Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.

MDR1 deficiency impairs mitochondrial homeostasis and promotes intestinal inflammation.

The multidrug resistance-1 (MDR1) gene encodes an ATP-dependent efflux transporter that is highly expressed in the colon. In mice, loss of MDR1 function results in colitis with similarities to human inflammatory bowel diseases (IBD). Here, we show that MDR1 has an unexpected protective role for the mitochondria where MDR1 deficiency results in mitochondrial dysfunction with increased mitochondrial reactive oxygen species (mROS) driving the development of colitis. Exogenous induction of mROS accelerates, while inhibition attenuates colitis in vivo; these effects are amplified in MDR1 deficiency. In human IBD, MDR1 is negatively correlated to SOD2 gene expression required for mROS detoxification. To provide direct evidential support, we deleted intestinal SOD2 gene in mice and showed an increased susceptibility to colitis. We exploited the genome-wide association data sets and found many (∼5%) of IBD susceptibility genes with direct roles in regulating mitochondria homeostasis. As MDR1 primarily protects against xenotoxins via its efflux function, our findings implicate a distinct mitochondrial toxin+genetic susceptibility interaction leading to mitochondrial dysfunction, a novel pathogenic mechanism that could offer many new therapeutic opportunities for IBD.

αv integrins on mesenchymal cells regulate skeletal and cardiac muscle fibrosis.

Mesenchymal cells expressing platelet-derived growth factor receptor beta (PDGFRß) are known to be important in fibrosis of organs such as the liver and kidney. Here we show that PDGFRß+ cells contribute to skeletal muscle and cardiac fibrosis via a mechanism that depends on αv integrins. Mice in which αv integrin is depleted in PDGFRß+ cells are protected from cardiotoxin and laceration-induced skeletal muscle fibrosis and angiotensin II-induced cardiac fibrosis. In addition, a small-molecule inhibitor of αv integrins attenuates fibrosis, even when pre-established, in both skeletal and cardiac muscle, and improves skeletal muscle function. αv integrin blockade also reduces TGFß activation in primary human skeletal muscle and cardiac PDGFRß+ cells, suggesting that αv integrin inhibitors may be effective for the treatment and prevention of a broad range of muscle fibroses.

Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback.

Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.

Low-dose dexamethasone as a treatment for women with heavy menstrual bleeding: protocol for response-adaptive randomised placebo-controlled dose-finding parallel group trial (DexFEM).

INTRODUCTION: Heavy menstrual bleeding (HMB) diminishes individual quality-of-life and poses substantial societal burden. In HMB endometrium, inactivation of cortisol (by enzyme 11ß hydroxysteroid dehydrogenase type 2 (11ßHSD2)), may cause local endometrial glucocorticoid deficiency and hence increased angiogenesis and impaired vasoconstriction. We propose that 'rescue' of luteal phase endometrial glucocorticoid deficiency could reduce menstrual bleeding. METHODS AND ANALYSIS: DexFEM is a double-blind response-adaptive parallel-group placebo-controlled trial in women with HMB (108 to be randomised), with active treatment the potent oral synthetic glucocorticoid dexamethasone, which is relatively resistant to 11ßHSD2 inactivation. Participants will be aged over 18 years, with mean measured menstrual blood loss (MBL) for two screening cycles ≥50 mL. The primary outcome is reduction in MBL from screening. Secondary end points are questionnaire assessments of treatment effect and acceptability. Treatment will be for 5 days in the mid-luteal phases of three treatment menstrual cycles. Six doses of low-dose dexamethasone (ranging from 0.2 to 0.9 mg twice daily) will be compared with placebo, to ascertain optimal dose, and whether this has advantage over placebo. Statistical efficiency is maximised by allowing randomisation probabilities to 'adapt' at five points during enrolment phase, based on the response data available so far, to favour doses expected to provide greatest additional information on the dose-response. Bayesian Normal Dynamic Linear Modelling, with baseline MBL included as covariate, will determine optimal dose (re reduction in MBL). Secondary end points will be analysed using generalised dynamic linear models. For each dose for all end points, a 95% credible interval will be calculated for effect versus placebo. ETHICS AND DISSEMINATION: Dexamethasone is widely used and hence well-characterised safety-wise. Ethical approval has been obtained from Scotland A Research Ethics Committee (12/SS/0147). Trial findings will be disseminated via open-access peer-reviewed publications, conferences, clinical networks, public lectures, and our websites. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT01769820; EudractCT 2012-003405-98.

