterozygous mice were used for the validation of IL7 expression in LyECs in the liver, for the development of liver LyEC isolation protocol, and generating liver ischemia/reperfusion (I/R) injury. Scanning electron microscopy was used for the structural analysis of LyECs. Changes in LyEC phenotypes in livers of mice with I/R were determined by RNA-seq analysis. Results: Through scRNA-seq analysis, we have identified IL7 as an exclusive marker for liver LyECs, with no overlap with other liver cell types. Based on IL7 expression in liver LyECs, we have established an LyEC isolation method and observed distinct cell surface structures of LyECs with fenestrae and cellular pores (ranging from 100 to 400 nm in diameter). Furthermore, we identified LyEC genes that undergo alterations during I/R liver injuries. Conclusions: This study not only identified IL7 as an exclusively expressed gene in liver LyECs, but also enhanced our understanding of LyEC structures and demonstrated transcriptomic changes in injured livers. Impact and implications: Understanding the lymphatic system in the liver is challenging because of the absence of specific markers for liver LyEC. This study has identified IL7 as a reliable marker for LyECs, enabling the development of an effective method for their isolation, elucidating their unique cell surface structure, and identifying LyEC genes that undergo changes during liver damage. The development of IL7 antibodies for detecting it in human liver specimens will further advance our understanding of the liver lymphatic system in the future.

Lymphatics in the liver for translational science.

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Berberine protects mice against type 2 diabetes by promoting PPARγ-FGF21-GLUT2-regulated insulin sensitivity and glucose/lipid homeostasis.

Type 2 diabetes (T2D) is a chronic, burdensome disease that is characterized by disordered insulin sensitivity and disturbed glucose/lipid homeostasis. Berberine (BBR) has multiple therapeutic actions on T2D, including regulation of glucose and lipid metabolism, improvement of insulin sensitivity and energy expenditure. Recently, the function of BBR on fibroblast growth factor 21 (FGF21) has been identified. However, if BBR ameliorates T2D through FGF21, the underlying mechanisms remain unknown. Herein, we used T2D wild type (WT) and FGF21 global knockout (FKO) mice [mouse T2D model: established by high-fat diet (HFD) feeding plus streptozotocin (STZ) injection], and hepatocyte-specific peroxisome proliferator activated receptor γ (PPARγ) deficient (PPARγHepKO) mice, and cultured human liver carcinoma cells line, HepG2 cells, to characterize the role of BBR in glucose/lipid metabolism and insulin sensitivity. We found that BBR activated FGF21 expression by up-regulating PPARγ expression at the cellular level. Meanwhile, BBR ameliorated glucosamine hydrochloride (Glcn)-induced insulin resistance and increased glucose transporter 2 (GLUT2) expression in a PPARγ/FGF21-dependent manner. In T2D mice, BBR up-regulated the expression of PPARγ, FGF21 and GLUT2 in the liver, and GLUT2 in the pancreas. BBR also reversed T2D-induced insulin resistance, liver lipid accumulation, and damage in liver and pancreas. However, FGF21 deficiency diminished these effects of BBR on diabetic mice. Altogether, our study demonstrates that the therapeutic effects of BBR on T2D were partly accomplished by activating PPARγ-FGF21-GLUT2 signaling pathway. The discovery of this new pathway provides a deeper understanding of the mechanism of BBR for T2D treatment.

The Sympathetic Nervous System Promotes Hepatic Lymphangiogenesis, which Is Protective Against Liver Fibrosis.

Liver is the largest lymph-producing organ. In cirrhotic patients, lymph production significantly increases concomitant with lymphangiogenesis. The aim of this study was to determine the mechanism of lymphangiogenesis in liver and its implication in liver fibrosis. Liver biopsies from portal hypertensive patients with portal-sinusoidal vascular disease (n = 22) and liver cirrhosis (n = 5) were evaluated for lymphangiogenesis and compared with controls (n = 9 and n = 6, respectively). For mechanistic studies, rats with partial portal vein ligation (PPVL) and bile duct ligation (BDL) were used. A gene profile data set (GSE77627), including 14 histologically normal liver, 18 idiopathic noncirrhotic portal hypertension, and 22 cirrhotic patients, was analyzed. Lymphangiogenesis was significantly increased in livers from patients with portal-sinusoidal vascular disease, cirrhotic patients, as well as PPVL and BDL rats. Importantly, Schwann cells of sympathetic nerves highly expressed vascular endothelial growth factor-C in PPVL rats. Vascular endothelial growth factor-C neutralizing antibody or sympathetic denervation significantly decreased lymphangiogenesis in livers of PPVL and BDL rats, which resulted in progression of liver fibrosis. Liver specimens from cirrhotic patients showed a positive correlation between sympathetic nerve/Schwann cell-positive areas and lymphatic vessel numbers, which was supported by gene set analysis from patients with noncirrhotic portal hypertension and cirrhotic patients. Sympathetic nerves promote hepatic lymphangiogenesis in noncirrhotic and cirrhotic livers. Increased hepatic lymphangiogenesis can be protective against liver fibrosis.

