Loss of Integrin αvβ8 in Murine Hepatocytes Accelerates Liver Regeneration
2018; Elsevier BV; Volume: 189; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2018.10.007
ISSN1525-2191
AutoresStephen N. Greenhalgh, Kylie P. Matchett, Richard S. Taylor, Katherine Huang, John T. Li, Koy Saeteurn, Mhairi Donnelly, Eilidh E.M. Simpson, Joshua L. Pollack, Amha Atakilit, Kenneth J. Simpson, Jacquelyn J. Maher, John P. Iredale, Dean Sheppard, Neil C. Henderson,
Tópico(s)Phagocytosis and Immune Regulation
ResumoRecent fate-mapping studies in mice have provided substantial evidence that mature adult hepatocytes are a major source of new hepatocytes after liver injury. In other systems, integrin αvβ8 has a major role in activating transforming growth factor (TGF)-β, a potent inhibitor of hepatocyte proliferation. We hypothesized that depletion of hepatocyte integrin αvβ8 would increase hepatocyte proliferation and accelerate liver regeneration after injury. Using Itgb8flox/flox;Alb-Cre mice to deplete hepatocyte αvβ8, after partial hepatectomy, hepatocyte proliferation and liver-to-body weight ratio were significantly increased in Itgb8flox/flox;Alb-Cre mice compared with control mice. Antibody-mediated blockade of hepatocyte αvβ8 in vitro, with assessment of TGF-β signaling pathways by real-time quantitative PCR array, supported the hypothesis that integrin αvβ8 inhibition alters hepatocyte TGF-β signaling toward a pro-regenerative phenotype. A diethylnitrosamine-induced model of hepatocellular carcinoma, used to examine the possibility that this pro-proliferative phenotype might be oncogenic, revealed no difference in either tumor number or size between Itgb8flox/flox;Alb-Cre and control mice. Immunohistochemistry for integrin αvβ8 in healthy and injured human liver demonstrated that human hepatocytes express integrin αvβ8. Depletion of hepatocyte integrin αvβ8 results in increased hepatocyte proliferation and accelerated liver regeneration after partial hepatectomy in mice. These data demonstrate that targeting integrin αvβ8 may represent a promising therapeutic strategy to drive liver regeneration in patients with a broad range of liver diseases. Recent fate-mapping studies in mice have provided substantial evidence that mature adult hepatocytes are a major source of new hepatocytes after liver injury. In other systems, integrin αvβ8 has a major role in activating transforming growth factor (TGF)-β, a potent inhibitor of hepatocyte proliferation. We hypothesized that depletion of hepatocyte integrin αvβ8 would increase hepatocyte proliferation and accelerate liver regeneration after injury. Using Itgb8flox/flox;Alb-Cre mice to deplete hepatocyte αvβ8, after partial hepatectomy, hepatocyte proliferation and liver-to-body weight ratio were significantly increased in Itgb8flox/flox;Alb-Cre mice compared with control mice. Antibody-mediated blockade of hepatocyte αvβ8 in vitro, with assessment of TGF-β signaling pathways by real-time quantitative PCR array, supported the hypothesis that integrin αvβ8 inhibition alters hepatocyte TGF-β signaling toward a pro-regenerative phenotype. A diethylnitrosamine-induced model of hepatocellular carcinoma, used to examine the possibility that this pro-proliferative phenotype might be oncogenic, revealed no difference in either tumor number or size between Itgb8flox/flox;Alb-Cre and control mice. Immunohistochemistry for integrin αvβ8 in healthy and injured human liver demonstrated that human hepatocytes express integrin αvβ8. Depletion of hepatocyte integrin αvβ8 results in increased hepatocyte proliferation and accelerated liver regeneration after partial hepatectomy in mice. These data demonstrate that targeting integrin αvβ8 may represent a promising therapeutic