Semaphorin 3E Secreted by Damaged Hepatocytes Regulates the Sinusoidal Regeneration and Liver Fibrosis during Liver Regeneration
2014; Elsevier BV; Volume: 184; Issue: 8 Linguagem: Inglês
10.1016/j.ajpath.2014.04.018
ISSN1525-2191
AutoresTomoki Yagai, Atsushi Miyajima, Minoru Tanaka,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoThe liver has a remarkable capacity to regenerate after injury. Although the regulatory mechanisms of hepatocytic regeneration have been a subject of intense study, the dynamism of the sinusoids, specialized blood vessels in the liver, remains largely unknown. Transient activation of hepatic stellate cells and hepatic sinusoidal endothelial cells, which constitute the sinusoids, contributes to liver regeneration during acute injury, whereas their sustained activation causes liver fibrosis during chronic injury. We focused on understanding the association between damaged hepatocytes and sinusoidal regeneration or liver fibrogenesis using a carbon tetrachloride–induced liver injury mouse model. Damaged hepatocytes rapidly expressed semaphorin 3E (Sema3e), which induced contraction of sinusoidal endothelial cells and thereby contributed to activating hepatic stellate cells for wound healing. In addition, ectopic and consecutive expression of Sema3e in hepatocytes by the hydrodynamic tail-vein injection method resulted in disorganized regeneration of sinusoids and sustained activation of hepatic stellate cells. In contrast, liver fibrosis ameliorated in Sema3e-knockout mice compared with wild-type mice in a chronic liver injury model. Our results indicate that Sema3e, secreted by damaged hepatocytes, affects sinusoidal regeneration in a paracrine manner during liver regeneration, suggesting that Sema3e is a novel therapeutic target in liver fibrogenesis. The liver has a remarkable capacity to regenerate after injury. Although the regulatory mechanisms of hepatocytic regeneration have been a subject of intense study, the dynamism of the sinusoids, specialized blood vessels in the liver, remains largely unknown. Transient activation of hepatic stellate cells and hepatic sinusoidal endothelial cells, which constitute the sinusoids, contributes to liver regeneration during acute injury, whereas their sustained activation causes liver fibrosis during chronic injury. We focused on understanding the association between damaged hepatocytes and sinusoidal regeneration or liver fibrogenesis using a carbon tetrachloride–induced liver injury mouse model. Damaged hepatocytes rapidly expressed semaphorin 3E (Sema3e), which induced contraction of sinusoidal endothelial cells and thereby contributed to activating hepatic stellate cells for wound healing. In addition, ectopic and consecutive expression of Sema3e in hepatocytes by the hydrodynamic tail-vein injection method resulted in disorganized regeneration of sinusoids and sustained activation of hepatic stellate cells. In contrast, liver fibrosis ameliorated in Sema3e-knockout mice compared with wild-type mice in a chronic liver injury model. Our results indicate that Sema3e, secreted by damaged hepatocytes, affects sinusoidal regeneration in a paracrine manner during liver regeneration, suggesting that Sema3e is a novel therapeutic target in liver fibrogenesis. The liver has a remarkable capacity to regenerate from surgical resection and damage caused by various insults, such as toxic chemicals and viral infection. Many injuries cause death of hepatocytes, which are liver parenchymal cells, followed by compensatory proliferation of the remaining hepatocytes to regenerate.1Taub R. Liver regeneration: from myth to mechanism.Nat Rev Mol Cell Biol. 2004; 5: 836-847Crossref PubMed Scopus (1220) Google Scholar Therefore, the mechanisms of liver regeneration are focused on hepatocytes,2Selden A.C. Hodgson H.J. Growth factors and the liver.Gut. 1991; 32: 601-603Crossref PubMed Scopus (11) Google Scholar, 3Michalopoulos G.K. DeFrances M.C. Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2842) Google Scholar whereas the regenerative process of sinusoids, unique capillary vessels in the liver, remains largely unknown. The hepatic sinusoid is composed of fenestrated sinusoidal endothelial cells (SECs) and hepatic stellate cells (HSCs). In general, the process of liver regeneration after injury is accompanied by sinusoid fibrogenesis. Although transient fibrogenesis is beneficial for wound healing by providing mechanical stability and a scaffold for hepatocytic regeneration,4Raghow R. The role of extracellular matrix in postinflammatory wound healing and fibrosis.FASEB J. 1994; 8: 823-831Crossref PubMed Scopus (361) Google Scholar prolonged fibrogenesis during chronic hepatitis often leads to the accumulation of extracellular matrix (ECM), resulting in nodule formation and alterations in hepatic function and blood flow. Therefore, liver fibrosis is a pathologic sign that results in severe hepatic diseases, such as cirrhosis and carcinogenesis.5Zhang D.Y. Friedman S.L. Fibrosis-dependent mechanisms of hepatocarcinogenesis.Hepatology. 2012; 56: 769-775Crossref PubMed Scopus (277) Google Scholar Previous studies have revealed that HSCs, characteristic pericytes that line the hepatic sinusoid, are a key cellular source for the ECM and that activated HSCs acquire myofibroblastic characteristics by secreting excess ECM protein, such as collagen types I and III.6Friedman S.L. Roll F.J. Boyles J. Bissell D.M. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver.Proc Natl Acad Sci U S A. 1985; 82: 8681-8685Crossref PubMed Scopus (735) Google Scholar In addition, SECs and HSCs cooperate to maintain the sinusoidal environment. For example, vascular endothelial growth factor (VEGF) secreted by HSCs maintains SEC homeostasis by preventing their capillarization.7DeLeve L.D. Wang X. Hu L. McCuskey M.K. McCuskey R.S. Rat liver sinusoidal endothelial cell phenotype is maintained by paracrine and autocrine regulation.Am J Physiol Gastrointest Liver Physiol. 2004; 287: G757-G763Crossref PubMed Scopus (206) Google Scholar Conversely, SECs revert activated HSCs to a quiescent status via nitric oxide synthesis.8Deleve L.D. Wang X. Guo Y. Sinusoidal endothelial cells prevent rat stellate cell activation and promote reversion to quiescence.Hepatology. 2008; 48: 920-930Crossref PubMed Scopus (238) Google Scholar These observations suggest that the reciprocal cell-to-cell communication between SECs and HSCs is critical for sinusoidal regeneration and liver fibrosis after liver injury and that SEC angiogenic factors could be the regulators of liver fibrogenesis through indirect activation of HSCs. Because liver injury is accompanied by inflammation and hepatocytic insults, inflammatory cells are involved in sinusoidal fibrogenesis.9Tanaka H. Leung P.S. Kenny T.P. Gershwin M.E. Bowlus C.L. Immunological orchestration of liver fibrosis.Clin Rev Allergy Immunol. 2012; 43: 220-229Crossref PubMed Scopus (20) Google Scholar In addition, molecules secreted from damaged hepatocytes contribute to the compensatory proliferation of surrounding hepatocytes.10Nishina T. Komazawa-Sakon S. Yanaka S. Piao X. Zheng D.M. Piao J.H. Kojima Y. Yamashina S. Sano E. Putoczki T. Doi T. Ueno T. Ezaki J. Ushio H. Ernst M. Tsumoto K. Okumura K. Nakano H. Interleukin-11 links oxidative stress and compensatory proliferation.Sci Signal. 2012; 5: ra5Crossref PubMed Scopus (73) Google Scholar, 11Li F. Huang Q. Chen J. Peng Y. Roop D.R. Bedford J.S. Li C.Y. Apoptotic cells activate the "phoenix rising" pathway to promote wound healing and tissue regeneration.Sci Signal. 