title: Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma.
abstract: OBJECTIVE
  To investigate the role of bone marrow-derived endothelial progenitor cells (EPCs) in the angiogenesis of hepatocellular carcinoma (HCC).
  
  
  METHODS
  The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein (GFP) + bone marrow cells. The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting. Serum and tissue levels of vascular endothelial growth factor (VEGF) and colony-stimulating factor (CSF) were quantified by enzyme-linked immunosorbent assay. The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction. The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry. The proportion of EPCs in vessels was then calculated.
  
  
  RESULTS
  The HCC model was successful established. The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively. These values are much higher than in the sham-operation group (0.11% ± 0.13%, 0.05% ± 0.11%, n = 9) at 14 d after modeling. At 21 d, the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%, 0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%, 0.12% ± 0.11% in control. Compared to the transient increase observed in controls, the higher level of circulating EPCs were induced by HCC. In addition, the level of serum VEGF and CSF increased gradually in HCC, reaching its peak 14 d after modeling, then slowly decreased. Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels. Under fluorescence microscopy, the bone-marrow (BM)-derived cells labeled with GFP were concentrated in the same area. The relative levels of CD133 and CD34 gene expression were elevated in tumors, around 5.0 and 3.8 times that of the tumor free area. In frozen liver sections from HCC mice, cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies. In tumor tissue, the double-positive cells were incorporated into vessel walls. In immunofluorescent staining. These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells (VECs) come partly from BM-derived EPCs. The proportion of GFP CD31 double positive VECs (out of all VECs) on day 21 was around 35.3% ± 21.2%. This is much higher than the value recorded on day 7 group (17.1% ± 8.9%). The expression of intercellular adhesion molecule 1, vascular adhesion molecule 1, and VEGF was higher in tumor areas than in tumor-free tissues.
  
  
  CONCLUSIONS
  Mobilized EPCs were found to participate in tumor vasculogenesis of HCC. Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.
authors.affiliation: Department of Hepatobiliary Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing, Jiangsu Province, China.
authors.forename: Xi-Tai
authors.initials: XT
authors.lastname: Sun
authors.forename: Xian-Wen
authors.initials: XW
authors.lastname: Yuan
authors.forename: Hai-Tao
authors.initials: HT
authors.lastname: Zhu
authors.forename: Zheng-Ming
authors.initials: ZM
authors.lastname: Deng
authors.forename: De-Cai
authors.initials: DC
authors.lastname: Yu
authors.forename: Xiang
authors.initials: X
authors.lastname: Zhou
authors.forename: Yi-Tao
authors.initials: YT
authors.lastname: Ding
mesh_terms: D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D018952:Antigens, CD34 / Q000235:genetics / Q000378:metabolism; D016026:Bone Marrow Transplantation; D006528:Carcinoma, Hepatocellular / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002465:Cell Movement; D002469:Cell Separation / Q000379:methods; D003114:Colony-Forming Units Assay; D003115:Colony-Stimulating Factors / Q000097:blood; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology* / Q000637:transplantation; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D007150:Immunohistochemistry; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008113:Liver Neoplasms / Q000098:blood supply* / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009389:Neovascularization, Pathologic; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000378:metabolism / Q000473:pathology*; D013997:Time Factors; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000097:blood; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D003115:Colony-Stimulating Factors; D006023:Glycoproteins; D010455:Peptides; C000606024:Prom1 protein, mouse; D019010:Vascular Cell Adhesion Molecule-1; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D018799:Intercellular Adhesion Molecule-1; D049452:Green Fluorescent Proteins; D040301:Vascular Endothelial Growth Factor Receptor-2