Proteinase Activated Receptor 1 Mediated Fibrosis in a Mouse Model of Liver Injury: A Role for Bone Marrow Derived Macrophages

Published onlineJournal ArticleResearch Support, Non-U.S. Gov'tThis is the final version of the article. Available from Public Library of Science via the DOI in this record.Liver fibrosis results from the co-ordinated actions of myofibroblasts and macrophages, a proportion of which are of bone marrow origin. The functional effect of such bone marrow-derived cells on liver fibrosis is unclear. We examine whether changing bone marrow genotype can down-regulate the liver's fibrotic response to injury and investigate mechanisms involved. Proteinase activated receptor 1 (PAR1) is up-regulated in fibrotic liver disease in humans, and deficiency of PAR1 is associated with reduced liver fibrosis in rodent models. In this study, recipient mice received bone marrow transplantation from PAR1-deficient or wild-type donors prior to carbon tetrachloride-induced liver fibrosis. Bone marrow transplantation alone from PAR1-deficient mice was able to confer significant reductions in hepatic collagen content and activated myofibroblast expansion on wild-type recipients. This effect was associated with a decrease in hepatic scar-associated macrophages and a reduction in macrophage recruitment from the bone marrow. In vitro, PAR1 signalling on bone marrow-derived macrophages directly induced their chemotaxis but did not stimulate proliferation. These data suggest that the bone marrow can modulate the fibrotic response of the liver to recurrent injury. PAR1 signalling can contribute to this response by mechanisms that include the regulation of macrophage recruitment.Funding for YNK SJF came from the MRC Clinical Research Training Fellowship (G0500428), www.mrc.ac.uk. For CJS RCC: Wellcome Trust Programme Grant (071124), www.wellcome.ac.uk. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Assessment of bone mineral density in tenofovir-treated patients with chronic hepatitis B:can the fracture risk assessment tool identify those at greatest risk?

Abstract BACKGROUND: Tenofovir disoproxil fumarate (TDF) is an established nucleotide analogue in the treatment of chronic hepatitis B. Bone mineral density loss has been described in TDF-treated patients with human immunodeficiency virus infection, but limited data exist for patients with chronic hepatitis B. Dual X-ray absorptiometry (DEXA) was used to determine bone mineral density changes in TDF-exposed patients. We evaluated the accuracy of the Fracture Risk Assessment Tool (FRAX) as an alternative to DEXA in clinical practice. METHODS: A total of 170 patients were studied: 122 were exposed to TDF, and 48 were controls. All patients underwent DEXA, and demographic details were recorded. FRAX scores (before and after DEXA) were calculated. RESULTS: TDF was associated with a lower hip T score (P = .02). On univariate and multivariate analysis, advancing age, smoking, lower body mass index, and TDF exposure were independent predictors of low bone mineral density. In addition, the pre-DEXA FRAX score was an accurate predictor of the post-DEXA FRAX treatment recommendation (100% sensitivity and 83% specificity), area under the curve 0.93 (95% CI, .87-.97, P < .001). CONCLUSIONS: TDF-treated patients with chronic hepatitis B have reduced bone mineral density, but the reduction is limited to 1 anatomical site. Age and advanced liver disease are additional contributing factors, underlining the importance of multifactorial fracture risk assessment. FRAX can accurately identify those at greatest risk of osteoporotic fractureP. T. F. K. thanks the Hepatology Services at Barts Health NHS Trust; Josephine Schulz, Louise Payaniandy, Deva Payaniandy, and Valerie Ross; and the Barts and The London Charity, for their ongoing support. M. J. W. M. thanks the Wellcome Trust, for postdoctoral training fellowship support, and the NIHR Biomedical Research Centre at Imperial College London, for infrastructure support

PAR1 activation induces monocyte/macrophage migration but not proliferation.

<p>(A–B) Migration assay of primary mouse BM-derived macrophages and a human monocyte cell line (THP-1) to SFLLRN demonstrating chemotaxis to the PAR1 agonist in both cell-types (MCP1 positive control). (C–D) SFLLRN has no pproliferative effect on primary mouse BM-derived macrophages (72 h) or a mouse macrophage cell line (RAW 264.7, 24 h) plated at the two different starting cell densities shown. (graphs show mean + SEM; p values as shown.)</p

Hepatic collagen I mRNA expression <i>in vivo</i>.

<p>A considerable reduction in COL1A1 gene expression was found in PAR1(−/−) mice subjected to carbon tetrachloride (CCl₄) liver injury compared to WT controls (A). A corresponding reduction in COL1A1 gene expression of lower magnitude was seen WT mice transplanted with PAR1(−/−) bone marrow (PAR1(−/−) BMT) compared to WT controls transplanted with WT bone marrow (WT BMT) (B). (graphs show mean + SEM, normalised to hypoxanthine guanine phosphoribosyl transferase housekeeping gene expression and against respective controls, p values as shown.)</p

Absence of PAR1 signalling on BM-derived cells is associated with a significant reduction in macrophage recruitment to the injured liver.

<p>Female C57/BL6 mice received BMT from male PAR1 knockout donors (PAR1(−/−)BMT, B&D) or from male C57/BL6 WT controls (WTBMT, A&C) before CCl₄ liver injury. In both groups, BM-derived myofibroblasts (αSMA, red, A&B) and hepatic macrophages (F4/80, red, C&D) are seen (examples indicated by white arrowheads, ×400 magnification). (E) Splenic tissue showing complete haematopoietic reconstitution of recipient mice (female) with male cells, validating the efficacy of the BMT protocol. In all panels BM-derived (male) cells are identified by Y chromosome <i>in situ</i> hybridisation (green dot, Y chrm), localised within nuclei (DAPI, blue). (F) Graph showing the relative proportion of hepatic myofibroblasts (αSMA) and macrophages (F4/80) of BM origin. There is a significant reduction of BM-derived macrophage infiltration into the liver with loss of PAR1 signalling. (n = 8 per group, mean + SEM, p values as shown.)</p

Hepatic PAR1 is up-regulated during liver injury and is expressed on liver macrophages.

<p>(A–B) In wild-type mice, there is a marked up-regulation of hepatic PAR1 (brown, ×100) after carbon CCl₄ injury on cells along the hepatic scars (black arrows) and within the lobule itself (black arrowheads). (C–E) PAR1 (green) is commonly co-localised to macrophages (C, F4/80, red), but only occasionally to myofibroblasts (D, αSMA, red) and hepatic endothelium (E, endomucin, red). (Fluorescence images at ×200 magnification, nuclei in blue.)</p

Primer nucleotide sequences used for real-time quantitative RT-PCR.

<p>Col 1A1, collagen Iα1; HPRT, hypoxanthine guanine phosphoribosyl transferase.</p

Details of primary antibodies used for immunohistochemistry.

<p>αSMA, α-smooth muscle actin; PAR1, proteinase activated receptor 1; SCMW, sodium citrate buffer (2.94 g/L) microwave,</p>*<p>used for total hepatic macrophage counting with light microscopy.</p>**<p>used for fluorescence immunodetection of macrophages in conjunction with <i>in situ</i> hybridisation for Y chromosome.</p>§<p>a kind gift from Dr Eleanor Mackie, Melbourne, Australia.</p