2010; 3: ra13Crossref PubMed Scopus (334) Google Scholar However, whether there is a direct association between damaged hepatocytes and sinusoidal regeneration or liver fibrogenesis is currently unknown. We found that semaphorin 3E (Sema3e) is up-regulated by 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding in a mouse model of chronic hepatitis.12Okabe M. Tsukahara Y. Tanaka M. Suzuki K. Saito S. Kamiya Y. Tsujimura T. Nakamura K. Miyajima A. Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver.Development. 2009; 136: 1951-1960Crossref PubMed Scopus (223) Google Scholar, 13Inagaki F.F. Tanaka M. Inagaki N.F. Yagai T. Sato Y. Sekiguchi K. Oyaizu N. Kokudo N. Miyajima A. Nephronectin is upregulated in acute and chronic hepatitis and aggravates liver injury by recruiting CD4 positive cells.Biochem Biophys Res Commun. 2013; 430: 751-756Crossref PubMed Scopus (17) Google Scholar Sema3e is a secretory protein that belongs to the class 3 semaphorin family14Yazdani U. Terman J.R. The semaphorins.Genome Biol. 2006; 7: 211Crossref PubMed Scopus (321) Google Scholar and plays a neurogenic and angiogenic repulsive role in development.15Zhou Y. Gunput R.A. Pasterkamp R.J. Semaphorin signaling: progress made and promises ahead.Trends Biochem Sci. 2008; 33: 161-170Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 16Gu C. Giraudo E. The role of semaphorins and their receptors in vascular development and cancer.Exp Cell Res. 2013; 319: 1306-1316Crossref PubMed Scopus (85) Google Scholar The receptors for semaphorins are plexins and neuropilins, and Sema3e specifically binds to plexin D1 (Plxnd1).17Gu C. Yoshida Y. Livet J. Reimert D.V. Mann F. Merte J. Henderson C.E. Jessell T.M. Kolodkin A.L. Ginty D.D. Semaphorin 3E and plexin-D1 control vascular pattern independently of neuropilins.Science. 2005; 307: 265-268Crossref PubMed Scopus (414) Google Scholar Sakurai et al18Sakurai A. Gavard J. Annas-Linhares Y. Basile J.R. Amornphimoltham P. Palmby T.R. Yagi H. Zhang F. Randazzo P.A. Li X. Weigert R. Gutkind J.S. Semaphorin 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and R-Ras.Mol Cell Biol. 2010; 30: 3086-3098Crossref PubMed Scopus (116) Google Scholar reported that Sema3e/Plxnd1 signaling initiates the antiangiogenic response by regulating Arf6 and R-Ras, inhibiting endothelial tip cell adhesion to the ECM, and retracting filopodia. Moreover, the Sema3e/Plxnd1 axis interferes with VEGF and VEGF receptor (VEGFR)-2 signaling via a feedback mechanism.19Kim J. Oh W.J. Gaiano N. Yoshida Y. Gu C. Semaphorin 3E-Plexin-D1 signaling regulates VEGF function in developmental angiogenesis via a feedback mechanism.Genes Dev. 2011; 25: 1399-1411Crossref PubMed Scopus (153) Google Scholar Indeed, Sema3e/Plxnd1 signaling plays an essential role in development because Sema3e-knockout (KO) mice display aberrant vascularization of intersomitic blood vessels.17Gu C. Yoshida Y. Livet J. Reimert D.V. Mann F. Merte J. Henderson C.E. Jessell T.M. Kolodkin A.L. Ginty D.D. Semaphorin 3E and plexin-D1 control vascular pattern independently of neuropilins.Science. 2005; 307: 265-268Crossref PubMed Scopus (414) Google Scholar However, the involvement of Sema3e/Plxnd1 signaling in liver regeneration and pathogenesis remains largely unknown. In this study, we focused on the mechanisms of sinusoidal regeneration after liver injury and found that Sema3e produced by damaged hepatocytes activates SECs via Plxnd1 and thereby plays a critical role in sinusoidal regeneration and liver fibrosis. C57BL/6 mice were obtained from CLEA Japan (Tokyo, Japan). Sema3e-KO mice were provided by Dr. Yutaka Yoshida (Cincinnati Children's Hospital Medical Center, Cincinnati, OH).17Gu C. Yoshida Y. Livet J. Reimert D.V. Mann F. Merte J. Henderson C.E. Jessell T.M. Kolodkin A.L. Ginty D.D. Semaphorin 3E and plexin-D1 control vascular pattern independently of neuropilins.Science. 