Vascular adhesion protein-1 blockade in primary sclerosing cholangitis: Open-label, multicenter, single-arm, phase II trial.

BACKGROUND: Primary sclerosing cholangitis is a progressive inflammatory liver disease characterized by biliary and liver fibrosis. Vascular adhesion protein-1 (VAP-1) is important in the inflammatory process driving liver fibrosis. We evaluated the safety and efficacy of VAP-1 blockade with a monoclonal antibody (timolumab, BTT1023) in patients with primary sclerosing cholangitis. METHODS: BUTEO was a prospective, single-arm, open-label, multicenter, phase II trial, conducted in 6 centers in the United Kingdom. Patients with primary sclerosing cholangitis aged 18-75 years had an alkaline phosphatase value of >1.5 times the upper limit of normal. The dose-confirmatory stage aimed to confirm the safety of timolumab through the incidence of dose-limiting toxicity and sufficient trough levels of circulating antibody to block VAP-1 function. The primary outcome of the dose-expansion portion of the trial was patient's response to timolumab at day 99, as measured by a reduction in serum alkaline phosphatase by 25% or more from baseline to day 99. RESULTS: Twenty-three patients were recruited: 7 into the initial dose-confirmatory stage and a further 16 into an expansion stage. Timolumab (8 mg/kg) was confirmed to be safe for the duration of administration with sufficient circulating levels. Only 2 of the 18 evaluable patients (11.1%) achieved a reduction in alkaline phosphatase levels of 25% or more, and both the proportion of circulating inflammatory cell populations and biomarkers of fibrosis remained unchanged from baseline. CONCLUSIONS: The BUTEO trial confirmed 8 mg/kg timolumab had no short-term safety signals and resulted in sufficient circulating levels of VAP-1 blocking timolumab. However, the trial was stopped after an interim assessment due to a lack of efficacy as determined by no significant change in serum liver tests.

Chronic activation of human cardiac fibroblasts in vitro attenuates the reversibility of the myofibroblast phenotype.

Activation of cardiac fibroblasts and differentiation to myofibroblasts underlies development of pathological cardiac fibrosis, leading to arrhythmias and heart failure. Myofibroblasts are characterised by increased α-smooth muscle actin (α-SMA) fibre expression, secretion of collagens and changes in proliferation. Transforming growth factor-beta (TGF-ß) and increased mechanical stress can initiate myofibroblast activation. Reversibility of the myofibroblast phenotype has been observed in murine cells but has not been explored in human cardiac fibroblasts. In this study, chronically activated adult primary human ventricular cardiac fibroblasts and human induced pluripotent stem cell derived cFbs (hiPSC-cFbs) were used to investigate the potential for reversal of the myofibroblast phenotype using either subculture on soft substrates or TGF-ß receptor inhibition. Culture on softer plates (25 or 2 kPa Young's modulus) did not alter proliferation or reduce expression of α-SMA and collagen 1. Similarly, culture of myofibroblasts in the presence of TGF-ß inhibitor did not reverse myofibroblasts back to a quiescent phenotype. Chronically activated hiPSC-cFbs also showed attenuated response to TGF-ß receptor inhibition and inability to reverse to quiescent fibroblast phenotype. Our data demonstrate substantial loss of TGF-ß signalling plasticity as well as a loss of feedback from the surrounding mechanical environment in chronically activated human myofibroblasts.

Targeting IL-36 improves age-related coronary microcirculatory dysfunction and attenuates myocardial ischemia/reperfusion injury in mice.

Following myocardial infarction (MI), elderly patients have a poorer prognosis than younger patients, which may be linked to increased coronary microvessel susceptibility to injury. Interleukin-36 (IL-36), a newly discovered proinflammatory member of the IL-1 superfamily, may mediate this injury, but its role in the injured heart is currently not known. We first demonstrated the presence of IL-36(α/ß) and its receptor (IL-36R) in ischemia/reperfusion-injured (IR-injured) mouse hearts and, interestingly, noted that expression of both increased with aging. An intravital model for imaging the adult and aged IR-injured beating heart in real time in vivo was used to demonstrate heightened basal and injury-induced neutrophil recruitment, and poorer blood flow, in the aged coronary microcirculation when compared with adult hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow, and reduced infarct size in both adult and aged mice. This may be mechanistically explained by attenuated endothelial oxidative damage and VCAM-1 expression in IL-36Ra-treated mice. Our findings of an enhanced age-related coronary microcirculatory dysfunction in reperfused hearts may explain the poorer outcomes in elderly patients following MI. Since targeting the IL-36/IL-36R pathway was vasculoprotective in aged hearts, it may potentially be a therapy for treating MI in the elderly population.

