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Thomas G Bird,
Wei-Yu Lu,
Luke Boulter,
Sabrina Gordon-Keylock,
Rachel A Ridgway,
Michael J Williams,
Jessica Taube,
James A Thomas,
Davina Wojtacha,
Adriana Gambardella,
Owen J Sansom, John P Iredale,
Stuart J Forbes
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ABSTRACT: Tissue progenitor cells are an attractive target for regenerative therapy. In various organs, bone marrow cell (BMC) therapy has shown promising preliminary results, but to date no definite mechanism has been demonstrated to account for the observed benefit in organ regeneration. Tissue injury and regeneration is invariably accompanied by macrophage infiltration, but their influence upon the progenitor cells is incompletely understood, and direct signaling pathways may be obscured by the multiple roles of macrophages during organ injury. We therefore examined a model without injury; a single i.v. injection of unfractionated BMCs in healthy mice. This induced ductular reactions (DRs) in healthy mice. We demonstrate that macrophages within the unfractionated BMCs are responsible for the production of DRs, engrafting in the recipient liver and localizing to the DRs. Engrafted macrophages produce the cytokine TWEAK (TNF-like weak inducer of apoptosis) in situ. We go on to show that recombinant TWEAK activates DRs and that BMC mediated DRs are TWEAK dependent. DRs are accompanied by liver growth, occur in the absence of liver tissue injury and hepatic progenitor cells can be isolated from the livers of mice with DRs. Overall these results reveal a hitherto undescribed mechanism linking macrophage infiltration to DRs in the liver and highlight a rationale for macrophage derived cell therapy in regenerative medicine.
Proceedings of the National Academy of Sciences 04/2013; · 9.68 Impact Factor
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Hepatology 02/2013; · 11.66 Impact Factor
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ABSTRACT: Fibrosis is a highly conserved wound healing response and represents the final common pathway of virtually all chronic inflammatory injuries. Over the past 3 decades detailed analysis of hepatic extracellular matrix synthesis and degradation using approaches incorporating human disease, experimental animal models and cell culture have highlighted the extraordinarily dynamic nature of tissue repair and remodelling in this solid organ. Furthermore emerging studies of fibrosis in other organs demonstrate that basic common mechanisms exist, suggesting that bidirectionality of the fibrotic process may not solely be the preserve of the liver. In this review we will examine the cellular and molecular mechanisms that govern extracellular matrix degradation and fibrosis resolution, and highlight how manipulation of these processes may result in the development of effective anti-fibrotic therapies.
Biochimica et Biophysica Acta 11/2012; · 4.66 Impact Factor
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Prakash Ramachandran,
Antonella Pellicoro,
Madeleine A Vernon,
Luke Boulter,
Rebecca L Aucott,
Aysha Ali,
Stephen N Hartland,
Victoria K Snowdon,
Andrea Cappon,
Timothy T Gordon-Walker,
Mike J Williams,
Donald R Dunbar,
Jonathan R Manning,
Nico van Rooijen,
Jonathan A Fallowfield,
Stuart J Forbes, John P Iredale
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ABSTRACT: 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.
Proceedings of the National Academy of Sciences 10/2012; · 9.68 Impact Factor
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Salvatore Pernagallo,
Olga Tura,
Mei Wu,
Kay Samuel,
Juan J Diaz-Mochon,
Anna Hansen,
Rong Zhang,
Melany Jackson,
Gareth J Padfield,
Patrick W F Hadoke,
Nicholas L Mills,
Marc L Turner, John P Iredale,
David C Hay,
Mark Bradley
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ABSTRACT: Rapid endothelisation is of critical importance in the prevention of adverse remodelling after device implantation. Currently, there is a need for alternative strategies to promote re-endothelialisation for intravascular stents and vascular grafts. Using polymer microarray technology 345 polymers are comprehensively assessed and a matrix is identified that specifically supports both progenitor and mature endothelial cell activity in vitro and in vivo while minimising platelet attachment.
Advanced healthcare materials. 09/2012; 1(5):646-56.
