Expression of Stromal Cell-Derived Factor-1 and of Its Receptor CXCR4 in Liver Regeneration from Oval Cells in Rat
2004; Elsevier BV; Volume: 165; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63248-8
ISSN1525-2191
AutoresPhilippe Mavier, Nadine Martin, Dominique Couchie, Anne-Marie Préaux, Yannick Laperche, Élie Serge Zafrani,
Tópico(s)Chemokine receptors and signaling
ResumoStromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway. Stromal cell-derived factor-1 is a chemokine that plays a major role during embryogenesis. Since stromal cell-derived factor-1 and its unique receptor CXCR4 are involved in the differentiation of progenitor cells, we studied the expression of this chemokine and of its receptor in hepatic regeneration from precursor oval cells. Hepatic regeneration was induced by treating rats with 2-acetylaminofluorene, and followed by partial hepatectomy. Oval cell accumulation, which predominated in periportal regions, reached a maximum at days 9 to 14 after hepatectomy and declined thereafter. Oval cells strongly expressed stromal cell-derived factor-1 protein and mRNA. CXCR4 mRNA hepatic level paralleled the number of oval cells and in situ hybridization showed CXCR4 mRNA expression by these cells. Treatment of rats with fucoidan, a sulfated polysaccharide which binds to stromal cell-derived factor-1 and blocks its biological effects, markedly decreased oval cell accumulation in five of the seven treated rats. In conclusion, our data demonstrate an expression of stromal cell-derived factor-1 and of its receptor CXCR4 in oval cells during hepatic regeneration and strongly suggest that stromal cell-derived factor-1 stimulates the proliferation of these precursor cells through an autocrine/paracrine pathway. The liver is an organ with an almost unlimited capacity to regenerate from mature differentiated cells.1Fausto N Hepatocytes break the rules of senescence in serial transplantation studies: is there a limit to their replicative capacity?.Am J Pathol. 1997; 151: 1187-1189PubMed Google Scholar However, in severe liver injury or after partial hepatectomy combined with inhibition of mature hepatocyte proliferation, regeneration is achieved through the expansion and differentiation of liver precursor cells, termed oval cells.2Sell S Heterogeneity and plasticity of hepatocyte lineage cells.Hepatology. 2001; 33: 738-750Crossref PubMed Scopus (376) Google Scholar These cells have been well characterized in rats in response to galactosamine D-induced massive necrosis,3Dabeva MD Shafritz DA Activation, proliferation, and differentiation of progenitor cells into hepatocytes in the D-galactosamine model of liver regeneration.Am J Pathol. 1993; 143: 1606-1620PubMed Google Scholar or after partial hepatectomy (PH) associated with treatment by 2-acetylaminofluorene (AAF).4Paku S Schnur J Nagy P Thorgeirsson SS Origin and structural evolution of the early proliferating oval cells in rat liver.Am J Pathol. 2001; 158: 1313-1323Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar They have also been reported in humans in fulminant hepatitis and in chronic liver diseases.5Lowes KN Brennan BA Yeoh GC Olynyk JK Oval cell numbers in human chronic liver diseases are directly related to disease severity.Am J Pathol. 1999; 154: 537-541Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 6Haque S Haruna Y Saito K Nalesnik MA Atillasoy E Thung SN Gerber MA Identification of bipotential progenitor cells in human liver regeneration.Lab Invest. 1996; 75: 699-705PubMed Google Scholar Oval cells are small, with a high nuclear-cytoplasmic ratio. They express hepatocellular and biliary markers, and can differentiate into hepatocytes3Dabeva MD Shafritz DA Activation, proliferation, and differentiation of progenitor cells into hepatocytes in the D-galactosamine model of liver regeneration.Am J Pathol. 