Suppressor CD4+ T cells expressing HLA-G are expanded in the peripheral blood from patients with acute decompensation of cirrhosis.

OBJECTIVE: Identifying components of immuneparesis, a hallmark of chronic liver failure, is crucial for our understanding of complications in cirrhosis. Various suppressor CD4+ T cells have been established as potent inhibitors of systemic immune activation. Here, we establish the presence, regulation and mechanism of action of a suppressive CD4+ T cell subset expressing human leucocyte antigen G (HLA-G) in patients with acute decompensation of cirrhosis (AD). DESIGN: Flow cytometry was used to determine the proportion and immunophenotype of CD4+HLA-G+ T cells from peripheral blood of 20 healthy controls (HCs) and 98 patients with cirrhosis (28 with stable cirrhosis (SC), 20 with chronic decompensated cirrhosis (CD) and 50 with AD). Transcriptional and functional signatures of cell-sorted CD4+HLA-G+ cells were delineated by NanoString technology and suppression assays, respectively. The role of immunosuppressive cytokine interleukin (IL)-35 in inducing this population was investigated through in vitro blockade experiments. Immunohistochemistry (IHC) and cultures of primary human Kupffer cells (KCs) were performed to assess cellular sources of IL-35. HLA-G-mediated T cell suppression was explored using neutralising antibodies targeting co-inhibitory pathways. RESULTS: Patients with AD were distinguished by an expansion of a CD4+HLA-G+CTLA-4+IL-35+ immunosuppressive population associated with disease severity, clinical course of AD, infectious complications and poor outcome. Transcriptomic analyses excluded the possibility that these were thymic-derived regulatory T cells. IHC analyses and in vitro cultures demonstrate that KCs represent a potent source of IL-35 which can induce the observed HLA-G+ phenotype. These exert cytotoxic T lymphocyte antigen-4-mediated impaired responses in T cells paralleled by an HLA-G-driven downregulation of T helper 17-related cytokines. CONCLUSION: We have identified a cytokine-driven peripherally derived suppressive population that may contribute to immuneparesis in AD.

Role of liver support systems in the management of post hepatectomy liver failure: A systematic review of the literature.

BACKGROUNDS/AIMS: Post-hepatectomy liver failure (PHLF) is a serious complication following liver resection, with limited treatment options, and is associated with high mortality. There is a need to evaluate the role of systems that support the function of the liver after PHLF. AIMS: The aim of this study was to review the literature and summarize the role of liver support systems (LSS) in the management of PHLF. Publications of interest were identified using systematically designed searches. Following screening, data from the relevant publications was extracted, and pooled where possible. FINDINGS: Systematic review identified nine studies, which used either Plasma Exchange (PE) or Molecular Adsorbent Recirculating System (MARS) as LSS after PHLF. Across all studies, the pooled 90-day mortality rate was 38% (95% CI: 9-70%). However, there was substantial heterogeneity, likely since studies used a variety of definitions for PHLF, and had different selection criteria for patient eligibility for LSS treatment. CONCLUSIONS: The current evidence is insufficient to recommend LSS for the routine management of severe PHLF, with the current literature consisting of only a limited number of studies. There is a definite need for larger, multicenter, prospective studies, evaluating the conventional and newer modalities of support systems, with a view to improve the outcomes in this group of patients.

Cardiovascular Effects of Unilateral Nephrectomy in Living Kidney Donors at 5 Years.

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Discovery of Highly Selective Inhibitors of Calmodulin-Dependent Kinases That Restore Insulin Sensitivity in the Diet-Induced Obesity in Vivo Mouse Model.