2005; 307: 265-268Crossref PubMed Scopus (414) Google Scholar The littermates were subjected to carbon tetrachloride (CCl4)–induced liver fibrosis. All animal experiments were performed in accordance with our institutional guidelines. Acute liver injury was induced by single i.p. injection of CCl4. CCl4 (Wako Pure Chemical, Osaka, Japan) was diluted in corn oil (Wako) to 20% and injected into mice at a dose of 1 mL/kg of CCl4. Liver fibrosis was induced by repeated injection of CCl4, twice per week for 4 weeks. Livers were harvested 3 days after the final CCl4 injection. Liver cryosections (8 μm) were mounted on glass slides and fixed with Zamboni fixative solution for 10 minutes for immunostaining. The fixed sections were incubated with 5% skim milk (w/v) in phosphate-buffered saline and then incubated with primary antibodies, followed by secondary antibodies. The antibodies used in this study are listed in Table 1. Images were captured using Observer Z1 with an AxioCam HRc (Zeiss, Oberkochen, Germany). The hematoxylin and eosin (H&E) staining was performed after immunostaining in some specific experiments as stated later. The cover glass on enclosed sections was eliminated carefully with adequate phosphate-buffered saline (Wako) and then stained with H&E (Muto Pure Chemicals, Tokyo, Japan). Sirius Red staining was performed followed by Bouin's solution (Sigma-Aldrich, St. Louis, MO) fixation, as described previously. In brief, nuclei were stained with Weigert's iron hematoxylin (Wako) and then stained with collagen and Direct Red 80 (Sigma-Aldrich).Table 1Antibodies Used in the StudyProteinSupplierSemaphorin 3E (Sema3e)Abgent (San Diego, CA)Stabilin-2 (Stab-2)Nonaka et al20Nonaka H. Tanaka M. Suzuki K. Miyajima A. Development of murine hepatic sinusoidal endothelial cells characterized by the expression of hyaluronan receptors.Dev Dyn. 2007; 236: 2258-2267Crossref PubMed Scopus (65) Google ScholarCD45BD Biosciences (San Diego, CA)Nerve growth factor receptor (NGFR, p75NTR)R&D Systems (Minneapolis, MN)Actin, α2, smooth muscle, aorta (α-SMA)Abcam (Cambridge, MA)CD16/CD32 (Fcγ III/II Receptor)BD BiosciencesKi-67eBioscience (San Diego, CA)Collagen IAbD Serotec (Kidlington, UK)PhalloidinInvitrogen (Carlsbad, CA)GAPDHMerck Millipore (Billerica, MA)α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p75NTR, p75 low-affinity neurotrophic growth factor receptor. Open table in a new tab α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p75NTR, p75 low-affinity neurotrophic growth factor receptor. Total RNA was isolated from mouse livers or hepatic cells using TRIzol reagent (Invitrogen). Reverse transcription to cDNA templates was performed using random primers and a High-Capacity cDNA Reverse-Transcription Kit (Applied Biosystems, Foster City, CA). Real-time RT-PCR experiments were conducted with a LightCycler 480 system and Universal Probe Library (Roche Diagnostics, Indianapolis, IN). The mouse ACTB gene assay in ProbeLibrary was used as the normalization control. The sequence information for the primer pairs used is listed in Table 2. Probes #63 and #27 were used for Sema3e and Plxnd1, respectively. Sema3e primers were used for experiments by analyzing the Sema3e expression. Sema3e vector primers were used to construct the expression vector.Table 2Primer Sequences Used for This StudyGeneSense primer sequenceAntisense primer sequenceSema3e5′-GGGGCAGATGTCCTTTTGA-3′5′-AGTCCAGCAAACAGCTCATTC-3′Plxnd15′-CTGGATGTCCATCTGCATGT-3′5′-CAGGAAGAACGGCTCACCTA-3′Construction