WO2005116066A1 - Peptides derived from decorin leucine rich repeats and uses thereof - Google Patents

Abstract

A first aspect of the invention provides a peptide consisting of from 5 to 40 amino acids from the portion of decorin which contains the leucine-rich repeats (LRRs), or a variant of said peptide. A second aspect of the invention provides a peptide consisting of no more than 250 amino acid residues comprising two or more peptides of the first aspect of the invention, or a peptide of the first aspect of the invention and additional peptide sequence not derived from decorin, or a variant of said peptide of no more than 250 amino acid residues. The peptides of the invention may be used for inhibiting undesirable angiogenesis, for example in the treatment of cancer.

Description

Peptides Derived From Decorin Leucine Rich Repeats and Uses Thereof

The present invention relates to compounds and uses thereof and, in particular, it relates to peptides and variants thereof and their use in inhibiting angiogenesis. The peptides and variants thereof may be used for treating cancer.

Angiogenesis, the formation of new blood vessels, is associated with many human diseases including cancer, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis and psoriasis. Both tumour growth and metastasis depend on angiogenesis stimulated by various angiogenic growth factors released by tumour cells. Thus, anti-angiogenesis is a promising alternative approach in anti-cancer drug development with several advantages over traditional cancer treatments including lower toxicity and less likelihood that the cancer will become resistant to treatment, and the potential for treating a wide range of tumours.

Decorin is a matrix proteoglycan belonging to the small leucine rich repeat (LRR) proteoglycans family. It contains ten LRR repeats at the C-terminal. It is mainly secreted by fibroblasts. Its normal physiological function is to fine tune the collagen fibrinogenesis. It has several binding domains for other matrix molecules such as fibronectin, thrombospondin, collagen, and growth factor TGF-beta, EGF receptor, as well as several metal ions.

Decorin binds to collagens Type I, II and IV and promotes the formation of fibres with increased stability. It has been found to have anti-angiogenic properties. It suppresses tumour cell mediated angiogenesis by inhibiting VEGF production by tumour cells (see, for example, Schonherr et al (1999) Eur J Cell Biol 78, 44-55; Stander et al (1999) Cell Tissue Res 296, 221- 227; Merle et al (1999) J Cell Biochem 75, 538-546; Nelimarkka et al (2001) Am J Pathol 158, 345-353; Davies et al (2001) Microvasc. Res 62, 26-42; and Grant et al (2002) Oncogene 21, 4765-4777).

WO 90/00194 reports that decorin can suppress cell proliferation. US Patent No 5,705,609 relates to the binding of decorin to TGF beta and describes peptides from the N-terminal region of decorin which inhibit the binding of decorin to TGF beta; it also describes fusions between maltose binding protein (MBP) and portions of decorin, some of which inhibit binding of decorin to TGF beta. US Patent No 6,277,812 Bl relates to the prevention or reduction of scarring by administering decorin to a wound. However, none of these publications describe the use of portions of decorin to inhibit angiogenesis, and none of them describe free peptides derived from the central or C-terminal portions of decorin.

The listing or discussion of any prior published document in this specification is not necessarily to be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Surprisingly, we have found that synthetic peptides containing portions of the central part of decorin function as angiogenic inhibitors. In particular, a peptide representing the complete fifth LRR (LRR5; peptide dLRR5, 26 amino acids) has a unique leucine organization and compared to all other LRRs in decorin, it has a longer sequence with extra amino acids and has the ability to bind collagen I but not TFG-beta, both molecules known to play a role in angiogenesis. Peptides based on LRR3, LRR4 and mutated version of LRR5 (including shortened peptides derived from LRR5) were also assessed for their anti-angiogenic functions. We have used various in vz^'trø angiogenesis assays to analyze the function of the decorin peptides. These assays include an endothehal cell migration assay, a matrigel tube formation assay and an apoptosis assay, relevant to the major events taking place during angiogenesis.

An object of the invention is the provision of anti-angiogenic peptides and variants thereof for use in the treatment of angiogenesis related diseases. Currently, a number of anti-angiogenic proteins are in clinical trials. The major limitations for use of proteins as pharmaceuticals are their lower bioavailability and biostability, difficulty to produce them in large quantity, immune responses from host, and the need to administer to patients by injection. These problems can be circumvented using small peptides and/or peptide mimetics. Peptides are also easily soluble in water and can potentially penetrate cell membrane and have the potential to be further developed into orally active or nasal-spray-type agents for therapeutic applications.

The peptides may be used as drugs in any diseases related to inbalanced angiogenesis including cancer, rheumatoid arthritis, psoriasis, infertility, delayed wound healing, ulcer, macular degeneration, diabetic retinopathy, and the like.

A first aspect of the invention provides a peptide consisting of from 5 to 40 amino acids from the portion of decorin which contains the leucine- rich repeats (LRRs), or a variant of said peptide.

The amino acid sequence of human decorin is shown in Figure 18, and the said portion in human decorin corresponds to amino acid residues 82 to 316. Equivalent portions from decorins from other species may also be used for the design and synthesis of peptides of the invention, and variants thereof. The GenBank accession number of human decorin is M98263 and for Swiss-Prot is P07585.

The peptide may be from 5 to 35 residues, 5 to 30 residues, for example, from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 residues. Peptides of from 10 to 30 residues are preferred, such as peptides of 12, 13, 21, 24 and 26 residues.

Typically, the peptides have amino acid sequence from the leucine rich repeats, LRRl, LRR2, LRR3, LRR4, LRR5, LRR6, LRR7, LRR8, LRR9 or LRR10. The LRRs of human decorin are shown in Figure 4 and, in particular, the amino acid residues of each in turn occupy residues: (1) 82 to 105; (2) 106 to 129; (3) 130 to 150; (4) 151 to 174; (5) 175 to 200; (6) 201 to 221; (7) 222 to 245; (8) 246 to 269; (9) 270 to 292; and (10) 293 to 316.

It is particularly preferred if the peptide consists of from 5 to 26 amino acids from LRR5 of decorin, or a variant of such a peptide. Suitable such peptides include QMIVIELGTNPLKSSGIENGAFQGMK (SEQ ID No 2); QMIVIELGTNPLK (SEQ ID No 4); SSGIENGAFQGMK (SEQ ID No 5); or LGTNPLKSSGIE (SEQ ID No 6) or smaller portions thereof, or a variant of said peptide. Preferably, the peptide has from 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acid residues from LRR5, or a variant of such a peptide.

It is also preferred if the peptide consists of from 5 to 24 amino acids from LRR4 of decorin, or a variant of said peptide. Such a peptide includes TLQELRAHENEITKVRKVTFNGLN (SEQ ID No 8), or a portion thereof, or a variant thereof. Preferably, the peptide has from 6, 1, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 23 amino acid residues from LRR4, or a variant of such a peptide.

It is also preferred if the peptide consists of from 5 to 21 amino acids from LRR3 of decorin, or a variant thereof. Such a peptide includes KLERLYLSKNQLKELPEKMPK (SEQ ID No 7), or a portion thereof, or a variant thereof. Preferably, the peptide has from 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19 or 20 amino acid residues from LRR3, or a variant of such a peptide.

The invention also provides a peptide consisting of no more than 250 amino acid residues comprising two or more peptides as defined above, or one or more peptides as defined above and additional peptide sequence not derived from decorin, or a variant of said peptide of no more than 250 amino acid residues. It is particularly preferred that the additional peptide sequence is not that of bacterial maltose binding protein (MBP).

It will be appreciated that two or more of the same or different peptides as defined above may be joined to form a larger peptide, provided that the peptide does not exceed 250 (and preferably does not exceed 200, 150, 100, 90, 80, 70, 60, or 50) amino acid residues. Preferably, the same peptide is repeated two or more times in tandem. Preferably, the decorin peptide portion comprises no more than 100 amino acid residues (when peptides of the first aspect of the invention are present in tandem) and preferably the decorin peptide portion contains no more than 80 or 70 or 60 or 50 or 40 or 30 or 20 or 10 amino acid residues. A preferred embodiment of this aspect of the invention is a peptide comprising two human LRR5 peptides in tandem joined by a linker of between 1 and 20 amino acid residues, preferably between 1 and 10. It will be appreciated that in addition to the peptide or variant thereof based on the decorin amino acid sequence, the peptide of this embodiment may comprise additional amino acid residues whose sequence is not based on decorin. The additional sequence may be based on other known protein sequences such as another anti-angiogenic peptide or a scaffold peptide or a peptide to target the peptide to tumour blood vessels such as one containing the amino acid sequence RDG or may be based on non-natural sequences. The additional amino acid residues may be N-terminal to the decorin-based peptide, or may be C-terminal to the decorin-based peptide, or both such that the additional sequence flanks the decorin-based peptide. Typically, there are between 1 and 10 additional amino acid residues, but there may be as many as 80. Typically, in this embodiment, the total size of the peptide does not exceed 80, or 70, or 60, or 50, or 40, or 30, or 20 or 15 amino acid residues. Variants of the peptides of this embodiment are also included within the scope of the invention.

The peptides of the invention typically and preferably comprise amino acids in the L-configuration, but see below. The peptides of the invention, or variants thereof, may be cyclised (ie do not have a free N- or C-terminal), or they may be linear or branched chain.

Variants of the abovementioned peptides of the invention include, but are not limited to, ones in which up to 40% of the decorin amino acid sequence of the decorin-containing portion are replaced with other amino acids, for example 30%, 20% or 10%. Typically, one or two or three or four or five or six or seven amino acids from the decorin sequence are replaced with another amino acid. The replacement may be with another naturally- occurring amino acid, or it may be with a non-naturally-occurring amino acid. Typical replacements include the replacement of an L-amino acid with a corresponding D-amino acid, or the replacement of one amino acid with a conservative amino acid, such as within the groups A,V; F, Y, W; T, S; I, L, V; D, E; and Q, N, H, although non-conservative substitutions are also contemplated.