1993; 143: 1606-1620PubMed Google Scholar or biliary cells.7Germain L Noel M Gourdeau H Marceau N Promotion of growth and differentiation of rat ductular oval cells in primary culture.Cancer Res. 1988; 48: 368-378PubMed Google Scholar They initially expand in the periportal region where they form ductular structures, and radiate toward the center of the lobule. In the adult liver, they can be viewed as the counterpart of bipotent hepatoblasts which, in the fetus, differentiate into both types of epithelial cells. The origin of oval cells is not fully elucidated. It is generally accepted that oval cells are the progeny of quiescent facultative stem cells which persist in the portal region of adult liver in terminal hepatic ductules (Hering ducts).8Theise ND Saxena R Portmann BC Thung SN Yee H Chiriboga L Kumar A Crawford JM The canals of Hering and hepatic stem cells in humans.Hepatology. 1999; 30: 1425-1433Crossref PubMed Scopus (603) Google Scholar It has been suggested that oval cells also derive from bone marrow stem cells, most likely hematopoietic stem cells, which might migrate into the liver, and differentiate into oval cells to give rise to hepatocytes or biliary cells.9Petersen BE Bowen WC Patrene KD Mars WM Sullivan AK Murase N Boggs SS Greenberger JS Goff JP Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2172) Google Scholar However, this assumption has been challenged in a recent study showing that mouse oval cells do not originate in bone marrow.10Wang X Foster M Al-Dhalimy M Lagasse E Finegold M Grompe M The origin and liver repopulating capacity of murine oval cells.Proc Natl Acad Sci USA. 2003; 100: 11881-11888Crossref PubMed Scopus (364) Google Scholar The mechanisms responsible for the activation, expansion, and differentiation of oval cells are still largely unknown, but soluble factors as well as direct contacts with their matricial and cellular microenvironment might dictate their fate.11Matsusaka S Tsujimura T Toyosaka A Nakasho K Sugihara A Okamoto E Uematsu K Terada N Role of c-kit receptor tyrosine kinase in development of oval cells in the rat 2-acetylaminofluorene/partial hepatectomy model.Hepatology. 1999; 29: 670-676Crossref PubMed Scopus (76) Google Scholar, 12Park DY Suh KS Transforming growth factor-beta1 protein, proliferation and apoptosis of oval cells in acetylaminofluorene-induced rat liver regeneration.J Korean Med Sci. 1999; 14: 531-538PubMed Google Scholar, 13Nagy P Bisgaard HC Santoni-Rugiu E Thorgeirsson SS In vivo infusion of growth factors enhances the mitogenic response of rat hepatic ductal (oval) cells after administration of 2-acetylaminofluorene.Hepatology. 1996; 23: 71-79Crossref PubMed Google Scholar, 14Hu Z Evarts RP Fujio K Marsden ER Thorgeirsson SS Expression of hepatocyte growth factor and c-met genes during hepatic differentiation and liver development in the rat.Am J Pathol. 1993; 142: 1823-1830PubMed Google Scholar, 15Fujio K Evarts RP Hu Z Marsden ER Thorgeirsson SS Expression of stem cell factor and its receptor, c-kit, during liver regeneration from putative stem cells in adult rat.Lab Invest. 1994; 70: 511-516PubMed Google Scholar, 16Bustos M Sangro B Alzuguren P Gil AG Ruiz J Beraza N Qian C Garcia-Pardo A Prieto J Liver damage using suicide genes: a model for oval cell activation.Am J Pathol. 2000; 157: 549-559Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 17Yin L Lynch D Sell S Participation of different cell types in the restitutive response of the rat liver to periportal injury induced by allyl alcohol.J Hepatol. 1999; 31: 497-507Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar Stromal cell-derived factor-1 (SDF-1) is a member of the CXC chemokine family18Luster AD Chemokines: chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3219) Google Scholar that binds to the seven-span transmembrane G-protein-coupled CXCR4 receptor, which has SDF-1 as its unique ligand.19Murdoch C CXCR4: chemokine receptor extraordinaire.Immunol Rev. 2000; 177: 175-184Crossref PubMed Scopus (237) Google Scholar CXCR4 is expressed by most leukocyte populations, endothelial cells, as well as by epithelial and carcinomatous cells.18Luster AD Chemokines: chemotactic cytokines that mediate inflammation.N Engl J Med. 1998; 