Brief Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Sep 21, 2012; 18(35): 4925-4933
Published online Sep 21, 2012. doi: 10.3748/wjg.v18.i35.4925
Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma
Xi-Tai Sun, Xian-Wen Yuan, Hai-Tao Zhu, Zheng-Ming Deng, De-Cai Yu, Xiang Zhou, Yi-Tao Ding
Xi-Tai Sun, Xian-Wen Yuan, Hai-Tao Zhu, Zheng-Ming Deng, De-Cai Yu, Yi-Tao Ding, Department of Hepatobiliary Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, Nanjing 210008, Jiangsu Province, China
Xi-Tai Sun, Xian-Wen Yuan, Hai-Tao Zhu, Zheng-Ming Deng, De-Cai Yu, Yi-Tao Ding, Institute of Hepatobiliary Surgery, Nanjing University, Nanjing 210008, Jiangsu Province, China
Hai-Tao Zhu, Zheng-Ming Deng, School of Medicine, Nanjing University, Nanjing 210008, Jiangsu Province, China
Xiang Zhou, Department of Pathology, the Affiliated Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing 210008, Jiangsu Province, China
Author contributions: Sun XT and Ding YT designed the research; Yuan XW, Zhu HT, Deng ZM, Yu DC and Zhou X performed the research; Sun XT analyzed the data and wrote the paper.
Supported by The National Natural Science Foundation of China, No. 30972904; Jiangsu Provincial Key Medical Center for Hepatobiliary Disease, No. ZX200605
Correspondence to: Yi-Tao Ding, Professor, Department of Hepatobiliary Surgery, the Affiliated Drum Tower Hospital of Nanjing University Medical School, No. 321 Zhongshan Road, Nanjing 210008, Jiangsu Province, China. yitaoding@hotmail.com
Telephone: +86-25-83304616 Fax: +86-25-83317016
Received: December 30, 2011
Revised: May 16, 2012
Accepted: May 26, 2012
Published online: September 21, 2012
Abstract

AIM: 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.

CONCLUSION: 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.

Keywords: Hepatocellular carcinoma; Angiogenesis; Endothelial progenitor cells; Bone-marrow cells; Orthotropic hepatic cancer model