strategy to drive liver regeneration in patients with a broad range of liver diseases. Although the liver has a unique ability to regenerate, in many cases of liver disease this regenerative capacity is overwhelmed. A successful pro-regenerative therapy for the liver could have widespread application, reducing the need for transplantation in both acute and chronic liver failure, and potentially allowing more patients with primary or metastatic liver cancer to be treated successfully. Recent fate-mapping studies in mice have provided strong evidence that, in most murine models of liver injury and regeneration, restoration of liver mass occurs predominantly through self-duplication of hepatocytes.1Malato Y. Naqvi S. Schürmann N. Ng R. Wang B. Zape J. Kay M.A. Grimm D. Willenbring H. Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.J Clin Invest. 2011; 121: 4850-4860Crossref PubMed Scopus (330) Google Scholar, 2Yanger K. Knigin D. Zong Y. Maggs L. Gu G. Akiyama H. Pikarsky E. Stanger B.Z. Adult hepatocytes are generated by self-duplication rather than stem cell differentiation.Cell Stem Cell. 2014; 15: 340-349Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar Hence, identifying targets that promote proliferation and expansion of the preexistent hepatocyte population represents an attractive therapeutic approach to drive liver regeneration. Transforming growth factor (TGF)-β has pleiotropic roles in liver disease. In addition to its role as a major proinflammatory cytokine,3Dooley S. ten Dijke P. TGF-beta in progression of liver disease.Cell Tissue Res. 2012; 347: 245-256Crossref PubMed Scopus (514) Google Scholar TGF-β is also a potent repressor of hepatocyte proliferation.4Han C. Bowen W.C. Li G. Demetris A.J. Michalopoulos G.K. Wu T. Cytosolic phospholipase A2alpha and peroxisome proliferator-activated receptor gamma signaling pathway counteracts transforming growth factor beta-mediated inhibition of primary and transformed hepatocyte growth.Hepatology. 2010; 52: 644-655Crossref PubMed Scopus (25) Google Scholar, 5McMahon J.B. Richards W.L. del Campo A.A. Song M.K. Thorgeirsson S.S. Differential effects of transforming growth factor-beta on proliferation of normal and malignant rat liver epithelial cells in culture.Cancer Res. 1986; 46: 4665-4671PubMed Google Scholar, 6Russell W.E. Coffey R.J. Ouellette A.J. Moses H.L. Type beta transforming growth factor reversibly inhibits the early proliferative response to partial hepatectomy in the rat.Proc Natl Acad Sci U S A. 1988; 85: 5126-5130Crossref PubMed Scopus (370) Google Scholar, 7Ichikawa T. Zhang Y.Q. Kogure K. Hasegawa Y. Takagi H. Mori M. Kojima I. Transforming growth factor beta and activin tonically inhibit DNA synthesis in the rat liver.Hepatology. 2001; 34: 918-925Crossref PubMed Scopus (77) Google Scholar Therefore, in principle, TGF-β inhibition appears an attractive therapeutic strategy to promote hepatocyte proliferation and liver regeneration. An ideal therapy would target TGF-β with precision, allowing hepatocytes to escape the mito-inhibitory effects of TGF-β, while not abrogating the positive effects of TGF-β on extracellular matrix production and vascular remodeling during the regenerative process.8Michalopoulos G.K. Hepatostat: liver regeneration and normal liver tissue maintenance.Hepatology. 