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Gastroenterology 08/2012; 143(4):890-2. · 11.68 Impact Factor
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Wenli Zhou,
Zara Hannoun,
Ellis Jaffray,
Claire N Medine,
James R Black,
Sebastian Greenhough,
Liang Zhu,
James A Ross,
Stuart Forbes,
Ian Wilmut, John P Iredale,
Ronald T Hay,
David C Hay
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ABSTRACT: The coordination of signalling pathways within the cell is vital for normal human development and post-natal tissue homeostasis. Gene expression and function is therefore tightly controlled at a number of levels. We investigated the role that post-translational modifications play during human hepatocyte differentiation. In particular, we examined the role of the small ubiquitin-like modifier (SUMO) proteins in this process. We used a human embryonic stem cell (hESC)-based model of hepatocyte differentiation to follow changes in protein SUMOylation. Moreover, to confirm the results derived from our cell-based system, we performed in vitro conjugation assays to characterise SUMO modification of a key liver-enriched transcription factor, HNF4α. Our analyses indicate that SUMOylation plays an important role during hepatocellular differentiation and this is mediated, in part, through regulation of the stability of HNF4α in a ubiquitin-dependent manner. Our study provides a better understanding of SUMOylation during human hepatocyte differentiation and maturation. Moreover, we believe the results will stimulate interest in the differentiation and phenotypic regulation of other somatic cell types.
Journal of Cell Science 04/2012; 125(Pt 15):3630-5. · 6.11 Impact Factor
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Luke Boulter,
Olivier Govaere,
Tom G Bird,
Sorina Radulescu,
Prakash Ramachandran,
Antonella Pellicoro,
Rachel A Ridgway,
Sang Soo Seo,
Bart Spee,
Nico Van Rooijen,
Owen J Sansom, John P Iredale,
Sally Lowell,
Tania Roskams,
Stuart J Forbes
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ABSTRACT: During chronic injury a population of bipotent hepatic progenitor cells (HPCs) become activated to regenerate both cholangiocytes and hepatocytes. Here we show in human diseased liver and mouse models of the ductular reaction that Notch and Wnt signaling direct specification of HPCs via their interactions with activated myofibroblasts or macrophages. In particular, we found that during biliary regeneration, expression of Jagged 1 (a Notch ligand) by myofibroblasts promoted Notch signaling in HPCs and thus their biliary specification to cholangiocytes. Alternatively, during hepatocyte regeneration, macrophage engulfment of hepatocyte debris induced Wnt3a expression. This resulted in canonical Wnt signaling in nearby HPCs, thus maintaining expression of Numb (a cell fate determinant) within these cells and the promotion of their specification to hepatocytes. By these two pathways adult parenchymal regeneration during chronic liver injury is promoted.
Nature medicine 03/2012; 18(4):572-9. · 27.14 Impact Factor
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Journal of Hepatology 02/2012; 56(6):1417-9. · 9.26 Impact Factor
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Zoi Michailidou,
Sophie Turban,
Eileen Miller,
Xiantong Zou,
Joerg Schrader,
Peter J. Ratcliffe,
Patrick W. F. Hadoke,
Brian R. Walker, John P. Iredale,
Nicholas M. Morton,
Jonathan R. Seckl
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ABSTRACT: In obesity, rapidly expanding adipose tissue becomes hypoxic, precipitating inflammation, fibrosis, and insulin resistance.
Compensatory angiogenesis may prevent these events. Mice lacking the intracellular glucocorticoid-amplifying enzyme 11β-hydroxysteroid
dehydrogenase type 1 (11βHSD1−/−) have “healthier” adipose tissue distribution and resist metabolic disease with diet-induced obesity. Here we show that adipose
tissues of 11βHSD1−/− mice exhibit attenuated hypoxia, induction of hypoxia-inducible factor (HIF-1α) activation of the TGF-β/Smad3/α-smooth muscle
actin (α-SMA) signaling pathway, and fibrogenesis despite similar fat accretion with diet-induced obesity. Moreover, augmented
11βHSD1−/− adipose tissue angiogenesis is associated with enhanced peroxisome proliferator-activated receptor γ (PPARγ)-inducible expression
of the potent angiogenic factors VEGF-A, apelin, and angiopoietin-like protein 4. Improved adipose angiogenesis and reduced
fibrosis provide a novel mechanism whereby suppression of intracellular glucocorticoid regeneration promotes safer fat expansion
with weight gain.