338: 436-445Crossref PubMed Scopus (3219) Google Scholar, 19Murdoch C CXCR4: chemokine receptor extraordinaire.Immunol Rev. 2000; 177: 175-184Crossref PubMed Scopus (237) Google Scholar Stromal cell-derived factor-1 is expressed in a broad range of tissues and is a potent chemo-attractant for a variety of cells including hematopoietic stem cells, lymphocytes, and monocytes.19Murdoch C CXCR4: chemokine receptor extraordinaire.Immunol Rev. 2000; 177: 175-184Crossref PubMed Scopus (237) Google Scholar Targeted destruction of its gene as well as of its receptor has shown the role of the SDF-1/CXCR4 couple during embryogenesis and organogenesis.20Ma Q Jones D Borghesani PR Segal RA Nagasawa T Kishimoto T Bronson RT Springer TA Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice.Proc Natl Acad Sci USA. 1998; 95: 9448-9453Crossref PubMed Scopus (1402) Google Scholar It might be involved in the differentiation of progenitor cells to a more mature form, which is illustrated by the supporting role of SDF-1 during B-lymphopoiesis20Ma Q Jones D Borghesani PR Segal RA Nagasawa T Kishimoto T Bronson RT Springer TA Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice.Proc Natl Acad Sci USA. 1998; 95: 9448-9453Crossref PubMed Scopus (1402) Google Scholar and by the observation that, in epithelia renewing from precursor cells, CXCR4 is expressed by the less differentiated cells, eg, lung type II alveolar cells,19Murdoch C CXCR4: chemokine receptor extraordinaire.Immunol Rev. 2000; 177: 175-184Crossref PubMed Scopus (237) Google Scholar basal crypt cells of the gut.21Jordan NJ Kolios G Abbot SE Sinai MA Thompson DA Petraki K Westwick J Expression of functional CXCR4 chemokine receptors on human colonic epithelial cells.J Clin Invest. 1999; 104: 1061-1069Crossref PubMed Scopus (155) Google Scholar Since hepatic precursor cells can give rise to mature epithelial cells in the adult liver, the aim of the present work was to study the expression and the role of the SDF-1/CXCR4 couple in a model of liver regeneration from oval cells. Male Fischer F-344 rats (Charles River IFFA CREDO, L'arbresle, France) and male Wistar rats (Centre d'Elevage Roger Janvier, Le Genest Saint Isle, France) were purchased at the age of 7 weeks (170 g). Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators. Liver regeneration from oval cells was induced in Fischer F-344 rats by treating the animals with AAF followed by a PH (AAF/PH model), according to a protocol previously described.14Hu Z Evarts RP Fujio K Marsden ER Thorgeirsson SS Expression of hepatocyte growth factor and c-met genes during hepatic differentiation and liver development in the rat.Am J Pathol. 1993; 142: 1823-1830PubMed Google Scholar In brief, rats were fed a diet containing 0.02% AAF (Altromin, Lage, Germany) for 7 days followed by a two-third hepatectomy (day 0) under diethyl ether anesthesia. The administration of AAF was then continued for 5 days. Animals were sacrificed under sodium pentobarbital anesthesia by exsanguination before treatment, the day of surgery (day 0) and at days 1, 5, 9, 14, or 21 after PH. A second group of rats fed a standard pelleted chow underwent PH only (PH model). These animals were sacrificed at the days above indicated in the AAF/PH model. In both groups, the whole liver was divided in two parts. One piece was fixed in buffered formalin, then embedded in paraffin for histopathological, immunohistochemical, and in situ hybridization studies; the other part was quickly frozen in liquid nitrogen and stored at −70°C until use. Wistar rats fed an AAF-containing diet underwent a two-third hepatectomy after 7 days. Immediately after surgery, the animals received an i.p. injection of 25 mg/kg fucoidan (Sigma Chemical Company, St. Louis, MO) dissolved in sterile pyrogen-free phosphate-buffered saline. Injections were repeated at the same dosage 8 hours after surgery and twice daily thereafter. Control animals received only the solvent. The animals were maintained on the AAF diet and sacrificed 4 days after surgery. One piece