Endothelial precursor cells as a model of tumor endothelium: characterization and comparison with mature endothelial cells.

Human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC) have been the standards for cell-based assays in the field of angiogenesis research and in antiangiogenic drug discovery. These normal mature endothelial cells may not be most representative of human tumor endothelial cells. Human AC133+/CD34+ bone marrow progenitor cells were established in cell culture media containing vascular endothelial growth factor, basic fibroblast growth factor (bFGF), and heparin to drive differentiation toward the endothelial phenotype. The resulting cells designated endothelial precursor cells (EPC) have many of the same functional properties as mature endothelial cells represented by HUVEC and HMVEC. By SAGE analysis, the genes expressed by EPC are more similar to the genes expressed by endothelial cells isolated from fresh surgical specimens of human tumors than are the genes expressed by HUVEC and HMVEC. Analysis of several cell surface markers by flow cytometry showed that EPC, HUVEC, and HMVEC have similar expression of P1H12, vascular endothelial growth factor 2, and endoglin but that EPC have much lower expression of ICAM1, ICAM2, VCAM1, and thrombomodulin than do HUVEC and HMVEC. The EPC generated can form tubes/networks on Matrigel, migrate through porous membranes, and invade through thin layers of Matrigel similarly to HUVEC and HMVEC. However, in a coculture assay using human SKOV3 ovarian cancer cell clusters in collagen as a stimulus for invasion through Matrigel, EPC were able to invade into the malignant cell cluster, whereas HMVEC were not able to invade the malignant cell cluster. In vivo, a Matrigel plug assay where human EPC were suspended in the Matrigel allowed tube/network formation by human EPC to be carried out in a murine host. EPC may be a better model of human tumor endothelial cells than HUVEC and HMVEC and, thus, may provide an improved cell-based model for second generation antineoplastic antiangiogenic drug discovery.

[1]  S. Rafii,et al.  Vascular Trauma Induces Rapid but Transient Mobilization of VEGFR2+AC133+ Endothelial Precursor Cells , 2001, Circulation research.

[2]  P. Gerwins,et al.  Function of fibroblast growth factors and vascular endothelial growth factors and their receptors in angiogenesis. , 2000, Critical reviews in oncology/hematology.

[3]  D. Ribatti,et al.  Bone marrow angiogenesis in patients with active multiple myeloma. , 2001, Seminars in oncology.

[4]  K. Kinzler,et al.  Genes expressed in human tumor endothelium. , 2000, Science.

[5]  Simon C Watkins,et al.  Isolation and identification of fresh tumor-derived endothelial cells from a murine RIF-1 fibrosarcoma. , 1994, Cancer research.

[6]  J. Folkman.  Tumor angiogenesis: therapeutic implications. , 1971, The New England journal of medicine.

[7]  J. Bischoff,et al.  AC133-2, a Novel Isoform of Human AC133 Stem Cell Antigen* 210 , 2002, The Journal of Biological Chemistry.

[8]  M. Shibuya,et al.  Tumor‐infiltrating endothelial cells and endothelial precursor cells in inflammatory breast cancer , 2002, International journal of cancer.

[9]  S. Rafii,et al.  Impaired recruitment of bone-marrow–derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth , 2001, Nature Medicine.

[10]  B. Teicher,et al.  Myofibroblasts enable invasion of endothelial cells into three-dimensional tumor cell clusters: a novel in vitro tumor model , 2003, Cancer Chemotherapy and Pharmacology.

[11]  J. Regnard,et al.  An In Vitro Model for the Study of Human Bone Marrow Angiogenesis: Role of Hematopoietic Cytokines , 2000, Laboratory Investigation.

[12]  J. Folkman,et al.  ISOLATION OF A TUMOR FACTOR RESPONSIBLE FOR ANGIOGENESIS , 1971, The Journal of experimental medicine.

[13]  R. Warnke,et al.  A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning. , 1997, Blood.

[14]  L. Chow,et al.  Characterization of rare human papillomavirus type 11 mRNAs coding for regulatory and structural proteins, using the polymerase chain reaction. , 1989, Virology.

[15]  Nasim Akhtar,et al.  Angiogenesis Assays: Problems and Pitfalls , 2004, Cancer and Metastasis Reviews.