Polymorphisms in the region of the calmodulin-dependent kinase isoform D (CaMK1D) gene are associated with increased incidence of diabetes, with the most common polymorphism resulting in increased recognition by transcription factors and increased protein expression. While reducing CaMK1D expression has a potentially beneficial effect on glucose processing in human hepatocytes, there are no known selective inhibitors of CaMK1 kinases that can be used to validate or translate these findings. Here we describe the development of a series of potent, selective, and drug-like CaMK1 inhibitors that are able to provide significant free target cover in mouse models and are therefore useful as in vivo tool compounds. Our results show that a lead compound from this series improves insulin sensitivity and glucose control in the diet-induced obesity mouse model after both acute and chronic administration, providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.

Clinical Potential of Targeting Fibroblast Growth Factor-23 and αKlotho in the Treatment of Uremic Cardiomyopathy.

Chronic kidney disease is highly prevalent, affecting 10% to 15% of the adult population worldwide and is associated with increased cardiovascular morbidity and mortality. As chronic kidney disease worsens, a unique cardiovascular phenotype develops characterized by heart muscle disease, increased arterial stiffness, atherosclerosis, and hypertension. Cardiovascular risk is multifaceted, but most cardiovascular deaths in patients with advanced chronic kidney disease are caused by heart failure and sudden cardiac death. While the exact drivers of these deaths are unknown, they are believed to be caused by uremic cardiomyopathy: a specific pattern of myocardial hypertrophy, fibrosis, with both diastolic and systolic dysfunction. Although the pathogenesis of uremic cardiomyopathy is likely to be multifactorial, accumulating evidence suggests increased production of fibroblast growth factor-23 and αKlotho deficiency as potential major drivers of cardiac remodeling in patients with uremic cardiomyopathy. In this article we review the increasing understanding of the physiology and clinical aspects of uremic cardiomyopathy and the rapidly increasing knowledge of the biology of both fibroblast growth factor-23 and αKlotho. Finally, we discuss how dissection of these pathological processes is aiding the development of therapeutic options, including small molecules and antibodies, directly aimed at improving the cardiovascular outcomes of patients with chronic kidney disease and end-stage renal disease.

The Role of Myeloid-Derived Cells in the Progression of Liver Disease.

Control of homeostasis and rapid response to tissue damage in the liver is orchestrated by crosstalk between resident and infiltrating inflammatory cells. A crucial role for myeloid cells during hepatic injury and repair has emerged where resident Kupffer cells, circulating monocytes, macrophages, dendritic cells and neutrophils control local tissue inflammation and regenerative function to maintain tissue architecture. Studies in humans and rodents have revealed a heterogeneous population of myeloid cells that respond to the local environment by either promoting regeneration or driving the inflammatory processes that can lead to hepatitis, fibrogenesis, and the development of cirrhosis and malignancy. Such plasticity of myeloid cell responses presents unique challenges for therapeutic intervention strategies and a greater understanding of the underlying mechanisms is needed. Here we review the role of myeloid cells in the establishment and progression of liver disease and highlight key pathways that have become the focus for current and future therapeutic strategies.

Intrahepatic macrophage populations in the pathophysiology of primary sclerosing cholangitis.

BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by progressive inflammatory and fibrotic injury to the biliary tree. We sought to further delineate the contribution of macrophage lineages in PSC pathobiology. METHODS: Human liver tissues and/or blood samples from patients with PSC, primary biliary cholangitis, other non-cholestatic/non-autoimmune diseases, including alcohol-related liver disease and non-alcoholic steatohepatitis, as well as normal liver, were sourced from our liver transplantation program. Liver fibrosis was studied using Van Gieson staining, while the frequencies of infiltrating monocyte and macrophage lineages, both in the circulation and the liver, were investigated by flow cytometry, including the expression of TGR-5, a G protein-coupled receptor (GPBAR1/TGR-5). RESULTS: Significantly higher frequencies of CD68+CD206+ macrophages were detected in the livers of patients with PSC (median 19.17%; IQR 7.25-32.8%; n = 15) compared to those of patients with other liver diseases (median 12.05%; IQR 5.61-16.03%; n = 12; p = 0.0373). CD16+ monocytes, including both intermediate (CD14+CD16++) and non-classical (CD14dimCD16++) monocytes, were preferentially recruited into chronically diseased livers, with the highest recruitment ratios in PSC (median 15.83%; IQR 9.66-29.5%; n = 15), compared to other liver diseases (median 6.66%; IQR 2.88-11.64%, n = 14, p = 0.0152). The expression of TGR-5 on CD68+ intrahepatic macrophages was increased in chronic liver disease; TGR-5 expression on intrahepatic macrophages was highest in PSC (median 36.32%; IQR 17.71-63.61%; n = 6) and most TGR-5+ macrophages were CD68+CD206+ macrophages. CONCLUSIONS: Underlying a potential role for macrophages in PSC pathobiology, we demonstrate, using patient-derived tissue, increased CD16+ monocyte recruitment and a higher frequency of CD68+CD206+ macrophages in the livers of patients with PSC; the CD68+CD206+ macrophage subset was associated with significantly higher TGR-5 expression in PSC. LAY SUMMARY: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease associated with progressive inflammation of the bile duct, leading to fibrosis and end-stage liver disease. In this study we explore the role of a type of immune cell, the macrophage, in contributing to PSC as a disease, hoping that our findings direct scientists towards new treatment targets. Our findings based on human liver and blood analyses demonstrate a greater frequency of a particular subset of immune cell, the CD68+CD206+ macrophage, with significantly higher TGR-5 expression on this subset in PSC.

Inhibition of semicarbazide-sensitive amine oxidase reduces atherosclerosis in apolipoprotein E-deficient mice.

Inflammation, oxidative stress, and formation of advanced glycated end products (AGEs) and advanced lipoxidation end products (ALEs) are important for atherosclerosis. Vascular adhesion protein-1 (VAP-1) participates in inflammation and has semicarbazide-sensitive amine oxidase (SSAO) activity, which catalyzes oxidative deamination to produce hydrogen peroxide and aldehydes, leading to generation of AGEs and ALEs. However, the effect of VAP-1/SSAO inhibition on atherosclerosis remains controversial, and no studies used coronary angiography to evaluate if plasma VAP-1/SSAO is a biomarker for coronary artery disease (CAD). Here, we examined if plasma VAP-1/SSAO is a biomarker for CAD diagnosed by coronary angiography in humans and investigated the effect of VAP-1/SSAO inhibition by a specific inhibitor PXS-4728A on atherosclerosis in cell and animal models. In the study, VAP-1/SSAO expression was increased in plaques in humans and in apolipoprotein E (ApoE)-deficient mice, and colocalized with vascular endothelial cells and smooth muscle cells (SMCs). Patients with CAD had higher plasma VAP-1/SSAO than those without CAD. Plasma VAP-1/SSAO was positively associated with the extent of CAD. In ApoE-deficient mice, VAP-1/SSAO inhibition reduced atheroma and decreased oxidative stress. VAP-1/SSAO inhibition attenuated the expression of adhesion molecules, chemoattractant proteins, and proinflammatory cytokines in the aorta, and suppressed monocyte adhesion and transmigration across human umbilical vein endothelial cells. Consequently, the expression of markers for macrophage recruitment and activation in plaques was decreased by VAP-1/SSAO inhibition. Besides, VAP-1/SSAO inhibition suppressed proliferation and migration of A7r5 SMC. Our data suggest that plasma VAP-1/SSAO is a novel biomarker for the presence and the extent of CAD in humans. VAP-1/SSAO inhibition by PXS-4728A is a potential treatment for atherosclerosis.

MerTK expressing hepatic macrophages promote the resolution of inflammation in acute liver failure.