of liver was fixed in buffered formalin, and then embedded in paraffin for histopathological and immunohistochemical evaluation. Three μm-thick sections of paraffin-embedded liver tissue were stained with hematein-eosin and picrosirius red for collagen with hematein counterstain. Oval cells, when present, predominated in periportal areas. Their size was slightly larger than that of epithelial cells lining interlobular bile ducts and was smaller than that of hepatocytes (Figure 1b). They were isolated or formed ductular structures, tortuous or not, with either clearly visible or poorly defined lumens (Figure 1b). Oval cell accumulation was semi-quantitatively evaluated as minimal when either individual oval cells or ductular structures were strictly limited to periportal areas (ie, involvement of less than one-third of the lobules), marked when isolated oval cells or ductules extended into mediolobular and centrilobular areas (ie, involvement of at least one-half of the lobules), and moderate when the pattern was intermediate between minimal and marked. For immunohistochemistry, antigen retrieval was performed in 0.01 mol/L citrate buffer at pH 6.0 in a 750 W microwave oven (3 × 5 minutes) after dewaxing and rehydration of 4 μm-thick sections. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. A three-step method using streptavidin-biotin-complexes (ABC) conjugated with peroxidase (Vectastain kit, Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine (DAB +, DAKO SA, Trappes, France) as a chromogen was performed according to the manufacturers' instructions. Sections were incubated with an anti-SDF-1 mouse monoclonal antibody (K15C IgG 2a)22Coulomb-L'Herminé A Amara A Schiff C Durand-Gasselin I Foussat A Delaunay T Chaouat G Capron F Ledee N Galanaud P Arenzana-Seisdedos F Emilie D Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells.Proc Natl Acad Sci USA. 1999; 96: 8585-8590Crossref PubMed Scopus (92) Google Scholar (a gift from Dr. F. Arenzana-Seisdedos, Institut Pasteur, Paris, France) for 1 hour at room temperature at a final concentration of 20 μg/ml. This antibody, which recognizes an epitope encompassing the amino-terminal end of the chemokine, has been demonstrated to specifically react with SDF-1α and SDF-1β. Sections were counterstained with Mayer's hematoxylin and mounted with an aqueous medium. Controls consisted in omission of the anti-SDF-1 antibody or its replacement by an isotype-matched control antibody (IgG 2a anti-human granzyme B, Novocastra Laboratories Ltd, Newcastle upon Tyne, UK). Rabbit polyclonal antibodies against alphafetoprotein (DAKO SA), an oval cell marker,23Petropoulos CJ Yaswen P Panzica M Fausto N Cell lineages in liver carcinogenesis: possible clues from studies of the distribution of alpha-fetoprotein RNA sequences in cell populations isolated from normal, regenerating, and preneoplastic rat livers.Cancer Res. 1985; 45: 5762-5768PubMed Google Scholar and Ki67 (Novocastra Laboratories Ltd) were incubated for 1 hour at room temperature and revealed by the three-step method described above. Complementary RNA probes were synthesized using a digoxigenin RNA labeling kit (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions. The complementary RNA probe specific for CXCR4 was obtained from the mouse I.M.A.G.E. cDNA clone 3385804 (UK HGMP Resource Centre, Cambridge, UK) digested with EcoRI (antisense probe) or NotI (sense probe) and transcribed from the T3 or T7 promoters, respectively. For SDF-1, probes were transcribed from an I.M.A.G.E cDNA clone 3483088 (UK HGMP Resource Centre) fragment obtained by PCR and subcloned into pCR4-TOPO plasmid. PCR was performed using forward 5′-CGCTGTGCTGGCCCTGGTGC-3′ and reverse 5′-CGCCCCCTGCAGGACAAGCC-3′, which map to positions 50–69 and 797–778 of the rat sequence (GI: 19923685). The amplified cDNA was ligated into pCR4-TOPO plasmid using a TOPO TA Cloning kit (Invitrogen, Cergy Pontoise, France) in the conditions recommended by the supplier. Double-strand sequencing of the insert was performed using the