Timothy T. Gordon-Walker

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.

Matrix stiffness modulates proliferation, chemotherapeutic response, and dormancy in hepatocellular carcinoma cells

There is increasing evidence that the physical environment is a critical mediator of tumor behavior. Hepatocellular carcinoma (HCC) develops within an altered biomechanical environment, and increasing matrix stiffness is a strong predictor of HCC development. The aim of this study was to establish whether changes in matrix stiffness, which are characteristic of inflammation and fibrosis, regulate HCC cell proliferation and chemotherapeutic response. Using an in vitro system of “mechanically tunable” matrix‐coated polyacrylamide gels, matrix stiffness was modeled across a pathophysiologically relevant range, corresponding to values encountered in normal and fibrotic livers. Increasing matrix stiffness was found to promote HCC cell proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7‐fold and 12.2‐fold higher, respectively, when the cells were cultured on stiff (12 kPa) versus soft (1 kPa) supports. This was associated with stiffness‐dependent regulation of basal and hepatocyte growth factor–stimulated mitogenic signaling through extracellular signal‐regulated kinase, protein kinase B (PKB/Akt), and signal transducer and activator of transcription 3. β1‐Integrin and focal adhesion kinase were found to modulate stiffness‐dependent HCC cell proliferation. Following treatment with cisplatin, we observed reduced apoptosis in HCC cells cultured on stiff versus soft (physiological) supports. Interestingly, however, surviving cells from soft supports had significantly higher clonogenic capacity than surviving cells from a stiff microenvironment. This was associated with enhanced expression of cancer stem cell markers, including clusters of differentiation 44 (CD44), CD133, c‐kit, cysteine‐X‐cysteine receptor 4, octamer‐4 (CXCR4), and NANOG. Conclusion: Increasing matrix stiffness promotes proliferation and chemotherapeutic resistance, whereas a soft environment induces reversible cellular dormancy and stem cell characteristics in HCC. This has implications for both the treatment of primary HCC and the prevention of tumor outgrowth from disseminated tumor cells. (HEPATOLOGY 2011;)

Macrophage therapy for murine liver fibrosis recruits host effector cells improving fibrosis, regeneration, and function.

Clinical studies of bone marrow (BM) cell therapy for liver cirrhosis are under way but the mechanisms of benefit remain undefined. Cells of the monocyte‐macrophage lineage have key roles in the development and resolution of liver fibrosis. Therefore, we tested the therapeutic effects of these cells on murine liver fibrosis. Advanced liver fibrosis was induced in female mice by chronic administration of carbon tetrachloride. Unmanipulated, syngeneic macrophages, their specific BM precursors, or unfractionated BM cells were delivered during liver injury. Mediators of inflammation, fibrosis, and regeneration were measured. Donor cells were tracked by sex‐mismatch and green fluorescent protein expression. BM‐derived macrophage (BMM) delivery resulted in early chemokine up‐regulation with hepatic recruitment of endogenous macrophages and neutrophils. These cells delivered matrix metalloproteinases‐13 and ‐9, respectively, into the hepatic scar. The effector cell infiltrate was accompanied by increased levels of the antiinflammatory cytokine interleukin 10. A reduction in hepatic myofibroblasts was followed by reduced fibrosis detected 4 weeks after macrophage infusion. Serum albumin levels were elevated at this time. Up‐ regulation of the liver progenitor cell mitogen tumor necrosis factor‐like weak inducer of apoptosis (TWEAK) preceded expansion of the progenitor cell compartment. Increased expression of colony stimulating factor‐1, insulin‐like growth factor‐1, and vascular endothelial growth factor also followed BMM delivery. In contrast to the effects of differentiated macrophages, liver fibrosis was not significantly altered by the application of macrophage precursors and was exacerbated by whole BM. Conclusion: Macrophage cell therapy improves clinically relevant parameters in experimental chronic liver injury. Paracrine signaling to endogenous cells amplifies the effect. The benefits from this single, defined cell type suggest clinical potential. (HEPATOLOGY 2011;)

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)

OC-157 Physiological changes in matrix stiffness modulate hepatic progenitor cell morphology, proliferation and differentiation

