BACKGROUND
interference with tumor vessel formation and blood supply has become a well-recognized approach of cancer therapy  <cit> , as epitomized by the recent fda approval of antiangiogenic drugs such as the humanized anti vascular endothelial growth factor  antibody avastin®  <cit> .
vascularization is required for tumor growth and metastasis, and constitutes an important step in the control of cancer progression  <cit> .
experimental evidence correlates tumor vascularization with high malignancy and poor prognosis, and shows that elevated levels of angiogenic factors, such as vegf and basic fibroblast growth factor , are associated with tumor progression  <cit> .
moreover, expansive growth of metastasis appears to be linked to the ability to stimulate endothelial cell growth  <cit> .
tumor-induced vessels are ultrastructurally abnormal and differ from normal vasculature.
they lack functional pericytes and are unusually permeable  <cit> .
these abnormalities reflect the pathological nature of their induction, and underpin the novel therapeutic strategies directed against the vascular elements of the tumor stroma to selectively affect tumor vasculature and inhibit tumor growth  <cit> .
in recent years an increasing body of evidence suggests that tumor vasculature expresses unique markers that distinguish it from normal vasculature.
numerous efforts have been aimed at the molecular characterization of tumor associated endothelial cells  in the search for differences between tumor and normal tissue ec.
a variety of techniques have been applied to study ec in vivo, isolated from ex vivo specimens or cultured in vitro.
the in vivo phage display technology was used to identify molecules selectively expressed on tumor endothelium  <cit> , while other approaches exploited the in vivo proteomic analysis to detect antigens accessible from the vasculature  <cit> .
these techniques have allowed, mostly through studies conducted in experimental tumor models, the identification of vascular-specific motifs expressed by different tumor types and during different stages of carcinogenesis.
gene expression of ec isolated from ex vivo human cancer and normal specimens, analyzed by means of serial analysis of gene expression  or microarray, revealed differences at the transcriptional level in tumor and normal tissues  <cit> .
in vitro models have been widely used to identify the specific functions of the endothelium and to understand the molecular modifications that might occur during angiogenesis.
in these studies ec were exposed to different microenvironmental conditions, such as angiogenic growth factors and matrix proteins, shear stress, hypoxia, or tumor conditioned media  <cit> .
all of these approaches led to the identification of mechanisms and molecules that are potentially involved in the formation of blood vessels.
despite these findings, attempts to discover tumor ec markers have always been hampered by technical difficulties in isolating functionally intact and phenotypically stable ec from tumor tissues.
in fact, all the in vitro models have used cultures of immortalized ec and/or primary ec from human umbilical vein .
we developed a method to isolate and culture ec from tumor specimens , by which we were able to show that tdec in vitro maintain several of the features described for tumor vasculature and that they differ from ec isolated from normal tissue   <cit> .
these findings suggested that tdec and ha-ec might represent useful tools to study tumor vessel properties and, ultimately, to identify tumor vasculature markers.
here we describe the isolation of ec from human specimens and the characterization of the transcriptional profiles of ec derived from ovarian carcinoma  and ha-ec by means of microarray technology.
through the comparison of hoc-ec and ha-ec gene expression we were able to demonstrate that tumor-associated ec differ from those in normal tissues, thus proving that molecular differences are maintained in vitro.
we successfully identified molecules that are expressed selectively by the tumor vasculature in vivo, thus providing distinctive features to be exploited to selectively target tumor vasculature.
RESULTS
characterization of endothelial cells  isolated from human specimens
endothelial cells were reproducibly isolated from human tissue specimens: hoc-ec from ovarian carcinoma, hkc-ec from kidney carcinoma, ha-ec from adrenal gland and hsk-ec from skin.
before use, their endothelial origin was established.
endothelial cell cultures typically created a homogeneous monolayer of elongated cells in close contact and maintained an uniform morphology after few passages in culture; they formed capillary-like cord structures when plated on matrigel .
their endothelial origin was established by positive immunostaining for platelet-endothelial cell adhesion molecule- <dig> , von willebrand factor  and vascular e-cadherin  .
in addition, no staining was observed when the cells were analyzed for the expression of alpha-smooth muscle actin , a typical non-endothelial marker .
flow cytometry studies assessed the ability to uptake low density lipoproteins , a characteristic of endothelial cells, and confirmed the positivity for cd31/pecam- <dig> .
percentages of positive cells analyzed by immunohistochemistry evaluating a total of  <dig> cells/population.
in parentheses are the number of independent human ec populations evaluated .
shown are the range of values obtained for the different endothelial cell types.
* population showing positivity values below 65% were not utilized.
nt=not determined
the purity of the isolated ec was comparable to that of ec from umbilical vein , which were used as the reference population for ec .
microarray results, performed on ha-ec and hoc-ec, confirmed the endothelial origin of the isolated cells .
the typical endothelial genes expressed at high levels include col8a <dig>  col18a <dig>  cyr <dig>  endoglin, e-selectin, hif1a, icam- <dig>  jam <dig> junctional adhesion molecule, neuropilin- <dig>  par- <dig> thrombin receptor, p-selectin, tek endothelial tyrosine kinase, vegfr- <dig>  and vegfr- <dig>  among these, the presence of transcripts for cd31/pecam- <dig>  ve-cadherin and vwf, whose protein expression was shown by immunohistochemical methods , is worth emphasizing.
three hoc-ec and two ha-ec populations were investigated by affymetrix genechip® human genome u95a arrays.
this list of highly expressed endothelial genes was extracted from the microarray results by keywords search within the complete probeset list annotated using source batch search .
genome wide transcriptional differences: tumor vs. normal tissue derived ec
comparison of the transcriptional profiles of hoc-ec and ha-ec  showed that ec from tumor and normal tissue have distinct and characteristic expression patterns that are maintained in vitro.
according to our selection criteria,  <dig> gene transcripts, corresponding to  <dig> probe sets, were more expressed by hoc-ec than by ha-ec.
the genes belong to the different functional classes that are listed in table  <dig>
list of the  <dig> gene transcipts more expressed by hoc-ec than ha-ec.
three hoc-ec and two ha-ec populations were investigated by affymetrix genechip® human genome u95a arrays and analyzed searchingfor intrinsic differences, as described in methods.