BigDyeTM Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) in an automated Applied Biosystems sequencer model 3100 Genetic Analyser (Applied Biosystems). The complementary RNA probes specific for SDF-1 were transcribed from T7 and T3 promoters of the plasmid digested with SpeI (antisense probe) or NotI (sense probe). The size of the RNA probes (750 bp for CXCR4 and 740 bp for SDF-1) was checked on 2% agarose gel. The digoxigenin-labeled RNA probes were applied to a nylon membrane positively charged to determine the labeling efficiency, using a DIG Nucleic Acid Detection kit (Roche) according to the supplier's instructions. Four μm-thick sections mounted on superfrost + slides (CML, Nemours, France) and stored at room temperature were treated as previously described.24Holic N Suzuki T Corlu A Couchie D Chobert MN Guguen-Guillouzo C Laperche Y Differential expression of the rat gamma-glutamyl transpeptidase gene promoters along with differentiation of hepatoblasts into biliary or hepatocytic lineage.Am J Pathol. 2000; 157: 537-548Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar They were hybridized overnight at 52°C in a humid chamber with sense and antisense probes in parallel. Hybridization buffer contained 50% desionized formamide, 4X SSC, (0.6 mol/L NaCl, 60 mmol/L Na+ citrate) 1X Denhardt's solution, 250 μg/ml yeast tRNA, and 5 ng/μl (CXCR4) or 8 ng/μl (SDF-1) digoxigenin-labeled oligonucleotide probes. After hybridization, sections were rinsed in 2X SSC and in 1X SSC for 30 minutes each at 52°C, then twice in 0.1X SSC for 30 minutes at 37°C. Detection of the probes was carried out using a DIG Nucleic Acid Detection kit (Roche). After a blocking step in bovine serum albumin and levamisole treatment, sections were incubated for 60 minutes with anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. Enzymatic activity was revealed after 3 hours in nitroblue tetrazolium 5-bromo-4-chloro-3-indolylphosphate (NBT-BCIP). Sections were counterstained in light green and mounted with Eukitt. Double-staining of CXCR4 mRNA and of alphafetoprotein was performed on the same section. After CXCR4 mRNA detection by in situ hybridization as above described, sections were incubated with the anti-alphafetoprotein rabbit polyclonal antibody. The immunohistochemical reaction was performed with the Envision Dual Link System (DAKO SA) conjugated with peroxidase for 30 minutes, 3,3′-diaminobenzidine being used as a chromogene during 10 minutes. CXCR4 mRNA signal was dark blue, whereas alphafetoprotein appeared in brown. Total RNA was isolated from rat liver.25Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar The amount and quality of RNA samples were determined by measuring optical density at 260 nm and analyzing 28S rRNA on an agarose gel, respectively.24Holic N Suzuki T Corlu A Couchie D Chobert MN Guguen-Guillouzo C Laperche Y Differential expression of the rat gamma-glutamyl transpeptidase gene promoters along with differentiation of hepatoblasts into biliary or hepatocytic lineage.Am J Pathol. 2000; 157: 537-548Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Total RNA (5 μg) was reverse-transcribed from an oligo(dT) primer using a first-strand synthesis kit (Stratagene Europe, Amsterdam, The Netherlands) in the conditions recommended by the supplier. Amplification of the first cDNA strand was carried out using a LightCycler Instrument and the LightCycler FastStart DNA Master SYBR Green I kit (Roche Diagnostics), as previously described.26Couchie D Holic N Chobert MN Corlu A Laperche Y In vitro differentiation of WB-F344 rat liver epithelial cells into the biliary lineage.Differentiation. 