title: Bone marrow progenitor cells do not contribute to liver fibrogenic cells.
abstract: OBJECTIVE
  To investigate the contribution of bone marrow (BM) cells to hepatic fibrosis.
  
  
  METHODS
  To establish a model of chimerism, C57Bl/6 female mice were subjected to full-body irradiation (7 Gy) resulting in BM myeloablation. BM mononuclear cells obtained from male transgenic mice expressing enhanced green fluorescent protein (GFP) were used for reconstitution. Engraftment was confirmed by flow cytometry. To induce liver injury, chimeric animals received carbon tetrachloride (CCl(4)) 0.5 mL/kg intraperitoneally twice a week for 30 d (CCl(4) 30 d) and age-matched controls received saline (Saline 30 d). At the end of this period, animals were sacrificed for post mortem analysis. Liver samples were stained with hematoxylin and eosin to observe liver architectural changes and with Sirius red for collagen quantification by morphometric analysis. α-smooth muscle actin (α-SMA) was analyzed by confocal microscopy to identify GFP+ cells with myofibroblast (MF) characteristics. Liver tissue, BM and peripheral blood were collected and prepared for flow cytometric analysis using specific markers for detection of hepatic stellate cells (HSCs) and precursors from the BM.
  
  
  RESULTS
  Injury to the liver induced changes in the hepatic parenchymal architecture, as reflected by the presence of inflammatory infiltrate and an increase in collagen deposition (Saline 30 d = 11.10% ± 1.12% vs CCl(4) 30 d = 12.60% ± 0.73%, P = 0.0329). Confocal microscopy revealed increased reactivity against α-SMA in CCl(4) 30 d compared to Saline 30 d, but there was no co-localization with GFP+ cells, suggesting that cells from BM do not differentiate to MFs. Liver flow cytometric analysis showed a significant increase of CD45+/GFP+ cells in liver tissue (Saline 30 d = 3.2% ± 2.2% vs CCl(4) 30 d = 5.8% ± 1.3%, P = 0.0458), suggesting that this increase was due to inflammatory cell infiltration (neutrophils and monocytes). There was also a significant increase of common myeloid progenitor cells (CD117+/CD45+) in the livers of CCl(4)-treated animals (Saline 30 d = 2.16% ± 1.80% vs CCl(4) 30 d = 5.60% ± 1.30%, P = 0.0142). In addition the GFP-/CD38+/CD45- subpopulation was significantly increased in the CCl(4) 30 d group compared to the Saline 30 d group (17.5% ± 3.9% vs 9.3% ± 2.4%, P = 0.004), indicating that the increase in the activated HSC subpopulation was not of BM origin.
  
  
  CONCLUSIONS
  BM progenitor cells do not contribute to fibrosis, but there is a high recruitment of inflammatory cells that stimulates HSCs and MFs of liver origin.
authors.affiliation: Bruno Diaz Paredes, Lanuza Alaby Pinheiro Faccioli, Luiz Fernando Quintanilha, Karina Dutra Asensi, Camila Zaverucha do Valle, Christina Maeda Takiya, Antonio Carlos Campos de Carvalho, Regina Coeli dos Santos Goldenberg, Carlos Chagas Filho Biophysics Institute, Rio de Janeiro 21941-902, Brazil.
authors.forename: Bruno Diaz
authors.initials: BD
authors.lastname: Paredes
authors.forename: Lanuza Alaby Pinheiro
authors.initials: LA
authors.lastname: Faccioli
authors.forename: Luiz Fernando
authors.initials: LF
authors.lastname: Quintanilha
authors.forename: Karina Dutra
authors.initials: KD
authors.lastname: Asensi
authors.forename: Camila Zaverucha
authors.initials: CZ
authors.lastname: do Valle
authors.forename: Paulo César
authors.initials: PC
authors.lastname: Canary
authors.forename: Christina Maeda
authors.initials: CM
authors.lastname: Takiya
authors.forename: Antonio Carlos Campos
authors.initials: AC
authors.lastname: de Carvalho
authors.forename: Regina Coeli Dos Santos
authors.initials: RC
authors.lastname: Goldenberg
keywords: Bone marrow; Chimeric mice; Fibrosis; Green fluorescent protein+ cells; Liver; Progenitor cells