for Sema3e expression vector5′-AGCTAGCCCCTGGAGGGAAGTACTAA-3′5′-GTCGACTCCTAGGTTCCTCAGCCGCC-3′ Open table in a new tab A single-cell suspension was obtained from the liver by a modified collagenase perfusion method, as described previously.21Takase H.M. Itoh T. Ino S. Wang T. Koji T. Akira S. Takikawa Y. Miyajima A. FGF7 is a functional niche signal required for stimulation of adult liver progenitor cells that support liver regeneration.Genes Dev. 2013; 27: 169-181Crossref PubMed Scopus (100) Google Scholar In brief, liver specimens were perfused with a basic perfusion solution containing 0.5 g/L of collagenase-Yakult (Yakult Pharmaceutical Industry Co. Ltd., Tokyo, Japan) and 50 mg/L of DNase I (Sigma-Aldrich). The digested liver was passed through a 70-μm cell strainer. After centrifugation at 60 × g for 1 minute, the precipitated cells were used as hepatocytes after Percoll (GE Healthcare, Piscataway, NJ) density centrifugation. The supernatant was transferred to a new tube and centrifuged at 120 × g for 2 minutes repeatedly until no pellet was visible. The final supernatant was centrifuged at 340 × g for 5 minutes, and the precipitated cells were used as non-parenchymal cells for cell isolation. Aliquots of cells were blocked with anti-FcγR antibody, co-stained with fluorescence- and/or biotin-conjugated antibodies, and then incubated with allophycocyanin-conjugated streptavidin (BD Biosciences, San Diego, CA) if needed. The samples were sorted by Moflo XDP (Beckman-Coulter, Fullerton, CA) or autoMACS pro (Miltenyi Biotec, Bergisch Gladbach, Germany) with anti-allophycocyanin microbeads. Dead cells were excluded by propidium iodide staining. Primary hepatocytes separated by Percoll were seeded in type I collagen–coated 6-well dishes (BD Biosciences) at 5 × 105 per well with William's Medium E (Life Technologies, Carlsbad, CA) that contained 10% fetal bovine serum (JRH Biosciences, Lenexa, KS). After 3 hours (0 hours), unattached hepatocytes were washed out, and dimethyl sulfoxide (vehicle) or CCl4 dissolved in dimethyl sulfoxide was added to the culture medium at a final concentration of 2.0 mmol/L. Then, total RNA was extracted from hepatocytes at 0, 3, 6, and 24 hours after CCl4 administration. Isolated primary SECs were seeded in dishes coated with collagen type I-C (Nitta gelatin, Osaka, Japan) with Dulbecco's modified Eagle's medium/Ham's nutrient mixture F-12 (Sigma-Aldrich). After 12 hours, recombinant mouse Sema3e (R&D Systems) was added to the culture medium for a final concentration of 500 ng/mL. After incubation for 30 minutes, the morphologic status of SECs was analyzed by immunocytochemistry using fluorescein-conjugated phalloidin and Hoechst stain. We used the pLIVE vector and TransIT-EE Hydrodynamic Delivery solution (Mirus Bio, Madison, WI) to introduce Sema3e cDNA into 8-week-old mice by hydrodynamic tail-vein injection (HTVi). The primer pairs used for the expression vector are listed in Table 2. The vascular density was determined by analyzing stabilin (Stab)-2–positive area in the fields, including the central vein (CV). Four independent images of liver sections at ×200 magnification per animal were analyzed using the ImageJ software version 1.46r (NIH, Bethesda, MD). The parenchymal area was evaluated by subtracting vascular luminal area from the total field area and used for calculation. The fibrosis area was assessed by analyzing the Sirius Red–stained collagen areas in the liver sections at ×50 magnification. Ki-67–positive hepatocytes were counted using In Cell Analyzer 2000 (GE Healthcare), as described previously.22Miyaoka Y. Ebato K. Kato H. Arakawa S. Shimizu S. Miyajima A. Hypertrophy and unconventional cell division of hepatocytes underlie liver regeneration.Curr Biol. 2012; 22: 1166-1175Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar Serum alanine aminotransferase (ALT) and albumin were measured at the Oriental Yeast Company (Tokyo, Japan) or by the Transaminase CII-test Wako (Wako). Hydroxyproline content was measured as described previously.23Reddy G.K. Enwemeka C.S. A simplified method for the analysis of hydroxyproline in biological tissues.Clin Biochem. 