Development of Scaffold-Free Three-Dimensional Cholangiocyte Organoids to Study the Progression of Primary Sclerosing Cholangitis.

Organoids are novel in vitro models to study intercellular cross talk between the different types of cells in disease pathophysiology. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), scaffold-free multicellular three-dimensional cholangiocyte organoids (3D-CHOs) were developed using primary liver cells derived from normal subjects and patients with PSC. Human liver samples from healthy donors and patients with PSC were used to isolate primary cholangiocytes [epithelial cell adhesion molecule (EpCam)+/ cytokeratin-19+], liver endothelial cells (CD31+), and hepatic stellate cells (HSCs; CD31-/CD68-/desmin+/vitamin A+). 3D-CHOs were formed using cholangiocytes, HSCs, and liver endothelial cells, and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence, quantitative RT-PCR, and transmission electron microscopy. Transcription profiles for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, and SCTR), fibrosis (ACTA2, COL1A1, DESMIN, and TGFß1), angiogenesis (PECAM, VEGF, CDH5, and vWF), and inflammation (IL-6 and TNF-α) confirmed PSC phenotypes of 3D-CHOs. Because cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal immunofluorescence was used to demonstrate localization of the neurokinin-1 receptor within cytokeratin-19+ cholangiocytes and desmin+ HSCs. Moreover, 3D-CHOs from patients with PSC confirmed PSC phenotypes with up-regulated neurokinin-1 receptor, tachykinin precursor 1, and down-regulated membrane metalloendopeptidase. Scaffold-free multicellular 3D-CHOs showed superiority as an in vitro model in mimicking PSC in vivo phenotypes compared with two-dimensional cell culture, which can be used in PSC disease-related research.

Growth Hormone Signaling in Liver Diseases: Therapeutic Potentials and Controversies.

Growth hormone (GH) and downstream insulin-like growth factor 1 (IGF1) signaling mediate growth and metabolism. GH deficiency causes short stature or dwarfism, and excess GH causes acromegaly. Although the association of GH/IGF1 signaling with liver diseases has been suggested previously, current studies are controversial and the functional roles of GH/IGF1 signaling are still undefined. GH supplementation therapy showed promising therapeutic effects in some patients, such as non-alcoholic fatty liver disease, but inhibition of GH signaling may be beneficial for other liver diseases, such as hepatocellular carcinoma. The functional roles of GH/IGF1 signaling and the effects of agonists/antagonists targeting this signaling may differ depending on the liver injury or animal models. This review summarizes current controversial studies of GH/IGF1 signaling in liver diseases and discusses therapeutic potentials of GH therapy.

Three-Dimensional Organoids as a Model to Study Nonalcoholic Fatty Liver Disease.

Despite the rising prevalence of nonalcoholic fatty liver disease (NAFLD), the underlying disease pathophysiology remains unclear. There is a great need for an efficient and reliable "human" in vitro model to study NAFLD and the progression to nonalcoholic steatohepatitis (NASH), which will soon become the leading indication for liver transplantation. Here, we review the recent developments in the use of three-dimensional (3D) liver organoids as a model to study NAFLD and NASH pathophysiology and possible treatments. Various techniques that are currently used to make liver organoids are discussed, such as the use of induced pluripotent stem cells versus primary cell lines and human versus murine cells. Moreover, methods for inducing lipid droplet accumulation and fibrosis to model NAFLD are explored. Finally, the limitations specific to the 3D organoid model for NAFLD/NASH are reviewed, highlighting the need for further development of multilineage models to include hepatic nonparenchymal cells and immune cells. The ultimate goal is to be able to accurately recapitulate the complex liver microenvironment in which NAFLD develops and progresses to NASH.

The Effects of Taurocholic Acid on Biliary Damage and Liver Fibrosis Are Mediated by Calcitonin-Gene-Related Peptide Signaling.