2017; 65: 1384-1392Crossref PubMed Scopus (242) Google Scholar, 9Michalopoulos G.K. Liver regeneration.J Cell Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1154) Google Scholar Furthermore, pan–TGF-β blockade may result in a number of unwanted, off-target effects, such as induction of autoimmunity and hepatocarcinogenesis.10Kulkarni A.B. Huh C.G. Becker D. Geiser A. Lyght M. Flanders K.C. Roberts A.B. Sporn M.B. Ward J.M. Karlsson S. Transforming growth factor beta 1 null mutation in mice causes excessive inflammatory response and early death.Proc Natl Acad Sci U S A. 1993; 90: 770-774Crossref PubMed Scopus (1649) Google Scholar, 11Im Y.H. Kim H.T. Kim I.Y. Factor V.M. Hahm K.B. Anzano M. Jang J.J. Flanders K. Haines D.C. Thorgeirsson S.S. Sizeland A. Kim S.J. Heterozygous mice for the transforming growth factor-beta type II receptor gene have increased susceptibility to hepatocellular carcinogenesis.Cancer Res. 2001; 61: 6665-6668PubMed Google Scholar, 12Kanzler S. Meyer E. Lohse A.W. Schirmacher P. Henninger J. Galle P.R. Blessing M. Hepatocellular expression of a dominant-negative mutant TGF-beta type II receptor accelerates chemically induced hepatocarcinogenesis.Oncogene. 2001; 20: 5015-5024Crossref PubMed Scopus (60) Google Scholar Therefore, a more nuanced, selective approach that targets the TGF-β pathway to promote liver regeneration is required. TGF-β is predominantly stored within the extracellular matrix in a latent state, and much of the regulation of TGF-β function results from precise, temporally and spatially restricted, extracellular activation of this latent complex.13Worthington J.J. Klementowicz J.E. Travis M.A. TGFbeta: a sleeping giant awoken by integrins.Trends Biochem Sci. 2011; 36: 47-54Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar The αv integrins, transmembrane heterodimeric proteins comprising an αv subunit and one of five β subunits, bind to an arginine-glycine-aspartate (RGD) motif present on the tip of an exposed loop within the latency-associated peptide that maintains TGF-β in an inactive state.14Hynes R.O. Integrins: bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6868) Google Scholar All five αv integrins have been shown to interact with the RGD motif present in the latency-associated peptide.15Munger J.S. Huang X. Kawakatsu H. Griffiths M.J. Dalton S.L. Wu J. Pittet J.F. Kaminski N. Garat C. Matthay M.A. Rifkin D.B. Sheppard D. The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis.Cell. 1999; 96: 319-328Abstract Full Text Full Text PDF PubMed Scopus (1644) Google Scholar, 16Mu D. Cambier S. Fjellbirkeland L. Baron J.L. Munger J.S. Kawakatsu H. Sheppard D. Broaddus V.C. Nishimura S.L. The integrin alpha(v)beta8 mediates epithelial homeostasis through MT1-MMP-dependent activation of TGF-beta1.J Cell Biol. 2002; 157: 493-507Crossref PubMed Scopus (593) Google Scholar, 17Asano Y. Ihn H. Yamane K. Jinnin M. Mimura Y. Tamaki K. Increased expression of integrin alpha(v)beta3 contributes to the establishment of autocrine TGF-beta signaling in scleroderma fibroblasts.J Immunol. 2005; 175: 7708-7718Crossref PubMed Scopus (195) Google Scholar, 18Asano Y. Ihn H. Yamane K. Jinnin M. Mimura Y. Tamaki K. Involvement of alphavbeta5 integrin-mediated activation of latent transforming growth factor beta1 in autocrine transforming growth factor beta signaling in systemic sclerosis fibroblasts.Arthritis Rheum. 2005; 52: 2897-2905Crossref PubMed Scopus (115) Google Scholar, 19Reed N.I. Jo H. Chen C. Tsujino K. Arnold T.D. DeGrado W.F. Sheppard D. The alphavbeta1 integrin plays a critical in vivo role in tissue fibrosis.Sci Transl Med. 2015; 7: 288ra79Crossref PubMed Scopus (190) Google Scholar This integrin–RGD interaction, in the presence of mechanical force supplied by the integrin-expressing cell, enables the release of the active TGF-β homodimer.20Shi M. Zhu J. Wang R. Chen X. Mi L. Walz T. Springer T.A. Latent TGF-beta structure and activation.Nature. 