Victoria K. Snowdon

Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis

Although macrophages are widely recognized to have a profibrotic role in inflammation, we have used a highly tractable CCl4-induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11Bhi F4/80int Ly-6Clo macrophage subset was most abundant in livers during maximal fibrosis resolution and represented the principle matrix metalloproteinase (MMP) -expressing subset. Depletion of this population in CD11B promoter–diphtheria toxin receptor (CD11B-DTR) transgenic mice caused a failure of scar remodeling. Adoptive transfer and in situ labeling experiments showed that these restorative macrophages derive from recruited Ly-6Chi monocytes, a common origin with profibrotic Ly-6Chi macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6Clo subset, compared with Ly-6Chi macrophages, showed a phenotype outside the M1/M2 classification, with increased expression of MMPs, growth factors, and phagocytosis-related genes, including Mmp9, Mmp12, insulin-like growth factor 1 (Igf1), and Glycoprotein (transmembrane) nmb (Gpnmb). Confocal microscopy confirmed the postphagocytic nature of restorative macrophages. Furthermore, the restorative macrophage phenotype was recapitulated in vitro by the phagocytosis of cellular debris with associated activation of the ERK signaling cascade. Critically, induced phagocytic behavior in vivo, through administration of liposomes, increased restorative macrophage number and accelerated fibrosis resolution, offering a therapeutic strategy to this orphan pathological process.

Elastin accumulation is regulated at the level of degradation by macrophage metalloelastase (MMP‐12) during experimental liver fibrosis

Elastin has been linked to maturity of liver fibrosis. To date, the regulation of elastin secretion and its degradation in liver fibrosis has not been characterized. The aim of this work was to define elastin accumulation and the role of the paradigm elastase macrophage metalloelastase (MMP‐12) in its turnover during fibrosis. Liver fibrosis was induced by either intraperitoneal injections of carbon tetrachloride (CCl4) for up to 12 weeks (rat and mouse) or oral administration of thioacetamide (TAA) for 1 year (mouse). Elastin synthesis, deposition, and degradation were investigated by immunohistochemistry, quantitative polymerase chain reaction (qPCR), western blotting, and casein zymography. The regulation of MMP‐12 elastin degradation was defined mechanistically using CD11b‐DTR and MMP‐12 knockout mice. In a CCl4 model of fibrosis in rat, elastin deposition was significantly increased only in advanced fibrosis. Tropoelastin expression increased with duration of injury. MMP‐12 protein levels were only modestly changed and in coimmunoprecipitation experiments MMP‐12 was bound in greater quantities to its inhibitor TIMP‐1 in advanced versus early fibrosis. Immunohistochemistry and macrophage depletion experiments indicated that macrophages were the sole source of MMP‐12. Exposure of CCl4 in MMP‐12−/− mice led to a similar degree of overall fibrosis compared to wildtype (WT) but increased perisinusoidal elastin. Conversely, oral administration of TAA caused both higher elastin accumulation and higher fibrosis in MMP‐12−/− mice compared with WT. Conclusion: Elastin is regulated at the level of degradation during liver fibrosis. Macrophage‐derived MMP‐12 regulates elastin degradation even in progressive experimental liver fibrosis. These observations have important implications for the design of antifibrotic therapies. (HEPATOLOGY 2012;55:1965–1975)

Relaxin modulates human and rat hepatic myofibroblast function and ameliorates portal hypertension in vivo.

Active myofibroblast (MF) contraction contributes significantly to the increased intrahepatic vascular resistance that is the primary cause of portal hypertension (PHT) in cirrhosis. We sought proof of concept for direct therapeutic targeting of the dynamic component of PHT and markers of MF activation using short‐term administration of the peptide hormone relaxin (RLN). We defined the portal hypotensive effect in rat models of sinusoidal PHT and the expression, activity, and function of the RLN‐receptor signaling axis in human liver MFs. The effects of RLN were studied after 8 and 16 weeks carbon tetrachloride intoxication, following bile duct ligation, and in tissue culture models. Hemodynamic changes were analyzed by direct cannulation, perivascular flowprobe, indocyanine green imaging, and functional magnetic resonance imaging. Serum and hepatic nitric oxide (NO) levels were determined by immunoassay. Hepatic inflammation was assessed by histology and serum markers and fibrosis by collagen proportionate area. Gene expression was analyzed by quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR) and western blotting and hepatic stellate cell (HSC)‐MF contractility by gel contraction assay. Increased expression of RLN receptor (RXFP1) was shown in HSC‐MFs and fibrotic liver diseases in both rats and humans. RLN induced a selective and significant reduction in portal pressure in pathologically distinct PHT models, through augmentation of intrahepatic NO signaling and a dramatic reduction in contractile filament expression in HSC‐MFs. Critical for translation, RLN did not induce systemic hypotension even in advanced cirrhosis models. Portal blood flow and hepatic oxygenation were increased by RLN in early cirrhosis. Treatment of human HSC‐MFs with RLN inhibited contractility and induced an antifibrogenic phenotype in an RXFP1‐dependent manner. Conclusion: We identified RXFP1 as a potential new therapeutic target for PHT and MF activation status. (Hepatology 2014;59:1492‐1504)