Introduction Liver injury is associated with changes in the biochemical and physical properties of the extracellular matrix (ECM). Hepatic progenitor cell (HPC) activation occurs in the context of severe liver injury. ECM stiffness has been shown to direct differentiation in mesenchymal stem cells. However, the effect of mechanical factors, such as ECM stiffness on HPC responses is poorly characterised. We examined the effect of ECM stiffness on HPC proliferation and differentiation. Methods Experiments were undertaken using a murine HPC line (BMOL) and primary murine HPCs. Cell culture experiments were performed using a system of laminin-coated polyacrylamide (PA) gel supports of variable stiffness. The stiffness of the PA supports (expressed as shear modulus) was altered across a physiological range (1–12 kPa) corresponding to values encountered in normal and fibrotic livers. Results Increasing matrix stiffness is associated with enhanced cell spreading. BMOL cells cultured on stiff (12kPa) supports develop prominent actin stress fibres. The projected surface area (mean±SEM) of BMOL cells culture on soft (1kPa) supports was 378±21 μm2 compared to 687±47 μm2 for BMOL cells cultured on stiff (12 kPa) supports (p<0.001). Cell proliferation (Ki67 positivity) increased as a function of increasing matrix stiffness. The proliferative index (PI) of BMOL cells cultured on 2.5 kPa and 12 kPa supports was 7.1-fold (p<0.01) and 11.8-fold higher (p<0.001), respectively, than cells cultured on 1kPa supports. Similarly, in experiments with primary cells, the PI of murine HPCs was 1.7-fold higher (p<0.05) when cells were cultured on stiff (12 kPa) vs soft (1 kPa) supports. Quantitative PCR revealed that BMOL cells cultured on soft (1 kPa) supports up-regulate hepatocyte markers, including; albumin (1.5-fold, p<0.01) and CYP7A1 (1.6-fold, p<0.01), and down-regulate the HPC/biliary marker cytokeratin-19 (0.6-fold, p<0.01), relative to cells on stiff (12kPa) supports. There was no significant change in expression of the biliary epithelial cell markers aquaporin-1 and γ-glutamyl-transferase. Conclusion Physiological changes in ECM stiffness lead to alterations in HPC morphology, proliferation and differentiation. Increased ECM stiffness (as would be encountered in an injured or fibrotic liver) promotes HPC proliferation and expression of the HPC/biliary marker cytokeratin-19. In contrast, a low-stiffness environment is associated with a reduction in cell proliferation and up-regulation of hepatocyte-specific markers. These results suggest that mechanical factors, such as ECM stiffness might regulate HPC responses following liver injury. Competing interests None declared.

P38 Matrix stiffness regulates proliferation, differentiation and chemotherapeutic responsiveness in hepatocellular carcinoma

Introduction The majority (80%) of hepatocellular carcinomas (HCC) develop within the context of advanced liver fibrosis and cirrhosis. Recent studies with ultrasound elastography have demonstrated that increased liver stiffness is a strong predictor of HCC. Aim To establish whether alterations in matrix stiffness regulate the phenotype and chemotherapeutic response of HCC cells. Method Experiments were conducted using a system of ligand-coated polyacrylamide gels of variable stiffness. Matrix stiffness (expressed as shear modulus) was modelled across a physiologically-relevant range (1–12 kPa), corresponding to values encountered in normal and fibrotic livers. Experiments were conducted in two HCC cells lines (Huh7/ HepG2). Results In each cell type, there was a consistent morphological response to changes in matrix stiffness. There was increased cell spreading on stiff gels in association with both stress-fibre and mature focal adhesion formation. Increasing matrix stiffness promoted cellular proliferation. The proliferative index (assessed by Ki67 staining) of Huh7 and HepG2 cells was 2.7-fold (p<0.001) and 12.2-fold (p<0.001) higher, respectively, when the cells were cultured on stiff (12 kPa) vs soft (1 kPa) supports. Cells cultured on soft supports developed a quiescent (dormant) phenotype with marked reduction in cyclinD1/ D3 expression, without upregulation of p21/p27. We postulated that altered sensitivity to mitogenic growth factors mediates the stiffness-dependent regulation of proliferation. Matrix stiffness modulated both the magnitude and time-course of mitogenic signalling in response to HGF, with lower baseline and HGF-induced FAK, ERK and STAT3 pathway activation. Increasing stiffness results in upregulation of mesenchymal markers (including N-cadherin and vimentin), consistent with mesenchymal shift, and down-regulation of differentiated hepatocyte markers (including albumin, α-1-antitrypsin and HNF4). Following treatment with cisplatin, cells cultured on soft supports were more susceptible to apoptosis (PARP/ Caspase-3 cleavage). However, in both Huh7 and HepG2 cells, surviving cells from soft supports had 2.2-fold (p<0.05) and 2.4-fold (p<0.001) higher clonogenic capacity respectively, than surviving cells from stiff supports. This was associated with upregulation of cancer stem cell markers (Oct4, NANOG, CD44, CD133, c-kit and CXCR4). Conclusion HCC is a tumour that develops within an altered biomechanical niche. Increasing matrix stiffness regulates HCC mitogenic signalling, proliferation, differentiation and chemotherapeutic resistance. However, a soft microenvironment (as may be encountered by disseminated tumour cells) promotes stem cell characteristics following chemotherapy. This provides a possible explanation for the failure of systemic chemotherapy both in relation to treatment of primary HCCs and the eradication of disseminated tumour cells that give rise to metastases. The selective targeting of the cytoskeleton represents a potentially novel approach to the treatment of HCC.