Journal of Biological Chemistry 02/2012; 287(6):4188-4197. · 4.77 Impact Factor
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ABSTRACT: The liver is a highly resilient organ that possesses enormous regenerative capacity. This is mediated mainly
through the most abundant cell type found in the liver, the hepatocyte.When the regenerative capacity of the hepatocyte
is compromised, during chronic or acute liver injury, hepatic progenitor cells (HPCs) are activated to
replace the damaged tissue. The HPC resides in a laminin-rich environment; as HPCs differentiate toward a hepatic
or biliary fate, the extracellular matrix (ECM) composition changes, influencing cell behavior. To assess the
impact that the biological ECM and the synthetic ECM have on the maintenance of hepatic stem cell gene expression,
a murine hepatic stem cell line was employed.We demonstrate that hepatic stem cell gene expression could
be maintained using a biological or synthetic substratum, but not on plastic alone.
BIORESEARCH OPEN ACCESS. 01/2012; 1(1):50-53.
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Antonella Pellicoro,
Rebecca L Aucott,
Prakash Ramachandran,
Andrew J Robson,
Jonathan A Fallowfield,
Victoria K Snowdon,
Stephen N Hartland,
Madeleine Vernon,
Jeremy S Duffield,
R Christopher Benyon,
Stuart J Forbes, John P Iredale
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ABSTRACT: 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 (CCl(4) ) 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 CCl(4) 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 CCl(4) 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 12/2011; 55(6):1965-75. · 11.66 Impact Factor
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Zoi Michailidou,
Sophie Turban,
Eileen Miller,
Xiantong Zou,
Joerg Schrader,
Peter J Ratcliffe,
Patrick W F Hadoke,
Brian R Walker, John P Iredale,
Nicholas M Morton,
Jonathan R Seckl
[show abstract]
[hide abstract]
ABSTRACT: In obesity, rapidly expanding adipose tissue becomes hypoxic, precipitating inflammation, fibrosis, and insulin resistance. Compensatory angiogenesis may prevent these events. Mice lacking the intracellular glucocorticoid-amplifying enzyme 11β-hydroxysteroid dehydrogenase type 1 (11βHSD1(-/-)) have "healthier" adipose tissue distribution and resist metabolic disease with diet-induced obesity. Here we show that adipose tissues of 11βHSD1(-/-) mice exhibit attenuated hypoxia, induction of hypoxia-inducible factor (HIF-1α) activation of the TGF-β/Smad3/α-smooth muscle actin (α-SMA) signaling pathway, and fibrogenesis despite similar fat accretion with diet-induced obesity. Moreover, augmented 11βHSD1(-/-) adipose tissue angiogenesis is associated with enhanced peroxisome proliferator-activated receptor γ (PPARγ)-inducible expression of the potent angiogenic factors VEGF-A, apelin, and angiopoietin-like protein 4. Improved adipose angiogenesis and reduced fibrosis provide a novel mechanism whereby suppression of intracellular glucocorticoid regeneration promotes safer fat expansion with weight gain.
Journal of Biological Chemistry 12/2011; 287(6):4188-97. · 4.77 Impact Factor
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Michael A Gibbons,
Alison C MacKinnon,
Prakash Ramachandran,
Kevin Dhaliwal,
Rodger Duffin,
Alexander T Phythian-Adams,
Nico van Rooijen,
Christopher Haslett,
Sarah E Howie,
A John Simpson,
Nikhil Hirani,
Jack Gauldie, John P Iredale,
Tariq Sethi,
Stuart J Forbes
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ABSTRACT: Idiopathic pulmonary fibrosis (IPF) is a devastating disease. Antiinflammatory therapies, including corticosteroids, are of no benefit. The role of monocytes and macrophages is therefore controversial.