A variant of a peptide also included those which contain one or more non- peptide linkages in place of a peptide bond. Variants also include peptides in which the N- or C-termini, or both, are blocked in order to confer resistance to hydrolysis. Similarly, one or more side chains of the amino acid residues may be modified to confer such resistance or other desirable properties. Strategies to improve the stability and bioavailability of peptides, and make them more useful as drugs, are well known in the art. In particular, reference is made to Adessi & Soto (2002) Current Medicinal Chemistry 9, 963-978, incorporated herein by reference, which describes such strategies in detail.

Variants of peptides fall into several classes which may conveniently be termed "modified peptides", "pseudopeptides" and "peptide mimetics".

Examples of modified peptides are where the N- and/or C-terminal ends are modified, for example by amino acylation or carboxy amidation or by the inclusion of a pyroglutamate group at the N-terminus. Other modified peptides are cyclisation of the peptide (ie where the -NH₂ and -COOH termini are condensed to form a peptide bond), amide nitrogen alkylation, D-amino acid substitution and side chain modifications.

Pseudopeptides are where there is alteration or replacement of some of the atoms participating in the peptide backbone resulting in molecules that are only partially peptides. These include amide bond surrogates (eg a replacement of the -CONH- group with -CH₂CH₂-. -CH₂NH-, -CH=CH-, -CH₂S-, -CH₂SO-, -CHCH₃S- and so on), peptoids and aza peptides. Peptoids are where the side chain which is attached to the I-carbon in the peptide structure is "switched" by one position to the amide nitrogen. Azapeptides are molecules in which one or more I-carbon atoms have been replaced by nitrogen atoms. When all the I-carbons are replaced, the molecule is called an azatide.

The peptides and variants of the invention typically have anti-angiogenic activity which may be assessed either in vitro or in vivo. Preferably, the peptide or variant has anti-angiogenic activity both in vitro and in vivo. Anti-angiogenic activity may be assessed in vivo using model systems where new blood vessel formation is assessed such as in xenograft tumour models. Anti-angiogenic activity may be assessed in vitro using, for example, a VEGF -induced endothehal cell migration assay such as the HUVEC assay described in the Example below. Typically, the peptide or variant of the invention may bind any one or more of collagen, gelatin or fibronectin and tests for determining binding to these molecules are known in the art.

The peptide M-hdLRR5 (SEQ ID No 6; also termed LRR5M) is able to inhibit tube formation. The peptide C-hdLRR5 (SEQ ID No 5; also termed LRR5C) is able to inhibit endothehal cell migration. Both are believed to be useful for inhibiting angiogenesis. Thus, preferred peptide of the invention are these peptides and peptides of up to 40 amino acid residues containing these amino acid sequences, or variants of the peptides.

It will therefore be appreciated that the peptides and variants of the invention include those which have substantially the same activity in the in vitro assays as the dLRR5 peptide (SEQ ID No 2), for example in the HUVEC migration assay of the Example. Suitably, the peptide mimic comprises a moiety which has substantially the same charge distribution and/or spatial configuration as any one of the specific peptides discussed above, in particular QMIVIELGTNPLKSSGIENGAFQGMK (SEQ ID No

2).

Some of the variants may be non-peptide mimics of the peptides and include molecules which have the same charge distribution and/or spatial configuration as any one of the peptides specific peptides discussed above, in particular QMIVIELGTNPLKSSGIENGAFQGMK (SEQ ID No 2).

The peptides and variants of the invention typically have a molecular mass of from around 800 to 10000, typically around 2000 to 5000.

When the compound of the invention is a peptide it may be synthesised using well known methods in the art. For example, peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9- fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N- dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl .ester (functionalising agent). The peptide-to-resin cleavable linked agent used is the acid-labile 4- hydroxymethyl-phenoxy acetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N,N-dicyclohexyl-carbodiimide/ 1 -hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50%) scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice depending on the constituent amino acids of the peptide being synthesised. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent titration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB), electrospray or matrix-assisted laser desorption ionization mass spectrometric analysis. Thus a further aspect of the invention provides a method of making a peptide or variant thereof of the invention comprising chemically synthesising said peptide or variant.

Alternatively, when the peptide of the invention is of a suitable size, such as greater than about 50 residues in length, it may be desirable to produce the peptide by recombinant DNA technology. Thus a further aspect of the invention provides a method of making a peptide the method comprising expressing the said peptide from a polynucleotide or an expression vector or in a host cell as discussed in more detail below.

The peptides of the invention may be encoded by a suitable polynucleotide which may be obtained or synthesised by methods well known in the art, for example as described in Sambrook & Russell (2001) "Molecular cloning, a laboratory manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

The polynucleotide, typically DNA, is then expressed in a suitable host to produce a peptide comprising the compound of the invention. Thus, the DNA encoding the peptide constituting the compound of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the peptide of the invention.

The DNA encoding the peptide constituting the compound of the invention may be joined to a wide variety of other DNA sequences for introduction into an appropriate host. The companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired. Generally, the ! 12

DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector. The vector is then introduced into the host through standard techniques. Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the peptide, which can then be recovered.

Many expression systems are known, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells.

The present invention also relates to a host cell transformed with a polynucleotide vector construct of the present invention. The host cell can be either prokaryotic or eukaryotic. Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RRl available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343). Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line. Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650. In addition to the transformed host cells themselves, the present invention also contemplates a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.

The peptides of the invention are useful in inhibiting angiogenesis, for example in a human or animal patient. Thus a further aspect of the invention provides the peptide or variants of the invention for use as a medicament. The invention also provides pharmaceutical compositions comprising the peptide or variant of the invention and a pharmaceutically acceptable carrier.

The compositions or formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (peptide or variant of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in- oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste. A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.

It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.

It will be appreciated that some of the compounds of the invention will be in the form of salts.

Salts which may be conveniently used in therapy include physiologically acceptable base salts, for example, derived from an appropriate base, such as an alkali metal (eg sodium), alkaline earth metal (eg magnesium) salts, ammonium and NX ⁺ (wherein X is Cμ alkyl) salts. Physiologically acceptable acid salts include hydrochloride, sulphate, mesylate, besylate, phosphate and glutamate.

Salts according to the invention may be prepared in conventional manner, for example by reaction of the parent compound with an appropriate base to form the corresponding base salt, or with an appropriate acid to form the corresponding acid salt.

The aforementioned compounds of the invention or a formulation thereof may be administered by any conventional method including oral and parenteral (eg subcutaneous or intramuscular) injection. The treatment may consist of a single dose or a plurality of doses over a period of time.

Whilst it is possible for a compound of the invention to be administered alone, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers. The carrier(s) must be acceptable in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.

It is particularly preferred if the peptides or variants thereof are delivered across the oral mucosa (including buccal mucosa, sub-lingual mucosa or gingival mucosa) or administered by intravenous or subcutaneous or intramuscular injection in liquid form (eg in an aqueous solution).

The dose or doses of the peptide or variants thereof of the invention may be determined by the physician and may vary depending on the condition to be treated. Typically the dose is effective to ameliorate the symptoms of the disease or condition to a useful extent.

A further aspect of the invention provides a method of inhibiting angiogenesis in a patient, the method comprising administering a peptide or variant of the invention, or a polynucleotide or an expression vector encoding said peptide. Typically, the patient has or is susceptible to a condition in which there is undesirable angiogenesis, or in which it is desirable to inhibit angiogenesis. Undesirable angiogenesis is associated with a variety of diseases and medical conditions. These include cancer, certain infectious diseases, certain autoimmune disorders, vascular malformations, DiGeorge Syndrome, HHT, cavernous hemangioma, atherosclerosis, transplant arteriopathy, obesity, psoriasis, warts, allergic dermatitis, scar keloids, pyogenic granulomas, blistering disease, Kaposi sarcoma in AIDS patients, persistent hyperplastic vitreous syndrome, diabetic retinopathy, retinopathy of prematurity, choroidal neovascularisation, primary pulmonary hypertension, asthma, nasal polyps, inflammatory bowel and periodontal disease, ascites, peritoneal adhesions, endometriosis, uterine bleeding, ovarian cysts, ovarian hyperstimulation, arthritis, synovitis, osteomyelitis and osteophyte formation (see, for example, Carmeliet (2003 Nature Medicine 9, 653-660, incorporated herein by reference). Thus a further aspect of the invention includes a method of treating a patient who has or is susceptible to any of these diseases or conditions, particularly cancer, diabetic retinopathy, macular degeneration, rheumatoid arthritis, ulcers, endometriosis and psoriasis, the method comprising administering to the patient an effective amount of a peptide or variant of the invention or a polynucleotide or expression vector encoding the same.

Still further aspects of the invention include the use of a peptide or variant of the invention or a polynucleotide or expression vector encoding the same in the manufacture of a medicament for treating undesirable angiogenesis in a patient; and the use of a peptide or variant of the invention or a polynucleotide or expression vector encoding the same in the manufacture of a medicament for treating a patient who has or is susceptible to any of cancer, diabetic retinopathy, macular degeneration, rheumatoid arthritis, ulcers, endometriosis and psoriasis. The invention will now be described in more detail with reference to the following Examples and Figures wherein:

Figure 1. Decorin inhibits EC tube formation on matrigel. 1A. Representative photos showing the inhibitory effect of two types of decorin: the native proteoglycan from bovine cartilage (DCN) and the recombinant human decorin (rhDCN) in the EC tube formation assay in the presence of 15 ng/ml VEGF, 15 ng/ml bFGF or 10% FBS. The assay was done using growth factor reduced matrigel. IB. Bar diagram showing the quantification of the inhibitory effect.

