WO2007017065A2

WO2007017065A2 - Angiogenesis inhibition based upon icam 1 or junb expression levels

Info

Publication number: WO2007017065A2
Application number: PCT/EP2006/007092
Authority: WO
Inventors: Manon Van Engeland; Arjan W. Griffioen
Original assignee: Oncomethylome Sciences Sa
Priority date: 2005-07-27
Filing date: 2006-07-19
Publication date: 2007-02-15
Also published as: WO2007017065A3; EP1907856A2

Abstract

Methods for determining angiogenesis associated with a disease in a subject include the steps of, in a suitable test sample, determining expression levels of at least one gene selected from junB and ICAM 1. A statistically significant decrease in, or low level of, expression of one or both of the genes indicates the presence of angiogenesis associated with the disease in the subject. Preferably, the test sample includes endothelial cells. The method may further include determining the expression of THBSl and/or IGFBP3. Methods of determining angiogenesis in a subject may also include determining the level of acetylated histone H3 in the promote region of the IGFBP3 gene in a test sample. Pharmaceutical compositions for use in inhibiting angiogenesis include a DNA methyltransf erase inhibitor together with a pharmaceutically acceptable carrier and are in a form suitable for metronomic dosing. They may alternatively or additionally include a component allowing targeting to endothelial cells. Methods of preventing or inhibiting angiogenesis associated with a disease in a subject involve administering a therapeutically effective amount of a DNA methyltransf erase inhibitor. Treatment regimes, microarrays and screening methods are also described.

Description

ANGIOGENESIS INHIBITION

Introduction

The process of tumor angiogenesis requires intricate regulation at the molecular level . The rapid identification of novel genes involved in the generation of new vasculature is expected to contribute to the understanding of tumor angiogenesis [1-5] . Little, however, is known on how the expression of the key players of tumor angiogenesis is regulated.

Involvement of histone deacetylases (HDACs) in tumor angiogenesis by silencing tumor suppressor genes and altering VEGF signalling has been reported recently [6, 7] . In addition to histone deacetylation, methylation of histones and DNA is involved in organization of chromatin in gene promoter regions [8] . DNA methylation by DNA methyltransferases (DNMTs) in conjunction with the action of methyl -binding proteins, histone hypoacetylation and histone methylation, contributes to changes in chromatin organization with suppressive effects on transcriptional activator complexes [9] . These phenomena are essential in regulating many biological processes including genomic imprinting, X chromosome inactivation and establishment of tissue specific gene expression [10, 11] . Aberrant epigenetic regulation has been observed in cancer cells and includes alterations in DNA methylation and modifications of histones H3 and H4 [12-14] . Changes in histone modifications, like histone deacetylation and methylation, aberrant hypermethylation of CpG islands within the promoter regions of tumor suppressor genes and subsequent inappropriate gene silencing is found in virtually every type of human cancer and affects genes involved in proliferation, apoptosis, DNA repair and metastasis [15-17] .

Involvement of DNMTs in regulating EC gene expression during tumor EC growth has not been reported thus far.

The reversible nature of epigenetic gene silencing in tumor cells creates a target for therapeutic strategies in cancer [18] . DNMT- as well as HDAC inhibitors can reactivate epigenetically silenced tumor suppressor genes and decrease tumor cell growth in vitro and in vivo. Because of these characteristics, these drugs are currently being tested in clinical trials [19-21] .

Description of the invention

The present invention is based around the discovery that epigenetics is not only key in regulation of tumor growth at the level of tumor suppressor genes, but also directly in angiogenesis by interfering with the expression of "angiosuppressor" genes. Such genes, in analogy to tumor suppressor genes, may have been suppressed in endothelial cells to promote angiogenesis.

The present invention relates to methods and compositions useful for diagnosing and treating diseases associated with angiogenesis and is based around the unexpected finding of new markers associated with angiogenesis suppression and also the finding that angiogenesis can be inhibited using DNMT inhibitors .

Diagnostic Methods of the Invention Accordingly, in a first aspect the invention provides a method of diagnosing a disease associated with angiogenesis in a subject comprising, in a test sample, determining expression levels of at least one gene selected from JUNB (jun B proto oncogene) and ICAMl (intercellular adhesions molecule 1 (CD54) ) , wherein a statistically significant decrease in expression or a low level of expression indicates the presence of the disease associated with angiogenesis in the subject. Thus, the methods allow determination of angiogenesis associated with a particular disease state.

Note that all genes are listed utilising standard nomenclature approved by the human genome organisation to ensure that each symbol is unique. Accession numbers providing full sequence information and further details can be found at www.gene.ucl.ac.uk/nomenclature.

The method is most preferably an in vitro method carried out on an isolated sample. In one embodiment the method may also include the step of obtaining the sample.

Prior to the present invention, it was not known that the expression of these genes is linked to angiogenesis and thus the incidence of diseases associated with angiogenesis and is down-regulated as an indication of the relevant disease. Generally, the method is utilised in order to prognose, diagnose or monitor the progression of the disease and to allow suitable treatment if the presence of the disease is suspected. The test sample is most preferably a tissue sample, taken from the subject. In a most preferred embodiment, the sample comprises, consists essentially of, or consists of endothelial cells. However, any other suitable test sample in which expression of the novel markers of the present invention can be measured to indicate the presence of an angiogenesis related disease are included within the scope of the invention.

The decreased level of expression may be statistically significant in order to provide a reliable test for monitoring the disease associated with angiogenesis. Any method for determining whether the expression level of the gene is significantly altered may be utilised. Such methods are well known in the art and routinely employed. For example, statistical analyses may be performed using an analysis of variance test . A typical P value for use in such a method would be P values of < 0.05 when determining whether the relative expression is statistically significant. A change in expression may be deemed significant if there is at least a 10% increase or decrease for example. The test may be made more selective by making the change at least about 15%, 20%, 25%, 30%, 35%, 40% 50%, 60%, 70%, 80%, 90% or 95% for example, in order to be considered statistically significant.

In a preferred embodiment, the decreased level of expression is determined with reference to a control sample. This control sample preferably comprises, consists essentially of or consists of non-activated (that is to say non- angiogenic) endothelial cells in the subject. Alternatively, the control sample is taken from the same tissue as that under test at an earlier time point. This is particularly relevant for monitoring progression of a disease associated with angiogenesis and in order to ensure that treatment has been effective to prevent progression of the disease.

It may not, however, be essential to directly compare expression to a control sample taken from the same subject. Low levels of gene expression may be detected as compared to levels of expression determined in other subjects. A large number of samples may be tested and the results accumulated in order to provide a "baseline" or "normal" level of expression, below which a subject is considered to be at risk from, or suffering from, the relevant disease associated with angiogenesis.

Suitable additional controls may also be included to ensure that the test is working properly, such as measuring expression of a suitable reference gene in both test and control samples.

In a most preferred embodiment, the subject is a human subject. Generally, the subject will be a patient wherein a potential angiogenesis related disease is suspected and the method may be used to determine if indeed there is a potentially dangerous condition developing.

The method may be carried out by determining expression of at least one of the genes listed, both of which represent novel markers linked to angiogenesis related disease. In one embodiment, expression levels of these and other genes is measured. This may be done utilising microarray technology for example (as described in more detail in the experimental section below) , which provides a convenient method of analysing expression of multiple genes at the same time and from a single test sample. Microarray technology is well known in the art and commercial entities will prepare and supply suitable arrays as required. Preferably, all genes are assessed in the same test sample to prevent inter-sample viability.

In a preferred embodiment, the method further comprises determining the expression of THBSl (thrombospondin 1) and/or IGFBP3 (insulin-like growth factor binding protein 3) , wherein a statistically significant decrease in expression of one or both of these genes is also indicative of the presence of the disease associated with angiogenesis in the subject.

In one embodiment, the levels of gene expression are determined using RT-PCR. Reverse transcriptase polymerase chain reaction is a well known technique in the art which relies upon the enzyme reverse transcriptase to reverse transcribe mRNA to form cDNA, which can then be amplified in a standard PCR reaction. Protocols and kits for carrying out RT-PCR are extremely well known to those of skill in the art and are commercially available.

In a preferred embodiment, the RT-PCR is carried out in real time and in a quantitative manner. Real time quantitative RT-PCR has been thoroughly described in the literature (see

Gibson et al for an early example of the technique) and a variety of techniques are possible. Examples include use of Taqtnan, Molecular Beacons, LightCycler (Roche) , Scorpion and Amplifluour systems. All of these systems are commercially available and well characterised, and may allow multiplexing (that is, the determination of expression of multiple genes) which is particularly advantageous in the method of the present invention.

These techniques produce a fluorescent read-out that can be continuously monitored. Real-time techniques are advantageous because they keep the reaction in a "single tube" . This means there is no need for downstream analysis in order to obtain results, leading to more rapidly obtained results. Furthermore keeping the reaction in a "single tube" environment reduces the risk of cross contamination and allows a quantitative output from the methods of the invention. This may be particularly important in the clinical setting of the present invention.

As an example, real-time quantification of PCR reactions can be accomplished using the TaqMan^® system (Applied Biosystems) , see Holland et al ; Detection of specific polymerase chain reaction product by utilising the 5 '-3' exonuclease activity of Thermus aquaticus DNA polymerase; Proc. Natl. Acad. Sci . USA 88, 7276-7280 (1991) (67), Gelmini et al . Quantitative polymerase chain reaction-based homogeneous assay with flurogenic probes to measure C-Erbb-2 oncogene amplification. Clin. Chem. 43, 752-758 (1997) (68) and Livak et al . Towards fully automated genome wide polymorphism screening. Nat. Genet. 9, 341-342 (1995) (69), incorporated herein by reference. Taqman^® probes are widely commercially available, and the Taqman^® system (Applied Biosystems) is well known in the art. Taqman^® probes anneal between the upstream and downstream primer in a PCR reaction. They contain a 5 ^■ -fluorophore and a 3 '-quencher. During amplification the 5 '-3' exonuclease activity of the Taq polymerase cleaves the fluorophore off the probe. Since the fluorophore is no longer in close proximity to the quencher, the fluorophore will be allowed to fluoresce. The resulting fluorescence may be measured, and is in direct proportion to the amount of target sequence that is being amplified.

In the Molecular Beacon system, see Tyagi & Kramer. Molecular beacons - probes that fluoresce upon hybridization. Nat. Biotechnol . 14, 303-308 (1996) (65) and Tyagi et al . Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16, 49-53 (1998) (70) both of which are incorporated by reference herein, the beacons are hairpin-shaped probes with an internally quenched fluorophore whose fluorescence is restored when bound to its target. The loop portion acts as the probe while the stem is formed by complimentary "arm" sequences at the ends of the beacon. A fluorophore and quenching moiety are attached at opposite ends, the stem keeping each of the moieties in close proximity, causing the fluorophore to be quenched by energy transfer. When the beacon detects its target, it undergoes a conformational change forcing the stem apart, thus separating the fluorophore and quencher. This causes the energy transfer to be disrupted to restore fluorescence.