2002; 69: 209-215Crossref PubMed Scopus (44) Google Scholar The following set of primers was used for CXCR4 cDNA amplification: forward 5′-CTGCATCATCATCTCCAAGC-3′ and reverse 5′-GGAAAGGATCTTGAGGCTGG-3′, which map to positions 648–667 and 981–962 of the rat sequence (GI: 1906612), two regions that are identical in rat and mouse. Negative controls without the RNA template or reverse transcriptase were included in each experiment. The melting curve, obtained at the end of each PCR, showed that the PCR products were free of primer dimers. In each PCR experiment, eight dilutions of mouse CXCR4 cDNA clone I.M.A.G.E. were used as external standards. The level of the CXCR4 sequence in cDNA samples was deduced from the standard curve. The amplified product was analyzed by hybridization to a 5′ end-labeled oligonucleotide (5′-CCCTCGCCTTCTTCCACTGTTGCCTGAACC-3′) complementary to CXCR4 mRNA. Hybridization was performed in Rapid-hyb buffer (Amersham Pharmacia Biotech Europe GmbH, Saclay, France). The blot was washed at 42°C in 0.1% sodium dodecyl sulfate, 0.1X SSC and visualized with a PhosphorFluorImager Storm 840 (Molecular Dynamics, Sunnyvale, CA). In the AAF/PH model, rare oval cells were observed in the periportal areas at day 1 after PH. Their number progressively increased with time. Oval cell accumulation was moderate at day 5 and marked at days 9 and 14 after PH, oval cells being individual or forming numerous periportal ductular structures extending into mediolobular areas (Figure 1b). Oval cell accumulation was milder at day 21 after PH. Biliary epithelial cells lining the interlobular bile ducts, individual oval cells as well as oval cells forming ductular structures strongly expressed SDF-1 protein, with an increased labeling at the periphery of the cell cytoplasm (Figure 1d). On serial sections, most SDF-1-labeled cells also expressed alphafetoprotein, confirming that they were indeed oval cells (Figure 1e). Stromal cell-derived factor-1 protein was also weakly detected in the cytoplasm of rare hepatocytes at days 9 and 14 after PH. No staining was detected in control sections incubated with a secondary antibody in the absence of anti-SDF-1 antibody or with an isotype-matched control antibody (data not shown). In situ hybridization with antisense SDF-1 riboprobe was performed in livers obtained at days 5, 9, and 14 after PH. There was marked cytoplasmic SDF-1 mRNA expression in individual intralobular oval cells as well as in oval cells forming periportal ductular structures (Figure 1, g and i). As shown above for the protein, SDF-1 mRNA-positive cells also expressed alphafetoprotein, as detected by in situ hybridization and immunohistochemistry performed on serial sections (Figure 1, i and j). By contrast, SDF-1 mRNA expression was not detected in biliary cells lining interlobular bile ducts (Figure 1h), and a low signal, barely detected in endothelial cells, could not be clearly distinguished from the background. In situ hybridization with sense SDF-1 riboprobe disclosed a very mild labeling of the structures labeled with antisense riboprobe. In normal rat liver as well as in hepatectomized liver without AAF treatment (PH model) at days 1, 5, 9, 14, and 21 after surgery, SDF-1 was immunohistochemically detected on biliary cells lining interlobular bile ducts and in very rare periportal ductules (Figure 1c). Stromal cell-derived factor-1 was not expressed by sinusoidal cells, portal mesenchymal cells, or endothelial cells lining hepatic arterioles and portal or hepatic venules. Stromal cell-derived factor-1 was weakly detected in rare hepatocytes in livers obtained at days 9, 14, and 21 after PH. Scarce intrasinusoidal mononuclear cells were also positive. Controls after omission of anti-SDF-1 antibody were negative. In situ hybridization with antisense SDF-1 riboprobe did not disclose any labeling in normal rats as well as in the PH model at days 9, 14, and 21 after surgery (Figure 1f). In the AAF/PH model, hepatic CXCR4 mRNA expression, assessed by quantitative RT-PCR, was low at day 0, increased to reach a maximal intensity at day 9 post-PH and decreased thereafter, thus paralleling the number of oval cells (Figure 2). In the PH model, CXCR4 mRNA expression remained low and constant throughout the study period. In situ hybridization with antisense CXCR4 riboprobes was performed in livers obtained in normal rats and at days 1, 9, and 14 after PH in the AAF/PH and PH models. In the