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  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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  • C12Q2600/00—Oligonucleotides characterized by their use
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  • G01N2800/00—Detection or diagnosis of diseases
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  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/20—Dermatological disorders

Definitions

• 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.
• HDACs histone deacetylases
• methylation of histones and DNA is involved in organization of chromatin in gene promoter regions [8] .
• DNMTs DNA methyltransferases
• 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] .
• 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 .
• 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.
• JUNB jun B proto oncogene
• ICAMl intercellular adhesions molecule 1 (CD54)
• the method is most preferably an in vitro method carried out on an isolated sample.
• the method may also include the step of obtaining the sample.
• test sample is most preferably a tissue sample, taken from the subject.
• sample comprises, consists essentially of, or consists of endothelial cells.
• 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.
• 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.
• 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.
• 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.
• the subject is a human subject.
• 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.
• 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.
• 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.
• THBSl thrombospondin 1
• IGFBP3 insulin-like growth factor binding protein 3
• 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.
• 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
• 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.
• 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.
• 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.
• 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.
• Fluorophores that may possibly be used in the method of the invention include, by way of example, FAM, HEXTM, NEDTM, ROXTM, Texas RedTM etc.
• Quenchers for example Dabcyl and TAMRA are well known quencher molecules that may ⁇ be used in the method of the invention.
• 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.
• 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.
• 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.
• 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.
• tumour angiogenesis is relevant for and/or contributes to tumour growth
• 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.
• promoter hypermethylation is not responsible for silencing of the genes listed above which are down regulated in diseases linked with angiogenesis.
• studies carried out by the inventors have shown that inactivation of the genes in activated EC occurs in correlation with promoter histone H3 acetylation patterns.
• 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.
• 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 .
• 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.
• the method is most preferably an in vitro method carried out on an isolated sample.
• the method may also include the step of obtaining the sample.
• 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.
• 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.
• the sample comprises endothelial cells.
• 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.
• 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.
• histone H3 acetylation is compared to histone H3 acetylation of the same genes in a control endothelial cell sample.
• 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.
• H3 acetylation 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 .
• the subject is a human subject.
• 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 histone H3 acetylation patterns of at least IGFBP3 is determined.
• 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.
• 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).
• ChIP chromatin immunoprecipitation
• the disease which is diagnosed may be any disease which is dependent upon angiogenesis for its progression.
• 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.
• DNA methyltransferase inhibitors can have an indirect effect on tumour growth by inhibiting angiogenesis.
• 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.
• DNA methyltransferase inhibitors allow 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.
• 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.
• tumour angiogenesis is relevant for tumour growth
• 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.
• 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.
• 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) .
• the DNA methyltransferase gene is (human) DNMTl.
• the agent may be a direct inhibitor of DNMTs.
• modified nucleotides such as phosphorothioate modified oligonucleotides (fig 6 of ref 63) and nucleosides and nucleotides such as cytidine analogues.
• 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 trasbarine)
• 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
• SAM S-Adenosyl -Methionine
• SAH S-adenosyl- homocysteine
• SAH S-adenosyl- homocysteine
• SAH S-adenosyl- homocysteine
• SAH S-adenosyl- homocysteine
• SAH S-aden
• 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) .
• organohalogenated compounds such as chloroform etc, procianamide
• intercalating agents such as mitomycin C, 4- aminobiphenyl etc
• inorganic salts of arsenic and selenium such as kanamycin, hygromycin and cefotaxim (63) .
• 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.
• the pharmaceutical composition additionally comprises a histone deacetylase inhibitor.
• a histone deacetylase inhibitor 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.
• HDAC inhibitors are also shown to have a profound effect on EC apoptosis (see Experimental section for further details) .
• 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) .
• TSA trichostatin A
• SBHA suberoyl hydroxamic
• 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.
• 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) .
• active agent in this case DNA methyltransferase inhibitor potentially with a histone deacetylase inhibitor in addition
• typical concentrations of active ingredients are of the micromolar scale in order to achieve optimal effects.
• anti-angiogenic effects can be seen at concentrations of active ingredient which are on the nanomolar scale.
• 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.
• 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.