BACKGROUND & AIMS: Cholangiocytes are the target cells of liver diseases that are characterized by biliary senescence (evidenced by enhanced levels of senescence-associated secretory phenotype, SASP, e.g., TGF-ß1), and liver inflammation and fibrosis accompanied by altered bile acid (BA) homeostasis. Taurocholic acid (TC) stimulates biliary hyperplasia by activation of 3',5'-cyclic cyclic adenosine monophosphate (cAMP) signaling, thereby preventing biliary damage (caused by cholinergic/adrenergic denervation) through enhanced liver angiogenesis. Also: (i) α-calcitonin gene-related peptide (α-CGRP, which activates the calcitonin receptor-like receptor, CRLR), stimulates biliary proliferation/senescence and liver fibrosis by enhanced biliary secretion of SASPs; and (ii) knock-out of α-CGRP reduces these phenotypes by decreased cAMP levels in cholestatic models. We aimed to demonstrate that TC effects on liver phenotypes are dependent on changes in the α-CGRP/CALCRL/cAMP/PKA/ERK1/2/TGF-ß1/VEGF axis. METHODS: Wild-type and α-CGRP-/- mice were fed with a control (BAC) or TC diet for 1 or 2 wk. We measured: (i) CGRP levels by both ELISA kits in serum and by qPCR in isolated cholangiocytes (CALCA gene for α-CGRP); (ii) CALCRL immunoreactivity by immunohistochemistry (IHC) in liver sections; (iii) liver histology, intrahepatic biliary mass, biliary senescence (by ß-GAL staining and double immunofluorescence (IF) for p16/CK19), and liver fibrosis (by Red Sirius staining and double IF for collagen/CK19 in liver sections), as well as by qPCR for senescence markers in isolated cholangiocytes; and (iv) phosphorylation of PKA/ERK1/2, immunoreactivity of TGF-ß1/TGF- ßRI and angiogenic factors by IHC/immunofluorescence in liver sections and qPCR in isolated cholangiocytes. We measured changes in BA composition in total liver by liquid chromatography/mass spectrometry. RESULTS: TC feeding increased CALCA expression, biliary damage, and liver inflammation and fibrosis, as well as phenotypes that were associated with enhanced immunoreactivity of the PKA/ERK1/2/TGF-ß1/TGF-ßRI/VEGF axis compared to BAC-fed mice and phenotypes that were reversed in α-CGRP-/- mice fed TC coupled with changes in hepatic BA composition. CONCLUSION: Modulation of the TC/ α-CGRP/CALCRL/PKA/ERK1/2/TGF-ß1/VEGF axis may be important in the management of cholangiopathies characterized by BA accumulation.

Molecular Mechanisms Linking Risk Factors to Cholangiocarcinoma Development.

The poor prognosis of cholangiocarcinoma in humans is related to several factors, such as (i) the heterogeneity of the disease, (ii) the late onset of symptoms and (iii) the limited comprehension of the carcinogenic pathways determining neoplastic changes, which all limit the pursuit of appropriate treatment. Several risk factors have been recognized, including different infective, immune-mediated, and dysmorphogenic disorders of the biliary tree. In this review, we report the details of possible mechanisms that lead a specific premalignant pathological condition to become cholangiocarcinoma. For instance, during liver fluke infection, factors secreted from the worms may play a major role in pathogenesis. In primary sclerosing cholangitis, deregulation of histamine and bile-acid signaling may determine important changes in cellular pathways. The study of these molecular events may also shed some light on the pathogenesis of sporadic (unrelated to risk factors) forms of cholangiocarcinoma, which represent the majority (nearly 75%) of cases.

The Functional Roles of Immune Cells in Primary Liver Cancer.