[16]  H. Rayburn,et al.  Expression of thrombomodulin and consequences of thrombomodulin deficiency during healing of cutaneous wounds. , 1999, The American journal of pathology.

[17]  P. Dell’Era,et al.  Basic fibroblast growth factor-induced angiogenic phenotype in mouse endothelium. A study of aortic and microvascular endothelial cell lines. , 1997, Arteriosclerosis, thrombosis, and vascular biology.

[18]  David J Mooney,et al.  Engineering and Characterization of Functional Human Microvessels in Immunodeficient Mice , 2001, Laboratory Investigation.

[19]  J. Isner,et al.  Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. , 1999, Circulation research.

[20]  Giovanni Martinelli,et al.  Continuous infusion of endostatin inhibits differentiation, mobilization, and clonogenic potential of endothelial cell progenitors. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[21]  C. Verfaillie,et al.  Origin of endothelial progenitors in human postnatal bone marrow. , 2002, The Journal of clinical investigation.

[22]  X. Bian,et al.  Correlation of bFGF, FGFR-1 and VEGF expression with vascularity and malignancy of human astrocytomas. , 2000, Analytical and quantitative cytology and histology.

[23]  R. Auerbach,et al.  Angiogenesis inhibition: a review. , 1994, Pharmacology & therapeutics.

[24]  D. Soligo,et al.  Differentiation and expansion of endothelial cells from human bone marrow CD133+ cells , 2001, British journal of haematology.

[25]  R. Reichman,et al.  Isolation and Propagation of Human Papillomavirus Type 16 in Human Xenografts Implanted in the Severe Combined Immunodeficiency Mouse , 1998, Journal of Virology.

[26]  J. Isner,et al.  VEGF contributes to postnatal neovascularization by mobilizing bone marrow‐derived endothelial progenitor cells , 1999, The EMBO journal.

[27]  S. Rafii,et al.  Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors. , 2000, Blood.

[28]  R. Auerbach,et al.  In vitro and in vivo differentiation into B cells, T cells, and myeloid cells of primitive yolk sac hematopoietic precursor cells expanded > 100-fold by coculture with a clonal yolk sac endothelial cell line. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[29]  A. Luttun,et al.  Vascular progenitors: from biology to treatment. , 2002, Trends in cardiovascular medicine.

[30]  C. Esmon.  Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[31]  A. Sparks,et al.  Using the transcriptome to annotate the genome , 2002, Nature Biotechnology.

[32]  H. Wakasugi,et al.  Induction of vasculogenesis in breast cancer models , 2002, British Journal of Cancer.

[33]  N. Datson,et al.  MicroSAGE: a modified procedure for serial analysis of gene expression in limited amounts of tissue. , 1999, Nucleic acids research.

[34]  Takayuki Asahara,et al.  Isolation of Putative Progenitor Endothelial Cells for Angiogenesis , 1997, Science.

[35]  James A. Thomson,et al.  Hematopoietic colony-forming cells derived from human embryonic stem cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[36]  R. Jain,et al.  During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium. , 1996, Nature medicine.

[37]  K. Müller,et al.  Expression of the endothelial markers PECAM-1, vWf, and CD34 in vivo and in vitro. , 2002, Experimental and molecular pathology.

[38]  S. Rafii,et al.  Circulating endothelial precursors: mystery, reality, and promise. , 2000, The Journal of clinical investigation.

[39]  W. Kamps,et al.  Mobilized human CD34+ hematopoietic stem cells enhance tumor growth in a nonobese diabetic/severe combined immunodeficient mouse model of human non-Hodgkin's lymphoma. , 2001, Cancer research.

[40]  R. Hebbel,et al.  Origins of circulating endothelial cells and endothelial outgrowth from blood. , 2000, The Journal of clinical investigation.

[41]  S. Rafii,et al.  Recruitment of Stem and Progenitor Cells from the Bone Marrow Niche Requires MMP-9 Mediated Release of Kit-Ligand , 2002, Cell.

[42]  J. Folkman,et al.  Relation of vascular proliferation to tumor growth. , 1976, International review of experimental pathology.

[43]  J. Folkman,et al.  Proceedings: Tumor angiogenesis factor. , 1974, Cancer research.

[44]  K. Pantel,et al.  In vitro differentiation of endothelial cells from AC133-positive progenitor cells. , 1999, Blood.