• the pharmaceutical composition of the invention is in a form suitable for metronomic dosing.
• the composition comprises a component allowing targeting to endothelial cells.
• 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) .
• angiogenesis inhibitors can be administered, and are effective, at lower concentrations than other drugs that target tumour cells directly.
• a composition according to the invention wherein the concentration of the DNA methyltransferase inhibitor is no more than about 1 ⁇ M.
• 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.
• suitable carriers include tablets, capsules and liquids.
• 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.
• 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.
• 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 .
• 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.
• the methods of the invention involve preventing or inhibiting angiogenesis associated with a disease.
• 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.
• the methods of the invention involve preventing or inhibiting angiogenesis associated with a disease by increasing expression of the relevant gene(s) .
• the subject is a human.
• 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.
• 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.
• any increase in expression will be beneficial for treatment of the angiogenesis related disease.
• 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.
• compositions according to the invention are used in these methods. Therefore, the discussion above relating to the pharmaceutical compositions also applies mutatis mutandis to these aspects of the invention.
• the method may comprise, consist essentially of or consist of metronomic dosing of the relevant composition.
• the disease may be any disease which depends upon angiogenesis for its progression.
• the disease associated with angiogenesis which is treated according to the invention is selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.
• tumour angiogenesis is relevant for tumour growth
• Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.
• 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.
• the medicament is useful for preventing or inhibiting angiogenesis associated with a disease .
• 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.
• the medicament is useful for preventing or inhibiting angiogenesis associated with a disease by increasing expression of the relevant gene (s) .
• 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.
• the medicament comprises a pharmaceutical composition of the invention (see the relevant section above) .
• the medicament may be designed to be administered by metronomic dosing.
• the disease may be any disease which depends upon angiogenesis for its progression.
• the disease associated with angiogenesis is selected from cancer, atherosclerosis, rheumatoid arthritis, endometriosis, diabetic retinopathy and psoriasis.
• tumour angiogenesis is relevant for tumour growth
• Non-limiting examples include cancers of the lung, breast, kidney, cervix, pancreas, ovaries, and head and neck.
• 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.
• 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.
• 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 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 ICAMl
• THBSl 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.
• 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 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
• 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.
• the array includes probes which represent each and every one of the genes listed (i.e. JUNB,
• 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.
• 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.
• 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;
• 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.
• 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.
• control sample is taken from non-activated (non-angiogenic) endothelial cells in the same non-human animal .
• 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;
• the methods are useful for identifying compounds capable of preventing or inhibiting angiogenesis associated with a disease.
• 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 .
• tumour angiogenesis is relevant for tumour growth
• 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.
• 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.
• 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) .
• Fig. 3 presents the effects of DAC, zebularine and TSA on
• 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 .
• scale bar 100 ⁇ m
• b Average sprout lengths ( ⁇ SEM) out of 3 experiments (*p ⁇ 0.046).
• 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 Measurement of 5-methylcytosine content as a percentage of the total cytosine pool .
• 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.
• DNMT- and HDAC inhibitors directly decrease EC growth and angiogenesis, thereby exhibiting direct angiostatic effects, e.g. by reactivation of epigenetically silenced "angiosuppressor" genes.
• DNMT DNA methyltransferase
• DAC 5- aza-2 ' -deoxycytidine
• HDAC histone deacetylase
• TSA histone deacetylase
• DAC, zebularine and TSA inhibit tumor growth and 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) .
• DNMT DNA methyltransferase
• HDAC histone deacetylase
• TSA histone deacetylase
• 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) .
• 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) .
• 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) .
• 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) .
• TSA 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 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.
• 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 ⁇ M (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.
• 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 ⁇ M.
• DAC vascular endothelial cell line
• 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.
• 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) .
• the HDAC inhibitor TSA caused a strong concentration dependent induction of apoptosis and total cell death (Fig. 2G) .
• BCEs bovine capillary ECs
• VEGF-induced vascular sprouting was observed in control explants (Fig. 4A) .
• Fig. 4B Tube formation was significantly inhibited.
• Average total sprout length of rings treated with 2 ⁇ M 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 chick chorio allantoic membrane
• assay a model for developmental angiogenesis
• 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) .