2011; 474: 343-349Crossref PubMed Scopus (686) Google Scholar Inhibition of myofibroblast αv integrins in mice reduces fibrosis in multiple organs via a reduction in TGF-β activation.21Henderson N.C. Arnold T.D. Katamura Y. Giacomini M.M. Rodriguez J.D. McCarty J.H. Pellicoro A. Raschperger E. Betsholtz C. Ruminski P.G. Griggs D.W. Prinsen M.J. Maher J.J. Iredale J.P. Lacy-Hulbert A. Adams R.H. Sheppard D. Targeting of alphav integrin identifies a core molecular pathway that regulates fibrosis in several organs.Nat Med. 2013; 19: 1617-1624Crossref PubMed Scopus (568) Google Scholar Furthermore, combined global knockout of integrins αvβ6 and αvβ8 phenocopies the developmental effects of loss of TGF-β–1 and –3.22Aluwihare P. Mu Z. Zhao Z. Yu D. Weinreb P.H. Horan G.S. Violette S.M. Munger J.S. Mice that lack activity of alphavbeta6- and alphavbeta8-integrins reproduce the abnormalities of Tgfb1- and Tgfb3-null mice.J Cell Sci. 2009; 122: 227-232Crossref PubMed Scopus (170) Google Scholar In the liver, expression of integrin αvβ6 appears restricted to activated cholangiocytes, transitional hepatocytes, and oval cells during biliary and portal fibrosis.23Popov Y. Patsenker E. Stickel F. Zaks J. Bhaskar K.R. Niedobitek G. Kolb A. Friess H. Schuppan D. Integrin alphavbeta6 is a marker of the progression of biliary and portal liver fibrosis and a novel target for antifibrotic therapies.J Hepatol. 2008; 48: 453-464Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 24Peng Z.W. Ikenaga N. Liu S.B. Sverdlov D.Y. Vaid K.A. Dixit R. Weinreb P.H. Violette S. Sheppard D. Schuppan D. Popov Y. Integrin alphavbeta6 critically regulates hepatic progenitor cell function and promotes ductular reaction, fibrosis, and tumorigenesis.Hepatology. 2016; 63: 217-232Crossref PubMed Scopus (77) Google Scholar Conversely, αvβ8 expression by hepatic cell types has not been well characterized. Integrin αvβ8 has been shown to play an important role in TGF-β activation in other systems, including dendritic cells,25Fenton T.M. Kelly A. Shuttleworth E.E. Smedley C. Atakilit A. Powrie F. Campbell S. Nishimura S.L. Sheppard D. Levison S. Worthington J.J. Lehtinen M.J. Travis M.A. Inflammatory cues enhance TGFbeta activation by distinct subsets of human intestinal dendritic cells via integrin alphavbeta8.Mucosal Immunol. 2017; 10: 624-634Crossref PubMed Scopus (40) Google Scholar, 26Reboldi A. Arnon T.I. Rodda L.B. Atakilit A. Sheppard D. Cyster J.G. IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches.Science. 2016; 352: aaf4822Crossref PubMed Scopus (185) Google Scholar, 27Travis M.A. Reizis B. Melton A.C. Masteller E. Tang Q. Proctor J.M. Wang Y. Bernstein X. Huang X. Reichardt L.F. Bluestone J.A. Sheppard D. Loss of integrin alpha(v)beta8 on dendritic cells causes autoimmunity and colitis in mice.Nature. 2007; 449: 361-365Crossref PubMed Scopus (405) Google Scholar regulatory T cells,28Worthington J.J. Kelly A. Smedley C. Bauché D. Campbell S. Marie J.C. Travis M.A. Integrin alphavbeta8-mediated TGF-beta activation by effector regulatory T cells is essential for suppression of T-cell-mediated inflammation.Immunity. 2015; 42: 903-915Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar neuroepithelium,29Arnold T.D. Niaudet C. Pang M.-F. Siegenthaler J. Gaengel K. Jung B. Ferrero G.M. Mukouyama Y.-S. Fuxe J. Akhurst R. Betsholtz C. Sheppard D. Reichardt L.F. Excessive vascular sprouting underlies cerebral hemorrhage in mice lacking alphaVbeta8-TGFbeta signaling in the brain.Development. 