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

UNLABELLED: 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.

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 CCl(4)-induced model of reversible hepatic fibrosis to identify and characterize the macrophage phenotype responsible for tissue remodeling: the hitherto elusive restorative macrophage. This CD11B(hi) F4/80(int) Ly-6C(lo) 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-6C(hi) monocytes, a common origin with profibrotic Ly-6C(hi) macrophages, indicative of a phenotypic switch in vivo conferring proresolution properties. Microarray profiling of the Ly-6C(lo) subset, compared with Ly-6C(hi) 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.

Improving detection of cystic fibrosis related liver disease using liver fibrosis assessment tools.

Background & aims: Cystic Fibrosis related liver disease (CFLD) is the 3rd largest cause of death in Cystic Fibrosis (CF). As advances in pulmonary therapies have increased life-expectancy, CFLD has become more prevalent. Current guidelines may underdiagnose liver fibrosis, particularly in its early stages. Newer modalities for the assessment of fibrosis may provide a more accurate assessment. FibroScan is validated in assessing fibrosis for several aetiologies including alcohol and fatty liver, the CFLD cohort have an entirely different phenotype so the cut off values are not transferrable. We appraised fibrosis assessment tools to improve diagnosis of CFLD. Methods: A prospective cohort (n = 114) of patients from the Manchester Adult Cystic Fibrosis Centre, UK were identified at annual assessment. Demographic data including co-morbidity, CFTR genotyping, biochemistry and imaging were used alongside current guidelines to group into CFLD and CF without evidence of liver disease. All patients underwent liver stiffness measurement (LSM) and assessment of serum-based fibrosis biomarker panels. A new diagnostic criterion was created and validated in a second, independent cohort. Results: 12 of 114 patient classified as CFLD according to the European Cystic Fibrosis Society best practice guidelines. No specific risk factors for development of CFLD were identified. Liver enzymes were elevated in patients with CFLD. Serum biomarker panels did not improve diagnostic criteria. LSM accurately predicted CFLD. A new diagnostic criterion was proposed and validated in a separate cohort, accurately predicating CFLD in 10 of 32 patients (31 %). Conclusion: We present a cohort of patients with CF assessed for the presence of liver fibrosis using blood biomarkers and LSM based platforms. We propose a new, simplified diagnostic criteria, capable of accurately predicting liver disease in patients with CF.Clinical trials number: NCT04277819.

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

UNLABELLED: 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.

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

UNLABELLED: 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. ß1-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;).

Hepatocellular carcinoma and the Fontan circulation: Clinical presentation and outcomes.

BACKGROUND: Fontan-associated liver disease (FALD) is universal in patients with a Fontan circulation. Hepatocellular carcinoma (HCC) is one of its severe expressions, and, though rare, frequently fatal. The purpose of this study was to describe the clinical presentation, risk factors, and outcomes of HCC in patients with a Fontan circulation. METHODS: A multicenter case series of Fontan patients with a diagnosis of HCC formed the basis of this study. The case series was extended by published cases and case reports. Clinical presentation, tumor characteristics, laboratory and hemodynamic findings as well as treatment types and outcomes, were described. RESULTS: Fifty-four Fontan patients (50% female) with a diagnosis of HCC were included. Mean age at HCC diagnosis was 30 ± 9.4 years and mean duration from Fontan surgery to HCC diagnosis was 21.6 ± 7.4 years. Median HCC size at the time of diagnosis was 4 cm with a range of 1 to 22 cm. The tumor was located in the right hepatic lobe in 65% of the patients. Fifty-one percent had liver cirrhosis at the time of HCC diagnosis. Fifty percent of the patients had no symptoms related to HCC and alpha-fetoprotein was normal in 26% of the cases. Twenty-six patients (48%) died during a median follow-up duration of 10.6 (range 1-50) months. CONCLUSIONS: HCC in Fontan patients occurs at a young age with a 1-year survival rate of only 50%. Meticulous liver surveillance is crucial to detect small tumors in the early stage.

Fontan-associated liver disease: A review.

The Fontan procedure has led to increased long-term survival of patients with single ventricle congenital heart disease. Hemodynamic changes associated with the Fontan circulation, including elevated central venous pressure and diminished cardiac output are responsible for the development of Fontan-associated liver disease (FALD). Liver fibrosis is a universal feature following the Fontan operation. The incidence of both liver cirrhosis and hepatocellular carcinoma (HCC) increases with the duration of the Fontan circulation. The staging of liver fibrosis in FALD requires a multi-modality approach involving clinical assessment, biochemical/hematological parameters, non-invasive fibrosis scores, radiological imaging, elastography, and liver histology. Patients with a failing Fontan circulation who have evidence of significant hepatic congestion require careful hemodynamic assessment to optimize the Fontan pathway and physiology. This may necessitate percutaneous or surgical intervention, or heart transplantation. Combined heart-liver transplantation may be required in patients with clinical, imaging, or biopsy evidence of advanced liver cirrhosis, particularly if there is evidence of hepatic decompensation or localized HCC. Patients with suspected liver cirrhosis should be enrolled into HCC surveillance and require endoscopic variceal assessment. There is a clear need to establish local/national registries for Fontan patients with standardized guidelines for the management of FALD, bringing together the expertise of professional bodies representing both cardiologists and hepatologists.

Exercise-Induced Systemic Venous Hypertension in the Fontan Circulation.

Increasingly end-organ injury is being demonstrated late after institution of the Fontan circulation, particularly liver fibrosis and cirrhosis. The exact mechanisms for these late phenomena remain largely elusive. Hypothesizing that exercise induces precipitous systemic venous hypertension and insufficient cardiac output for the exercise demand, that is, a possible mechanism for end-organ injury, we sought to demonstrate the dynamic exercise responses in systemic venous perfusion (SVP) and concurrent end-organ perfusion. Ten stable Fontan patients and 9 control subjects underwent incremental cycle ergometry-based cardiopulmonary exercise testing. SVP was monitored in the right upper limb, and regional tissue oxygen saturation was monitored in the brain and kidney using near-infrared spectroscopy. SVP rose profoundly in concert with workload in the Fontan group, described by the regression equation 15.97 + 0.073 watts per mm Hg. In contrast, SVP did not change in healthy controls. Regional renal (p <0.01) and cerebral tissue saturations (p <0.001) were significantly lower and decrease more rapidly in Fontan patients. We conclude that in a stable group of adult patients with Fontan circulation, high-intensity exercise was associated with systemic venous hypertension and reduced systemic oxygen delivery. This physiological substrate has the potential to contribute to end-organ injury.