microarray results reveal an alteration in cell-cell and cell-matrix interaction as shown by the enhanced expression of molecules such as desmoglein  <dig>  n- and ob-cadherin, protocadherin- <dig> and - <dig> and p-selectin .
in keeping with the notion that the extracellular matrix in tumor differs from that in normal tissue  <cit>  glypican- <dig>  laminin alpha  <dig>  tenascin c, versican, as well as different types of collagens  and thrombospondin- <dig>  were expressed at higher levels by hoc-ec than by ha-ec .
moreover, alterations of cytoskeletal protein involved in cell motility, for instance the actin and myosin related molecules myom <dig>  myl <dig> and ripx, were also observed .
the peptidase family is the most represented among the enzymatic classes of genes expressed by hoc-ec .
it includes carboxypeptidase a <dig>  carboxypeptidase e, cathepsin z, membrane metallo-endopeptidase, serinprotease  <dig>  and urokinase-plasminogen activator.
this is consistent with the concept that peptidases are very important for the activation of pro-enzymes or protein precursors, as well as in the degradation of the extracellular matrix  <cit> .
most of the listed genes are involved in signaling and comprise molecules previously shown to be involved in angiogenesis and other vascular functions, such as adrenomedullin receptor, cd <dig>  endothelin- <dig> and - <dig>  endothelin receptor-a and tissue factor .
consistent with the notion that the vascular and nervous systems share similar signals and principles  <cit> ,  <dig> members of the gaba receptors family and several molecules involved in synaptic signaling, as well as elements involved in brain differentiation and axon elongation, were expressed at higher levels by hoc-ec .
four members of the solute carrier family  were over-expressed in hoc-ec, suggesting an alteration in the extra-intracellular exchange of metabolites .
among the transporters, the enhanced expression of the atp-binding cassette family members abca <dig> and abcb <dig>  should be emphasized.
abcb <dig>  best known as mdr/p-glycoprotein, is involved in drug removal from cells.
higher expression also affected genes involved in transcriptional response, such as members of the zinc finger protein family  and of the homeobox family  .
smad <dig>  and bambi , involved and acting as inhibitors of tgf-beta signaling, were also more expressed.
among the class of cell cycle regulators, the high expression of the anti-apoptotic protein bcl <dig> is noteworthy .
selected transcripts are expressed by tumor derived endothelial cells
in order to identify novel markers of tumor endothelium our attention focused on transcripts that are highly expressed by tumor-derived endothelium , but whose products and functions were not previously reported as being involved in tumor angiogenesis or associated to vascular functions.
among them we distinguished four genes on the basis of their structural or functional characteristics, such as the presence of transmembrane domains and/or enzymatic activities: disintegrin and metalloproteinase domain  <dig> , fibroblast activation protein alpha , transmembrane glycoprotein nmb , and serine protease  <dig> or mesotrypsin .
the relative expression in hoc-ec compared to ha-ec was  <dig>  for adam <dig>   <dig>  for fap,  <dig>  for gpnmb and  <dig>  for prss <dig>  as calculated from microarray data.
these results were validated by real-time pcr in ec isolated from six neoplastic tissues  and from four non-neoplastic tissues  different from those investigated by microarray.
these tumor or normal tissue derived ec are described in table  <dig>
to mimic the different environments in which ec might be embedded in vivo – either subjected to persistent stimulation imposed by the tumor cells or not, ec were investigated both in the presence  or the absence  of the angiogenic factors vegf, fgf-  <dig>  of epidermal growth factor  and of fibronectin.
under both conditions the expression of adam <dig>  fap, gpnmb and prss <dig> genes was much higher in ec isolated from tumor specimens, as shown by the fold difference values .
as specified by the p values, statistical analysis demonstrated a significant disparity in the expression levels for each of the genes being investigated .
these results confirm microarray findings and demonstrate that such a diversity is indeed an intrinsic characteristic associated to the tumor or normal tissue origin of the ec, regardless of the culture conditions.
sarcoglycan epsilon , chosen as an internal control of a gene expressed to a much lesser extent by hoc-ec, confirmed the microarray results .
expression in non endothelial cells
having shown that adam <dig>  fap, gpnmb, and prss <dig> were expressed by a panel of ec, with the highest expression found in tdec, we next investigated their expression in other cell types.
the gene expression analysis revealed that they could be expressed to different extents also by other stromal cells, such as fibroblasts  and smooth muscle cells , but in general not by tumor cells .
specifically, their expression in the carcinoma cell lines  was not or was barely detectable  .
this lack of expression was supported by the northern blot results of a panel of tumor cell lines represented on the multiple tissue expression array .
moreover, the reference database for gene expression analysis  confirmed that the tumor cell lines did not show expression of the selected genes.
conversely, the expression of fap and gpnmb was higher in both the hufb and malme  <dig> fibroblasts than in ec, and fap was also expressed at a higher level by the uasmc smooth muscle cells.
adam <dig> and prss <dig> were instead expressed to a lesser extent by all the stromal cells analyzed .
expression in healthy tissues
tissue expression of adam <dig> and gpnmb was assessed using a multiple tissue expression  array: a panel of human mrnas in dot-blot format from  <dig> normal adult tissues, seven fetal tissues, and eight cell lines.
as shown in figure  <dig>  adam <dig> and gpnmb were expressed by a limited subset of tissues, while tumor cells did not express either one of them.
in particular, adam <dig> expression was highest in cardiovascular tissues and fetal heart, as well as in central nervous system  samples, while it was much lower in fetal brain .
the expression of gpnmb was highest in cardiovascular tissues and fetal heart, as well as in placenta, while it was detected to a much lesser extent in lung, uterus, and bladder tissue.