2014; 141: 4489-4499Crossref PubMed Scopus (74) Google Scholar and in fibroinflammatory airway disease.30Minagawa S. Lou J. Seed R.I. Cormier A. Wu S. Cheng Y. Murray L. Tsui P. Connor J. Herbst R. Govaerts C. Barker T. Cambier S. Yanagisawa H. Goodsell A. Hashimoto M. Brand O.J. Cheng R. Ma R. McKnelly K.J. Wen W. Hill A. Jablons D. Wolters P. Kitamura H. Araya J. Barczak A.J. Erle D.J. Reichardt L.F. Marks J.D. Baron J.L. Nishimura S.L. Selective targeting of TGF-beta activation to treat fibroinflammatory airway disease.Sci Transl Med. 2014; 6: 241ra79Crossref PubMed Scopus (71) Google Scholar Further, integrin αvβ8 inhibits proliferation of lung epithelium via TGF-β activation.31Fjellbirkeland L. Cambier S. Broaddus V.C. Hill A. Brunetta P. Dolganov G. Jablons D. Nishimura S.L. Integrin alphavbeta8-mediated activation of transforming growth factor-beta inhibits human airway epithelial proliferation in intact bronchial tissue.Am J Pathol. 2003; 163: 533-542Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar Therefore, given the important role of αvβ8 in mediating TGF-β activation in other organ systems and pathologic processes, we investigated the role of hepatocyte integrin αvβ8 in the context of liver regeneration. We hypothesized that depletion of integrin αvβ8 from hepatocytes would reduce local activation of TGF-β and would result in increased hepatocyte proliferation and accelerated liver regeneration after liver injury. Albumin-Cre (Alb-Cre) mice32Postic C. Shiota M. Niswender K.D. Jetton T.L. Chen Y. Moates J.M. Shelton K.D. Lindner J. Cherrington A.D. Magnuson M.A. Dual roles for glucokinase in glucose homeostasis as determined by liver and pancreatic beta cell-specific gene knock-outs using Cre recombinase.J Biol Chem. 1999; 274: 305-315Crossref PubMed Scopus (1024) Google Scholar were obtained from The Jackson Laboratory (Bar Harbor, ME), crossed with Itgb8flox/flox mice33Proctor J.M. Zang K. Wang D. Wang R. Reichardt L.F. Vascular development of the brain requires beta8 integrin expression in the neuroepithelium.J Neurosci. 2005; 25: 9940-9948Crossref PubMed Scopus (137) Google Scholar obtained from Louis F. Reichardt (University of California, San Francisco, San Francisco, CA), and the resulting Itgb8flox/flox;Alb-Cre mice were maintained on a C57BL/6 background. Pdgfrb-Cre mice (also on a C57BL/6 background) were obtained from Ralf H. Adams (Max Planck Institute for Molecular Biomedicine and University of Münster, Münster, Germany).34Foo S.S. Turner C.J. Adams S. Compagni A. Aubyn D. Kogata N. Lindblom P. Shani M. Zicha D. Adams R.H. Ephrin-B2 controls cell motility and adhesion during blood-vessel-wall assembly.Cell. 2006; 124: 161-173Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar Mice used for all experiments were 8 to 16 weeks old and housed under specific pathogen–free conditions in the Animal Barrier Facility of the University of California, San Francisco, or the University of Edinburgh, UK. Genotyping of all mice was performed by PCR. Sample size was determined statistically before experimentation. Age- and sex-matched littermate controls were used for all experiments. Investigators (S.N.G., K.P.M., K.H., K.S., M.C.D., E.E.M.S., and N.C.H.) were blinded to mouse genotype, and experimental order was decided randomly. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco, or performed in accordance with the UK Home Office regulations. Two-thirds of the liver was surgically removed under isoflurane anesthesia as previously described.35Mitchell C. Willenbring H. A reproducible and well-tolerated method for 2/3 partial hepatectomy in mice.Nat Protoc. 2008; 3: 1167-1170Crossref PubMed Scopus (363) Google Scholar All surgeries were performed in the first half of the day. To label proliferating hepatocytes, 5-bromo-2′-deoxyuridine (BrdU; 10280879001; Sigma-Aldrich, Gillingham, UK) was injected 2 hours before liver harvest (100 mg/kg intraperitoneally). Mice and livers were weighed at harvest to calculate liver-to-body weight ratio. Male mice were injected with diethylnitrosamine (25 mg/kg intraperitoneally; Sigma-Aldrich) at 12 to 14 days. Mice were sacrificed at 40 weeks, and macroscopic tumors were counted and