Figure 2. LRR5 domain in decorin core protein potently inhibits EC tube formation on matrigel. 2 A. List of peptides used in this study. 2B. Representative photographs of tube formation assay using growth factor reduced matrigel. Each sample was incubated in media containing 0.5% FBS plus 100 μM peptide in the presence of 15 ng/ml VEGF. 2C. Quantitation of tube formation inhibition by LRR peptides. LRR5 dose dependently inhibited VEGF induced tube formation.

Figure 3. LRR5 inhibits both VEGF and bFGF induced HUVEC migration. 3A. LRR5 inhibits EC migration in a dose dependent manner. VEGF₁₆₅ (1 ng/ml) was used as chemotactic agent. 3B. bFGF (10 ng/ml) was used as chemoattactant. The % migration in Y axis is the mean value from three sets of experiments.

Figure 4. LRR5 does not inhibit EC proliferation. 4A. EC proliferation induced by 10 ng/ml VEGF. LRR5 does not inhibit proliferation up to 10 μM concentration while at 100 μM there was a mild inhibition. 4B. EC proliferation induced by 10 ng/ml bFGF. LRR5 does not have any effect on bFGF stimulated EC proliferation.

Figure 5. LRR5 induces HUVEC apoptosis through the caspase dependent pathway. 5A. Observation of fragmented chromosomes under microscope by staining DNA with Hoechst dye 33258. White arrows indicate apoptotic cells with fragmented chromosomes. 5B. Quantitation of apoptosis using the cell death ELISA assay. The experiment is done in the presence or absence of 10 ng/ml VEGF and/or 10 μM z-VAD-fmk, a pan caspase inhibitor. LRR5 induced apoptosis even in the presence of VEGF, but this effect is almost completely inhibited by z-VAD-fmk.

Figure 6. Middle region of LRR5 potently inhibits EC tube formation. 6A. Representative photographs of anti-tube formation activity of LRR5N, LRR5M and LRR5C. Peptide LRR5M potently inhibits VEGF stimulated EC tube formation on growth factor reduced matrigel in a dose dependent manner. 6B. Quantitation of tubes formed in the presence of 15 ng/ml VEGF. 6C. Quantitation of tubes formed in the presence of 15 ng/ml bFGF. The values are mean tube lengths from two repeated experiments.

Figure 7. Effect of LRR5 shorter peptides on HUVEC migration. 7A. The anti-migration effect of LRR5N, LRR5M and LRR5C peptides when stimulated with 10 ng/ml VEGF. LRR5C dose dependently inhibits VEGF stimulated EC migration. 7B. The anti-migration effect of LRR5N, LRR5M and LRR5C peptides when stimulated with 10 ng/ml bFGF. LRR5C does not inhibit bFGF stimulated EC migration while peptide LRR5M is effective. The results are mean values from three different experiments. Figure 8. LRR5 shorter peptides do not inhibit EC proliferation. 8A. EC proliferation stimulated by VEGF at 10 ng/ml. Peptide LRR5N and LRR5C at concentrations 10 μM and above have mild inhibition in VEGF stimulated EC proliferation. 8B. EC proliferation stimulated by bFGF at 10 ng/ml. None of the peptides had any significant effect on bFGF stimulated EC proliferation. The results are mean values from three experiments.

Figure 9. Different activities of LRR5 sub-regions in inducing EC apoptosis. 9A. Representative photographs of apoptotic cells after TUTSEEL staining. The photos shown here are the effect of peptides in the presence of 10 ng/ml VEGF. LRR5, LRR5N and LRR5M are effective while LRR5C is not very effective. 9B. Quantitation of apoptosis using the cell death ELISA assay measuring fragmented DNA. The experiment is done in the presence or absence of 10 ng/ml VEGF and/or 10 μM z-VAD-fmk, a pan caspase inhibitor. LRR5, LRR5N and LRR5M induce apoptosis even in the presence of VEGF, but this effect is almost completely inhibited by z-VAD-fmk.

Figure 10. Effect of LRR5 and its related peptides in inhibiting EC attachment to fibronectin. EC attachment to fibronectin is inhibited by DCN, LRR5 and all three shorter peptides. LRR5 and LRR5N are more potent when compared to LRR5M and LRR5C. The values presented here are the mean of three repeated experiments.

Figure 11. LRR5 and LRR5M effectively inhibited VEGF stimulated FAK Y397 phosphorylation in HUVECs. IP: immunoprecipitation; WB: Western blot. Figure 12. Peptides LRR5 and LRR5M effectively inhibited VEGF induced paxillin relocation to focal adhesions. HUVECs were fixed, permeabilized and stained for paxillin. The concentration of peptides was 100 μM and recombinant DCN 100 nM. Peptide treated samples were all in the presence of 10 ng/ml VEGF. Experiment was repeated for three times and representative pictures are shown. Focal adhesions are indicated by white arrowheads. Images were obtained using Olympus 1x70 confocal microscope.

Figure 13. LRR5 and LRR5M effectively inhibited VEGF induced actin stress fiber formation. HUVEC cells were fixed and stained for filamentous actin with phalloidin TRITC conjugate. Peptide treated samples were all in the presence of 10 ng/ml VEGF. Experiment was repeated for three times. Pictures are representatives of 20 different microscopy locations in each sample. Nucleus is counter stained blue with Hoechst dye 33258. Actin stress fibers are indicated by white arrowheads. Images were obtained using a Zeiss fluorescent microscope with 63 OX magnification.

Figure 14. CD spectra of LRR5, its scrambled and three shorter derivatives. The concentration of peptides was 1 mM.

Figure 15 shows that peptide hdLRR5 (SEQ ID No 2; also termed LRR5) inhibits VEGF induced HUVEC migration. 1. No growth factor control; 2. lOng/ml VEGF; 3-8: lOng/ml VEGF plus decorin hdLRR5 peptide at concentrations of lOOμM; lOμM; IμM; lOOnM; lOnM; InM.

Figure 16 shows that peptide hdLRR5 inhibits tube formation in matrigel. Figure 17 shows that decorin peptide dLRR5 induce EC apoptosis as determined by TUNEL labeling. Green fluorescences (which cannot be seen in the black and white picture) indicate apoptotic cells.

Figure 18 shows the human decorin protein sequence (SEQ ID No 1) and annotation. The signal peptide (residues 1 to 16) is underlined. The pro- peptide (residues 17-30) is shown in italic. Mature decorin is residues 31- 359. The LRRs are shown in alternative upper case and lower case starting at position 82. LRRS is in bold and underlined (residues 175 to 200). The glycosaminoglycan (GAG) binding site is at S34 shown in bold upper case and underlined. Residues N211, N262 and N303 are three sites for binding to oligosaccharide carbohydrates (N-linked glycosylation) and are underlined. An independent N-terminal collagen I binding domain is at residues 48 to 72. LRR6 binds EGFR. LRR3 binds fibronectin and thrombospondin. C-terminal domain (residues 309-359) also binds fibronectin. The N-terminal domain also binds fibrinogen in the presence ofZn⁺⁺.

Figure 19 shows that hdLRR5 (SEQ ID No 2) induces endothehal cell apoptosis at high concentration (100 TM). Apoptotic cells are indicated by arrows.

Figure 20 shows the results of tube formation assays. Decorin peptides inhibit tube formation in matrigel dLRR5 is SEQ ID No 2; sd LRR5 is SEQ ID No 3; dLRR5 M is SEQ ID No 6; dLRR5 N is SEQ ID No 4; dLRR5C is SEQ ID No 5; decorin is SEQ ID No 2.

Figure 21 shows the results of the effect of decorin peptides on HUVEC migration. 1. No Growth Factor (-); 2. 10 ng/ml VEGF (+); 3. 3 Tg/ml Decorin (+); 4. 1 TM LRR3 (+); 5. 1 TM LRR4 (+); 6. 1 TM LRR5 (+); 7. 1 TM Scramble LRR5 (+).

Decorin is SEQ ID No 1; LRR3 is SEQ ID No 6; LRR4 is SEQ ID No 7; > LRR5 is SEQ ID No 2; and Scramble LRR5 is SEQ ID No 3.

Example 1: Peptides derived from human decorin core protein leucine Rich Repeat 5 potently inhibit angiogenesis by multiple mechanisms

i The abbreviations used in this Example are: DCN: decorin; LRR: Leucine Rich Repeat; VEGF: Vascular Endothehal Growth Factor; bFGF: basic Fibroblast Growth Factor; TGF: Transforming Growth Factor; TNF: Tumor Necrosis Factor; FBS: Fetal Bovine Serum; BSA: Bovine Serum Albumin; GAG: glycosamino glycan; PG: proteoglycan; PBS: Phosphate-Buffered Saline; FITC: Fluorescein Isothiocyanate; TUNEL: Terminal Deoxynucleotidyl Transferase-Mediated dUTP nick-end-labeling; HUVECs: Human Umbilical Vascular Endothehal Cells; FAK: Focal Adhesion Kinase; EC: endothehal cell, CD: Circular Dichroism.