Effects of acute glucocorticoid blockade on metabolic dysfunction in patients with Type 2 diabetes with and without fatty liver.

To investigate the potential of therapies which reduce glucocorticoid action in patients with Type 2 diabetes we performed a randomized, double-blinded, placebo-controlled crossover study of acute glucocorticoid blockade, using the glucocorticoid receptor antagonist RU38486 (mifepristone) and cortisol biosynthesis inhibitor (metyrapone), in 14 men with Type 2 diabetes. Stable isotope dilution methodologies were used to measure the rates of appearance of glucose, glycerol, and free fatty acids (FFAs), including during a low-dose (10 mU·m⁻² ·min⁻¹) hyperinsulinemic clamp, and subgroup analysis was conducted in patients with high or low liver fat content measured by magnetic resonance spectroscopy (n = 7/group). Glucocorticoid blockade lowered fasting glucose and insulin levels and improved insulin sensitivity of FFA and glycerol turnover and hepatic glucose production. Among this population with Type 2 diabetes high liver fat was associated with hyperinsulinemia, higher fasting glucose levels, peripheral and hepatic insulin resistance, and impaired suppression of FFA oxidation and FFA and glycerol turnover during hyperinsulinemia. Glucocorticoid blockade had similar effects in those with and without high liver fat. Longer term treatments targeting glucocorticoid action may be useful in Type 2 diabetes with and without fatty liver.

Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification.

Liver fibrosis: a bidirectional model of fibrogenesis and resolution.

Liver fibrosis is the generic response to chronic injury of varying aetiologies. A number of common mechanisms link this response to the pathogenesis of fibrosis in other organs. While long thought to be relentlessly progressive, there is now excellent evidence in both human liver disease and animal models that hepatic fibrosis is potentially reversible. The liver therefore provides an excellent bidirectional model for the study of fibrogenesis and fibrosis resolution. In this article, we will review the evidence for the reversibility of liver fibrosis. We will highlight some of the mechanisms responsible for fibrogenesis and fibrosis regression, focussing on the role of hepatic myofibroblast activation and apoptosis, the importance of matrix metalloproteinases and their tissue inhibitors and the central involvement of hepatic macrophages in orchestrating this process. Finally, we will briefly discuss what renders liver fibrosis irreversible and how this accumulating knowledge base could lead to badly needed anti-fibrotic therapies in the future.

Acute liver failure in Scotland between 1992 and 2009; incidence, aetiology and outcome.

AIM: To describe incidence, aetiology and outcome data for Scotland since the inception of the Scottish Liver Transplant Unit (SLTU) in 1992. BACKGROUND: Acute liver failure (ALF) is a rare but frequently fatal condition. Few studies have adequate patient numbers to draw convincing conclusions over demographic features, aetiology and outcome. DESIGN: Statistical analysis of prospectively collected data on aetiology, demographic, clinical and outcome of all admissions, including those with ALF, to the SLTU. METHODS: Incidence data presented for admissions and ALF. Descriptive frequencies for aetiology, clinical, demographic and outcome data presented; including split analysis for paracetamol and non-paracetamol aetiologies. Univariate and multivariate analysis of admission factors predictive of outcome is described. RESULTS: Nine hundred and forty-nine patients were admitted to the SLTU between 1992 and 2009. Five hundred and twenty-four patients had ALF. The annual incidence of ALF in the Scottish population is 0.62 per 100,000 and paracetamol overdose (POD) was the largest causative factor; responsible for 0.43 cases of ALF per 100,000 population per year. The odds ratio (OR) of transplantation or death was 0.47 in the POD group compared to other aetiologies; yet of not being a transplant candidate having met the Kings College Hospital poor prognostic criteria OR was 4.9. Of admissions listed for transplant 76.0% were transplanted. Of those listed and not transplanted mortality was approaching 100% and 76.1% of those transplanted survived to discharge. CONCLUSION: This large, prospective, single centre study with a defined geographical area and well-recorded population provides accurate data regarding ALF between 1992 and 2009.