measured. Whole blood was collected immediately after death and allowed to clot, and serum was obtained by centrifugation (9391 × g for 5 minutes twice). Samples were frozen at −20°C pending analysis. Serum albumin, total bilirubin, alanine transaminase, and alkaline phosphatase measurements were determined by using commercial kits (Alpha Laboratories, Eastleigh, UK, for albumin, bilirubin, and alanine transaminase; Randox Laboratories, Crumlin, County Antrim, UK, for alkaline phosphatase) adapted for use on a Cobas Fara centrifugal analyzer (Roche Diagnostics, Welwyn Garden City, UK). Liver samples were fixed overnight at room temperature in either methacarn (60% methanol, 30% chloroform, 10% acetic acid) for BrdU immunohistochemistry (IHC) or 10% neutral-buffered formalin. Samples were then paraffin-embedded before sectioning. No image processing was performed before quantitative analysis. Images presented in figures were contrast-enhanced by adjusting intensity minima and maxima. Images to be compared were processed identically and in a manner that preserved the visibility of dim and bright structures in the original image. Endogenous peroxidases were quenched with 0.3% H2O2 in methanol, followed by consecutive 10-minute incubation steps with 0.1% trypsin (T7168; Sigma-Aldrich), warm 1.8 mol/L HCl, and 0.1 mol/L sodium tetraborate decahydrate (S9640; Sigma-Aldrich). Blocking and subsequent incubation steps used the Mouse on Mouse Elite Peroxidase Kit (PK2200; Vector Laboratories, Peterborough, UK). Primary antibody was mouse anti-BrdU (dilution 1:40; M0744; Dako, Agilent Technologies, Cheadle, UK). Detection was performed by using the Elite Vectastain ABC kit (PK7100; Vector Laboratories) and 3,3′-diaminobenzidine (K3468; Dako) before counterstaining, dehydration, and mounting. For each liver sample, approximately 3000 hepatocytes were counted to calculate the percentage of proliferating hepatocytes. Antigen retrieval was performed with Tris-EDTA [platelet-derived growth factor receptor (PDGFR)β only], endogenous peroxidases were quenched with 3% H2O2, Avidin/Biotin block was applied (SP-2001; Vector Laboratories) before blocking with 20% goat serum (GR1/PDGFRβ) or rabbit serum (F4/80). Primary antibodies were applied for 2 hours at room temperature (PDGFRβ, dilution 1:500; ab32570; Abcam, Cambridge, UK) or overnight at 4°C (GR1, dilution 1:750; MAB1037; R&D, Abingdon, UK; F4/80, dilution 1:200; ab6640; Abcam). Secondary antibody (PDGFRβ–biotinylated goat anti-rabbit, dilution 1:1000; BA-1000; Vector Laboratories; GR1–biotinylated goat anti-rat, dilution 1:1000; BA-9401; F4/80–biotinylated rabbit anti-rat, dilution 1:200; BA-4001) was applied for 30 minutes at room temperature. Detection was performed by using the Elite Vectastain ABC kit and 3,3′-diaminobenzidine, before counterstaining, dehydration, and mounting. For each sample, 10 sequential fields were acquired at ×20 magnification, and the percentage of positive staining was calculated by using FIJI.36Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Preibisch S. Rueden C. Saalfeld S. Schmid B. Tinevez J.-Y. White D.J. Hartenstein V. Eliceiri K. Tomancak P. Cardona A. Fiji: an open-source platform for biological-image analysis.Nat Methods. 