S-nitrosylation of EMMPRIN influences the migration of HSCs and MMP activity in liver fibrosis.

The mechanism of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of liver fibrosis has not been clarified. This study aims to investigate the role of EMMPRIN S-nitrosylation (SNO) in the regulation of hepatic stellate cell (HSC) migration and matrix metalloproteinase (MMP) activities in liver fibrosis. The results from the tissue microarrays and rat/mouse liver tissues suggest that EMMPRIN mRNA and protein levels in the fibrotic livers are lower than those in the corresponding normal control livers, but higher SNO level of EMMPRIN in fibrotic liver area was shown by immunohistochemistry, immunofluorescence staining, and biotin-switch assay conversely in vivo. Primary EMMPRIN comes from hepatocytes and liver sinus epithelial cells (LSECs) rather than quiescent HSCs. To mimic the uptake of extrinsic EMMPRIN, supernatants from mouse primary hepatocytes/293 cells transfected with EMMPRIN wild-type plasmids (WT) and EMMPRIN SNO site (cysteine 87) mutation plasmids (MUT) were collected and added to JS-1/LX2 cell medium. The MUT EMMPRIN diminishes SNO successfully, enhances the activities of MMP2 and MMP9, and subsequently increases HSC migration. In conclusion, SNO of EMMPRIN influences HSC migration and MMP activities in liver fibrosis. This finding may shed light on the possible regulatory mechanism of MMPs in ECM accumulation in liver fibrosis.

Encapsulation of an Antioxidant in Redox-Sensitive Self-Assembled Albumin Nanoparticles for the Treatment of Hepatitis.

Hepatitis is an inflammation of the liver caused by the inadequate elimination of reactive oxygen species (ROS) derived from Kupffer cells. Edaravone is clinically used as an antioxidant but shows poor liver distribution. Herein, we report on the design of a Kupffer cell-oriented nanoantioxidant based on a disulfide cross-linked albumin nanoparticle containing encapsulated edaravone (EeNA) as a therapeutic for the treatment of hepatitis. Since the edaravone is bound to albumin, this results in a soluble and stable form of edaravone in water. Exchanging the intramolecular disulfide bonds to intermolecular disulfide bridges of albumin molecules allowed the preparation of a redox responsive albumin nanoparticle that is stable in the blood circulation but can release drugs into cells. Consequently, EeNA was fabricated by the nanoscale self-assembly of edaravone and albumin nanoparticles without the additives that are contained in commercially available edaravone preparations. EeNA retained its nanostructure under serum conditions, but the encapsulated edaravone was released efficiently under intracellular reducing conditions in macrophages. The EeNA was largely distributed in the liver and subsequently internalized into Kupffer cells within 60 min after injection in a concanavalin-A-induced hepatitis mouse. The survival rate of the hepatitis mice was significantly improved by EeNA due to the suppression of liver necrosis and oxidative stress by scavenging excessive ROS. Moreover, even through the postadministration, EeNA showed an excellent hepatoprotective action as well. In conclusion, EeNA has the potential for use as a nanotherapeutic against various types of hepatitis because of its Kupffer cell targeting ability and redox characteristics.

An optimized visualization and quantitative protocol for in-depth evaluation of lymphatic vessel architecture in the liver.