.
these northern blot results for adam <dig> and gpnmb expression were confirmed by the refexa database  <cit> .
data from refexa  <cit>  and literature report the expression of prss <dig> by brain, colon and pancreas  <cit> , while fap expression is described only in the activated stromal fibroblast of tumors  <cit> .
expression by tumor vasculature in vivo
in order to investigate the in vivo localization of adam <dig>  gpnmb and prss <dig>  human tumor tissues were analyzed by in situ hybridization.
endothelial expression of the selected genes was confirmed in the tumor samples analyzed.
as shown in figure  <dig>  the hybridization signals for adam <dig>  gpnmb, and prss <dig> localized around the blood vessels, and exclusively in the vascular walls.
according to the in vitro results, tumor cells did not express the transcripts, as shown by the absence of staining in the parenchyma of the tissues .
noteworthy was the finding that stromal compartments  lacked the expression of either gene.
no staining was observed in normal brain and alzheimer's-diseased brain .
it is remarkable that adam <dig> and prss <dig> transcripts, whose expression in cns was revealed by the mte array and/or reported by refexa  <cit> , were not detected in normal or diseased brain, except for blood vessels in medulloblastoma or metastatic adenocarcinoma .
sense probes did not hybridize with any of the tissue sections analyzed .
discussion
targeting the process of tumor neovascularization has become a promising anti-cancer strategy.
this is particularly evident when vascular targeting agents are combined with conventional chemotherapy and radiotherapy, as demonstrated by recent reports and clinical trials  <cit> .
however, the success of this approach depends on the availability of suitable endothelial markers that can be used to selectively target the tumor vascular compartment or to deliver a ligand-directed effector to the endothelium  <cit> .
herein, we successfully combined a method to isolate and culture endothelial cells  from human specimens of tumor  and non-neoplastic  origin with oligonucleotide microarray analysis to compare their gene expression.
we first showed that the tdec transcriptional profile differs from that of ndec, and, thereafter identified specific molecular features of tumor-derived endothelium.
our laboratory previously established a procedure  <cit>  to derive ec of high purity from human specimens, their origin assessed by immunohistochemical staining for the three endothelial markers cd31/pecam- <dig>  ve-cadherin and vwf.
accordingly, microarray results confirmed their expression, but also revealed the expression of many other genes annotated as typically endothelial through source  <cit> .
these included e-selectin, vegfr- <dig>  and vegfr- <dig>  further confirming our previous findings  <cit> .
despite being less successful with tumors  than with non neoplastic , the reproducible yield of ec from human specimens enabled us to explore molecular differences between tumor and normal endothelium.
the comparative transcriptomic analysis of ec from ovarian carcinoma  and adrenal gland  revealed the higher expression of  <dig> gene transcripts by hoc-ec.
the finding that tumor and normal tissue ec have distinct gene expression patterns concurs with other investigations, which describe genes that are preferentially expressed by endothelium from malignant cancer  <cit> .
in these studies, sage  <cit>  or microarray analyses  <cit>  were executed on ec freshly obtained by immunoselection of ex vivo tissue samples.
endothelial cells in culture often lose their tissue-specificity and their specialized properties  <cit> .
our results, nonetheless, demonstrate that we were able to preserve in vitro some of the phenotypes that distinguish the diverse endothelia.
likewise, they confirm our previous findings  <cit>  and agree with other reports  <cit>  showing that, despite being in culture, ec isolated from different vascular beds possess characteristic gene expression profiles.
here we describe for the first time a much higher expression of adam <dig>  fap, gnpnb and prss <dig> in ec derived from six tumor samples than in ec from four normal tissue specimens examined by real-time pcr, whose origin differed from that investigated by microarray analysis.
the differences we detected between tdec and ndec were maintained whether or not they were exposed to a combination of pro-angiogenic factors that presumably mimic the different environmental conditions under which ec might be embedded in vivo .
these results suggest that the transcriptional differences are not culture condition artifacts, but, rather, true differences that distinguish ec of different origin .
despite having mainly utilized ec from ovarian cancer and from adrenal glands as the non-neoplastic counterpart, the results hold true for ec isolated from kidney cancer and skin specimens .
these observations, would seem to indicate that the ec characteristics we observed are not organ-specific, but, rather, exclusive of tdec.
this is in agreement with a recent report  <cit>  that compared ec freshly isolated from ovarian cancer and normal ovary in which several of the genes identified as distinctive of the tumor associated vasculature showed conserved expression across the ec from other tumor types, such as colon  <cit> , brain  <cit>  and breast  <cit> .
cultures of ec are valuable tools for biochemical and functional analysis, but they do not reflect the physiological situation entirely.
all the information should be considered critically from the standpoint of what might occur in blood vessels in vivo.
to this end, in situ hybridization results confirmed adam <dig>  gnpnb and prss <dig> expression associated to blood vessels in tumor tissues, but the lack of their expression in cancer cells .
overall, these findings support the premise that endothelial markers identified in vitro are likely to have in vivo relevance, and bear out the validity of our experimental setting designed to reproduce situations that might occur in vivo.
adam <dig> belongs to the cellular disintegrins, a family of membrane-anchored proteins that are potential regulators of cell-to-cell and cell-to-matrix interactions  <cit>  and for which a specific binding interaction with alphavbeta <dig> integrin has been demonstrated  <cit> .
our mte array results showed that adam <dig> is expressed in fetal and adult heart, and also confirmed its previously reported expression by fetal and adult brain  <cit> .
however, our in situ hybridization finding  contradicted the latter, which implies that the expression of adam <dig> might indeed be much higher in tumor ec, including blood vessels in medulloblastoma, a highly malignant brain tumor, than in the brain.
further studies, aimed to produce antibodies to adam <dig>  are necessary to prove its suitability as a tumor vascular target.
our results also revealed that adam <dig> was not expressed in cancer cells.
this observation is consistent with the data available from refexa and other recent reports  <cit> , which describe hypermethylation of the adam <dig> promoter that silences its expression in breast and pancreatic cell lines and in the corresponding primary tumors.
our evidence of a high expression of adam <dig> by tdec suggests that this protein might be important for establishing contacts and promoting adhesive functions during the pathological processes leading to the formation of new blood vessels within the tumor.
in addition, it is intriguing to speculate that this protein – through its ability to bind alphavbeta <dig> – could play some specialized role in the maintenance of vascular functions within the tumor.
in keeping with this hypothesis is the fact that alphavbeta <dig> is expressed by ec and has been shown to be involved in tumor neo-vascularization  <cit> .
gpnmb is a type i membrane protein identical to the hematopoietic growth factor inducible neurokinin- <dig> type   <cit>  sharing similarities with neurokinin- <dig> , a receptor for tachykinins and a member of seven transmembrane g protein coupled receptors.
hgfin was shown to interact with substance p , which has been reported to induce ec proliferation and increase vascular density in an in vivo model of inflammation, and these effects could be inhibited by an nk <dig> receptor antagonist  <cit> .