To define the role of monocytes and macrophages during lung fibrogenesis and resolution, and explore the phenotype of the cells involved.
We used multiple in vivo depletional strategies, backed up by adoptive transfer techniques. Further studies were performed on samples from patients with IPF.
Depletion of lung macrophages during fibrogenesis reduced pulmonary fibrosis as measured by lung collagen (P = 0.0079); fibrosis score (P = 0.0051); and quantitative polymerase chain reaction for surrogate markers of fibrosis Col1 (P = 0.0083) and a-smooth muscle actin (P = 0.0349). There was an associated reduction in markers of the profibrotic alternative macrophage activation phenotype, Ym1 (P = 0.0179), and Arginase 1. The alternative macrophage marker CD163 was expressed on lung macrophages from patients with IPF. Depletion of Ly6Chi circulating monocytes reduced pulmonary fibrosis (P = 0.0052) and the number of Ym1- positive alternatively activated lung macrophages (P = 0.0310). Their adoptive transfer during fibrogenesis exacerbated fibrosis (P = 0.0304); however, adoptively transferred CD45.1 Ly6Chi cells were not found in the lungs of recipient CD45.2 mice.
We demonstrate the importance of circulating monocytes and lung macrophages during pulmonary fibrosis, and emphasize the importance of the alternatively activated macrophage phenotype. We show that Ly6Chi monocytes facilitate the progression of pulmonary fibrosis, but are not obviously engrafted into lungs thereafter. Finally, we provide empirical data to suggest that macrophages may have a resolution-promoting role during the reversible phase of bleomycin-induced pulmonary fibrosis.
American Journal of Respiratory and Critical Care Medicine 06/2011; 184(5):569-81. · 11.08 Impact Factor
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Gastroenterology 03/2011; 140(5):1395-8. · 11.68 Impact Factor
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David C Hay,
Salvatore Pernagallo,
Juan Jose Diaz-Mochon,
Claire N Medine,
Sebastian Greenhough,
Zara Hannoun,
Joerg Schrader,
James R Black,
Judy Fletcher,
Donna Dalgetty,
Alexandra I Thompson,
Philip N Newsome,
Stuart J Forbes,
James A Ross,
Mark Bradley, John P Iredale
[show abstract]
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ABSTRACT: Maintaining stable differentiated somatic cell function in culture is essential to a range of biological endeavors. However, current technologies, employing, for example, primary hepatic cell culture (essential to the development of a bio-artificial liver and improved drug and toxicology testing), are limited by supply, expense, and functional instability even on biological cell culture substrata. As such, novel biologically active substrates manufacturable to GMP standards have the potential to improve cell culture-based assay applications. Currently hepatic endoderm (HE) generated from pluripotent stem cells is a genotypically diverse, cheap, and stable source of "hepatocytes"; however, HE routine applications are limited due to phenotypic instability in culture. Therefore a manufacturable subcellular matrix capable of supporting long-term differentiated cell function would represent a step forward in developing scalable and phenotypically stable hESC-derived hepatocytes. Adopting an unbiased approach we screened polymer microarrays and identified a polyurethane matrix which promoted HE viability, hepatocellular gene expression, drug-inducible metabolism, and function. Moreover, the polyurethane supported, when coated on a clinically approved bio-artificial liver matrix, long-term hepatocyte function and growth. In conclusion, our data suggest that an unbiased screening approach can identify cell culture substrate(s) that enhance the phenotypic stability of primary and stem cell-derived cell resources.
