The Role of Circulating Endothelial Progenitor Cells ... - IngentaConnect

Current Stem Cell Research & Therapy, 2011, 6, 115-121

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The Role of Circulating Endothelial Progenitor Cells in Tumor Angiogenesis Julie A. Mund1-3 and Jamie Case*,1-3 1

Department of Pediatrics, 2Herman B Wells Center for Pediatric Research, 3Indiana University Melvin and Bren Simon Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana, USA Abstract: There is great controversy over the origin and definition of murine endothelial progenitor cells (EPCs). EPCs are reportedly important for the repair and remodeling of the vasculature and are implicated in tumor angiogenesis. Many conflicting reports exist as to whether these EPCs arise from the bone marrow hematopoietic compartment or whether they are non-hematopoietic in origin, and these differences could be attributed to the wide variance in assays used to identify the cells and time points at which the data are collected. Recently, circulating murine EPCs have been characterized as CD45-CD13+CD117+FLK-1+ expressing cells via flow cytometry and this phenotype varies from prior descriptions. This review will focus on the changing phenotypic definition of murine circulating EPCs and the evidence that has been published in support of the lineage of origin of circulating EPCs and the role EPCs play in tumor angiogenesis in the adult mouse.

Keywords: Endothelium, hematopoiesis, murine endothelial progenitor cell, myeloid cells, tumor angiogenesis. INTRODUCTION Circulating endothelial progenitor cells (EPCs) have been implicated in the replacement of damaged cells and the formation of new blood vessels in vivo. These cells have been reported to have an impact on normal vascular endothelial turnover, in addition to playing a controversial role in tumor angiogenesis [1-17]. Since their discovery in 1997 by Asahara et al., circulating EPCs have been described utilizing various methods to ascertain their function and role in adult murine tumor angiogenesis [18]. Many differences in the definition of a circulating endothelial cell (CEC) are present in the literature as well as use of different nomenclature when referring to an EPC, making this area of study quite confusing. Thus, extrapolating the results of studies defining the role of EPCs in a particular area of vascular repair or regeneration in the murine system has not always been straightforward to translate for examination of the role of EPCs in human cardiovascular disease or cancer. One of the challenges when studying the murine EPC is the controversy over the existence of an adult bone marrow (BM)-derived hemangioblast, a cell capable of creating both endothelial and hematopoietic lineages. Evidence to support the presence of a hemangioblast has been reported for the developing murine embryo [19] and in certain adult murine models of retinal vascular regeneration and repair [20-22]. Very recent data suggests that hematopoietic cells in mice [23-25] and zebrafish [26, 27] are directly derived from endothelial cells (ECs) rather than a hemangioblast, but it remains unknown and controversial whether endothelium can *Address correspondence to this author at the Department of Pediatrics, Indiana University School of Medicine, 1044 West Walnut Street, R4-470, Indianapolis, Indiana 46202, USA; Tel: +1 317 278 7928; E-mail: [email protected]

1574-888X/11 $58.00+.00

be derived from either the hematopoietic system or a hemangioblast precursor. Part of the controversy in this area is that studies have been published reporting both a hematopoietic and non-hematopoietic origin of EPCs and their ability to incorporate into growing tumors [1, 6, 11, 14, 28, 29]. By delving further into the published tumor angiogenesis studies, this review will critically focus on the evidence put forth to support a hematopoietic or non-hematopoietic origin for the EPCs, and more importantly, whether these cells become fully functional and long-lasting ECs at sites of tumor formation. ELUCIDATION OF MURINE ADULT CIRCULATING EPCs Utilizing magnetic separation technology to isolate blood borne cells, Asahara et al., made the sentinel discovery of both murine and human circulating EPCs [18]. Murine EPCs represented about 1% of the mononuclear cell (MNC) fraction and were isolated by fetal liver kinase-1 (FLK-1) coated beads then plated for culture, in addition to being injected into mice that had undergone femoral arterial ligation to induce hind-limb ischemia. After 4 weeks post injection, putative ECs from the donor mice were found incorporated into capillaries and small arteries of the host injured tissue [18] and this evidence formed the basis for promoting the concept of postnatal vasculogenesis in a murine adult model of tissue ischemia. Subsequently, a flow cytometry panel consisting of monoclonal antibodies to CD45, FLK-1, CD105, VECadherin, CD31 and CD34 was developed to assess CECs in immunodeficient mice bearing human lymphoma implants [30]. A phenotypic distinction was then made between mature CECs and EPCs by proposing that EPCs expressed the murine stem and progenitor cell markers stem cell antigen-1 (SCA-1) and CD117 (i.e. c-kit), which were not expressed by mature CECs [6, 31-34]. As more research was conducted © 2011 Bentham Science Publishers Ltd.