Excessive angiogenesis is involved in many human diseases and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports of whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrate that not only purified decorin, but also the 26 residue Leucine Rich Repeat 5 (LRR5) of decorin core protein, functions as potent angiogenesis inhibitor by inhibiting both Vascular Endothehal Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF) induced angiogenesis. Peptide LRR5 inhibited multiple aspects of angiogenesis including Vascular Endothehal Growth Factor (VEGF) stimulated endothehal cell (EC) migration, tube formation on matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that peptides derived from different sub-regions of LRR5 inhibited different aspects of angiogenesis with the middle region (LRR5M, 12 residues) inhibited endothelial cell tube formation up to 1000 times more potently than LRR5. While the C-terminal region (LRR5C) potently inhibited VEGF stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose dependently similar to LRR5 through a caspase dependent pathway, LRR5C has no such function. We further shown that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF induced focal adhesion kinase activation and the assembly of focal adhesions as well as actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to matrix. Circular Dichroism studies revealed that LRR5 undergoes an inter-conversion between 3_]0 helix and β-sheet structure in solution, a characteristic potentially important for its antiangiogenic activity. Peptides LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into antiangiogenic drugs.

Proteins carry out their functions by interacting with other molecules through their functional domains. These domains vary in size, composition and structure. In recent years, a lot of efforts have been made to develop proteins for therapeutic applications. However, use of proteins as drugs, has limitations due to their poor bioavailability, antigenicity, unfavorable pharmacokinetics and inconsistency in bioactivity from batch to batch productions (1). In contrast, small peptides have the advantage of easy antigenicity, high solubility in water and improved bioavailability with potential oral delivery (2). Such peptides can represent a complete or partial protein functional modules as well as possible protein-protein interaction sites (3-6).

Angiogenesis, the formation of new blood vessels from existing vasculature, is a multistep process involving degradation of extracellular matrix, endothelial cell proliferation and migration, capillary tube formation and matrix remodeling (7, 8). Many proteins including growth factors and their cell surface receptors, extracellular matrix molecules, integrins, matrix metalloproteases and their inhibitors are involved in this process. Several endogenous proteins such as endostatin and angiostatin, fragments of collagen and plasminogen respectively, are potent inhibitors of angiogenesis (9, 10). As excessive angiogenesis is involved in many human diseases, developing angiogenesis inhibitors is an important area of drug development.

Decorin, a small dermatan sulfate proteoglycan (PG), is a ubiquitous component of extracellular matrix, where it is preferentially found in association with collagen fibrils. Its major functions include regulation of collagen fibrinogenesis, maintenance of tissue integrity via binding with fibronectin and thrombospondin as well as serving as a reservoir of Transforming Growth Factor β (TGFβl) (11-14). Decorin inhibits cell adhesion and spreading on fibronectin (15). It also inhibits the growth of various tumor cells with a diverse histogenetic background when either overexpressed in tumor cells or provided as a recombinant protein primarily via a protracted inactivation of the epidermal growth factor receptor (EGFR) tyrosine kinase (16,17). Decorin expression is induced in endothelial cells during angiogenesis, but not when migration and may support the formation of fibrillar pericellular matrix that stabilizes the differentiated endothelial phenotype during the later stages of angiogenesis (18). However, there have been conflicting reports of decorin as an angiogenesis inhibitor. Decorin was reported to indirectly suppress tumor angiogenesis through inhibition of VEGF production by tumor cells (19). Increased expression of decorin in endothelial cells through retroviral transduction inhibited EC migration (18). In addition, purified decorin inhibited endothelial cell migration and tube formation in collagen I lattices when ECs were grown on decorin coated surfaces (20). On the other hand, recombinant decorin was reported to have no effect on endothelial cell tube formation on matrigel (19) and decorin expression in EA.hy926 ECs by adenovirus transduction promoted tube formation in collagen lattices while inhibiting EC apoptosis (21).

Structurally decorin belongs to a growing family of the small leucine rich repeat PGs. It contains a core protein of 359 amino acids linked to a single chondroitin sulfate or dermatan sulfate glycosamino glycans (GAGs) (11). The mature protein is highly conserved across species and consists of a central domain harboring ten Leucine Rich Repeats (LRRs) flanked by disulfide bonded terminal sequences. The amino terminus contains a single attachment site for GAG, whereas the central domain contains three potentially N-linked glycosylation sites. LRRs are involved in protein- protein interactions and have been found in a large number of proteins including PGs such as biglycan, fibromodulin and lumican (22). Decorin binds to collagen mainly through LRR 4 and 5 of the core protein (23). In addition, a high-affinity binding site for TGFβ is located between LRR3 and 5 (24). Decorin binding to TGFβ prevents the binding of TGFβ to its receptor and regulates TGFβ mediated cellular signaling (25). Recently, it was reported that biologically active decorin is a monomer in solution and,

proteins, growth factors, and cell surface receptors (26). However, crystal structure analysis of decorin indicated that it is a stable dimer with large interfaces (27).

In an effort to characterize the role of decorin core protein and its LRR repeats in angiogenesis and develop small peptide angiogenic inhibitors, we analyzed decorin core protein and the role of LRR3, 4 and 5 in angiogenesis using a synthetic peptide approach. The 26 residue LRR5 peptide was found to be a potent angiogenesis inhibitor and inhibited VEGF, bFGF as well as serum induced angiogenesis. LRR5 inhibits angiogenesis through multiple mechanisms including inhibition of VEGF induced EC tube formation on matrigel, EC migration as well as EC attachment to fibronectin. In addition, it also induced EC apoptosis through a caspase dependent pathway. Functional mapping identified sub-regions of LRR5 that inhibited different aspects of angiogenesis. Importantly, the central 12 residue (LRR5M) specifically inhibited EC tube formation up to 1000 times more potently than LRR5. While the C-terminal 13 residue (LRR5C) retained the ability to inhibit EC migration, the N-terminal 13 residue (LRR5N) is most effective in inhibiting EC attachment to fibronectin. Furthermore, both LRR5N and LRR5M induced EC apoptosis, with LRR5M as effective as LRR5. Further mechanistic study indicated that the abilities of LRR5 and LRR5M to inhibit EC tube formation is linked to their inhibition of VEGF induced FAK phosphorylation at Y397 and subsequent assembly of focal adhesions and actin stress fibers in ECs.

EXPERIMENTAL PROCEDURES

Reagents and Antibodies - Recombinant human VEGFι₆₅ ^and recombinant human decorin (rhDCN) were purchased from R&D Systems Inc.

Λ fl«_non_{« n}i ;_n T TC Λ Λ τ-> _ —- - τ-> -π-> r- ^π Purified decorin (DCN) from bovine articular cartilage, gelatin and Giemsa solution were from Sigma. Falcon cell culture inserts (8.0 μm pore size) and matrigel were from BD Biosciences (Bedford, MA, USA). Human plasma fibronectin was from Invitrogen. Endothelial culture media CSC were from Cell Systems (California, USA). Pan-caspase inhibitor z-VAD- fmk was from Merck. Sigma bovine articular DCN protein concentration was determined using Bio-Rad protein Coomassie Brilliant Blue G-250 dye binding assay and Sigma bovine serum albumin as standard. DCN purity was checked by both native and SDS PAGE and shown to be more than 95%) pure. The endotoxin levels in DCN and rhDCN were determined using ET0200 kit from Sigma.

Peptide Synthesis - Peptides were synthesized by solid phase synthesis using Perseptive Biosystems automated peptide synthesizer adopting FMOC (Fluorenyl methoxy carbonyl) chemistry in-house or procured from commercial source (SynPep, California, USA). Peptides were purified by reversed phase HPLC, freeze dried and stored at -20°C until used.

Cell culture - Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords and cultured in CSC complete medium at 37°C in a humidified 5% C0₂ atmosphere. All cells used for the experiments were from passages 3-6.

Endothelial Cell Tube Formation Assay - 2.5 ^χ l0⁴ HUVECs were pre- incubated with various concentrations of peptides (from 1 nM to 100 μM) for 30 min before seeded onto the solidified growth factor reduced matrigel in 96-well plate. After incubating in media with or without 10 ng/ml VEGF or bFGF at 37°C for 4-6 h, cells were fixed and tubes formation was analyzed by light microscopy (XI 00). Four random fields were chosen in each well and the total tube length was quantified by using NIH ImageJ 1.32 software.

Cell Migration Assay - Migration assay was performed using Falcon cell culture inserts as described previously with modifications (28). Briefly, HUVECs were starved overnight, trypsinized and suspended at a final concentration of 3^χ l0⁵ cells/ml. Various concentrations of peptides were pre-incubated with the 2-3x 10 cells cells for 30 min at 37°C before seeding onto the gelatin coated cell culture inserts. VEGF (10 ng/ml) was placed into the lower chamber. The assembled cell culture insert chamber was then incubated at 37°C for 10-12h. After removing the non-migrating cells with a cotton swab, migrated cells on the lower surface of the culture inserts were fixed, stained with 0.4% Giemsa and photographed under a light microscope ( 200). Five random fields were chosen in each insert and the cell number was quantified using the NIH ImageJ 1.32 software.

Proliferation and Cytotoxicity Analyses - Proliferation and cytotoxicity were determined using the non-radioactive EZ4U cell proliferation and cytotoxicity assay kit according to manufacturer's instructions (Biomedica, Vienna, Austria). The method was based on the fact that living cells are capable of reducing less coloured tetrazolium salts into intensely coloured Formazan derivatives. This reduction process requires functional mitochondria, which are inactivated within a few minutes after cell death. In a typical 96 well plate assay, we used around 3000 HUVECs/well and the absorbance of Formazan at 460 nm was represented as mitochondrial activity. The cells were incubated in CSC medium plus 2% FBS with or without 10 ng/ml VEGF or bFGF and various concentrations of peptides (1 nM to 100 μM) for 48 h. For cytotoxicity analyses, HUVECs were incubated with peptides for 6 h while 48 h peptide incubation was used to measure proliferation. Cell Attachment Assay - HUVECs were pretreated with different peptides for 20 min before plated onto fibronectin coated 24-well plate and incubated for 30 min at 37°C. Attached cells were then fixed and stained with Giemsa. The number of cells attached were quantified in 5 random fields per well. The assay was done in duplicate and repeated three times on separate days.