the action of sp via the nk <dig> receptor may be a direct effect, since nk <dig> receptors have previously been localized to endothelium  <cit> , and both sp and selective nk <dig> agonists were shown to enhance in vitro ec proliferation  <cit> .
substance p in vivo also contributes to vascular permeability, plasma extravasation, and edema  <cit> , all features that characterize the abnormal vasculature of tumors.
our evidence of a high expression of gpnmb by tdec suggests that gpnmb may be a tumor stroma receptor for sp, involved in tumor angiogenic events and in determining the distinctive features of tumor vasculature.
corroborating this premise are data suggesting that, in addition to nk <dig> receptors, other mechanisms contribute in mediating in vivo sp effects on ec  <cit> .
the prss <dig> gene encodes for a trypsinogen whose functions have not yet been fully elucidated  <cit> .
prss <dig> transcript, whose expression in cns is reported by refexa, was not detected in normal or alzheimer's-diseased brain, except for blood vessels in a brain metastasis.
this finding might mean that the expression of prss <dig> is indeed characteristic of tumor ec, as seen for adam <dig>  our search in the refexa and source databases revealed that prss <dig> was not expressed by tumor cells, with the exception of colon carcinoma.
it seems very plausible that prss <dig> produced by tdec may contribute to tumor angiogenesis, invasion and metastasis through its peptidase activity.
this notion upholds evidence that the migration and invasion of ec in the tumor tissue requires the activation of pro-enzymes  and protein precursors  <cit> .
to this end, it is noteworthy that other peptidases  were among the genes we found to be highly expressed by tumor-derived endothelium.
in particular, upa is capable of initiating the process of extracellular matrix degradation through the activation of plasminogen and matrix metalloproteases; in addition, it has been shown to be required for the migration of ec during the process of angiogenesis  <cit> .
it is interesting that the expression of the upa receptor  was also seen to be higher in hoc-ec than in ha-ec , a finding that would implicate the upa-upar system in tumor driven angiogenesis.
our results show that the cell surface protease fap was also expressed at significantly higher levels by tdec than by normal ec.
fap was originally reported to be an antigen recognized by f <dig> antibody in cultured fibroblasts  <cit> .
accordingly, its expression in our tests was found to be much higher in cultured fibroblasts  and in smooth muscle cells .
nonetheless, while normal adult tissues are generally fap-negative, its expression is detectable in the stroma of over 90% of carcinomas  <cit> .
such behavior suggested its suitability for stromal targeting and/or cancer detection and therapy, and in recent years has underpinned the development of a humanized anti-fap antibody  and the implementation of clinical trials  <cit> .
our results uphold the notion that fap is a valid target for therapy, be it as stromal or as vascular marker.
in keeping with our results, the in vivo expression of fap on blood vessels was recently demonstrated by ghersi et al.
in invasive breast ductal carcinoma  <cit> .
many of the transcripts we found to be expressed at a high level by ec from neoplastic tissues were previously reported as being involved in tumor angiogenesis or expressed by tumor ec.
in this regard, it is worth listing the various types of matrix proteins that we detected, namely: collagens alpha- <dig> type i, alpha- <dig> type iii, v, xiii and xv, glypican- <dig>  laminin alpha <dig>  and tenascin c. our results confirm similar findings from other laboratories  <cit>  and denote a remodeling of the extracellular matrix known to promote blood vessel sprouting and growth.
in keeping with the experimental data, and as an example, tenascin c was shown to be expressed in vivo and to colocalize with microvessels in the stroma of non-small cell lung cancer  <cit> .
the expression of cell adhesion proteins was also altered in tdec.
this may be explained by the fact that migrating and proliferating ec must "loosen up", that is, detach and then create new interactions.
our results implicate cd <dig> and cadherins in this process, and confirm previous reports showing n-cadherin over-expression on proliferative state ec  <cit> , and enhanced expression of cd <dig> on the vasculature of solid tumors compared to normal tissue  <cit> .
noteworthy is the finding that cd <dig> expression was up-regulated when the tdec were exposed to angiogenic growth factors , in accordance with previous results showing that fgf- <dig> and vegf stimulate cd <dig> expression in ec  <cit> .
interestingly, abca <dig> and abcb <dig>  were also more abundantly expressed by tdec, reflecting the previously reported higher expression of p-glycoprotein  <dig> in ec from glioma than from normal brain  <cit> .
the atp-binding cassette transporters  are responsible for the efflux of chemotherapeutic agents in cancer cells  <cit> .
these data suggest that the multidrug resistance phenomenon in tumors may be due to both ec and tumor cells.
molecules such as smad <dig>  bambi and bcl <dig> are listed among the transcripts that are more highly expressed by hoc-ec than by ha-ec.
smad <dig> and bambi interfere with the tgfβ pathway and may potentially block the anti-proliferative action of tgfβ  <cit> .
their expression may induce an enhanced proliferation of the tdec, which is one of the steps of the angiogenic process.
bcl <dig> is one of the most widely recognized anti-apoptotic factors, whose upregulation enhances ec survival and intratumoral angiogenesis, thus promoting tumor growth  <cit> .
emerging studies suggest that the vascular and the nervous systems share the same molecular signals in their development.
the involvement of netrins, semaphorins, robo/slit and vegf families in both angiogenesis and neurogenesis has been demonstrated in recent years  <cit> .
in keeping with these findings, our results revealed that tdec express numerous molecules that are associated with the cns or are implicated in brain differentiation and axon guidance.
in particular, adora2b was identified as a novel netrin- <dig> receptor and reported to mediate axon outgrowth  <cit> .
in a similar way – and considering that netrin- <dig> is pro-angiogenic  <cit>  – our results allow speculatation that adora2b could mediate the sprouting of vessels during tumor neovascularization.
CONCLUSIONS
targeting angiogenesis and vasculature represents a promising option to control tumor growth and dissemination.
to exploit this opportunity, however, selective targets in tumor associated vasculature need to be identified.
we describe a model consisting of in vitro cultured endothelial cells isolated from human normal and neoplastic specimens that maintained their respective molecular phenotype and enabled us to determine molecular differences between tumor and normal tissue derived ec.
specifically, we identified four transcripts whose expression is much higher in endothelial cells from tumors than from normal tissue.
the in vivo expression in cancer-associated blood vessels was confirmed by in situ hybridization of human specimens.
this work evinces some of the distinctive features of tumor-derived endothelium, and identifies potential markers that may provide the groundwork for novel therapeutic strategies.
future studies are needed to clarify the functions and roles of these molecules in tumor progression and vascularization.