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on methods to discriminate CECs and EPCs, a natural progression of antibody choices evolved. One more widely used phenotypic definition of a murine EPC is a cell that can be characterized as CD45-CD13+FLK-1+CD117+7AAD- [28, 31, 35-37]. According to this definition, the EPC is a nonhematopoietic cell, as only CD45- cells are gated upon. Interestingly, Shaked et al., reported distinct genetic heterogeneity amongst mouse strains for the resting levels of CECs and EPCs. The number of CECs for mice on a 129 background is 11-32 cells/l peripheral blood (PB) compared to less than 5 cells/l PB for mice from a C57BL/6 background. The number of CEPs is 3-7 cells/l PB for 129 mice where C57BL/6 mice have less than 1 cell/l PB. This could possibly account for some of the variability presented by other groups that have published on the raw numbers and their ability to incorporate into vessels [31]. In fact, it should be noted that mice from a C57BL/6 background displayed significantly lower numbers of both CECs and EPCs as compared to mice from a 129 background, and to date, the majority of the published data defining a role for EPCs in vascular regeneration or repair are conducted with C57BL/6 mice [31]. One group has cultured adult murine blood outgrowth endothelial cells (BOECs) from the systemic circulation, albeit with a relatively low success rate per experiment (1128% outgrowth cell emergence without vascular endothelial growth factor (VEGF) pre-treatment) [38]. The recovery of PECAM-1 + cells, which were hypothesized to be the BOEC was low, only 139 (±72) per 1mL of mouse blood [38]. These cells were able to form capillary like tubes using an in vitro Matrigel assay, and via flow cytometry, were shown to be CD14- and CD11b-, yet positive for vascular cell adhesion molecule (VCAM). Furthermore, these cells were able to be expanded in vitro and retained the same phenotypic and functional qualities as low passage cells [38]. Hebbel et al., also tracked the homing of human BOECs after tail vein injection into NOD/SCID mice [39]. Initially the cells were found to home to the lungs with the help of E-selectin, Pselectin and 4 integrin as shown by blocking these molecules and inhibiting the cells from lodging in the lung [39]. Nonetheless, the BOECs were able to expand in vivo in all of the major organs including the BM, liver, spleen and brain. Thus, some evidence is presented for the existence of a CEC with proliferative potential in the murine circulation. Another group has produced EPCs from C57BL/6 fetal blood cells that upon transplantation facilitated hematopoietic cell reconstitution after preparative whole body irradiation [40, 41]. In addition to total cell count recovery, the hematopoietic stem cell (HSC) fractions expressing CD117 and CD117/SCA-1 were also increased numerically after EPC injection. The ability of the EPCs to facilitate the HSC repopulation of the whole blood counts post-transplant was significantly hindered by the co-injection of a VECadherin antibody, suggesting that active angiogenesis was required for the hematopoietic supportive activity [41]. In support of a critical role of endothelium for hematopoietic engraftment, Hooper et al., [42] reported that cell signaling via the vascular endothelial growth factor receptor 2 (VEGFR-2) in BM ECs was required to promote optimal hematopoietic reconstitution following transplantation.