2012; 9: 676-682Crossref PubMed Scopus (30462) Google Scholar Antigen retrieval was performed with Tris-EDTA (β8 integrin subunit) or sodium citrate (cleaved caspase-3), endogenous peroxidases were quenched with 3% H2O2 before blocking with 20% horse serum. Primary antibody (β8 integrin subunit, dilution 1:500; ab80673; Abcam; Cleaved Caspase-3, dilution 1:1000; 9664; Cell Signaling Technology, Leiden, The Netherlands) was applied overnight at 4°C. Detection was performed by using the ImmPRESS Polymerized Reporter Enzyme Staining System (MP7401; Vector Laboratories) and 3,3′-diaminobenzidine before counterstaining, dehydration, and mounting. Sections were baked at 55°C overnight before de-waxing and rehydration. Slides were then placed in Harris Hematoxylin (Thermo Fisher Scientific, Paisley, UK) for 5 minutes. After washing, slides were placed in 1% acid alcohol for 5 seconds, followed by Scott's tap water for 2 minutes. Slides were then transferred to Eosin Y solution (Thermo Fisher Scientific) for 2 minutes, followed by washing, dehydration, and mounting. For quantification of mitotic figures, a minimum of 1000 hepatocytes were counted per sample. Primary mouse hepatocytes were isolated by retrograde perfusion of the liver with Liver Perfusion Medium (Thermo Fisher Scientific), followed by Liver Digest Medium (Thermo Fisher Scientific) at 37°C. When hepatocytes were visually dispersed within the liver capsule, the liver was removed to a sterile dish and minced with scissors to release the crude cell isolate. The cells were then suspended in Dulbecco's modified Eagle's medium/F-12 (Thermo Fisher Scientific) and pelleted twice. Hepatocytes were purified from the washed pellets by resuspension in culture medium and centrifugation through 50% equilibrated Percoll (GE Healthcare Life Sciences, Little Chalfont, UK). Primary hepatocytes were isolated as described in the section above, resuspended in low-serum medium [Dulbecco's modified Eagle's medium (Thermo Fisher Scientific), 2.5% fetal bovine serum (Thermo Fisher Scientific), 2% l-glutamine (Thermo Fisher Scientific), 1% penicillin streptomycin (Thermo Fisher Scientific)], and plated onto collagen-coated wells (Collagen Type I; Millipore, Watford, UK) in a 6-well plate at a density of 500,000 cells per well. Either β8 integrin subunit blocking antibody26Reboldi A. Arnon T.I. Rodda L.B. Atakilit A. Sheppard D. Cyster J.G. IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches.Science. 2016; 352: aaf4822Crossref PubMed Scopus (185) Google Scholar or nonbinding control antibody were added at 20 μg/mL, and samples were incubated for 24 hours at 37°C in 5% CO2. Wells were then washed with phosphate-buffered saline (PBS), and cells were lysed as described in the following section. RNA was isolated from whole mouse liver, primary hepatocytes, or liver sinusoidal endothelial cells by using a RNeasy Mini Kit (whole liver, hepatocytes) or Rneasy Plus Micro Kit (liver sinusoidal endothelial cells; Qiagen, Manchester, UK). cDNA transcription and real-time quantitative PCR (qPCR) were performed by using a SYBR-GreenER Two-Step qRT-PCR kit (Invitrogen, Thermo Fisher Scientific) or QuantiTect Reverse Transcription and SYBR Green PCR Kits (Qiagen). Samples were amplified on an ABI 7900HT thermocycler (Applied Biosystems, Thermo Fisher Scientific) and normalized to Actb and/or Gapdh expression. Primers used were as follows: Actb, 5′TGTTACCAACTGGGACGACA-3′ (forward) and 5′-GGGGTGTTGAAGGTCTCAAA-3′ (reverse); Itgb8, 5′-CTGAAGAAATACCCCGTGGA-3′ (forward) and 5′-ATGGGGAGGCATACAGTCT-3′ (reverse). Quantitect Primer Assays (249990; Qiagen) were used for the following genes: Ccna2 (QT00102151), Ccnb1 (QT00152040), Ccnd1 (QT00154595), Ccne1 (QT00103495), Cdkn1a (QT00137053), Cdkn1b (QT01058708), Gapdh (QT01658692), and Plat (QT00133630). To assess TGF-β signaling, a custom RT2 Profiler PCR array (330171; Qiagen) was designed which contained primer sequences for the genes shown in Supplemental Table S1. RNA was isolated after primary hepatocyte culture as described in the section above and reversed transcribed by using the RT2 First Strand Kit (330401; Qiagen). qPCR was performed by using RT2 SYBR Green ROX qPCR Mastermix (330522; Qiagen) on an ABI 7900HT