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Tumor Targeting with Transgenic Endothelial Cells Recent Advances in Angiogenesis and Antiangiogenesis, 2009 93 mobilized by the chemokine-gradient and finally (vi) is able to home preferentially to the tumor and not to other organs. 2. SOURCES OF ENDOTHELIAL CELLS BOEC or ECFC can be propagated from peripheral blood, cord blood and bone marrow aspirates after isolation of mononuclear cells, attachment to a collagen type-I matrix and stimulation with the two prominent angiogenic factors vascular growth factor (VEGF) and basic fibroblast growth factor (bFGF) [16,17]. BOEC can be easily propagated from peripheral blood samples within 1 month of propagation in adequate numbers required for cell therapy and importantly have a well characterized uniform endothelial cell phenotype (CD31 + , CD146 + , KDR + , CD45 - , CD133 - , CD34 - ). BOEC undergo regularly cellular senescence and show no chromosomal abnormalities and importantly still have the potential to home to the ischemic myocardium of Bone marrow Hemangioblast CD117+ KDR+ CD133+ CD34+ Blood vessel tissue stem cell CD45low CD34+ CD31+ CD146+ CD45low CD34+ KDR+ CD133+ nude rats [18]. BOEC clones presumably originate from endothelial progenitor cells EPC also named circulating endothelial progenitors (CEP, see Fig. 1). This cell type has been shown to differentiate from bone marrow progenitor cells and to incorporate into blood vessels of tumors [5,19]. Meanwhile, endothelial cells have also been differentiated ex-vivo from hematopoietic and mesenchymal stem cells and from tissue-resident stem cells. Moreover, monocytes have been shown to adhere on fibronectin-coated material and transdifferentiate under VEGF stimulation into “Hill colonies” [20] now named Colony Forming Unit Endothelial Cells (CFU-EC). Despite characteristics of monocytes (expression of CD14 and CD45) CFU-EC express endothelial surface markers, have a low proliferative capacity in-vitro and are still able to phagocyte bacteria [17]. This cell type does not incorporate in newly formed blood vessels, but homes to sites of ischemia [17]. In contrast, BOEC have been shown to incorporate in newly formed blood vessels of matrigel plugs in nude mice and retain a high proliferative potential [17]. EPC/CEP are very Sources of EPC HPC CD45+ CD34+ CD133+ KDR+ CFU-EC EPC/CEP CD45low CD34+ CD133+ ECFC KDR+ BOEC CEC CD146+ CD31+ CD34 + CD133low EPC/CEP CD45low CD34+ CD133+ ECFC KDR+ BOEC CD14+ CD45+ CD34 low CD31+ CD45low CD34- CD133- KDR+/CD31+ CD45low CD34- CD133- KDR+/CD31+ Fig. (1). There are various sources of endothelial cells or precursors for ex-vivo manipulation: (I) hematopoietic stem cells which are found in very rare numbers also in peripheral blood and to a higher extent in the bone-marrow, (II) circulating endothelial progenitor cells or” (EPC/CEP,) giving rise to “blood outgrowth endothelial cells” (BOEC) or “endothelial colony forming cells” (ECFC) under tissue culture conditions, (III) myeloid cells (CFU-EC) and (IV) circulating mature endothelial cells (CEC), derived from the endothelial layer of blood vessels. All cell types present in peripheral blood can differentiate towards an endothelial cell phenotype under appropriate culture conditions.
94 Recent Advances in Angiogenesis and Antiangiogenesis, 2009 Untergasser and Gunsilius rare cell-types that can only be found at lowest frequencies in the circulation of healthy persons and tumor patients [21]. They can be identified by their complex phenotype CD45 low , CD34 + , CD133 + and KDR + . These markers are by no way specific for this cell-type and can be also found on subsets of hematopoietic stem cells [22] or tumor stem cells [23]. Hitherto, no single reliable marker for EPC has been discovered. Apart from the rare EPC cell-type, more abundant circulating endothelial cells (CEC) have been monitored in peripheral blood samples after damage of the vasculature [24]. CEC are mature endothelial cells expressing CD146 and therefore have a low proliferative potential. CEC are elevated in patients with advanced malignancies and vasculopathies [25] and monitoring of CEC has been proposed as surrogate marker for therapy-induced effects [26]. EPC Transgenic EPC Transposase Chromosomal DNA Transposon Transposon CUT Based on essential premises for genetic modification and for use in cell-based therapies, i.e. (i) a characterized homogenous cell population, (ii) a high proliferative capacity and (iii) the capacity of terminal endothelial differentiation, BOEC seem the most appropriate cell-type for cell-based therpies targeting the tumor. 3. GENETIC MODIFICATION OF ENDOTHELIAL CELLS Adenovirus PASTE permanent expression Adenoviruses can infect non-dividing cells (see Fig. 2). The adenoviral double stranded DNA is not incorporated into the host-genome and remains episomal as separate extra-chromosomal element in the Adenoviral transfection EPC transgenic EPC Viral DNA Plasmid GENE GENE GENE GENE GENE RECOMBINATION transient expression transgenic EPC Fig. (2). Viral and non-viral systems for the genetic manipulation of endothelial cells. Recombinant adenoviruses can be generated by homologue recombination of the plasmid containing the gene of interest with the adenoviral DNA. These viruses are used to infect endothelial cells, that in turn transiently express the transgene GFP. The Sleeping Beauty (SB) transposon carrying the gene of interest is "cut" out of the plasmid vector by the engine of this machine (the transposase) and then "pasted" directly into the chromosome of endothelial cells, that in turn permanently express GFP. GFP GFP
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Page 4 and 5: FOREWORD It has been known for a ve
Page 6 and 7: single proangiogenic protein. Howev
Page 8 and 9: v Vito Pistoia Head, Laboratory of
Page 10 and 11: 2 Recent Advances in Angiogenesis a
Page 12 and 13: 10 Recent Advances in Angiogenesis
Page 16 and 17: The Role of Mesenchymal Stem Cells
Page 22 and 23: Thymus and Angiogenesis Recent Adva
Page 32 and 33: Role of Thymidine Recent Advances i
Page 34 and 35: Role of Stromal Cells Recent Advanc
Page 44 and 45: Tumor Vascular Disrupting Agents Re
Page 50 and 51: Tumour Vascular Disrupting Agents R
Page 52: Index Recent Advances in Angiogenes

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