Mund and Case

It is well recognized that tumor cells must recruit adequate blood flow to permit rapid tumor growth. Indeed, antiangiogenic strategies have been devised and are now used as adjunct clinical therapies in patients with tumors and a significant amount of information on the role of EPCs in vascular regeneration has been derived by evaluating tumor angiogenesis [1, 2, 6, 36]. These studies have presented evidence for both an EC and a hematopoietic cell being injected into the host and incorporating into the expanding vasculature of the tumors under the broad term, EPC. SUPPORT FOR A BONE MARROW ORIGIN OF PURPORTED EPCs Shortly after the initial discovery of EPCs, Isner et al., presented data using sorted SCA-1+ PB cells as the EC enriched fraction (10.7±1% of circulating cells) and plated them for 5 days in culture producing spindle shaped cells that stained positive for lectin binding and ingestion of acetylated low-density lipoprotein (acLDL) [43]. Using a murine hind-limb ischemia model to show the mobilization of EPCs into the bloodstream in response to experimentally induced ischemia [43] and Tie2--galactosidase labeled transgenic murine BM expressing cells, this group reported that the BM cells home to sites of ischemic tissue and provide enhanced neo-vascularization [43]. Given the prevailing opinion at that time that only ECs were able to express Tie2, Isner and colleagues interpreted the recruitment of the BM cells into the ischemic site as evidence to support a marrow EPC origin of some cells that displayed endothelial properties. More recently, Naldini and collegues have described multiple cell fractions (including a subset of HSCs, ECs, and even a monocyte population that display Tie2 expression) that display distinct pro-angiogenic qualities, and are recruited to the tumor neovasculature [5, 44, 45]. These cells of monocyte/macrophage origin are able to incorporate into newly forming vessels of tumors (usually in a periendothelial location) but fail to show co-staining with EC markers [45]. The genetic elimination of these cells from the BM compartment also prohibited the vascularization of implanted human glioma cells, in addition to causing significant tumor regression, indicating the importance of these myeloid cells in modulating tumor angiogenesis [45]. Ruzinova et al., also reported evidence for a BM origin of EPCs. Mice with mutations in the Id family of genes display abnormalities in tumor angiogenesis that resulted in increased vascular density, but tumor vessels that formed were surrounded by extensive cellular necrosis [12]. It should be noted that wild-type BM transplantation restored angiogenesis in the Id mutant mice. Id mutant mice were shown to have the mutations confined to the tumor ECs in those malignancies possessing large areas of cellular necrosis or hemorrhage. ECs from tumors without necrosis or hemorrhage were negative for Id3 mRNA and Id1 protein. Of interest, transplantation of LacZ+ labeled whole BM cells restored angiogenesis function in Id- mutant mice, with both BM derived EPCs and myeloid cells being shown to incorporate within vasculature in spontaneously occurring tumors [7, 12]. BM derived ECs found within tumors were defined as cells co-expressing CD31 and LacZ, with some cells also staining positive for VEGFR-2, which was attributed to an EPC subset [12]. Thus, Ruzinova and colleagues proposed

Bone Marrow Derived Cells in Tumor Angiogenesis

that BM derived EPCs play an important role in formation of tumor angiogenesis. A further study by the same group showed BM derived EPCs incorporating into spontaneous prostate tumors only when the tumors were poorly differentiated [46]. Id3-/- Id3+/- transgenic adenocarcinoma of the mouse prostate (TRAMP) mice had postponed tumor growth compared to wild-type. One confusing piece of data was that well developed tumors had very little BM derived EC contribution (2.6% blue ECs) [46]. Also, given that most of the BM derived blood vessels seen in the poorly differentiated tumors were surrounded by BM derived hematopoietic cells, it is hard to prove that these cells are actually incorporated into the endothelium and not just adjacent. A paper published in 2010 by Dudley et al., refutes these claims using the same TRAMP model and citing that the BM is home to pro-angiogenic myelomonocytic cells, but not ECs [47]. Dudley et al., found GFP+ cells that were co-localized with either CD31, SMA, and LYVE-1 in the perivascular region, however none were found on the luminal surface of the blood vessels [47]. When plated, the GFP+ tumor derived cells grew out colonies similar to the monocytic colony forming unit endothelial cell (CFU-EC) that is found in humans, but no EC colonies [47]. This study used microscopy, cell culture and FACS to determine the identity of the cells within the tumors and found that they were not endothelial [47]. Integrin 4 has been shown to play an important role in the engraftment and homing of HSCs in the BM niche [43, 48-50]. Losordo et al., recently reported that the majority of murine BM MNCs express 4 and that all double positive SCA-1 and FLK-1 cells were also CD45+ [16]. These cells, which represent approximately 1% of the CD45+ MNCs, were subsequently isolated, plated and EPC colonies were defined as those adherent cells ingesting DiI-acLDL and displaying positive isolectin B4 staining [16]. Subsequently, using a murine hind-limb ischemia model, transplanted donor Tie2/LacZ+ BM MNCs were shown to incorporate into newly forming vessels with the interpretation that marrow EPCs were contributing to the repair [16]. Since BM derived cells of a myeloid lineage also display all of the criteria that were used to define the EPCs, one could question whether the term endothelial could be definitively added to the characterization of these cells [44, 45]. Slayton et al., also used SCA-1, FLK-1 double positive BM cells as putative EPCs to repopulate BM sinusoidal vessels after exposure to total body lethal irradiation [14]. These donor derived cells lined the endosteal niche and the surviving structures first showing a spreading down the healthiest structures to restore the BM. Interestingly, cell surface CD31 (PECAM) was expressed throughout the BM hematopoietic compartment at many stages of blood cell development, and thus Slayton et al., noted that CD31 does not serve as a suitable marker to specifically identify sinusoidal endothelium [14]. Another recent study conducted by Nolan et al., revealed that GFP labeled BM cells were mobilized and became incorporated into developing blood vessels within growing tumors, and reported that BM cells peaked at 3-7 days [1]. Many of the GFP+ BM derived cells recruited to the tumors stained positive for VE-cadherin and displayed low level