Apoptosis Assay - Apoptosis was analyzed by morphological detection of chromatin fragmentation as described before (29). HUVECs grown to 60-70%o confluence in 4-well-chamber slides were incubated with peptides for 48 h in 0.5% FBS in presence or absence of 10 ng/ml VEGF.. Cells were fixed, stained with Hoechst dye 33258 and observed under a fluorescent microscope (^χ630). Alternatively, TUNEL staining was performed using the ApoAlert DNA fragmentation assay kit (Clontech, USA). Apoptosis was quantified using a cell death detection ELISA kit (Roche Applied Sciences, Germany). Briefly, 20,000 HUVECs/well were seeded onto 0.2% gelatin coated 24 well plates for 24 h prior to treatment with peptides. Cells were treated with different concentrations of peptides, with or without 10 ng/ml VEGF for 24 h before harvested using trypsin. DNA fragmentation was measured by quantitation of cytosolic oligonucleosome bound DNA according to the manufacturer's instructions. The cytosolic fraction (13,000 x g) of HUVECs treated with peptides in the presence and absence of 10 ng/ml VEGF was used as antigen source in the Sandwich ELISA with anti- human histone antibody coated to the plate and a secondary anti-DNA antibody coupled to peroxidase for detection. The effect of peptides in inducing apoptosis was represented as absorbance value at 405 nm. The experiment was also performed in the presence of 10 μM benzyloxycarbonyl- Val-Ala-Asp (OMe) fuoromethylketone (Z-VAD- FMK), a pan-caspase inhibitor. Solid-phase Binding Assay - Ninety-six well microtiter plates were coated with 100 μg/ml collagen I or fibronectin solutions overnight at 4°C. Control wells were coated with 0.1% BSA in PBS. After rinsing with 0.15 M NaCl, 0.05% (v/v) Tween-20, the wells were pre-incubated with 0.1%) (w/v) BSA for 2 h before incubating with varying concentrations of FITC-labeled peptides for 4 h at room temperature. Florescence bound to wells was determined using Spectramax^R Gemini XS dual scanning spectrofluorometer (Molecular Devices, Germany). Saturation graphs were obtained using single site binding, non-linear regression analysis by Graph Pad Prism 4 (GraphPad Software Inc., USA). All analyses were done in triplicates.

Western blot and Immunoprecipitation - Starved HUVECs were treated with 30 μM peptides individually for 2 h before exposed to 15 ng/ml VEGF₁₆₅ or 30 min. The cells were lysed in cell lysis buffer (Cell Signaling Technology, USA). After adjustment for protein concentration with Bradford method, 100 μg of proteins from each sample were used for Western blot analysis. The blot was probed with primary rabbit anti-FAK antibody (Upstate Biotechnologies, USA) and horseradish peroxidase- labeled secondary antibody. The products were visualized by enhanced chemiluminescence reagent (Pierce, USA). On the other hand, 500 μg of proteins from the same samples were immunoprecipitated with rabbit anti- FAK antibody at 4°C overnight and probed with primary mouse anti- phospho-FAK (Y397) antibody (Chemicon, USA).

Analysis of Focal Adhesions and Actin Stress Fibers in HUVECs - Cells were grown to near confluence on chamber slides. VEGF (10 ng/ml) and peptides (100 μM) were added into the wells and incubated for 90 min at 37°C. Cells were fixed, permeabilized and stained with a mouse monoclonal antibody against paxillin (Upstate technology, USA) and FITC- labeled anti-mouse IgG (Santa Cruz Biotechnology, USA). Slides were observed under confocal microscope (Olympus 1X70). TRITC-coηjugated phalloidin (Sigma) was used to stain the actin cytoskeleton and Hoechst dye 33258 was used to counter stain the nucleus.

Circular Dichroism Spectral Analysis - Circular Dichroism (CD) spectra of all peptides were recorded on a JASCO J-810 spectropolarimeter using a 1 mm path length quartz cell at room temperature in 10 mM phosphate buffer pH 7.0 at 1 mM concentration. The spectra were recorded between 190 - 260 nm with a scan speed of 50 nm/min and were the average of 3 scans. Base line was subtracted for final representation.

RESULTS

Antiangiogenic Effect of Decorin and LRR5

There have been conflicting reports regarding the role of decorin in inhibiting angiogenesis when used as a soluble factor. To clarify the role of decorin on EC tube formation, we tested purified decorin from bovine articular cartilage as well as recombinant human decorin expressed in insect cells using HUVECs tube formation assay on matrigel. Both decorins were found to potently inhibit VEGF as well as bFGF stimulated HUVEC tube formation on growth factor reduced matrigel, with purified bovine articular decorin more potent than recombinant human decorin (Fig. 1A and IB). Both decorin preparations inhibited VEGF induced tube formation more potently than bFGF induced tube formation. This paper therefore mainly focuses on VEGF induced angiogenesis in subsequence studies. No endotoxin contamination was found in the decorin preparations (refer to methods section), confirming that the antiangiogenic activity is a specific activity of decorin (data not shown). LRR domains in decorin core protein play important roles in decorin function and LRR3-5 region has been shown to be the main region binding collagen I as well as TGFβ (14, 25). We set out to test if individual LRR3, 4 and 5 can inhibit HUVEC tube formation using a synthetic peptide approach. Figure 2A lists all the peptides used in this study. Peptide LRR5 effectively inhibited EC tube formation dose dependently while LRR3 and LRR4 at lOOμM only had marginal effect (Fig. 2B and 2C). Importantly, when LRR5 amino acid sequence was scrambled (LRR5S), the inhibition effect was completely lost. These peptides were not cytotoxic to ECs when tested up to 100 μM as shown in Table 1. Therefore, LRR5 can specifically inhibits VEGF induced EC tube foπnation on matrigel in a sequence dependent manner and functions as an angiogenic inhibitor. As angiogenesis involves multiple steps, we tested the LRR peptides for their ability to inhibit EC proliferation and migration. As shown in Figure 3, peptide LRR5 potently inhibited VEGF stimulated EC migration in a dose- dependent as well as sequence-dependent manner with an estimated ED₅o between 100 nM and 1 μM while LRR5S had no such activity. Purified bovine decorin also potently inhibited EC migration at nM concentration (Fig. 3A). Both decorin and LRR5 also inhibited bFGF induced EC migration (Fig. 3B), but much less effectively when compared to VEGF stimulation.

In contrast, neither decorin nor LRR5 had significant inhibitory effect on VEGF or bFGF stimulated EC proliferation, with LRR5 only has mild inhibition effect at 100 μM (Fig. 4A and 4B).

Induction of apoptosis in endothelial cells is one of the mechanisms utilized by antiangiogenic molecules to inhibit angiogenesis. To test whether LRR5 has the ability to induce apoptosis in HUVECs, apoptosis was analyzed by morphological detection of chromatin fragmentation, TUNEL staining as well as quantitative ELISA measurement of fragmented DNA in peptide treated cells. As shown in Figure 5A, LRR5 specifically induced apoptosis in HUVECs in a concentration dependent manner. At 100 μM, many cells with fragmented chromatins were observed. TUNEL staining also confirmed the morphological observation (Fig. 5B). ELISA measurement of DNA fragmentation indicated that LRR5 potently induced apoptosis in the presence or absence of VEGF at 100 μM, and this inhibition is caspase- dependent as the pan caspase inhibitor z-VAD-fmk effectively abolished its apoptosis inducing activity (Fig. 5C). Decorin at 100 nM did not induce EC apoptosis (Fig. 5C).

In summary, decorin and its core protein LRR5 domain are potent angiogenesis inhibitors. LRR5 peptide inhibits angiogenesis through multiple mechanisms including inhibition of EC tube formation, chemotactic migration, and induction of apoptosis. The antiangiogenic activity of LRR5 is both sequence and dose dependent.

Different Regions of LRR5 Mediate Different Antiangiogenic Functions

To map the sub-regions within LRR5 that are responsible for its antiangiogenic activity and obtain shorter peptide angiogenic inhibitors, the 26 residue -LRR5 was further truncated into three overlapping peptides representing the N-terminal (LRR5N, 13 residues), middle (LRR5M, 12 residues) and C-terminal (LRR5C, 13 residues) portion of LRR5 (Fig. 2A). Interestingly, LRR5M peptide inhibited VEGF induced HUVEC tube formation on matrigel much more potently than LRR5 peptide (Fig. 6A). While LRR5 inhibited tube formation in a dose dependent manner with ED₅₀ at around 100 nM, LRR5M completely inhibited tube foπnation at 10 nM concentration and its ED₅₀ is estimated to be 100 pM which is up to 1000 times more potent comparing to LRR5 (Fig. 6B). In contrast, peptides LRR5N and LRR5C cannot inhibit VEGF induced EC tube formation effectively. The LRR5 peptides also inhibited bFGF induced tube formation in a dose dependent manner, with the exception of LRR5C which is inactive (Fig. 6C). Consistent results were observed when experiments were repeated with different batches of HUVECs (data not shown).