The liver lymphatic system is essential for maintaining tissue fluid balance and immune function. The detailed structure of lymphatic vessels (LVs) in the liver remains to be fully demonstrated. The aim of this study is to reveal LV structures in normal and diseased livers by developing a tissue-clearing and coimmunolabeling protocol optimized for the tissue size and the processing time for three-dimensional (3-D) visualization and quantification of LVs in the liver. We showed that our optimized protocol enables in-depth exploration of lymphatic networks in the liver, consisting of LVs along the portal tract (deep lymphatic system) and within the collagenous Glisson's capsule (superficial lymphatic system) in different species. With this protocol, we have shown 3-D LVs configurations in relation to blood vessels and bile ducts in cholestatic mouse livers, in which LVs were highly dilated and predominantly found around highly proliferating bile ducts and peribiliary vascular plexuses in the portal tract. We also established a quantification method using a 3-D volume-rendering approach. We observed a 1.6-fold (P < 0.05) increase in the average diameter of LVs and a 2.4-fold increase (P < 0.05) in the average branch number of LVs in cholestatic/fibrotic livers compared with control livers. Furthermore, cholestatic/fibrotic livers showed a 4.3-fold increase (P < 0.05) in total volume of LVs compared with control livers. Our optimized protocol and quantification method demonstrate an efficient and simple liver tissue-clearing procedure that allows the comprehensive analysis of liver lymphatic system.NEW & NOTEWORTHY This article showed a comprehensive 3-D-structural analysis of liver lymphatic vessel (LV) in normal and diseased livers in relation to blood vessels and bile ducts. In addition to the LVs highly localized at the portal tract, we revealed capsular LVs in mouse, rat, and human livers. In cholestatic livers, LVs are significantly increased and dilated compared with normal livers. Our optimized protocol provides detailed spatial information for LVs remodeling in normal and pathological conditions.

Endotheliopathy of liver sinusoidal endothelial cells in liver disease.

Liver is the largest solid organ in the abdominal cavity, with sinusoid occupying about half of its volume. Under liver disease, hemodynamics in the liver tissue dynamically change, resulting in injury to liver sinusoidal endothelial cells (LSECs). We discuss the injury of LSECs in liver diseases in this article. Generally, in noninflamed tissues, vascular endothelial cells maintain quiescence of circulating leukocytes, and unnecessary blood clotting is inhibited by multiple antithrombotic factors produced by the endothelial cells. In the setting of inflammation, injured endothelial cells lose these functions, defined as inflammatory endotheliopathy. In chronic hepatitis C, inflammatory endotheliopathy in LSECs contributes to platelet accumulation in the liver tissue, and the improvement of thrombocytopenia by splenectomy is attenuated in cases with severe hepatic inflammation. In COVID-19, LSEC endotheliopathy induced by interleukin (IL)-6 trans-signaling promotes neutrophil accumulation and platelet microthrombosis in the liver sinusoids, resulting in liver injury. IL-6 trans-signaling promotes the expression of intercellular adhesion molecule-1, chemokine (C-X-C motif) ligand (CXCL1), and CXCL2, which are the neutrophil chemotactic mediators, and P-selectin, E-selectin, and von Willebrand factor, which are involved in platelet adhesion to endothelial cells, in LSECs. Restoring LSECs function is important for ameliorating liver injury. Prevention of endotheliopathy is a potential therapeutic strategy in liver disease.

Protocol for enrichment, purification, and cytocentrifugation of mouse liver endothelial cells.

Liver endothelial cells (LECs) are critical in maintaining liver homeostasis. To understand the mechanistic processes occurring in these cells, high-quality isolation protocols must be in place. Here, we present a protocol for LEC enrichment, subsequent LEC purification using fluorescence-assisted cell sorting, and cytocentrifugation of sorted LECs for imaging. We describe steps for isolation of LEC-enriched population from mouse livers, immunolabeling and sorting, and cytospin and immunostaining. We then mention procedures for downstream analysis. For complete details on the use and execution of this protocol, please refer to Drzewiecki et al. (2021).1.

Portal Hypertension in Alcohol-Associated Hepatitis.

Purpose of Review: This review article will examine portal hypertension in alcoholic hepatitis (AH) from both a basic mechanistic and a clinical perspective. Recent Findings: Alcoholic hepatitis is a major public health problem in the USA, accounting for over 300,000 hospital admissions in a recent year of data (Jinjuvadia et al. J Clin Gastroenterol. 60;49:506-511). Portal hypertension is a key consequence of AH and a driver of liver-related morbidity and mortality. Alcohol may directly mediate portal hypertension via multiple possible mechanisms, including increased portal inflow, increased intrahepatic vasoconstriction, inflammation, and changes in the liver vasculature such as perisinusoidal fibrosis and phlebosclerosis. Summary: Portal hypertension is a key consequence of AH and a critical area for future research.