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CD31 expression. These CD31+ cells also stained positive for Isolectin B4 and represent 31±8.3% of GFP+ cells found in early tumors. In later stages of tumor formation (days 1014), GFP- ECs were found to be the main component in vessel formation with donor chimeric vessels dramatically decreased by four weeks post injection (approximately 1%) [1]. The authors conclude that BM-derived EPCs are necessary for neovascularization of growing tumors by differentiating into ECs and incorporating into early vessels and that EPCs may herald the influx of host ECs to provide long-term tumor vascularization. In 2008, Shaked et al., transplanted lethally irradiated mice with GFP+ BM cells and allowed for BM reconstitution of the host [28]. The mice were then injected with Lewis Lung Carcinoma cells (LLC), a spontaneous murine lung cancer. After the LLC tumors reached 500mm3, a single injection of paclitaxel was given to some mice and subsequent analysis of the tumors revealed donor GFP+ cell incorporation, thus suggesting a BM derived cell role in tumor angiogenesis. Later these tumors were recovered and digested into a single cell suspension and analyzed for the presence of donor ECs, Tie2 expressing monocytes (TEMs; GFP+CD45 + Tie2+CD11b+ [45]) and hemangiocytes (GFP+CD45+CXCR4+VEGFR1 + [51]). Tumors from mice injected with paclitaxel had increased CEPs, TEMs, and hemangiocytes. Paclitaxel is a plant alkaloid that has been found to mobilize CECs within hours of injection [28, 52], but is primarily used as a chemotherapeutic agent against many types of cancer including leukemia, lymphoma, breast cancer and melanoma [53]. When paclitaxel was administered along with the VEGF blocking reagent DC101, a significant decrease in tumor size and number of viable vessels was observed [28]. This study was interpreted as evidence that CEPs were mobilized into the blood (baseline 1-2 CEP/l PB; 24 hrs post injection 6-10 CEP/l PB) by an injection of paclitaxel, whereas a different chemotherapeutic, gemcitabine, did not cause an increase in these circulating cells [28]. This increase in BM CEPs after injection of paclitaxel was thought to occur due to a rapid release of stromal derived factor-1 (SDF-1) that is stored in platelets and released to mobilize BM cells that express CXCR4 [51]. Thus, the strongest evidence that supports a BM origin for EPCs comes from studies employing the use of BM transplantation of labeled cells that are demonstrated to incorporate as putative ECs in the growing tumor vasculature [1, 12, 14, 28, 43]. The abnormal tumor and tumor vascular growth in mice with ECs lacking certain Id proteins that is normalized through engraftment of wild-type BM cells has been used as an argument suggesting marrow-derived EPCs can be recruited to rescue abnormal endothelial functions [12, 28]. One complicating issue that was not consistently addressed was whether the putative EPCs were CD45+ (hematopoietic) or CD45- (non-hematopoietic) [30, 31, 33, 36]. SUPPORT AGAINST BONE MARROW CONTRIBUTION TO TUMOR ANGIOGENESIS AND VASCULAR ENDOTHELIUM Some groups report that BM cells are neither involved with tumor angiogenesis nor become incorporated into the vascular endothelium [4, 9, 10, 13]. In 2003, De Palma et al.,