Any suitable fluorophore is included within the scope of the invention. Fluorophores that may possibly be used in the method of the invention include, by way of example, FAM, HEX™, NED™, ROX™, Texas Red™ etc. Quenchers, for example Dabcyl and TAMRA are well known quencher molecules that may^¬ be used in the method of the invention. However, the invention is not limited to these specific examples. EDANS and DABCYL form a particularly efficient fluorophore/quencher pair (65) , as do fluorescein/DABCYL (66) .

A further real-time fluorescence based system which may be incorporated in the methods of the invention is Zeneca's Scorpion system, see Detection of PCR products using self- probing amplicons and fluorescence by Whitcombe et al . Nature Biotechnology 17, 804 - 807 (01 Aug 1999) (64) . This reference is incorporated into the application in its entirety. The method is based on a primer with a tail attached to its 5' end by a linker that prevents copying of the 5' extension. The probe element is designed so that it hybridizes to its target only when the target site has been incorporated into the same molecule by extension of the tailed primer. This method produces a rapid and reliable signal, because probe-target binding is kinetically favoured over intrastrand secondary structures .

Amplifluour primers (as described in US Patent 5,866,336 to Nazarenko and WO98/02449 both of which are incorporated herein by reference) rely upon a similar principle to molecular beacons. However, in this case, the hairpin structure is part of the amplification primer itself. The primer binds to a nucleic acid strand and directs synthesis and thus becomes part of the amplification product. When the complementary strand is synthesised amplification occurs through the hairpin structure. This separates the fluorophore and quencher molecules, thus leading to generation of fluorescence as amplification proceeds.

The disease which is diagnosed may be any disease which is dependent upon angiogenesis for its progression. In one embodiment, the disease associated with angiogenesis which is diagnosed according to the methods of the invention is selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.

All cancers in which tumour angiogenesis is relevant for and/or contributes to tumour growth are included within the scope of the invention, since reduced expression of the genes listed is indicative of activated endothelial cells (EC) leading to angiogenesis. Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.

It has been surprisingly shown herein that promoter hypermethylation is not responsible for silencing of the genes listed above which are down regulated in diseases linked with angiogenesis. In contrast, studies carried out by the inventors (see the experimental section below) have shown that inactivation of the genes in activated EC occurs in correlation with promoter histone H3 acetylation patterns. Moreover, specific DNA methyltransferase inhibitors and histone deacetylase inhibitors can induce re- expression of the genes which is also in accordance with the histone H3 acetylation patterns.

Accordingly, the invention provides, in a second aspect, a method of diagnosing a disease associated with angiogenesis in a subject comprising, in a test sample, determining the histone acetylation patterns, preferably the histone H3 acetylation patterns, of at least one gene selected from IGFBP3, THBSl, JUNB and ICAMl, wherein a statistically significant decrease in, or low level of, acetylation of at least one gene indicates the presence of a disease associated with angiogenesis . Thus, by determining the level of acetylated histone H3 in the promoter region of the relevant gene, the presence of angiogenesis associated with a disease may be determined.

As mentioned above, all genes are listed utilising standard nomenclature approved by the human genome organisation to ensure that each symbol is unique. Accession numbers providing full sequence information and further details can be found at www.gene.ucl.ac.uk/nomenclature.

Prior to the present invention, it was not known that histone acetylation patterns are responsible for controlling the expression of these genes, which in turn is linked to the incidence of diseases associated with angiogenesis and that acetylation is decreased as an indication of the relevant disease. Generally, the method is utilised in order to prognose, diagnose or monitor the progression of the disease and to allow suitable treatment if the presence of the disease is suspected. The test sample is most preferably a tissue sample, taken from the subject. In a most preferred embodiment, the sample comprises endothelial cells. However, any other suitable test sample in which histone H3 acetylation patterns of the novel markers of the present invention can be measured to indicate the presence of an angiogenesis related disease are included within the scope of the invention.

The decreased level of histone acetylation may be statistically significant in order to provide a reliable test for monitoring the disease associated with angiogenesis. Any method for determining whether the level of histone acetylation of the gene is significantly altered may be utilised. Such methods are well known in the art and routinely employed. For example, statistical analyses may be performed using an analysis of variance test . A typical P value for use in such a method would be P values of < 0.05 when determining whether the relative level of histone acetylation is statistically significant. A change in acetylation may be deemed significant if there is at least a 10% increase or decrease for example. The test may be made more selective by making the change at least 15%, 20%, 25%, 30%, 35%, 40% or 50%, for example, in order to be considered statistically significant.

In a preferred embodiment, the decreased level of histone H3 acetylation is determined with reference to a control sample. This control sample is preferably taken from non- activated (that is to say non-angiogenic) endothelial cells in the subject. Thus, histone H3 acetylation is compared to histone H3 acetylation of the same genes in a control endothelial cell sample.

Alternatively, the control sample is taken from the same tissue as that under test at an earlier time point. This is particularly relevant for monitoring progression of a disease associated with angiogenesis and in order to ensure that treatment has been effective to prevent progression of the disease.

It may not, however, be essential to directly compare histone (H3) acetylation to a control sample taken from the same subject. Low levels of histone (H3) acetylation may be detected as compared to levels of histone (H3) acetylation determined in other subjects. A large number of samples may be tested and the results accumulated in order to provide a "baseline" or "normal" level of histone (H3) acetylation, below which a subject is considered to be at risk from or suffering from the relevant disease associated with angiogenesis . Suitable additional controls may also be included to ensure that the test is working properly, such as measuring histone H3 acetylation levels of a suitable reference gene in both test and control samples .

In a most preferred embodiment, the subject is a human subject. Generally the subject will be a patient wherein a potential angiogenesis related disease is suspected and the method may be used to determine if indeed there is a potentially dangerous condition developing.

In a preferred embodiment, the histone H3 acetylation patterns of at least IGFBP3 is determined. In a further preferred embodiment, the promoter regions of the genes is assessed for histone H3 acetylation patterns. The promoter region is typically most important for determining expression levels of the relevant genes, since changes in acetylation patterns determine the accessibility of the transcription initiation sites for transcription factors and other elements of the transcription machinery.

In one embodiment, the acetylation patterns of the genes are assessed using chromatin immunoprecipitation (ChIP) . Chromatin immunoprecipitation is a well known technique in the art which relies upon cross-linking of the binding protein to the DNA, followed by isolation, shearing of the DNA, antibody detection and isolation by precipitation. The isolated DNA is then released from the binding protein by reversing the cross-linking and is amplified by PCR to determine where the binding protein was bound. (Metivier, R. et al . , Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment of cofactors on a natural target promoter, Cell 2003, 115(6) P751-63). Thus, most preferably, the levels of acetylated histone H3 in the promoter region of the IGFBP3 gene are determined by ChIP.

The disease which is diagnosed may be any disease which is dependent upon angiogenesis for its progression. In one embodiment, the disease associated with angiogenesis which is diagnosed according to the methods of the invention is selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis. All cancers in which tumour angiogenesis is relevant for tumour growth are included within the scope of the invention, since reduced histone H3 acetylation, leading to reduced expression, of the genes listed is indicative of activated endothelial cells (EC) leading to angiogenesis. Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.

Compositions of the invention It has also been surprisingly discovered that DNA methyltransferase inhibitors can have an indirect effect on tumour growth by inhibiting angiogenesis. Previously, the known activity of DNA methyltransferase inhibitors has been directly on tumour cells themselves; by reducing levels of methylation of certain tumour suppressor genes their expression is increased which thus prevents development of the tumour. Some of these drugs are now in clinical trial (19-21) .

The newly discovered effect of DNA methyltransferase inhibitors allows additional therapeutic routes to be opened, by allowing both direct and indirect targeting of tumours, with indirect therapy being provided by preventing activation of EC. In addition, DNA methyltransferase inhibitors may now be used to treat a whole range of angiogenesis related diseases (that is, diseases which rely upon angiogenesis for their progression) . These include, for example, cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.

All cancers in which tumour angiogenesis is relevant for tumour growth are included within the scope of the invention, since treatment with suitable DNA methyltransferase inhibitors may be used to suppress angiogenesis and thereby treat the disease. Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.

Accordingly, in a third aspect, the invention provides a pharmaceutical composition for use in inhibiting angiogenesis comprising a DNA methyltransferase inhibitor together with a pharmaceutically acceptable carrier.

The effect of the composition is to increase the level of gene expression of the genes whose expression is reduced in angiogenesis related diseases to the levels of gene expression found in normal endothelial cells. However, it may be that any increase in expression will be beneficial for treatment of the disease. The DNA methyltransferase inhibitor is provided in a therapeutically relevant amount to ensure that a controlled increase in gene expression is achieved.

The effects may be achieved by, for example, inhibiting EC growth (proliferation) and/or EC migration and/or EC sprouting .

The DNA methyltransferase inhibitor may, in one embodiment, be one which reduces expression of DNMT genes, such as suitable antisense molecules, or siRNA molecules which mediate RNAi for example. The design of a suitable siRNA molecule is within the capability of the skilled person and suitable molecules can be made to order by commercial entities (see for example, www.ambion.com) . Preferably, the DNA methyltransferase gene is (human) DNMTl.

Alternatively, the agent may be a direct inhibitor of DNMTs. Examples include modified nucleotides such as phosphorothioate modified oligonucleotides (fig 6 of ref 63) and nucleosides and nucleotides such as cytidine analogues.

Suitable examples of cytidine analogues include 5- azacytidine, 5-aza-2 ' -deoxycytidine, 5-fluouro-2 ' - deoxycytidine, pseudoisocytidine, 5 , 6-dihydro-5-azacytidine, l-jS-D-arabinofuranosyl-5-azacytosine (known as fazabarine)

(see figure 4 of ref (63)).

In another embodiment, the DNA methyltransferase inhibitor comprises Decitabine. Full details of this drug can be found at www.supergen.com for example.

Additional DNMT inhibitors include S-Adenosyl -Methionine (SAM) related compounds like ethyl group donors such as L- ethionine and non-alkylating agents such as S-adenosyl- homocysteine (SAH) , sinefungin, (S) -6-methyl-6-deaminosine fungin, 6-deaminosinefungin, N4 -adenosyl -N4-methyl -2 , 4- diaminobutanoic acid, 5 ' -methylthio-5 ' -deoxyadenosine

(MTA) and 5 ' -amino-5 ^■ -deoxyadenosine (ref 63).

Further agents which may alter DNA methylation and which may, therefore, be useful in the present compositions include organohalogenated compounds such as chloroform etc, procianamide, intercalating agents such as mitomycin C, 4- aminobiphenyl etc, inorganic salts of arsenic and selenium and antibiotics such as kanamycin, hygromycin and cefotaxim (63) . However, any suitable DNA methyltransferase inhibitor which is capable of increasing the expression of at least one of the genes listed above, and thus can contribute to the treatment of a disease associated with angiogenesis, is included within the scope of the invention.

In one embodiment, the pharmaceutical composition additionally comprises a histone deacetylase inhibitor. Such an agent may complement the effect of the DNA methyltransferase inhibitor, especially in view of the importance of histone H3 acetylation for determining expression levels of the genes reported herein.