Overexpression of NgBR inhibits high-fat diet-induced atherosclerosis in ApoE-deficiency mice.

BACKGROUND: Hyperlipidemia (hypercholesterolemia and/or hypertriglyceridemia) is a risk factor for atherosclerosis. Nogo-B receptor (NgBR) plays important roles in hepatic steatosis and cholesterol transport. However, the effect of NgBR overexpression on atherosclerosis remains unknown. MATERIALS AND METHODS: Apolipoprotein E deficient (ApoE-/-) mice infected with adeno-associated virus (AAV)-NgBR expression vector were fed a high-fat diet for 12 weeks, followed by determination of atherosclerosis and the involved mechanisms. RESULTS: We determined that high expression of NgBR by AAV injection mainly occurs in the liver and it can substantially inhibit en face and aortic root sinus lesions. NgBR overexpression also reduced levels of inflammatory factors in the aortic root and serum, and levels of cholesterol, triglyceride, and free fatty acids in the liver and serum. Mechanistically, NgBR overexpression increased the expression of scavenger receptor type BI and the genes for bile acid synthesis, and decreased the expression of cholesterol synthesis genes by reducing sterol regulatory element-binding protein 2 maturation in the liver, thereby reducing hypercholesterolemia. In addition, NgBR overexpression activated AMP-activated protein kinase α via the Ca2+ signaling pathway, which inhibited fat synthesis and improved hypertriglyceridemia. CONCLUSIONS: Taken together, our study demonstrates that overexpression of NgBR enhanced cholesterol metabolism and inhibited cholesterol/fatty acid synthesis to reduce hyperlipidemia, and reduced vascular inflammation, thereby inhibiting atherosclerosis in ApoE-/- mice. Our study indicates that NgBR might be a potential target for atherosclerosis treatment.

The evolving role of liver sinusoidal endothelial cells in liver health and disease.

LSECs are a unique population of endothelial cells within the liver and are recognized as key regulators of liver homeostasis. LSECs also play a key role in liver disease, as dysregulation of their quiescent phenotype promotes pathological processes within the liver including inflammation, microvascular thrombosis, fibrosis, and portal hypertension. Recent technical advances in single-cell analysis have characterized distinct subpopulations of the LSECs themselves with a high resolution and defined their gene expression profile and phenotype, broadening our understanding of their mechanistic role in liver biology. This article will review 4 broad advances in our understanding of LSEC biology in general: (1) LSEC heterogeneity, (2) LSEC aging and senescence, (3) LSEC role in liver regeneration, and (4) LSEC role in liver inflammation and will then review the role of LSECs in various liver pathologies including fibrosis, DILI, alcohol-associated liver disease, NASH, viral hepatitis, liver transplant rejection, and ischemia reperfusion injury. The review will conclude with a discussion of gaps in knowledge and areas for future research.

Hepatic lymphatic vascular system in health and disease.

In recent years, significant advances have been made in the study of lymphatic vessels with the identification of their specific markers and the development of research tools that have accelerated our understanding of their role in tissue homeostasis and disease pathogenesis in many organs. Compared to other organs, the lymphatic system in the liver is understudied despite its obvious importance for hepatic physiology and pathophysiology. In this review, we describe fundamental aspects of the hepatic lymphatic system and its role in a range of liver-related pathological conditions such as portal hypertension, ascites formation, malignant tumours, liver transplantation, congenital liver diseases, non-alcoholic fatty liver disease, and hepatic encephalopathy. The article concludes with a discussion regarding the modulation of lymphangiogenesis as a potential therapeutic strategy for liver diseases.

Inhibition of high-fat diet-induced obesity via reduction of ER-resident protein Nogo occurs through multiple mechanisms.