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were the first to give evidence that BM cells did not contribute to vascular endothelium [4]. They transduced lineagenegative BM derived cells with lentiviral vectors expressing GFP from various promoters, including Tie2 (for EC-specific marking) and ubiquitously active regulatory sequences, and then injected the labeled BM cells into lethally irradiated mice to ensure engraftment. Tie2-GFP marrow from transgenic mice was used as a control against issues of the efficiency of ex vivo transduction. Once the BM had been reconstituted, LLC, mammary carcinoma (TS/A) or melanoma (B16) cells were subcutaneously injected and tumor growth was monitored [4]. In mice who had received the Tie2-GFP BM, the GFP+ cells were small, expressed CD45 and were located mainly on the periphery of the tumors [4]. The conclusion was that these cells were a marrow derived hematopoietic cell, not an EC. Very rarely was a Tie2-GFP+ cell expressing CD31 found in tumor vessels (1 in 6000 vessels). This indicates the lack of a BM derived EC having significant involvement in tumor angiogenesis [4]. Another early study by Machein et al., looked at the BM cell contribution in gliomas [9]. Two LacZ reporter mice were employed, either expressing from the ubiquitous Rosa26 locus or Tie2 promoter. The BM from these tranegenic mice was transplanted into lethally irradiated recipient mice and then allowed to engraft for 6 weeks to 6 months. After this time, GL261 murine glioma cells were injected either subcutaneously or in situ and tumor growth was monitored at both early and late time points [9]. In subcutaneous tumors in Rosa26-LacZ transplanted mice, LacZ+ cells were found in areas of necrosis, but none co-stained with EC markers [9]. In the Tie2-LacZ transplanted mice, some intracerebral tumors were found to have LacZ+ cells in the tumor stroma or into the peripheral areas of the tumor vascular structures and stained positive for CD105 and CD31, these however were not integrated into the tumor endothelium and most stained for CD45 [9]. These findings led the authors to determine that BM cells do not incorporate directly into the tumor vasculature. Gothert and colleagues [10] refuted the claim that BM derived cells contribute to tumor endothelium by creating endothelial specific transgenic mice in which transgene activity could be inducible by tamoxifen administration. The authors demonstrated that predicted reporter gene expression was observed in all blood vessel endothelium, including the vessels recruited to growing implanted tumors. However, when the BM of the induced transgenic mice was transplanted into non-transgenic recipient animals bearing implanted tumors, no contribution of the BM cells to the tumor endothelium was observed. [10]. The authors concluded that BM cells do not contribute to tumor endothelium and proposed that their data demonstrated the lineage relationship between pre-existing endothelium and newly generated tumor ECs. A later study by Purhonen et al., [13] confirmed with earlier reports[4, 9, 45] that the apparent reported abundance of BM derived cells in tumor angiogenesis may be perivascular in location rather than part of the endothelial intima and called for a reconsideration of the role of EPCs as vascular reparative cells [13]. In this study, BM cells were labeled with GFP, DsRed.T3, or -gal and BM transplants were

Mund and Case

conducted to permit identification of marrow-derived EPCs into the bloodstream or into various sites of angiogenesis. Regardless of the methods used, BM cells expressing VEGF receptors were not mobilized into the blood stream by VEGF. Using a variety of tumor cell types, all BM derived cells were shown to be either in a stromal or perivascular location in the growing tumor endothelium [13] and BM cells never became incorporated as ECs in any blood vessels. Using confocal microscopy, it became evident to the investigators that large numbers of BM derived cells were recruited to a perivascular location in many of the tumors, however, these cells did not become endothelium. These observations led the investigator to conclude that these BM derived ECs do not directly incorporate into forming tumor vessels [13]. To test the role of circulating EPCs in a non-myeloablative setting, a parabiotic mouse model was constructed wherein one animal was transgenic for GFP expression and the other was not. After approximately 12 weeks of a shared systemic circulation and clear evidence of cross engraftment of marrow cells, no GFP+ ECs were found in the tumor bearing parabiotic animals, confirming the results obtained previously using BM transplantation techniques. WHAT HAVE WE LEARNED AND WHERE ARE WE GOING WITH EPC DEFINITIONS? Most of the data that has been published thus far is insufficient on its own to determine whether EPCs are being incorporated as ECs into the tumor vasculature. In fact, due to the lack of a specific marker to identify a circulating EPC, one can still debate whether it exists at all. Currently the attempted definition of these cells requires a complex combination of both positive and negative markers (Table 1). At present, there are conflicting recommendations as to which markers are optimum for the elucidation of EPCs from hematopoietic progenitor cells in mice. For instance CD34 (which is routinely used as a human HSC marker) is known to be variably expressed in mice depending on the stage of development [54]. CD31, a routine EC marker, is also found to be expressed throughout the BM hematopoietic compartment, thus diminishing the use of this antigen as an exclusive marker [14]. Furthermore, CD117 and SCA-1 are hematopoietic stem and progenitor cell markers, however these antigens are often used for EPC identification [55, 56]. CD45 is known to be exclusively expressed on nucleated blood cells, however, even this antigen is thought to be a marker of EPCs in some reports [30, 31, 33]. In addition, the necessary cell culture assays have not been performed to truly show the reported cells being capable of becoming ECs. At present no data has been provided to show actual incorporation of a BM derived cell into a tumor vessel, thus leading to many possible explanations. Many photomicrographs have been published that show BM derived cells near the vessel, however the greater resolution found with the use of confocal microscopy has revealed that most of the BM derived cells were in fact perivascular and therefore not incorporating into the vessel [13]. Evidence that BM derived cells are primarily recruited into tumor vasculature within the first 10 days and then dramatically declines, does not support the conversion of the BM cells into long term ECs