 interference with tumor vessel formation and blood supply has become a well-recognized approach of cancer therapy, as epitomized by the recent fda approval of antiangiogenic drugs such as the humanized anti vascular endothelial growth factor antibody avastin®. vascularization is required for tumor growth and metastasis, and constitutes an important step in the control of cancer progression. experimental evidence correlates tumor vascularization with high malignancy and poor prognosis, and shows that elevated levels of angiogenic factors, such as vegf and basic fibroblast growth factor, are associated with tumor progression. moreover, expansive growth of metastasis appears to be linked to the ability to stimulate endothelial cell growth. tumor-induced vessels are ultrastructurally abnormal and differ from normal vasculature. they lack functional pericytes and are unusually permeable. these abnormalities reflect the pathological nature of their induction, and underpin the novel therapeutic strategies directed against the vascular elements of the tumor stroma to selectively affect tumor vasculature and inhibit tumor growth. in recent years an increasing body of evidence suggests that tumor vasculature expresses unique markers that distinguish it from normal vasculature. numerous efforts have been aimed at the molecular characterization of tumor associated endothelial cells in the search for differences between tumor and normal tissue ec. a variety of techniques have been applied to study ec in vivo, isolated from ex vivo specimens or cultured in vitro. the in vivo phage display technology was used to identify molecules selectively expressed on tumor endothelium, while other approaches exploited the in vivo proteomic analysis to detect antigens accessible from the vasculature. these techniques have allowed, mostly through studies conducted in experimental tumor models, the identification of vascular-specific motifs expressed by different tumor types and during different stages of carcinogenesis. gene expression of ec isolated from ex vivo human cancer and normal specimens, analyzed by means of serial analysis of gene expression or microarray, revealed differences at the transcriptional level in tumor and normal tissues. in vitro models have been widely used to identify the specific functions of the endothelium and to understand the molecular modifications that might occur during angiogenesis. in these studies ec were exposed to different microenvironmental conditions, such as angiogenic growth factors and matrix proteins, shear stress, hypoxia, or tumor conditioned media. all of these approaches led to the identification of mechanisms and molecules that are potentially involved in the formation of blood vessels. despite these findings, attempts to discover tumor ec markers have always been hampered by technical difficulties in isolating functionally intact and phenotypically stable ec from tumor tissues. in fact, all the in vitro models have used cultures of immortalized ec and/or primary ec from human umbilical vein. we developed a method to isolate and culture ec from tumor specimens, by which we were able to show that tdec in vitro maintain several of the features described for tumor vasculature and that they differ from ec isolated from normal tissue. these findings suggested that tdec and ha-ec might represent useful tools to study tumor vessel properties and, ultimately, to identify tumor vasculature markers. here we describe the isolation of ec from human specimens and the characterization of the transcriptional profiles of ec derived from ovarian carcinoma and ha-ec by means of microarray technology. through the comparison of hoc-ec and ha-ec gene expression we were able to demonstrate that tumor-associated ec differ from those in normal tissues, thus proving that molecular differences are maintained in vitro. we successfully identified molecules that are expressed selectively by the tumor vasculature in vivo, thus providing distinctive features to be exploited to selectively target tumor vasculature.  characterization of endothelial cells isolated from human specimens endothelial cells were reproducibly isolated from human tissue specimens: hoc-ec from ovarian carcinoma, hkc-ec from kidney carcinoma, ha-ec from adrenal gland and hsk-ec from skin. before use, their endothelial origin was established. endothelial cell cultures typically created a homogeneous monolayer of elongated cells in close contact and maintained an uniform morphology after few passages in culture; they formed capillary-like cord structures when plated on matrigel. their endothelial origin was established by positive immunostaining for platelet-endothelial cell adhesion molecule-, von willebrand factor and vascular e-cadherin. in addition, no staining was observed when the cells were analyzed for the expression of alpha-smooth muscle actin, a typical non-endothelial marker. flow cytometry studies assessed the ability to uptake low density lipoproteins, a characteristic of endothelial cells, and confirmed the positivity for cd31/pecam-. percentages of positive cells analyzed by immunohistochemistry evaluating a total of cells/population. in parentheses are the number of independent human ec populations evaluated. shown are the range of values obtained for the different endothelial cell types. * population showing positivity values below 65% were not utilized. nt=not determined the purity of the isolated ec was comparable to that of ec from umbilical vein, which were used as the reference population for ec. microarray results, performed on ha-ec and hoc-ec, confirmed the endothelial origin of the isolated cells. the typical endothelial genes expressed at high levels include col8a col18a cyr endoglin, e-selectin, hif1a, icam- jam junctional adhesion molecule, neuropilin- par- thrombin receptor, p-selectin, tek endothelial tyrosine kinase, vegfr- and vegfr- among these, the presence of transcripts for cd31/pecam- ve-cadherin and vwf, whose protein expression was shown by immunohistochemical methods, is worth emphasizing. three hoc-ec and two ha-ec populations were investigated by affymetrix genechip® human genome u95a arrays. this list of highly expressed endothelial genes was extracted from the microarray results by keywords search within the complete probeset list annotated using source batch search. genome wide transcriptional differences: tumor vs. normal tissue derived ec comparison of the transcriptional profiles of hoc-ec and ha-ec showed that ec from tumor and normal tissue have distinct and characteristic expression patterns that are maintained in vitro. according to our selection criteria, gene transcripts, corresponding to probe sets, were more expressed by hoc-ec than by ha-ec. the genes belong to the different functional classes that are listed in table list of the gene transcipts more expressed by hoc-ec than ha-ec. three hoc-ec and two ha-ec populations were investigated by affymetrix genechip® human genome u95a arrays and analyzed searchingfor intrinsic differences, as described in methods. microarray results reveal an alteration in cell-cell and cell-matrix interaction as shown by the enhanced expression of molecules such as desmoglein n- and ob-cadherin, protocadherin- and - and p-selectin. in keeping with the notion that the extracellular matrix in tumor differs from that in normal tissue glypican- laminin alpha tenascin c, versican, as well as different types of collagens and thrombospondin- were expressed at higher levels by hoc-ec than by ha-ec. moreover, alterations of cytoskeletal protein involved in cell motility, for instance the actin and myosin related molecules myom myl and ripx, were also observed. the peptidase family is the most represented among the enzymatic