• TSA also had angiostatic activity in the CAMs (32% inhibition of microvessel formation at 400 ⁇ M, p ⁇ 0.001, Fig. 4D) .
• 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.
• total genomic 5-methylcytosine content was quantified by high-performance capillary electrophoresis [26] .
• angiogenesis inhibiting genes could be explained by the reexpression of angiogenesis inhibiting genes in activated EC.
• all of these genes contain promoter CpG islands.
• 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) .
• meaningful differences in promoter methylation patterns of these genes between silenced- and activated EC seemed to be not present .
• 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.
• tumor angiogenesis An important aspect of tumor growth is tumor angiogenesis .
• 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.
• 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.
• 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] .
• 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.
• 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.
• the cell cycle inhibitors pl4ARF [37] , pl5INK4b [38, 39] and pl6INK4a [40] thereby reducing tumor cell growth.
• DNMT- and HDAC inhibitors in addition to their inhibitory effects on tumor cell growth, might indirectly exhibit angiostatic effects in vivo.
• 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] .
• pl6INK4a and p73 [16] which negatively regulate VEGF expression [41, 42]
• 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
• 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.
• DNMTs have additional transcriptional repressor functions apart from their methylation ability.
• DNMTs bind to HDACs and can repress gene transcription through histone deacetylase activity [55, 56] .
• 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 .
• HUVEC Human umbilical vein endothelial cells
• 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 37 0 C, 5% CO 2 . 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.
• bFGF basic Fibroblast Growth Factor
• VEGF Vascular Endothelial Growth Factor
• 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 ⁇ mol/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) .
• PHA phycohaemagglutinin
• Bovine capillary endothelial cells were kindly provided by Dr. M. Furie (State University of New York, Stony Brook, USA) and were cultured on gelatin coated flasks in MEM- ⁇ (Life Technologies, Breda, the Netherlands) supplemented with 10% FCS and 2 mM L-glutamin and antibiotics.
• DNMT DNA methyltransferase
• DAC 5-aza-2 ' -deoxycytidine
• HDAC histone deacetylase
• TSA histone deacetylase
• 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.
• mice 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] .
• 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 ⁇ Ci [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 -20 0 C.
• 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 0 C.
• DNA extraction buffer 45 mM Na2HPO4, 2.5 mM citric acid and 0.1% Triton X-100
• PI Propidium iodide
• HUVEC migration was measured using the wound assay [24] .
• 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.
• BCE bovine capillary EC
• 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] .
• 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 ⁇ -MEM (Life Technologies, Breda, The Netherlands), 1 volume 11.76 mg/ml sodium bicarbonate, 20 ng/ml bFGF and 20 ng/ml VEGF.
• CAM assay was performed as previously described [58] . CAMs were treated by daily addition of sterile saline
• HUVECs 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 ⁇ M) 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.) .
• 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.
• 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'
• Genomic DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega) . Bisulfite modification of genomic DNA was carried out as described previously [62] .
• 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) :
• IGFBP3 (-251; -29) 5'-GGGTATATTTTGGTTTTTGTAGA-S'
• IGFBP3 (-53; +189) 5'-GTGTTTTGGGTTATTTYGGTT-S'
• IGFBP3 (+167; +602 ) 5 ' -GTTGATTTTGTTGGTGTTGTTT-3 ' (SEQ ID NO:17)
• 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'
• IGFBP3 (-283; -123) 5 ' -GTGCTGAGGTGGCCTGGAGT- 3 '
• IGFBP3 (-198; -65) 5' -CGAGGAGCAGGTGCCCG- 3 '
• IGFBP3 (-2; +134) 5 ' -CCAGATGCGAGCACTGCG-3 '
• CDKN2/pl6/MTSl gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res, 1995. 55 (20) : p. 4525-30.
• Tumor angiogenesis is accompanied by a decreased inflammatory response of tumor- associated endothelium. Blood, 1996. 88(2): p. 667-73.
• Fuks, F., et al . , Dnmt3a binds deacetylases and is recruited by a sequence-specific repressor to silence transcription. Embo J, 2001. 20(10) : p. 2536-44.
• BPI Bactericidal/permeability-increasing protein

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