When the three shorter peptides were analyzed for their ability to inhibit VEGF induced HUVEC migration, the 13 residue LRR5C peptide was as potent as LRR5 (Fig. 7A). Both LRR5 and LRR5C inhibited VEGF induced EC migration in a dose dependent manner with ED₅₀ between 100 nM and 1 μM. Although some inhibitions were observed with LRR5N, the effects were not dose dependent while LRR5M is inactive. In comparison, LRR5C is ineffective in inhibiting bFGF induced EC migration while LRR5M has only mild inhibitive effect at high concentrations (Fig. 7B).

Consistent with the insignificant inhibition of LRR5 on EC proliferation (Fig. 4A and 4B), LRR5N and LRR5C are only mildly inhibitive to VEGF stimulated EC proliferation at high concentrations while none of the shorter peptides inhibited bFGF stimulated EC proliferation (Fig. 8A and 8B).

When the three shorter peptides were tested for their ability to induce EC apoptosis, LRR5M is as active as LRR5 while LRR5N is less active (Figs. 5B; 9A and 9B). Similar to LRR5, both shorter peptides induced EC apoptosis in a caspase-dependent manner. In contrast, LRR5C lacks the apoptosis induction function.

In summary, the above results indicate that different sub-regions of LRR5 inhibit different aspects of angiogenesis. While the middle region inhibits EC tube formation, the C-terminal region is responsible for inhibiting EC migration towards VEGF. Both the N-terminal region (LRR5N) and the middle region (LRR5M) are involved in inducing EC apoptosis through the caspase mediated apoptosis pathway.

LRR5 Inhibited Angiogenesis Not by Interference of EC Attachment to Matrix

Decorin is known to bind many ECM proteins including fibronectin and collagen as well as growth factors such as TGFβ (11-15). To test if LRR5 and its shorter peptides also bind these proteins, various matrix proteins including gelatin, collagen I, fibronectin, growth factors (TGFβl, TGFβ2, VEGF and bFGF), fibrinogen and BSA were immobilized onto nitrocellulose membranes and probed with FITC-labeled peptides. All peptides could bind directly to the matrix proteins gelatin, collagen I and fibronectin, but not the other proteins tested (data not shown). Their binding affinities to fibronectin and collagen I were then determined using a solid phase binding assay. Peptide LRR5 and all the three shorter peptides have slightly higher binding affinity for collagen I compared with fibronectin, but the binding affinities are generally quite low with Kd in μM or even lower mM range (Table 2).

EC attachment and adhesion to ECM are critical for the progression of angiogenesis. Both migration and tube formation are initiated by attachment, and if the peptide acts as an anti-attachment factor, then both cell migration and tube formation could be inhibited. Since LRR5 and its related peptides all bind fibronectin and collagen I, they could interfere with EC's attachment to ECM and therefore inhibit angiogenesis. Indeed, when cells were pre-incubated with the peptides for 30 min before seeded onto fibronectin coated 96-wells, the number of cells attached were significantly reduced (Fig. 10). Significant inhibitions were observed with LRR5 and LRR5N even at concentration as low as 10 nM which was comparable with that of purified decorin. In contrast, peptides LRR5M and LRR5C were less effective in this function and only had obvious inhibitory effect at μM concentrations. Similarly, these peptides also inhibited EC attachment to collagen I dose dependently (data not shown). Based on these results, it is obvious that the ability of LRR5 to inhibit EC tube formation and migration is not due to its interference of EC's attachment to fibronectin as LRR5N which is as effective as LRR5 in inhibiting EC attachment to fibronectin, is not effective in inhibiting tube formation or migration.

LRR5 and LRR5M Inhibit VEGF Stimulated FAK Phosphorylation, Localization of Paxillin Into Focal Adhesions As Well As Formation of Actin Stress Fibers

To understand the differential functions of LRR5M and LRR5C peptides, additional mechanistic studies related to FAK were conducted. FAK is an important component of the focal adhesion complex which attaches cells to the extracellular matrix and is essential for cell migration, adhesion, survival and cell cycle control (30). It is a non-receptor tyrosine kinase, which integrates growth factor and integrin signals to promote cell migration. Proper migration of cells depends on both actin filament dynamics and adhesion complex remodeling. It has been reported that one of the mechanisms of VEGF stimulated EC migration is by stimulating FAK phosphorylation which activates FAK and initiates the assembly and rearrangements of focal adhesions (31). Activated FAK then phosphorylate and activate other focal adhesion associated proteins including paxillin which is immediately recruited into focal adhesion complexes. This is followed by an accumulation of actin stress fibers associated with new actin polymerization that interact with focal adhesion-associated paxillins (32). FAK is then inactivated by dephosphorylation and cleaved by cellular proteases, leading to the disassembly of focal adhesions as well as actin filament. During apoptosis, FAK is cleaved by caspases, generating FRNK, a C-terminal fragment of FAK that inhibits FAK function (33). As shown in Figure 11, both LRR5 and LRR5M suppressed VEGF stimulated FAK phosphorylation. LRR5M was more effective in inhibiting FAK phosphorylation compared with LRR5, consistent with its more potent activity in inhibiting EC tube formation and apoptosis induction. In contrast, LRR5C which is very effective at inhibiting EC migration, but not active in inhibiting tube formation and induction of apoptosis did not show any inhibitory effect on FAK phosphorylation. Rather, there is a mild stimulation of FAK phosphorylation by this peptide.

VEGF induced paxillin recruitment into focal adhesions, an indication of the formation and rearrangement of focal adhesions, was also effectively blocked by both LRR5 and LRR5M. As shown in Figure 12, the large number of VEGF induced patched paxillin staining (indicated by arrows) was almost completely abolished upon treatment with LRR5 and LRR5M, with LRR5M being more effective. Purified decorin also inhibited focal adhesion formation at 100 nM. While peptide LRR5N reduced the formation of focal adhesions to a much lesser extent, LRR5C and LRR5S were inactive (Fig. 12).

Consistently, LRR5 and LRR5M potently suppressed VEGF induced actin stress fiber formation as shown by immunofluorescent staining of actin filaments by TRITC-conjugated phalloidin (Fig. 13). While LRR5N was less effective, LRR5C was essentially ineffective in this function.

The above three experiments correlated well with each other to reveal that inhibition of EC tube formation on matrigel and induction EC apoptosis by peptides LRR5 and LRR5M are linked with their ability to suppress VEGF induced FAK phosphorylation, paxillin relocation into focal adhesions and actin stress fiber formation. The more potent LRR5M in inhibiting EC tube formation and induction of EC apoptosis is also more potent in suppressing FAK phosphorylation at Y397.

CD Spectral Studies Revealed a Stable Structure for LRR5 in Solution To study the structural basis of the peptide' s function, CD analyses of the peptides were carried out. The CD spectra of peptide LRR5 at 1 mM concentration showed a negative maximum around 212 nm and a negative shoulder around 222 nm indicating the possibility of a 3₁₀ helical structure (Fig. 14). Addition of trifluroethanol (TFE) at different ratios from 5 to 80 percent of TFE/water did not reveal any difference in LRR5 CD spectra, indicating LRR5 peptide has a stable helical structure (34). Furthermore, a weak bend near 208, and two negative minima at 212 and 222 nm could be indicative of this peptide undergoing an inter-conversion of 3₁₀ and β-sheet structure at pH 7.0. A similar absorption pattern was observed for peptide , LRR5N with a wide negative minima ranging from 209-240 nm and an intense sharp minima at 209 nm, inferring that this peptide also undergoes an inter-conversion between helix and β-sheet or β-turn structure. However, the structure of LRR5N is not stable since addition of TFE improves the helical shape by eliminating β-sheet (data not shown). Scrambled peptide LRR5S showed a strong negative absorption near 210 nm and weak negative absorption near 222-225 nm indicating that a 3_]0 helical structure. The CD spectra of LRR5C and LRR5M both showed a negative absorption at 202-205 nm which is an indication of type I β-turn.

DISCUSSION

Although decorin has been reported to have antiangiogenic activity under certain circumstances, we showed here for the first time that purified decorin can inhibit VEGF, bFGF as well as serum induced HUVEC tube formation on matrigel. Purified bovine articular decorin inhibited EC tube formation more potently than recombinant human decorin expressed in insect cells (Fig. 1). Furthermore, a peptide representing LRR5 domain of the core protein (26 residues) functions as a potent angiogenesis inhibitor by inhibiting multiple aspects of angiogenesis including EC tube formation on matrigel, VEGF or bFGF stimulated migration, attachment to fibronectin as well as inducing EC apoptosis. We further demonstrated that different sub- regions of LRR5 inhibit different aspects of angiogenesis. The middle region (LRR5M) is responsible for inhibiting EC tube formation. The C- terminal region (LRR5C) specifically inhibits chemotactic EC migration towards VEGF, but has no effect on bFGF induced EC migration. Both the N-terminal and middle regions are involved in inducing EC apoptosis. On the other hand, the N-terminal region (LRR5N) is more effective in inhibiting EC attachment to fibronectin. And we showed that the ability of LRR5 and LRR5M to inhibit VEGF induced EC tube formation is linked with their suppression of VEGF induced FAK activation, assembly of focal adhesions as well as actin stress fiber formation, but not through interference of EC attachment to matrix proteins. A summary of the LRR5 peptides is shown in Table 3.

VEGF is the most important angiogenic factor which induces EC proliferation, migration, focal adhesion complex formation and reorganization, remodeling of the actin cytoskeleton into stress fibers as well as tube formation, all necessary components of an angiogenic response. Except proliferation, LRR5 inhibited all aspects of VEGF induced angiogenesis. The correlation of potent anti-tube formation activity of LRR5 and LRR5M with their ability to inhibit VEGF induced FAK activation, recruitment of paxillin into focal adhesions and actin stress fiber formation strongly suggests that the FAK signaling pathways play an important role in VEGF induced EC tube formation on matrigel. The more potent anti-tube formation activity of LRR5M correlated with its more potent inhibition of FAK activation.