Hepatic fibrosis and cirrhosis in the Fontan circulation: a detailed morphological study.

AIMS: To describe the histological features of the liver in patients with a Fontan circulation. METHODS: Specimens from liver biopsies carried out as part of preoperative assessment prior to extracardiac cavopulmonary conversion of an older style Fontan were examined and scored semi-quantitatively for pertinent histological features. To support the use of the scoring, biopsy specimens were also ranked by eye for severity to allow correlation with assigned scores. RESULTS: Liver biopsy specimens from 18 patients with a Fontan circulation were assessed. All specimens showed sinusoidal fibrosis. In 17 cases there was at least fibrous spur formation, with 14 showing bridging fibrosis and 2 showing frank cirrhosis. In 17 cases at least some of the dense or sinusoidal fibrosis was orcein positive, although a larger proportion of the dense fibrous bands were orcein positive compared with the sinusoidal component. All specimens showed marked sinusoidal dilatation, and 14 showed bile ductular proliferation; 1 showed minimal iron deposition, and 1 showed mild lobular lymphocytic inflammation. There was no cholestasis or evidence of hepatocellular damage. Similar appearances were observed in 2 patients with severe tricuspid regurgitation. DISCUSSION: The histological features of the liver in patients with a Fontan circulation are similar to those described in cardiac sclerosis. Sinusoidal dilatation and sinusoidal fibrosis are marked in the Fontan series. The presence of a significant amount of orcein negative sinusoidal fibrosis suggests there may be a remediable component, although the dense fibrous bands are predominantly orcein positive, suggesting chronicity and permanence. No inflammation or hepatocellular damage is evident, suggesting that fibrosis may be mediated by a non-inflammatory mechanism.

Case report of an arterioportal fistula, presenting with accelerated decompensation and sepsis, twenty-six years after initial liver biopsy.

We describe the case of a 55-year-old lady who presented with accelerated hepatic decompensation from an arterioportal fistula (APF). There is histological evidence the APF proceeded a percutaneous liver biopsy performed 26 years ago. She had shown no symptoms or signs of liver disease in the intervening period. The clinical presentation initially was that of portal hypertension but evolved into a systemic inflammatory response syndrome associated with renal and liver failure. We describe how the APF was embolised by interventional radiology and how the timing of this decision was a balance between reversing abnormal haemodynamics and trying to avoid instrumentation of a potentially septic environment. This unusual case reflects the relationship between portal hypertension, sepsis and renal failure.

Influence of cytokine and ICAM-1 gene polymorphisms on susceptibility to chronic pancreatitis.

AIMS: To test the hypothesis that single nucleotide polymorphisms (SNPs) within genes (or their promoter regions) encoding cytokines, growth factors, and intercellular adhesion molecules modulate the risk of development of chronic pancreatitis (CP). METHODS: DNA was extracted from peripheral blood leucocytes or formalin fixed, paraffin wax embedded tissue from 53 patients with CP and 266 healthy controls. SNPs within the interleukin 1beta (IL-1beta), IL-6, IL-8, tumour necrosis factor alpha (TNFalpha) and vascular endothelial growth factor (VEGF) gene promoter regions and the transforming growth factor beta1 (TGFbeta1) and intercellular cell adhesion molecule 1 (ICAM-1) genes were genotyped by the amplification refractory mutation system polymerase chain reaction or 5' nuclease (Taqman) techniques. Patient-control comparisons were made using 2 x 2 contingency tables and chi2 analyses. RESULTS: A non-significant decrease in the frequency of the IL-8 -251 AA genotype and a non-significant increase in the frequency of the ICAM-1 +469 GA genotype was seen in patients compared with controls. No associations were identified between SNPs in the promoter regions of the IL-1beta, IL-6, or TNFalpha proinflammatory cytokines genes or the TGFbeta1 and VEGF genes and susceptibility to CP. CONCLUSIONS: This preliminary study suggests that genetic polymorphism within several cytokine genes is unlikely to influence susceptibility to CP, but the possible role of IL-8 and ICAM-1 polymorphisms in the development of this disease requires further investigation.