classes of genes expressed by hoc-ec. it includes carboxypeptidase a carboxypeptidase e, cathepsin z, membrane metallo-endopeptidase, serinprotease and urokinase-plasminogen activator. this is consistent with the concept that peptidases are very important for the activation of pro-enzymes or protein precursors, as well as in the degradation of the extracellular matrix. most of the listed genes are involved in signaling and comprise molecules previously shown to be involved in angiogenesis and other vascular functions, such as adrenomedullin receptor, cd endothelin- and - endothelin receptor-a and tissue factor. consistent with the notion that the vascular and nervous systems share similar signals and principles, members of the gaba receptors family and several molecules involved in synaptic signaling, as well as elements involved in brain differentiation and axon elongation, were expressed at higher levels by hoc-ec. four members of the solute carrier family were over-expressed in hoc-ec, suggesting an alteration in the extra-intracellular exchange of metabolites. among the transporters, the enhanced expression of the atp-binding cassette family members abca and abcb should be emphasized. abcb best known as mdr/p-glycoprotein, is involved in drug removal from cells. higher expression also affected genes involved in transcriptional response, such as members of the zinc finger protein family and of the homeobox family. smad and bambi, involved and acting as inhibitors of tgf-beta signaling, were also more expressed. among the class of cell cycle regulators, the high expression of the anti-apoptotic protein bcl is noteworthy. selected transcripts are expressed by tumor derived endothelial cells in order to identify novel markers of tumor endothelium our attention focused on transcripts that are highly expressed by tumor-derived endothelium, but whose products and functions were not previously reported as being involved in tumor angiogenesis or associated to vascular functions. among them we distinguished four genes on the basis of their structural or functional characteristics, such as the presence of transmembrane domains and/or enzymatic activities: disintegrin and metalloproteinase domain, fibroblast activation protein alpha, transmembrane glycoprotein nmb, and serine protease or mesotrypsin. the relative expression in hoc-ec compared to ha-ec was for adam for fap, for gpnmb and for prss as calculated from microarray data. these results were validated by real-time pcr in ec isolated from six neoplastic tissues and from four non-neoplastic tissues different from those investigated by microarray. these tumor or normal tissue derived ec are described in table to mimic the different environments in which ec might be embedded in vivo – either subjected to persistent stimulation imposed by the tumor cells or not, ec were investigated both in the presence or the absence of the angiogenic factors vegf, fgf- of epidermal growth factor and of fibronectin. under both conditions the expression of adam fap, gpnmb and prss genes was much higher in ec isolated from tumor specimens, as shown by the fold difference values. as specified by the p values, statistical analysis demonstrated a significant disparity in the expression levels for each of the genes being investigated. these results confirm microarray findings and demonstrate that such a diversity is indeed an intrinsic characteristic associated to the tumor or normal tissue origin of the ec, regardless of the culture conditions. sarcoglycan epsilon, chosen as an internal control of a gene expressed to a much lesser extent by hoc-ec, confirmed the microarray results. expression in non endothelial cells having shown that adam fap, gpnmb, and prss were expressed by a panel of ec, with the highest expression found in tdec, we next investigated their expression in other cell types. the gene expression analysis revealed that they could be expressed to different extents also by other stromal cells, such as fibroblasts and smooth muscle cells, but in general not by tumor cells. specifically, their expression in the carcinoma cell lines was not or was barely detectable. this lack of expression was supported by the northern blot results of a panel of tumor cell lines represented on the multiple tissue expression array. moreover, the reference database for gene expression analysis confirmed that the tumor cell lines did not show expression of the selected genes. conversely, the expression of fap and gpnmb was higher in both the hufb and malme fibroblasts than in ec, and fap was also expressed at a higher level by the uasmc smooth muscle cells. adam and prss were instead expressed to a lesser extent by all the stromal cells analyzed. expression in healthy tissues tissue expression of adam and gpnmb was assessed using a multiple tissue expression array: a panel of human mrnas in dot-blot format from normal adult tissues, seven fetal tissues, and eight cell lines. as shown in figure adam and gpnmb were expressed by a limited subset of tissues, while tumor cells did not express either one of them. in particular, adam expression was highest in cardiovascular tissues and fetal heart, as well as in central nervous system samples, while it was much lower in fetal brain. the expression of gpnmb was highest in cardiovascular tissues and fetal heart, as well as in placenta, while it was detected to a much lesser extent in lung, uterus, and bladder tissue.. these northern blot results for adam and gpnmb expression were confirmed by the refexa database. data from refexa and literature report the expression of prss by brain, colon and pancreas, while fap expression is described only in the activated stromal fibroblast of tumors. expression by tumor vasculature in vivo in order to investigate the in vivo localization of adam gpnmb and prss human tumor tissues were analyzed by in situ hybridization. endothelial expression of the selected genes was confirmed in the tumor samples analyzed. as shown in figure the hybridization signals for adam gpnmb, and prss localized around the blood vessels, and exclusively in the vascular walls. according to the in vitro results, tumor cells did not express the transcripts, as shown by the absence of staining in the parenchyma of the tissues. noteworthy was the finding that stromal compartments lacked the expression of either gene. no staining was observed in normal brain and alzheimer's-diseased brain. it is remarkable that adam and prss transcripts, whose expression in cns was revealed by the mte array and/or reported by refexa, were not detected in normal or diseased brain, except for blood vessels in medulloblastoma or metastatic adenocarcinoma. sense probes did not hybridize with any of the tissue sections analyzed. discussion targeting the process of tumor neovascularization has become a promising anti-cancer strategy. this is particularly evident when vascular targeting agents are combined with conventional chemotherapy and radiotherapy, as demonstrated by recent reports and clinical trials. however, the success of this approach depends on the availability of suitable endothelial markers that can be used to selectively target the tumor vascular compartment or to deliver a ligand-directed effector to the endothelium. herein, we successfully combined a method to isolate and culture endothelial cells from human specimens of tumor and non-neoplastic origin with oligonucleotide microarray analysis to compare their gene expression. we first showed that the tdec transcriptional profile differs from that of ndec, and, thereafter identified specific molecular features of tumor-derived endothelium. our laboratory previously established a procedure to derive ec of high purity from human specimens, their origin