PMO-129 Relaxin reduces portal hypertension through stimulation of hepatic nitric oxide production

Introduction We have previously reported that the multifunctional hormone relaxin (RLX) downregulated the activation state and contractility of hepatic myofibroblasts and reduced portal hypertension (PHT) in cirrhotic rats (Fallowfield J et al BASL 2010). RLX has been shown to induce a range of haemodynamic effects in different organs and species, largely through effects on nitric oxide (NO). In cirrhosis, there is hepatic NO deficiency and hyporesponsiveness. We postulated that the effects of RLX on PHT were, at least in part, mediated by activation of the NO pathway. Methods Cirrhosis and PHT was induced in age-matched male Sprague-Dawley rats by 8 weeks biweekly i.p. CCl4, before randomisation to the following groups: (1) recombinant human H2-relaxin (H2-RLX) s.c. for 72 h; (2) placebo s.c. for 72 h; (3) H2-RLX s.c. + L-NAME p.o. for 72 h; (4) placebo s.c. + L-NAME p.o. for 72 h; n=5–10/group. NO levels in serum were determined by quantitative immunoassay for total nitrite and hepatic NO bioavailability by cGMP immunoassay. Relative levels of Ser473 phosphorylated Akt (p-Akt) and Ser1179 phosphorylated eNOS (p-eNOS) protein in whole liver extracts were quantified by Western blotting. Rho-kinase activity was assessed by phosphorylation of the endogenous Rho-kinase substrate moesin (Thr558). Portal pressure (PP) and mean arterial pressure (MAP) were measured under general anaesthesia by direct cannulation. Results Rats treated with CCl4 for 8 weeks developed micronodular cirrhosis, splenomegaly and PHT. There was no difference in mean serum nitrite levels between H2-RLX and placebo treated rats. However, H2-RLX increased hepatic cGMP production (p<0.01) and upregulated expression of p-Akt (p<0.05) and p-eNOS (p<0.05) protein. In contrast, there was no difference in p-moesin levels. H2-RLX treated animals had a lower mean PP than placebo controls (11.6±0.3 mm Hg [95% CI 10.97 to 12.81] vs 9.2±0.6 mm Hg [7.66 to 10.7]; p=0.008) and decreased spleen size (p=0.01). The portal hypotensive effect of H2-RLX was abrogated by co-administration of the NOS inhibitor L-NAME (11.42±0.35 mm Hg [10.44 to 12.4]; p=0.004 vs H2-RLX). MAP was comparable in RLX and placebo treated animals that also received L-NAME. Conclusion A reduction in NO bioavailability is considered to be a major factor increasing intrahepatic vascular tone in cirrhosis. Our data indicate that H2-RLX was capable of stimulating intrinsic (but not systemic) NO generation in fibrotic liver by activating the Akt/eNOS/cGMP pathway. Furthermore, inhibition of this axis with L-NAME ablated the portal hypotensive effect of H2-RLX, suggesting that it could represent a novel liver-specific NO donor in cirrhotic PHT. Competing interests None declared.

OP10 Relaxin inhibits human myofibroblast contractility and modulates portal hypertension in vivo