In addition to effects on EC growth and EC migration, HDAC inhibitors are also shown to have a profound effect on EC apoptosis (see Experimental section for further details) .

In a preferred embodiment, the histone deacetylase (HDAC) inhibitor comprises at least one of trichostatin A (TSA) , suberoyl hydroxamic acid (SBHA), 6- (3- chlorophenylureido) caproic hydroxamic acid (3-Cl-UCHA), m- carboxycinnamic acid bishydroxylamide (CBHA) , suberoylanilide hydroxamic acid (SAHA) , azelaic bishydroxamic acid (ABHA) , pyroxamide, scriptaid, aromatic sulfonamides bearing a hydroxamic acid group, oxamflatin, trapoxin, cyclic-hydroxamic-acid containing peptides, FR901228, MS-275, MGCD0103 (see www.methylgene.com), short- chain fatty acids and N-acetyldinaline (63) .

In a pharmaceutical composition incorporating a suitable DNA methyltransferase inhibitor and potentially additionally a histone deacetylase, preferred compositions include pharmaceutically acceptable carriers including, for example, non-toxic salts, sterile water or the like. A suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration. The carrier may also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, somobility or the like. Pharmaceutical compositions which permit sustained or delayed release following administration may also be used.

One important distinguishing feature of anti-angiogenesis treatments, as compared to, for example, chemotherapeutic agents which treat tumours directly, is that effects are seen with lower levels of active agent (in this case DNA methyltransferase inhibitor potentially with a histone deacetylase inhibitor in addition) . Thus, for demethylation in cancer cells, typical concentrations of active ingredients are of the micromolar scale in order to achieve optimal effects. As shown in the experimental section below, anti-angiogenic effects can be seen at concentrations of active ingredient which are on the nanomolar scale.

Additionally, metronomic dosing has been shown to be effective for inhibiting angiogenesis related diseases. Thus, in contrast to traditional therapy, lower amounts of the pharmaceutical composition of the invention may be utilised in a form which can be administered in small doses on a continuous or regular basis. This has the beneficial effect of reducing side effects such as neurotoxicity and damage to proliferating cells in healthy tissues, which can pose a serious constraint on the use of chemotherapy.

There is the added beneficial effect that efficacy can be maintained without a requirement for episodic treatment and recovery intervals before treatment can be continued.

Metronomic dosing is particularly useful for treating angiogenesis related diseases due to the genetic stability of the endothelial cells that form blood vessels. It is well established that tumour-associated endothelial cells proliferate during chronic angiogenesis in tumours, albeit at lower frequencies than the tumour cells themselves.

Apparently because of their lower rate of cell division, replication of these endothelial cells is only weakly disrupted by the episodic regimens of standard chemotherapeutic protocols. In contrast metronomic dosing of a suitable pharmaceutical composition according to the invention may lead to more positive effects by preventing activation of endothelial cells.

The pharmaceutical compositions of the invention may be used together with other standard chemotherapeutic treatments which target tumour cells directly. Non limiting examples include paclitaxel, cyclaphosphomide and 5-tumor-uracil (5- FU) and pharmaceutically acceptable derivatives thereof including salts, etc.

Thus, in one embodiment, the pharmaceutical composition of the invention is in a form suitable for metronomic dosing. Preferably, the composition comprises a component allowing targeting to endothelial cells. In one embodiment, this is achieved by utilizing RGD peptides in the compositions of the invention (see Pharmacokinetics and biodistribution of RGD-targeted doxorubicin-loaded nanoparticles in tumor- bearing mice. Bibby DC, Talmadge JE, Dalai MK, Kurz SG, Chytil KM, Barry SE, Shand DG, Steiert M. , Int J Pharm. 2005 Apr 11;293 (1-2) :281-90) .

As mentioned above, angiogenesis inhibitors can be administered, and are effective, at lower concentrations than other drugs that target tumour cells directly. Accordingly, in one embodiment, a composition according to the invention is provided wherein the concentration of the DNA methyltransferase inhibitor is no more than about 1 μM. In further embodiments, the concentration of the DNA methyltransferase inhibitor in the composition is no more than about 500 nM, preferably no more than about 250 nM, no more than about 100 nM, no more than about 50 nM, no more than about 40 nM, 30 nM, 20 nM, 10 nM, 5nM, etc.

The therapeutic agent may, for example, be encapsulated and/or combined with suitable carriers in solid dosage forms for oral administration which would be well known to those of skill in the art or alternatively with suitable carriers for administration in an aerosol spray. Examples of oral dosage forms include tablets, capsules and liquids.

Alternatively, the therapeutic agent may be administered parenterally. Specific examples include intradermal injection, subcutaneous injection (which may advantageously give slower absorption of the therapeutic agent) , intramuscular injection (which can provide more rapid absorption) , intravenous delivery (meaning the drug does not need to be absorbed into the blood stream from elsewhere) , sublingual delivery (for example by dissolving of a tablet under the tongue or by a sublingual spray) , rectal delivery, vaginal delivery, topical delivery, transdermal delivery and inhalation.

In a pharmaceutical composition incorporating a suitable DNA methyltransferase inhibitor and possibly also a histone deacetylase, preferred compositions include pharmaceutically acceptable carriers including, for example, non-toxic salts, sterile water or the like. A suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration. The carrier may also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, somobility or the like. Pharmaceutical compositions which permit sustained or delayed release following administration may also be used.

Furthermore, as would be appreciated by the skilled practitioner, the specific dosage regime may be calculated according to the body surface area of the patient or the volume of body space to be occupied, dependent on the particular route of administration to be used. The amount of the composition actually administered will, however, be determined by a medical practitioner based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the age, weight and response of the individual .

Methods of Treatment In a further related aspect, the invention provides a method of treating a disease associated with angiogenesis and/or preventing, restricting, inhibiting or decreasing any of EC growth and/or EC proliferation and/or EC sprouting and/or EC migration in a subject comprising administering a therapeutically effective amount of a DNA methyltransferase inhibitor to the subject in order to treat said disease. Thus, the methods of the invention involve preventing or inhibiting angiogenesis associated with a disease.

In a still further aspect, the invention also provides a method of treating a disease associated with angiogenesis and/or preventing, restricting, inhibiting or decreasing any of EC growth and/or EC proliferation and/or EC sprouting and/or EC migration in a subject comprising administering a therapeutically effective amount of a DNA methyltransferase inhibitor to the subject such that expression of at least one gene selected from JUNB, ICAMl, THBSl and IGFBP3 is increased. Thus, the methods of the invention involve preventing or inhibiting angiogenesis associated with a disease by increasing expression of the relevant gene(s) .

Preferably, the subject is a human. Generally, the subject will be one who has been diagnosed with a disease associated with angiogenesis; for example using the methods according to the present invention. In a preferred embodiment, the effect of the treatment is to increase the level of gene expression to the levels of gene expression found in normal non-activated (non-angiogenic) endothelial cells. However, it may be that any increase in expression will be beneficial for treatment of the angiogenesis related disease. Of course, the DNA methyltransferase inhibitor or histone deacetylase inhibitor is provided in a therapeutically relevant amount to ensure that a controlled increase in gene expression is achieved.

In a most preferred embodiment, the compositions according to the invention (see "Compositions of the Invention" section above) are used in these methods. Therefore, the discussion above relating to the pharmaceutical compositions also applies mutatis mutandis to these aspects of the invention. For example, the method may comprise, consist essentially of or consist of metronomic dosing of the relevant composition.

As aforementioned, the disease may be any disease which depends upon angiogenesis for its progression. In preferred embodiments, the disease associated with angiogenesis which is treated according to the invention is selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.

All cancers in which tumour angiogenesis is relevant for tumour growth are included within the scope of the invention, since the anti -angiogenic effects of the DNA methyltransferase inhibitor should have a beneficial effect for the subject. Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.

Similarly, the invention also provides for use of a DNA methyltransferase inhibitor in the manufacture of a medicament for treating a disease associated with angiogenesis in a subject. Thus, the medicament is useful for preventing or inhibiting angiogenesis associated with a disease .

Additionally, the invention provides for the use of a DNA methyltransferase inhibitor and/or a histone deacetylase inhibitor in the manufacture of a medicament for treating a disease associated with angiogenesis in a subject by increasing expression of at least one gene selected from JUNB, ICAMl, THBSl and IGFBP3. Thus, the medicament is useful for preventing or inhibiting angiogenesis associated with a disease by increasing expression of the relevant gene (s) .

The discussion of the methods of treatment according to the invention apply mutatis mutandis to the use aspects of the invention.

In a preferred embodiment, the medicament causes the level of gene expression to be increased to the levels of gene expression found in non activated (non-angiogenic) endothelial cells.

Most preferably, the medicament comprises a pharmaceutical composition of the invention (see the relevant section above) . For example, the medicament may be designed to be administered by metronomic dosing.

The disease may be any disease which depends upon angiogenesis for its progression. In separate embodiments, the disease associated with angiogenesis is selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.

All cancers in which tumour angiogenesis is relevant for tumour growth are included within the scope of the invention, since the anti-angiogenic effects of the DNA methyltransferase inhibitor (and possibly histone deacetylase inhibitor) should have a beneficial effect for the subject. Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.

The advantages of metronomic dosing are described in more detail above, with respect to treatment of angiogenesis related diseases. In accordance with this, the invention further provides a treatment regime for treating a disease associated with angiogenesis comprising metronomic dosing of a pharmaceutical composition according to the invention.

In separate embodiments, the treatment regime is used to treat a disease associated with angiogenesis which is selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis, although any disease which depends upon angiogenesis for its progression may be treated. All cancers in which tumour angiogenesis is relevant for tumour growth are included within the scope of the invention, since the anti-angiogenic effects of the DNA methyltransferase inhibitor (and possibly histone deacetylase inhibitor) should have a beneficial effect for the subject. Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.

Microarrays

Since there are multiple novel markers whose reduced expression has been found in the present invention to be indicative of a disease associated with angiogenesis, there is benefit in being able to screen for expression of this panel of genes in a simple and rapid manner. Accordingly, the invention provides, in a further aspect, a microarray for use in the methods of the invention which involve determining levels of expression of genes, comprising probes immobilised on a solid support hybridizing with transcripts or parts thereof of at least one gene selected from JUNB,

ICAMl, THBSl and IGFBP3.

Microarrays and their means of manufacture are well known and can be manufactured to order by commercial entities such as Affymetrix, for example.

The probes are the sequences which are immobilized onto the array, by known methods, and which represent selected sequences from the genes of interest, in this case the novel markers whose reduced expression has been found in the present invention to be indicative of a disease associated with angiogenesis. Probe selection and array design lie at the heart of the reliability, sensitivity, specificity, and versatility of the microarrays of the invention. The methods for selecting suitable probes would be readily apparent for one of skill in the art and may involve optimization using data collected from multiple databases, bioinformatics tools, and experiment -trained computer models.

The key elements of probe selection and design are common to the production of all arrays, regardless of their intended application and as such would be well known to one of skill in the art. Strategies to optimize probe hybridization, for example, are invariably included in the process of probe selection. Hybridization under particular pH, salt, and temperature conditions may be optimized by taking into account melting temperatures and using empirical rules that correlate with desired hybridization behaviours.