assessed by immunohistochemical staining for the three endothelial markers cd31/pecam- ve-cadherin and vwf. accordingly, microarray results confirmed their expression, but also revealed the expression of many other genes annotated as typically endothelial through source. these included e-selectin, vegfr- and vegfr- further confirming our previous findings. despite being less successful with tumors than with non neoplastic, the reproducible yield of ec from human specimens enabled us to explore molecular differences between tumor and normal endothelium. the comparative transcriptomic analysis of ec from ovarian carcinoma and adrenal gland revealed the higher expression of gene transcripts by hoc-ec. the finding that tumor and normal tissue ec have distinct gene expression patterns concurs with other investigations, which describe genes that are preferentially expressed by endothelium from malignant cancer. in these studies, sage or microarray analyses were executed on ec freshly obtained by immunoselection of ex vivo tissue samples. endothelial cells in culture often lose their tissue-specificity and their specialized properties. our results, nonetheless, demonstrate that we were able to preserve in vitro some of the phenotypes that distinguish the diverse endothelia. likewise, they confirm our previous findings and agree with other reports showing that, despite being in culture, ec isolated from different vascular beds possess characteristic gene expression profiles. here we describe for the first time a much higher expression of adam fap, gnpnb and prss in ec derived from six tumor samples than in ec from four normal tissue specimens examined by real-time pcr, whose origin differed from that investigated by microarray analysis. the differences we detected between tdec and ndec were maintained whether or not they were exposed to a combination of pro-angiogenic factors that presumably mimic the different environmental conditions under which ec might be embedded in vivo. these results suggest that the transcriptional differences are not culture condition artifacts, but, rather, true differences that distinguish ec of different origin. despite having mainly utilized ec from ovarian cancer and from adrenal glands as the non-neoplastic counterpart, the results hold true for ec isolated from kidney cancer and skin specimens. these observations, would seem to indicate that the ec characteristics we observed are not organ-specific, but, rather, exclusive of tdec. this is in agreement with a recent report that compared ec freshly isolated from ovarian cancer and normal ovary in which several of the genes identified as distinctive of the tumor associated vasculature showed conserved expression across the ec from other tumor types, such as colon, brain and breast. cultures of ec are valuable tools for biochemical and functional analysis, but they do not reflect the physiological situation entirely. all the information should be considered critically from the standpoint of what might occur in blood vessels in vivo. to this end, in situ hybridization results confirmed adam gnpnb and prss expression associated to blood vessels in tumor tissues, but the lack of their expression in cancer cells. overall, these findings support the premise that endothelial markers identified in vitro are likely to have in vivo relevance, and bear out the validity of our experimental setting designed to reproduce situations that might occur in vivo. adam belongs to the cellular disintegrins, a family of membrane-anchored proteins that are potential regulators of cell-to-cell and cell-to-matrix interactions and for which a specific binding interaction with alphavbeta integrin has been demonstrated. our mte array results showed that adam is expressed in fetal and adult heart, and also confirmed its previously reported expression by fetal and adult brain. however, our in situ hybridization finding contradicted the latter, which implies that the expression of adam might indeed be much higher in tumor ec, including blood vessels in medulloblastoma, a highly malignant brain tumor, than in the brain. further studies, aimed to produce antibodies to adam are necessary to prove its suitability as a tumor vascular target. our results also revealed that adam was not expressed in cancer cells. this observation is consistent with the data available from refexa and other recent reports, which describe hypermethylation of the adam promoter that silences its expression in breast and pancreatic cell lines and in the corresponding primary tumors. our evidence of a high expression of adam by tdec suggests that this protein might be important for establishing contacts and promoting adhesive functions during the pathological processes leading to the formation of new blood vessels within the tumor. in addition, it is intriguing to speculate that this protein – through its ability to bind alphavbeta – could play some specialized role in the maintenance of vascular functions within the tumor. in keeping with this hypothesis is the fact that alphavbeta is expressed by ec and has been shown to be involved in tumor neo-vascularization. gpnmb is a type i membrane protein identical to the hematopoietic growth factor inducible neurokinin- type sharing similarities with neurokinin-, a receptor for tachykinins and a member of seven transmembrane g protein coupled receptors. hgfin was shown to interact with substance p, which has been reported to induce ec proliferation and increase vascular density in an in vivo model of inflammation, and these effects could be inhibited by an nk receptor antagonist. the action of sp via the nk receptor may be a direct effect, since nk receptors have previously been localized to endothelium, and both sp and selective nk agonists were shown to enhance in vitro ec proliferation. substance p in vivo also contributes to vascular permeability, plasma extravasation, and edema, all features that characterize the abnormal vasculature of tumors. our evidence of a high expression of gpnmb by tdec suggests that gpnmb may be a tumor stroma receptor for sp, involved in tumor angiogenic events and in determining the distinctive features of tumor vasculature. corroborating this premise are data suggesting that, in addition to nk receptors, other mechanisms contribute in mediating in vivo sp effects on ec. the prss gene encodes for a trypsinogen whose functions have not yet been fully elucidated. prss transcript, whose expression in cns is reported by refexa, was not detected in normal or alzheimer's-diseased brain, except for blood vessels in a brain metastasis. this finding might mean that the expression of prss is indeed characteristic of tumor ec, as seen for adam our search in the refexa and source databases revealed that prss was not expressed by tumor cells, with the exception of colon carcinoma. it seems very plausible that prss produced by tdec may contribute to tumor angiogenesis, invasion and metastasis through its peptidase activity. this notion upholds evidence that the migration and invasion of ec in the tumor tissue requires the activation of pro-enzymes and protein precursors. to this end, it is noteworthy that other peptidases were among the genes we found to be highly expressed by tumor-derived endothelium. in particular, upa is capable of initiating the process of extracellular matrix degradation through the activation of plasminogen and matrix metalloproteases; in addition, it has been shown to be required for the migration of ec during the process of angiogenesis. it is interesting that the expression of the upa receptor was also seen to be higher in hoc-ec than in ha-ec, a finding that would implicate the upa-upar system in tumor driven angiogenesis. our results show that the cell surface