OBJECTIVE: Acute liver failure (ALF) is characterised by overwhelming hepatocyte death and liver inflammation with massive infiltration of myeloid cells in necrotic areas. The mechanisms underlying resolution of acute hepatic inflammation are largely unknown. Here, we aimed to investigate the impact of Mer tyrosine kinase (MerTK) during ALF and also examine how the microenvironmental mediator, secretory leucocyte protease inhibitor (SLPI), governs this response. DESIGN: Flow cytometry, immunohistochemistry, confocal imaging and gene expression analyses determined the phenotype, functional/transcriptomic profile and tissue topography of MerTK+ monocytes/macrophages in ALF, healthy and disease controls. The temporal evolution of macrophage MerTK expression and its impact on resolution was examined in APAP-induced acute liver injury using wild-type (WT) and Mer-deficient (Mer-/-) mice. SLPI effects on hepatic myeloid cells were determined in vitro and in vivo using APAP-treated WT mice. RESULTS: We demonstrate a significant expansion of resolution-like MerTK+HLA-DRhigh cells in circulatory and tissue compartments of patients with ALF. Compared with WT mice which show an increase of MerTK+MHCIIhigh macrophages during the resolution phase in ALF, APAP-treated Mer-/- mice exhibit persistent liver injury and inflammation, characterised by a decreased proportion of resident Kupffer cells and increased number of neutrophils. Both in vitro and in APAP-treated mice, SLPI reprogrammes myeloid cells towards resolution responses through induction of a MerTK+HLA-DRhigh phenotype which promotes neutrophil apoptosis and their subsequent clearance. CONCLUSIONS: We identify a hepatoprotective, MerTK+, macrophage phenotype that evolves during the resolution phase following ALF and represents a novel immunotherapeutic target to promote resolution responses following acute liver injury.

Vascular adhesion protein-1 is elevated in primary sclerosing cholangitis, is predictive of clinical outcome and facilitates recruitment of gut-tropic lymphocytes to liver in a substrate-dependent manner.

OBJECTIVE: Primary sclerosing cholangitis (PSC) is the classical hepatobiliary manifestation of IBD. This clinical association is linked pathologically to the recruitment of mucosal T cells to the liver, via vascular adhesion protein (VAP)-1-dependent enzyme activity. Our aim was to examine the expression, function and enzymatic activation of the ectoenzyme VAP-1 in patients with PSC. DESIGN: We examined VAP-1 expression in patients with PSC, correlated levels with clinical characteristics and determined the functional consequences of enzyme activation by specific enzyme substrates on hepatic endothelium. RESULTS: The intrahepatic enzyme activity of VAP-1 was elevated in PSC versus immune-mediated disease controls and non-diseased liver (p<0.001). The adhesion of gut-tropic α4ß7+lymphocytes to hepatic endothelial cells in vitro under flow was attenuated by 50% following administration of the VAP-1 inhibitor semicarbazide (p<0.01). Of a number of natural VAP-1 substrates tested, cysteamine-which can be secreted by inflamed colonic epithelium and gut bacteria-was the most efficient (yielded the highest enzymatic rate) and efficacious in its ability to induce expression of functional mucosal addressin cell adhesion molecule-1 on hepatic endothelium. In a prospectively evaluated patient cohort with PSC, elevated serum soluble (s)VAP-1 levels predicted poorer transplant-free survival for patients, independently (HR: 3.85, p=0.003) and additively (HR: 2.02, p=0.012) of the presence of liver cirrhosis. CONCLUSIONS: VAP-1 expression is increased in PSC, facilitates adhesion of gut-tropic lymphocytes to liver endothelium in a substrate-dependent manner, and elevated levels of its circulating form predict clinical outcome in patients.

SCARF-1 promotes adhesion of CD4+ T cells to human hepatic sinusoidal endothelium under conditions of shear stress.

Liver-resident cells are constantly exposed to gut-derived antigens via portal blood and, as a consequence, they express a unique repertoire of scavenger receptors. Whilst there is increasing evidence that the gut contributes to chronic inflammatory liver disease, the role of scavenger receptors in regulating liver inflammation remains limited. Here, we describe for the first time the expression of scavenger receptor class F, member 1 (SCARF-1) on hepatic sinusoidal endothelial cells (HSEC). We report that SCARF-1 shows a highly localised expression pattern and co-localised with endothelial markers on sinusoidal endothelium. Analysis of chronically inflamed liver tissue demonstrated accumulation of SCARF-1 at sites of CD4+ T cell aggregation. We then studied the regulation and functional role of SCARF-1 in HSEC and showed that SCARF-1 expression by HSEC is regulated by proinflammatory cytokines and bacterial lipopolysaccharide (LPS). Furthermore, SCARF-1 expression by HSEC, induced by proinflammatory and gut-derived factors acts as a novel adhesion molecule, present in adhesive cup structures, that specifically supports CD4+ T cells under conditions of physiological shear stress. In conclusion, we show that SCARF-1 contributes to lymphocyte subset adhesion to primary human HSEC and could play an important role in regulating the inflammatory response during chronic liver disease.