The GeneChip arrays produced by Affymetrix involve a Perfect Match/Mismatch probe strategy. For each probe designed to be perfectly complementary to a target sequence, a partner probe is generated that is identical except for a single base mismatch in its centre. These probe pairs, called the

"Perfect Match probe (PM)" and the "Mismatch probe (MM)", allow the quantitation and subtraction of signals caused by non-specific cross-hybridization. The difference in hybridization signals between the partners, as well as their intensity ratios, serve as indicators of specific target abundance. Such an array design may be applicable to, and incorporated into, the arrays of the present invention. In order to ensure specificity of the probes in terms of accurately representing the genes listed above, the microarray preferably comprises at least 10, 20, 30 or 40 probes representing each gene on the array. However, other numbers of probes may be utilised provided that the expression of each gene which is selected to form part of the array can be accurately and specifically measured.

In a preferred embodiment, the array includes probes which represent each and every one of the genes listed (i.e. JUNB,

ICAMl, THBSl and IGFBP3) . However, this may not be necessary in order for accurate diagnosis. Probes representing only 1, 2 or 3 of the genes may be utilised in the array. Accordingly, in one preferred embodiment, the microarray comprises probes representing transcripts of at least the ICAMl and/or JUNB genes.

Each probe is preferably at least about 20, 30 or 35 nucleotides in length such that a probe of sufficient length to ensure sensitivity and specificity of hybridization is provided. However, any length of probe may be utilised within the scope of the invention, provided that accurate results are achieved in terms of detecting expression of the genes which represent novel markers whose reduced expression has been found in the present invention to be indicative of a disease associated with angiogenesis . Possible lengths for the probes include at least about 10 nucleotides and up to about 250 nucleotides and preferably between about 20 and about 50 nucleotides.

Compound Screening Methods By identifying the novel markers whose reduced expression has been found in the present invention to be indicative of a disease associated with angiogenesis, new treatments for these diseases may be discovered. Thus, the expression pattern of these genes may be used as a research tool to identify new compounds and possible therapeutic agents etc. which may be used to treat, prevent or control diseases associated with angiogenesis.

Accordingly, in a still further aspect, the invention provides a method of identifying a compound capable of treating or reducing the effects or progression of a disease associated with angiogenesis comprising the steps of;

(a) administering the compound to an experimental non-human animal having said angiogenesis associated disease;

(b) generating an expression profile of a panel of genes comprising at least one of JUNB, ICAMl, THBSl and IGFBP3

(c) comparing the expression profile obtained in (b) with the expression profile of a corresponding panel of genes expressed in a control endothelial cell sample from a subject not having the angiogenesis associated disease; wherein a positive correlation of the expression profiles is indicative that the compound is capable of treating or reducing the effects or progression of the angiogenesis associated disease. Thus, the methods are useful for identifying compounds capable of preventing or inhibiting angiogenesis associated with a disease.

The non-human animal may be any suitable animal, such as a mouse, rat or monkey for example. The experimental animal may be sacrificed following the testing. In one embodiment, the control sample is taken from an experimental non-human animal which does not have said angiogenesis related disease. The non-human animal providing the control sample will be the same species as the test non-human animal, and preferably closely related.

Preferably, the control sample is taken from non-activated (non-angiogenic) endothelial cells in the same non-human animal .

Similarly, the invention also provides an in vitro method of identifying a compound capable of treating or reducing the effects or progression of a disease associated with angiogenesis comprising the steps of; (a) administering the compound to an endothelial cell sample taken from a subject having said angiogenesis associated disease;

(b) generating an expression profile of a panel of genes comprising at least one of JUNB, ICAMl, THBSl and IGFBP3 ; (c) comparing the expression profile obtained in (b) with the expression profile of a corresponding panel of genes expressed in a control endothelial cell sample from a subject not having the angiogenesis associated disease; wherein a positive correlation of the expression profiles is indicative that the compound is capable of treating or reducing the effects or progression of the angiogenesis associated disease. Thus, the methods are useful for identifying compounds capable of preventing or inhibiting angiogenesis associated with a disease.

For both of these compound screening methods, the disease associated with angiogenesis may be any disease which depends upon angiogenesis for its progression and is preferably selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis .

All cancers in which tumour angiogenesis is relevant for tumour growth are included within the scope of the invention, since discovering compounds with anti-angiogenic effects should lead to new treatments for these diseases. Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.

The invention also provides the compounds identified by the methods described above. These compounds may be formulated into suitable pharmaceutical compositions, including suitable carriers, for administration to a patient in need thereof. Details of suitable pharmaceutical compositions are provided above, and it is envisaged that some of the specific DNMT (and HDAC) inhibitors may prove to act positively in the screening methods according to this aspect of the invention.

In addition, there is also provided a method of treating a disease associated with angiogenesis comprising administering to a subject in need thereof a therapeutically effective amount of a compound which has been identified using the screening methods of the invention. The invention will now be further described, by way of example, with reference to the following experimental section and figures in which:

Fig. 1 shows that DAC, zebularine and TSA inhibit tumor growth and angiogenesis in mice. a, Tumor growth inhibition of B16F10 mouse melanoma tumors in C57BL/6 mice by DAC, zebularine and TSA treatment . Data are expressed as mean tumor volume (mm3 ± SEM) , *p<0.0001. b, Cryosections of tumors from control mice and treated mice stained with CD31 antibody for microvessel density assessment (scale bar = 100 μm) . c, Quantification of microvessel density as mean number of vessels per mm2 (± SEM, *p<0.0001); d, Tumor growth curves of human LS174T colon carcinoma in athymic mice either or not treated daily with zebularine (*p<0.007) .

Fig. 2 represents results showing that DAC, zebularine and TSA inhibit EC growth characteristics. a-c, Dose-response curves of DAC, zebularine and TSA on growth factor- induced and spontaneous proliferation of HUVEC and b.END5 endothelioma cells respectively, after 72 hours of treatment . d, Kinetic analysis of the response of tumor- conditioned HUVEC after 24, 48 and 72 hours of treatment with DAC. Data are expressed as mean relative proliferation compared to untreated cultures values (±SEM) of 4 independent triplicate experiments (*p<0.037, **p<0.005, ***p<0.001) . e-g, Dose-response curves of DAC (e) , zebularine (f) and TSA (g) on apoptosis (solid symbols) and total cell death (open symbols) of growth factor- stimulated HUVEC. HUVEC cultured in the presence of 1% serum was used as a positive control for apoptosis (τapoptosis, V total cell death) . Data are represented as mean values (± SEM) of 3 (DAC, zebularine) or 6 (TSA) independent triplicate experiments (*p<0.05, **p<0.006, ***p<0.001).

Fig. 3 presents the effects of DAC, zebularine and TSA on

EC migration and angiogenesis in vitro. a, b, Relative wound width of dose ranges of DAC (a) and TSA (b) treated cultures as compared to untreated cultures are shown. Data are represented as mean values (± SEM) of 5 independent experiments (*p<0.05, **p<0.01,

***p<0.001) . c, Sprouting of BCEs cultured on gelatin-coated

Cytodex-3 beads into a collagen matrix. Sprout formation was induced by bFGF and VEGF (control) . d-f, Quantification of results plotted as mean values

(± SEM) of relative sprouting compared to untreated BCE from 3 independent experiments (*p<0.037, #p<0.046).

Fig. 4 shows that DAC, zebularine and TSA inhibit angiogenesis in a human ex vivo model and in the chick chorioallantoic membrane (CAM) in vivo model. a, Photographs of human vessel rings (diameter 1 mm) embedded in a collagen matrix after 13 days of culture in medium containing bFGF and VEGF, in absence or presence of DAC, zebularine or TSA . In the right panel a higher magnification of the sprouts is shown (scale bar = 100 μm) . b. Average sprout lengths (± SEM) out of 3 experiments (*p<0.046). c. CAMs treated daily with saline (control), DAC (5 mM) , zebularine or TSA from day 10 to day 13. d. Mean vessel density values (± SEM) of CAMs treated with DAC (5 μM, n=9; 5 mM n=7) , zebularine (n=4) or TSA (n=5) , *p<0.023, **p<0.001, ***p<0.0001.

Fig. 5 shows DNMTl activity, global 5 -methylcytosine content, and chromatin modifications of the THBSl, JunB, ICAMl and IGFBP3 promoters in ECs. a. DNMTl activity in quiescent HUVEC (HUVEC-), activated HUVEC (HUVEC+) , activated HUVEC treated with DAC, zebularine (zeb.) or TSA, B16F10 and HCT116. Results are represented as mean values (± SEM) of 3 independent experiments (*p<0.05 versus HUVEC-, #p<0.05 versus HUVEC+) . b. Measurement of 5-methylcytosine content as a percentage of the total cytosine pool . c. Relative mRNA expression of THBSl, JunB, ICAMl and IGFBP3 measured by semi-quantitative real-time RT-PCR in quiescent HUVEC (H-), activated HUVEC (H+), and activated HUVEC treated with DAC or TSA. Results are plotted as mean values (± SEM) of relative mRNA expression compared to H- from 3 independent experiments (*p<0.04 vs. H-, **p<0.02 vs. H-, #p<0.05 vs. H+, ##p<0.03 vs. H+) . d. Genomic bisulfite sequencing of 5'CpG islands of THBSl, JunB, ICAMl and IGFBP3. In each clone, the methylation status of each CpG dinucleotide is represented as a box. If a box is shaded, the position is methylated, if white, it is not. Numbers indicate the position relative to transcriptional start site. e. Chromatin immunoprecipitation (ChIP) of the IGFBP3 CpG island with anti-acetylated histone H3 antibody. The numbers on the right indicate the location of the DNA fragments amplified by PCR done on the DNA recovered from ChIP experiments and correspond with the horizontal bars below the schematic IGFBP3 promoter CpG island in (d) . For each primer set, PCR was performed on non- immunoprecipitated (input) DNA, immunoprecipitated DNA (Ac-H3 Ab) and a no-antibody (no Ab) control DNA.

Fig. 6 is a model of the anti -tumor effects of DNMT- and HDAC inhibitors in vivo.

1. Inhibition of tumor cell growth by reactivation of epigenetically silenced tumor suppressor genes. 2. Release of transcriptional repression of angiogenesis inhibiting tumor suppressor genes in the tumor cells might result in indirect angiostatic effects.

3. DNMT- and HDAC inhibitors directly decrease EC growth and angiogenesis, thereby exhibiting direct angiostatic effects, e.g. by reactivation of epigenetically silenced "angiosuppressor" genes.

Experimental Section

Tumor growth is regulated by epigenetic events. Here, we show that the DNA methyltransferase (DNMT) inhibitors 5- aza-2 ' -deoxycytidine (DAC) and zebularine, as well as the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) markedly inhibit angiogenesis as well. We demonstrate that these compounds are anti-proliferative for activated endothelial cells (EC) , and inhibit angiogenesis in vitro, ex vivo and in vivo. Both DAC and TSA induced reexpression of the angiogenesis inhibiting genes THBSl, JUNB, ICAMl and IGFBP3, which are suppressed in activated EC. Although DNMT activity and overall genomic methylation levels were increased in activated EC, promoter hypermethylation did not seem to be responsible for silencing of these genes.