1996; 29: 225-229Crossref PubMed Scopus (1034) Google Scholar Statistical analysis was performed using the unpaired two-tailed Student's t-test. Gene expression in multiple liver cell fractions was compared by one-way analysis of variance and subsequent Tukey's tests. P < 0.05 was considered statistically significant. The i.p. injection of CCl4 produces a conventional liver injury model to study liver regeneration and subsequent fibrosis. We treated mice with CCl4 and monitored the status of hepatocytes and SECs to investigate sinusoidal regeneration after liver injury. We have reported previously that Stab-2, a scavenger receptor, and receptors II (CD32) and III (CD16) for Fc fragment of IgG (FcγRs) are highly expressed in SECs and make it possible to distinguish SECs from other kinds of endothelial cells.20Nonaka H. Tanaka M. Suzuki K. Miyajima A. Development of murine hepatic sinusoidal endothelial cells characterized by the expression of hyaluronan receptors.Dev Dyn. 2007; 236: 2258-2267Crossref PubMed Scopus (65) Google Scholar, 24Nonaka H. Sugano S. Miyajima A. Serial analysis of gene expression in sinusoidal endothelial cells from normal and injured mouse liver.Biochem Biophys Res Commun. 2004; 324: 15-24Crossref PubMed Scopus (16) Google Scholar Frozen liver sections were subjected to immunohistochemistry (IHC) using anti–Stab-2 or anti-FcγR antibodies, followed by H&E staining to visualize SECs in CCl4-treated liver. Sinusoids in normal liver (0 hours) extended from the CV in a high-density radial pattern (Figure 1, A and B, and Supplemental Figure S1, A and B). Because CCl4 is metabolized by cytochrome P450 in hepatocytes surrounding the CV to produce toxic free radicals as intermediate metabolites,25Weddle C.C. Hornbrook K.R. McCay P.B. Lipid peroxidation and alteration of membrane lipids in isolated hepatocytes exposed to carbon tetrachloride.J Biol Chem. 1976; 251: 4973-4978PubMed Google Scholar administering CCl4 causes massive hepatocytic death around the CV. H&E staining revealed mild and obvious degeneration of hepatocytes at 24 and 48 hours after CCl4 treatment, respectively (Figure 1, C and E, and Supplemental Figure S1, C and E). The sinusoidal structure was disorganized 24 hours after CCl4 treatment, and most SECs in the degenerated region (Figure 1) seemed to be contracted, suggesting that some substantial alterations occurred in the SECs (Figure 1, C and D, and Supplemental Figure S1, C and D). The degenerated region became more obvious, and the arrangement of SECs remained disorganized 48 hours after CCl4 treatment (Figure 1, E and F, and Supplemental Figure S1, E and F). Degeneration of hepatocytes decreased and hepatocytes regenerated 72 and 96 hours after CCl4 treatment, but immune cells accumulated around the CV (Figure 1, G and I, and Supplemental Figure S1, G and I). In contrast, SECs returned to their original position and morphologic status during these phases (Figure 1, H and J, and Supplemental Figure S1, H and J). These data suggest that the process of sinusoidal regeneration would be completed by 72 hours after CCl4-induced injury in mice. Because a sign of contraction was observed in SECs at 24 hours after CCl4 treatment, it was supposed that some angiogenesis-related factors might be involved in this