Models and mechanisms of fibrosis resolution

Liver fibrosis and its end stage, cirrhosis, represent the final common pathway of virtually all chronic liver diseases. As our understanding of the pathogenesis of liver fibrosis has progressed, it has become evident that the liver provides a useful generic model of inflammation and repair, demonstrating interplay between the epithelial, inflammatory, myofibroblast and extracellular matrix components of the mammalian wound healing response. In this review, the paradigm that liver fibrosis is a potentially reversible process-demonstrating both fibrosis (scarring) and resolution with remodeling and restitution of normal or near-normal tissue architecture-will be explored. The remarkable progress in unraveling the complexities of liver fibrosis has been due to developments in technologies including the isolation of discrete liver cell populations which have facilitated studies of their behavior in tissue culture and in vivo. More recently, animal models that mimic chronic liver diseases have been established. These models are tractable and can be applied in gene knockout and transgenic mice. This article will highlight recent studies that reveal key mechanisms mediating the regression of liver fibrosis which have derived from the use of such complementary animal and human model systems and describe how our greater understanding of this dynamic process is likely to inform the development of directed and effective anti-fibrotic approaches.

Serelaxin as a potential treatment for renal dysfunction in cirrhosis: Preclinical evaluation and results of a randomized phase 2 trial

Background Chronic liver scarring from any cause leads to cirrhosis, portal hypertension, and a progressive decline in renal blood flow and renal function. Extreme renal vasoconstriction characterizes hepatorenal syndrome, a functional and potentially reversible form of acute kidney injury in patients with advanced cirrhosis, but current therapy with systemic vasoconstrictors is ineffective in a substantial proportion of patients and is limited by ischemic adverse events. Serelaxin (recombinant human relaxin-2) is a peptide molecule with anti-fibrotic and vasoprotective properties that binds to relaxin family peptide receptor-1 (RXFP1) and has been shown to increase renal perfusion in healthy human volunteers. We hypothesized that serelaxin could ameliorate renal vasoconstriction and renal dysfunction in patients with cirrhosis and portal hypertension. Methods and findings To establish preclinical proof of concept, we developed two independent rat models of cirrhosis that were characterized by progressive reduction in renal blood flow and glomerular filtration rate and showed evidence of renal endothelial dysfunction. We then set out to further explore and validate our hypothesis in a phase 2 randomized open-label parallel-group study in male and female patients with alcohol-related cirrhosis and portal hypertension. Forty patients were randomized 1:1 to treatment with serelaxin intravenous (i.v.) infusion (for 60 min at 80 μg/kg/d and then 60 min at 30 μg/kg/d) or terlipressin (single 2-mg i.v. bolus), and the regional hemodynamic effects were quantified by phase contrast magnetic resonance angiography at baseline and after 120 min. The primary endpoint was the change from baseline in total renal artery blood flow. Therapeutic targeting of renal vasoconstriction with serelaxin in the rat models increased kidney perfusion, oxygenation, and function through reduction in renal vascular resistance, reversal of endothelial dysfunction, and increased activation of the AKT/eNOS/NO signaling pathway in the kidney. In the randomized clinical study, infusion of serelaxin for 120 min increased total renal arterial blood flow by 65% (95% CI 40%, 95%; p < 0.001) from baseline. Administration of serelaxin was safe and well tolerated, with no detrimental effect on systemic blood pressure or hepatic perfusion. The clinical study’s main limitations were the relatively small sample size and stable, well-compensated population. Conclusions Our mechanistic findings in rat models and exploratory study in human cirrhosis suggest the therapeutic potential of selective renal vasodilation using serelaxin as a new treatment for renal dysfunction in cirrhosis, although further validation in patients with more advanced cirrhosis and renal dysfunction is required. Trial registration ClinicalTrials.gov NCT01640964

Noninvasive Evaluation of Portal Hypertension: Emerging Tools and Techniques

Portal hypertension is the main cause of complications in patients with cirrhosis. However, evaluating the development and progression of portal hypertension represents a challenge for clinicians. There has been considerable focus on the potential role of noninvasive markers of portal hypertension that could be used to stratify patients with respect to the stage of portal hypertension and to monitor disease progression or treatment response in a longitudinal manner without having to undertake repeated invasive assessment. The pathogenesis of portal hypertension is increasingly understood and emerging knowledge of the vascular processes that underpin portal hypertension has paved the way for exploring novel biomarkers of vascular injury, angiogenesis, and endothelial dysfunction. In this paper we focus on the pathogenesis of portal hypertension and potential non-invasive biomarkers with particular emphasis on serum analytes.