Although FAK signaling pathways are also involved in VEGF induced EC migration, LRR5 most likely inhibited EC migration through a FAK independent signaling pathway as LRR5C which inhibited EC migration did not suppress FAK activation. It has recently been reported that integrin- linked kinase (ILK), a signaling protein that interact with the cytoplasmic domains of integrin, also plays an important role in HUVEC vessel morphogenesis induced by VEGF and VEGF induced HUVEC chemotactic migration was suppressed by the inhibition of ILK (35). ILK also mediates actin filament rearrangements and cell migration and invasion through PI3K/Akt/Racl signaling (36). Whether LRR5C also inhibit VEGF stimulated HUVEC chemotactic migration through inhibition of ILK signaling pathway warrants future investigation.

Cell migration involves the dynamic assembly and disassembly of both focal adhesions and actin filaments. Although LRR5C did not inhibit VEGF induced focal adhesion assembly and actin stress fiber formation, whether it interferes with the disassembly or dynamic changes of them still need to be investigated. Furthermore, the dynamic cycles of FAK phosphorylation and dephosphorylation are essential of the dynamic changes of focal adhesions. As LRR5C slightly stimulated FAK phosphorylation, whether this peptide affects the dynamic changes of FAK phosphorylation/dephophorylation need further investigation.

Cell migration can be triggered not only through matrix mediated integrin- clustering and FAK activation, but also through growth factor/receptor activated multiple intracellular signaling pathways. In addition to FAK pathway, many other pathways such as PI3 kinase activation, p38- MAPK activation, Akt activation, induction of NO release, etc have also been linked with VEGF induced EC migration (32). It is possible that LRR5C inhibited VEGF induced EC migration through interfering with one of these pathways.

The signaling pathways that are involved in mediating EC tube formation are not thoroughly understood. As tube formation requires multiple processes including cell-cell adhesion, cell-matrix adhesion, cell fusion, apoptosis, as well as EC differentiation in addition to cell migration, inhibition of FAK activation most likely affected one or more of these processes which lead to inhibition of tube formation. Our data clearly indicated that inhibiting VEGF induced FAK signaling pathway in ECs is linked with the ability of LRR5 and LRR5M peptides to inhibit tube formation on matrigel. Peptides LRR5C and LRR5N, which could not or weakly inhibit VEGF induced FAK signaling pathways (as shown by FAK activation, paxillin relocation into focal adhesion complex as well as actin stress fiber formation), did not inhibit tube formation effectively.

It is well known that FAK signaling is involved not only in growth factor and integrin mediated cell migration, but also in cell proliferation, apoptosis as well as cell adhesion and spreading (37). Our results clearly indicate that the inhibition of FAK phosphorylation at Y397 by peptides LRR5 and LRR5M is linked to their ability to inhibit tube formation as well as induction of EC apoptosis through the caspase dependent pathway. Cell attachment and adhesion to matrix is a critical step in angiogenesis. It is known that cell adhesion and spreading on fibronectin and TSP1 substrates are inhibited by decorin (13, 15). Although LRR5 and LRR5N efficiently interfered with HUVEC attachment to fibronectin at low concentration, it seems that inhibition of EC attachment to matrix is not a critical mechanism by which LRR5 inhibited EC tube formation. This is because LRR5M potently inhibited EC tube formation at concentrations in which it is not very efficient in inhibiting HUVEC attachment to fibronectin (Figs. 6 and 10).

The binding affinities of the peptides to collagen I and fibronectin are low in the range of low mM to μM. Using FITC-labeled peptides, we observed that LRR5 and its related peptides do not enter cells or bind to cell surface (data not shown), indicating that they most likely function in the extracellular matrix. Although LRR3-5 region of decorin core protein has been reported to bind TGFβ, a growth factor involved in angiogenesis, LRR5 did not bind this growth factor when tested in binding conditions described in this work (data not shown). Most likely, the antiangiogenic activity of LRR5 is independent of decorin's ability to bind TGFβ.

Peptide therapeutics is increasingly making their way into clinical application. The advantage for peptides are their small sizes, simple to synthesis, lack of toxicity and immune reaction to host system, viable for chemical modifications (2). Several antiangiogenic peptides have been obtained from various domains of endogenous proteins including the Pex domain of MMP-2, lg domains of VEGFR1, TSR domains of TSP1 as well as endostatin (5, 28, 38, 39). This work represents the first report of antiangiogenic peptides derived from LRR domains.

It has been recently reviewed that antiangiogenic peptides tend to have high incidence of hydrophobic residues and fold as antiparallel β- sheet (40). A potent angiogenesis inhibitor, the 33 amino acids Anginex peptide, has been designed containing a β-sheet which is the bioactive conformation (41, 42). The 26 residue LRR5 peptide also has a β-sheet in addition to a stable 3₁₀ helix structure in solution as indicated by CD spectrum (Fig. 14). While preparing for this manuscript, the crystal structure of the dimeric protein core of decorin was reported (27) indicating that LRR VI of bovine decorin (equivalent to human LRR5 in this work) contains a 3₁₀ helix and a β-sheet, consistent with our CD results. LRR5 is at the center of the bananashaped decorin monomer molecule with the 3_J0 helix exposed to the external surface and the β-sheet at the dimerization interfaces. Amongst all the LRRs in decorin core protein, only LRR5 has the 3₁₀ helix structure (27). In addition, in many bioactive peptides, the presence of β-turn is critical for its function (43). All peptides described here also have β-turn structure which may be important for their biological activities.

In summary, LRR5 domain of decorin core protein shows potent antiangiogenic effect through inhibiting EC tube formation, migration, attachment to matrix as well as inducing EC apoptosis. Furthermore, different sub-regions of LRR5 affect different aspects of angiogenesis: LRR5M inhibits tube formation; LRR5C inhibits migration and LRR5N is most effective in inhibiting EC attachment to matrix. Both LRR5N and LRR5M are involved in inducing EC apoptosis. We further showed that the anti-tube formation activities of LRR5 and LRR5M are linked with their ability to suppress VEGF stimulated FAK phosphorylation at Y3_.97 as well as focal adhesion and actin stress fiber formation. LRR5M is much more potent in inhibiting tube formation than LRR5 and the parent molecule decorin.

Table 1. Cytotoxicity of LRR5 and its shorter peptides to ECs. The numbers presented are absorbance values at 460 nm using the EZ4U kit, measuring mitochondria enzyme activity. In this assay condition, a decrease in absorbance indicates reduction of live cells in the sample well which represents cytotoxicity. Mean of three experiments are presented. Around 3000 HUVECs/well were used in the 96 well plate assay.

Table 2. Kd values for the binding of LRR5 and its related peptides with collagen I and fibronectin. SE: standard error.

Table 3. Summary of antiangiogenic activities of LRR5 peptides. NE: not effective.

References for Example 1

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Example 2: Decorin peptides inhibit angiogenesis

Peptide synthesis The peptides were synthesized using a peptide synthesizer (Applied Biosystems, Inc., model 473 A) and then purified by HPLC C18 reverse- phase column using an acetonitrile gradient in 0.1% trifluoroacetic acid.

1. QMIVIELGTNPLKSSGIENGAFQGMK (hdLRR5) (26-mer) (SEQ ID No 2)

2. TALQLKSIGKMMSGIGVNPIFGEENQ (Scrambled dhLRR5) (26- mer) (SEQ ID No 3)

3. QMIVIELGTNPLK (N-hdLRR5) (13-mer) (SEQ ID No 4)

4. SSGIENGAFQGMK (C-hdLRR5) (13-mer) (SEQ ID No 5)

5. LGTNPLKSSGIE (M-hdLRR5) (12-mer) (SEQ ID No 6)

6. KLERLYLSKNQLKELPEKMPK (hdLRR3) (23-mer) (SEQ ID No 7)

7. TLQELRAHENEITKVRKVTFNGLN (hdLRR4) (26-mer) (SEQ ID No 8).

Angiogenesis assays

HUVEC migration assay

Cell migration assay was performed using 8Tm cell culture insert (Falcon) in 24-well format. HUVECs were pre-starved in culture medium + 2% serum for 3-5h before the experiment. Cell culture inserts were pre-coated with 0.2% gelatin or fibronectin (50ug/ml) on both sides and dried. Starved HUVECs were then incubated with all the various peptides individually for 30 mins at 37°C, 5%o C0₂ incubator.

The cells (3.0 x lO⁴ cells/insert) were then plated onto the top surface of cell culture insert in 24-well culture plate. Chemoattractant (VEGF or bFGF) containing medium was added to the bottom well and cells incubated for 6-14h. Cells at the upper surface of the inserts were gently removed with cotton bar. Cells at the bottom side of the inserts were fixed with 70% ethanol for 10-15 mins and the inserts were then dried. Cells were stained with 0.04%) Giemsa solution for 10 mins for analyses under microscope. Migrated cells were counted using the NIH Image Software.

Matrigel tube formation assay

Matrigel (from Chemicon or BD) was pre-coated onto the well surfaces of 96-well plate. HUVECs (, 1.5-2X10⁴ /well, passages < 6) were harvested and resuspended in medium (Ml 99 or CSC medium without growth factor) with 1% fetal bovine serum. Cells were then incubated with all the various peptides individually at various concentrations at 37°C cell culture incubator for 30 minutes before plating onto the 96-well and incubated for 12-16 hours. Tube formations were observed from 7-8h after cell plating. Angiogenesis were measured by taking 5 random field and the tube length and area were determined using the NIH Imager software.