Primary liver cancer includes hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Incidence of liver cancer has been increasing in recent years, and the 5-year survival is <20%. HCC and CCA are often accompanied with a dense stroma coupled with infiltrated immune cells, which is referred to as the tumor microenvironment. Populations of specific immune cells, such as high density of CD163+ macrophages and low density of CD8+ T cells, are associated with prognosis and survival rates in both HCC and CCA. Immune cells in the tumor microenvironment can be a therapeutic target for liver cancer treatments. Previous studies have introduced immunotherapy using immune checkpoint inhibitors, pulsed dendritic cells, or transduced T cells, to enhance cytotoxicity of immune cells and inhibit tumor growth. This review summarizes current understanding of the roles of immune cells in primary liver cancer covering HCC and CCA.

Cyclic AMP Signaling in Biliary Proliferation: A Possible Target for Cholangiocarcinoma Treatment?

Cholangiocarcinoma is a lethal disease with scarce response to current systemic therapy. The rare occurrence and large heterogeneity of this cancer, together with poor knowledge of its molecular mechanisms, are elements contributing to the difficulties in finding an appropriate cure. Cholangiocytes (and their cellular precursors) are considered the liver component giving rise to cholangiocarcinoma. These cells respond to several hormones, neuropeptides and molecular stimuli employing the cAMP/PKA system for the translation of messages in the intracellular space. For instance, in physiological conditions, stimulation of the secretin receptor determines an increase of intracellular levels of cAMP, thus activating a series of molecular events, finally determining in bicarbonate-enriched choleresis. However, activation of the same receptor during cholangiocytes' injury promotes cellular growth again, using cAMP as the second messenger. Since several scientific pieces of evidence link cAMP signaling system to cholangiocytes' proliferation, the possible changes of this pathway during cancer growth also seem relevant. In this review, we summarize the current findings regarding the cAMP pathway and its role in biliary normal and neoplastic cell proliferation. Perspectives for targeting the cAMP machinery in cholangiocarcinoma therapy are also discussed.

Feedback Signaling between Cholangiopathies, Ductular Reaction, and Non-Alcoholic Fatty Liver Disease.

Fatty liver diseases, such as non-alcoholic fatty liver disease (NAFLD), are global health disparities, particularly in the United States, as a result of cultural eating habits and lifestyle. Pathological studies on NAFLD have been mostly focused on hepatocytes and other inflammatory cell types; however, the impact of other biliary epithelial cells (i.e., cholangiocytes) in the promotion of NAFLD is growing. This review article will discuss how cholestatic injury and cholangiocyte activity/ductular reaction influence NAFLD progression. Furthermore, this review will provide informative details regarding the fundamental properties of cholangiocytes and bile acid signaling that can influence NAFLD. Lastly, studies relating to the pathogenesis of NAFLD, cholangiopathies, and ductular reaction will be analyzed to help gain insight for potential therapies.

Current Advances in Basic and Translational Research of Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a type of biliary tract cancer emerging from the biliary tree. CCA is the second most common primary liver cancer after hepatocellular carcinoma and is highly aggressive resulting in poor prognosis and patient survival. Treatment options for CCA patients are limited since early diagnosis is challenging, and the efficacy of chemotherapy or radiotherapy is also limited because CCA is a heterogeneous malignancy. Basic research is important for CCA to establish novel diagnostic testing and more effective therapies. Previous studies have introduced new techniques and methodologies for animal models, in vitro models, and biomarkers. Recent experimental strategies include patient-derived xenograft, syngeneic mouse models, and CCA organoids to mimic heterogeneous CCA characteristics of each patient or three-dimensional cellular architecture in vitro. Recent studies have identified various novel CCA biomarkers, especially non-coding RNAs that were associated with poor prognosis or metastases in CCA patients. This review summarizes current advances and limitations in basic and translational studies of CCA.

Organoids and Spheroids as Models for Studying Cholestatic Liver Injury and Cholangiocarcinoma.