Editorial: measuring inflammatory and fibrotic components of portal hypertension - a noninvasive hepatic venous pressure gradient?

The gold standard measurement for portal hypertension is the hepatic venous pressure gradient (HVPG). The prognostic value of HVPG in chronic liver disease (CLD) is well-documented. An HVPG >10 mmHg (‘clinically significant PHT’) is predictive of variceal development and reduction in HVPG with therapy to <12 mmHg or >20% from baseline has been shown to reduce variceal re-bleeding rates. 2 However, HVPG is an invasive and highly specialised technique. A ‘noninvasive HVPG’ would be extremely valuable to assess prognosis, disease progression and response to drug therapy in CLD. Baveno VI Consensus recommendations included the use of noninvasive methods for screening and surveillance of varices and PHT in CLD. Liver stiffness measurement (LSM) by transient elastography (TE) and platelet count were proposed to identify patients with compensated CLD in whom screening endoscopy could be safely avoided. However, TE has known limitations and LSM does not correlate well with HVPG above 12 mmHg. It is therefore with interest that Sandahl et al. report a novel serum biomarker test – sCD163 combined with enhanced liver fibrosis (ELF) score – to predict CSPH. This approach comprises a macrophage activation marker reflecting the influence of inflammation on HVPG 8 and another marker validated for measuring fibrosis, such that together they might capture both dynamic and structural components of raised intrahepatic vascular resistance (IHVR). This two-centre study using independent estimation (EC, n = 80) and validation (VC, n = 80) cohorts of cirrhotic patients undergoing HVPG measurement and a small control group (n = 21) showed that sCD163 and ELF components (hyaluronic acid, amino-terminal propeptide of type-III procollagen and tissue inhibitor of matrix metalloproteinase-1) were all significantly associated with risk of CSPH. Moreover, the combined sCD163-fibrosis PHT score had an AUROC of 0.91 (EC) and 0.9 (VC) for predicting CSPH. A high cutoff of 3.6 to rule-in CSPH identified 68% of patients with CSPH (PPV 0.99, NPV 0.27 in EC; PPV 0.93, NPV 0.73 in VC) and a low cut-off of 1.4 to rule-out CSPH (NPV 1.0, PPV 0.93 in EC; NPV 0.94, PPV 0.89 in VC). Furthermore, in the VC a score higher than 3.6 was associated with increased 4-year mortality. The authors should be commended on developing a methodology to assess clinically significant PHT based on the underlying pathobiology of PHT. The diagnostic accuracy of the sCD163-fibrosis score for clinically significant PHT is good and, notably, the low cut-off value has a very high NPV which in a real-world clinical setting may be useful for identifying patients who could avoid endoscopy with a low risk of decompensation. However, it is not yet clear whether this test is predictive of clinical outcomes or whether it is sensitive enough to detect dynamic changes in HVPG over time or in response to therapy (sCD163 values did not change after TIPSS insertion). In the ongoing search for the most responsive and reliable biomarker test for PHT it would be informative to explore other markers/combinations, including factors associated with endothelial dysfunction (e.g. vWF), also implicated in PHT. Prospective studies are required to define the clinical utility and cost-effectiveness of this test.

OC-031 Relaxin Modulates Cirrhosis-induced Renal Vascular Endothelial Dysfunction