TUNEL Apoptosis Assay was performed using the ApoAlert kit from Clontech. Briefly, HUVECs were seed into 4 well chamber slide with and treated with the peptides for 24 hrs. After fixing the cells with 4% paraformaldehyde, cells were incubated with nucleotide mix containing FITC-labelled UTP and TdT. Cells were counter-stained with propidium iodide (PI).

Alternatively, apoptosis was analyzed by directly DNA staining with Hoechst dye 33258 (500 ng/ml) after incubating with the peptides at various concentrations and fix the cells with acetone methanol (1 : 1) for 10 mins. Apoptotic cells were counted in random fields under a microscope (630 x magnification, 5 fields per sample).

Results

The results are shown in Figures 15 to 17. Figure 15 shows that Peptide hdLRR5 (SEQ ID No 2) inhibits VEGF induced HUVEC migration. Figure 16 shows that peptide hdLRR5 (SEQ ID No 2) inhibits tube formation in matrigel. Figure 17 shows that decorin peptide hdLRR5 induce EC apoptosis as determined by TUNEL labeling.

A 26 amino acids peptide corresponding to LRR5 of human decorin (ie hdLRR5; SEQ ID No 2) can inhibit tube formation in in vitro matrigel angiogenesis assay and inhibit endothelial cell migration (HUVECs) in response to VEGF. LRR5 was picked due to its unique L (leucine) organization comparing to all other LRRs in decorin, longer sequence with extra amino acids, potential ability to bind collagen and TGF-beta.

The decorin peptides tested are not cytotoxic to endothelial cells. Concentration ranges between lOpM to 1 mM were tested for its effect on metabolic status of the endothelial cell. No cytotoxicity was observed on cultured endothelial cells even at the concentration of 1 mM. The cytotoxicity levels are at par with other known angiogenesis inhibitors such as angiostatin and endostatin.

We have also shown that hdLRR5 (SEQ ID No 2) binds nitrocellulose- bound collagen (data not shown).

Example 3: Additional peptides Additional modified versions of LRR5:

IVIELGTNPLKSSGIENGAFQGMK (SEQ ID No 9) lELGTNPLKSSGIENGAFQGMK (SEQ ID No 10) LGTNPLKSSGIENGAFQGMK (SEQ ID No 11) TNPLKSSGIENGAFQGMK (SEQ ID No 12) PLKSSGIENGAFQGMK (SEQ ID No 13) LKSSGIENGAFQGMK (SEQ ID No 14) GIENGAFQGMK (SEQ ID No 15) ENGAFQGMK (SEQ ID No 16)

Additional alanine scans mutation of peptides: Based on M-LRR5:

AGTNPLKSSGIE (12-mer) (SEQ ID No 17) LATNPLKSSGIE (12-mer) (SEQ ID No 18) LGANPLKSSGIE (12-mer) (SEQ ID No 19) LGTAPLKSSGIE (12-mer) (SEQ ID No 20) LGTNALKSSGIE (12-mer) (SEQ ID No 21) LGTNPAKSSGIE (12-mer) (SEQ ID No 22) LGTNPLASSGIE (12-mer) (SEQ ID No 23) LGTNPLKASGIE (12-mer) (SEQ ID No 24) LGTNPLKSAGIE (12-mer) (SEQ ID No 25) LGTNPLKSSAIE (12-mer) (SEQ ID No 26) LGTNPLKSSGAE (12-mer) (SEQ ID No 27) LGTNPLKSSGIA (12-mer) (SEQ ID No 28)

Based on C-LRR5:

ASGIENGAFQGMK (13-mer) (SEQ ID No 29) SAGIENGAFQGMK (SEQ ID No 30) SSAIENGAFQGMK (SEQ ID No -31) SSGAENGAFQGMK (SEQ ID No 32) SSGIANGAFQGMK (SEQ ID No 33) SSGIEAGAFQGMK (SEQ ID No 34) SSGIENAAFQGMK (SEQ ID No 35) SSGIENGAAQGMK (SEQ ID No 36) SSGIENGAFAGMK (SEQ ID No 37) SSGIENGAFQAMK (SEQ ID No 38) SSGIENGAFQGAK (SEQ ID No 39) SSGIENGAFQGMA (SEQ ID No 40)

Similar sequences in other vertebrate species: QMIVVELGTNPLKSSGIENGAFQGMK (horse, sheep, cow, pig, dog

LRR5) (SEQ ID o 41)

NVLVIELGGNPLKNSGIENGAFQGLK (mouse LRR5) (SEQ ID No 42)

RMIVIELGGNPLKNSGIENGALQGMK (rat LRR5) (SEQ ID No 43)

QVIVLELGTNPLKSSGIENGAFQGMK (chicken LRR5) (SEQ ID No 44)

NVIVMELGSNPLSSSGVDNGAFADLK (zebrafish LRR5) (SEQ ID No

45)

Example 4: Treatment of cancer

A patient suffering from cancer is administered intravenously the peptide QMIVIELGTNPLKSSGIENGAFQGMK (SEQ ID No 2).

Claims

1. A peptide consisting of from 5 to 40 amino acids from the portion of decorin which contains the leucine-rich repeats (LRRs), or a variant of said peptide.

2. A peptide according to Claim 1 consisting of from 5 to 30 amino acids from the portion of decorin which contains the leucine-rich repeats (LRRs), or a variant of said peptide.

3. A peptide according to Claim 1 consisting of from 5 to 26 amino acids from the portion of decorin which contains the leucine-rich repeats (LRRs), or a variant of said peptide.

4. A peptide according to Claim 3 consisting of from 5 to 26 amino acids from LRR5 of decorin, or a variant of such a peptide.

5. A peptide according to Claim 4 which has the amino acid sequence QMIVIELGTNPLKSSGIENGAFQGMK (SEQ ID No 2); QMIVIELGTNPLK (SEQ ID No 4); or SSGIENGAFQGMK (SEQ ID No 5); LGTNPLKSSGIE (SEQ ID No 6), or a variant of said peptide.

6. A peptide according to any of Claims 1 to 3 consisting of from 5 to 24 amino acids from LRR4 of decorin, or a variant of said peptide.

7. A peptide according to Claim 6 which has the amino acid sequence TLQELRAHENEITKVRKVTFNGLN (SEQ ID No 8), or a variant thereof.

8. A peptide according to any of Claims 1 to 3 consisting of from 5 to 21 amino acids from LRR3 of decorin, or a variant thereof.

9. A peptide according to Claim 8 which has the amino acid sequence KLERLYLSKNQLKELPEKMPK (SEQ ID No 7), or a variant thereof.

10. A peptide consisting of no more than 250 amino acid residues comprising two or more peptides as defined in any of Claims 1 to 9, or a peptide as defined in any of Claims 1 to 9 and additional peptide sequence not derived from decorin, or a variant of said peptide of no more than 250 amino acid residues.

11. A peptide according to Claim 10 consisting of no more than 100 amino acid residues, or a variant thereof.

12. A variant of a peptide as defined in any of the preceding claims wherein in the variant one or two or three or four amino acids from the decorin sequence are replaced with another amino acid.

13. A variant according to Claim 12 wherein one or more of the replaced amino acids are non-naturally occurring amino acids.

14. A variant of a peptide as defined in any one of Claims 1 to 11 wherein the variant contains one or more non-peptide linkages in place of a peptide bond.

15. A peptide as defined in any of Claims 1 to 11 which is a cyclic peptide.

16. A polynucleotide encoding a peptide as defined in any of Claims 1 to 11.

17. An expression vector comprising a polynucleotide according to Claim 16.

18. A host cell comprising a polynucleotide according to Claim 16 or an expression vector according to Claiml7.

19. A method of making a peptide or variant thereof according to any of Claims 1 to 15 comprising chemically synthesising said peptide or variant.

20. A method of making a peptide as defined in any of Claims 1 to 11, the method comprising expressing the said peptide from a polynucleotide according to Claim 16 or an expression vector according to Claim 17 or in a host cell according to Claim 18.

21. A pharmaceutical composition comprising a peptide or variant thereof according to any one of Claims 1 to 15 and a pharmaceutically acceptable carrier.

22. A peptide or variant thereof according to any one of Claims 1 to 15 for use as a medicament.

23. A method of inhibiting angiogenesis in a patient, the method comprising administering a peptide or variant thereof according to any of Claims 1 to 15, or a polynucleotide according to Claim 16 or an expression vector according to Claim 17.

24. A method according to Claim 23 wherein the patient has or is susceptible to a condition in which there is undesirable angiogenesis.

25. A method of treating a patient who has or is susceptible to any of cancer, diabetic retinopathy, macular degeneration, rheumatoid arthritis, ulcers, endometriosis and psoriasis, the method comprising administering to the patient an effective amount of a peptide or variant thereof according to any of Claims 1 to 15, or a polynucleotide according to Claim 16 or an expression vector according to Claim 17.

26. Use of a peptide or variant thereof according to any of Claims 1 to 15, or a polynucleotide according to Claim 16 or an expression vector according to Claim 17, in the manufacture of a medicament for treating undesirable angiogenesis in a patient.

27. Use of a peptide or variant thereof according to any of Claims 1 to 15, or a polynucleotide according to Claim 16 or an expression vector according to Claim 17, in the manufacture of a medicament for treating a patient who has or is susceptible to any of cancer, diabetic retinopathy, macular degeneration, rheumatoid arthritis, ulcers, endometriosis and psoriasis.