Investigating the safety and activity of the use of BTT1023 (Timolumab), in the treatment of patients with primary sclerosing cholangitis (BUTEO): A single-arm, two-stage, open-label, multi-centre, phase II clinical trial protocol.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a progressive inflammatory liver disease characterised by relentless liver fibrosis and a high unmet need for new therapies. Preventing fibrosis represents an important area of interest in the development of vital new drugs. Vascular adhesion protein-1 (VAP-1) drives inflammation in liver disease, and provision of an antibody against VAP-1 blunts fibrosis in murine models of liver injury. METHODS AND ANALYSIS: BUTEO is a single-arm, two-stage, open-label, multi-centre, phase II clinical trial. Up to 59 patients will receive treatment with anti-VAP monoclonal antibody, BTT1023, over a 78-day treatment period. Adults with PSC and a serum alkaline phosphatase (ALP) of at least 1.5 times the upper limit of normal will be included. Our primary outcome measure is a reduction in ALP by >25% from baseline to Day 99. Secondary outcome measures include safety and tolerability, changes pre therapy/post therapy in circulating serum VAP-1 as well as imaging findings. The first patient participant was recruited on 08 September 2015. ETHICS AND DISSEMINATION: This protocol has been approved by the Research Ethics Committee (REC, reference 14/EM/1272). The first REC approval date was 06 January 2015 with three subsequent approved amendments. This article refers to protocol V3.0, dated 16 March 2016. Results will be disseminated via peer-reviewed publication and presentation at international conferences. TRIAL REGISTRATION: The trial is registered with the European Medicines agency (EudraCT: 2014-002393-37), the National Institute for Health Research (Portfolio ID: 18051) and ISRCTN: 11233255. The clinicaltrials.gov identifier is NCT02239211. Pre-results.

Impaired Transmigration of Myeloid-Derived Suppressor Cells across Human Sinusoidal Endothelium Is Associated with Decreased Expression of CD13.

Human monocytic myeloid-derived suppressor cells (MO-MDSCs) within the hepatic compartment suppress inflammation and impair immune surveillance in liver cancer. It is currently not known whether recruitment of MO-MDSCs from blood via hepatic sinusoidal endothelium (HSEC) contributes to their enrichment within the hepatic compartment. We compared the transmigratory potential of MO-MDSCs and monocytes after adhesion to hepatic endothelial monolayers in flow-based assays that mimic in vivo shear stress in the sinusoids. Despite comparable binding to HSEC monolayers, proportionally fewer MO-MDSCs underwent transendothelial migration, indicating that the final steps of extravasation, where actin polymerization plays an important role, are impaired in MO-MDSCs. In this article, we found reduced levels of CD13 on MO-MDSCs, which has recently been reported to control cell motility in monocytes, alongside reduced VLA-4 expression, an integrin predominantly involved in adherence to the apical side of the endothelium. CD13 and VLA-4 blocking and activating Abs were used in flow-based adhesion assays, live-cell imaging of motility, and actin polymerization studies to confirm a role for CD13 in impaired MO-MDSC transmigration. These findings indicate that CD13 significantly contributes to tissue infiltration by MO-MDSCs and monocytes, thereby contributing to the pathogenesis of hepatic inflammation.

CD151 supports VCAM-1-mediated lymphocyte adhesion to liver endothelium and is upregulated in chronic liver disease and hepatocellular carcinoma.