Stem cell research 03/2011; 6(2):92-102. · 3.39 Impact Factor
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James A Thomas,
Caroline Pope,
Davina Wojtacha,
Andrew J Robson,
Timothy T Gordon-Walker,
Stephen Hartland,
Prakash Ramachandran,
Marielle Van Deemter,
David A Hume, John P Iredale,
Stuart J Forbes
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ABSTRACT: 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 03/2011; 53(6):2003-15. · 11.66 Impact Factor
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ABSTRACT: The pathological mechanisms that distinguish simple steatosis from steatohepatitis (or NASH, with consequent risk of cirrhosis and hepatocellular cancer) remain incompletely defined. Whereas both a methionine- and choline-deficient diet (MCDD) and a choline-deficient diet (CDD) lead to hepatic triglyceride accumulation, MCDD alone is associated with hepatic insulin resistance and inflammation (steatohepatitis). We used metabolic tracer techniques, including stable isotope ([¹³C₄]palmitate) dilution and mass isotopomer distribution analysis (MIDA) of [¹³C₂]acetate, to define differences in intrahepatic fatty acid metabolism that could explain the contrasting effect of MCDD and CDD on NASH in C57Bl6 mice. Compared with control-supplemented (CS) diet, liver triglyceride pool sizes were similarly elevated in CDD and MCDD groups (24.37 ± 2.4, 45.94 ± 3.9, and 43.30 ± 3.5 μmol/liver for CS, CDD, and MCDD, respectively), but intrahepatic neutrophil infiltration and plasma alanine aminotransferase (31 ± 3, 48 ± 4, 231 ± 79 U/l, P < 0.05) were elevated only in MCDD mice. However, despite loss of peripheral fat in MCDD mice, neither the rate of appearance of palmitate (27.2 ± 3.5, 26.3 ± 2.3, and 28.3 ± 3.5 μmol·kg⁻¹·min⁻¹) nor the contribution of circulating fatty acids to the liver triglyceride pool differed between groups. Unlike CDD, MCDD had a defect in hepatic triglyceride export that was confirmed using intravenous tyloxapol (142 ± 21, 122 ± 15, and 80 ± 7 mg·kg⁻¹·h⁻¹, P < 0.05). Moreover, hepatic de novo lipogenesis was significantly elevated in the MCDD group only (1.4 ± 0.3, 2.3 ± 0.4, and 3.4 ± 0.4 μmol/day, P < 0.01). These findings suggest that important alterations in hepatic fatty acid metabolism may promote the development of steatohepatitis. Similar mechanisms may predispose to hepatocyte damage in human NASH.
AJP Endocrinology and Metabolism 02/2011; 300(2):E402-9. · 4.75 Impact Factor
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Catherine M Payne,
Kay Samuel,
Anne Pryde,
Jason King,
David Brownstein,
Joerg Schrader,
Claire N Medine,
Stuart J Forbes, John P Iredale,
Philip N Newsome,
David C Hay
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ABSTRACT: Human embryonic stem cells (hESCs) can be efficiently differentiated to hepatocyte-like cells (HLCs) in vitro and demonstrate many of the functions and gene expression found in the adult liver.
In this study, we assess the therapeutic value of HLCs in long-term cell-based therapies in vivo.
hESC-derived HLCs were injected into the spleen of acutely injured NODscid(IL-2Rγ) null mice and analysed at various time points post-transplantation up to 3 months.
Large clusters of human cells engrafted in the spleen after 3 days and had expanded considerably by 31 days. At these time points, we identified human cells expressing parenchymal hepatocyte markers, exhibiting biliary duct-like structures and expressing myofibroblast markers. Three months after transplantation, we could detect human HLCs that were positive for albumin and CK18 by immunostaining and human DNA by fluorescent in situ hybridisation. Moreover, we could detect secretion of human serum albumin by enzyme-linked immunoabsorbant assay.
We observed the persistence, engraftment and function of HLCs in vivo up to 3 months post-translation; however, all murine recipients developed large splenic and liver tumours that contained endodermal and mesodermal cell types. Although our studies demonstrate that hESC-derived HLCs have the potential to play an important role in cell-based therapies, current methodologies and transplantation strategies require substantial refinement before they can be deployed safely.
Liver international: official journal of the International Association for the Study of the Liver 02/2011; 31(2):254-62. · 3.82 Impact Factor
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Journal of Hepatology 12/2010; 54(5):853-5. · 9.26 Impact Factor