thermocycler, normalized to Actb and Gapdh expression. Adhesion was assessed by using a colorimetric ECM Cell Adhesion Array Kit (ECM540; Millipore) according to the manufacturer's instructions. Primary mouse hepatocytes were isolated as described above, plated in triplicate at 50,000 cells per well, and incubated for 2 hours at 37°C in 5% CO2. Absorbance was measured at 570 nm by using a Synergy HT microplate reader (BioTek, Swindon, UK). Relative absorbance was calculated by standardizing to absorbance in the Collagen I well, before calculation of mean relative absorbance for each extracellular matrix protein for each sample. Primary mouse hepatocytes were isolated as above and plated at 10,000 cells per well in 24-well plates (353847; Primaria; Corning, St David's Park, UK) in Dulbecco's modified Eagle's medium/F-12 supplemented with 15 mmol/L HEPES (H3375; Sigma-Aldrich), 10% fetal bovine serum, 1% insulin-transferrin-selenium (41400-045; Thermo Fisher Scientific), and 1% penicillin streptomycin. Cells were allowed to adhere for 4 hours before washing with PBS. Cells were then cultured for 48 hours in a low-serum version of the plating medium, containing only 0.5% fetal bovine serum. The β8 integrin subunit blocking antibody or nonbinding control antibody was added at 20 μg/mL. Growth factors [hepatocyte growth factor (HGF), PHG0254, and epidermal growth factor (EGF), PMG8044; Thermo Fisher Scientific] were added at 40 ng/mL. Culture medium, antibodies, and growth factors were refreshed at 24 hours, at which time 10 μmol/L EdU (5-ethynyl-2′-deoxyuridine; C10640; Thermo Fisher Scientific) was added. After the 48-hour culture period, cells were washed with PBS-BSA [PBS supplemented with 1% bovine serum albumin (BSA); A8806; Sigma-Aldrich] and then fixed by using 4% paraformaldehyde in PBS for 15 minutes at room temperature. Proliferating hepatocytes were detected by using the Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (C10640; Thermo Fisher Scientific). Briefly, fixed cells were washed with PBS-BSA and incubated in 0.5% Triton X-100 (T8787; Sigma-Aldrich) in PBS for 20 minutes at room temperature. After washing, the Click-iT Plus reaction cocktail was added, and cells were incubated for 30 minutes at room temperature and protected from light. The cells were washed again and then incubated in 5 μg/mL Hoechst 33342 (Thermo Fisher Scientific) for 30 minutes at room temperature and protected from light. Finally, cells were washed with PBS and imaged. Imaging was performed by using an LSM780 confocal microscope system (Carl Zeiss Ltd, Cambridge, UK). Tiled images were acquired, with three non-overlapping areas of 18 μm2 imaged per well. Imaris version 8.4.1 (Bitplane AG, Zurich, Switzerland) was used to identify the total (Hoechst-positive) nuclei number and the number of EdU-positive nuclei, and the percentage of proliferating nuclei was calculated. Sample preparation, labeling, and array hybridizations were performed by using the Agilent GE 4 × 44 Mouse microarray platform (Agilent Technologies, Palo Alto, CA). Total RNA quality was assessed by using a Pico Chip on an Agilent 2100 Bioanalyzer, and RNA was amplified and labeled with cyanine 3–cytidine-5′-triphosphate by using the Agilent Technologies low RNA input fluorescent linear amplification kits according to the manufacturer's protocol. Labeled cRNA was assessed by using the Nanodrop ND-100 (Nanodrop Technologies, Inc., Wilmington DE), and equal amounts of cyanine 3-labeled target were hybridized to Agilent whole mouse genome 4 × 44K Ink-jet arrays (G4122F; Agilent Technologies). Hybridizations were performed for 14 hours, according to the manufacturer's protocol. Arrays were scanned by using the Agilent Technologies mi
Referência(s)