protease fap was also expressed at significantly higher levels by tdec than by normal ec. fap was originally reported to be an antigen recognized by f antibody in cultured fibroblasts. accordingly, its expression in our tests was found to be much higher in cultured fibroblasts and in smooth muscle cells. nonetheless, while normal adult tissues are generally fap-negative, its expression is detectable in the stroma of over 90% of carcinomas. such behavior suggested its suitability for stromal targeting and/or cancer detection and therapy, and in recent years has underpinned the development of a humanized anti-fap antibody and the implementation of clinical trials. our results uphold the notion that fap is a valid target for therapy, be it as stromal or as vascular marker. in keeping with our results, the in vivo expression of fap on blood vessels was recently demonstrated by ghersi et al. in invasive breast ductal carcinoma. many of the transcripts we found to be expressed at a high level by ec from neoplastic tissues were previously reported as being involved in tumor angiogenesis or expressed by tumor ec. in this regard, it is worth listing the various types of matrix proteins that we detected, namely: collagens alpha- type i, alpha- type iii, v, xiii and xv, glypican- laminin alpha and tenascin c. our results confirm similar findings from other laboratories and denote a remodeling of the extracellular matrix known to promote blood vessel sprouting and growth. in keeping with the experimental data, and as an example, tenascin c was shown to be expressed in vivo and to colocalize with microvessels in the stroma of non-small cell lung cancer. the expression of cell adhesion proteins was also altered in tdec. this may be explained by the fact that migrating and proliferating ec must "loosen up", that is, detach and then create new interactions. our results implicate cd and cadherins in this process, and confirm previous reports showing n-cadherin over-expression on proliferative state ec, and enhanced expression of cd on the vasculature of solid tumors compared to normal tissue. noteworthy is the finding that cd expression was up-regulated when the tdec were exposed to angiogenic growth factors, in accordance with previous results showing that fgf- and vegf stimulate cd expression in ec. interestingly, abca and abcb were also more abundantly expressed by tdec, reflecting the previously reported higher expression of p-glycoprotein in ec from glioma than from normal brain. the atp-binding cassette transporters are responsible for the efflux of chemotherapeutic agents in cancer cells. these data suggest that the multidrug resistance phenomenon in tumors may be due to both ec and tumor cells. molecules such as smad bambi and bcl are listed among the transcripts that are more highly expressed by hoc-ec than by ha-ec. smad and bambi interfere with the tgfβ pathway and may potentially block the anti-proliferative action of tgfβ. their expression may induce an enhanced proliferation of the tdec, which is one of the steps of the angiogenic process. bcl is one of the most widely recognized anti-apoptotic factors, whose upregulation enhances ec survival and intratumoral angiogenesis, thus promoting tumor growth. emerging studies suggest that the vascular and the nervous systems share the same molecular signals in their development. the involvement of netrins, semaphorins, robo/slit and vegf families in both angiogenesis and neurogenesis has been demonstrated in recent years. in keeping with these findings, our results revealed that tdec express numerous molecules that are associated with the cns or are implicated in brain differentiation and axon guidance. in particular, adora2b was identified as a novel netrin- receptor and reported to mediate axon outgrowth. in a similar way – and considering that netrin- is pro-angiogenic – our results allow speculatation that adora2b could mediate the sprouting of vessels during tumor neovascularization.  targeting angiogenesis and vasculature represents a promising option to control tumor growth and dissemination. to exploit this opportunity, however, selective targets in tumor associated vasculature need to be identified. we describe a model consisting of in vitro cultured endothelial cells isolated from human normal and neoplastic specimens that maintained their respective molecular phenotype and enabled us to determine molecular differences between tumor and normal tissue derived ec. specifically, we identified four transcripts whose expression is much higher in endothelial cells from tumors than from normal tissue. the in vivo expression in cancer-associated blood vessels was confirmed by in situ hybridization of human specimens. this work evinces some of the distinctive features of tumor-derived endothelium, and identifies potential markers that may provide the groundwork for novel therapeutic strategies. future studies are needed to clarify the functions and roles of these molecules in tumor progression and vascularization. 
our efforts focused on the molecular characterization of endothelial cells in the search for selective markers of tumor vasculature that might be helpful for the development of effective therapeutic approaches. evidence suggests that tumor vasculature expresses unique markers that distinguish it from normal vasculature. we investigated by microarray analysis the gene expression profiles of ec purified and cultured from tumor and normal tissue specimens. comparative transcriptomic analysis allowed us to determine molecular differences of tumor and normal tissue-derived endothelium and to identify novel markers that might be exploited to selectively target tumor vasculature. we found distinct transcriptional features characterizing the ec of different origin, and identified transcripts highly expressed by hoc-ec. these findings elucidate some of the molecular features of the tumor endothelium. in vivo investigation by in situ hybridization established that adam gpnmb and prss expression is localized on blood vessels of human cancer specimens. targeting tumor angiogenesis and vasculature is a promising strategy for the inhibition of tumor growth and dissemination. in vitro experiments confirmed the higher expression of the selected genes in tumor-derived endothelium with no expression in tumor cells.
 targeting tumor angiogenesis and vasculature is a promising strategy for the inhibition of tumor growth and dissemination. evidence suggests that tumor vasculature expresses unique markers that distinguish it from normal vasculature. our efforts focused on the molecular characterization of endothelial cells in the search for selective markers of tumor vasculature that might be helpful for the development of effective therapeutic approaches.  we investigated by microarray analysis the gene expression profiles of ec purified and cultured from tumor and normal tissue specimens. we found distinct transcriptional features characterizing the ec of different origin, and identified transcripts highly expressed by hoc-ec. we analyzed four of these genes, adam fap, gpnmb and prss which were not previously known to be expressed by endothelium. in vitro experiments confirmed the higher expression of the selected genes in tumor-derived endothelium with no expression in tumor cells. in vivo investigation by in situ hybridization established that adam gpnmb and prss expression is localized on blood vessels of human cancer specimens.  these findings elucidate some of the molecular features of the tumor endothelium. comparative transcriptomic analysis allowed us to determine molecular differences of tumor and normal tissue-derived endothelium and to identify novel markers that might be exploited to selectively target tumor vasculature. 