CD151, a member of the tetraspanin family of receptors, is a lateral organizer and modulator of activity of several families of transmembrane proteins. It has been implicated in the development and progression of several cancers, but its role in chronic inflammatory disease is less well understood. Here we show that CD151 is upregulated by distinct microenvironmental signals in a range of chronic inflammatory liver diseases and in primary liver cancer, in which it supports lymphocyte recruitment. CD151 was highly expressed in endothelial cells of the hepatic sinusoids and neovessels developing in fibrotic septa and tumor margins. Primary cultures of human hepatic sinusoidal endothelial cells (HSECs) expressed CD151 at the cell membrane and in intracellular vesicles. CD151 was upregulated by VEGF and HepG2 conditioned media but not by proinflammatory cytokines. Confocal microscopy confirmed that CD151 colocalized with the endothelial adhesion molecule/immunoglobulin superfamily member, VCAM-1. Functional flow-based adhesion assays with primary human lymphocytes and HSECs demonstrated a 40% reduction of lymphocyte adhesion with CD151 blockade. Inhibition of lymphocyte adhesion was similar between VCAM-1 blockade and a combination of CD151/VCAM-1 blockade, suggesting a collaborative role between the two receptors. These studies demonstrate that CD151 is upregulated within the liver during chronic inflammation, where it supports lymphocyte recruitment via liver endothelium. We propose that CD151 regulates the activity of VCAM-1 during lymphocyte recruitment to the human liver and could be a novel anti-inflammatory target in chronic liver disease and hepatocellular cancer prevention.NEW & NOTEWORTHY Chronic hepatitis is characterized by lymphocyte accumulation in liver tissue, which drives fibrosis and carcinogenesis. Here, we demonstrate for the first time that the tetraspanin CD151 supports lymphocyte adhesion to liver endothelium. We show that CD151 is upregulated in chronic liver disease and hepatocellular carcinoma (HCC) and is regulated on endothelium by tissue remodeling and procarcinogenic factors. These regulatory and functional studies identify CD151 as a potential therapeutic target to treat liver fibrosis and HCC.

Human liver sinusoidal endothelial cells promote intracellular crawling of lymphocytes during recruitment: A new step in migration.

The recruitment of lymphocytes via the hepatic sinusoidal channels and positioning within liver tissue is a critical event in the development and persistence of chronic inflammatory liver diseases. The hepatic sinusoid is a unique vascular bed lined by hepatic sinusoidal endothelial cells (HSECs), a functionally and phenotypically distinct subpopulation of endothelial cells. Using flow-based adhesion assays to study the migration of lymphocytes across primary human HSECs, we found that lymphocytes enter into HSECs, confirmed by electron microscopy demonstrating clear intracellular localization of lymphocytes in vitro and by studies in human liver tissues. Stimulation by interferon-γ increased intracellular localization of lymphocytes within HSECs. Furthermore, using confocal imaging and time-lapse recordings, we demonstrated "intracellular crawling" of lymphocytes entering into one endothelial cell from another. This required the expression of intracellular adhesion molecule-1 and stabilin-1 and was facilitated by the junctional complexes between HSECs. CONCLUSION: Lymphocyte migration is facilitated by the unique structure of HSECs. Intracellular crawling may contribute to optimal lymphocyte positioning in liver tissue during chronic hepatitis. (Hepatology 2017;65:294-309).

Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury.

Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1(+) macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP(+) fibrogenic cells. Stabilin-1(-/-) macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6C(lo) monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1(+) monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage-specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1(+) macrophages shape the tissue microenvironment during liver injury and healing.

Intestinal CCL25 expression is increased in colitis and correlates with inflammatory activity.

CCL25-mediated activation of CCR9 is critical for mucosal lymphocyte recruitment to the intestine. In immune-mediated liver injury complicating inflammatory bowel disease, intrahepatic activation of this pathway allows mucosal lymphocytes to be recruited to the liver, driving hepatobiliary destruction in primary sclerosing cholangitis (PSC). However, in mice and healthy humans CCL25 expression is restricted to the small bowel, whereas few data exist on activation of this pathway in the inflamed colon despite the vast majority of PSC patients having ulcerative colitis. Herein, we show that colonic CCL25 expression is not only upregulated in patients with active colitis, but strongly correlates with endoscopic Mayo score and mucosal TNFα expression. Moreover, approximately 90% (CD4(+)) and 30% (CD8(+)) of tissue-infiltrating T-cells in colitis were identified as CCR9(+) effector lymphocytes, compared to <10% of T-cells being CCR9(+) in normal colon. Sorted CCR9(+) lymphocytes also demonstrated enhanced cellular adhesion to stimulated hepatic sinusoidal endothelium compared with their CCR9(-) counterparts when under flow. Collectively, these results suggest that CCR9/CCL25 interactions are not only involved in colitis pathogenesis but also correlate with colonic inflammatory burden; further supporting the existence of overlapping mucosal lymphocyte recruitment pathways between the inflamed colon and liver.