Obesity is a risk factor for insulin resistance, type 2 diabetes, and cardiovascular diseases. Reticulon-4 (Nogo) is an endoplasmic reticulum-resident protein with unclear functions in obesity. Herein, we investigated the effect of Nogo on obesity and associated metabolic disorders. Human serum samples were collected to explore the relationship between circulating Nogo-B and body mass index value. Nogo-deficient and WT littermate control mice were fed normal chow or high-fat diet (HFD) for 14 weeks, and HFD-induced obese C57BL/6J mice were injected scrambled or Nogo siRNA for 2 weeks. We found that in human and mouse serum, Nogo-B was positively correlated to body mass index/bodyweight and lipid profiles. Reduced Nogo (by genetic deletion or siRNA transfection) protected mice against HFD-induced obesity and related metabolic disorders. We demonstrate that Nogo deficiency reversed HFD-induced whitening of brown adipose tissue, thereby increasing thermogenesis. It also ameliorated lipid accumulation in tissues by activating the adiponectin-adiponectin receptor 1-AMP-activated kinase α signaling axis. Finally, Nogo deficiency potently reduced HFD-induced serum proinflammatory cytokines and infiltration of macrophages into metabolic organs, which is related to enhanced NF-κB p65 degradation via the lysosome pathway. Collectively, our study suggests that reduced levels of Nogo protect mice against HFD-induced obesity by increasing thermogenesis and energy metabolism while inhibiting NF-κB-mediated inflammation. Our results indicate that inhibition of Nogo may be a potential strategy for obesity treatment.

Nitric oxide facilitates the targeting Kupffer cells of a nano-antioxidant for the treatment of NASH.

Kupffer cells are a key source of reactive oxygen species (ROS) and are implicated in the development of steatohepatitis and fibrosis in nonalcoholic steatohepatitis (NASH). We recently developed a polythiolated and mannosylated human serum albumin (SH-Man-HSA), a nano-antioxidant that targets Kupffer cells, in which the mannosyl units on albumin allows their specific uptake by Kupffer cells via the mannose receptor C type 1 (MRC1), and in which the polythiolation confers antioxidant activity. The aim of this study was to investigate the therapeutic potential of SH-Man-HSA in NASH model mice. In livers from mice and/or patients with NASH, we observed a reduced blood flow in the liver lobes and the down-regulation in MRC1 expression in Kupffer cells, and SH-Man-HSA alone failed to improve the pathological phenotype in NASH. However, the administration of a nitric oxide (NO) donor restored hepatic blood flow and increased the expression of the mannose receptor C type 2 (MRC2) instead of MRC1. Consequently, treatment with a combination of SH-Man-HSA and an NO donor improved oxidative stress-associated pathology. Finally, we developed a hybrid type of nano-antioxidant (SNO-Man-HSA) via the S-nitrosation of SH-Man-HSA. This nanomedicine efficiently delivered both NO and thiol groups to the liver, with a hepatoprotective effect that was comparable to the combination therapy of SH-Man-HSA and an NO donor. These findings suggest that SNO-Man-HSA has the potential for functioning as a novel nano-therapy for the treatment of NASH.

Covid-19 and Liver Injury: Role of Inflammatory Endotheliopathy, Platelet Dysfunction, and Thrombosis.

Liver injury, characterized predominantly by elevated aspartate aminotransferase and alanine aminotransferase, is a common feature of coronavirus disease 2019 (COVID-19) symptoms caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). Additionally, SARS-CoV-2 infection is associated with acute-on-chronic liver failure in patients with cirrhosis and has a notably elevated mortality in patients with alcohol-related liver disease compared to other etiologies. Direct viral infection of the liver with SARS-CoV-2 remains controversial, and alternative pathophysiologic explanations for its hepatic effects are an area of active investigation. In this review, we discuss the effects of SARS-CoV-2 and the inflammatory environment it creates on endothelial cells and platelets more generally and then with a hepatic focus. In doing this, we present vascular inflammation and thrombosis as a potential mechanism of liver injury and liver-related complications in COVID-19.

Portal hypertension in cirrhosis: Pathophysiological mechanisms and therapy.

Portal hypertension, defined as increased pressure in the portal vein, develops as a consequence of increased intrahepatic vascular resistance due to the dysregulation of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs), frequently arising from chronic liver diseases. Extrahepatic haemodynamic changes contribute to the aggravation of portal hypertension. The pathogenic complexity of portal hypertension and the unsuccessful translation of preclinical studies have impeded the development of effective therapeutics for patients with cirrhosis, while counteracting hepatic and extrahepatic mechanisms also pose a major obstacle to effective treatment. In this review article, we will discuss the following topics: i) cellular and molecular mechanisms of portal hypertension, focusing on dysregulation of LSECs, HSCs and hepatic microvascular thrombosis, as well as changes in the extrahepatic vasculature, since these are the major contributors to portal hypertension; ii) translational/clinical advances in our knowledge of portal hypertension; and iii) future directions.