Inactivation of IGFBP3 in activated EC and reexpression by DAC and TSA occurred in conjunction with promoter histone H3 acetylation patterns. Our data indicate that growth inhibitory genes are epigenetically silenced in activated EC and that DNMT inhibitors decrease EC growth by reactivating these genes through methylation-independent effects. We propose a dual action of DNMT- and HDAC inhibitors on tumor angiogenesis, both indirectly via tumor cells and directly on tumor EC.

Results

DAC, zebularine and TSA inhibit tumor growth and angiogenesis in vivo

To investigate the role of epigenetics in tumor angiogenesis in vivo, B16F10 tumor bearing mice were treated with the DNA methyltransferase (DNMT) inhibitors 5-aza-2 ' -deoxycytidine (DAC) or zebularine, or with the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) . On day 9 after inoculation, when established tumors of approximately 100 mm3 were visible, treatment was started.

DAC treatment (10 mg/kg, i.p., daily) resulted in a significant abrogation of tumor growth (p<0.0001), causing almost full stasis over the treatment period (Fig. IA) . To determine whether the inhibition of tumor growth was associated with suppressed angiogenesis, microvessel density was determined immunohistochemically by staining for CD31 (Fig. IB, C). Microvessel density was significantly lower in tumors of treated mice (47% inhibition compared to untreated control tumors, p<0.0001) . The inhibitory activity of DNMT suppression on angiogenesis and tumor growth was confirmed by treating B16F10 tumor bearing mice with the DAC-analogue zebularine, a compound recently found to have similar functional activity but with a lower toxicity profile [22, 23] . Similar results for both tumor growth and microvessel density were observed for zebularine (1000 mg/kg, i.p., daily) (Fig. 1) . Also in the xenograft tumor model of human LS174T colon carcinoma in athymic mice, treatment with zebularine resulted in almost 50% inhibited tumor growth as compared to tumor growth in control mice (p<0.007, Fig. ID) . A similar reduction in microvessel density was observed in these tumors (data not shown) .

Treatment with the HDAC inhibitor TSA was used in the same model. TSA (1 mg/kg, i.p., daily) also significantly inhibited tumor growth (p<0.0001) by approximately 60%, as compared to tumors in untreated mice. Similar to DAC and zebularine, TSA significantly reduced microvessel density (52% inhibition, p<0.0001), as compared to untreated tumors (Fig. 1C) .

DAC, zebularine and TSA inhibit growth characteristics of

EC

To explore whether DNMT- and HDAC inhibitors have direct effects on EC growth, DAC, zebularine and TSA were tested for their ability to inhibit proliferation of cultured HUVEC using the [3H] -thymidine incorporation assay. In these assays, tumor conditions were mimicked by culture in the presence of bFGF, VEGF and tumor cell line conditioned medium. DAC exhibited a concentration dependent inhibition of HUVEC proliferation. While the dose for half-maximal response (ED50) of DAC was about 100 nM, maximal inhibition of proliferation (67%, p<0.001) was observed at 1 μM (Fig. 2A) . Similarly, zebularine also inhibited HUVEC proliferation in a concentration dependent way (Fig. 2B) . HUVEC proliferation in reaction to treatment with the HDAC inhibitor TSA elicited a bi-phasic response. At 10 nM, TSA displayed a slight but significant stimulatory effect on proliferation (9% upregulation, p<0.037), whereas at concentrations above 100 nM, TSA exhibited inhibitory effects (Fig. 2C) , reaching an ED50 at 200 nM and a maximal inhibition of more than 95% at 1 μM.

Kinetic studies on the response of EC to DAC revealed that a 72 -hour exposure resulted in stronger responses as compared with treatment for 48 and 24 hours (Fig. 2D) . In contrast, for TSA, similar inhibitory effects were observed after treatment for 72, 48 and 24 hours (data not shown) .

Similar anti-proliferative effects of DAC, zebularine and TSA were obtained using HUVEC stimulated with bFGF or VEGF alone, as well as in the human microvascular endothelial cell line (HMEC) (data not shown) . In addition, DNMT- and HDAC inhibitors had similar growth-inhibitory potency against mouse EC (b.END5 brain endothelioma cells, Fig. 2A-C) .

To determine whether inhibition of EC growth was caused by inducing cell death, the percentage of dying cells in general, as well as the percentage of cells undergoing apoptosis was quantified by flow cytometry [24] after treatment for 3 days with or without DAC, zebularine or TSA. At growth inhibitory concentrations, the DNMT inhibitor DAC did not significantly affect EC apoptosis or total cell death, as measured by the percentage of cells with subdiploid DNA content (Fig. 2E) . Similar results were observed for zebularine, although a small percentage of EC underwent apoptosis at the highest concentration tested (p<0.05, Fig. 2F) . In contrast to the DNMT inhibitors, the HDAC inhibitor TSA caused a strong concentration dependent induction of apoptosis and total cell death (Fig. 2G) .

Effects of DAC, zebularine and TSA on EC migration, tube formation and angiogenesis in vitro

To assess the effects of DNMT- and HDAC inhibitors on EC migration, which is a key event in EC sprouting, the wound assay was used [24] . Confluent monolayers of activated HUVEC were treated for 3 days with DAC, zebularine or TSA after which they were wounded [24] . Migration of EC was not influenced by treatment with DAC at concentrations up to 1000 nM (Fig. 3A). Similar results were found for zebularine (data not shown) . In contrast to the DNMT inhibitors, TSA effectively inhibited migration of wounded confluent monolayers in a dose dependent manner (Fig. 3B) . While the maximal inhibition of approximately 70% was seen at 500 nM after 8 hours, significant effects (p<0.05) were already observed after 4 hours at doses as low as 300 nM.

To investigate effects of the DNMT- and HDAC inhibitors on in vitro 3 -dimensional EC tube formation, sprouting of bovine capillary ECs (BCEs) into a semi-natural collagen matrix was analyzed [24] . At increasing concentrations, DAC, zebularine and TSA all progressively inhibited growth factor- induced sprout formation of BCEs (Fig. 3C) , with ED₅₀ values of approximately 0.1 μM (p<0.037), 150 μM

(p<0.037) and 5 nM (p<0.037), respectively (Fig. 3D-F) .

An ex vivo model for human angiogenesis was developed by adapting the rat aortic ring assay using veins derived from the human peritoneum. After 10-13 days, bFGF and

VEGF- induced vascular sprouting was observed in control explants (Fig. 4A) . In rings treated with DNMT inhibitors, tube formation was significantly inhibited (Fig. 4B) . Average total sprout length of rings treated with 2 μM DAC was 2.6 (± 0.17) cm, compared to 4 (± 0.67) cm in the control rings (p<0.046) . Treatment with zebularine at 2 mM concentration completely inhibited capillary outgrowth (0 cm, p<0.046) . TSA at 500 nM decreased average total sprout length to 0.3 (± 0.17) cm (p<0.034) (Fig. 4B).

DAC, zebularine and TSA inhibit angiogenesis in vivo To study whether in vivo angiogenesis is perturbed by DNMT- and/or HDAC inhibitors, the chick chorio allantoic membrane (CAM) -assay, a model for developmental angiogenesis, was used. In CAMs onto which DAC was pipetted daily from day 10 through day 13, a profound inhibition of microvessel formation was observed, whereas larger preexisting vessels were apparently unaffected (Fig. 4C) . Anti -angiogenic effects of DAC were concentration dependent. Maximal responses of 40% angiogenesis inhibition were reached at 5 mM DAC, p<0.0001 (Fig. 4D) . These results were confirmed in zebularine- treated CAMs, in which maximal inhibition of microvessel formation was observed at 100 mM concentration (p<0.023) . TSA also had angiostatic activity in the CAMs (32% inhibition of microvessel formation at 400 μM, p<0.001, Fig. 4D) .

Activity and levels of DNA methyltransferase and 5- methylcytosine content in EC

The effects of DNMT inhibitors on EC proliferation and angiogenesis suggest that DNA methyltransferases are involved in regulation of EC growth and angiogenesis. Further proof for a role of these enzymes in regulating EC growth was obtained using a DNMT activity assay [25] . DNMT activity of activated- and quiescent EC, as well as B16F10 tumor cells was measured in this assay. HCT116 cells were used as a positive control [25] . Overall, DNMT activity levels in EC are lower as compared to HCT116 and B16F10 tumor cells (Fig. 5A) . In growth factor- stimulated EC, DNMT activity was significantly increased as compared to serum- deprived EC (2.6 fold increase, p<0.05). DAC treatment almost completely inhibited DNMT activity in growth factor- stimulated EC, as was observed for zebularine (Fig. 5A) . The HDAC inhibitor TSA reduced approximately 45% of DNMT activity, although this was not statistically significant (p<0.275). The induction of DNMT activity in activated EC was associated with increased DNMTl protein levels. Treatment with DNMT- and HDAC- inhibitors decreased DNMTl protein expression. To study whether the increased DNMT activity in activated EC is accompanied by increased "overall" genomic methylation levels, total genomic 5-methylcytosine content was quantified by high-performance capillary electrophoresis [26] . A small, but significant hypermethylation was displayed in activated HUVEC as compared to quiescent HUVEC. DAC almost completely erased genomic DNA methylation, whereas the DNMT inhibitor zebularine only caused a small reduction (Fig. 5B) . Treatment with TSA significantly decreased "overall" genomic methylation levels (p<0.004) .

Expression and chromatin modifications of THBSl, JunB, ICAMl and IGFBP3 in activated EC

The angiostatic effects of DNMT- and HDAC inhibitors could be explained by the reexpression of angiogenesis inhibiting genes in activated EC. We performed microarray experiments to identify genes that are silenced in activated HUVEC as compared with quiescent HUVEC, and upregulated by treatment of activated EC with DAC and TSA [27] . Analysis of these microarrays revealed the angiogenesis inhibitor thrombospondin 1 (THBSl) [28] , JUNB, a negative growth regulator and potential tumor suppressor [29] , intercellular adhesion molecule 1 (ICAMl) [30] and insulin-like growth factor binding protein 3 (IGFBP3) , an inhibitor of HUVEC proliferation [31] . As expected, all of these genes contain promoter CpG islands.

These genes were validated by quantitative real-time RT- PCR. Transcript levels of THBSl, JUNB, ICAMl and IGFBP3 were downregulated in activated EC as compared with quiescent EC. Treatment with DAC or TSA reactivated these genes (Fig. 5C) .

To study whether silencing of these angiogenesis inhibiting genes in activated EC is associated with DNA methylation, promoter CpG island methylation was evaluated using genomic bisulfite sequencing. The CpG islands in promoters of THBSl, JUNB, ICAMl and IGFBP3 contained only a few methylated CpG sites (Fig. 5D) . Furthermore, meaningful differences in promoter methylation patterns of these genes between silenced- and activated EC seemed to be not present .