AAF/PH model, no clear positive signal was observed at day 1. At days 9 and 14, there was marked cytoplasmic CXCR4 mRNA expression in individual intralobular oval cells as well as in oval cells forming ductular structures in periportal areas (Figure 3, a and b). Double-staining of CXCR4 mRNA and alphafetoprotein clearly showed that these cells corresponded to oval cells since CXCR4 mRNA and alphafetoprotein colocalized in occasional oval cells forming ductules (Figure 3f). This double-staining also demonstrated that many oval cells, predominantly located around portal areas and identified by their typical morphology and their organization in ductules, only expressed CXCR4 mRNA and not alphafetoprotein, whereas many oval cells forming ductular structures at distance from the portal areas only expressed alphafetoprotein and not CXCR4 mRNA (Figure 3e). CXCR4 mRNA expression could also be detected in the cytoplasm of some intrasinusoidal mononuclear cells. CXCR4 mRNA was not detected in biliary cells lining interlobular bile ducts (Figure 3a), and the low signal observed in endothelial cells was not clearly significant. In situ hybridization with sense CXCR4 riboprobe did not disclose any labeling (Figure 3c). In normal rats and in the PH model, no labeling could be detected in any cell type, either with the antisense or with the sense riboprobes (Figure 3d). However, these results do not exclude the existence in these livers of some CXCR4 mRNA-positive cells, for instance, inflammatory lymphoid cells not detected on the examined sections, which may account for the low but measurable level of CXCR4 mRNA detected by RT-PCR in the PH model (Figure 2). To determine whether the SDF-1/CXCR4 couple plays a role in oval cell accumulation, rats were injected for 4 days with fucoidan, a sulfated polysaccharide known to bind to SDF-1 and to inhibit its biological effects.27Sweeney EA Lortat-Jacob H Priestley GV Nakamoto B Papayannopoulou T Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice: involvement in mobilization of stem/progenitor cells.Blood. 2002; 99: 44-51Crossref PubMed Scopus (185) Google Scholar These experiments were performed in Wistar rats because preliminary trials showed that this strain tolerated this compound better than Fischer rats. Livers were examined after 4 days of a treatment which was well tolerated, as judged by the absence of difference in the weight of treated and control animals at the end of the experiment. Seven rats fed an AAF-containing diet received fucoidan after PH. The presence and number of oval cells were compared to those observed in six AAF/PH rats which did not receive fucoidan. As estimated after picrosirius red and hematein-eosin stainings, a similar degree of oval cell accumulation was observed in all untreated animals (Figure 3g). In five of the seven rats treated with fucoidan, oval cell number decreased by about one-third in one rat, one-half in two rats, two-thirds in one rat, and could not be seen in one animal (Figure 3h). In the two remaining rats, oval cell number was the same as in untreated animals. Similar results were observed when oval cells were identified by immunodetection of alphafetoprotein (Figure 3, i and j). In both animal groups, only rare hepatocytes expressed Ki67, a cell proliferation marker, indicating that fucoidan-treated animals had indeed taken AAF. In this study, we examined the hepatic expression of SDF-1 and of its receptor CXCR4 during liver regeneration from precursor cells. This study was conducted in a well-characterized model consisting in an AAF treatment associated with PH. In this model and in agreement with previous reports,14Hu Z Evarts RP Fujio K Marsden ER Thorgeirsson SS Expression of hepatocyte growth factor and c-met genes during hepatic differentiation and liver development in the rat.Am J Pathol. 1993; 142: 1823-1830PubMed Google Scholar periportal oval cell accumulation was moderate at day 5 after PH, reached a peak at days 9 to 14, and then decrease
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