Cholangiopathies, such as primary sclerosing cholangitis, biliary atresia, and cholangiocarcinoma, have limited experimental models. Not only cholangiocytes but also other hepatic cells including hepatic stellate cells and macrophages are involved in the pathophysiology of cholangiopathies, and these hepatic cells orchestrate the coordinated response against diseased conditions. Classic two-dimensional monolayer cell cultures do not resemble intercellular cell-to-cell interaction and communication; however, three-dimensional cell culture systems, such as organoids and spheroids, can mimic cellular interaction and architecture between hepatic cells. Previous studies have demonstrated the generation of hepatic or biliary organoids/spheroids using various cell sources including pluripotent stem cells, hepatic progenitor cells, primary cells from liver biopsies, and immortalized cell lines. Gene manipulation, such as transfection and transduction can be performed in organoids, and established organoids have functional characteristics which can be suitable for drug screening. This review summarizes current methodologies for organoid/spheroid formation and a potential for three-dimensional hepatic cell cultures as in vitro models of cholangiopathies.

Cholangiocarcinoma: bridging the translational gap from preclinical to clinical development and implications for future therapy.

Introduction: Cholangiocarcinoma (CCA) is a devastating liver tumor with a poor prognosis. While less than 50% of the patients with CCA may benefit from surgical resection, the rest undergoes chemotherapy with disappointing results (mean survival <2 years). Alternative pharmacological treatments are needed to improve the outcomes in patients with CCA.Areas covered: In this review, we discuss CCA-related (1) experimental systems used in preclinical studies; (2) pharmacological targets identified by genetic analysis; (3) results obtained in preliminary trials in human with their pros and cons; and (4) possible targeting of endocrinal modulation. A PubMed bibliographic search matching the term 'cholangiocarcinoma' with 'experimental model', 'preclinical model', 'genetic target', 'targeted therapy', 'clinical trial', or 'translational research' was conducted and manuscripts published between 2010 and 2020 were retrieved for reading and reviewing.Expert opinion: Several factors contribute to the translational gap between bench research and clinical practice in CCA. The tumor heterogeneity, lack of a preclinical model recapitulating the different features of CCA, and difficult patient enrollment in clinical trials are elements to consider for basic and clinical research in CCA. Establishment of international networks formed by experts in the field of CCA may improve future research and its translational findings on patients.

The interplay between mast cells, pineal gland, and circadian rhythm: Links between histamine, melatonin, and inflammatory mediators.

Our daily rhythmicity is controlled by a circadian clock with a specific set of genes located in the suprachiasmatic nucleus in the hypothalamus. Mast cells (MCs) are major effector cells that play a protective role against pathogens and inflammation. MC distribution and activation are associated with the circadian rhythm via two major pathways, IgE/FcεRI- and IL-33/ST2-mediated signaling. Furthermore, there is a robust oscillation between clock genes and MC-specific genes. Melatonin is a hormone derived from the amino acid tryptophan and is produced primarily in the pineal gland near the center of the brain, and histamine is a biologically active amine synthesized from the decarboxylation of the amino acid histidine by the L-histidine decarboxylase enzyme. Melatonin and histamine are previously reported to modulate circadian rhythms by pathways incorporating various modulators in which the nuclear factor-binding near the κ light-chain gene in B cells, NF-κB, is the common key factor. NF-κB interacts with the core clock genes and disrupts the production of pro-inflammatory cytokine mediators such as IL-6, IL-13, and TNF-α. Currently, there has been no study evaluating the interdependence between melatonin and histamine with respect to circadian oscillations in MCs. Accumulating evidence suggests that restoring circadian rhythms in MCs by targeting melatonin and histamine via NF-κB may be promising therapeutic strategy for MC-mediated inflammatory diseases. This review summarizes recent findings for circadian-mediated MC functional roles and activation paradigms, as well as the therapeutic potentials of targeting circadian-mediated melatonin and histamine signaling in MC-dependent inflammatory diseases.

Kupffer Cells: Inflammation Pathways and Cell-Cell Interactions in Alcohol-Associated Liver Disease.