We performed chromatin immunoprecipitation (ChIP) of the IGFBP3 5'CpG island to study whether gene silencing is associated with aberrant patterns of histone deacetylation. The region analyzed extended from 386 nucleotides upstream of the transcriptional start site to 323 nucleotides downstream, covering the area of greatest CpG density in the promoter and overlapping the region examined by genomic bisulfite sequencing. In the area from -2 to +323, acetylated histone H3 was observed in the transcriptionally active promoter of quiescent HUVEC, but was undetectable in activated HUVEC (Fig. 5E) . In cells treated with DAC or TSA, histone H3 acetylation reappeared in this promoter region. Thus, silencing of IGFBP3 in activated HUVEC occurred in conjunction with histone H3 deacetylation and reexpression by DAC and TSA was associated with reappearance of histone H3 acetylation.

Discussion

An important aspect of tumor growth is tumor angiogenesis . In this study, we investigated whether DNMT- and HDAC inhibitors target tumor angiogenesis, in particular whether DNMTs and HDACs regulate processes such as EC proliferation, migration and apoptosis. Our main question was whether DNMT- and HDAC inhibitors directly affect EC growth and angiogenesis, apart from potential indirect angiostatic activities in vivo via effects on tumor cells.

Although angiostatic effects of HDAC inhibitors have been described [6, 7] , nothing has previously been disclosed about the direct effects of DNMT inhibition on EC growth and angiogenesis.

We show that the DNMT inhibitor DAC decreases proliferation of activated HUVEC and mouse b . END5 brain endothelioma cells, an observation which was confirmed using zebularine, a recently described DAC analogue with great potential in clinical use [32] . The differences in the kinetics of the inhibitory effects of the DNMT- and HDAC inhibitors on EC growth, i.e. DAC and zebularine showed stronger anti-proliferative capacities after 72 hours of treatment, compared to 48 and 24 hours (Fig. 2D, data not shown) , correspond with the mechanism of action of these nucleoside analogues. Once these compounds are incorporated into the DNA during replication, they trap DNMTs during progression of the replication machinery, which, after some cell divisions, will eventually result in demethylation [33] .

The DNMT inhibitors did not influence EC apoptosis, while TSA did have a profound effect on EC apoptosis. The apoptosis inducing capacity of TSA in tumor EC has not been described before, although Kim et al . show an increased viability of HUVEC in culture medium from HepG2 cells transfected with HDACl vectors [6] . The DNMT- and HDAC inhibitors also had differential effects on EC migration. The inhibitory effect of TSA on EC migration is also described by Kim et al . [6] .

Our findings that DNMT- and HDAC inhibitors caused significant inhibition of tube formation in an in vitro model of angiogenesis proves that these agents directly inhibit EC sprouting, since in this assay ECs are not surrounded by tumor cells and therefore cannot be indirectly affected by DNMT- and HDAC inhibitors. Our data demonstrate that DNMT inhibitors act directly on activated EC and inhibit angiogenesis in vitro and in vivo. These findings suggest that DNMTs are involved in tumor EC growth, which is substantiated by the enhanced DNMT activity and expression levels in activated- as compared to quiescent EC. This is accompanied by a small but significantly increased overall genomic methylation level in activated EC (7% increase in activated EC versus quiescent EC) . Since the bulk of methylation occurs in the noncoding DNA, large differences in total methylcytosine content are not expected (Paz MF, Avila S, Fraga MF, Pollan M, Capella G, Peinado MA, Sanchez-Cespedes M, Herman JG, Esteller M. Germ-line variants in methyl-group metabolism genes and susceptibility to DNA methylation in normal tissues and human primary tumors. Cancer Res. 2002 Aug 1;62 (15) :4519-24) . Methylation differences in tumor cells as compared to normal counterparts are different; despite regional promoter hypermethylation of tumor suppressor genes, global demethylation of the genome occurs [13] . This hypomethylation in CpG depleted regions has been proposed to cause chromosomal instability and harmful expression of endogenous viral sequences. Therefore, it might not be surprising that this global hypomethylation is not found in activated EC as compared with quiescent EC. Anti-proliferative effects of DNMT- and HDAC inhibitors on non-tumor cells such as i.e. fibroblasts [34] and cells in an atherosclerotic plaque [35] have been shown. There is limited evidence on the role of epigenetics in regulating endothelial -specific gene expression, showing that DNA methylation plays an important role in the EC-specific expression of eNOS [36] .

However, nothing is currently known about the involvement of DNMTs or DNA methylation in the regulation of EC growth. Our results suggest that DNMTs, besides their central role in tumor cells, play an important role in regulation of EC proliferation and tube formation.

In two different mouse models (syngenic and xenograft models) DNMT inhibitors caused full abrogation of tumor growth over the treatment period. Both drugs showed potent angiostatic activity and the effective concentration of zebularine was about 100-fold higher than DAC, both in vitro and in vivo. This difference in effective dose is in agreement with results of Cheng et al . [23] and can be explained by the less efficient metabolic activation of zebularine as compared to DAC [22] , as well as by the fact that zebularine is probably incorporated into RNA as well as into DNA [23] . TSA also significantly inhibited tumor growth, although to a lesser extent. Several mechanisms can explain the anti-tumor activity of DNMT- and HDAC inhibitors. Firstly, the results in mice are likely to result from reactivation of epigenetically silenced tumor suppressor genes in the tumor cells, such as the cell cycle inhibitors pl4ARF [37] , pl5INK4b [38, 39] and pl6INK4a [40], thereby reducing tumor cell growth.

Secondly, by reexpression of tumor suppressor genes with angiogenesis inhibiting properties in tumor cells, DNMT- and HDAC inhibitors, in addition to their inhibitory effects on tumor cell growth, might indirectly exhibit angiostatic effects in vivo. Examples of angiogenesis inhibiting genes for which epigenetic regulation in tumor cells has been described are pl6INK4a and p73 [16] , which negatively regulate VEGF expression [41, 42] , as well as other angiogenesis inhibitors like the protease inhibitor maspin [43, 44], TIMP3 , which antagonizes matrix metalloproteinase activity and blocks binding of VEGF to VEGF receptor 2 [45, 46] and THBSl [47-49] . Interestingly, here we demonstrate an additional mechanism behind the anti-tumor activities of DNMT- and HDAC inhibitors, namely by exhibiting direct effects on tumor EC themselves, thereby decreasing EC growth and thus tumor angiogenesis.

This allows us to propose a model which suggests 3 mechanisms by which the anti-tumor effects of DNMT- and HDAC inhibitors can be explained in vivo (Fig. 6) .

A possibility is that, in analogy with epigenetic silencing of tumor suppressor genes in tumor cells, "angiosuppressor genes" are genes downregulated by epigenetic modifications in tumor EC. Thrombospondin 1 (THBSl) , JUNB, intercellular adhesion molecule 1 (ICAMl) and insulin-like growth factor binding protein 3 (IGFBP3) are candidate "angiosuppressor genes". IGFBP3 , a key regulator of cell growth and apoptosis, potently inhibits VEGF-mediated HUVEC proliferation [31] . The angiogenesis inhibitor THBSl blocks EC migration and induces EC apoptosis [28, 48] . JUNB negatively regulates cell growth by activating pl6INK4A and decreasing cyclin Dl expression [29] . ICAMl, an important EC adhesion molecule that mediates arrest and extravasation of leukocytes into tumors, is downregulated by angiogenic factors, which presents a mechanism to escape from immune surveillance [30, 50] . Our findings that IGFBP3 , THBSl, JUNB and ICAMl are suppressed in activated HUVEC and become reexpressed after DAC and TSA treatment indicate that these genes are silenced by epigenetic modifications in these cells. Previous studies demonstrate that silencing of IGFBP3 , THBSl and JUNB in tumor cells is associated with promoter methylation [51, 52] [47, 49] [53] . Also for ICAMl, a role of epigenetics in downregulation of this gene is suggested in ovarian adenocarcinoma, despite the lack of direct proof [54] . In activated EC, however, silencing of these genes seems not to be associated with promoter hypermethylation, as demonstrated by the lack of meaningful methylation differences between quiescent- and activated EC that could explain the silencing of the genes in activated EC. Despite the latter, a significant increase in total genomic 5-methylcytosine content was observed in activated EC. In contrast with promoter methylation patterns, histone H3 acetylation patterns of the IGFBP3 promoter correlated with silencing of IGFBP3 in activated HUVEC and reexpression by DAC and TSA (Fig. 5F) .

These data suggest that promoter histone deacetylation is responsible for downregulation of IGFBP3 in activated EC and that histone deacetylation is inhibited by the DNMT- and HDAC inhibitors, thereby reexpressing the gene.

DNMTs have additional transcriptional repressor functions apart from their methylation ability. Several studies demonstrate that DNMTs bind to HDACs and can repress gene transcription through histone deacetylase activity [55, 56] . Remarkably, in this situation, the methyltransferase activity of the DNMTs is dispensable for transcriptional silencing. Gene silencing of our candidate genes in activated HUVEC might be caused by methylation- independent transcriptional silencing effects of DNMTs. Reexpression by DNMT inhibitors could be the result of dissociating the direct link between DNMTs and HDACs, by removal of DNMTs from the transcription repressor complex at gene promoters .

Although previous publications show anti -angiogenic activities of HDAC inhibitors, histone acetylation or DNA methylation in promoters of tumor-conditioned EC has never been studied. We demonstrate that epigenetic modifications, besides their role in tumor cells, are involved in regulating expression of growth inhibitory genes in activated EC. The dual effects on both tumor cell growth and angiogenesis make epigenetic therapy promising for cancer treatment.

Methods

Cells, cultures and reagents

Human umbilical vein endothelial cells (HUVEC) were harvested from normal human umbilical cords by perfusion with 0.125% trypsin/EDTA. Harvested HUVECs were cultured in RPMI-1640 (Life Technologies, Breda, The Netherlands) supplemented with 20% heat inactivated human pooled serum (provided by the University Hospital Maastricht) , 2 mM L- glutamin (Life Technologies, Breda, The Netherlands) , 50 ng/ml streptomycin and 50 U/ml penicillin (ICN Biomedicals) in 0.2% gelatin coated tissue culture flasks at 37⁰C, 5% CO₂. Confluent cultures were sub cultured 1:3 and used for experiments between passage 2 and 4. Tumor conditions were mimicked by a 3 -day exposure to 10 ng/ml basic Fibroblast Growth Factor (bFGF; Peprotech, London, UK) , 10 ng/ml Vascular Endothelial Growth Factor (VEGF; Peprotech, London, UK) and, where indicated, 20% (v/v) of a 1:1 mixture of filtered culture supernatants of LS174T and CaCo-2 human colon carcinoma cell lines.

Mouse b.END5 brain endothelioma cells (ECACC, Salisbury, United Kingdom) were cultured in Dulbecco's MEM (Life Technologies) containing 10% fetal calf serum (FCS, Bio Whittaker, Verviers, Belgium) , 2 mM L-glutamin and 5 μmol/1 2 -mercaptoethanol (Sigma, st Louis, MO) .

Peripheral blood leukocytes, obtained from healthy individuals, were cultured in RPMI-1640 supplemented with 10% FCS, 2 mM L-glutamin and antibiotics. Leukocytes were activated for 3 days with phycohaemagglutinin (PHA) .