Introduction The peptide hormone relaxin (H2-RLN) has a broad range of biological activities including antifibrotic, anti-inflammatory and haemodynamic effects in a range of target tissues. Increased intrahepatic vascular resistance in cirrhosis is due to mechanical factors related to scarring, but also a dynamic component mediated by myofibroblast (MFB) contractility. We hypothesised that H2-RLN could modulate the dynamic attribute of portal hypertension (PHT) via a direct effect on hepatic stellate cell (HSC)-MFB mediated vasoconstriction. Aim To determine whether H2-RLN could target the contractile phenotype of activated HSC-MFBs in vitro, counterbalance the contractile response in vivo in a model of sinusoidal portal hypertension, and explore the mechanisms underlying its portal hypotensive effect. Method Gene expression in culture activated human HSCs was analysed by qRT-PCR and Western blotting. Collagen gel contraction assays were used to assess HSC-MFB contractility. Nitric oxide (NO) production was measured by Griess assay. Cirrhosis and portal hypertension was induced in age matched male Sprague–Dawley rats by 8 weeks bi-weekly i.p. CCl4, before randomisation to treatment with recombinant human H2-RLN or placebo delivered by s.c. osmotic minipump for 72 hrs (n=10 per group). Portal pressure was measured by direct cannulation. Serum H2-RLN levels were quantified by immunoassay. Liver fibrosis was measured after Sirius red staining by calculation of collagen proportionate area by digital image analysis. Finally, systemic arterial and portal pressure was measured simultaneously in groups of cirrhotic and control rats randomised to acute i.v. H2-RLN or placebo. Results Treatment of culture activated HSCs with H2-RLN downregulated expression of the major cytoskeletal protein, alpha-smooth muscle actin (alpha-SMA), in a dose dependent manner. In collagen gel contraction assays, H2-RLN inhibited HSC-MFB contraction, an effect shown by RNA interference to be mediated via RXFP-1 receptor signaling and increased NO production. Rats treated with CCl4 for 8 weeks developed micronodular cirrhosis, splenomegaly and portal hypertension. Treatment with s.c. H2-RLN for 72 h achieved physiologically relevant serum concentrations and decreased portal pressure by 24% (mean 12.8±0.8 mm Hg vs 9.8±0.3 mm Hg; p=0.002), whereas placebo had no significant effect. The observed haemodynamic response was independent of fibrosis regression. Hepatic expression of alpha-SMA and other intermediate filament proteins was markedly inhibited by H2-RLN, while eNOS activity was increased. Infusion of i.v. H2-RLN in cirrhotic rats rapidly depressed portal pressure (mean 28%±6) but not systemic pressure, compared to placebo which had minimal effect on either parameter. Conclusion We demonstrate for the first time that H2-RLN will effectively downregulate MFB contractile filament expression, contractile function and has a portal hypotensive effect in vivo. Our findings support the deployment of H2-RLN in clinical studies of cirrhosis and PHT.

OP07 Macrophage cell therapy causes the hepatic recruitment of host effector cells and improves structure and function in a murine model of chronic liver disease

Introduction Bone marrow (BM) cell populations have a number of roles in the development and resolution of chronic liver disease. Clinical trials of BM cell therapy have already begun. These have generally employed mixed cell populations often enriched for adult stem cells. Such cells may have a range of phenotypically diverse progeny. The identification of a defined cell type with beneficial effect will provide the basis of rational and predictable therapy. We have previously shown that macrophages are key mediators of scar remodelling. Iterative injury with carbon tetrachloride (CCl4) results in a well characterised model of murine hepatic fibrosis. Aim We sought to determine whether bone marrow derived macrophages (BMMs) could be used as cell therapy for liver fibrosis. Method Liver fibrosis was induced in female C57/Bl6 mice by 12 weeks i.p. carbon tetrachloride (CCl4). Macrophages were derived from the bone marrow of age-matched syngeneic mice cultured for 7 days under low adherence conditions in macrophage colony stimulating factor conditioned media. 8 weeks into the CCl4 injury protocol, mice received either 106 BMMs via the hepatic portal vein (n=8) or control medium (n=8). Serum was analysed for albumin and livers were analysed for mediators of inflammation, fibrosis and regeneration. To track donor cells, male (C57Bl/6) or transgenic green fluorescent protein+ (CBA) BMMs were delivered to strain-matched fibrotic wild type mice. Results BMMs were 88% F4/80+/CD11b+, possessed characteristic morphologic and phenotypic features, and expressed the chemokines MCP-1, MIP-1α and MIP-2. At 12 weeks, C57Bl/6 mice receiving the macrophage injection had 32% less fibrosis (mean±SEM: 2.5±0.4 vs 3.7±0.3%, p<0.05) and higher serum albumin levels (46±2.6 vs 39.9±0.86 g/l, p=0.05). Significant improvements in fibrosis and serum albumin were also demonstrated in CBA mice. Donor macrophages transiently engrafted the scar increasing hepatic levels of macrophage (MCP-1), and neutrophil (MIP-1α, MIP-2 and KC) chemoattractants (p<0.05). This enhanced recruitment of host macrophages and neutrophils to the hepatic scar areas with associated increases in MMP-13 and MMP-9 (p<0.05). A 60% reduction in myofibroblast staining (p<0.05) followed. The early influx of host leukocytes was accompanied by a 346% increase in hepatic levels of the anti-inflammatory cytokine IL-10. Donor BMMs expressed high levels of the progenitor cell mitogen TWEAK. Macrophage recipients upregulated hepatic TWEAK by 216% with a 40% increase in the number of liver progenitor cells (p<0.05). Hepatocyte proliferation was not significantly affected. Conclusion BMM therapy decreases fibrosis and increases regeneration improving clinically meaningful parameters of chronic liver disease in this model. The actions of the donor BMMs are amplified through paracrine signalling to numerically greater endogenous cell populations. Importantly, these effects are mediated by a single differentiated donor cell type, bringing clarity to the cause-effect relationship.