Chronic alcohol consumption is linked to the development of alcohol-associated liver disease (ALD). This disease is characterized by a clinical spectrum ranging from steatosis to hepatocellular carcinoma. Several cell types are involved in ALD progression, including hepatic macrophages. Kupffer cells (KCs) are the resident macrophages of the liver involved in the progression of ALD by activating pathways that lead to the production of cytokines and chemokines. In addition, KCs are involved in the production of reactive oxygen species. Reactive oxygen species are linked to the induction of oxidative stress and inflammation in the liver. These events are activated by the bacterial endotoxin, lipopolysaccharide, that is released from the gastrointestinal tract through the portal vein to the liver. Lipopolysaccharide is recognized by receptors on KCs that are responsible for triggering several pathways that activate proinflammatory cytokines involved in alcohol-induced liver injury. In addition, KCs activate hepatic stellate cells that are involved in liver fibrosis. Novel strategies to treat ALD aim at targeting Kupffer cells. These interventions modulate Kupffer cell activation or macrophage polarization. Evidence from mouse models and early clinical studies in patients with ALD injury supports the notion that pathogenic macrophage subsets can be successfully translated into novel treatment options for patients with this disease.

Concise Review: Functional Roles and Therapeutic Potentials of Long Non-coding RNAs in Cholangiopathies.

Long non-coding RNAs (lncRNAs) are RNAs with lengths exceeding 200 nucleotides that are not translated into proteins. It is well-known that small non-coding RNAs, such as microRNAs (miRNAs), regulate gene expression and play an important role in cholangiopathies. Recent studies have demonstrated that lncRNAs may also play a key role in the pathophysiology of cholangiopathies. Patients with cholangiopathies often develop cholangiocarcinoma (CCA), which is cholangiocyte-derived cancer, in the later stage. Cholangiocytes are a primary target of therapies for cholangiopathies and CCA development. Previous studies have demonstrated that expression levels of lncRNAs are altered in the liver of cholangiopathies or CCA tissues. Some lncRNAs regulate gene expression by inhibiting functions of miRNAs leading to diseased liver conditions or CCA progression, suggesting that lncRNAs could be a novel therapeutic target for those disorders. This review summarizes current understandings of functional roles of lncRNAs in cholangiopathies and seek their potentials for novel therapies.

Neuroendocrine Changes in Cholangiocarcinoma Growth.

Cholangiocarcinoma (CCA) is a highly aggressive malignancy that emerges from the biliary tree. There are three major classes of CCA-intrahepatic, hilar (perihilar), or distal (extrahepatic)-according to the location of tumor development. Although CCA tumors are mainly derived from biliary epithelia (i.e., cholangiocytes), CCA can be originated from other cells, such as hepatic progenitor cells and hepatocytes. This heterogeneity of CCA may be responsible for poor survival rates of patients, limited effects of chemotherapy and radiotherapy, and the lack of treatment options and novel therapies. Previous studies have identified a number of neuroendocrine mediators, such as hormones, neuropeptides, and neurotransmitters, as well as corresponding receptors. The mediator/receptor signaling pathways play a vital role in cholangiocyte proliferation, as well as CCA progression and metastases. Agonists or antagonists for candidate pathways may lead to the development of novel therapies for CCA patients. However, effects of mediators may differ between healthy or cancerous cholangiocytes, or between different subtypes of receptors. This review summarizes current understandings of neuroendocrine mediators and their functional roles in CCA.

Cholangiocarcinoma: novel therapeutic targets.

Introduction: Cholangiocarcinoma (CCA) is a liver cancer derived from the biliary tree with a less than 30% five-year survival rate. Early diagnosis of CCA is challenging and treatment options are limited. Some CCA patients have genetic mutations and several therapeutic drugs or antibodies have been introduced to target abnormally expressed proteins. However, CCA is heterogeneous and patients often present with drug resistance which is attributed to multiple mutations or other factors. Novel approaches and methodologies for CCA treatments are in demand.Area covered: This review summarizes current approaches for CCA treatments leading to the development of novel therapeutic drugs or tools for human CCA patients. A literature search was conducted in PubMed utilizing the combination of the searched term 'cholangiocarcinoma' with other keywords such as 'miRNA', 'FGFR', 'immunotherapy' or 'microenvironment'. Papers published within 2015-2019 were obtained for reading.Expert opinion: Preclinical studies have demonstrated promising therapeutic approaches that target various cells or pathways. Recent studies have revealed that hepatic cells coordinate to promote CCA tumor progression in the tumor microenvironment, which may be a new therapeutic target. Although further studies are required, novel therapeutic tools such as extracellular vesicles could be utilized to manage CCA and its microenvironment.