Bovine capillary endothelial cells (BCE) were kindly provided by Dr. M. Furie (State University of New York, Stony Brook, USA) and were cultured on gelatin coated flasks in MEM-α (Life Technologies, Breda, the Netherlands) supplemented with 10% FCS and 2 mM L-glutamin and antibiotics.

Mouse B16F10 melanoma cells (kindly provided by dr. J. Fidler, Houston, Texas) were cultured using Hank's MEM

(Life Technologies, Breda, The Netherlands) containing 5% FCS, 1% non-essential amino acids (Life Technologies), 1% sodium pyruvate (Life Technologies), 1.5% MEM vitamins (Life Technologies) , and 2% sodium bicarbonate (Life Technologies) .

Cells were treated with the DNA methyltransferase (DNMT) inhibitors 5-aza-2 ' -deoxycytidine (DAC) (Sigma, Zwijndrecht, the Netherlands) or zebularine (obtained from the NCI, Bethesda, US), or with the histone deacetylase (HDAC) inhibitor TSA (Wako, Neuss, Germany) . For treatment, culture medium was supplemented with DAC, zebularine or TSA for 72 hours, replacing drugs and culture medium every 24 hour.

For treatment of tumor-conditioned HUVEC with DNMT- or HDAC inhibitors, HUVEC cultures were exposure for 72 hours to 10 ng/ml bFGF, 10 ng/ml VEGF and human colon carcinoma cell lines supernatants, followed by 72 hours exposure to DAC, zebularine or TSA, replacing drugs and medium every 24 hours.

Mouse tumor models

The animal experiments were approved by the local ethical review committee. At day 0, 6-wk-old C57BL/6 mice

(obtained from Charles River) were inoculated with 1x105 B16F10 mouse melanoma cells subcutaneously on the right flank. Between day 6 and 9 thetumors became visible in all mice and treatments were initiated. In the LS174T xenograft model, nu/nu Swiss mice (Charles River) were inoculated with IxIO⁶ LS174T human colon carcinoma cells. Between day 10 and 14 the tumors became visible and treatment was initiated. DAC (n=5), at doses of 10 mg/kg, zebularine (n=5) , at doses of 1000 mg/kg [23] and TSA (n=5) , at doses of 1 mg/kg [6] , were administered daily by intraperitoneal injection in a solution of 0.9% saline for 7 days. Tumor volumes were measured daily, and calculated as follows: width2 x length x 0.52.

All mice were sacrificed 24 hours after the last treatment. Tumors were frozen in liquid nitrogen for histological analysis. Observed differences were tested for significance using the two-way ANOVA test. Vessels were stained with rat-anti mouse CD31 and peroxidase labelled goat anti-rat Ig. The microvessel density was evaluated as described previously [57] .

Proliferation and apoptosis measurement EC proliferation was measured using a [3H] thymidine incorporation assay. Tumor-conditioned HUVEC, seeded at 2000 cells per well, were exposed for 3 days to a concentration range DAC, zebularine or TSA. During the last 6 hours of the assay, the culture was pulsed with 0.3 μCi [methyl-3H] thymidine (Amersham Life Science) per well. Activity was measured using liquid scintillation. In each experiment, measurements were done in triplicate. Tumor-conditioned HUVEC cultured for 72 hours with DAC, zebularine or TSA were harvested by trypsinisation (0.125%) and fixed for at least two hours in 70% ethanol at -20⁰C. The cells were subsequently centrifuged at 1500 rpm for 5 minutes and resuspended in DNA extraction buffer (45 mM Na2HPO4, 2.5 mM citric acid and 0.1% Triton X-100) and incubated for 20' at 37 ⁰C. Propidium iodide (PI) (Brunschwig Chemie, Amsterdam, the Netherlands) was added to a final concentration of 20 μg/ml and the DNA profile was directly analyzed using flow cytometry (FACS-calibur, Becton Dickinson) [24] . Serum deprivation of HUVEC (3 days) was used as a positive control for apoptosis.

Migration measurement

HUVEC migration was measured using the wound assay [24] . In brief, confluent monolayers of tumor-conditioned HUVEC cultured with DAC, zebularine or TSA were wounded using a blunt glass pipette . Cultures were washed and medium and drugs were replaced. Wound width was measured in triplicate cultures at four predefined locations at start and after 2, 4, 6, 8 and 24 hours after wounding.

In vitro angiogenesis

Sprouting and tube formation of bovine capillary EC (BCE) was studied using cytodex-3 beads overgrown with EC in a 3 -dimensional gel, as previously described [58] . BCE were mixed with gelatin coated cytodex-3 microcarrier beads (Sigma, The Netherlands) and cultured for 48 hours in the presence of bFGF, VEGF, CaCo-2 and LS174T supernatants , followed by a 3 -day exposure to DAC, zebularine or TSA. Next, the beads were placed in a 3-dimentional gel and medium was applied on top of the gel containing 20 ng/ml bFGF, 10 ng/ml VEGF, and 20% of a 1:1 mixture of culture supernatants of LS174T and CaCo-2 human colon carcinoma cells, with or without DAC, zebularine or TSA at concentrations as indicated. After 24 hours photographs were taken and digitally analyzed.

Ex vivo human vessel ring assay The human vessel ring assay was based on the rat aortic ring assay [59, 60] . For this ex vivo angiogenesis assay, human veins (diameter 1 mm) derived from the peritoneum were carefully removed from the surrounding fibroadipous tissue and were cut into 1-mm-thick cross-sectional rings. Ring-shaped explants were embedded in a semi -natural matrix of collagen type I, prepared by mixing 8 volumes vitrogen-100 (Collagen Corporation, Fermont, CA, USA), 1 volume 10x concentrated α-MEM (Life Technologies, Breda, The Netherlands), 1 volume 11.76 mg/ml sodium bicarbonate, 20 ng/ml bFGF and 20 ng/ml VEGF. After gellation, medium (RPMI-1640, 20% HS, 2 mM L-glutamin, 50 ng/ml streptomycin, 50 U/ml penicillin and 0.25 μg/ml amphotericin B) was applied on top of the gel containing 20 ng/ml Bfgf and 20 ng/ml VEGF, in absence or presence of DAC (2 μM) , zebularine (2 mM) or TSA (0.5 μM) . Cultures were maintained at 37°C for up to 13 days, replacing drugs and medium every 2 days. Vascular sprouting from each ring was examined daily using an inverted microscope. Photographs were taken on day 13. The width of the tube formation area was measured at four different predefined places of the aortic ring.

Chorioallantoic membrane (CAM) assay

The CAM assay was performed as previously described [58] . CAMs were treated by daily addition of sterile saline

(0.9% NaCl), DAC (5 μM and 5 mM) , zebularine (100 mM) or TSA (400 μM) from day 10 to day 13. On day 14 the CAMs were photographed. Quantification of vascularization was performed by enumeration of intersections with 5 concentric rings that were superimposed on the photographs .

DNA methyltransferase assay and Western blot analysis HUVECs were cultured for 3 days in the presence of bFGF and VEGF. To avoid possible contamination of DNMT activity from tumor cell lines, colon tumor cell line supernatant was excluded. Tumor-conditioned HUVEC were treated for 3 days with DAC (200 nM) , zebularine (200 μM) or TSA (300 nM) . Serum deprivation of HUVEC was used as a model for silenced EC (HUVEC-) . DNA methyltransferase enzyme activity was measured as described [25] . Results are expressed as the mean disintegrations per minute (d.p.m.) .

Western Blot and activity assay: as descibied previously

(see p24, line 27 of #645820, Vertino, P.M., et al . , De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-) - methyltransferase. MoI Cell Biol, 1996. 16(8): p. 4555-65.

High performance liquid chromatography As described previously (Fraga, M. F., et al . , High- performance capillary electrophoretic method for the quantification of 5-methyl 2 ' -deoxycytidine in genomic DNA: application to plant, animal and human cancer tissues . Electrophoresis, 2002. 23(11): p. 1677-81.

Semi-quantitative real-time RT-PCR Total RNA isolation, cDNA synthesis and semi-quantitative real-time RT-PCR were performed as described previously [61] on tumor-conditioned HUVEC, treated for 3 days with or without DAC (200 nM) or TSA (300 nM) , and serum deprivation silenced HUVEC.

The sequences of the primers used for quantitative real- time RT-PCR were as follows, with the first primer listed being the forward primer and the second primer listed being the reverse primer: TSPl 5' -CATCTGCGGCATCTCCTGTG-3'

(SEQ ID N0:l) 5 ' -AGTCACTTTGCGGATGCTGTC-3 '

(SEQ ID NO: 2) JUNB 5 ' -AAGACCAAGAGCGCATCAAAG- 3 '

(SEQ ID NO: 3)

5' -CTTGTCCTCCAGGCGCG-S' (SEQ ID NO:4)

IGFBP3 5 ' -CTGTGGCCATGACTGAGGAAAG-S '

(SEQ ID NO: 5)

5' -TCCCTGAGCCTGACTTTGCC-3 '

(SEQ ID NO: 6) cyclophilin A 5 ' -CTCGAATAAGTTTGACTTGTGTTT-3 '

(SEQ ID NO: 7)

5 ' -CTAGGCATGGGAGGGAACA- 3 '

(SEQ ID NO: 8)

Bisulfite sequencing

Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega) . Bisulfite modification of genomic DNA was carried out as described previously [62] .

The sequences of the primers used for bisulfite sequencing were as follows, with the forward primer listed first and the reverse primer listed second (positions relative to transcription start site) :

TSPl (-119; +229) 5'-AGAGAGGAGTTTAGATTGG-B'

(SEQ ID NO: 9) 5 ' -CAAAAAAACTAAAACCTCAAC- 3 '

(SEQ ID NO: 10) JUNB (-10; +338) 5'-GGGATTTTGAGAGYGGTTAGG-B'

(SEQ ID NO:11)

5 ' -CCRTATCCCRTAACTATATATAAATC-S ' (SEQ ID N0:12)

IGFBP3 (-251; -29) 5'-GGGTATATTTTGGTTTTTGTAGA-S'

(SEQ ID NO: 13)

5' -AAAAACCRAAATAACCCAAAACAC-S'

(SEQ ID NO: 14) IGFBP3 (-53; +189) 5'-GTGTTTTGGGTTATTTYGGTT-S'

(SEQ ID NO: 15)

5 ' -AAACAACACCAACAAAATCAAC-3 '

(SEQ ID NO: 16)

IGFBP3 (+167; +602 ) 5 ' -GTTGATTTTGTTGGTGTTGTTT-3 ' (SEQ ID NO:17)

5 ' -CAACAACCCCCAAACCCTTC- 3 '

(SEQ ID NO:18)

ChIP assay ChIP assays were performed as previously described.