Bone Marrow Derived Cells in Tumor Angiogenesis

Table 1.


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Common Flow Cytometry and Immunohistochemistry Markers Utilized for the Identification of Endothelial Cells as Hematopoietic Study

EPC Definition

Supporting

Reported in Other Fields

References Isner et al. [43]

Tie-2+

[18]

Monocytes [4, 5]

Ruzinova et al. [7,12]

CD31+/FLK-1+

CD31: [1]

CD31: BM Niches [14]

FLK-1: [21, 24, 25] SCA-1 Losordo et al. [16]

Slayton et al. [14]

SCA-1+/ FLK1+/CD45+

SCA-1+/ FLK1+/CD31+

CD45: [53]

CD45: Hematopoietic

FLK-1: [21, 24, 25]

SCA-1: Hematopoietic

CD31: [1]


FLK-1: [21, 24, 25]

SCA-1: Hematopoietic

Nolan et al. [1]

VE-Cadherin+/CD31+

CD31: [1]


Shaked et al. [28]

CD45-/CD13+/FLK1+/CD117+

CD13: [50, 51]

CD13: Monocytes [52]

CD45: [53]

CD45: Hematopoietic

CD117: [28, 31, 36]

CD117: Hematopoietic [54]

FLK-1: [21, 24, 25]

[1]. The doubling time of tumor endothelium has been estimated at 2.4-13 days depending on the type and stage of the cancer being evaluated, which would allow for recruited BM GFP+ ECs to remain in the vessel if in fact they were truly incorporated [57]. Thus, some significant deficiencies exist with respect to solid evidence that BM cells of the hematopoietic lineage are recruited into a site of tumor cell growth to become ECs that form tumor supporting vasculature. CONCLUSIONS To date, the role of circulating EPCs has been blurred in conjunction with the role of hematopoietic cells in tumor angiogenesis. The most conclusive sets of evidence pertaining to the role of BM derived cells in tumor angiogenesis point to them as being myeloid pro-angiogenic helper cells that are perivascular in nature and fail to give rise to ECs [13]. The ECs that have been shown to incorporate into growing tumor vessels are not BM derived, but come from the host endothelium. More experiments need to be conducted using higher power confocal microscopy of endothelial monolayers from tumor vessels to determine where these ECs are coming from and the method of their incorporation into the tumor vasculature. Advanced flow cytometric acquisition and analysis methodologies should also be used, along with increased markers to further characterize the CECs and to allow for the exclusion of known myeloid cells such as the tumor-associated macrophages (TAMs) and the Tie-2 expressing monocytes which express many EC antigens [5, 44, 45]. By combining the strengths of the currently available assays, a better understanding of the role of both endothelial and hematopoietic cells in tumor angiogenesis will be possible.

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Received: April 28, 2010

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Revised: May 27, 2010

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Hobson B, Denekamp J. Endothelial proliferation in tumours and normal tissues: continuous labelling studies. Br J Cancer 1984; 49(4): 405-13.

Accepted: June 21, 2010