morphologic change. We found in our previous research about liver regeneration during chronic hepatitis that Sema3e is up-regulated in injured liver. Therefore, we examined the Sema3e expression pattern by quantitative RT-PCR (RT-qPCR) after CCl4 treatment and measured serum concentrations of ALT as a liver injury marker (Figure 2, A and B). As a result, Sema3e expression was negligible in normal liver but was up-regulated drastically at 24 hours after CCl4 treatment. Intriguingly, Sema3e expression then decreased sharply at 48 hours and returned to the basal level after 72 hours. These results suggest that up-regulation of Sema3e might be related with degeneration of hepatocytes, as evaluated by serum ALT (Figure 2, A and B), whereas rapid Sema3e down-regulation may represent completion of hepatocytic death. Therefore, we hypothesized that damaged hepatocytes could be a source for Sema3e. To address this hypothesis, we examined Sema3e expression in injured liver sections by IHC using an anti-Sema3e antibody. As expected, strong Sema3e signals were detected within the degenerating area around the CV 24 hours after CCl4 treatment (Figure 2C). To investigate whether the cells expressing Sema3e were hepatocytes, we stimulated hepatocytes isolated from normal liver with CCl4 in culture. Primary cultured hepatocytes became gradually swollen after CCl4 treatment, and most of the hepatocytes degenerated 24 hours after CCl4 challenge (Figure 2D). Real-time RT-PCR revealed that the expression level of Sema3e of CCl4-treated hepatocytes was significantly increased compared with that of vehicle-treated or nontreated hepatocytes and 3.7-fold higher than that of injured liver 24 hours after in vivo administration of CCl4 (whole liver), suggesting that damaged hepatocyte by oxidative stress is a major source of robust expression of Sema3e in CCl4 injury model (Figure 2E). Although Sema3e is expressed in immune cells,26Wanschel A. Seibert T. Hewing B. Ramkhelawon B. Ray T.D. van Gils J.M. Rayner K.J. Feig J.E. O'Brien E.R. Fisher E.A. Moore K.J. Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention.Arterioscler Thromb Vasc Biol. 2013; 33: 886-893Crossref PubMed Scopus (87) Google Scholar CD45+ blood cells isolated from CCl4-treated liver hardly expressed Sema3e (Supplemental Figure S2A). Moreover, Sema3e was not induced after a 70% partial hepatectomy, which was not accompanied by hepatocyte damage (Supplemental Figure S2B). These results strongly suggest that Sema3e is induced in hepatocytes in vitro and in vivo in a damage-dependent manner. We isolated each type of liver cell by fluorescence-activated cell sorting and analyzed Sema3e receptor (Plxnd1) expression by RT-qPCR to identify the cell type that responded to Sema3e in injured liver. Plxnd1 was predominantly expressed in Stab-2+ SECs (Figure 3A), suggesting that Sema3e secreted from degenerating hepatocytes could affect SECs in a paracrine fashion. Sema3e plays a role in repulsing the endothelial tip and inhibiting cell migration. However, the effect of Sema3e on SECs has not been examined. Therefore, we investigated the effect of Sema3e on freshly isolated SECs in culture. As a result, SEC filopodia retracted significantly in the presence of Sema3e (Figure 3, B and C), as reported previously in other endothelial cell lines.18Sakurai A. Gavard J. Annas-Linhares Y. Basile J.R. Amornphimoltham P. Palmby T.R. Yagi H. Zhang F. Randazzo P.A. Li X. Weigert R. Gutkind J.S. Semaphorin 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and R-Ras.Mol Cell Biol. 2010; 30: 3086-3098Crossref PubMed Scopus (116) Google Scholar, 27Fukushima Y. Okada M. Kataoka H. Hirashima M. Yoshida Y. Mann F. Gomi F. Nishida K. Nishikawa S. Uemura A. Sema3E-PlexinD1 signaling selectively suppresses disoriented angiogenesis in ischemic retinopathy in mice.J Clin Invest. 