Melatonin and circadian rhythms in liver diseases: Functional roles and potential therapies.

Circadian rhythms and clock gene expressions are regulated by the suprachiasmatic nucleus in the hypothalamus, and melatonin is produced in the pineal gland. Although the brain detects the light through retinas and regulates rhythms and melatonin secretion throughout the body, the liver has independent circadian rhythms and expressions as well as melatonin production. Previous studies indicate the association between circadian rhythms with various liver diseases, and disruption of rhythms or clock gene expression may promote liver steatosis, inflammation, or cancer development. It is well known that melatonin has strong antioxidant effects. Alcohol drinking or excess fatty acid accumulation produces reactive oxygen species and oxidative stress in the liver leading to liver injuries. Melatonin administration protects these oxidative stress-induced liver damage and improves liver conditions. Recent studies have demonstrated that melatonin administration is not limited to antioxidant effects and it has various other effects contributing to the management of liver conditions. Accumulating evidence suggests that restoring circadian rhythms or expressions as well as melatonin supplementation may be promising therapeutic strategies for liver diseases. This review summarizes recent findings for the functional roles and therapeutic potentials of circadian rhythms and melatonin in liver diseases.

Preclinical insights into cholangiopathies: disease modeling and emerging therapeutic targets.

INTRODUCTION: The common predominant clinical features of cholangiopathies such as primary sclerosing cholangitis (PSC), primary biliary cholangitis (PBC), and biliary atresia (BA) are biliary damage/senescence and liver fibrosis. Curative therapies are lacking, and liver transplantation is the only option. An understanding of the mechanisms and pathogenesis is needed to develop novel therapies. Previous studies have developed various disease-based research models and have identified candidate therapeutic targets. Areas covered: This review summarizes recent studies performed in preclinical models of cholangiopathies and the current understanding of the pathophysiology representing potential targets for novel therapies. A literature search was conducted in PubMed using the combination of the searched term 'cholangiopathies' with one or two keywords including 'model', 'cholangiocyte', 'animal', or 'fibrosis'. Papers published within five years were obtained. Expert opinion: Access to appropriate research models is a key challenge in cholangiopathy research; establishing more appropriate models for PBC is an important goal. Several preclinical studies have demonstrated promising results and have led to novel therapeutic approaches, especially for PSC. Further studies on the pathophysiology of PBC and BA are necessary to identify candidate targets. Innovative therapeutic approaches such as stem cell transplantation have been introduced, and those therapies could be applied to PSC, PBC, and BA.

Mechanisms of cholangiocyte responses to injury.

Cholangiocytes, epithelial cells that line the biliary epithelium, are the primary target cells for cholangiopathies including primary sclerosing cholangitis and primary biliary cholangitis. Quiescent cholangiocytes respond to biliary damage and acquire an activated neuroendocrine phenotype to maintain the homeostasis of the liver. The typical response of cholangiocytes is proliferation leading to bile duct hyperplasia, which is a characteristic of cholestatic liver diseases. Current studies have identified various signaling pathways that are associated with cholangiocyte proliferation/loss and liver fibrosis in cholangiopathies using human samples and rodent models. Although recent studies have demonstrated that extracellular vesicles and microRNAs could be mediators that regulate these messenger/receptor axes, further studies are required to confirm their roles. This review summarizes current studies of biliary response and cholangiocyte proliferation during cholestatic liver injury with particular emphasis on the secretin/secretin receptor axis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.