Apoptosis and proliferation of acinar and islet cells in chronic pancreatitis: evidence for differential cell loss mediating preservation of islet function.

BACKGROUND: Chronic pancreatitis is characterised clinically by early exocrine insufficiency, with diabetes mellitus occurring as a late phenomenon. This is mirrored pathologically by extensive acinar cell destruction and islet preservation. The mechanisms underlying this differential rate of cellular destruction are unknown. AIMS: To test the hypothesis that acinar loss and islet preservation in chronic pancreatitis occurs due to differential epithelial kinetics and investigate the role of inflammatory cells and cell cycle associated molecules. METHODS: Archival tissue from six chronic pancreatitis cases was compared with six normal controls using TUNEL and immunohistochemistry for CD3, CD20, CD68, MIB-1, Bcl-2, Bax, Fas, Fas ligand, retinoblastoma protein (Rb), and tissue inhibitor of metalloproteinases 1 (TIMP-1) and 2 (TIMP-2). RESULTS: The acinar cell apoptotic index (AI) and proliferation index were higher in chronic pancreatitis than controls. T lymphocytes diffusely infiltrated fibrous bands and acini but rarely islets. Acinar Bcl-2 expression exceeded islet expression in chronic pancreatitis and controls while Bax was strongly expressed by a subset of islet cells and weakly by centroacinar cells. Islet Fas and Fas ligand expression exceeded acinar expression in chronic pancreatitis and controls. Acinar Rb expression was higher in chronic pancreatitis than in controls. Islets in chronic pancreatitis and controls showed intense TIMP-1 and TIMP-2 expression. CONCLUSION: Apoptosis plays a significant role in acinar loss in chronic pancreatitis. Acinar Bcl-2 and islet Bax expression indicates complex AI control. Increased acinar Rb expression in chronic pancreatitis may differentially promote acinar loss. Fas ligand expression may be restricted to islet cell membranes through TIMP-1 expression and inhibit islet damage by promoting apoptosis of cytotoxic T lymphocytes.

Hepatic stellate cell behavior during resolution of liver injury.

Acute self-limiting and chronic liver injury are both associated with activation and proliferation of hepatic stellate cells (HSCs). In chronic injury, activated stellate cells are the major source of the collagens that comprise fibrosis and cirrhosis, as well as of the tissue inhibitors of metalloproteinases (TIMPs) which inhibit collagen degradation. Recovery from acute and chronic injury is characterized by apoptosis of activated HSCs, which removes extracellular matrix-producing cells that are also expressing TIMPs, thereby relieving the inhibition of matrix degradation. HSC apoptosis is regulated in progressive injury and counterbalances cell proliferation. Apoptosis probably also represents a default pathway for the HSCs. The survival of activated HSCs in liver injury is dependent on soluble growth factors and cytokines, and on components of the fibrotic matrix. Additionally, stimulation of death receptors expressed on HSCs can precipitate their apoptosis. Our increasing understanding of the process of stellate cell behavior in recovery from injury is likely to be important to the design of antifibrotic therapies.

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo.

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.

Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis and stimulating their apoptosis could be an effective treatment for liver fibrosis. METHODS: Activated HSCs, hepatocytes, and rats with liver fibrosis were treated with gliotoxin. RESULTS: Addition of gliotoxin to activated (alpha-smooth muscle actin positive) rat and human HSCs resulted in morphologic alterations typical of apoptosis. Within 2-3 hours of incubation, caspase 3 activity was markedly induced and caspase inhibitor 1 (Z-VAD-FMK)-sensitive oligonucleosome-length DNA fragments were detectable by gel electrophoresis of low molecular weight DNA. Apoptosis was widespread as judged by fluorescence-activated cell sorter analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in both rat and human HSCs at concentrations that had no effect on the viability of rat hepatocytes. Gliotoxin treatment significantly reduced the number of activated stellate cells and mean thickness of bridging fibrotic septae in livers from rats treated with carbon tetrachloride. CONCLUSIONS: These data demonstrate proof-of-concept that by up-regulating HSC apoptosis, the extent of fibrosis can be decreased in inflammatory liver injury.

Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors.

BACKGROUND: Activated hepatic stellate cells (HSC) are central to the pathogenesis of liver fibrosis, both as a source of fibrillar collagens that characterise fibrosis and matrix degrading metalloproteinases and their tissue inhibitors, the TIMPs. AIMS: To test the hypothesis that HSC apoptosis is critical to recovery from biliary fibrosis and that soluble growth factors may regulate HSC survival and apoptosis. METHODS: Rats (n=15) were subjected to bile duct ligation for 21 days, after which biliodigestive anastomosis was undertaken (n=13). Livers were harvested at fixed time points of recovery for periods of up to 42 days. Numbers of activated HSCs were quantified after alpha smooth muscle actin staining and HSC apoptosis was detected by terminal UDP-nick end labelling (TUNEL) staining and quantified at each time point. HSC apoptosis was quantified in vitro in the presence or absence of insulin-like growth factor (IGF)-1, IGF-2, platelet derived growth factor (PDGF), and transforming growth factor beta1 (TGF-beta1). RESULTS: Following biliodigestive anastomosis after 21 days of bile duct ligation, rat liver demonstrated a progressive resolution of biliary fibrosis over 42 days, associated with a fivefold decrease in activated HSC determined by alpha smooth muscle actin staining. TUNEL staining indicated that loss of activated HSC resulted from an increase in the rate of apoptosis during the first two days post biliodigestive anastomosis. Serum deprivation and culture in the presence of 50 microM cycloheximide was associated with an increase in HSC apoptosis which was significantly inhibited by addition of 10 ng/ml and 100 ng/ml IGF-1, respectively (0.05>p, n=5). In contrast, 1 and 10 ng/ml of TGF-beta1 caused a significant increase in HSC apoptosis compared with serum free controls (p<0.05, n=4). PDGF and IGF-2 were neutral with respect to their effect on HSC apoptosis. CONCLUSION: HSC apoptosis plays a critical role in the spontaneous recovery from biliary fibrosis. Both survival and apoptosis of HSC are regulated by growth factors expressed during fibrotic liver injury.

Liver fibrosis, the hepatic stellate cell and tissue inhibitors of metalloproteinases.

Liver fibrosis occurs as a consequence of net accumulation of matrix proteins (especially collagen types I and III) in response to liver injury. The pathogenesis of liver fibrosis is underpinned by the activation of hepatic stellate cells (HSC) to a myofibroblast like phenotype with a consequent increase in their synthesis of matrix proteins such as interstitial collagens that characterise fibrosis. In addition to this there is increasing evidence that liver fibrosis is a dynamic pathologic process in which altered matrix degradation may also play a major role. Extracellular degradation of matrix proteins is regulated by matrix metalloproteinases (MMPS)- produced by HSC--which in turn are regulated by several mechanisms which include regulation at the level of the gene (transcription and proenzyme synthesis), cleavage of the proenzyme to an active form and specific inhibition of activated forms by tissue inhibitors of metalloproteinases (TIMPS). Insights gained into the molecular regulation of HSC activation will lead to therapeutic approaches in treatment of hepatic fibrosis in the future, and could lead to reduced morbidity and mortality in patients with chronic liver injury.