Introduction Hepatorenal syndrome (HRS) is a feared complication of cirrhosis characterised by intense renal vasoconstriction. The pathophysiology remains unclear, pharmacotherapy is limited and mortality is high. We investigated vascular responsiveness and the pathogenesis of renal vasoconstriction in models of advanced rat cirrhosis. Additionally,we determined the mechanism of action of the vasoactive peptide relaxin (RLN), previously shown to increase renal blood flow (RBF) in experimental cirrhosis (Snowdon V et al ., BSG 2013). Methods We induced cirrhosis,reduced RBF and renal dysfunction in male SD rats by carbon tetrachloride (16 wk) or bile duct ligation (4 wk). Arteries from renal (renal, segmental, interlobar) and splanchnic circulation were isolated for functional assessment using wire myography. qPCR array for vasoactive signalling genes,western blot for eNOS signalling proteins and NOS activity assay were undertaken in cirrhotic and control kidneys. Markers of oxidative stress and inflammatory cytokines were measured in serum by ELISA. We studied the effects of s.c. infusion of recombinant human RLN(seralaxin;72 h, 4 µg/h) on these parameters. Doppler USS measured changes in cardiac output (CO) and renal arterial resistive index (RRI) in response to i.v. RLN (4 µg). Kidney endothelial morphology was assessed by electron microscopy, H+E and PAS stained kidney by light microscopy. Results In renal arteries from control and cirrhotic rats endothelial vasodilatation was eNOS-dependent. In cirrhotic rats endothelium-dependent relaxation (acetylcholine; 10–9–3 × 10–5 M) was dramatically reduced (p < 0.0001) in all renal arteries,with only a modest reduction seen in the mesenteric arteries. Endothelium-independent relaxation (sodium nitroprusside; 10–9–3 × 10–5 M) and vasoconstriction (phenylephrine; 10–9–3 × 10–5 M) were unaltered. In cirrhotic kidneys,total eNOS expression was up-regulated,as were arginase2 and caveolin1 (negative regulators of eNOS),and NOS activity was reduced (p < 0.05). Acute RLN had no effect on CO but decreased RRI (p < 0.05). Extended RLN restored endothelium-dependent relaxation, increased kidney NOS activity (p < 0.05), increased phosphorylated Akt and eNOS, and reduced serum TNFα levels. Conclusion Renal vascular endothelial dysfunction characterises experimental cirrhosis, through a reduction in renal eNOS activity. This impairment may contribute to the renal vasoconstriction seen in cirrhosis and is a promising target for therapeutic modulation. RLN treatment restored renal endothelial vasodilatation. The potential for recombinant forms of RLN as a haemodynamic modulator in human cirrhosis and HRS merits investigation in translational studies. Disclosure of Interest None Declared.

Relaxin is a renal vasodilator in experimental models of cirrhosis and a potential novel therapy for hepatorenal syndrome in man

Abstract Background Hepatorenal syndrome is a feared complication of cirrhosis, with a high mortality rate and limited treatment options. The hallmarks of the disorder are functional renal failure, normal kidney histological findings, and profound renal vasoconstriction. The hormone relaxin mediates haemodynamic adaptations to pregnancy including increased renal blood flow and glomerular filtration rate (GFR). We hypothesised that relaxin could be used to modulate renal blood flow in cirrhosis. Methods Cirrhosis was induced in male rats by 16 weeks of carbon tetrachloride (intraperitoneally) and decompensated biliary cirrhosis by bile duct ligation. The effect of acute intravenous or extended (72 h) subcutaneous relaxin versus placebo was determined by analysis of systemic haemodynamics, renal blood flow, GFR, and organ histology. The effect of co-treatment with L-NG-nitroarginine methyl ester (L-NAME) was measured in subgroups of relaxin or placebo treated rats. Blood oxygen dependent-MRI (BOLD-MRI) was used to non-invasively detect changes to renal oxygenation. Hepatic and renal expression of RXFP1 (relaxin receptor) was determined by IHC and quantitative PCR (qPCR). A qPCR array was used to quantify changes in renal vasodilator/vasoconstrictor gene expression in response to relaxin. Findings RXFP1 was detected in glomerular podocytes, renal pericytes, and renal, segmental, and interlobar arteries of cirrhotic rats. In carbon tetrachloride (CCl 4 ) induced cirrhosis, acute intravenous relaxin (4 μg) induced a 50% increase in renal blood flow after 60 min (p 4 (p 4 (p vs placebo, n=8) and 70% in bile duct ligated animals (p vs placebo, n=3). Mean arterial pressure was unaffected by relaxin. L-NAME (250 mg/L) orally abrogated the effect of relaxin on renal blood flow and GFR. Relative expression of vasoconstrictor genes in kidney was markedly reduced by relaxin treatment. Interpretation Relaxin increases renal blood flow in experimental models of cirrhosis. Crucially, relaxin also improves renal function and oxygenation and does not induce systemic hypotension even in advanced disease. These effects are modulated via augmentation of nitric oxide and downregulation of vasoconstrictors, pivotal in the pathogenesis of hepatorenal syndrome. Relaxin has potential as a novel therapy for hepatorenal syndrome, and further translational studies are warranted. Funding Wellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative.

1024 RELAXIN IS A RENAL VASODILATOR IN EXPERIMENTAL MODELS OF CIRRHOSIS AND A POTENTIAL NOVEL THERAPY FOR HEPATORENAL SYNDROME (HRS) IN HUMANS

Background Hepatorenal syndrome is a feared complication of cirrhosis, with a high mortality rate and limited treatment options. The hallmarks of the disorder are functional renal failure, normal kidney histological findings, and profound renal vasoconstriction. The hormone relaxin mediates haemodynamic adaptations to pregnancy including increased renal blood flow and glomerular filtration rate (GFR). We hypothesised that relaxin could be used to modulate renal blood flow in cirrhosis.