The sequences of the primers used for the ChIP assay were as follows, with the forward primer listed first and the reverse primer listed second (positions relative to transcription start site) : IGFBP3 (-386; -211) 5'-TCGCCGCAGGGAGACCT-S'

(SEQ ID NO: 19) 5 ' -GAGCCCGTCACCTTGTCGTC- 3 ' (SEQ ID NO: 20)

IGFBP3 (-283; -123) 5 ' -GTGCTGAGGTGGCCTGGAGT- 3 '

(SEQ ID NO: 21)

5' -CCGTGCTTCGCCCTGAG- 3 '

(SEQ ID NO:22)

IGFBP3 (-198; -65) 5' -CGAGGAGCAGGTGCCCG- 3 '

(SEQ ID NO:23)

5 ' -CGCGCCCAGGAGTGG- 3 '

(SEQ ID NO:24) IGFBP3 (-79; +34) 5 ' -CCACTCCTGGGCGCGC- 3 '

(SEQ ID NO: 25)

5 ' -CTGATCCTCAGCGCCCAG- 3 '

(SEQ ID NO: 26)

IGFBP3 (-2; +134) 5 ' -CCAGATGCGAGCACTGCG-3 '

(SEQ ID NO:27)

5 ' -CATGACGCCTGCAACCG-3 '

(SEQ ID NO: 28)

IGFBP3 (+68; +254) 5 ' -GTGTACTGTCGCCCCATCCC- 3 '

(SEQ ID NO: 29)

5 ' -CTCGCAGCGCACCACG- 3 '

(SEQ ID NO: 30)

IGFBP3 (+168; +323) 5' -CTGACTCTGCTGGTGCTGCTC-S'

(SEQ ID NO:31)

5 ' -CTCGCGCACCAGCTCCG- 3 '

(SEQ ID NO: 32)

Statistical analysis

All values are given as mean values + SEM. Statistical analysis for the tumor volumes was done by means of the two-way ANOVA test. The Student's t-test was used for statistical analyses of microvessel density levels in the mouse tumors and CAMs and for the migration assay. Statistical analyses of the proliferation, apoptosis, in vitro and ex vivo angiogenesis assays, DNMT activity assay, HPLC, as well as the semi-quantitative real-time RT-PCR were done using the Wilcoxon-Mann-Whitney rank sum test which was performed in SPSS 10.0.5. software. All values are two-sided and P-values <0.05 were considered statistically significant.

All references in the specification are specifically incorporated into the present disclosure by reference.

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Claims

- 75 -CLAIMS

1. A method of determining angiogenesis associated with a disease in a subject comprising, in a test sample, determining expression levels of at least one gene selected from junB and ICAM 1, wherein a statistically significant decrease in, or low level of, expression indicates the presence of angiogenesis associated with the disease in the subject.

2. The method according to claim 1 wherein the test sample comprises endothelial cells.

3. The method according to claim 1 or 2 wherein the level of expression is assessed with reference to a control sample .

4. The method according to claim 3 wherein the control sample is taken from non-activated endothelial cells.

5. The method according to claim 3 wherein the control sample is taken from the same tissue as the test sample at an earlier time point.

6. The method according to any one of claims 1 to 5 further comprising determining the expression of THBSl and/or IGFBP3 , wherein a statistically significant decrease in, or low level of, expression indicates the presence of angiogenesis in the subject.

7. The method according to any one of claims 1 to 6 wherein gene expression is determined using RT-PCR. - 76 -

8. The method according to claim 7 wherein real-time quantitative RT-PCR is utilised.

9. A method of determining angiogenesis associated with a disease in a subject comprising, in a test sample, determining the level of acetylated histone H3 in the promoter region of the IGFBP3 gene wherein a statistically significant decrease in, or low level of, acetylated histone H3 indicates the presence of angiogenesis associated with the disease.

10. The method according to claim 9 wherein the test sample is an endothelial cell sample.

11. The method according to any one of claims 9 to 12 wherein levels of acetylated histone H3 are compared to levels of acetylated histone H3 in the promoter region of the IGFBP3 gene in a control endothelial cell sample.

12. The method according to any one of claims 9 to 13 wherein the level of acetylated histone H3 is assessed using chromatin immunoprecipitation.

13. The method according to any preceding claim wherein the angiogenesis is associated with a disease selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.

14. The method according to claim 13, wherein the cancer is selected from cancer of the lung, breast, kidney, cervix, pancreas, ovaries, and/or head and neck. - 77 -

15. A pharmaceutical composition for use in inhibiting angiogenesis comprising a DNA methyltransferase inhibitor together with a pharmaceutically acceptable carrier and which is in a form suitable for metronomic dosing and/or comprises a component allowing targeting to endothelial cells.

16. The composition of claim 15 which prevents endothelial cell proliferation or growth

17. The composition of claim 15 or 16 wherein the DNA methyltransferase inhibitor comprises any one or more of antisense molecules, RNAi molecules or siRNA molecules which reduce expression of DNA methyltransferase genes.

18. The composition of claim 17 wherein the DNA methyltransferase gene is DNMTl.

19. The composition of claim 15 or claim 16 wherein the DNA methyltransferase inhibitor comprises any one or more of 5-azacytidine, 5-aza-2 ' -deoxycytidine, 5-fluouro-2 ' - deoxycytidine, pseudoisocytidine and 5, 6-dihydro-5- azacytidine, l-/3-D-arabinofuranosyl-5-azacytosine .

20. The composition of claim 15 or claim 16 wherein the DNA methyltransferase inhibitor comprises Decitabine.

21. The composition of claim 15 or claim 16 wherein the DNA methyltransferase inhibitor comprises any one or more of L- ethionine, S-adenosyl -homocysteine, sinefungin, (S) -6- methyl-6-deaminosine fungin, 6-deaminosinefungin, N4- - 78 -

adenosyl-N4 -methyl -2 , 4-diaminobutanoic acid, 5 ' -methylthio- 5 ' -deoxyadenosine and 5 ' -amino-5 ' -deoxyadenosine .

22. The composition of any one of claims 15 to 21 which additionally comprises a histone deacetylase inhibitor.

23. The composition of claim 22, wherein the histone deacetylase (HDAC) inhibitor comprises at least one of trichostatin A (TSA), suberoyl hydroxamic acid (SBHA), 6- (3- chlorophenylureido) caproic hydroxamic acid (3-Cl-UCHA), m- carboxycinnamic acid bishydroxylamide (CBHA) , suberoylanilide hydroxamic acid (SAHA) , azelaic bishydroxamic acid (ABHA) , pyroxamide, scriptaid, aromatic sulfonamides bearing a hydroxamic acid group, oxamflatin, trapoxin, cyclic-hydroxamic-acid containing peptides, FR901228, MS-275, MGCD0103, short-chain fatty acids and N- acetyldinaline .

24. The composition according to any one of claims 15 to 23 wherein the component allowing targeting to endothelial cells comprises RGD-peptides .

25. A composition according to any one of claims 15 to 24 wherein the concentration of the DNA methyltransferase inhibitor is no more than 1 μ.M.

26. A composition according to any one of claims 15 to 25wherein the concentration of the DNA methyltransferase inhibitor is no more than 500 nM. - 79 -

27. A composition according to any one of claims 15 to 26 wherein the concentration of the DNA methyltransferase inhibitor is no more than 250 nM.

28. A composition according to any one of claims 15 to 27 wherein the concentration of the DNA methyltransferase inhibitor is no more than 100 nM.

29. A composition according to any one of claims 15 to 28 wherein the concentration of the DNA methyltransferase inhibitor is no more than 50 nM.

30. A method of preventing or inhibiting angiogenesis associated with a disease in a subject comprising administering a therapeutically effective amount of a DNA methyltransferase inhibitor to the subject in order to prevent or inhibit angiogenesis.

31. A method of preventing or inhibiting angiogenesis associated with a disease in a subject comprising administering a therapeutically effective amount of a DNA methyltransferase inhibitor to the subject such that expression of at least one gene selected from JUNB, ICAMl, THBSl and IGFBP3 is increased.

32. The method according to claim 31 wherein the level of gene expression is increased to the levels of gene expression found in non-activated endothelial cells.

33. The method of any one of claims 30 to 32 wherein the composition of any one of claims 15 to 29 is utilised. - 80 -

34. The method according to any one of claims 30 to 33 wherein the angiogenesis is associated with a disease selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.

35. The method according to any one of claims 30 to 34 wherein endothelial cell proliferation or growth is prevented or inhibited.

36. Use of a DNA methyltransferase inhibitor in the manufacture of a medicament for preventing or inhibiting angiogenesis associated with a disease in a subject.

37. Use of a DNA methyltransferase inhibitor or a histone deacetylase inhibitor in the manufacture of a medicament for preventing angiogenesis associated with a disease in a subject by increasing expression of at least one gene selected from JUNB, ICAMl, THBSl and IGFBP3.

38. The use according to claim 37 wherein the level of gene expression is increased to the levels of gene expression found in non-activated endothelial cells.

39. The use according to any one of claims 36 to 38 wherein the medicament comprises a composition as defined in any one of claims 15 to 29.

40. The use according to any one of claims 36 to 39 wherein the angiogenesis is associated with a disease selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis. - 81 -

41. The use according to any one of claims 36 to 40 wherein endothelial cell proliferation or growth is prevented or inhibited.

42. A treatment regime for preventing or inhibiting angiogenesis comprising metronomic dosing of the composition of any one of claims 15 to 29.

43. The treatment regime of claim 42 wherein the angiogenesis is associated with a disease selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.

44. The treatment regime according to claim 42 or 43 wherein endothelial cell proliferation or growth is prevented or inhibited.

45. A microarray for use in the method of any one of claims 1 to 8 comprising probes immobilised on a solid support hybridizing with transcripts or parts thereof of at least two genes selected from JUNB, ICAMl, THBSl and IGFBP3

46. The microarray of claim 45 wherein there are at least 10 probes representing each gene.

47. The microarray of claim 45 or 46 wherein each probe is at least 20 nucleotides in length.

48. A method of identifying a compound capable of preventing or inhibiting angiogenesis associated with a disease comprising the steps of; - 82 -

(a) administering the compound to an experimental non- human animal having an angiogenesis associated disease;

(c) comparing the expression profile obtained in (b) with the expression profile of a corresponding panel of genes expressed in a control endothelial cell sample; wherein a positive correlation of the expression profiles is indicative that the compound is capable of preventing or inhibiting angiogenesis associated with a disease.

49. An in vitro method of identifying a compound capable of preventing or inhibiting angiogenesis associated with a disease comprising the steps of;

(a) administering the compound to an endothelial cell sample taken from a subject having an angiogenesis associated disease;

(b) generating an expression profile of a panel of genes comprising at least one of JUNB, ICAMl, THBSl and

IGFBP3 ;

(c) comparing the expression profile obtained in (b) with the expression profile of a corresponding panel of genes exposed in a control endothelial cell sample from a subject not having the angiogenesis associated disease; wherein a positive correlation of the expression profiles is indicative that the compound is capable of preventing or inhibiting angiogenesis associated with a disease.

50. The method of claim 48 wherein the control sample is taken from an experimental non-human animal which does not have said angiogenesis related disease. - 83 -

51. The method of claim 48 wherein the control sample is taken from non-activated endothelial cells in the same non- human animal .

52. The method according to any one of claims 48 to 51 wherein the disease associated with angiogenesis is selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.