2011; 121: 1974-1985Crossref PubMed Scopus (159) Google Scholar Given that Sema3e also induced retraction of SEC filopodia in vivo, transient expression of Sema3e from damaged hepatocytes could be involved in the morphologic contraction of SECs as observed 24 hours after CCl4 treatment (Figure 1D and Supplemental Figure S1D). This idea was supported by the result that SECs returned to their original morphologic status during reconstruction of sinusoids after the drastic decrease in Sema3e expression. To further verify Sema3e function in vivo, we examined the effect of prolonged Sema3e expression by HTVi, which is a method of delivering an expression vector into hepatocytes. We initially injected either Sema3e or a control expression vector into wild-type mice by HTVi. Then, each mouse was treated with either CCl4 or vehicle 3 days after HTVi. We first examined SEC by using H&E and IHC staining 1 day after CCl4 administration when endogenous Sema3e expression is drastically induced. We observed no obvious differences in the damaged area between control- and Sema3e-HTVi liver (Supplemental Figure S3). These results suggested that the additional overexpression of Sema3e was not effective because the amount of endogenous Sema3e was enough to affect sinusoidal contraction 1 day after liver injury. Then, we analyzed the livers 3 days after CCl4 administration when endogenous Sema3e expression is decreased (Figure 3D). Sinusoidal regeneration and SEC status were evaluated by IHC using an anti–Stab-2 antibody and vascular density, as represented by the ratio of the Stab-2–positive area to total parenchymal area (Figure 3, E and F). We confirmed that exogenous Sema3e expression by HTVi was maintained irrespective of CCl4 treatment (Supplemental Figure S4). Although the vascular system of Sema3e-HTVi livers tended to decrease compared with that of control-HTVi livers, no significant difference was observed in vehicle treatment (Figure 3F). The Sema3e-HTVi liver treated with CCl4 had markedly disoriented and contracted SECs, whereas radial arrays of SECs were reconstructed around the CV in control-HTVi liver (Figure 3E), which was similar to the image 24 hours after CCl4 treatment (Figure 1D and Supplemental Figure S1D). Consistent with this observation, the vascular system of the Sema3e-HTVi liver decreased significantly compared with that of the control-HTVi liver (Figure 3F). These results suggest that Sema3e is able to affect the morphologic status of SECs in regeneration process in vivo and in vitro. We examined whether regeneration of hepatocytes was affected after liver injury because inhibiting sinusoidal reconstruction in Sema3e-HTVi liver may affect blood flow in the liver. However, no significant differences were observed between control- and Sema3e-HTVi livers after H&E staining (Figure 4A) or by serum albumin concentration (Supplemental Figure S5). We next investigated the status of HSCs, another key component of the liver sinusoid. Because HSCs express p75 low-affinity neurotrophic growth factor receptor (p75NTR),28Suzuki K. Tanaka M. Watanabe N. Saito S. Nonaka H. Miyajima A. p75 Neurotrophin receptor is a marker for precursors of stellate cells and portal fibroblasts in mouse fetal liver.Gastroenterology. 2008; 135: 270-281.e273Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 29Passino M.A. Adams R.A. Sikorski S.L. Akassoglou K. Regulation of hepatic stellate cell differentiation by the neurotrophin receptor p75NTR.Science. 2007; 315: 1853-1856Crossref PubMed Scopus (148) Google Scholar SECs and HSCs in a regeneratin
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